The lung: Development, Aging and the Environment

Contents Preface, vii List of Contributors, ix Introduction, xiii Part I: Critical Events in Normal Lung Development and Aging 1 Lung Morphogenesis, Role of Growth Factors and Transcription Factors 3-12 Wellington V. Cardoso 2 Development of Airway Epithelium 13-32, Charles G. Plopper, Michelle V. Fanucchi 3 Development of the Airway Innervation 33-53, Malcolm P. Sparrow, Markus Weichselbaum, Jenny Tollet, Peter K. McFawn, John T. Fisher 4 Development of Alveoli 55-73, Stephen E. McGowan, Jeanne M. Snyder 5 Development of the Pulmonary Basement Membrane Zone 75-79, Michael J. Evans, Philip L. Sannes

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10 Development of the Pulmonary Surfactant System 149-167, Sandra Orgeig, Christopher B. Daniels, Lucy C. Sullivan 11 Development of the Pulmonary Immune System 169-176, Lisa A. Miller 12 Development of Antioxidant and Xenobiotic Metabolizing Enzyme Systems 177-185, Michelle V. Fanucchi 13 Compensatory Lung Growth: Relationship to Postnatal Lung Growth and Adaptation in Destructive Lung Disease 187-199, Connie C.W. Hsia, Robert L. Johnson Jr, Ewald R. Weibel 14 Pulmonary Transition at Birth 201-211, Stuart B. Hooper, Richard Harding 15 Normal Aging of the Lung 213-233, Kent E. Pinkerton, Francis H.Y. Green

6 Development of the Pulmonary Vasculature 81-103, Part II: Environmental Influences on Lung Rosemary Jones, Lynne M. Reid Development and Aging 7 Developmental Physiology of the Pulmonary Circulation 105-117, Steven H. Abman

16 Pulmonary Consequences of Preterm Birth 237-251, Kurt H. Albertine, Theodore J. Psysher

8 Development of Fluid Transport Pulmonary Epithelia 119-129, Jonathan H. Widdicombe

17 Role of Nutrition in Lung Development Before and After Birth 253-266, Richard Harding, Megan L. Cock, Cheryl A. Albuquerque

9 Physical, Endocrine and Growth Factors in Lung Development 131-148, Stuart B. Hooper, Megan J. Wallace

18 Influence of High Altitude on Lung Development and Function 267-275, David P. Johns, David W. Reid

vi

Contents

19 Genetic Factors Involved in Susceptibility to Lung Disease 277-289, Steven R. Kleeberger

25 Environmental Toxicants and Lung Development in Experimental Models 345-351, Michelle V. Fanucchi, Charles G. Plopper

20 Effects of Environmental Tobacco Smoke on Lung Development 291-299, Jesse Joad

26 Repair of Environmental Lung Injury During Development 353-362, Suzette Smiley-Jewell, Laura S. Van Winkle

21 Nicotine Exposure During Early Development: Effects on the Lung 301-309, Gert S. Maritz

27 Effects of Aging, Disease and the Environment on the Pulmonary Surfactant System 363-375, Sandra Orgeig, Christopher B. Daniels

22 Exposure to Allergens During Development 311-319, Laurel J. Gershwin

28 Environmental Determinants of Lung Aging 377-395, Francis H.Y. Green, Kent E. Pinkerton

23 Development of Atopy in Children 321-331, David B. Peden

Index, Pages 397-403

24 Effects of Air Pollution on Lung Function Development and Asthma Occurrence 333-343, Frank D. Gilliland, Rob McConnell

A color plate section follows the index

From the moment of birth, until the time of death, the lung is an essential organ that provides our bodies with oxygen and eliminates the carbon dioxide we produce. Our ability to reach our physical and mental potential throughout our entire life span is strongly influenced by the efficient functioning of our lungs. Over the last few decades it has become increasingly apparent that both environmental and genetic factors operating during early life can induce persistent alterations in the function and health of the respiratory system. The principal objectives of this book are, firstly, to concisely present current concepts of normal processes involved in the growth, maturation and aging of the lung, and secondly, to integrate the growing body of evidence relating to the influence o f environmental and genetic factors on the structure and function of the lung and on respiratory health in later life. A third objective is to identify future directions for research into factors influencing respiratory health. We consider that these are important objectives as respiratory illness is a major contributor to morbidity and death at all stages of life, from birth to senescence. For example, asthma affects millions of children and adults worldwide, with both the incidence of asthma and the number of asthma-related deaths increasing. In the USA alone, the number of asthma cases has been estimated at 12 million, with the estimated cost of asthma-related care rising from 6.2 billion dollars in 1990 to over 10 billion dollars in 1995. The dramatic increase in the incidence and cost of this chronic inflammatory disorder has resulted in enormous research funding being directed towards its prevention and treatment. With the increasing use of molecular and cellular technologies, our knowledge of biological processes involved in the development of the respiratory organs has expanded tremendously; as a result, new concepts regarding the control of lung development have rapidly evolved. In parallel with our greater understanding of normal development is the realization that a wide range of environ-

mental factors can impact upon the genetic program of lung development. Many such factors can result in persistent alterations in lung structure and function that can, in turn, lead to an increased susceptibility to respiratory illness throughout postnatal life. For example, an increasing body of epidemiological data suggests that early childhood events such as premature birth, early respiratory infections or exposure to allergens can predispose the individual to airway dysfunction and common respiratory disorders such as asthma and chronic obstructive airway disease (COPD), increasing the risk of death from respiratory causes. It is also evident that genetic polymorphisms can affect an individual's susceptibility to a range of environmental factots such as allergens, cigarette smoke, nutrient restriction and infection, and these are only now becoming better understood. With the increasing interest in early developmental origins of ill-health and the role of the environment in human biology, we believe it is timely to review the scientific literature relating to these important health issues. Our purpose is to integrate current knowledge of the impact of environmental factors that can influence lung development, susceptibility to respiratory illness and the rate of aging of the lung. Each of these aspects of lung biology is of direct relevance to an understanding of respiratory health, a matter that is likely to become increasingly important in an aging population. In preparing this book, we have aimed at making it accessible to not only those working in lung biology, but also to non-experts with a broad interest in human health. Our hope is that this book will be of value to all concerned with respiratory health, including thoracic physicians, respiratory scientists, members of the pharmaceutical industry, toxicological and environmental regulators, pediatricians, perinatologists and gerontologists.

Richard Harding, Kent E. Pinkerton, and Charles G. Plopper

List of Contributors

Steven H. Abman Department of Pediatrics University of Colorado School of Medicine The Children's Hospital Denver, CO USA Kurt H. Albertine Department of Pediatrics, Medicine, Neurobiology, and Anatomy University of Utah School of Medicine Salt Lake City, UT USA Cheryl A. Albuquerque Department of Obstetrics and Gynecology Santa Clara Valley Medical Center San Jose, CA 95128 USA

Christopher B. Daniels Department of Environmental Biology University of Adelaide Adelaide, SA Australia Michael I. Evans Department of Anatomy, Physiology & Cell Biology School of Veterinary Medicine University of California, Davis Davis, CA USA Michelle V. Fanucchi Department of Anatomy, Physiology & Cell Biology School of Veterinary Medicine University of California, Davis Davis, CA USA

Wellington V. Cardoso Pulmonary Center Boston University School of Medicine Boston, MA USA

John T. Fisher Department of Physiology Queens University Kingston Ontario Canada

Megan L. Cock Department of Physiology Monash University Clayton, VIC Australia

Laurel I. Gershwin School of Veterinary Medicine University of California, Davis Davis, CA USA

Frank D. Gilliland

Rosemary Jones

Department of Preventive Medicine USC Keck School of Medicine Los Angeles, CA USA

Harvard Medical School and Department of Anesthesia and Critical Care Massachusetts General Hospital Boston MA USA

Francis H.Y. Green Department of Pathology & Laboratory Medicine University of Calgary Calgary Alberta Canada Richard Harding Department of Physiology Monash University Clayton, VIC Australia Stuart B. Hooper Department of Physiology Monash University Clayton, VIC Australia Connie C.W. Hsia Department of Internal Medicine University of Texas Southwestern Medical Center Dallas, TX USA

Jesse Joad

Steven R. Kleeberger Laboratory of Pulmonary Pathobiology National Institute of Environmental Health Sciences Research Triangle Park NC USA Gert S. Maritz Department of Physiological Sciences University of the Western Cape Bellville South Africa Rob McConnell Department of Preventive Medicine USC Keck School of Medicine Los Angeles, CA USA Peter K. McFawn Department of Physiology University of Western Australia Nedlands, WA Australia

Department of Pediatrics School of Medicine University of California, Davis Davis CA USA

Stephen E. McGowan Department of Internal Medicine Veterans Affairs Research Service University of Iowa Iowa City, IA USA

David P. Johns Discipline of Medicine University of Tasmania Clinical School Hobart Tasmania Australia

Lisa A. Miller Department of Anatomy, Physiology & Cell Biology School of Veterinary Medicine University of California, Davis Davis, CA USA

Robert L. Johnson, Jr

Sandra Orgeig

Department of Internal Medicine University of Texas Southwestern Medical Center Dallas, TX USA

Department of Environment Biology University of Adelaide Adelaide, SA Australia

David B. Peden Division of Allergy, Immunology & Environmental Medicine The School of Medicine University of North Carolina Chapel Hill, NC USA Kent E. Pinkerton Center for Health and the Environment University of California, Davis Davis, CA USA Charles G. Plopper School of Veterinary Medicine University of California Davis, CA USA Theodore J. Pysher Department of Pathology University of Utah School of Medicine Primary Children's Medical Center Salt Lake City UT USA David W. Reid Discipline of Medicine University of Tasmania Clinical School Hobart Tasmania Australia tynne M. Reid Department of Pathology Harvard Medical School Children's Hospital Boston, MA USA Philip t. Sannes Department of Molecular Biomedical Sciences College of Veterinary Medicine North Carolina State University Raleigh, NC USA

Suzette Smiley-lewell Department of Anatomy, Physiology and Cell Biology School of Veterinary Medicine University of California, Davis Davis, CA USA Jeanne M. Snyder Department of Anatomy and Cell Biology University of Iowa College of Medicine Iowa City, IA USA Malcolm P. Sparrow Department of Medicine University of Western Australia Nedlands, WA Australia Lucy C. Sullivan Department of Microbiology and Immunology University of Melbourne Melbourne, VIC Australia Jenny Toilet Department of Physiology University of Western Australia Nedlands, WA Australia Laura S. Van Winkle Department of Anatomy, Physiology and Cell Biology School of Veterinary Medicine University of California, Davis Davis, CA USA Megan I. Wallace Department of Physiology Monash University Clayton, VIC Australia Ewald R. Weibel Institute of Anatomy University of Bern Bern Switzerland

Markus Weichselbaum Department of Medicine University of Western Australia Nedlands, WA Australia

Jonathan H. Widdicombe Department of Human Physiology University of California, Davis Davis, CA USA

The respiratory system is one of the most complex organ systems in the mammalian body. In the lower respiratory tract alone, well over forty distinct cell phenotypes have been identified. These cell populations are distributed throughout a very complex architectural framework which includes a series of branching contiguous tubular structures which terminate in an even more complex series of septated surfaces, the alveoli. The cell phenotypes are distributed in a highly heterogeneous fashion. Their phenotypic expression and functional interactions with other phenotypes and the associated matrix are unique to the microenvironment in which they reside. This book addresses two general questions. During the development of the respiratory system, what are the critical events that lead to the complex mature organ system? Secondly, what is the impact of environmental and genetic factors on these developmental events? Early in embryonic development, the respiratory system begins as an outpocketing of the primitive embryonic gut into the adjacent mesenchyme. This initial budding consists of two cell phenotypes, one epithelial in nature from the endoderm and the other mesenchymal from the surrounding splanchnopleure. The transformation from this small aggregation of cells, about the size of a period on this page, to the highly complex series of structures we know as the larynx, trachea, extrapulmonary bronchi, intrapulmonary bronchi, bronchioles and the alveolar gas exchange area involves tremendous change and growth. This can be best appreciated by realizing that for the average human adult, the surface area of the gas exchange area is similar to that of a tennis court. The key events that produce these changes are (a) overall growth, (b) branching morphogenesis, (c) cellular proliferation, (d) cellular differentiation and (e) matrix formation. In addition to enormous overall growth, the formation of this complex architecture is the result of branching morphogenesis of the budding epithelium into the surrounding matrix. The event of branching itself, and the fact that the cells forming this complex architecture remain the same size as the airways grow, requires a very high rate of cellular proliferation of both epithelial and mesenchymal components. In fact,

the process of branching morphogenesis itself relies heavily on differential proliferation of adjacent epithelial cell populations combined with focal programmed cell death. As the number and size of branches continues to grow and the number of cells continues to increase, many of the cellular populations in specific focal areas begin to differentiate for specific functional roles. The complex architecture that makes up the branching tubular tracheobronchial airway tree and septated alveolar gas exchange area relies on the differential formation and reorganization of the vast complex of matrix and fibrous connective tissue structures which are continually undergoing synthesis, reorganization, and, in some cases, removal. Epithelial tissue undergoes four general categories of branching morphogenesis during lung development: epithelium branches into mesenchyme to form airways, into mesenchyme to form airway secretory glands, into mesenchyme to form blood vessels, and, together with extracellular matrix, epithelium forms interalveolar septa. The growth of the tracheobronchial tree and associated blood vessels are the initial branching events; these two processes lead to the formation of the conducting airways and large vessels, pulmonary arteries, pulmonary veins, and bronchial arteries. These events occur early in gestation and are generally complete prior to the end of gestation. While the tracheobronchial airways grow and mature, branching morphogenesis forms the submucosal glands within the airway walls. The process of alveolarization, which produces interalveolar septa, is a branching event that involves not only epithelium, but vascular endothelium as well. Both cell types are in close interaction with fibroblasts and connective tissue elements for the reorganization of primary septa into secondary septa which leads to the formation of definitive alveoli. The very large increase in the number of cells required for the growth in size and complexity of the developing lung does not occur uniformly in the developmental process. It varies according to (a) tissue and cell compartment, (b) gestational age, (c) status of cellular differentiation and (d) the local microenvironment. In contrast to adults, in

which cell proliferation is very low, cell proliferation in the developing lungs of fetuses and infants is very high. The pattern, rate, and actual phenotypes undergoing active proliferation vary greatly within the developing lung, depending upon which tissue compartment and which cell population within that compartment are involved at any particular time. Active proliferation continues for a significant portion of in utero lung development and is highly dependent on the status of cellular differentiation occurring in the same site. The local microenvironment, determined by position within the airway or vascular trees, and the mix of cell types at each site, dictate the focal proliferation rate. These proliferative and morphogenetic events result in vast increases in almost all components of the respiratory system. This growth includes not only an increase in the volume of the lungs and trachea as a whole, but also increases in alveolar air space volume, capillary blood volume, and the volume of large airways and vessels. There are major increases in surface area including that on the epithelial side and that on the capillary endothelial side of the alveolar blood-air barrier. In addition, there are tremendous increases in the numbers of all structures, including alveoli, capillaries, and, of course, all of the cell components that make up the entire system. The processes of cellular differentiation and cellular proliferation appear to compete with each other during development. Cellular differentiation within the respiratory system begins primarily in late gestation and after birth, and involves a tremendous increase in the diversity of cell types. This includes expression of differentiated function for at least fifteen different epithelial phenotypes, including elements derived from the neuroectoderm, along with the endodermal derivatives from the gut. A wide variety of mesenchymal cells, including immune and inflammatory cells recruited from the circulation, can differentiate locally. As with the differentiation of most cell populations within the respiratory system, the differential expression of enzyme systems critical for interaction between the organism and its environment also show a primarily postnatal pattern of expression. This includes enzymes involved in bioactivation of a wide range of organic compounds and enzyme systems which manage antioxidant pools and Phase II detoxification systems which transform reactive metabolites into non-reactive excretable chemicals. These enzymes

include cytochrome P450 monooxygenases, epoxide hydrolase, glutathione pool regulation enzymes, glutathione s-transferases, glucuronyl transferases, SOD and GPx, catalase. In Part 2 of this book, it becomes abundantly clear that all of the processes and events involved in the development of the respiratory system are susceptible to perturbation by environmental influences. In considering the impact of different environmental influences on lung and airway development, we need to bear in mind that all of these developmental processes represent a continuum beginning early in embryonic life and continuing through old age to the death of the organism. It appears that a very restricted number of these events are critical for successful life outside the uterus. Birth disarranges some of these events, but does not substantially impede their progress. There appears to be a small number of these processes that during fetal life must reach a certain stage to permit viability of the newborn. The gas exchange area must have grown to sufficient size in relation to metabolic body size to promote adequate oxygenation. The blood-air barrier must be of sufficient area and be sufficiently thin to allow adequate exchange of gases between the alveolar air surface and the alveolar capillary blood. Pulmonary surfactant and associated proteins must be produced in sufficient quantity and with a wide enough distribution to ensure lowering of surface tension below that required to allow cyclic alveolar expansion and collapse with minimal expenditure of energy. An additional factor that needs careful consideration when evaluating the impact of environmental factors on the developing lung is that all of these processes, including timing of events and the pattern in which they unfold, is highly variable depending on the species. This becomes especially problematic when attempting to establish models for evaluating the level of risk which exposure to environmental factors may pose for the developing human lung and for identifying the mechanisms by which environmental factors modify developmental processes in the lung. Clearly further research and appropriate animal models are required before we can fully understand the important role that environmental and genetic factors play in the development of the human lung. It is hoped that such research will ultimately lead to a better quality of lifetime respiratory health and greater longevity for the human population.

Critical Events in Normal Lung Development and Aging

ISBN 0 12 324751 9

Part 1

Copyright © 2004 Elsevier

Chapter

Lung Morphogenesis, Role of Growth Factors and Transcription Factors W e l l i n g t o n V, ...C . ardoso

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Pulmonary Center, Boston University School of Medicine, Boston, MA, USA

INTRODUCTION

The term 'morphogenesis' refers to a coordinated series of molecular and cellular events that during development shape the structure of tissues and organs. In the lung this represents the process by which epithelial tubules and blood vessels are formed and patterned to ultimately generate the airways and alveoli. In most species this encompasses the pre- and postnatal period of life (Fig. 1.1). This chapter focuses on the mechanisms that regulate the initial events of lung morphogenesis and discusses how signaling molecules present in the early lung influence this process. Because most of the information available in the literature has been generated in mouse models, this species will be used as a reference throughout the text. Subjects such as regulation of lung cell differentiation and blood vessel formation are discussed in detail in other chapters and therefore will be only superficially reviewed here. Lung, thyroid, liver, and pancreas are examples of organs derived from the primitive foregut. 1 At an early stage of embryonic development, when gastrulation is completed, a single sheet of endodermal cells located outside the embryo invaginates to form the primitive gut tube. A number of transcription factors start to be expressed in the endoderm in overlapping but distinct domains along the anterior-posterior (A-P) axis of the gut tube. This roughly subdivides the gut endoderm into organ-specific domains in which specific cell fates are assigned. Subsequently, the endoderm in these areas undergoes bud morphogenesis to form organ primordia. Endodermal development is influenced not only by locally expressed transcription factors, but also by soluble factors that diffuse from adjacent cell layers to the endoderm. Soluble factors are critical in mediating paracrine interactions and establishing feedback loops that control The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

morphogenesis. It has been recently reported that a close contact between vascular endothelial cells and the gut endoderm is essential for induction of liver and pancreatic primordial buds. 2'3 Moreover, soluble factors secreted by developing structures in the neighborhood of the gut tube play a major role in the development of foregut derivatives. For example, fibroblast growth factor (Fgf) and bone morphogenetic protein (Bmp) signals from the heart and transverse septum (future diaphragm) are critical for the initial steps of hepatogenesis; 4'5 the notochord provides Fgf and activin signals necessary for initiation of a pancreatic program of gene expression by the endoderm. 6'7 The signals and mechanisms involved in specification of the endoderm to become lung are currently undetermined.

ONSET

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DEVELOPMENT

Primary lung buds are identified in humans around the fourth week of embryonic life; however, in species such as the mouse or rat, buds emerge much later, at midgestation (embryonic days E9.5 and E11.5, respectively). 8'9 Endoderreal buds form from each ventro-lateral side of the foregut and invade the adjacent mesoderm; these buds then grow caudally and ventrally, joining each other at the midline to form the primordial lung. At the site where the primary buds connect (future carina), the trachea develops. 1~ The endoderm generates a great variety of specialized cells that constitute the respiratory epithelium, such as alveolar type I and II cells, and airway secretory mucous, serous, Clara and ciliated cells, among others. The lung mesenchyme originates from cells of the lateral plate mesoderm (splanchnic mesoderm), which at an early developmental stage migrate to the primitive foregut. Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

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Mesodermal-derived mesenchyme of the distal lung generates blood vessels by vasculogenesis. 11 Vascular structures also form by angiogenesis from vessels that migrate from the aortic arches to the area of the nascent lung. Veins originate from the left atrium. As the lung develops, vascular and airway components become closer at the distal end of the respiratory tree to form the future alveolar-capillary barrier. Other mesenchymal-derived structures are cartilage that surrounds large airways, airway and vascular smooth muscle and the connective tissue of the lung. The pleura, like other mesothelial membranes such as the pericardium, originates from the somatic mesoderm.

Molecular regulation of lung bud initiation Little is known about the mechanisms that control the initial events of lung development. One of the earliest signs of lung development is local expression of thyroid transcription factor I (Ttfl) by the endoderm. 12 This homeoboxcontaining transcription factor, also known as Nkx2.1, or thyroid-specific enhancer binding protein-1 (T/ebp-1), is found in thyroid primordia, forebrain, pituitary gland, and the epithelium of the lung. Ttfl marks the sites where thyroid and lung will form (Fig. 1.2). Analysis of Ttfl knockout mice shows that this gene, although important for normal branching and differentiation, is not essential for initiation of bud morphogenesis of these organs. 13'14 Information from genetically altered mice also implicates signaling by retinoids, hepatocyte nuclear factor (Hnf3~), the zinc finger transcription factor Gli, and Fgf as critical regulators of this process. Retinoids are vitamin A derivatives that play an important role in development and homeostasis of a variety of organs. Retinoic acid (RA) is the active form of retinoids and exists as several isoforms, including all trans and 9-cis. R A results from a multi-step oxidation process from retinol (vitamin A). 15 Retinaldehyde dehydrogenase-2 (Raldh2) appears to be the critical enzyme in generating RA during organogenesis. 16-1s RA effects are mediated by two families of nuclear receptors, RARs and RXRs. These receptors, each comprising three isotypes (t~, ~ and y), heterodimerize to form the functional unit that transduces RA signaling. 15'19RARs and RXRs are expressed throughout lung development beginning at the earliest stages. 2~ High levels of Raldh2 and ubiquitous activation of a retinoic acid responsive element R A R E - l a c Z transgene in a reporter mouse at the onset of lung development (Fig. 1.2) suggest that RA signaling is highly active in the lung primordia. 22 Several studies implicate RA as an essential signal for lung bud initiation. Retinoid deprivation in vitamin A deficient rats or administration of retinoid antagonists in cultured embryos result in lung agenesis. 23'24 Disruption of RA signaling in double RAR t~ and [3 knockout mice results in unilateral lung agenesis and lung hypoplasia. 25 The mechanisms involved in these abnormalities have not been established. Hnf3[3, recently renamed Foxa2, is a member of the Hnf-3/winged helix/forkhead family of transcription factors. Hnf3~l plays an essential role in early gut morphogenesis.

In Hnf3~ null mutant mice the endoderm fails to invaginate and the gut tube does not form. Consequently development of all gut derivatives is impaired and embryos die by day E9.5. 26 Interestingly, in normal mice Hnf3~ expression is maintained in the lung epithelium throughout embryonic and adult life, where it controls expression of differentiation genes. 27 Glis are zinc finger transcription factors expressed in the foregut mesoderm and in the developing lung mesenchyme. 28'29 Glis are believed to transduce signaling by Sonic hedgehog (Shh, discussed below). Individually or in combination, the three members of this family (Glil-3) appear to play different roles in lung development. Remarkably, when Gli2 and Gli3 are simultaneously inactivated in knockout mice, no lungs or trachea are formed and other foregut derivatives such as stomach and pancreas are hypoplastic. 3~This phenotype is intriguing for two reasons: first, it is more severe than that found in Shh null mice, in which lungs are formed, 31 suggesting that Gli2 and 3 may be signals shared by other pathways besides Shh; secondly, Glis have not been shown to be locally expressed at prospective sites of lung formation and it is not known whether they induce local expression of soluble factors to act on the endoderm. Thus it is unclear how the lack of Glis so dramatically affects lung bud initiation. Presumably Glis are critical for maintaining Hnf3~ expression and consequently overall endodermal survival. Supporting this hypothesis, low levels of Hnf3 have been described in Gli2 and Gli3 null mice. 3~ Primary lung bud formation requires activation of Fgf signaling by foregut endodermal cells. Mice lacking Fgfl0 or its receptor Fgfr2b do not have lungs. 32'33 As discussed later, Fgfl0 induces budding by binding to and activating Fgfr2 signaling in the endoderm. Fgfr2 is expressed throughout the foregut endoderm, while Fgfl0 is found in the mesoderm at sites of prospective lung bud formation. 34'35 Fgfl0 is a chemoattractant and a proliferation factor for endodermal and endodermal-derived epithelial cells. 36'37 The mechanism elicited by Fgfl0-Fgfr2 appears to be a rather general strategy to form buds; disruption of Fgfr2b signaling dramatically affects development of other foregut derivatives including thyroid and pancreas, and structures such as the limbs. 38

Formation of the trachea There is morphological and genetic evidence suggesting that the trachea and lungs originate by independent processes. A classical study from Spooner and Wessels 1~ shows that in mice, formation of lung buds precedes tracheal formation. A striking observation from Fgfl0 knockout mice is the absence of lungs in the presence of a well-formed and apparently normal trachea. 32 Thus, the mechanism elicited by Fgfl0-Fgfr2 to generate buds seems to be dispensable to form the tracheal tube. Interestingly, Fgfr2 is highly expressed in the endoderm of the prospective trachea but, besides Fgfl0, no other Fgf with similar high-affinity binding for this receptor has been reported in this region.

Lung Morphogenesis

Several mechanisms have been proposed to explain how the tracheal tube forms and separates from the developing foregut. It is currently accepted that once lung buds form and fuse in the midline, a septum growing from caudal to cranial regions separates tracheal and esophageal compartments. Alternatively it is thought that separation occurs by fusion of endodermal ridges growing from each side of the foregut; as they meet in the midline, two tubes form. 39'4~ In addition, a mechanism involving local activation of programmed cell death in the endoderm has been proposed. 41 Tracheo-esophageal fistula, a relatively common abnormality of human tracheal development, results from partial to complete lack of separation of the respiratory tract from the esophagus. 42 This abnormality has been reported in a number of knockout mice, including Shh-/-31 (see below), Ttfl _/_13 and Gli2-/-; Gli3 +/_.30 Retinoids are also essential for normal tracheal development because in vitamin A deficient rat embryos and RARtx/I]2 double mutant null mice, tracheo-esophageal fistula is observed. 23'25

BRANCHING

MORPHOGENESIS

To generate the bronchial tree, primary buds undergo branching morphogenesis. This patterning event, also found in other tree-like developing structures, involves bud outgrowth, bud elongation and subdivision of the terminal units by reiterated budding or by formation of clefts between buds. In mice it starts at around day El0.5, as secondary buds arise, and extends up to day -~E17, when the distal lung expands to form saccules (Fig. 1.1). 9,43 The role of epithelial-mesenchymal interactions in regulating branching morphogenesis and differentiation has been well demonstrated by classical and recent studies using embryonic lung cultures. These studies have demonstrated that the respiratory epithelium is able to change its pattern of growth and differentiation when recombined

with mesenchyme of different origins. This is best exemplified by the ectopic induction of distal lung buds from the tracheal epithelium when trachea is cultured in the presence of distal lung mesenchyme. 1~ Diffusible factors in concert with transcription factors form local networks that mediate these epithelial-mesenchymal interactions.

FGF signaling as a driving mechanism for branching The fibroblast growth factor family comprises more than 20 ligands which signal via four tyrosine kinase receptors (Fgfrl-4). Interactions of ligand and receptor with heparan sulfate proteoglycans are fundamental to form a stable Fgf-Fgfr complex and to properly transduce Fgf signaling. 45 Fgfs are found in a wide variety of species and their role in epithelial branching has been remarkably conserved during evolution. In Drosophila, expression of the Fgf ligand branchless (Bnl) is detected in cells near tracheal epithelial tubules at prospective sites of budding. Bnl acts as a chemotactic factor for the epithelium. Bnl diffuses to bind and activate an Fgfr (breathless, btl) expressed by the epithelium; epithelial cells then elongate and migrate toward Bnlexpressing cells, resulting in bud formation. 46 An analogous mechanism involving Fgfl0 and Fgfr2 appears to control branching of the developing mouse lung. These regulators are already present at the onset of lung development and are essential for bud initiation. 3z During branching Fgfl0 is expressed in a dynamic fashion in the lung mesenchyme; transcripts have been localized to sites where distal epithelial buds will form (Figs 1.2 and 1.3). In turn Fgfr2, which binds to Fgfl0 with high affinity, is evenly expressed along the respiratory tract epithelium and is locally activated by Fgfl0. 35'47 Studies in organ culture show that a heparin bead soaked in recombinant Fgfl0 placed near a mesenchyme-free lung epithelial explant induces the epithelium to migrate and proliferate toward the bead. Moreover, if the dynamic pattern of Fgfl0 is disrupted by engrafting

Pseudoglandular ~'%'.'~ Saccular t i .....] Canalicular Illllmlll Alveolar

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Fig. 1.1. Stages of lung development in humans and mice. During the pseudoglandular stage most branching morphogenesis occurs, and the lung has a gland-like appearance with epithelial tubules separated by thick mesenchyme; during the canalicular stage, airway branching is completed, the mesenchyme becomes thinner leading to an approximation between the epithelial tubules and blood vessels. During the saccular stage the distal lung expands to form saccules, and type I and type II cells differentiate. During the alveolar stage, septation of saccules gives rise to mature alveoli, d: day; PN: postnatal; w: week; y: years. (Reproduced with permission from Malpel S, Cardoso WV. Lung development. In: Encyclopedia of Life Sciences, Volume 11, London: Nature Publishing Group, 2002; p. 164.)

Fig. 1.2. Molecular regulators of lung morphogenesis. Retinoic acid synthesis and utilization in the foregut at the onset of lung (lu) development: high levels of expression of Raldh2 (A) and ubiquitous activation of RARE-lacZ transgene (B). (C) Ttfl expression in lung and thyroid (th) primordia. During branching morphogenesis (D) Fgfr2b is expressed throughout the respiratory tract epithelium, while (E) Fgfl0 and (F) Bmp4 are localized to the distal mesenchyme and distal epithelium, respectively. (G) Distal epithelial expression of Shh in lung culture. (H) Fgfl0 is a chemoattractant factor for distal epithelial buds; an Fgfl0 bead engrafted onto lung organ culture is engulfed by distal buds (black arrowhead). (A, B) E9.5 days; (C) El0 days; (D-F) E11.5 days; (G, H) E11.5 + 24 h lung culture. A, C, D-G: whole mount in situ hybridization; B: X-gal staining. Arrowheads point to signal in each panel. (See Color plate 1.)

an Fgfl0-containing bead onto cultured embryonic lungs, the local 'static expression' of Fgfl0 deviates airway growth toward the bead and alters the branching pattern. 36'37 The unique pattern of expression of Fgfl0 in the early lung suggests that Fgfl0 is involved in the spatial control of lung bud formation in vertebrates. Fgfl 0 and the Drosophila branchless may be evolutionarily related and may regulate epithelial morphogenesis via a similar mechanism. However,

in contrast to mice, tracheal budding in flies does not involve cell proliferation and occurs solely by cell migration. 46 During branching, the mesenchyme seems to modulate the responses of the epithelium to Fgfl0. There are in vitro data showing that in the absence of mesenchyme, Fgfl0 induces budding in both proximal and distal epithelial rudiments. However, when the epithelium and mesenchyme are cultured together, Fgfl0 elicits budding only in

Lung Morphogenesis

distal lung. 36'37 This is intriguing since Fgfr2b is expressed in both proximal and distal epithelium at seemingly equal levels. 47 Presumably the proximal mesenchyme contains factors that inhibit Fgfr2 activation by Fgfl0 or prevent Fgfl 0-induced bud morphogenesis from occurring.

Control of branching morphogenesis

Fig. 1.3. Signaling molecules in early lung development. (A) Expression of Fgfl0 in the distal mesenchyme activates Fgfr2b signaling in the epithelium and budding (arrow) is initiated. (B) As the bud grows, the tip bud epithelium interacts with Fgfl0-expressing cells; Fgfl0 induces Bmp4 and epithelial cell proliferation is inhibited; Shh inhibits Fgfl0 expression to extinguish the chemoattractant source. (C) Fgfl 0 appears at different sites to induce a new generation of buds (arrow). A cleft is formed in between buds. Tgf[3 1 at the subepithelial mesenchyme prevents local budding by inhibiting Fgfl0 expression and inducing synthesis of extracellular matrix components, which stabilize the cleft. Other factors such as epidermal growth factors (EGO,hepatocyte growth factors (Hgf), Pdgf and Fgf7 are expressed in the distal mesenchyme in a more diffuse fashion and contribute to the overall growth of the branching tubules besides having other functions. Model based on Bellusci et al. 35 and Lebeche et al. 48 (See Color plate 2.)

As branching morphogenesis proceeds, the exchange of signals between epithelial and mesenchymal cell layers of nascent buds establishes feedback loops that control airway size, branching patterns and cell fate. Signaling molecules differentially expressed at the tips of the branching tubules form a distal signaling centre that is critical in controlling these events (Figs 1.2 and 1.3). Correct branching requires a precise control of Fgfl0 levels in time and space. There is evidence that at least some of the factors that regulate Fgfl0 expression are present in the epithelium of the developing bud. When embryonic lung mesenchymal cells are cultured in the absence of the epithelium, Fgfl0 mRNA levels markedly increase, 48 suggesting that the epithelium secretes diffusible factors that are inhibitory for Fgfl 0 gene expression. One of these factors seems to be Sonic hedgehog (Shh). Shh is an important signaling molecule expressed in a proximal-distal gradient with the highest levels at tips of the bud epithelium (Fig. 1.2). Its major receptor, Patched-1 (Ptc 1), and downstream mediators of Shh signaling, the transcription factors Glil-3, are expressed in the mesenchyme. 29'49 Shh induces mesenchymal cell proliferation and regulates the expression of a number of mesenchymal genes. 31 Shh, either as a recombinant protein in organ cultures or overexpressed in lungs of transgenic mice, inhibits Fgfl0 expression. 35'48 Lungs from Shh knockout mice show disrupted airway branching and resemble rudimentary sacs, but proximaldistal differentiation is preserved. Interestingly, in these mice Fgfl0 expression is no longer focal as in wild type, but becomes rather diffuse. 31 Thus, distal epithelial expression of Shh in the growing bud may function to locally inhibit Fgfl0 expression in the mesenchyme and prevent widespread distribution of Fgfl0 signals. Expression of different Glis in the lung occurs in somewhat overlapping domains; however, sites such as the subepithelial mesenchyme show the highest levels of Glil. 29 The role of Gli genes in lung bud initiation has been previously discussed. Disruption of Gli3 gene expression leads to defects in specific lobes of the lung. 29 Fgf signaling and airway branching are also controlled by a family of cysteine-rich proteins collectively called Sprouty (Spry). First described in Drosophila, it gained this name because Spry mutant flies show an increased number of tracheal branches. 5~ Spry is induced by the FGF ligand branchless at the bud tips and results in inhibition of lateral budding. In mice, four family members have been identified. 51 Spry2 and Spry4 are expressed in the developing distal lung in the epithelium and mesenchyme, respectively. 52-54 Disruption of Spry2 in lung cultures using anti-sense oligonucleotides shows a stimulatory effect on distal branching

: The~: E:Ung: Development; :Aging andthe Envi ~onment~

and differentiation. 52 By contrast, overexpression of Spry2 in the distal lung epithelium of transgenic mice inhibits branching and epithelial cell proliferation. 54 Bone morphogenetic protein-4 (Bmp4) is a member of the T g ~ superfamily of growth factors and is expressed during branching morphogenesis at high levels and in a dynamic fashion at the bud tip epithelium (Fig. 1.2). 55,56 Bmp4 signaling is transduced by type I and type II serinethreonine kinase receptors and Smad transcription factors. 57 In the developing lung Bmp4 appears to restrict cell proliferation and assign a distal cell fate to the bud epithelium. Targeted disruption of Bmp4 signaling in the lung epithelium of transgenic mice results in proximalization. In these mice, the peripheral lung is populated by proximal cell phenotypes, such as ciliated cells or secretory Clara cells. 56 In turn, overexpression of Bmp4 in the distal epithelium results in small lungs containing distal flat cells that resemble alveolar type I cells. 55 Fgfl0 controls Bmp4 levels. In organ cultures, Bmp4 expression is induced in the distal epithelium that surrounds an engrafted Fgfl0 bead. 37'48 Bmp4 antagonizes the Fgfl0 effects by inhibiting distal epithelial cell proliferation and preventing bud formation (Fig. 1.3). Bmp signaling is also controlled by antagonists such as Noggin, Chordin and the Cerberus-related factor Cerl, 56'57 all expressed in the developing lung. Noggin is a secreted molecule that binds to Bmp4 with high affinity and prevents it from binding to Bmp receptors. During branching Noggin is expressed at low levels in the distal lung mesenchyme until around day 13.5; Noggin is also expressed in the dorsal mesenchyme of the trachea in a pattern complementary to Bmp4. 56 Noggin null mutants, however, do not seem to have abnormal lungs. 58 Several other Tgf~ superfamily members are present in branching airways. Tgf~ 1 transcripts are uniformly expressed throughout the subepithelial mesenchyme, although the protein accumulates at sites of cleft formation and along proximal airways. 48's9 Tgffl 1 inhibits Fgfl0 expression and is a potent negative regulator of epithelial cell proliferation, differentiation and branching in lung organ cultures. 6~ Moreover, Tgf]] 1 induces synthesis of extracellular matrix which, when deposited in the epithelial-mesenchymal interface, is thought to prevent local branching (Fig. 1.3). 59 Recent data suggest that establishment of a distal signaling centre and branching are antagonized by RA signaling. In contrast to its role in primary lung bud initiation (see above), RA does not seem to be necessary for branching morphogenesis. As shown by an RARE-lacZ reporter mouse, the appearance of secondary buds and subsequent branching is marked by a sharp downregulation of RA signaling in the lung in both epithelium and mesenchyme. 22 Studies in RA-treated lung organ cultures show that preventing this downregulation from occurring with exogenous RA disrupts distal lung formation and maintains the lung in a proximallike immature stage. 61'62 In these cultures, levels and distribution of genes involved in distal lung formation are markedly altered; Fgfl0 expression is inhibited and Bmp4 signals at bud tips are low and diffuse. 22

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Another factor that is essential for airway branching and differentiation is Ttfl (Nkx2.1). Ttfl is expressed in the lung epithelium from its earliest stages; as branching proceeds, a proximal-distal gradient is identified with the highest levels at the tips. 12 Ttfl knockout mice have highly abnormal dilated hypoplastic lungs, airway branching is markedly disrupted and overall development appears to be arrested at early pseudoglandular stage. 13 The role of Hox proteins in lung pattern formation remains controversial. In Drosophila, mutation of these transcription factors results in dramatic changes in anterior-posterior specification of the body plan. 63 Many Hox genes are expressed in the developing lung, 64 however, only Hoxa5 shows lung abnormalities when inactivated in knockout mice. Lungs from Hoxa5-/- mice are immature and tracheal development is disrupted. 65 Left-right a s y m m e t r y One of the least understood and most intriguing patterning events in organogenesis is the establishment of left-right (L-R) symmetry. Left and right lungs have highly stereotypical but different branching patterns and number of lobes, which vary according to species. L-R patterning of the lung is linked to the general body plan and is actually initiated well before lungs are formed. Lefty-1 and-2, nodal and Pitx-2 have been identified as major regulators of L-R asymmetry in viscera. 66 When expression of these transcription factors is disrupted in mice, laterality defects known as pulmonary isomerisms are found. 67 These defects are characterized by abnormally symmetric lungs. In wild-type mice, the left and right lungs consist of one and four lobes, respectively; however, in Lefty knockout mice single-lobed lungs are found on each side. 67 Paradoxically, while branching is influenced by Lefty-1, this regulator is not expressed by the developing lung. Like Lefty-2 and nodal, Lefty-1 is expressed only during a short window of time around days E8-8.5, on the left side of the prospective floor plate and lateral plate mesoderm. This suggests that some patterning decisions have already occurred when organ primordia arise. Other signaling molecules such as Shh, RA, Gli and activin receptor IIb have been implicated in L-R asymmetry in the lung. 3~

REGULATION OF PROXIMAL-DISTAL DIFFERENTIATION Although defined cell phenotypes are mostly seen around the time of birth, morphological and molecular features of differentiation can be detected much earlier, when the respiratory tree is being formed. The first molecular marker of differentiation expressed by lung epithelial cells is the surfactant protein gene SP-C. SP-C has been detected in the mouse lung in the distal epithelium at around day E10.5, when secondary buds form. 7~Other surfactant protein genes, SP-A, SP-B and SP-D, are consistently detected 3-4 days later. 71-73 By this time, morphological differeflces between

Lung Morphogenesis distal (cuboidal) and proximal (tall columnar) cells are obvious. Differentiation of the distal epithelium into type I and type II cells occurs around the period of sacculation (in mice day - E l 7 ) . 9 Type II cells express surfactant protein genes and contain lamellar bodies, cytoplasmic inclusions that store surfactant material. Type I cells are characteristically flat and express the markers Aquaporin 5 and TI~. 74'75 Regulation of the type I cell phenotype is still little understood (see Chapter 9). It has been suggested that factors such as Bmp4 may favor formation of type I over a type II cell phenotype. 55 By contrast, Fgf7 inhibits type I cell markers and induces surfactant protein gene expression. 75,76 Signaling molecules involved in early patterning events have been shown to also act as regulators of epithelial differentiation at later developmental stages. Ttfl, Hnf3[3 and Gata6 induce surfactant protein gene expression in lung epithelial cell lines. 77 T g ~ l , besides inhibiting branching in vitro, prevents distal lung maturation when ectopically expressed in the distal lung epithelium of transgenic mice. TM The Hnf3/forkhead homologue transcription factor Hfh4 (Foxjl) is essential for the development of ciliated cells and also serves as a proximal marker of lung differentiation. Hfh4 is expressed in the mouse lung from day El4 to El5 onwards. 79 Loss or gain of function of Hfh4 in genetically altered mice leads, respectively, to absence or ectopic formation of ciliated cells in target organs. Hfh4 knockout mice, besides lacking ciliated cells, also display laterality defects in internal organs, s~ In Spc-Hfh4 transgenic mice, misexpression of Hfh4 in the distal lung leads to the appearance of ciliated cells in peripheral areas and suppression of surfactant protein expression, sl Clara cell 10kDa protein (CC10) is expressed by non-ciliated proximal epithelial cells of secretory nature; transcripts are first identified around day El6 in murine lungs. Ttfl, Hnf3 and C/EBP transcription factors have been reported to be regulators of CC10 expression. 73'82

CONCLUSIONS The recent use of molecular and genetic tools to approach classical questions in developmental biology has advanced the current understanding of how signaling molecules control organogenesis. While much information has been generated regarding early lung morphogenesis, less is known about the molecular regulation of alveolarization. This formidable patterning event that takes place in the distal lung subdivides the pre-existing saccules into smaller units, the mature alveoli, and greatly increases the surface area for gas exchange. Septation of the primary saccules appears to be dependent on interstitial myofibroblasts and seems to occur under tight control of elastin levels. There is evidence that signaling by platelet-derived growth factor (Pdgf), Fgf and retinoic acid is involved in this process. Pdgf signaling is necessary for the development of lung myofibroblasts; RA is thought to promote elastin synthesis while Fgfr3 and -4 control elastin gene expression; glucocorticoid hormones

9

oppose the RA effects. 83-86 During neonatal and adult life, expression of signaling molecules such as Tgf~s, Fgfs and Shh has been reported in the lung, presumably to maintain homeostasis. 87-89 There is evidence that at least some of these signals are recruited to mediate cellular activities during injury-repair. 87 Studies aimed at understanding the molecular basis of lung development may provide useful insights into lung regeneration that can be potentially applied to areas such as stem cell therapy and tissue engineering for lung diseases.

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38. Ohuchi H, Hori Y, Yamasaki M e t al. FGF10 acts as a major ligand for FGF receptor 2 IIIb in mouse multi-organ development. Biochem. Biophys. Res. Commun. 2000; 277:643-9. 39. Sutliff KS, Hutchins GM. Septation of the respiratory and digestive tracts in human embryos: crucial role of the tracheoesophageal sulcus. Anat. Rec. 1994; 238:237-47. 40. Zaw-Tun HA. The tracheoesophageal septum - Fact or fantasy? ActaAnat. 1982; 114:1-21. 41. Zhou, Hutson JM, Farmer PJ, Hasthorpe S etal. Apoptosis in tracheoesophageal embryogenesis in rat embryos with or without adriamycin treatment, ft. Pediatr. Surg. 1999; 34:872-5. 42. Landing BH, Dixon LG. Congenital malformations and genetic disorders of the respiratory tract (larynx, trachea, bronchi and lungs).Am. Rev. Respir. Dis. 1979; 120:151-85. 43. Hogan BLM. Morphogenesis. Cell 1999; 96:225-33. 44. Shannon JM. Induction of alveolar type II cell differentiation in fetal tracheal epithelium by grafted distal lung mesenchyme. Dev. Biol. 1994; 166:600-14. 45. Szebenyi G, Fallon JF. Fibroblast growth factors as multifunctional signalling factors. Int. Rev. Cytol. 1999; 185:45-106. 46. Sutherland D, Samakovlis C, Krasnow MA. Breathless encodes a Drosophila FGF homolog that controls tracheal cell migration and the pattern of branching. Cell 1996; 87:1091-101. 47. Cardoso WV, Ito A, Nogawa H et al. FGF-1 and FGF-7 induce distinct patterns of growth and differentiation in embryonic lung epithelium. Dev. Dyn. 1997; 208:398-405. 48. Lebeche D, Malpel S, Cardoso WV. Fibroblast growth factor interactions in the developing lung. Mech. Dev. 1999; 86:125-36. 49. Bellusci S, Furuta Y, Rush MG et al. Involvement of Sonic hedgehog (Shh) in mouse embryonic lung growth and morphogenesis. Development 1997; 124:53-63. 50. Hacohen N, Kramer S, Sutherland D et al. Sprouty encodes a novel antagonist of FGF signalling that patterns apical branching of the Drosophila airways. Cell 1998; 92:253-63. 51. Minowada G, Jarvis LA, Candace L e t al. Vertebrate sprouty genes are induced by FGF signalling and can cause' chondrodysplasia when overexpressed. Development 1999; 126:4465-75. 52. Tefft DT, Lee M, Smith S etal. Conserved function ofmSpry-2, a murine homolog of Drosophila sprouty, which negatively modulates respiratory organogenesis. Current Biol. 1999; 9:219-22. 53. DeMaximy AA, Nakatake Y, Moncada S et al. Cloning and expression pattern of a mouse homologue of Drosophila sprouty in the mouse embryo. Mech. Dev. 1999; 81:213-16. 54. Mailleux AA, Tefft D, Ndiaye D etal. Evidence that SPROUTY2 functions as an inhibitor of mouse embryonic lung growth and morphogenesis. Mech Dev. 2001; 102:81-94. 55. Bellusci S, Henderson R, Winnier Get al. Evidence from normal expression and targeted misexpression that bone morphogenetic protein-4 (Bmp-4) plays a role in mouse embryonic lung morphogenesis.Development 1996; 122:1693-702. 56. Weaver M, Yingling JM, Dunn NR etal. Bmp signalling regulates proximal-distal differentiation of endoderm in mouse lung development. Development 1999; 126:4005-15. 57. Massague J, Chen YG. Controlling TGF signalling. Genes Dev. 2000; 14:627-44. 58. Brunet LJ, McMahon JA, McMahon AP et al. Noggin, cartilage morphogenesis, and joint formation in the mammalian skeleton. Science 1998; 280:1455-7. 59. Heine UI, Munoz EF, Flanders KC etal. Colocalization of TGF-[31 and collagen I and III, fibronectin, and glycosaminoglycans during lung branching morphogenesis. Development 1990; 109:29-36. 60. Serra R, Moses HL. pRb is necessary for inhibition of N-myc expression by TGF-1 in embryonic lung organ cultures. Development 1995; 121:3057-66.

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Development of Airway Epithelium Charles G. Plopper* and Michelle V. Fanucchi SchoolUepartm ent ot An atomy, Ph ys,ology a n d Cell B,o, ogy, of Veterinary Med. icine, Uni:versitg:of:California, uaws, c,~, ua,~ r ' ~

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INTRODUCTION

A number of developmental processes are involved in the establishment of the tracheobronchial airway tree. The pattern of branching of the airways including the angle of branching and the proportions of daughter branches in relation to parent airway appear to be established relatively early by the process of branching morphogenesis. As summarized in detail in Chapter 1, this process is initiated with the earliest formation of respiratory tract structures in the thorax in the embryonic period and continues for a substantial period of time during early gestation. It is heavily dependent on epithelial-mesenchymal contact and continual interaction to regulate the rate and pattern of formation. The composition of the wall of the airways in adults varies substantially between different segments, with most of the differences being highly polarized from more proximal airways to more distal airways. The major components of the wall include: (1) the surface lining epithelium with its associated derivative, the submucosal gland, (2) the basement membrane zone, including basal lamina and an extended population of fibroblasts, and (3) bundles of smooth muscle and cartilage. The distribution of all these components varies substantially within the airway tree in adults. The entire wall is invested with a large number of nerves that appear to be in two separate distributional patterns, one associated with the epithelial surface and another associated with the glands and smooth muscle in the submucosa and adventitia. As detailed in Chapter 3, the formation of *To whom correspondence should be addressed. The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

the nerves occurs early in development, once the pattern of the airway tree has been laid down. The presence of nerves in the wall, however, does not establish that they have processes that extend into the epithelial compartment or directly to the smooth muscle. It is not clear when this occurs, but it apparently occurs during the differentiation process. Chapter 3 also addresses airway smooth muscle and establishes that it is differentiated early in development once the basic pattern of the wall airway has been laid down. Once the basic geometric pattern of the airways has been established, they undergo substantial enlargement through longitudinal and circumferential growth. The great increase in cell and tissue mass necessary to accomplish growth relies on active proliferation of resident cell populations and the ability of the same cell populations to synthesize and secrete matrix components. How these processes are established and regulated and how they are balanced with forces promoting differentiation of the same cell populations is not understood. Further, these complex processes continue for a substantial period of time after birth. The temporal pattern for the differentiation processes varies significantly by species, but always moves in a proximal to distal direction with time. What this means is that during pre- and postnatal development of the airways, different airway generations will be in different stages of development. At any given time point, more proximal airway generations will be more differentiated than the more distal generations. Because the other aspects of airway development have been defined in Chapters 1 and 3, this chapter will emphasize the epithelium and its pattern of differentiation and what is known about regulation of the differentiation process. Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

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PHENOTYPIC ADULTS

This chapter is organized on the premise that understanding the development of cellularly and architecturally complex organ systems such as the respiratory system, especially in the case of tracheobronchial airways, requires definition of changes based on specific airway sites and clear distinction of the timing of events in these sites. One of the major considerations in evaluating the potential toxicity of environmental contaminants for the developmental process is understanding which of the compartments is in which stage of development and differentiation at the time of exposure. Further understanding of airway development and the mechanisms that regulate it needs to be based on a clear understanding of the architectural organization and microenvironment-related characteristics that are expressed in differentiated systems in adults and on how these microenvironments respond to toxic stressors in adults. Previous studies have clearly established that two of the major classes of respiratory toxicants, oxidant air pollutants and bioactivated polyaromatic hydrocarbons, produce patterns of acute cytotoxicity that are highly site- and cell-selective. To define the complexity of the respiratory toxic response, we have compared multiple sites with profoundly different responses to oxidant air pollutants and bioactivated cytotoxicants" respiratory mucosa and olfactory mucosa in the nasal cavity, the trachea, and proximal, mid-level bronchi and distal bronchioles in the lungs. 1-14 It is now well-recognized that the respiratory system of adult mammals contains over 40 different cell phenotypes

~i~:~ i~i~ii/i(~i~ii~~,i::~i ~i~i~:~:i~~I~I i ~i~~):~ii!i,~:i~i~:~i~:i~~!~i~/i~( ~/i~~ ~ii~ ~ ~I~,~I~~:I:~)~:~i:~)~:~i!~i:i:~!i~!/~'~i~!i)~i~,!/~

distributed within a large number of distinct microenvironments. Using microdissection, we have defined the complexity and microenvironment-dependent nature of phenotypic expression for potential target cell populations within different airway sites. 2'15-3z Virtually every aspect of the composition of the wall of the airways, including epithe' lium and glands, smooth muscle, and cartilage vary by species. This is especially true for the airway epithelium, which is highly varied in any one species depending on precisely where in the airway tree the cell populations are examined for these characteristics: the composition of the cell populations lining the luminal surface (Table 2.1), the composition of the secretory product found within these epithelial populations (Tables 2.1-2.3) where the same phenotypes are present in many different airways, their relative abundance and proportion of the luminal surface occupied by the specific phenotypes may vary widely. What this means is that the organization of tracheal epithelium is very different from that of terminal and respiratory bronchioles in the same animal. When different species are compared on an airway by airway basis it is clear that the same types of variability exist. In fact in many species, individuals that are free of respiratory disease have very different cell populations in the same microenvironment compared to healthy individuals of other species. This is also true for the potential for the metabolism of xenobiotics either by an activation system (cytochrome P-450 monooxygenases) or a variety of detoxification and antioxidant systems (see Chapter 12). This also applies to the distribution of submucosal glands, with many species having glands extensively down the airway tree as far as

Table 2.1. Carbohydrate content of tracheal epithelium. Carbohydrate content PAS

AB

HID

+

+

-

+

+

+

-

+

+

+

-

Mucous

+

+

+

+

Clara

I::

Species

Cell type

Abundance

Hamster

Clara

:::

Mucous

+

Rat

Serous

:::

Mucous Mouse

Mucous

Rabbit Dog

Mucous

++

+

+

+

Cat

Mucous

++

+

+

+ and-

Serous

+

ND

ND

ND

Mucous

-H-

+

+

+ and-

+ +

+ and+

Pig Sheep

Mucous

++

i:~ M o n k e y

Mucous

++

+ +

Mucous

III

+

ii i~i:~Huma:n I . . . .

: ....

+:

:i : + a n d _

Development

of A i r w a y

Epithelium

Table 2.2. Carbohydrate content of tracheal submucosal glands. i 84~ii :i/:i

:~iii~/!:ii~I i

Carbohydrate content Species

Abundance

Hamster Rat

+ +

Mouse

+

Rabbit Dog

+ ++

Cat Pig

', ~ ~ ',

~ ~~

Secretory cell

PAS

AB

HID

MUCOUS

+

+

m

+

+ and

Serous

+

Mucous

+

Serous

+

Mucous Mucous Serous Mucous Serous Mucous Serous Mucous

+

+

+ a n d _ : : ~:~i

+

+

4--

small bronchioles whereas in other species they are restricted to the most proximal portions of the trachea. Cartilage is not a prominent feature of the conducting airways distal to the trachea in most species the size of rabbits or smaller, but is found extensively throughout the intrapulmonary airways in larger species, including humans. The distribution and organization of smooth muscle appear to be relatively site-specific. The complexity of the cellular organization within even a restricted portion (bronchial airways) of a complex organ such as the lungs emphasizes the need for highly precise sampling methodology. This need is further emphasized by the wide variability in local exposure dose created by the architectural complexity of the tracheobronchial airway tree itself. 5'9'33 As would be expected from a highly complex cellular organization, the metabolic potential of cell populations in different microenvironments within the respiratory system varies widely. The principal enzyme system for xenobiotic bioactivation, the cytochrome P-450 monooxygenases, has broad variability in isozyme expression, substrate specificity and level of activity. 2'34-41 This is also true for the enzyme systems involved in detoxification, especially the glutathione S-transferases and epoxide hydrolases. 35'4~ The cells in each of these different microenvironments also manage their glutathione pools very differently. 9'41'44'45 The pattern of heterogeneity of metabolic function appears to be relatively unique for each species of mammal. Inflammatory responses

.... iii:~

_

+ +

....

+ .

: -]-

+

.

.

.

+ and' : : + :

generated by acute exposure to oxidant air pollutants also vary greatly by site within the tracheobronchial airway tree. 3'46 The biological uniqueness of the cell populations in local airway microenvironments is further emphasized by the fact that when epithelial populations are cultured with the surrounding matrix intact, they maintain the same phenotypic expression and response to toxicants that would be expected if they were still resident within the intact animal. 1~ This complexity emphasizes the need for precise sampling to establish meaningful cellular and metabolic profiles and to validate them for patterns of cytotoxicity. Cultures have been used for definition of local cytotoxicity, 12,17'30 metabolism, 49 maintenance of biological function in vitro 10'12 and definition of local exposure dose. 5'9'33 They have even been validated for obtaining nucleic acids for definition of gene expression at the level of the local microenvironment.50

OVERALL

DEVELOPMENT

OF

AIRWAYS

Early branching morphogenesis As outlined in Chapter 1, the early formation of the airways and the subsequent development of submucosal glands are produced by the process of branching morphogenesis. In essence, this involves the differential growth of an epithelial tube into an associated mesenchymal derivative

Table 2.3.

Lectin reactivity of airway epithelium and submucosai glands.

?~)::~,~:~/:::~i:i!/!ii::::(i i:~!~.... :! : .... : :: : ?i: ::i::~::::~::~)~:: :

84184184184 ~ ~ ~ ,

:i

LCA

WGA

Man GIc GalNAc

NANA GalNAc

-

: :::

~:i~:~: :~:~ ~i~

BSAI::

~:

SBA :i ~:~:::::~

~

::

DBA

Gai-

Gal

GalNAc

Gal GalNAc

Gal GalNAc

Gal :::::: : GalNAc

:I :

::::::: P N A : :

UEAi)I:::I:::::

GalNAc

Species/cell type

Sugar specificity

:::

::i:::

In a i r w a y surface e p i t h e l i u m Human bronchi

Mucous

+

+

~,::

:: ::

Sheep trachea

M u c o u s M1

-

::',

++

:I:

Mucous M2

-

',::

++

::'

Mucous M3

:':

::;

-

-

-

+

+

-(+++)

+

-

-(-H-I-) a

--(-H--l-)

bronchiole Rat trachea

Mucous

-

Rhesus trachea

Mucous

~,:::

,,,

i

::II

l'l|

O f a i r w a y s u b m u c o s a l glands Human bronchi

Mucous

Sheep

Serous Mucous M4

Mouse trachea

Serous Mucous

+ ; ' :

Mucous

++

Serous Rhesus trachea

+

:::

: ', : ',

:::

-H-

+

++

++ ++ +

_ -(+++)

-

-

:::: ::: fill

1111

IIII

:III

-

++

++

-(+-H-)

-

(::::)

-

-

Serous Rat trachea

+

: : :

.

.

.

.

'...................:::~:::~::: ::: = :i ~ : :~::~:::::::: :

.el :'~,,, m

!

|

Mucous

::::

::::

:::

-

-(::::)

-(::::)

:::I

Serous

++

++

+

-

-(++)

-(++)

+

|

.....:: ::

.....

Abbreviations: LCA, Lotus tetragonolobus; WGA, wheat germ agglutinin; SBA, Glycine max; BSA 1, Bandeirea simplicifolia i; DBAI ii Dolichos biflorus; PNA, Arachis hypogea; RCA, Ricinus communis; UEA1, Ulex europeus; Man, mannose; GIc, glucose; GalNAcI: ::~::

N-acetylgalactosamine; NANA, N-acetylneuraminic acid (sialic acid); Gal, galactose; Fuc, fucose. aReaction in parenthesis is after neuraminidase treatment.

containing both cells and matrix. The composition of the matrix appears to dictate where the growing tube will divide. The bifurcation process itself is produced by focal differences in proliferation and programmed cell death to produce rapid growth in areas adjacent to sites of no growth. The no growth sites appear to be associated with bands of newly formed collagen and elastin. Each branching of this growing tube is regulated by a variety of cytokines and growth factors. Subsequent development of other components that form the wall in adults occurs at later times in specific airways. This development appears to move in a proximal to distal pattern following the branching of the epithelial tube. For the formation of the airway tree, this process is thought to be complete prior to birth and varies from species to species as to the percentage of gestation during which the process is complete. As outlined in Chapter 1, subsequent branching produces alveolar septation in alveolar spaces. Once the general pattern of the tree has been established, subsequent developmental processes are essentially growth in two directions: either longitudinally

.

.

.

.

:::::

to extend the length of the tube or circumferentially to increase its diameter. What regulates these processes and how they are associated with differentiation and growth of the wall constituents are not yet clear. This would be of particular signifcance given the substantial impact that the size and angles of the airways have on the flow of air during the respiratory cycle. In most mammalian species, the majority of the growth of the airways is a postnatal event. This suggests that for an extended period after birth, these growth events are susceptible to perturbations by environmental contaminants.

Respiratory bronchioles The most distal airways, located at the junction between the gas exchange area and tracheobronchial airway tree, form an extensive transitional zone in the human lung. This zone, exceeding three generations of branching in humans, is characterized by intermixing of alveolar epithelium, simple cuboidal epithelium mixed with the pseudostratified cuboidal epithelium (with basal, mucus and ciliated cells)

. . . .

found in more proximal airways (see 51 for a review). The respiratory bronchioles are extensive (exceeding three generations) in humans, macaques, dogs, cats and ferrets. In rhesus monkeys, and possibly in other primates, including humans, the two epithelial populations, bronchiolar and alveolar, are distributed on opposite sides of the airway in relation to the position of the pulmonary arteriole. 52 A pseudostratified population with ciliated cells lines numerous generations of respiratory bronchioles on the side adjacent to the pulmonary arteriole. The alveolarized areas are surrounded by a simple cuboidal bronchiolar epithelial population on the side opposite the arteriole. In the majority of mammalian species, the bronchiolar epithelium occupies the proximal portion of the transitional bronchiole, and alveolar gas exchange epithelium lines the distal portion. This is the case for mice, hamsters, rats, guinea pigs, rabbits, pigs, sheep, cattle and horses, s3 The composition of the peribronchiolar region associated with Clara cells includes the presence of smooth muscle adjacent to the basal lamina, extensive collagen interspersed with elastin and few capillaries. Those capillaries that are present are not closely associated with the epithelial basal lamina. The principal vessel in the area is the pulmonary arteriole. In contrast, the alveolar portions of this transitional zone generally include a substantial capillary bed closely applied to the basal lamina of the alveolar epithelial populations. Although the matrix composition of the alveolar gas exchange portions of the lung have been studied in some detail, the same is not true for the matrix associated with the bronchioles. 54 In fetal animals, where the majority of epithelial cells are poorly differentiated or undifferentiated, the boundary between the epithelium lining presumptive distal conducting airway and that lining future gas exchange regions is defined relatively easily in some species. 54-57 The distinguishing features include differences in epithelial configuration and modifications in the surrounding mesenchymaUy derived components. Most of these components, including smooth muscle and fibroblast-like cells, appear to mature somewhat more quickly than do the associated epithelium. 52'58 The morphogenesis of the respiratory bronchiole during fetal lung development has been studied in detail in only one species: rhesus monkeys. 58 The respiratory bronchiole begins as a tube lined by glycogen-filled cuboidal cells intermixed with an occasional ciliated cell. Alveolarization begins in the most proximal aspect of the respiratory bronchiole, at approximately 60% gestation in rhesus monkeys and in humans. The alveolarization appears as a formation of outpocketings into surrounding extracellular matrix. The outpocketings, which are lined by cuboidal epithelium, occur only on the side of the potential respiratory bronchiole opposite the pulmonary arteriole. They begin at the same time that secondary septa are forming in the distal acinus. Outpocketing or alveolarization occurs over a very short period (5 days) in rhesus monkeys. As alveolarization progresses from proximal to distal in the potential respiratory bronchiole, the epithelial cells also differentiate. By

~

i i i 84 !~

:

~ii~

i~i ~ 84

ii

67% gestation, ciliated cells are confined to the epithelium adjacent to the pulmonary arteriole, and the cytodifferentiation of the epithelial cells characteristic of alveoli is beginning in the outpocketings. Contacts between epithelium and underlying fibroblastic cells are observed for a very brief period in regions of respiratory bronchiole development. Epithelium of proximal generations of respiratory bronchiole differentiates earlier than more distal generations, but much later than in the trachea.

Submucosal glands The developmental events involved in the formation of submucosal glands have been well described for a number of species, including rats, 59 opossums, 6~ ferrets, 6x rhesus monkeys 62 and humans. 63'64 The sequence of events in humans has been characterized sub-grossly 65'66 and histologically. 63'64'67'68 The ultrastructure and histochemistry of gland development have been characterized in more detail in rhesus monkeys. 62 In rhesus monkeys, most of the process occurs in the fetus between the end of the pseudoglandular stage and the beginning of the terminal sac stage of development. Gland development implies four phases summarized in Fig. 2.1: (a) the formation of buds by projections of undifferentiated cells from the maturing surface epithelium, (b) the outgrowth and branching of these buds into cylinders of undifferentiated cells, (c) the differentiation of mucus cells in proximal tubules associated with proliferation of tubules and acini, and with undifferentiated cells distally, and (d) differentiation of serous cells in peripheral tubules and acini, with continued proliferation in most distal areas. The cells forming gland buds are not basal cells, as first thought, but rather an undifferentiated cell similar to the surface epithelium (Fig. 2.1). 62 Connective tissue appears to play a role in this process, as evidenced primarily through the presence of cartilage plates in the areas of initial bud formation. Glands appear first at the junction of cartilage plate and smooth muscle, followed by areas over cartilage plates and then in the area over smooth muscle. The secretory cell population differentiates in a centrifugal pattern, with nearly mature cells lining proximal tubules and immature cells in more distal portions. Mucus cells in the proximal portion of the gland develop before serous cells. Glandular mucus cells and serous cells differentiate at different times during development and through a different sequence of events. 62

EPITHELIAL D I F F E R E N T I A T I O N Overview Of the over 40 different cellular phenotypes that have been identified in the lungs of adult mammals, the differentiation of the epithelial cells lining the air passages appears to be the most critical in the successful function of the lung. At least eight of these cell phenotypes line the tracheobronchial

The Lung: Development,

Aging and the Environment

Fig. 2.1. Morphogenesis of submucosal glands in the trachea of rhesus monkeys. In very young fetuses (72 days gestational age (DGA)), the initial phase is projection of buds (B) from the luminal (L) surface epithelium into the surrounding matrix accompanied by an invagination (arrow) of the surface epithelium. As fetuses age (80 and 87 DGA) the buds extend further into submucosal connective tissue, with an apparent lumen (arrowhead) and continue until the formation of a cylindrical projection. In mid-gestation (105 DGA), the tube (T) branches extensively into the matrix with a patent lumen (arrow) apparent throughout. In late gestation (125 DGA) and early postnatal age (12 DPN), differentiation between the duct (D) and more peripheral secretory structures including proximal tubes (P) and large numbers of secretory acini (A) are evident. Continuing growth includes expansion of a center area's proximal tubular structures and marked enlargement of the ducts in adults.

conducting airways, including ciliated cells, basal cells, mucus goblet cells, serous cells, Clara cells, small mucus granule cells, brush cells, neuroendocrine cells, and a number of undifferentiated or partially differentiated phenotypes that have not been well characterized. The abundance and distribution of these cell types within the conducting airway tree vary by position within the tree and by species. The pattern of differentiation of the tracheal epithelial lining has been characterized for a large number of species. 69 The general pattern appears to be the same for most species in terms of which phenotypes are identified earliest during development and which differentiate later (summarized in Fig. 2.2). The critical difference between differentiation of these epithelial cells during development

is the percentage of intrauterine life in which the differentiation occurs. The epithelium of the trachea is the earliest of all the epithelial populations to differentiate. In some species it is relatively differentiated prior to birth, and in other species the majority of the differentiation occurs postnatally. In most species, with the possible exception of ferrets, ciliated cells differentiate first. Nonciliated cells with secretory granules appear next. Basal and small mucus granule cells appear last. There is a polarity in the differentiation of ciliated cells in the trachea, with the epithelium over the smooth muscle undergoing ciliogenesis earlier than that on the cartilaginous side. The reverse appears to be true for the nonciliated secretory cells, with secretory granules appearing on the cartilaginous side of the trachea first.

Development of Airway Epithelium

DIFFERENTIATION

OF T R A C H E A L E P I T H E L I U M

Neuroendocrine (small granule) . . . . . . . . . . . . . . . Differentiating . . . . . . . . . . . . . .9 ciliate

.

\

M a t u r e ciliated " 99 : 9

Undifferentiated Columnar

~

9 9

Differentiating 9 secretory

9 1 4 9 1 4 9

/

\

9 1 4 9 1 4 9 1 4 9 1 4 9 1 4 9 1 4 9 1 4 9 1 4 9

M a t u r e secretory--

9 9

e w w,,w

.

w wwv,,w.l,w

w~ uw ww.,.,,wwv.w..,,w

o

I I e

"Differentiating . . . . . . . . . . . . . . . . .

/

kM a t u r e basal

basal Mouse

I - ............ 2 5 % . . . . . . . . . . ( 5 days)

Hamster

I-. ............ 1 2 . 5 % ....... ( 2 days)

Rat

i - ............. 2 0 % . . . . . . . . . . (4.5 days)

Rabbit

I-- ............ 4 0 % ......... (13 days)

Rhesus M o n k e y

j . ............................. 7 0 % ............... ~ : ~ (118 days)

Human

~. ..............................

75% . . . . . . . . . . . . . . . . . .

(26 wks)

Species

Duration of Gestation ( % ) (Days)

Birth

Fig. 2.2. Diagrammatic comparison of the pattern of differentiation and maturation of the principal cell phenotypes observed in the tracheobronchial airways, with an emphasis on epithelium in trachea. This compares the timing of events in relation to parturition (open arrow) for each species. The dotted line indicates the duration of time during gestation from the initial observation of the formation of cilia to parturition. It is represented both in terms of the percentage of gestation over which differentiation occurs in utero and the actual number of days it takes. For most species, a significant portion of differentiation is postnatal. The proportion of time during gestation when these events occur is very species-specific.

Pattern in trachea and bronchi The ultrastructural features of overall tracheal epithelial differentiation in developing fetuses have been described in rabbits, 7~ mice, 7] hamsters 72'73 and rats. 68 In view of the diversity in the airways in different species, small laboratory mammals may not be adequate models for the study of human tracheobronchial epithelium. 6s The most extensive study of the development of the mucus cell was performed on the trachea of rhesus monkeys TM and is reviewed here. Gestation for rhesus monkeys averages 168 days, with the stages of lung development as follows: embryonic period, 21-55 days gestational age (DGA); pseudoglandular, 56-80 DGA; canalicular, 80-130 DGA; and terminal sac, 131 DGA to term. 75 In the youngest fetuses, all cells appear as illustrated in Fig. 2.3A. The cells are columnar and the apices of most of

the cells reach the luminal surface. Nuclei have little heterochromatin, and the cytoplasm is filled from base to apex with glycogen. The few organelles present are located in the apex of the cell and include short narrow strands of granular endoplasmic reticulum (GER), small spherical mitochondria and a small Golgi apparatus located adjacent to the lateral surface of the cell (Fig. 2.3A). These cells were present in the epithelial lining in the youngest animals in the embryonic stage to the middle of the canalicular phase. Near the end of the embryonic period, many cells similar to those at younger ages, but containing larger numbers of apical organelles, are observed. The organelles include spherical mitochondria and increased amounts of GER with dilated cisternae. The cisternae of the Golgi apparatus are dilated and surrounded by enlarged membrane-bound vacuoles, and glycogen is concentrated near the nucleus and

it

Fig. 2.3. Differentiation of mucus goblet cells in trachea of fetal rhesus monkeys: (A) early in gestation (46 DGA) columnar cells contain large pools of glycogen (Gly) and a central nucleus (N); (B) with continued age, columnar cells taper at the base and begin to accumulate membranebound secretory granules (arrowheads) in apical cytoplasm near the luminal surface (62 DGA); (C) by approximately 50% of gestation (90 DGA) columnar cells have markedly tapered bases, numerous apical secretory granules (arrowhead) and little cytoplasmic glycogen; (D) in the perinatal period (141 DGA) many of the cells have a prominent Golgi apparatus (Go) and an abundance of secretory granules in their apical cytoplasm (arrowhead).

intermixed with the organelles. These cells are observed in fetuses up to early in the canalicular stage. Through most of the pseudoglandular stage, most nonciliated cells had increased numbers of apical membrane-bound secretory granules containing a flocculent matrix, with a small electron-dense spherical core (Fig. 2.3B). Most of the remaining cytoplasm is still filled with glycogen. The cytoplasm surrounding the glycogen is more electron-dense than in younger ages and occupies more of the apical portion of the cell. The nuclei exhibit prominent nucleoli and small patches of heterochromatin. The mitochondria exhibit noncircular profiles and appear to be tubular. The amount of GER appears to be the same as in younger ages, but the cisternae were no longer dilated. The Golgi apparatus is surrounded by vacuoles of various sizes. The luminal surface of these cells is covered by long, regular microvilli. In somewhat older animals (late pseudoglandular), the apices of a large proportion of the secretory cells are filled with spherical granules (Fig. 2.3C). Most of these cells have abundant cytoplasmic glycogen, most of which is basal to the nucleus. Apical to the nucleus, glycogen is interspersed among organelles and granules. The cells appear more fusiform than at younger ages, being wide at the luminal side and narrow at the base (Fig. 2.3C).

In fetuses from midcanalicular stage and older, secretory cells containing cytoplasmic glycogen were rare and, when observed, the glycogen content was minimal. From this time to parturition, only two forms of secretory cells are observed. Both cells have little cytoplasmic glycogen, and the cytoplasm was condensed. There is a distinct variation in the abundance of apical secretory granules in these cells, ranging from very few, in cells with a narrow cytoplasm and few organelles, to an abundance of these granules in other cells (Fig. 2.3D). The cytoplasm of these cells contains small mitochondria and varying amounts of GER. The Golgi apparatus is located on the apical side of the nucleus and show variable degrees of activity. In cells with more granules (Fig. 2.3D), the Golgi apparatus is larger, has more cisternal stacks, and has larger and more numerous adjacent vesicles. Long, regular microvilli are a characteristic feature of the surface of the secretory cells. There is considerable variability in the abundance of these cellular forms between 105 days and parturition. At earlier ages, they are of approximately equal abundance. Near parturition, most of the secretory cells resemble that in Fig. 2.3D. Some of the cells have an even larger percentage of their cytoplasm occupied by granules than illustrated in Fig. 2.3D.

Development

In the postnatal period, most of the secretory cells have an abundance of electron-lucent granules filling their apical cytoplasm. The majority of these granules have small electron-dense cores. A few have large electron-dense biphasic cores, as observed in the adult. In general, the nucleus and its surrounding cytoplasm are restricted to the basal portion of the cell and the Golgi apparatus, and other organelles occupy a small percentage of the cytoplasm. Up to 134 days after birth, there are, however, a few secretory cells the cytoplasm of which contained abundant organelles and a variable number of secretory granules, as is observed in the late fetal period (Fig. 2.3D). By 134 days of postnatal age, nearly all the secretory cells have a configuration similar to that observed in adults. The cytoplasm is filled with electron-lucent secretory granules that appeared to distend the cell's cytoplasm. The nucleus is compressed at the basal portion of the cell, and organelles are minimal. In most cases, the cytoplasmic granules contain a biphasic core. The central part of the core is the most electron-dense portion of the granules. Differentiation of mucus glycoprotein biosynthesis and secretion develops slightly behind the other aspects of

of A i r w a y

El~itheliun7

2i

cellular differentiation. Prior to the presence of secretory granules, the principal material reacting with periodic acid-Schiff (PAS) is the large store of glycogen surrounding the nucleus (Fig. 2.4). Once granules appear, based on ultrastructure, the contents of the granules resemble that in adults (Fig. 2.4). These granules are not only PAS positive, but also positive for alcian blue (AB), indicating acidic groups, and for high iron diamine (HID) indicating that they are sulfated. The range of distribution of these patterns of stain reactivity varies by airway depending on the mix in the adult. In core granules, the sulfated material is generally identified in the center, whereas in uncored granules it tends to be on the periphery. As the cells fill with secretory product the distribution of staining reaction tends to follow closely that of the granules. The pattern by which sugars are expressed during differentiation is quite variable, depending on the specific end group (Table 2.4). The majority of sugars are expressed during the early phases of biosynthesis and granule formation. Others are expressed much later and all are generally, if they will be expressed in the adult, present in cells with a differentiated secretory apparatus by birth. With the exception of N-acetylgalactosamine (bound by PNA

Fig. 2.4. Comparison of the distribution of reaction products for alcian blue/periodic acid-Schiff (AB/PAS) and high iron diamine (HID) in the tracheal epithelium of fetal rhesus monkeys during the period when granule formation is first observed (62 DGA). The granules found in the apex of the cell are primarily AB/PAS positive (A) (arrow). Highly PAS positive material is found on the apical and basal portions of the nucleus (arrowhead). The majority (but not all) of the AB positive material is also sulfated (B) (arrow).

i ~ii~h ~i:!!~i~~i ~ g iii:ID e~/o!p

~ !~ ~ i !!!~i~g~ ~ing !a!!~ i~ !!:i hi~ I~!IE!!~:V iroi~i m ~ ~!~ii~i~iil~!~ii84 i!~!ii:84~i!iii ill i~~iiif:i~i!ii ~i~:i!ilii!iiiI!!~i:ii~i~ili~~i ii ~i~iii~i~!i!!i i il~!:~i~IIi84 iI~84184

Table 2.4. Lectin reactivity in developing rhesus monkey trachea. i ~i~i~ili!i:i~/!i!~~iil~:ii:i~ii:iii!~~?i :~iiiii:i~~~i~ :~ i~84184 iii!~ii::i! i84 ~ii/i ii iI Days:igestational age

Lectin

50

LCA UEA 1 SWGA BSA 1 PNA DBA

-

80

- : + + + + + . . . . . . + + + . . .

90

135

155

18dPN

+ + +

+ + +

+ + + + -

+ + + + -

+ .

~ i ii: i:

.

.

Abbreviations: see Table 2.3.

lectin), all others are more prominent in postnatal animals than in neonatal. This particular terminal sugar appears to be expressed in the early phases of mucus cell differentiation and its expression is suppressed, at least in rhesus monkeys, in late gestation and early postnatal life. Use of monoclonal antibodies established against mucus antigens indicates that very early in differentiation the composition of the core proteins for the secretory product may not be incorporated into the granules which initially form (Fig. 2.5). Their incorporation appears to be somewhat later during the differentiation process. Tracheobronchial epithelium continually renews itself. To identify the progenitor cell types that are involved in the self-renewal in vivo, the traditional approach is to carry out mitotic index and nuclear labeling studies. For the nuclear labeling study, the incorporation of [3H] thymidine or bromodeoxyuridine is used. Using these approaches, most of the data suggest that less than 1% of the epithelial

A

cell population is involved in cell proliferation. 76-8~ Both basal and secretory cell types are capable of incorporating these nucleotide precursors and mitosis, whereas ciliated cells are considered to be terminally differentiated and incapable of division. 81 In fact, only under exceptional circumstances are the ciliated cells of isolated hamster trachea capable of synthesizing DNA, as evidenced by the incorporation of [3H] thymidine. 8z Differentiation and proliferation normally are inversely related. Based on this view, a number of investigators 83'84 suggest that it is the basal cell type that serves as the stem cells, or the progenitor cell type that is involved in normal maintenance as well as in the regeneration and redifferentiation of bronchial epithelium after injury. However, this view is inconsistent with data obtained from the developmental studies and studies of injury/repair. In the developing tracheas of a number of animal species, including humans and nonhuman primates, basal cells are derived from an undifferentiated columnar epithelium. 85 Furthermore, the appearance of the basal cell type in the tracheal surface lining layer occurs after the appearance of ciliated and nonciliated secretory cell types. 86 Furthermore, in the growing intrapulmonary airways, 87'88 the basal cell type is not found in the smallest airway. 85 In the injury models, such as the mechanical and toxic gases exposure models, hyperproliferation is seen in the secretory cell type, but not in the basal cell type. 80'89-91 This indicates that it is less likely for the basal cell type to serve as a progenitor cell type that initiates the growth of airway epithelium and the repair of epithelial damage. 85 Studies of the repopulation of epithelial ceils on denuded tracheal grafts have been used to assess the "progenitor" nature of various bronchial epithelial cell types. Denuded tracheal grafts are usually produced by removing the lining epithelial layer by repeated freezing and thawing of tracheal grafts. 92 Using this technique, combined with the cell

B

Fig. 2.5 Comparison of the distribution of immunoreactive mucin with an antibody that reacts with all mucus cells in adult rhesus monkeys and the distribution of AB/PAS positive material from serial sections of trachea of fetal rhesus monkey when secretory granules are just beginning to form (50 DGA). (A) The immunoreactive secretory product is in highly focal areas of a small number of positive cells (arrows). (B) On section serial to (A), it is clear that these sites are also positive for PAS (arrows). Cartilage (C) is negative. (E) epithelium.

:

:

:

(

: Devil

separation technique, the mucociliary epithelium can be repopulated in the denuded tracheal graft by enriched basal cell population from rabbits and rats. 93'94 These experiments clearly demonstrate the pluripotent nature of the basal cell type. However, there are several deficiencies in these experiments. First of all, the definition of basal cell type is based on the ultrastructural picture and the immunohistochemical stain. It is well known, though, that secretory cells lose their differentiated features upon isolation and culturing in vitro. The degranulated secretory cells may resemble the basal cell type, and the morphologic tools used in these studies cannot distinguish satisfactorily the basal one from the degranulated secretory cell type in dissociated and isolated cell preparations. Furthermore, for the preparations in these studies, the purity of basal cell type population is only 90%. Using flow cytometry to isolate basal cells, it has been found that basal cells from rat trachea had a colonyforming efficiency of 0.6%, whereas secretory cells and unsorted cells had efficiencies of 3.4 and 2.6%, respectively.95 From these results, it may be concluded that basal cells have less proliferative activity than secretory cells. It is therefore difficult to conclude from these tracheal graft repopulation studies that basal cell type is the progenitor cell type responsible for the initiation of airway epithelial cell growth and the repair in response to injury.

Pattern in bronchioles The process of cytodifferentiation of the nonciliated cells of distal bronchioles entails substantial rearrangement, loss and biogenesis of cellular organelles. Up to late fetal age, terminal bronchioles are lined by simple cuboidal to columnar epithelium composed of glycogen-filled nonciliated cells with few organelles. The shifts in cellular components with time for species in which the predominant cellular constituent in adults is agranular (smooth) endoplasmic reticulum (AER), such as in mice, hamsters, rats and rabbits, are summarized in Fig. 2.6. The pattern is essentially similar for these species. What varies from species to species is the timing of these events. The first event is a dramatic loss in cytoplasmic glycogen. In rabbits, this drop is from ---70% of cytoplasmic volume to less than 10% in adults. A similar substantial loss occurs in rats, hamsters and mice. In rabbits, this loss begins immediately prior to birth and continues for up to 4 weeks of postnatal age. 96 A similar change occurs in mice. 97 In rats, the loss of cytoplasmic glycogen begins at birth and drops to adult levels within the first week of postnatal life. 98 In hamsters, cytoplasmic glycogen is not detectable immediately after birth. 99 Associated with the drop in cellular glycogen is a substantial biogenesis of membranous organelles, especially AER. Smooth endoplasmic reticulum is not detected in nonciliated cells until immediately prior to birth in rabbits (Fig. 2.6). 1~176 At birth, fewer than 20% of the cells contain more than 10% AER. By 2 weeks, in almost 70% of the nonciliated cells, AER occupies more than 10% (up to 50%) of the cell volume. The adult configuration is reached at approximately 28 days postnatally in rabbits. In mice, the adult configuration of

Epiih ii m84 AER i s reached at approximately 3 weeks postnatally. 97 Granular endoplasmic reticulum in prenatal animals is approximately twice as abundant in rabbit Clara cells as it is in rats (Fig. 2.6). 1~ The decrease in cellular abundance of GER occurs gradually in rabbits and is still double the adult configuration (2% of cell volume) at 4 weeks postnataUy, but in rats the level decreases by 50% immediately postpartum and is at or near the adult configuration (less than 1%) by 10 days postnatally. The situation for rats and mice appears similar to that for rabbits, but for hamsters GER is near the adult configuration immediately postpartum. 99'1~ Secretory granule appearance also varies by species. The earliest at which secretory granules are detected in the Clara cells of rabbits and mice is within the first week of postnatal life, whereas in rats and hamsters granules are abundant prenatally. In rabbits as well as mice, granule abundance resembling adult levels occurs by 21 days postnatally. In rats, granule abundance reaches adult abundance by 7 days postnatally and is at adult configuration immediately postpartum in the hamster. The only species in which Clara cell differentiation has been characterized fully where the adult Clara cell population does not have an abundance of AER is rhesus monkeys. 52 In that species, the loss of cytoplasmic glycogen and an increase, rather than a decrease, in GER occurs over a substantial period both prenatally and postnatally. Studies in humans suggest that developmental events for Clara cells are similar to those in rhesus monkeys, but may extend longer than the 6 months to a year (postnatally) required for differentiation of all the nonciliated cells in terminal respiratory bronchioles of monkeys. As summarized in Chapter 12, the expression of cytochrome P-450 monooxygenases (CYP) in Clara cells during their differentiation has been evaluated in a number of species. 1~176 Protein for the NADPH P-450 reductase and CYP2B is detected earliest, with the reductase somewhat later than CYP2B in rabbits. CYP4B is detected 2-3 days of age later (see Chapter 12). The initial distribution is in the most apical border of a small percentage of the nonciliated cell population. During the period in which the amount of detectable protein increases, the distribution changes in two ways. First, an immunologically detectable protein is found in an increasing proportion of the nonciliated cells as animals become older. Second, the distribution of detectable protein within an individual cell increases from the apex to the base with increasing age. The youngest age at which intracellular protein can be detected immunohistochemically varies substantially between these species (rat, rabbit, monkey). Protein becomes detectable in hamsters approximately 3-4 days prior to birth and reaches the distribution and intensity observed in the adult by 3 days postnatally. CYP4B is not detectable before 1 day postnatally, but is at adult levels shortly thereafter. In rabbits and rats, the timing is somewhat different. NADPH reductase is found initially just prior to birth in rabbits, and CYP2B and 4B are not observed until after birth. All these proteins have an adult distribution and intensity by 28 days postnatally. In rats, CYP2B, CYP4B and NADPH reductase are detected in the first 2-3

T h e Lung: D e v e l o p m e n t ,

A g i n g and the E n v i r o n m e n t

27 DGA 100 I

80 60

40 20

0-.099

.1-.199

.2-.299

.3-.399

.4-.499

>.5

Late Gestational Age 10o-

1-2 D Postnatal

80" 60" 4020"

~

i1,, 0-.099

.1-.199

.2-.299

9 .3-.399

...... ,4-.499

>.5

Perinatal Period 2 weeks 10080 6040

0..099

.1-.199

.2-.299

,3-.399

1-2 Weeks Postnatal

.4-.499

,

,, ,

>.5

,

Adult

100 9

~

M

(12-17 w e e k s )

80 60 40 20

0-.099

.1-.199

.2-.299

.3-.399

.4-.499

>.5

Volume Fraction (vol/vol) of Agranular Endoplasmic Reticulum in Clara Cell Cytoplasm 4 Weeks and Adult Fig. 2.6. Diagrammatic comparison of Clara cellular organization during pre- and postnatal differentiation with correlation to morphometric analysis of cytoplasmic content of smooth endoplasmic reticulum from morphometric measurements in lungs of rabbit. The morphometric data (histograms) compare the percent of bronchiolar Clara cells with different amounts of smooth endoplasmic reticulum in developing and adult rabbits. AER, agranular (smooth) endoplasmic reticulum; BL, basal lamina; G, Golgi apparatus; GER, granular (rough) endoplasmic reticulum; Gly, glycogen; M, mitochondria; Gr, secretory granule; Nu, nucleus.

days of postnatal life and are apparently at adult densities and distributions by 21 days postnatally. CYP1AI is not detectable prenatally in rats but can be detected in increasing, but small, amounts until it reaches adult levels at approximately

21 days postnatally. Intracellular expression of protein precedes the appearance and increase in the abundance of AER by 2-4 days in each of these species. Activity for these proteins is first detected approximately 2-3 days after the

Development

of A i r w a y

Epithelium

Table 2.5. D e v e l o p m e n t of agranular endoplasmic reticulum (AER) P-450 reductase and monooxygenase enzymes in the rabbit lung.

Amount of enzyme activity 27-28 DGA

1-2 DPN

7 DPN

Assay

28 DPN

Adult

64.5

100

% of adult value

AERa'b P-450 reductase Immunohistochemistry b,c Western blot P-450 isozyme 2B

Immunohistochemistryb Western blot r~-Tll P-450 ;,sv._yme 4B immunohistochemistry b ~A~--..L--_"

14 DPN

L~--L

0.2

8.2

8.2

30.1

+ +

0:i~::~ ~ ~ ~ ~i~=

+

0

.

.

protein is immunologically detectable within Clara cells. The activity studies have been done with whole lung homogenates and reflect potential activity from other cell populations as well as from Clara cells. While both the AER abundance and antigenic protein intensity reach the adult configuration in ---3-4 weeks after birth in rats and rabbits, the activity for these isozymes is still considerably below that for adults. This suggests that the functionality of these proteins continues to increase after the protein density and organelle composition have reached adult levels of expression. Table 2.5 summarizes the relationship between changes in AER abundance, expression of immunoreactive protein, and microsomal P-450 activity for rabbits. The timing for rats is somewhat shifted to the left for postnatal time points and to the right for perinatal ones, compared with rabbits. The pattern of expression of Clara cell secretory protein is similar to that of the cytochrome P-450 monooxygenase system in relation to the appearance of cellular organelles. While there is substantial interspecies variability in the timing of expression, the general pattern is similar, at least for the four species studied in most detail: rats, rabbits, hamsters and mice. 97'1~176176176 The protein appears earliest in the central or apical portion of a few cells per bronchiole, and the number of cells in which antigen can be detected

+-H'+

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+

+ .

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++

++

+

0 0

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.

.

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.

.

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.

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increases with increasing age. In hamsters, the secretory protein antigen can be detected in a number of cells by the beginning of the last trimester of pregnancy and reaches the adult configuration in terms of density and number of cells labeled at about 3-4 days postnatally. In rats, a small proportion of the cells are labeled prenatally, and the distribution observed in the adult is present at about 7 days postnatally. This adult configuration occurs between 3 and 4 weeks in rabbits, and the earliest detectable signal in the Clara cells is immediately prior to birth. The timing in mice and rats is similar to that in rabbits. Immunoreactive protein has also been detected in late gestation in human fetuses, but at what age the distribution resembles adults has not been determined. Intracellular expression of the protein follows the changes in GER and is closely related to the first appearance and increase in the abundance of secretory granules. Western blotting of this protein indicates that it is present earlier in lung homogenate than its appearance in bronchiolar Clara cells suggests. This is because secretory cells in proximal airways express the protein much earlier and, in general, are more differentiated in the perinatal period than are secretory cells of bronchioles. In hamsters, the situation is inverse, with the bronchioles differentiating in this respect prior to the bronchi.

REGULATION

OF

DIFFERENTIATION

Trachea and bronchi Defining the mechanisms regulating maturation of fetal and neonatal airway epithelium is an area of active research with studies focused primarily on (a) in vitro studies, using isolated cell populations, and (b) genetically manipulated mice. The factors controlling differentiation and the mechanisms for these controls are poorly understood, especially in vivo. Three areas of regulatory control have been explored in some detail: the interleukins, vitamin A and growth factors, and transcription factors. In vivo studies have been minimal for direct treatment. However, the effects of epidermal growth factor (EGF) on lung development have been examined in rhesus monkeys. EGF treatment in utero markedly stimulates the maturation of the tracheal secretory apparatus, including both the tracheal surface and submucosal glands. 1~ The secretory apparatus is more differentiated in that there are more mucus cells, increased secretory product stored in the epithelium and glands, and increased quantities of secretory product in lavage and amniotic fluid. By contrast, treatment with triamcinalone, a glucocorticoid, induces maturation of the gas exchange area, 11~but does not affect the maturation of the secretory apparatus (Table 2.6). Based on studies primarily in mice, it appears that most of the differentiation of proximal airway epithelium is under the regulation of two transcriptional factors, the homeodomain transcriptional factor NKX2.1 (otherwise known as TTF-1) and winged-helix family transcription factor HNF-3/forkhead homolog-4 (HFH-4). TM The former appears to promote the initial branching of the tracheal bud but also promotes other aspects of tracheal differentiation, possibly via the FGF pathway. The key regulatory mechanism by which these transcription factors modulate differentiation of epithelium in proximal airways is not clearly understood. There appears to be a few key differentiation processes that may be regulated by specific transcription factors. Some studies have suggested that NKX2.1 promotes differentiaTable 2.6. Effect of EGF and Triamcinalone Acetonide (TAC) on total glycoconjugate detectable in the trachea of fetal rhesus monkeys.

Days gestational age 128 150 128 150

150 p < 0.05

Total secretory product (mm 3x 103/mm 2) (~+ 1 SD)

Treatment None None EGF TAC (11 mg/day) T A C (I O:mg/day)

0.48 + 0.37 1.36 a+ 0.33 1.77a+0.28 1.27 + 1,35 0.75 _+0i34:

control.

tion of Clara cells, but others suggest that tracheal mucous cell formation is independent of this transcription factor. 112 These developmental studies were based on mice after gene manipulation; however, healthy adult mice normally do not have differentiated mucous goblet cells. In contrast, it appears that HNF-3/forkhead homolog-4 transcription factor is critical for the differentiation of ciliated cells in respiratory epithelial populations. 113'114A number of epithelial-mesenchymal interactions appear to be critical to the differentiation of the epithelium; two operate through HNF-3 and GATA6 and may serve as regulators of cell-cell communication activities. The two interleukins that appear to have the most impact on transdifferentiation of airway epithelium in proximal airways of mice from a Clara cell phenotype to a mucus cell phenotype are IL9 and ILl3.11s'116 When the ILl3 gene is attached to a promoter for the Clara cell 10 KDa protein, it actively promotes the expression of mucus cells of the proximal airways of mice. 116-118 In all these cases, the airways of transgenic mice have three characteristic features: mucus metaplasia, eosinophilic inflammation and airway hyperresponsiveness. In vivo studies have also shown that inhibition of ILl3 blocks allergen induced effects on the airways of mice. On the other hand, direct administration of ILl3 to the airways can produce the same effects as allergen. 119'12~For both IL9 and ILl3, a variety of cellular responses occurs, including elevation of eosinophils, lymphocytes, mast cells and subepithelial collagen in airway w a l l s . 115'119'120 While most of these events appear to be regulated through the ILl3 R alpha 1 subunit in combination with the IL4 receptor, they neither appear to be modulated by factors that regulate the matrix changes nor influence inflammatory cell populations. 118 Defining exactly how ILl3 produces these changes has been difficult. It is clear that for poorly differentiated airway epithelial cells in vitro, ILl3 induces a dramatically different pattern of gene expression than is observed in airway smooth muscle cells or lung fibroblasts. 121 The four major transcription factors increased were OTF2, HSP factor 4, Id-3 and NRF-1. There was elevated phosphorylation of STAT 6 and some increases in factors related to extracellular matrix production. Tracheobronchial epithelial cells, like many other epithelial cells, lose their differentiated functions upon culturing in vitro. However, the loss of differentiated functions- at least in primary tracheobronchial epithelial c u l t u r e - is transient. In repopulation studies using cultured cells, 92 undifferentiated rabbit tracheal epithelial cells maintained long term in culture are able to repopulate the grafts and form a new mucociliary epithelium. 122' 123 Epithelial cells, despite dedifferentiation in culture, apparently maintain their intrinsic differentiated potential, which is expressed if an appropriate environment is provided: hormonal requirements, vitamin A supplement and collagen gel substratum. 124-127 Based on the amino acid and carbohydrate composition analyses of the in vitro secretory products as compared with the in vivo mucin products purified from sputum and epithelial cell layer, it appears that cultured tracheal epithelial cells from a number of species are able to

Development of Airway Epithelium

secrete authentic m u c i n . 128-131 Critical to use of in vitro models for epithelial differentiation has been the development of the Whitcutt chamber to grow airway epithelial cells between air and a liquid medium interface, 132-136based on the premise that airway epithelial cells in vivo are usually located between air and a liquid interface. Using this chamber, columnarized formation of cultured epithelial cells was observed, and with further development of mucociliary differentiation in culture, 122'123 including both human and monkey tracheobronchial epithelial cells. 124'125'135'136 Scanning electron microscopy demonstrates extensive ciliary features on the culture surface, and transmission electron microscopy has demonstrated the formation of abundant mucus-secreting granules and the columnarized features with a two- to four-cell layer. The basal cell layer is compressed and resembles basal cells in vivo. Tracheobronchial epithelium is a vitamin A-targeted tissue. 137-14~ The epithelium requires vitamin A for the preservation and induction of the expression of differentiated functions. Keratinizing squamous metaplasia of mucociliary epithelium occurs with vitamin A deficiency along with a reduction in the synthesis of mucus glycoproteins. The administration of vitamin A or its synthetic derivatives (retinoids) reverses this phenomenon. Excess vitamin A can convert stratified skin epithelium in chick embryos to an epithelium containing mucus-secreting granules. 141 Vitamin A treatment enhances the proliferation of small mucus-granule cell type in primary hamster tracheal epithelial cultures. 142-144 However, vitamin A does enhance DNA synthesis of basal cells of keratinocyte cultures. 145 There is no evidence that vitamin A inhibits squamous cell proliferation. Vitamin A and its derivatives clearly play a role in the differentiation and expression of mucin genes in human tracheal bronchial epithelial cells. 146 At least four mucin genes (MUC2, MUC5 AC, MUC5 B MUC7) are retinoic - (RA) or retinol-dependent while MUCi, MUC4 and MUC8 are not. Regulation of mucin genes by retinoic acid appears to be mediated by retinoic acid receptors RAR~ and y. Two other regulators of cellular function interact closely with RA to modulate mucin genes: thyroid hormone (T3) and EGF. T 3 inhibits mucin gene expression, particularly MUC5 AC, apparently through competitive inhibition of receptor responses through the thyroid receptors by inhibiting gene transcription. While EGF is thought to stimulate mucin expression and secretion in cultured airways of some species, especially the rat, it has an inhibitory effect in human bronchial cells in culture; it is suggested that EGF's impact may in fact be retinoic aciddependent.

Regulation in bronchioles Factors regulating Clara cell differentiation are not well understood. The postnatal nature of the majority of the cytodifferentiation process in most species suggests that it is independent of the hormones associated with pregnancy and parturition. The fact that the timing varies by as much as 2-3 weeks in different species would further suggest that

the process may be under regulation of a variety of factors that act in different temporal sequences and with different levels of influence in different species. A number of mediators have been shown to stimulate cytodifferentiation of type II alveolar epithelial cell and produce architectural rearrangements of lung connective tissue elements to promote gas exchange, including corticosteroids, thyroid hormone, EGF and cAMP. 147 Whether all these mediators influence Clara cell differentiation is not known. The best studied are the glucocorticoids, especially dexamethasone. Treatment in the perinatal period retards Clara cell differentiation as evidenced by an increase in cytoplasmic glycogen and minimal alterations in organelles in both rats and mice. 148'149 Dexamethasone administered either prenatally or immediately postnatally elevates the surfactant protein messenger RNA (mRNA) levels in lungs of rats of all ages, producing this elevation in both alveolar type II cells and Clara cells. 15~ Glucocorticoid administered to pregnant rabbits appears to have a stimulatory effect on the differentiation of secretory potential in fetal Clara cells by elevating the amount of the uteroglobin-like Clara cell secretory protein. 151'152 Dexamethasone administered to pregnant rabbits also has a stimulatory effect on the pulmonary cytochrome P-450 system in fetuses, based on measurements of whole lung microsomes. 153-155 While glycogenolysis is retarded by dexamethasone treatment, glycogen, epinephrine and 8-bromo-cAMP produce a rapid drop in Clara cell glycogen content. 98 One of the factors that appears to have the most impact on Clara cell differentiation is injury during the developmental period, in which normal differentiation occurs. Normal differentiation is characterized by loss of glycogen and appearance of secretory granules, and by differentiation of Clara cells into ciliated cells, even in the absence of frank injury to either ciliated or Clara cells. Postnatal exposure to compounds that injure the respiratory system retard Clara cell differentiation. Hyperoxia during the early postnatal period inhibits differentiation. 156'157 Injury by treatment with 4-ipomeanol impedes Clara cell differentiation even for a short term after treatment is discontinued. 158 Not only are Clara cells in postnatal animals more susceptible to injury than in adults, but the expression of the P-450 system in the post-treatment period is markedly reduced. In rats, exposure to cigarette smoke of either the pregnant mother or the newborn accelerates the appearance of one cytochrome P-450 monooxygenase isozyme, CYP 1A 1, but not CYP2B. 1~ The increased P-450 expression is primarily in the Clara cell population and is not found in either alveolar type II cells or in the vascular endothelium, both targets for inducers in adult animals. Other factors besides postnatal hyperoxia, including maternal undernutrition during the last 5 days of pregnancy, retard Clara cell differentiation, but these effects appear to be reversible with time.98,156-158 There is considerable indirect evidence to suggest that a number of growth factors, including TGF-~, EGF, basic fibroblast growth factor (FGF), insulin-like growth factors,

and platelet-derived growth factor, may play roles in regulating bronchiolar epithelial differentiation. 159'16~The EGF receptor (EGFr) has been detected in bronchiolar epithelium throughout pre- and postnatal lung development in rats and humans. 161'162 EGFr has also been detected in human lung at midgestation 163'164 and has been detected in human and rat fetal lung extracts. 161'165 Both ligands of EGFr, as well as T G F - ~ and EGF, have been detected immunohistochemically in bronchiolar epithelium in a number of species. EGF is barely detectable in bronchiolar epithelium of fetal humans (first and second trimesters), but is present in postnatal human lung. 166 EGF has been reported in homogenates of lung from late fetal (21-day gestational age) and adult rats, 167 and immunoreactive protein has been detected in bronchiolar epithelium throughout fetal development in lambs and mice. 168'169 TGF-[3 has been detected in bronchiolar epithelium of mid-gestational humans. 17~ It can be extracted and mRNA can be detected in fetal rat lung homogenates. 171 Plateletderived growth factor receptor has also been detected in bronchiolar epithelium during most of the prenatal stages of lung development. 163'164 Basic F G F and its receptor are found in bronchiolar epithelium during most of fetal rat lung development. 172 Both the F G F receptor and the protein appear to colocalize in the epithelium and adjacent interstitial compartments. There is some suggestion that insulin-like growth factors are involved in aspects of epithelial development in bronchioles. 173 These growth factors may play a role in autocrine regulation because both receptors and the proteins themselves appear within the bronchiolar epithelium. They may also play a paracrine role because growth factor protein appears to be distributed to interstitial cell components, fibroblasts, and smooth muscle surrounding bronchiolar epithelium, during various stages of lung development. At present, there is no direct evidence that any of these factors influence bronchiolar epithelial maturation. There is, however, evidence that pharmacological doses of E G F alter branching morphogenesis in mice, TM enhances differentiation of alveolar type II cells in fetal rabbits, monkeys and sheep, 69'175'176 and alter the differentiation of tracheal epithelium in rhesus monkeys. 1~

REFERENCES 1. Paige RC, Wong V, Plopper CG. Long-term exposure to ozone increases acute pulmonary centriacinar injury by 1-nitronaphthalene. II. Quantitative histopathology. J. Pharmacol. Exp. Ther. 2000; 295:942-50. 2. Fanucchi MV, Murphy ME, Buckpitt AR et al. Pulmonary cytochrome P-450 monooxygenase and Clara cell differentiation in mice.Am. J. Respir. Cell Mol. Biol. 1997; 17:302-14. 3. Hyde DM, Hubbard WC, Wong V e t al. Ozone-induced acute tracheobronchial epithelial injury: relationship to granulocyte emigration in the lung. Am. J. Respir. Cell Mol. Biol. 1992; 6:481-97. 4. Paige R, Wong V, Plopper C. Dose-related airway-selective epithelial toxicity of 1-nitronaphthalene in rats. Toxicol. Appl. Pharmacol. 1997; 147:224-33.

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158. Massaro GD, McCoy L, Massaro D. Hyperoxia reversibly suppresses development of bronchiolar epithelium. Am. J. Physiol. 1986; 251:R1045-50. 159. Jetten AM. Growth and differentiation factors in tracheobronchial epithelium. Am. J. Physiol. 1991; 260:L361-73. 160. Kelley J. Cytokines of the lung. Am. Rev. Respir. Dis. 1990~ 141:765-88. 161. Strandjord TP, Clark JG, Madtes DK. Expression of TGF-ot, EGF, and EGF receptor in fetal rat lung. Lung Cell Mol. Physiol. 1994; 267:L384-9. 162. Johnson MD, Gray ME, Carpenter G etal. Ontogeny of epidermal growth factor receptor and lipocortin-1 in fetal and neonatal human lungs. Hum. Pathol. 1990; 21:182-91. 163. Han RN, Liu J, Tanswell AK etal. Ontogeny of plateletderived growth factor receptor in fetal rat lung. Microsc. Res. Tech. 1993; 26:381-8. 164. Caniggia I, Liu J, Han R etal. Fetal lung epithelial cells express receptors for platelet-derived growth factor. Am. J. Resp. Cell Mol. Biol. 1993; 9:54-63. 165. Nexo E, Kryger-Baggesen N. The receptor for epidermal growth factor is present in human fetal kidney, liver and lung. Regul. Pept. 1989; 26:1-8. 166. Stahlman MT, Orth DN, Gray ME. Immunocytochemical localization of epidermal growth factor in the developing human respiratory system and in acute and chronic lung disease in the neonate. Lab. Invest. 1989; 60:539-47. 167. Raaberg L, Seier Poulsen S, Nexo E. Epidermal growth factor in the rat lung. Histochemistry 1991; 95:471-5. 168. Johnson MD, Gray ME, Carpenter G e t al. Ontogeny of epidermal growth factor receptor/kinase and of lipocortin-1 in the ovine lung. Pediatr. Res. 1989; 25:535-41. 169. Snead M, Luo W, Oliver P e t al. Localization of epidermal growth factor precursor in tooth and lung during embryonic mouse development. Dev. Biol. 1989; 134:420-9. 170. Strandjord TP, Clark JG, Hodson WA etal. Expression of transforming growth factor alpha in mid-gestation human fetal lung.Am. J. Respir. Cell Mol. Biol. 1993; 8:266-72. 171. Kida K, Utsuyama M, Takizawa T etal. Changes in lung morphologic features and elasticity caused by streptozotocininduced diabetes mellitus in growing rats. Am. Rev. Respir. Dis. 1983; 128:125-31. 172. Han R, Liu J, Tanswell A et al. Expression of basic fibroblast growth factor and receptor: immunolocalization studies in developing rat fetal lung. Pediatr. Res. 1992; 31:435-40. 173. Stiles AD, D'Ercole AJ. The insulin-like growth factors and the lung.Am. J. Respir. Cell Mol. Biol. 1990; 3:93-100. 174. Warburton D, Seth R, Shum L e t al. Epigenetic role of epidermal growth factor expression and signalling in embryonic mouse lung morphogenesis. Dev. Biol. 1992; 149:123-33. 175. Catterton WZ, Escobedo MB, Sexson WR et al. Effect of epidermal growth factor on lung maturation in fetal rabbits. Pediatr. Res. 1979; 13:104-8. 176. Sundell HW, Gray ME, Serenius FS et al. Effects of epidermal growth factor on lung maturation in fetal lambs. Am. J. Pathol. 1980; 100:707-26.

Development of the Airway Innervation

Chapter 3

Malcolm P. Sparrow* and Markus Weichselbaum Asthma and Allergy Research Institute, Department of Medicine, University of Australia, WA, Australia .

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The innervation and the airway smooth muscle (ASM) have recently been recognized as dominant, integral components of the developing lung: both are present in the epithelial tubules of the embryonic lung bud shortly after it evaginates from the foregut. Whereas the ASM is functionally mature shortly after it is laid down, the growth and maturation of the innervation largely follows the morphological stages of lung development. In the human lung, the primary pattern of branching of the bronchial tree is established during the pseudoglandular stage, followed by elongation of airways with increased vascularization of the lung periphery in the canalicular stage. The development within the acinus (thinning of the epithelial cells, expansion of the air space) occurs during the saccular stage. Until recently, knowledge of the overall innervation of the fetal airway was restricted because nerves were mostly detected only in thin sections by light microscopy, revealing cross-sections or short lengths of a nerve pathway that may be many centimetres in length, or by electron microscopy. Using such methods, a limited number of ganglia and nerve bundles were described in the peribronchial region of large airways of human fetal lungs *To whom correspondence should be addressed. The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

from 9 to 40 weeks gestation. 1 In recent years, the use of confocal microscopy has revealed many hundreds of ganglia, together with their connecting nerve pathways and fine branches supplying the ASM. A result of using thin sections was that studies of the functional neurophysiologic behaviour of airway afferents and efferents preceded detailed knowledge of the morphology of airway innervation. In this chapter our purpose is twofold. First, to provide an overview of the spectacular advances in the knowledge of the development of airway innervation due to new imaging technologies, and secondly, to review the functional behaviour of the efferent nerves associated with the lower airways.

ANATOMY, MORPHOLOGY AND DISTRIBUTION

This section describes recent morphological insights into the ontogeny of the pulmonary innervation in relation to the developing airways. Neural tissue is a dominant feature of the fetal lung and undergoes dramatic development during gestation. The stages of maturation have recently been graphically captured using confocal microscopy. Immunofluorescently stained whole lungs, lobes and airway segments have been scanned by optical sectioning through the entire Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

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thickness of the airway wall, using markers of neural tissue in conjunction with markers for ASM and epithelial tubules. From the three-dimensional information obtained, overviews and detailed images of the network of nerves and ganglia that envelop the lung primordia have been prepared. As lung development proceeds through gestation to postnatal life, comprehensive maps of the pathways of the nerves to their target tissues have provided unique views of the airway innervation. The picture that emerges is that neural tissue and ASM are an integral part of the lung from its inception, and persist in a dynamic state throughout gestation and into postnatal life and late adulthood. We present evidence for this beginning in the embryonic lung of the mouse, then proceeding through gestation in the human and the pig lung. Origin o f t h e i n n e r v a t i o n - t h e f e t a l m o u s e lung We first describe the development of the innervation of the fetal mouse lung from embryonic days 10-14, the early pseudoglandular stage. In this period, branching morphogenesis is at its peak, and every 24 h of gestation sees a striking change in lung structure and in the maturation of the innervation that accompanies branching. In mice, two lung buds begin to evaginate from the foregut at embryonic day 10 (El0), 2 whereas in humans and most mammals the lung develops from a single lung bud. Neural crest-derived cells (NCC) are present in the foregut prior to the formation of lung buds and have been assumed to migrate into the lung where they differentiate into intrinsic pulmonary neurons. 3 This migration has recently been demonstrated in the mouse lung by immunostaining whole mounts of foregut including the lung buds and imaging them using

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confocal laser scanning microscopy, from El0 and thereafter (pseudoglandular stage). 4 NCC are identified with antibodies to protein gene product 9.5 (PGP 9.5; a general neural marker) and NCC-specific markers, including phox2b and p75 NTR. phox2b is a transcription factor located in NCC nuclei. 5 p75 NTR is a low-affinity trk-receptor and is present in the membranes of NCC and their nerve processes. 4-6 Neurons are also identified with an antibody to PGP 9.57 which stains mature neurons and nerve fibres but not precursors (i.e. NCC). At El0, PGP 9.5- and p75-positive nerve fibres run along the dorsal side of the foregut. Among these fibres are many migrating NCC with phox2b-positive nuclei (Fig. 3.1a) and p75NTR-positive membranes (Fig. 3.1b) with vagal processes on either side of the foregut. At this early stage, the emerging lung buds are largely free of NCC, albeit a few solitary NCC at the base of the lung buds with occasional processes directed into the bud (Fig. 3.1b, lower inset). Some NCC in the foregut have matured sufficiently to show PGP 9.5 staining, whereas the cells in the lung buds remain negative for this neuronal marker. By E l l , the neural tissue along the foregut condenses into two large nerve trunks, the vagus nerves, which stain strongly for PGP 9.5. 4 Neural processes positive for PGP 9.5 and p75 NTR reach from the vagi to the trachea and primary bronchi (Fig. 3.2a). The vagi are comprised of neural processes and many migrating NCC (Fig. 3.2b). These processes are likely to comprise both afferent fibres originating from vagal and spinal sensory ganglia, and preganglionic efferents that will ultimately synapse on NCC once they have completed migration. Neural processes from the vagi to the primary bronchi (Fig. 3.2e) and the dorsal trachea contain

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Fig. 3.2. Mouse lung at embryonic day 11. (a) A confocal projection showing a ventral view of the right upper half of an E11 mouse lung stained for nerves (black) with the protein gene product 9.5 (PGP 9.5). This also stained the undifferentiated epithelium of the tubules and growing end buds (grey). The ASM that covers the tubules is stained with e~-actin (dark grey). The carina lies at the top of the figure. The left vagus (V) sends out nerve processes to the ASM covering the left lobar bronchus. Some extend towards the mesenchymal cap. Inset: videomicrograph ventral side. (b) A single optical section through the vagus nerve shows that it contains NCC and many axons running between them (stained with an antibody to p75 NTRwhich is positive for cell membranes and axons). (c) Nerve fibres going from the vagus into the lung (see (a)) comprise processes and NCC (stained for p75NTR).

migrating NCC. Many NCC are present on the dorsal trachea, located over the trachealis muscle and some are on the ventral surface of the proximal primary bronchi, in the process of aggregating into large ganglia. By El2 the lobular organization of the lung is complete, with one large left lobe and four smaller right lobes. A large nerve plexus is present on the ventral side of the lung on the hilum (Fig. 3.3a) which originates from the vagus 4 and is comprised of nerve fibres and large ganglia-like clusters of NCC, with numerous cells in each cluster. From these ganglia, nerves positive for PGP 9.5 (Fig. 3.3b) and p75 NTR (Fig. 3.3e) extend along the bronchi, following the smooth muscle-covered tubules. NCC also migrate along these nerve tracts but lag behind the

growth of the nerve axons (Fig. 3.3e). Superimposed confocal projections of both the neural tissue and the ASM reveal their close relationship. By El3 the neuronal precursors lying over the dorsal trachea have matured to form a PGP 9.5-positive network of thin nerve trunks interconnected by small ganglia, giving fine fibres that penetrate the smooth muscle layer. By E14 this plexus is more extensive comprising larger ganglia and more numerous thick nerve trunks with multiple connections to the vagi (Fig. 3.4a). Small nerves from the ganglia branch into many fine varicose fibres that run along the smooth muscle bundles. The ganglia vary greatly in size, and many large ganglia contain over 100 cell bodies positive for PGP9.5 (Fig. 3.4a) and their nuclei positive for phox2b

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Fig. 3.3. Mouse lung at embryonic day 12. (a) A confocal projection (ventral view) showing the Iobular organization (the accessory lobe and the vagi have been removed) with the epithelial tubules in longitudinal section. The first two laterals of the left lobe reveal the end buds in the process of dividing. The undifferentiated epithelium of the tubules, and particularly their end buds are immunoreactive to PGP 9.5 (grey-black). PGP 9.5 diffusely stains ganglia (black) connected by nerve trunks and fibres in the ventral hilum (long arrow). (b) PGP 9.5-positive nerve fibres issue from the large ganglion (long arrow) at the base of the left pulmonary bronchi and reach along the left lobar bronchus (short arrows) and along some of the laterals but no PGP 9.5-positive cells and ganglia are present along the tubules. (c) The large ganglion at the base of the left lobar bronchus (long arrow) contains many NCCs with phox2b-positive nuclei (black) and p75NTR-positive membranes (grey). The NCC migrate along the p75 NTR nerve fibres (grey, short arrows) that grow along the lobar bronchus and laterals. The cells lag behind the growth of fibres; the majority have only reached as far as the first lateral and a few as far as the second lateral. ((a) and (b) can be viewed in colour, see Ref.4, courtesy American Association of Anatomists.)

(Fig. 3.4b). The axons in the nerve bundles connecting the ganglia stain strongly for GFRtxl, the receptor for Glialderived neurotrophic factor (see below). The innervation from the vagus to the main ganglia lying on the dorsal trachea, ventral hilum and left lobe are shown schematically in Fig. 3.4e. During this early pseudoglandular phase, most nerves follow the smooth muscle-covered tubules, but some nerves course through the mesenchyme toward the lung cap, where they form varicose terminal arborizations by E13. 4 Among the first neurotransmitters to appear in the foregut is CGRP at El2. s By El3, nNOS can be demonstrated in the lung by NADPH-diaphorase activity in nerves associated with the airways and blood vessels. At El5, immunostaining reveals the presence of nNOS in neurons and fibres on the trachea, and from the hilum to the bronchioles. 9 Glial-derived neurotrophic factor (GDNF) has been identified as the most important neurotrophic factor in the development of the enteric nervous system 1~ and there is increasing evidence to suggest that G D N F is of similar importance during lung development. 11 In the gut of mice lacking G D N F or RET (receptor for GDNF), all neurons below the oesophagus and proximal stomach are absent 12 but it is not known whether neurons of the lung are affected. In cultured explants of left lung lobes at El2, neurons survive and display proliferation, differentiation and continued migration along the developing smooth musclecovered tubules. 11 In the presence of serum, a characteristic of these explants is the formation of a layer of tx-actin-positive cells (possibly smooth muscle precursors) that grows out from the lung periphery and attracts nerves that grow onto

this layer. When cultured in GDNF-supplemented medium, the amount of neural tissue on this layer increases 14-fold. The neural tissue consists of a high-density network of nerve trunks and large ganglia and comprises many PGP 9.5-positive cells, indicating that migration, proliferation and differentiation of neuronal precursors as well as neurite extension have taken place as a result of stimulation by GDNF. This suggests that GDNF is a chemoattractant to both nerves and NCC. GDNF-impregnated beads attract nerves growing out from cultured lung explants and in some instances NCC surround the treated beads. The membranes and nerve processes of the NCC are positive for the GDNFreceptor, GFRtxl (Fig. 3.4b), suggesting that nerves and NCC are guided by GDNF. The presence of GDNF-mRNA has been demonstrated in the mesenchyme adjacent to the fetal mouse epithelial tubules 13 possibly in the smooth muscle, which thus may play an important role to attract nerve fibres and migrating NCC.

MAPPING THE FETAL PIG AND

INNERVATION: HUMAN LUNG

The rapid development seen in mice during the pseudoglandular stage from days El0 to E14 contrasts with that of large mammals where the equivalent time period lasts from 3 to 8 weeks in pig and 5 to 17 weeks in human. 14'15 In mice at El4 and subsequently, the application of confocal microscopy becomes more difficult. The signal emission is reduced at increasing depth of scanning, which is a consequence of

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Fig. 3.4. Mouse trachea at embryonic day 14. (a) PGP 9.5-positive (black) network of ganglia connected by thick bundles to the vagus (lower right). Nerves from ganglia spread over smooth muscle on the surface of trachea (upper part of panel). (b) Ganglia with phox2b-positive nuclei (white) and nerve trunks staining for GFR0d (grey) lying over the dorsal trachea. (c) Scheme showing the innervation from the vagus to ganglia lying on the dorsal trachea and ventral hilum. Main nerve trunks to the lobes arise from the latter. Oblique ventral view. tr, trachea; fo, foregut; ga, ganglia; I.vag, left vagus; br, bronchus.

the increased tissue thickness and density, and the associated decrease of antibody penetration. These problems can be overcome by removal of the lung cap, mesenchyme and pulmonary vascular tissue leaving the bronchial tree fully exposed, which is only feasible in larger mammals. 16-18 Thus, the entire bronchial tree can be progressively scanned with the confocal microscope at high resolution. Using this approach montages of near-complete bronchial trees in the pseudoglandular stage about 6 m m in length from fetal pigs 14'16 (Fig. 3.5), and smaller lengths of subsegmental airways from fetal humans 17 have been assembled. These clearly display the organization of nerves and ganglia and their relationship to the ASM, the glands and blood vessels; fine detail is also shown at selected sites. 16 Thus, the development of the innervation from the embryonic lung bud to postnatal life is revealed. The structural characteristics and distribution of the nerves are similar in the three species (mouse, pig and human) at comparable developmental stages, and likewise the ASM. The muscle bundles are orientated around the airways perpendicular to their long axis from the trachea to the base of the epithelial buds, and this arrangement persists into

postnatal life. The innervation of the porcine and human bronchial tree from the adventitia to the epithelium has been reported from early gestation to postnatal life. 16-19

Pseudoglandular stage The main characteristics of this stage are: (i)

Chains of forming ganglia connected by thick nerve trunks to each other and to the vagus lying over the ASM of the dorsal trachea and the ventral surface of the hilum. (ii) In general, two thick main nerve trunks extend from the hilum along each airway to the growing tips. These lie above the ASM supported by the mesenchyme. In the fetal pig at 5.5 weeks gestation, proximal trunks (~-50ktm in diam.) run about 40-60~tm above the ASM, progressively thinning distally over a length of 4 mm to --20 ktm in diam. and 15-20 ktm from the ASM. The nerve trunks terminate as thin bundles in the collar of ASM that surrounds the epithelial buds. 16'2~ All along the length of the trunks branches descend towards the smooth muscle, and break up into small

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Fig. 3.5. (a) Montage showing dorsal view of the bronchial tree of fetal pig lung at 5.8 weeks gestation (pseudoglandular stage) with nerves and ganglia stained for synaptic vesicle protein (SV2) and for ASM with smooth muscle myosin. It has been optically sectioned to show the outline of the airway wall. Nerve trunks run down the length of the airways to the epithelial buds. Arrow shows a large ganglion. Ganglia were seen at airway bifurcations and at the branching points of the nerve trunks. An immature ganglion (boxed region) is shown in (b). (b) Two major nerve trunks stained with SV2 give rise to a fine network of varicose processes overlying the ASM. At this stage the varicose fibres are randomly distributed on and in the smooth muscle, some located within 1 pm from the muscle cells. The accumulation of cell bodies (arrow) is a precursor ganglion present at the bifurcation point of the airway. The cell profiles in the ganglion can be distinguished by the SV2-positive nerve fibres lying around them. ((a) and (b) from Ref.16, courtesy American Thoracic Society.) (c) The innervation and ASM in the developing airways of a fetal human lung at 58 days of gestation (pseudoglandular stage). The field shows branching epithelial tubules in the periphery of a lobe. Nerves and ganglia are stained for PGP 9.5 and form a network overlying the ASM stained for 0~-actin. The circumferential arrangement of the muscle bundles around the epithelial tubule is faintly seen in each of the above, where it is perpendicular to the long axis of the tubule. (See Color plate 3.)

Development of the Airway lnnervation

bundles. From these, fine varicose fibres issue that spread over the muscle ending in arborizations --1 ktm from muscle cells, suggesting functional innervation. At this stage the varicose fibres are randomly distributed on and in the smooth muscle (Fig. 3.5b) but later become oriented along the smooth muscle bundles. 16'18 (iii) Immature ganglia are present along the main trunks from which nerve branches radiate out to connect to many other smaller ganglia that form a network covering the airway wall. Fig. 3.5c shows this innervation in the distal airways of a fetal human lung at 7.5 weeks gestation. The mean distance between ganglia TM is 64_+ 18 ktm (n-87), very similar to the pig (70ktm at comparable gestation). Ganglia also lie at most airway branch points and give rise to smaller trunks that follow the airways as they proceed distally. Proximal ganglia are large (e.g. > 300 cell bodies at 5.5 weeks gestation) whereas distal ganglia are small and ultimately comprise a few neurons. Individual neurons within the ganglia show different intensities of staining with PGP 9.5, indicating variance in their type or maturity. (iv) PGP 9.5 diffusely stains nerve trunks, but many cell profiles of Schwann cells remain unstained (revealed using an antibody to the Schwann cell marker S-100). Staining for synaptic vesicle protein 2 (SV2), a component of the membranes of the vesicles in the varicosities, reveals individual varicose fibres in the nerve trunks indicating that vesicle traffic is prolific at this stage of development. 16'2~This abundance of SV2-positive fibres decreases with ongoing maturation; by postnatal life, varicose fibres are restricted to the distal nerve bundles and the fine fibres that lie on and in the ASM. Staining for neurofilament sharply defines a small proportion of individual fibres in a trunk; these can be traced along the tubules where several terminate in the collar of smooth muscle that surrounds the base of the epithelial bud. 2~ The low proportion of neurofilament-positive fibres in the nerve trunks may reflect the level of maturity of these nerves, since the proportion of neurofilament-positive neural tissue increases as gestation progresses. TM

Canalieular stage With airway growth there is increasing spatial separation of the ganglia. The large ganglia lying on the central airways that form nodes at nerve junctions undergo a fourfold increase in separation to --254 ktm. The ganglia vary greatly in s i z e - large ones are 1201.tin at their greatest width and contain as many as 200 neurons of--11 t.tm diameter. Many of those lying on the trunks gradually become displaced laterally to become attached by a stem, with nerves radiating out from them over the airway. TMThe bronchial vasculature becomes more prominent with arterioles running adjacent to the trunks and around the ganglia. Nerve fibres penetrate the submucosal glands. By midterm, ganglia have condensed and become compact and spherical. Fig. 3.6a shows a montage of nerve tracts in the subsegmental airways of

an 18-week fetal human lung. 17 Large nerve trunks run the entire length of airways reducing in diameter from 45 to <20 ktm distally, with many ganglia attached to them from which nerves issue to connect with a network of smaller ganglia lying closer to the airway surface. A high-power view (Fig. 3.6b) shows a fine plexus of nerves containing many small ganglia lying close to the ASM. Mucosal nerves are now abundant; they arise from branches of the adventitial nerves that penetrate the ASM layer at intervals where they run in parallel bundles in the lamina propria along the length of the airway. 17 The development of the mucosal vascular circulation is now well advanced. 19 At this point the lung is well endowed with the beginnings of a neural network that can serve the afferent and efferent functions of the vagus nerve. Most neurotransmitters make their appearance during the canalicular stage (in human fetal lung 16-26 weeks, 15 pigs 7-13 weeks, TMrats 18-19 days and mice 16.6-17.4 days21). In rats at day El7, calcitonin gene-related peptide (CGRP) is present in neuroendocrine bodies (NEB) in the epithelium. At 18 days, CGRP nerve fibres are present in the trachea, stem bronchi and proximal intrapulmonary airways, mainly lying below the epithelium, and by days E19-20 fine fibres are seen on the bronchial smooth muscle and around blood vessels in the adventitia. 22 In mouse lung, nitrergic neurons can be detected in the airways as early as El3 by using an assay for N A D P H diaphorase; by El5, nNOS expression is present in airway neurons and fibres. 9 Functional cholinergic transmission has been demonstrated (as airway narrowing) in the pseudoglandular stage indicating that some fibres are already cholinergic. 2~ In humans, evidence for the presence of cholinergic neurons by 10-12 weeks was reported using acetylcholinesterase 3 which may not be a reliable marker for the presence of acetylcholine (Aeh). 23 Choline acetyltransferase (CHAT) is a specific marker for ACh, 24 and in fetal pig lungs both ChAT-positive neurons and fibres are present in the trachea and on the ASM of the peripheral airways at the early canalicular stage. ChAT does not stain the very fine terminal varicose fibres that SV2 reveals, which may indicate that it is not sensitive enough to detect very low levels of ACh. TM In human lung, vasoactive intestinal peptide (VIP) and substance P (SP) positive fibres are present at 16 weeks in ASM, and thin fibres containing CGRP start to ascend from the basement membrane of the epithelium. 3 The latter are likely to be the sensory C-fibres seen in postnatal life. 19'25'26 Substance P and CGRP are present in the axons of the nerve trunks running in the airway adventitia at midterm TM and by the beginning of the saccular stage VIP, T H and NPY are also present.

Saccular stage In humans, the saccular stage runs through most of the third trimester, when further maturation of the ganglia and nerves occurs. The processes of glial cells increasingly surround neurons in the ganglia, and the axons in nerve trunks

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Fig. 3.6. (a) A montage showing the peribronchial innervation of a segmental bronchus and its branches from a human fetal lung (18 weeks gestation, canalicular stage). Nerves are dark grey (PGP 9.5 stain) and smooth muscle light grey (0~-actin). Nerve trunks extend to the most distal airways. Ganglia are present along the trunks and at the divisions of nerve bundles. The inset shows a higher power projection of a ganglion at the junction of several nerve trunks (arrow). (b) A higher power view of the straight region on the lower right-hand side of the montage showing the disposition of the nerves, ganglia and ASM. PGP 9.5 stained a plexus of fine nerves containing many small ganglia. Arterioles of the bronchial circulation can be seen accompanying the larger nerve trunks (see Ref.17, courtesy American Thoracic Society). (See Color plate 4.)

Development of the Airway Innervation

and bundles. TM This glial ensheathment may contribute to restricting intraganglionic communication between adjacent neurons. 27 Separation of the ganglia greatly increases as the airways lengthen and widen. Neurotransmitters are fully expressed now with strong immunostaining of neurons and their axons. TM Neurofilament is now expressed in many neurons and axons. The perikarya are located mainly in the periphery of the ganglion with many neurite structures in the centre. Neurons appear to contain one major axon and therefore correspond to Dogiel type 1 neurons. Some neurons show strong PGP9.5 staining of the nucleus only while others exhibit a faint homogeneous staining throughout the perikaryon. TM The bronchial mucosal circulation, which is rudimentary at the end of the pseudoglandular stage, increases in complexity during the canalicular stage, becoming a welldeveloped network of microvessels. The mucosa is now richly innervated with nerve bundles and varicose fibres running the length of the airways which stain for NOS, SP, CGRP, VIP, T H and NPY in fetal pig lung. TM The presence of the neuropeptides SP and CGRP is indicative of an afferent population of nerves. Although it appears that many nerve bundles follow arterioles, immunohistochemical evidence suggests the opposite, as the neural tissue can be stained earlier (i.e. in the pseudoglandular stage) than the bronchial vessels that are first demonstrable in the canalicular stage, where they run contiguously with the nerves.

Function of the airway innervation during fetal life Whether the innervation plays a functional role during specific events in fetal lung development is unknown. With the recent insight into the organization of the nerves in the bronchial tree, it should be feasible to study neurotransmission in the ganglia and nerves in the lung either excised from the fetus, or in situ. At birth, a range of sensory reflexes is present (e.g. Hering-Breuer reflex). It is possible that some afferent mechanoreceptors, such as slowly adapting stretch receptors, located in or adjacent to the ASM, may already be firing at a low rate in the fetal lung as it is expanded with liquid. It seems reasonable to assume that well before birth afferent and efferent nerves are capable of function, although little is known about the pathways of afferent nerves and their receptors in fetal airways. Afferent fibres must have been present when NCC and nerve processes migrated from the vagi at the formation of the lung bud. They doubtless make up a major part of the nerve trunks in the fetal lung since more than 70% of the nerves in the vagus that innervate the lung are afferent. 28 These are the fibres that pass through the ganglia as they extend distally to their sensory receptor endings. Markers of C-fibre sensory nerves (i.e. SP and CGRP) are seen in fibres in lamina propria of rats at mid-term 22 and in the epithelium in humans, 3 but the apical plexus of C-fibre nerve endings in the epithelium that constitute the receptive fields i n postnatal pigs (see Fig. 3.7b) and humans 19were not observed. Surprisingly, mechanoreceptors which are large tree-like arborizations 100-200 ~tm in length have not been recognized

in the fetal airways, but more focussed searching in the ASM and lamina propria of the trachea, bronchi and bronchioles may reveal them.

Summary: ontogeny of lung innervation before birth Neural tissue and ASM are integral components of the primordial lung in which epithelial tubules (the future bronchial tree) are enveloped in a network of precursor ganglia and loose bundles of nerve fibres. These ganglia comprise fiat patches of NCC that have migrated along nerve processes that issue from the vagi. They lie over the wall of the epithelial tubules supported by the mesenchyme, and are interconnected by nerve bundles. ASM is laid down at the base of the epithelial buds that are sites of new tubule growth which occurs through an epithelial-rnesenchymal interaction. 29 Thus, as the epithelial tubules elongate, the ASM forms a continuous layer that extends from the trachea to the growing tips. Small nerve bundles branch from the nerve network and descend to the ASM; the ASM is functionally mature shortly after it is formed as the terminal tubules show rhythmic contractions in situ. 3~ G D N F is a likely neurotrophic factor that acts as a chemoattractant for nerves in lung explants, 11 and G D N F receptors (GFR~I) are present on the nerve processes in vivo but whether G D N F is expressed by ASM is not yet known. By the end of the pseudoglandular stage, when branching is virtually complete, most precursor neural tissue has completed proliferating, and is differentiating into mature neurons. In the canalicular stage, ganglia develop a more compact, spherical shape, and come to lie away from the nerve bundles. Arterioles of the bronchial circulation appear adjacent to the nerve trunks and nerve bundles. The mucosal innervation becomes established followed by the mucosal vasculature. The chemical coding of neurons and their fibres occurs during this stage. In the saccutar stage (most of the third trimester), lung growth is rapid with greater spatial separation of the ganglia, and their neurons become progressively ensheathed by glial cell processes as do the axons in nerve trunks and bundles.

POSTNATAL LUNG Early postnatal period to late adulthood: Overviews and higher power views obtained b y 3D imaging of the adventitial (peribronchial) and mucosal innervation have recently been obtained using whole mounts of airway from young pigs, 16'19 humans, 17 mice 4'11 and rats. 33 The peribronchial nerve plexus has been designated as 'extrachondrial' and 'subchondrial' in the bronchi, where the main trunks lie outside the cartilage plates with a lesser layer of nerves between the cartilage and the ASM. 34 In the bronchioles the distinction disappears. Since only a single nerve plexus originates over the ASM in the fetal lung, the peribronchial (adventitial) nerves can be regarded as a single entity. With postnatal growth this plexus continues to comprise two major nerve trunks running the length of the airways.

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Fig. 3.7. (a) Confocal projection showing SP immunoreactive nerves in the bronchial epithelium of a young pig. Nerve endings have one or more enlarged terminal varicosities (arrows). Some nerves have curved profiles (arrow heads) where they encircle goblet cells (not stained). Image depth from lumen 21 l~m. Bar 50 t~m. (b) Projected cross-sections show SP nerves in the epithelium arranged in two plexi. An apical plexus lies immediately below the luminal surface (lumen, L) with fibres descending to the base of the epithelium to form a second lateral layer. Crosssections were reconstructed from a confocal z-series imaged from the lumen surface with optical sections 0.5 l~m. Loss of resolution occurs in reconstructing side views, making the nerves appear thicker than they are. (c, d) Confocal projections of the nerves overlying the ASM viewed from the adventitial surface of the bronchus of an adult mouse (c) and of a 54-year human (d). Varicose nerves run along the ASM bundles which lie around the circumference arranged perpendicular to the long axis of the airway (muscle bundles not shown to avoid obscuring nerves). (To view double staining in colour see Refs4'18). (e) Varicose nerves in close proximity to pulmonary neuroendocrine cells (NEC) in the epithelium of a human bronchus. Both nerves and NEC are revealed with PGP9.5. '0 ~ lumen view, 90 ~ side view. The latter shows that the cell body of the NEC stands at the base of the epithelium and the apex at the lumen. Processes extend along the basement membrane and others upwards toward the lumen.

D e v e l o p m e n t of the A i r w a y I n n e r v a t i o n

Large bundles frequently branch off and rejoin the main trunk or lesser branches. 16'34The density of the nerve fibres in this plexus is sufficiently great as to give the appearance of a continuous network. 16 However in the terminal airways the nerves thin out revealing arborizations of varicose fibres. In the fetus some nerve bundles from this adventitial plexus penetrate between the bundles of the ASM to initiate the development of the mucosal plexus. 17 After birth in pigs, large nerve bundles run the length of the lamina propria lying close to the ASM border. Smaller bundles comprising both sensory and motor nerves 19'35 branch off to run parallel with the bronchial arterioles while others continue upward to enter the epithelium where they form a basal plexus of fibres lying just above the basement membrane. In pigs and humans, fibres ascend from this basal plexus between the epithelial cells to within a few microns of the lumen where they arborize forming thin (---1 ~tm diam.) varicose fibres that spread out (up to 120 ~tm in length) over the apical epithelium (Fig. 3.7a). Each varicose fibre terminates in one of more swollen varicosities (--3 ~m diam.) which contain SP and C G R P . 19'26 These are the sensory endings of the C-fibres 19'35 which respond to chemical and mechanical stimuli. 36 They have been demonstrated immunochemically in thin sections in most species in which they contain SP, CGRP and neurokinin A. 37'38 When confocal projections of the SP nerves in the epithelium of pigs are reconstructed, the basal and apical plexuses are readily seen (Fig. 3.7b). A dense plexus of varicose fibres containing SP in the epithelium of rat trachea have been demonstrated, although an apical plexus was not distinguished. 33 Although recent advances made using 3D imaging techniques have given new insight into airway innervation, the classic drawings of lower airway nerves, neurons and afferent mechanoreceptors in infants, rabbits and dogs form the basis of our current understanding of lower airway innervation. 34'39 In the last 20 years, the use of specific antibodies to most neurotransmitters of the autonomic nerves have enabled identification of the chemical coding of nerves in the airway wall. The lack of a suitable antibody to stain parasympathetic cholinergic nerves is a major problem; staining for acetylcholinesterase is not specific. 23 A recently introduced antibody to ChAT 24 lacks the sensitivity to reveal fine varicose nerve fibres. TMAn antibody to vesicular ACh transporter protein has been successfully used in the intestine of rats. 4~Notwithstanding, a large body of information has been obtained on the neurotransmitters found in the neurons in the parasympathetic ganglia, and in nerves of the lower airways. There are many recent specialized reviews available on neurotransmitters in the nerves in ASM, 41 glands and goblets cells, 42 immune tissue in the airway wall,43 bronchial vasculature, 44 as well as their suggested roles in cotransmission and neuromodulation. 45'46

Density of innervation With the realization that nerves are abundant in the fetal and postnatal airways in at least five species of mammals

Table 3.1. Density of innervation of single axons (mm/mm 2) in the mucosa and ASM of rat and pig.

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epithelium 190,000t.tm2 (courtesyJ Lamb, Universityof Western Australia); PGP 9.5, a pan-neuronal marker; SP, substance P.

(humans, 17 pigs, 16 mice, 4 rats 33 and dogsl8), the question can be p o s e d - what is their functional significance? Firstly, the innervation needs to be quantified so that comparisons can be made between tissues (e.g. ASM and epithelium) and species (e.g. rat and pig). Nerve densities in the trachea of postnatal rats 33 and bronchi/bronchioles of pigs 16'19 have been estimated by a point counting method (Table 3.1). The studies show that the total innervation to the ASM is --2-fold more dense than in the epithelium, and the densities in pigs are about twice that of rats. SP nerves comprise 90% (rat 33) and 94% (pig 19) of the total epithelial nerves (stained using PGP 9.5). While nerves have not been quantified in the fetal lung, montages of the fetal airway innervation 4'16'17 show that their abundance approaches that of postnatal life. Bybirth, the varicose fibres covering the ASM are oriented in the direction of the muscle bundles, with single fibres running along most muscle bundles in the young pig, 16 adult mouse (Fig. 3.7c), human infant 17 and adult (Fig. 3.7d). Thus nerve density is maintained during the considerable growth that occurs during development, indicating that nerves continue to extend over the expanding surface of the airway wall as growth proceeds. This arrangement of nerve fibres running along the muscle bundle is probably the main determinant of the ultimate nerve density attained in ASM.

Efferent nerves: long preganglionic fibres and short postganglionic fibres In the adult lungs of large mammals, the distribution and morphology of the innervation becomes increasingly difficult to characterize because of the sheer size of the lung and airway tree. The thickness of the layers of tissue, particularly connective tissue and cartilage, makes the

adventitial nerves and ganglia difficult to expose. Furthermore, the density of ganglia becomes reduced with growth so that they become difficult to locate. Indeed, ganglia are considered to be absent beyond the third-generation airways, a view largely based on evidence from early reports. 34'47'48 From this, it is assumed that long postganglionic nerves run from the ganglia in the central airways along the length of the bronchial tree to the terminal airways. 27'34 The central ganglia may therefore play a key role in regulating airway function; 27 however, this view is not compatible with studies on the development of the airway innervation. 14'16'17 Ganglia are shown to extend to the 9-10th generation in the canalicular stage in 18-week fetal human lungs (see Fig. 3.6a). In the lung of mid-gestation fetal pigs, ganglia extend to distal generations of 50~tm diameter, which is the limit of dissection. 14'1s These ganglia are mature (proximal) or maturing (distal) and we have no reason to believe that they might disappear from the lungs (e.g. through apoptosis) at this late stage of development. It is reasonable to assume that ganglia lie chiefly in a thin layer surrounding the airway wall; the extent of their separation with lung growth is then a function of the increase in the surface area of the airway wall and the increase in length of the airways of the bronchial tree. It should be possible to obtain estimates for these parameters to determine ganglia separation in the adult, and thereby the probability of finding them in thin sections. In summary, studying the ontogeny of neural development reveals long preganglionic fibres and short postganglionic fibres which is consistent with other tissues innervated by autonomic nerves.

Orientation of airway smooth muscle bundles In the fetal lungs of humans, pigs and mice, muscle bundles encircle the airways and lie perpendicular to the long axis of the airways from the trachea to the terminal airways. This orientation is maintained into postnatal life in these species as well as in postnatal rat and young dog. TMAn ultrastructural study reporting that ASM has a pitch of approximately 30 ~ in adult humans 49 is at variance with these wide-ranging confocal microscopic studies in which large areas from the central and distal airways have been scanned. 5~At branching points, the orientation of some ASM bundles varies widely, presumably to suit the local airway architecture, and occasionally bundles lie almost parallel with the length of the airway. 5~

Innervation of pulmonary neuroendocrine cells Pulmonary neuroendocrine cells arise from undifferentiated epithelial cells of endodermal origin that line the tubules in the fetal lung early in gestation. 51 NEC occur both as solitary cells and as clusters in conjunction with Clara cells called NEB 5e'53 and are distributed from the nasal respiratory mucosa 54 to the terminal airways and alveoli) 3 In humans, differentiated NEC and NEB are present by 10 weeks gestation. 55'56 The NEC are bottle- or flaskshaped, and reach from the basement membrane to the

lumen. They can be distinguished by their profile of bioactive amines and peptides, namely serotonin, calcitonin, CGRP, chromogranin A and bombesin. 57 NEB may play a role as hypoxia-sensitive chemoreceptors 58 as an O2-sensitive K § channel coupled to an 0 2 sensory protein has been demonstrated in their membrane at the luminal surface) 9 They may also be involved in regulating epithelial cell growth and regeneration through a paracrine mechanism whereby their bioactive peptides are released into the surrounding epithelium and lamina propria. 6~ Ultrastructural studies have shown that some NEC and NEB become innervated in fetal life. 51'5L56 Nerve terminals with synaptic contacts have been described at the base of the NEC in infant bronchial epithelium. 61 In fetal human lungs at mid-gestation, cholinergic axon terminals lie deep within NEB. s6 Some terminals exhibit vesicle profiles indicative of adrenergic fibres and form gap junctions with adjacent cells within the NEB. However, the majority of axons are sensory, as demonstrated by a loss of NEB innervation after unilateral vagotomy. 62 In rats, labelling neurons in the nodose ganglia with DiI enabled tracing of sensory afferents to NEB. 63 These nerves do not contain the sensory neuropeptide CGRP in contrast to the afferents that supply the C-fibre endings in the epithelium. NEC are abundant in adult human lungs and are homogeneously distributed in the epithelium; 64 the density is - 2 5 0 per mm 2 which is higher than previously reported. 65 Solitary NEC exhibit a predominantly flask-like shape (Fig. 3.7e); the base of the cell bodies is located at the basement membrane and long processes extend along the basement membrane while other processes extend upwards to the luminal surface. Patches of nerves were present in the epithelium, some lying in close apposition to NEC (Fig. 3.7e) suggesting functional innervation. CGRP was present in the processes of 20% of all NEC revealed with PGP 9.5 and bombesin. Three-dimensional animated renditions of NEC illustrate the extraordinary complexity of the processes and localized distribution of the CGRP and other markers. In humans, NEB decrease in frequency with age and are rare in adult lung. 65 The physiological role of the innervation to NEC and NEB is not well understood. It has been proposed that the nerve endings at the base of the NEC subserve an axon reflex, presumably in the NEB itself and possibly to deeper tissues such as ASM. 55 There may also be local reflex connections through peripheral ganglia. Hypoxia detected by the 0 2 sensor in the NEC is presumed to release mediators that stimulate vagal afferents, but no central nervous reflexes have been identified. Whether they exert only intraganglionic effects remains to be shown. Recordings from single afferent fibres from NEB have not been made, and all studies on the effect of hypoxia on vagal afferents from C-fibres, rapidly adapting and slowly adapting receptors have been negative. 66 Advances in microscopic techniques may shed light on the morphological basis for many of the suggested functions of NEC innervation.

Development of the Airway Innervation

O N T O G E N Y OF AIRWAY S M O O T H MUSCLE F U N C T I O N : F U N C T I O N A L CONSEQUENCES The functional consequences of ASM activation are typically considered in relation to the effects of airway narrowing on airway resistance. 67 Increased resistance leading to increased work of breathing and eventual respiratory failure are two features associated with inflammatory neonatal lung diseases, such as bronchopulmonary dysplasia (BPD). 68'69 The latter consists of a range of pathophysiologic changes including non-specific airway hyperresponsiveness. 6s-7~ That ASM innervation is involved in these events can be inferred from the action of muscarinic antagonists o r ~2 agonists, both of which cause bronchodilation in BPD. 71-73 Indeed, the action of muscarinic antagonists suggests that the activity of vagal innervation to ASM, which relies on excitatory cholinergic pathways, is significantly increased. 72 Although the pathophysiologic approach to understanding ASM function illustrated above has shed much light on the ontogeny of airway function, considerable data suggest that the ability to regulate the contractile function of ASM is a normal characteristic of both neonatal and prenatal life (see below). Furthermore, elucidation of mechanisms involved in the signal transduction and the control of intracellular contractile machinery of smooth muscle have begun to improve understanding of the ontogeny of ASM function. The following section highlights the functional consequences of ASM activation on the pre- and postnatal lung, and the insight provided by recent advances in muscarinic receptor biology and signal transduction mechanisms controlling the molecular machinery responsible for smooth muscle shortening.

Prenatal ASM function Some evidence was present in the 1930s for contractile function in human fetal airways. TM This observation, however, was largely forgotten until a series of elegant studies of fetal airway narrowing. 75-83 These began with studies showing contractile function in airways from first trimester human airways studied in vitro 76-84 and were followed by a series of studies on fetal airways from porcine and human airways. 77-83 These reports highlighted both the ability of airways to narrow in response to cholinergic agonists, and the loss of airway contractile function in the presence of the non-specific muscarinic receptor antagonist, atropine. In the fetal pig (8g) during the pseudoglandular phase, the airways exhibit contractile activity (term---1.3kg); 81 furthermore, airway narrowing is observed during exposure to cholinergic agonists and to field stimulation, both of which are blocked by atropine. The response to field stimulation was also eliminated by tetrodotoxin, suggesting that the functional cholinergic innervation appeared very early in the developmental sequence of events related to the lung. 81 Perhaps the greatest challenge of studies describing fetal bronchial motility is the interpretation of the physiologic role of the precocial development ofvagal innervation to ASM.

The peristalsis-like waves of activity observed in these preparations were reminiscent of those reported for the gut, but without a functional correlate. It was speculated that contractile activity may indeed be important for normal lung development by providing local stretch of the airway wall. 5~ Stretch is both a powerful and important stimulus to lung growth 85-88 (see Chapter 9) and contractions of the fetal airways may be part of a coordinated series of events that lead to appropriate alveolar development. 81'85-88 As outlined above with respect to the innervation of the preand postnatal lung, there is an abundance of evidence showing the presence of innervation to the airways as well as considerable ASM. 79'81'89-91 Vagal innervation to the lung is also thought to be critically important for the successful transition from fetal life to air breathing at birth, as it has been shown that the successful transition to air breathing at birth is dependent on intact vagal innervation to the lung 92 and that loss of innervation in utero leads to altered surfactant function. 93 These studies illustrate other potentially critical physiologic roles for prenatal vagal innervation.

Postnatal A S M function Studies of the postnatal function of ASM predate those described above for fetal airways and as such, they were somewhat preoccupied with demonstrating the presence and impact of innervation in V/VO,94-99 o r in V/I/'O.77-79'100-104'104-112 Direct activation of vagal efferent nerves to ASM in the newborn narrows and stiffens the airways. 83'96'113-115Considering the increased compliance of neonatal airways 82'1~ in vitro, it is surprising that the change in lung resistance in vivo is not greater than that seen in the adult (for review seellS). The physiologic wisdom of this dichotomy is reflected by both in vitro and in vivo studies. In the former, ASM contraction is sufficient to retard the normal collapse or narrowing of airways subjected to collapsing pressure. 83'113,115 The response to maximal activation of vagal efferent fibres or to injected cholinergic agonists in vivo are modest in the newborn. 96'109'119 This may reflect enhanced parenchymal interdependence in the newborn 12~ which could provide some level of protection against airway closure compared to the adult. TM

REFLEX CONTROL OF ASM IN THE NEWBORN LUNG The ASM of the newborn is activated in response to various stimuli, 67 including chemoreceptor 95 or irritant stimuli. 125-127 Reflex responses rely upon the function of peripheral receptors, both within and outside the lung, integration of such feedback by medullary centres and neurotransmission from vagal nuclei and ganglia to the airways. 118 There is little insight into the impact of such integrated behaviour in the fetal lung. In the newborn, studies performed in vivo show that chemoreceptor stimulation by inhalation of CO 2 results in a modest reflex bronchoconstriction, 95 presumably reflecting

46

The: ~ungi:: Deve/0pmen:t:: Agi ng anti'it:he'Environment I

the increased drive to breathe associated with chemoreflex stimulation. 128 Indeed, chemoreflex stimulation appears to cause a parallel output to both respiratory skeletal and smooth muscles. 128 Unlike the adult, 129'13~hypoxia does not generally cause an increase in bronchomotor tone in the newborn. 95'131 This probably reflects the fact that in the newborn hypoxia can cause a decrease in respiratory drive due to a concomitant decrease in metabolic rate. 123'132'133 Therefore, the lack of bronchomotor effects associated with hypoxia in the newborn 95'131 is consistent with the parallel output hypothesis for respiratory skeletal and ASMs. 128 The reflex responses to irritant C-fibre stimuli, such as capsaicin or lactic acid, are suggestive of protective airway responses designed to limit airway injury. Although the former represents the experimental strategy of using a vanilloid compound to activate the capsaicin receptor/vanilloid receptor 1 or VR1,134'135 the latter stimulus is more physiological, 126'127'135'136 especially in the newborn. 126 The reflex narrowing and stiffening of tracheobronchial airways 125-127 causes inhaled irritants to be impacted at large airway branch points and protects the airways from dynamic collapse during rapid shallow breathing. In the newborn lamb, C-fibre afferents also alter the pattern of breathing and laryngeal muscle responses during irritant stimuli such as capsaicin or pulmonary edema. 137'138 Thus, the neonatal response to lung irritation relies on the integrated efferent control of both upper and lower airway muscles, which is initiated by C-fibre afferents. Whether such reflex actions are present in the fetus remains to be seen. One might speculate that such control would be present, since the control of laryngeal aperture is an active component of fetal breathing that influences the egress of pulmonary fetal liquid (see Chapter 9).

MUSCARINIC RECEPTORS IN THE LUNG Muscarinic receptors are members of the heterotrimeric G-protein-coupled receptor (GPCR) family for which five muscarinic receptor subtypes have been cloned. 139-141 Even numbered muscarinic GPCRs (M2 and M4) are coupled to Gi/G o and exert their action through inhibitory effects on adenylate cyclase, whereas odd numbered muscarinic GPCRs (M1, M3 and M5) are coupled to Gq/G11. In ASM cells, the latter act through stimulatory effects on phospholipase C and inositol phosphate generation 118'141 to cause airway narrowing. Desensitization of GPCR-mediated effects is due to G protein receptor kinases (GRKs) and protein kinase C . 142 It is widely recognized that muscarinic receptors play a critical role in the cholinergic responses of the heart, lung and CNS. 118'141'143-146 Muscarinic receptor antagonists provide some insight into receptor subtype distribution, but they lack a high degree of selectivity and therefore possess unwanted side effects. 118'119'141'147 This continues to hamper the ability to assign specific actions to specific muscarinic

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receptors, or to identify specific interactions between muscarinic receptor subtypes. Mutant mice have been engineered to lack specific muscarinic receptor subtypes, and so the question of the physiologic role of different receptor subtypes can be addressed. 141'143-146 Muscarinic receptors are important in asthma, where an exaggerated vagally mediated reflex bronchoconstriction and airway hyperresponsiveness to muscarinic acetylcholine receptor (mAChR) agonists are present. 14s-15~Furthermore, the magnitude of neural muscarinic activation to the lung plays a critical role in determining whether airway closure occurs. 124 These findings and others 15~ highlight the exaggerated vagal efferent activity in the asthmatic and the importance of understanding the control of signal transduction in ASM. Based on the effects of "selective" pharmacological antagonists in vivo the cardiopulmonary actions of ACh in the newborn display some differences from the adult. 119 This, plus in vitro studies have suggested differences in receptor subtype expression or action compared to the adult. 119'151 Three of the five cloned muscarinic receptors have been classified as having cardiopulmonary actions. 152-154 The M1, M2 and M3 receptors have all been linked to the vagal efferent pathway to ASM 11s and M4 has been found in lung tissue. 155-157M1 receptors have been suggested to be located on neural tissue 152'153 and in airway sympathetic ganglia of some species. 152'153 M1 receptors have also been suggested to be present in parasympathetic ganglia of some species, 158'159 including humans, 153'154'16~but not of others. 152'161'162 Prejunctional M2 receptors presumed to be located on post-ganglionic vagal axons are autoinhibitory and reduce the magnitude of vagally mediated bronchoconstriction in viT)o. 152'159'163-168 Viral infection 169 and experimentally induced airway inflammation cause a downregulation of prejunctional M2 receptors that lead to exaggerated vagally mediated bronchoconstriction. 170' 171 M2 receptors of myocardial tissue are thought to mediate the bradycardia associated with vagal stimulation. 141'146 The M3 receptor is thought to mediate contraction of ASM and mucus secretion, 154'162'168'172and it is the receptor subtype target for anti-cholinergic bronchodilators. 72' 152- 154' 173 Use of these non-specific antagonists causes bronchodilation in normals, asthmatics, COPD patients and in infants with 1 bronchopulmonary dysplasia. 72' 152,53,173,174 The M3 receptor is also linked to vasodilation mediated by NO. 141 M4 receptors have been detected in rabbit lung, but their functional significance remains unknown. 154-157

Muscarinic receptors: development and pharmacological antagonists The developmental physiology of muscarinic receptors has relied on selective antagonists and suggests that M receptor differentiation is present but is developmentally regulated. 119'151 Typically such approaches compare the ability of several "selective" muscarinic antagonists to differentially inhibit airway vs cardiac events associated with vagal stimulation in vivo. 119'147 Indeed, it is only by comparing multiple

Development of the Airway Innervation

antagonists that one can begin to assign function based on selective antagonists in vivo, especially with M3 and M1 antagonists.159'161'163'164 Physiologic studies of neonatal ASM have suggested that muscarinic receptor subtypes are at least partially differentiated at birth. 119'151 Studies using semi-selective muscarinic receptor antagonists suggest that M3 receptors in ASM are most likely responsible for airway narrowing. 119 However, M2 receptor responses are either absent or sufficiently weak that the autoinhibitory function of prejunctional M2 receptors on ACh release which is normally seen in the adult is not apparent. 119 This is supported by indirect studies of M2 receptor pharmacology. 119'151M1 receptors do not appear to be functionally significant. 119'151Notwithstanding the above, much remains to be learned about the pre- and postnatal maturation of muscarinic receptor subtypes. For example, studies of cardiac tissue have suggested that mRNA or protein for various muscarinic receptors are present in areas that were traditionally thought to be exclusively M2 receptor in function. These types of molecular studies have yet to be carried out on pre- or postnatal airways. Indeed, the recent availability of M2 and M3 receptor knockout mutant mice lacking receptors for these subtypes provides a unique opportunity to explore the role of muscarinic receptor function in the developing lung.

Control of A S M excitability via Ca 2+ sensitivity The primary mechanism responsible for the activation of smooth muscle is an increase in the intracellular calcium concentration. 175'176In smooth muscle, cytosolic Ca2+, binds to calmodulin (CAM) which, as a regulatory unit, activates myosin light chain kinase (MLCK) to phosphorylate the 20 kDa myosin light chain (MLC20) at serine 19.175 Phosphorylation of the MLC allows the actin-activated myosin ATPase to cause contraction (reviewed in Refs175'177'178). Physiologic control of the sensitivity of the contractile machinery to cytosolic Ca2+ is an important regulatory mechanism that is part of pharmacomechanical coupling of smooth muscle. 175-178Thus, some agonists increase the sensitivity of the contractile machinery such that for a given cytosolic Ca 2+ a greater force is obtained (reviewed in Refs175-178). An example of an agonist that causes calcium sensitization is that of txl-adrenergic agonists, which cause a greater force to be generated in response to a given cytosolic [Ca2§ in vascular smooth muscle (i.e. an enhanced force/Ca 2§ ratio). 179'18~Muscarinic agonists exert a similar physiologic effect in guinea pig ileum muscle TM and tracheal smooth muscle. 182'183 Interestingly, tracheal smooth muscle displays a developmentally regulated contractile responses to catecholamines in the newborn which is lost in the adult, where only relaxant effects are present. 184-186The response is mediated by t~-adrenergic receptors (tz 1 and t~2) and in addition to postnatal changes in adrenergic receptor populations Ca2§ sensitization may be present. 179'18~Although calcium sensitization of ASM during development has not been explored there is recent evidence of developmental regulation in cerebral blood vessels. 187 The molecular mechanisms

responsible for Ca 2+ sensitization in ASM are inadequately explored, especially in light of the recently discovered functions of the small, monomeric GTPases (Ras p21-related superfamily), in particular the Rho subfamily. 175'176'188-191

Monomeric GTPases Small GTPases (N21 kDa, hence p21 proteins) act as molecular switches in regulating cell function. 176'188'192 The archetypical and best studied of these monomeric G-proteins are the Ras GTPases due to their role in cancer. 188'190'191 Other monomeric G-proteins include members of the Rho family, RhoA, Rac and Cdc42.176 These proteins bind GDP/GTP and, when activated, stimulate protein kinases. 176'188'192 RhoA activates Rho-associated kinase (ROK) while Racl and mammalian Cdc42 activate p21-activated kinase (PAK). 19e These families of small GTPases are inactive when bound to GDP, but are active when bound to GTP. The conversion from the inactive to active form is mediated by guanine exchange factors (GEFs) and further regulation is provided by GTPase activating proteins (GAPs) that enhance the rate of hydrolysis of GTP to GDP. 176'188'192 Binding of GTP allows monomeric G-proteins to activate their associated kinase. Inactivation of p21 is by GAPs which induce GTPase activity and hydrolysis of the bound GTP to GDp.176,188,192

Although activated Rho, Rac and Cdc42 have been associated with cytoskeletal and motility events in nonmuscular tissue (see Ref.176), Rho, Rac, Cdc42 and their associated kinases ROK and PAK have been found to act in smooth muscle. 176'189'193 Although six isoforms of PAK have been identified, which isoforms might be involved in smooth muscle control is not clear.

D o P A K and/or R O K cause Ca 2+ sensitization in ASM? The most well-defined mechanism of Ca e+ sensitization acts via inhibition of MLC phosphatase, which in turn increases the phosphorylation of the regulatory MLC20175'176'179'180'194 and therefore, force generation. 175'176'178'18~ Inhibition of MLC phosphatase has been described in tracheal homogenares from adult animals but the mechanism of this response has not been explored; Ca 2+ sensitization itself is thought to be a fundamental feature of smooth muscle and the mechanisms can vary between smooth muscle from different tissues.175,176,178 Both PAK and ROK can produce CaZ+-independent contractions of skinned smooth muscle fibres but apparently through independent mechanisms. 176'189'193 The skinned fibre preparation allows direct access to the cytoskeletal apparatus and control of the free Ca 2§ by permeabilizing the membrane with triton. In vascular smooth muscle, ROK induces Ca 2§ sensitization by phosphorylating and inactivating MLC phosphatase as well as directly phosphorylating MLC kinase. 176'189'193'195 Interestingly, inhibition of ROK reduces arterial pressure in hypertensive rats, suggesting that such a pathway could be important in regulating vascular smooth muscle tone. 195 ROK has been suggested to be

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involved in contraction of guinea pig tracheal smooth muscle. 195 However, this was based on: (1) putative inhibitots of ROK, rather than measurements of the effects of R O K added to skinned fibres; (2) unreported data for the effect of R O K inhibitors; 195 and (3) a specificity of the inhibitor that remains to be fully determined across smooth muscle types. Thus, the role of R O K remains to be fully elucidated in both adult and neonatal ASM. The addition of constitutively active PAK to tritonskinned taenia coli produces CaZ+-independent contractions; however, the mechanism of action is different from that of ROK. PAK does not alter the phosphorylation of MLC, 189'193 but appears to act through a novel mechanism involving the phosphorylation of caldesmon (CalD) by PAK. Phosphorylation of CalD removes the well-described CalDdependent interference with actin stimulation of myosin ATPase. 193 The presence and role of Ca2+-sensitization varies across the many diverse types of smooth muscle. Although CaZ+-sensitization is thought to be important in ASM, the role of PAK vs R O K has not been well assessed during pre- or postnatal development. If these kinases cause Ca2+-sensitization in ASM, it raises the possibility that such regulatory proteins may also be important in airway hyperresponsiveness in the newborn and adult. Developmental regulation of such regulatory pathways may be significant. The impact of PAK on skinned ASM has been recently assessed in the adult. 196 Western blots show that PAK is present in ASM, 196'197and that it can indeed induce both Ca2+-independent and Ca2+-sensitized contractions. 196 Furthermore, preliminary evidence suggests that PAK may induce Ca2+-independent contractions in neonatal tracheal smooth muscle (see Fig. 3.8). The roles for PAK or

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Fig. 3.8. Illustration of the effect of constitutively active p21-activated kinase (PAK) on force generation in skinned neonatal tracheal smooth muscle (canine). Force increased during exposure to near-maximal calcium concentration (pCa=4.4, indicated by horizontal bar) followed by relaxation during exposure to nominally 0 calcium (upward artifacts on force tracing represent change in bath media). Addition of PAK (indicated by horizontal bar) during nominal 0 calcium (pCa > 9) resulted in a slow increase in force that amounted to approximately 30-40% of the maximal response in this fibre. Force (vertical bar) and time (horizontal bar) calibrations as indicated. (Tracing from McFawn and Fisher, unpublished observations.)

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ROK may be particularly important in the newborn where significant contractile effects of ct-adrenergic agonists are present. 184-186

ACKNOWLEDGEMENTS Supported by the Raine Medical Foundation of Western Australia, the National Health and Medical Research Council of Australia and a bequest from the late Annie Phillips (M. Sparrow), by the Canadian Institutes of Health Research (CIHR) and the Ontario Thoracic Society (J. Fisher), and a joint Fellowship from the Canadian Thoracic Society/ Glaxo/CIHR (P. Mc Fawn).

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:::::....

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77. Sparrow MP, Mitchell HW. Contraction of smooth muscle of pig airway tissues from before birth to maturity. J. Appl. Physiol. 1990; 68:468-77. 78. Mitchell HW, Sparrow MP, Tagliaferri RP. Inhibitory and excitatory responses to field stimulation in fetal and adult pig airway. Pediatr. Res. 1990; 28:69-74. 79. Booth RJ, Sparrow MP, Mitchell HW. Early maturation of force production in pig tracheal smooth muscle during fetal development. Am. J. Respir. Cell Mol. Biol. 1992; 7:590-7. 80. Sparrow MP, Warwick SP, Mitchell HW. Fetal airway motor tone in prenatal lung development of the pig. Eur. Respir. J. 1994; 7:1416-24. 81. Sparrow MP, Warwick SP, Everett AW. Innervation and function of the distal airways in the developing bronchial tree of fetal pig lung. Am. J. Respir. Cell Mol. Biol. 1995; 13:518-25. 82. McFawn PK, Mitchell HW. Bronchial compliance and wall structure during development of the immature human and pig lung. Eur. Respir. J. 1997; 10:27-34. 83. McFawn PK, Mitchell HW. Effect of transmural pressure on preloads and collapse of immature bronchi. Eur. Respir. J. 1997; 10:322-9. 84. Richards IS, Kulkarni A, Brooks SM. Human fetal tracheal smooth muscle produces spontaneous electromechanical oscillations that are Caz+ dependent and cholinergically potentiated. Dev. Pharmacol. Ther. 1991; 16:22-8. 85. Liu M, Skinner SJ, Xu Jet al. Stimulation of fetal rat lung cell proliferation in vitro by mechanical stretch. Am. J. Physiol. 1992; 263:L376-83. 86. Liu M, Qin Y, Liu J e t al. Mechanical strain induces pp60src activation and translocation to cytoskeleton in fetal rat lung cells.J. Biol. Chem. 1996; 271:7066-71. 87. Liu M, Tanswell AK, Post M. Mechanical force-induced signal transduction in lung cells. Am. J. Physiol. 1999; 277:L667-83. 88. Liu M, Post M. Invited review: mechanochemical signal transduction in the fetal lung. J. Appl. Physiol. 2000; 89:2078-84. 89. Weichselbaum M, Everett AW, Sparrow MP. Mapping the innervation of the bronchial tree in fetal and postnatal pig lung using antibodies to PGP 9.5 and SV2. Am. J. Respir. Cell Mol. Biol. 1996; 15:703-10. 90. Sparrow MP, Weichselbaum M. Structure and function of the adventitial and mucosal nerve plexuses of the bronchial tree in the developing lung. Clin. Exp. Pharmacol. Physiol. 1997; 24:261-8. 91. Weichselbaum M, Sparrow MP. A confocal microscopic study of the formation of ganglia in the airways of fetal pig lung. Am. J. Respir. Cell Mol. Biol. 1999; 21:607-20. 92. Wong KA, Bano A, Rigaux A et al. Pulmonary vagal innervation is required to establish adequate alveolar ventilation in the newborn lamb.J. Appl. Physiol. 1998; 85:849-59. 93. Hasan SU, Lalani S, Remmers JE. Significance of vagal innervation in perinatal breathing and gas exchange. Respir. Physiol. 2000; 119:133-41. 94. Schwieler GH, Douglas JS, Bouhuys A. Postnatal development of autonomic efferent innervation in the rabbit. Am. J. Physiol. 1970; 219:391-7. 95. Waldron MA, Fisher JT. Differential effects of CO z and hypoxia on bronchomotor tone in the newborn dog. Respir. Physiol. 1988; 72:271-82. 96. Fisher JT, Brundage KL, Waldron MA et al. Vagal cholinergic innervation of the airways in newborn cat and dog. J. Appl. Physiol. 1990; 69:1525-31. 97. Tepper RS. Maturation affects the maximal pulmonary response to methacholine in rabbits. Pediatr. Pulmonol. 1993; 16:48-53.

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98. Tepper RS, Gunst SJ, Doerschuk CM et al. Effect of transpulmonary pressure on airway closure in immature and mature rabbits.J. Appl. Physiol. 1995; 78:505-12. 99. Tepper RS, Du T, Styhler A et al. Increased maximal pulmonary response to methacholine and airway smooth muscle in immature compared with mature rabbits. Am. J. Respir. Crit. Care Med. 1995; 151:836-40. 100. Hayashi S, Toda N. Age-related alterations in the response of rabbit tracheal smooth muscle to agents. J. Pharmacol. Exp. Ther. 1980; 214:675-81. 101. Duncan PG, Douglas JS. Influences of gender and maturation on responses of guinea-pig airway tissues to LTD4. Eur. J. Pharmacol. 1985; 112:423-7. 102. Panitch HB, Allen JL, Ryan JP etal. A comparison of preterm and adult airway smooth muscle mechanics. J. Appl. Physiol. 1989; 66:1760-5. 103. Murphy TM, Mitchell RW, Blake JS etal. Expression of airway contractile properties and acetylcholinesterase activity in swine.J. Appl. Physiol. 1989; 67:174-80. 104. Mitchell RW, Murphy TM, Kelly E etal. Maturation of acetylcholinesterase expression in tracheal smooth muscle contraction.Am. J. Physiol. 1990; 259:L130-5. 105. Murphy TM, Mitchell RW, Phillips IJ. Ontogenic expression of acetylcholinesterase activity in trachealis of young swine. Am. J. Physiol. Lung Cell Mol. Physiol. 1991; 261:L322-6. 106. Sauder RA, McNicol KJ, Stecenko AA. Effect of age on lung mechanics and airway reactivity in lambs. J. Appl. Physiol. 1986; 61:2074-80. 107. Ikeda K, Mitchell RW, Guest KA et al. Ontogeny of shortening velocity in porcine trachealis. Am. J. Physiol. 1992; 262:L280-5. 108. Murphy TM, Mitchell RW, Halayko A et al. Effect of maturational changes in myosin content and morphometry on airway smooth muscle contraction. Am. J. Physiol. 1991; 260:L471-80. 109. Fisher JT. Airway smooth muscle contraction at birth: in vivo versus in vitro comparisons to the adult. Can. J. Physiol. Pharmacol. 1992; 70:590-6. 110. Stevens EL, Uyehara CFT, Southgate WM et al. Furosemide differentially relaxes airway and vascular smooth muscle in fetal, newborn, and adult guinea pigs. Am. Rev. Respir. D/s. 1992; 146:1192-7. 111. Southgate WM, Pichoff BE, Stevens EL etal. Ontogeny of epithelial modulation of airway smooth muscle function in the guinea pig. Pediatr. Pulmonol. 1993; 15:105-10. 112. Fayon M, Ben-Jebria A, Elleau Cet al. Human airway smooth muscle responsiveness in neonatal lung specimens. Am. J. Physiol. 1994; 267:L180-6. 113. Penn RB, Wolfson MR, Shaffer TH. Effect of tracheal smooth muscle tone on collapsibility of immature airways. J. Appl. Physiol. 1988; 65:863-9. 114. Mitchell HW, McFawn PK, Sparrow MP. Increased narrowing of bronchial segments from immature pigs. Eur. Resp. J. 1992; 5:207-12. 115. Bhutani VK, Koslo RJ, Shaffer TH. The effects of tracheal smooth muscle tone on neonatal airway collapsibility. Pediatr. Res. 1986; 20:492-5. 116. Bhutani VK, Rubenstein SD, Shaffer TH. Pressure-volume relationships of tracheae in fetal, newborn, and adult rabbits. Respir. Physiol. 1981; 43:221-31. 117. Bhutani VK, Rubenstein SD, Shaffer TH. Pressure induced deformation in immature airways. Pediatr. Res. 1981; 15:829-32. 118. Fisher JT, Haxhiu MA, Martin RJ. Regulation of lower airway function. In: Polin RA, Fox WW (eds), Fetal and Neonatal Physiology. Philadelphia, PA: W.B. Saunders. 1998, pp. 1060-70.

119. Fisher JT, Brundage KL, Anderson JW. Cardiopulmonary actions of muscarinic receptor subtypes in the newborn dog. Can. jT. Physiol. Pharmacol. 1995; 74:603-13. 120. Fisher JT, Mortola JP. Statics of the respiratory system in newborn mammals. Respir. Physiol. 1980; 41:155-72. 121. Fisher JT, Mortola JP. Statics of the respiratory system and growth: an experimental and allometric approach. Am..7. Physiol. 1981; 241:R336-41. 122. Mortola JP. Dynamics of breathing in newborn mammals. Physiol. Rev. 1987; 67:187-243. 123. Mortola JP. Respiratory physiology of newborn mammals: a comparative perspective. Baltimore: The Johns Hopkins University Press, 2001. 124. Fisher JT, McFawn PK, Allen MA etal. Unpublished observations. 125. Anderson JW, Fisher JT. Capsaicin-induced reflex bronchoconstriction in the newborn. Respir. Physiol. 1993; 93:13-27. 126. Nault MA, Vincent SG, Fisher JT. Mechanisms of capsaicinand lactic acid-induced bronchoconstriction in the newborn dog.J. Physiol. (Lond.) 1999; 515:567-78. 127. Marantz MJ, Vincent SG, Fisher JT. Role of vagal C-fiber afferents in the bronchomotor response to lactic acid in the newborn dog. J. Appl. Physiol. 2001; 60:2311-18. 128. Richardson CA, Herbert DA, Mitchell RA. Modulation of pulmonary stretch receptors and airway resistance by parasympathetic efferents. J. Appl. Physiol. 1984; 57:1842-9. 129. Nadel JA, Widdicombe JG. Effect of changes in blood gas tensions and carotid sinus pressure on tracheal volume and total lung resistance to airflow. J. Physiol. 1962; 163:13-33. 130. Green M, Widdicombe JG. The effect of ventilation of dogs with different gas mixtures on airway calibre and lung mechanics.J. Physiol. (Lond.) 1966; 186:363-81. 131. Fisher JT, Waldron MA, Armstrong CJ. Effects ofhypoxia on lung mechanics in the newborn cat. Can. J. Physiol. Pharmacol. 1987; 65:1234-8. 132. Mortola JP, Tenney SM. Effects of hyperoxia on ventilatory and metabolic rates of newborn mice. Respir. Physiol. 1986; 63:267-74. 133. Mortola JP, Rezzonico R. Metabolic and ventilatory rates in newborn kittens during acute hypoxia. Respir. Physiol. 1988; 73:55-68. 134. Caterina MJ, Schumacher MA, Tominaga M et al. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 1997; 389:816-24. 135. Lee LY, Pisarri TE. Afferent properties and reflex functions of bronchopulmonary C-fibers. Respir. Physiol. 2001; 125:47-65. 136. Lee LY, Morton RF, Lundberg JM. Pulmonary chemoreflexes elicited by intravenous injection of lactic acid in anesthetized rats. J. Appl. Physiol. 1996; 81:2349-57. 137. Diaz V, Dorion D, Kianicka Iet al. Vagal afferents and active upper airway closure during pulmonary edema in lambs. J. Appl. Physiol. 1999; 86:1561-9. 138. Diaz V, Dorion D, Renolleau S e t al. Effects of capsaicin pretreatment on expiratory laryngeal closure during pulmonary edema in lambs. J. Appl. Physiol. 1999; 86:1570-7. 139. Hosey MM. Diversity of structure, signalling and regulation within the family of muscarinic cholinergic receptors. FASEBJ. 1992; 6:845-52. 140. Hulme EC, Birdsall NJM, Buckley NJ. Muscarinic receptor subtypes. Annu. Rev. Pharmacol. Toxicol. 1990; 30:633-73. 141. Wess J. Molecular biology of muscarinic acetylcholine receptors. Crit. Rev. Neurobiol. 1996; 10:69-99. 142. Pitcher JA, Freedman NJ, Lefkowitz RJ. G protein-coupled receptor kinases. Annu. Rev. Biochem. 1998; 67:653-92. 143. Gomeza J, Zhang L, Kostenis E etal. Enhancement of D1 dopamine receptor-mediated locomotor stimulation in

M(4) muscarinic acetylcholine receptor knockout mice. Proc. Natl. Acad. Sci. USA 1999; 96:10483-8. 144. Gomeza J, Shannon H, Kostenis E et al. Pronounced pharmacologic deficits in M2 muscarinic acetylcholine receptor knockout mice. Proc. Natl. Acad. Sci. USA 1999; 96:1692-7. 145. Shapiro MS, Loose MD, Hamilton SE etal. Assignment of muscarinic receptor subtypes mediating G-protein modulation of Ca(2+) channels by using knockout mice. Proc. Natl. Acad. Sci. USA 1999; 96:10899-904. 146. Stengel PW, Gomeza J, Wess J etal. M(2) and M(4) receptor knockout mice: muscarinic receptor function in cardiac and smooth muscle in vitro. J. Pharmacol. Exp. Ther. 2000; 292:877-85. 147. Fisher JT, Froese AB, Brundage KL. Bases physiologiques de l'utilisation des antagonistes muscariniques dans les dysplasies bronchopulmonaires. Arch. Pediatr. 1995; 2 (Suppl. 2):163S-71S. 148. Julia-Serda G, Molfino NA, Chapman KR et al. Heterogeneous airway tone in asthmatic subjects. J. Appl. Physiol. 1992; 73:2328-32. 149. Molfino NA, Slutsky AS, Hoffstein Vet al. Changes in crosssectional airway areas induced by methacholine, histamine, and LTC4 in asthmatic subjects. Am. Rev. Respir. Dis. 1992; 146:514-80. 150. Molfino NA, Slutsky AS, Julia-Serda G et al. Assessment of airway tone in asthma: comparison between double lung transplant patients and healthy subjects. Am. Rev. Respir. DIS. 1993; 148:1238-43. 151. Haxhiu-Poskurica B, Ernsberger P, Haxhiu MA et al. Development of cholinergic innervation and muscarinic receptor subtypes in piglet trachea. Am. J. Physiol. 1993; 264: L606-14. 152. Maclagan J, Barnes PJ. Muscarinic pharmacology of the airways. TIPS 1989; 10 (Suppl. Dec.):88-92. 153. Barnes PJ. Muscarinic receptor subtypes: implications for therapy.Agents Actions 1993; 43 (Suppl.):243-52. 154. Barnes PJ. Muscarinic receptor subtypes in airways. Life Sci. 1993; 52:521-7. 155. Lazareno S, Buckley NJ, Roberts FF. Characterization of muscarinic M 4 binding sites in rabbit lung, chicken heart, and NG108-15 cells. Mol. Pharmacol. 1990; 38:805-15. 156. D6rje F, Levey AI, Brann MR. Immunological detection of muscarinic receptor subtype proteins (ml-m5) in rabbit peripheral tissues. Mol. Pharmacol. 1991; 40:459-62. 157. Mak JCW, Haddad E-B, Buckley NJ etal. Visualization of muscarinic m 4 mRNA and M 4 receptor subtype in rabbit lung. Life Sci. 1993; 53:1501-8. 158. Maclagan J, Fryer AD, Faulkner D. Identification of M 1 muscarinic receptors in pulmonary sympathetic nerves in the guinea-pig by use of pirenzepine. Br. J. Pharmacol. 1989; 97:499-505. 159. Beck KC, Vettermann J, Flavahan NA et al. Muscarinic M1 receptors mediate the increase in pulmonary resistance during vagus nerve stimulation in dogs. Am. Rev. Respir. DIS. 1987; 136:1135-9. 160. Lammers J, Minette P, McCusker M etal. The role of pirenzepine-sensitive (M1) muscarinic receptors in vagally mediated bronchoconstriction in humans. Am. Rev. Respir. Dis. 1989; 139:446-9. 161. Maclagan J, Faulkner D. Effect of pirenzepine and gallamine on cardiac and pulmonary muscarinic receptors in the rabbit. Br. J. Pharmacol. 1989; 97:506-12. 162. Eltze M, Galvan M. Involvement of muscarinic M z and M3, but not of M 1 and M 4 receptors in vagally stimulated contractions of rabbit bronchus/trachea. Pulmonary Pharmacol. 1994; 7:109-20. 163. Fryer AD, Maclagan J. Muscarinic inhibitory receptors in pulmonary parasympathetic nerves in the guinea-pig. Br. J. Pharmacol. 1984; 83:973-8.

164. Blaber LC, Fryer AD, Maclagan J. Neuronal muscarinic receptors attenuate vagally induced contraction of feline bronchial smooth muscle. Br. J. Pharmacol. 1985; 86:723-8. 165. Faulkner D, Fryer AD, Maclagan J. Postganglionic muscarinic inhibitory receptors in pulmonary parasympathetic nerves in the guinea-pig. Br. J. Pharmacol. 1986; 88:181-7. 166. Ito Y, Yoshitomi T. Autoregulation of acetylcholine release from vagus nerve terminals through activation of muscarinic receptors in the dog trachea. Br. J. Pharmacol. 1988; 93:636-46. 167. Minette PA, Barnes PJ. Prejunctional inhibitory muscarinic receptors on cholinergic nerves in human and guinea pig airways.J. Appl. Physiol. 1988; 64:2532-7. 168. Watson N, Barnes PJ, Maclagan J. Actions of methocitramine, a muscarinic M2 receptor antagonist, on muscarinic and nicotinic cholinoceptors in guinea-pig airways in vivo and in vitro. Br. J. Pharmacol. 1992; 105:107-12. 169. Fryer AD, Jacoby DB. Parainfluenza virus infection damages inhibitory M z muscarinic receptors on pulmonary parasympathetic nerves in the guinea pig. Br. J. Pharmacol. 1991; 102:267-71. 170. Schultheis AH, Bassett DJP, Fryer AD. Ozone-induced airway hyperresponsiveness and loss of neuronal M2 muscarinic receptor function.J. Appl. Physiol. 1994; 76:1088-97. 171. Gambone LM, Elbon CL, Fryer AD. Ozone-induced loss of neuronal M z muscarinic receptor function is prevented by cyclophosphamide.J. Appl. Physiol. 1994; 77:1492-9. 172. Janssen LJ, Daniel EE. Pre- and postjunctional muscarinic receptors in canine bronchi. Am. J. Physiol. (Lung Cell Mol. Physiol.) 1990; 259:L304-14. 173. Gross NJ. Ipratropium bromide. N. Engl. J. Med. 1988; 319: 486-94. 174. DeTroyer A, Yernault JC, Rodenstein D. Effects of vagal blockade on lung mechanics in normal man. J. Appl. Physiol. 1979; 46:217-26. 175. Somlyo AP, Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 1994; 372:231-6. 176. Somlyo AP, Somlyo AV. From pharmacomechanical coupling to G-proteins and myosin phosphatase [published erratum appears in Acta Physiol. Scand. 1999; 165(4):423]. Acta. Physiol. Scand. 1998; 164:437-48. 177. Somlyo AP, Himpens B. Cell calcium and its regulation in smooth muscle. FASEB J. 1989; 3:2266-76. 178. Walsh MP. Regulation of vascular smooth muscle tone. Can. J. Physiol. Pharmacol. 1994; 72:919-36. 179. Kitazawa T, Gaylinn BD, Denney GH etal. G-proteinmediated Ca2+ sensitization of smooth muscle contraction through myosin light chain phosphorylation. J. Biol. Chem. 1991; 266:1708-15. 180. Masuo M, Reardon S, Ikebe M et al. A novel mechanism for the CaZ+-sensitizing effect of protein kinase C on vascular smooth muscle: inhibition of myosin light chain phosphatase. J. Gen. Physiol. 1994; 104:265-86. 181. Kitazawa T, Somlyo AP. Desensitization and muscarinic re-sensitization of force and myosin light chain phosphorylation to cytoplasmic Ca2+ in smooth muscle. Biochem. Biophys. Res. Commun. 1990; 172:1291-7. 182. Gerthoffer WT. Regulation of the contractile element of airway smooth muscle. Am. J. Physiol. 1991; 261 :L 15-28. 183. Gunst SJ, Gerthoffer WT, al Hassani MH. Ca2+ sensitivity of contractile activation during muscarinic stimulation of tracheal muscle. Am. J. Physiol. 1992; 263:C1258-65. 184. Pandya KH. Postnatal developmental changes in adrenergic receptor responses of the dog tracheal muscle. Arch. Int. Pharmacodyn. Ther. 1977; 230:53-64. 185. Watanabe H, Vincent SG, Fisher JT. Alpha adrenergic receptor contractile responses in the newborn dog. Can. J. Physiol. Pharmacol. 1994; 72 (Suppl. 1):483.

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186. Watanabe H, Fisher JT. Alpha adrenergic receptors mediate contractile responses to catecholamines in neonatal canine airway smooth muscle. Am. J. Respir. Crit. Care Med. 1995; 151 :A438 [Abstract]. 187. Nauli SM, Ally A, Zhang L et al. Maturation attenuates the effects of cGMP on contraction, [Ca2+]i and Ca2+ sensitivity in ovine basilar arteries. Gen. Pharmacol. 2000; 35:107-18. 188. Macara IG, Lounsbury KM, Richards SA etal. The Ras superfamily of GTPases. FASEB J. 1996; 10:625-30. 189. van Eyk JE, Arrell DK, Foster DB et al. Different molecular mechanisms for Rho family GTPase-dependent, Ca2+independent contraction of smooth muscle. J. Biol. Chem. 1998; 273:23433-9. 190. Schmitz AA, Govek EE, Bottner B etal. Rho GTPases: signaling, migration, and invasion. Exp. Cell Res. 2000; 261:1-12. 191. Bishop AL, Hall A. Rho GTPases and their effector proteins. Biochem. J. 2000; 348 (Pt 2):241-55. 192. Manser E, Leung T, Salihuddin H etal. A brain serine/ threonine protein kinase activated by Cdc42 and Racl. Nature 1994; 367(6458):40-6.

193. Foster DB, Shen LH, Kelly Jet al. Phosphorylation of caldesmon by p21-activated kinase. Implications for the Ca(2+) sensitivity of smooth muscle contraction. J. Biol. Chem. 2000; 275:1959-65. 194. Kubota Y, Nomura M, Kamm KE etal. GTPgammaSdependent regulation of smooth muscle contractile elements. Am. J. Physiol. (Cell Physiol.) 1992; 262:C405-10. 195. Uehata M, Ishizaki T, Satoh H et al. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 1997; 389(6654) :990-4. 196. McFawn PK, Shen L, Vincent SG et al. Calcium-independent contraction and sensitisation of airway smooth muscle by p21-activated protein kinase. Am. J. Physiol. (Lung Cell Mol. PhysioL) 2003; 284:L863-70. 197. Dechert MA, Holder JM, Gerthoffer WT. p21-activated kinase 1 participates in tracheal smooth muscle cell migration by signaling to p38 Mapk. Am. J. Physiol. Cell Physiol. 2001; 281:C123-32.

Chapter 4

Development of Aiveoli Stephen E. McGowan Department of Internal Medicine, Veterans Affairs Research Service, University of Iowa, iowa City, IA, USA

Jeanne M. Snyder* Department of Anatomy and Cell Biology, University of Iowa College of Medicine, Iowa City, IA, USA

INTRODUCTION

The respiratory system consists of the trachea, the conducting airways and the gas-exchange portion of the lung, which includes the respiratory bronchioles, alveolar ducts, alveolar sacs and alveoli. 1 Alveoli are tiny, thin-walled sacs that facilitate the exchange of gases between capillaries in the alveolar wall and air brought into the lung during inspiration. The alveolar surface area available for gas exchange is enormous, approximately 100 m 2 in an adult human, and represents the contribution of approximately 300• 106 alveoli. 1-3 The structure of an individual lung alveolus is deceptively simple. The alveolar wall in the adult lung consists of a narrow connective tissue core that contains fibroblasts, myofibroblasts and capillary endothelial cells plus extracellular matrix (ECM) components, most importantly, elastin. The alveolar epithelium is made up of two cell types, namely alveolar type I cells and alveolar type II cells. Alveolar type I cells are thin, flattened cells that, together with the capillary endothelial cell and the fused basal laminae of the capillary and epithelium, form the actual gas-blood exchange barrier. Alveolar type I cells cover about 90% of the alveolar surface area. 3 The alveolar type II cell is a roughly cuboidal cell, frequently located in the corner of an alveolus, that occupies less than 10% of the alveolar surface area. The alveolar type II cell is a stem cell for renewal of the damaged alveolar epithelium since alveolar type I cells do not divide. 4 Alveolar type II cells secrete pulmonary surfactant, a lipoprotein substance that spreads on the alveolar aqueous *To whom correspondence should be addressed. The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

lining layer and reduces its surface tension 5 (see Chapter 10). Adequate amounts of properly functioning pulmonary surfactant are required for normal lung function. 5 In the human, the formation of pulmonary alveoli begins during late gestation and continues after birth. 1-3 In other species, alveolarization is either predominantly postnatal (i.e. in rats and mice) or predominantly prenatal (i.e. in guinea pigs and rabbits). 6 Regardless of the timing, alveolarization seems to occur via a roughly similar process in all vertebrates. An impairment of alveolarization has been implicated in the pathogenesis of bronchopulmonary dysplasia (BPD), a disease that affects prematurely born human infants. 7 Damage to existing alveoli, with a resulting decrease in gasexchange surface area, is involved in the pathogenesis of emphysema. 8 Thus, a better understanding of the cellular and biochemical events involved in alveolarization, as well as the factors that regulate the formation of new alveoli, could lead to improved treatments for several serious pulmonary conditions. During the past 10 years, a clearer understanding of many of the factors that regulate alveolarization has emerged, in large part the result of genetic studies performed in mice. Alveolarization is a relatively late event in lung development, occurring after branching morphogenesis has laid down the conducting airway system. However, some of the regulatory factors that control alveolarization are also involved in lung bud formation and branching morphogenesis. Therefore, it is useful to review briefly the regulation of the early events in the embryogenesis of the lung in order to gain greater insight into the processes involved in the formation of alveoli in the developing lung. Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

The Lung:: D~w:.~iopment, A.oin~, ,~nd the Environmer~t :

O V E R V I E W OF EARLY L U N G DEVELOPMENT

STAGES OF FETAL L U N G DEVELOPMENT

Lung bud formation

Pseudoglandular phase

The lung begins as a diverticulum from the embryonic foregut, at about 4 weeks post-fertilization in the human. 1 The foregut and its diverticula (such as the lung) are lined with epithelial cells that are derived from the endoderm germ layer. The lung diverticulum is covered with splanchnic mesoderm that gives rise to the connective tissue components of the lung (cartilage, smooth muscle, blood vessels, etc.). Factors that initiate the formation of the lung diverticulum remain obscure but signals between splanchnic mesoderm and foregut endoderm may initiate lung bud formation. 9 Studies in rodents suggest that transcription factors downstream from the Sonic hedgehog signaling cascade (Gli-2, -3 and Foxfl)regulate the formation of the lung diverticulum. 1~ There is also compelling evidence of a role for fibroblast growth factor 10 (FGF-10) and its receptor (FGFR2) in lung diverticulum formation. 12'13 The lung diverticulum displays aspects of the right/left symmetry, such that lung buds in the human initially form three diverticula in the right lung bud and two in the left that correspond to the lobes of the adult lungs. The control of right/left asymmetry has been studied by transgene studies in mice, which also have an asymmetric lung structure. 9 Several transcription factors, including lefty-1, lefty-2, nodal and Pitx-2, are probably involved in determining lung asymmetry. 9'14 In addition retinoic acid receptors (RARs), which are also nuclear transcription factors, may be involved, since mice in which the RAR~ and RAR[3 genes have both been deleted are missing the left lung. 15

This phase of lung development, characterized by repeated branching, occurs in the human from about 6-16 weeks of gestation. 1 It is thought that almost all the pulmonary conducting airways are created during this process, about 20-22 orders of airways in the human. 19 Differentiation of the epithelium of the conducting airways also commences during this early phase of lung development. 1 The most distal aspects of the branching duct system will eventually be remodeled to give rise to the alveolar region of the lung. Epithelial cells in the distal region remain undifferentiated as tall columnar cells with no specialized features other than large pools of intracellular glycogen (Fig. 4.1). 20

Branehing morphogenesis Early lung development is characterized by branching morphogenesis, in which the endoderm-lined ducts of the lung buds undergo dichotomous branching giving rise to the primary bronchi, then the secondary (lobular) bronchi, the tertiary (segmental) bronchi and so forth. 16 Branching morphogenesis involves the stabilization of the linear portion of the distal ducts, the creation of a cleft region at the rounded tips of the ducts and growth on either side of the cleft, a process that results in branching of the terminal portion of the duct. The regulation of branching morphogenesis involves epithelial-mesenchymal interactions via ECM components and growth factors. 16 In the developing lung, growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factors I and II (IGF-I and IGF-II), hepatic growth factor (HGF), vascular endothelial growth factor (VEGF) as well as retinoic acid have been shown to regulate branching morphogenesis. 16'17 Other mediators of branching morphogenesis in the developing lung include transcription factors such as HNF-3[3, HFH-4 and TTF-1.18

Canalicular phase Remodeling of the distal portions of the branching duct system to form gas-exchange units begins during this stage, which in the human lasts from 16 to 24 weeks of gestation. 1 This stage is characterized by increasing numbers of capillaries between the terminal ducts and the beginning of the differentiation of the presumptive alveolar epithelium. 2~ Some of the distal epithelial cells become more cuboidal and begin to synthesize and release surfactant; these type II cells are characterized by decreased intracellular glycogen pools, microvilli on their apical surface, and the appearance of lamellar bodies. 2~ At the same time, capillaries in the interstitium induce the overlying epithelial cells to flatten and differentiate into alveolar type I cells. Factors that regulate angiogenesis in the developing lung include VEGF and its receptors as well as FGFs and their receptors. 21 Type I and type II alveolar epithelial cells (AECs) differentiate in response to a number of regulatory factors 22 (see also Chapter 9). Thus, some aspects of alveolar development, in particular AEC differentiation, begin during the canalicular stage of lung development. However, in the human, true alveoli do not begin to form until much closer to birth. 1

Saccular phase During this phase, which occurs from about 24 weeks of gestation to term in the human, the terminal portions of the duct system (the terminal sacs) lengthen and may undergo further branching to give rise to alveolar ducts and alveolar sacs. 1 In the human, it is estimated that 15-18% of true alveoli form late in gestation, but the bulk are formed after birth. 1 Premature infants whose lungs are in the late canalicular/early saccular stage of lung development at the time of birth can survive without alveoli, in response to surfactant therapy and ventilation, although they are at risk of developing BPD. 7 The regulation of alveolarization is poorly understood, probably because there are no easily measured end points. Investigators who study alveolarization have relied primarily

Development

of Alveoli

on morphological techniques such as measurements of alveolar number and dimensions and the surface area available for gas exchange. 6 While there are well-defined markers for AEC differentiation, primarily those associated with type II cell differentiation and surfactant production, there are few biochemical markers for alveolarization per se, with the possible exception of elastin production. 23

C H R O N O L O G Y OF M O R P H O L O G I C CHANGES DURING ALVEOLARIZATION The process of alveolarization has been described in detail in rats and humans. 1 During the late canalicular and terminal saccular stages of prenatal lung development, when the conducting airways have stopped branching and are enlarging at their distal termini, there is a progressive loss of the mesenchymal cells that separate capillaries from the epithelium lining the future air spaces. This yields a rudimentary gas-exchange surface that can support respiration. Shortly after birth, in both rats and humans, the surface area of the air-blood interface begins to increase markedly as the terminal saccules become alveolar ducts and these in turn give rise to alveolar sacs and the alveoli.

Secondary septation Alveolar formation in the developing lung has been divided into several phases" the first is termed secondary septation

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Fig. 4.1. Light micrographs of methylene blue-stained, epoxy sections of human fetal lung tissue at different stages of development (550x): (A) Lung tissue from a 14-week-gestational-age fetus. Lung tissue in the pseudoglandular stage of lung development is characterized by abundant connective tissue and a few branching ducts that are lined by a columnar epithelium. There are very few capillaries in the tissue and none are associated with the ductal epithelium. (B) Lung tissue from a 24-week-gestational-age fetus. At the canalicular stage of development, the ducts are more numerous and their epithelial cells are cuboidal. Capillaries are more abundant than in previous stages and are closely associated with the ductal epithelium. Differentiated type II cells are first observed at this stage of development in the human fetus. (C) Lung tissue from a 40-week-gestational-age fetus. In the terminal sac stage of lung development, the number of capillaries is greatly increased and the relative amount of connective tissue greatly decreased when compared to previous stages of lung development. (Reproduced with permission from Mallampalli RK, Acarregui MJ, Snyder JM. Differentiation of the alveolar epithelium in the fetal lung. In: McDonald JA (ed.), Lung Growth and Development. New York: Marcel Dekker, 1997, pp. 119-62.)

new alveolar septa by septation of the terminal sacs. Septation is initiated by the protrusion of secondary crests from primary septa. The primary septa, which are the walls of the terminal sacs, are comprised of a central core that contains fibroblasts and other connective tissue components, surrounded on each side by capillaries and the epithelium of the terminal s a c . 24 These same components are also found in the secondary crest. Capillaries are interconnected in the primary septa, but the two capillary layers formed in the secondary septa during septation initially have few interconnections. On postnatal days 2-4 in the rat, fibroblasts proliferate at the site of origin of the secondary crest, and this causes the secondary septum to lengthen and project perpendicularly into the alveolar sac. 25 The secondary crests are usually adjacent to elastic fibers and arise where the capillaries in the primary septum can be folded u p . 1'26 The tips of the secondary crests are occupied by myofibroblastic cells that are located next to elastic fibers. 27 After postnatal day 4 in the rat, fibroblast proliferation decreases in the proximal aspect of the secondary septum, but persists at a higher level in the distal septal t i p s y The increase in the height of the secondary septae is accompanied by an increase in the number of lamellar bodies in type II AECs and a corresponding increase in surfactant secretion. 1 Levels of surfactant produced in the lung apparently increase in parallel with increasing surface area in the distal portion of the developing lung. 28

::T:he Lung: Developmenti: Aging and: the

Growth Alveolarization continues through the third postnatal week in rats and until 2 years in humans. 1 The lung then enters the third phase of alveolar formation, a growth phase in which the gas-exchange surface area increases to the 0.7th power of lung volume, a factor consistent with an isotropic expansion of the alveoli. 24 This process is accompanied by an increase in the volume of air space in the lung, at the expense of alveolar septal tissue mass. During the growth phase of alveolarization, the volume density of alveolar type I and type II epithelial cells increases roughly in proportion to the increase in lung volume. 1 In mammals, this phase of lung development usually ends prior to the termination of the growth of long bones and increase in lean body mass. However, in mice and rats, remodeling in the sub-pleural regions of the lung may continue throughout life. 3~

that alveolar septation starts in utero. 35 Postnatal alveolar septation occurs at a more constant rate in humans than in rats; children do not undergo the rapid burst of septal outgrowth that occurs after birth in rats. In children, septation ends between 2 and 5 years of age. 1 In humans, peripheral lung desmosine can be detected during the final 10 weeks of gestation, but the major, ---&fold increase in its concentration occurs during the first 2 years of life, during alveolar secondary septal formation. 36 During this same period, lung hydroxyproline, a marker for collagen, increases ---3-fold when adjusted to dry lung weight. 33 Despite the differences in the kinetics and duration of the alveolar phases in the two species, the proportional enlargement of the alveolar surface area after birth is very similar in rats (21.4-fold) and humans (20.5-fold). 35 Some mammals, such as the rabbit and guinea pig, develop the majority of their alveoli prior to birth and the majority of the internal surface area of the lung is acquired prenatally. 37 Unlike humans, monkeys develop nearly all their alveoli prior to birth (26.2 x 106cm -2 in newborns compared to 26.6 x 106 cm -2 in the adult). 3s Similarly, in sheep, alveolar septation primarily occurs prenatally. 39'4~Secondary septa in the sheep fetus double in number during the final quarter of gestation and alveolarization is accompanied by both thinning of the alveolar septa and maturation of capillaries, events which occur primarily postnatally in humans and rats. Alveolarization in the sheep fetus is accompanied by a 20-30% increase in parenchymal elastin and an approximately 10% increase in parenchymal collagen over the same interval. 41'42

INTERSPECIES COMPARISON OF ALVEOLARIZATION

DEVELOPMENT OF THE ALVEOLAR EPITHELIUM

Rats and mice

The two types of AECs, the type I and type II cells, 2~ both arise from the endoderm-derived epithelial cells that line the distal portion of the branching duct system in the developing lung (Fig. 4.2). The differentiation of AECs commences in the human fetus prior to the formation of true alveoli, which is primarily a postnatal event.

Remodeling of the alveolar wall The second phase of alveolarization is marked by further lengthening and thinning of the secondary septae, primarily via the loss of interstitial mesenchymal cells and extensive capillary remodeling. 1 During this remodeling phase, the original dual capillary system becomes a single capillary system in which the capillaries are interconnected through a process termed intussusceptive microvascular growth. 29 This involves the fusion of adjacent capillaries by interconnecting endothelium-lined tissue pillars. During the period of exuberant new septal formation, the surface area of the lung increases to the 1.6th power of lung volume. 24

At birth, the gas-exchanging regions in rats, mice (and humans) consist primarily of immature terminal saccules with some secondary septa. In rats, alveolar septation occurs during the first 3 postnatal weeks, followed by a period in which the alveolar surface increases through the enlargement of pre-existing alveoli, without the formation of new alveoli. 1 Elastin is a critical structural protein in the primary and secondary septa. Tropoelastin (TE, the soluble precursor of elastin) mRNA is present in the lung during the pseudoglandular stage, but only in the walls of airways and blood vessels. 23 Elastin synthesis in the primary alveolar septa begins during the canalicular stage of lung development. 31 Alveolar septation is accompanied by a ---4-6-fold increase in parenchymal desmosine (a molecule that is unique to elastin and is a marker for elastin fiber deposition) and a --~6-fold increase in hydroxyproline residues, which are primarily found in collagen. 32'33 Postnatal elastin accumulation has also been studied in mice, in which the most abundant deposition of cross-linked elastin occurs between postnatal days 9 and 20. 34 In humans, the lungs at birth contain ---50x 106 alveoli (---18% of the alveolar number found in adults), suggesting

Alveolar epithelium stem cells Alveolar type II cells can divide and give rise to new type II cells or differentiate into type I cells. 43-45 There is little evidence that type I cells can divide; therefore, most investigators consider the type I cell to be a terminally differentiated cell type. When the lung epithelium is injured, alveolar type II cells divide and cover the injured area and this is followed by the gradual reappearance of type I cells. 4'44'45 Some investigators have hypothesized that during fetal lung development, undifferentiated epithelial cells first differentiate into alveolar type II cells, followed by the differentiation of some of these cells into alveolar type I cells.46'47 When isolated type II cells are maintained in vitro on tissue culture plastic surfaces under particular physicochemical conditions, they rapidly take on the phenotypic characteristics of alveolar type I cells. 48'49

Development

Fig. 4.2. Electron micrograph of lung tissue from a human newborn. The alveolar type II cell is filled with lamellar bodies. The thin cytoplasm of an alveolar type I cell covers the basement membrane that is shared with a capillary endothelial cell. R, red blood cell; A, alveolar lumen; C, capillary lumen; Ib, lamellar body; mv, microvilli; thick arrow, type I cell cytoplasm; arrowhead, endothelial cell cytoplasm. Bar equals 1 l~m. (Reproduced with permission from Mallampalli RK, Acarregui MJ, Snyder JM. Differentiation of the alveolar epithelium in the fetal lung. In: McDonald JA (ed.), Lung Growth and Development. New York: Marcel Dekker, 1997, pp. 119-62.)

Regulation of alveolar epithelial cell differentiation Numerous factors have been shown to regulate the differentiation of the alveolar epithelium in the fetal lung. As reported in several reviews 9'18'22'5~ these factors include transcription factors, hormones, growth factors, regulatory agents such as cyclic AMP, neurotransmitters and physical factors such as stretch. Many AEC differentiation factors also regulate the process of alveolarization itself and the role of some of these factors in the formation of alveoli will be discussed at the end of this chapter. Epithelial-mesenchymal interactions are well-established mediators of lung development and are also likely to be specifically involved in alveolarization. 16'53 The composition of the ECM beneath the alveolar epithelium has dramatic effects on the differentiated state of AECs) 4 In particular, laminin substrata seem to promote the retention of alveolar type II cell differentiation in vitro. 55 In addition, the growth of the pulmonary vasculature, in particular the capillary network of the septa, influences the differentiation state of the overlying epithelium, probably promoting an alveolar type I cell phenotype in the cells that directly overlie capillaries. TM Finally, it has been reported that alveolar type II cells are localized preferentially over cables of elastin in the alveolar wall. 57

Differentiation of the alveolar type I cell The alveolar type I cell is an important component of the air-blood barrier, as it overlies capillaries in the alveolar wall and comprises most of its surface area (-90%). In the past, studies of the structure/function of the type I cells

of Alveoli

were difficult because very few biochemical markers characteristic of this cell type were known. A few markers for the type I cell had been proposed, for example, binding to certain lectins. 5s'59 However, several investigators have recently described proteins that are predominantly expressed in the lung in type I AECs. 49'5s Tl-t~ and HT1 are proteins that were originally identified based on monoclonal antibodies that recognized type I cell-specific proteins. 49'6~ Tl-o~ is the best-characterized marker and is a plasma membrane protein. 6~ It is expressed only in alveolar type I cells in the lung, but is also present in epithelial cells of the ciliary body in the eye and of the choroid plexus of the brain, sites of expression suggestive that Tl-o~ is involved in fluid transport. 6~ Tl-ct is induced in type II cells undergoing a phenotypic transition to a type I-like appearance during culture in vitro. 49 Another important class of proteins expressed in type I AECs are the water channels, aquaporins. 62 Aquaporins are a family of water channels that are expressed ubiquitously; however, in the distal portion of the human lung, aquaporins 4 and 5 seem to be relatively restricted to the alveolar type I cell while aquaporin 3 is expressed in the alveolar type II cell. 63 Other antigens that are either relatively specific to the type I cell or else are highly enriched in this cell type include an intercellular adhesion molecule, ICAM-I, which may be involved in establishing and maintaining the flattened phenotype of the alveolar type I cell. 64 Gap junctions are thought to exist between adjacent alveolar type I and II cells in the alveolar wall. 65 The expression of relatively unique complexes of connexins have recently been described in type I and type II AECs. 66 Finally, several recent reports have documented that type I cells are characterized by abundant caveoli and the expression of caveolin 1 protein while alveolar type II cells possess few caveoli and express little caveolin 1 protein. 67 Caveoli are plasma membrane structures that may be important in mediating the transport of materials across a cell and also may concentrate signaling mediators. 68

Differentiation of alveolar type H cells Although type II AECs, which produce pulmonary surfactant, 5 occupy only a small portion of the alveolar surface area (---7%),3 there are about twice as many of them as type I cells. 69 Type II cell differentiation begins as early as 24 weeks gestation in the human (and about day E18 in the mouse fetus). 1'18 Surfactantphospholipids Surfactant is comprised of about 80% phospholipids, 10% cholesterol and 10% protein. 7~ The most abundant class of phospholipids in pulmonary surfactant is phosphatidylcholine, in particular dipalmitoylphosphatidylcholine (DPPC), which is the primary surface tension lowering component in surfactant. 7~ The next most abundant phospholipid is phosphatidylglycerol (PG), an anionic phospholipid. Interestingly, the surfactant initially produced by the fetal type II cell contains another anionic phospholipid, phosphatidylinositol (PI), rather

T:he::Lung:: Development, Aging and the Environment .....

than PG. 7~ As gestation proceeds, the relative amount of PI decreases while that of PG increases. 7~ Various other phospholipids are also present in characteristic amounts in pulmonary surfactant. 7~ Cholesterol is also present, but its functional significance is not clear. 72 The synthesis of surfactant phospholipids in differentiating alveolar type II cells is accompanied by several morphologic changes in the type II cell (Fig. 4.3). 20 First, lamellar bodies, which are the intracellular storage form of pulmonary surfactant, appear in the cytoplasm. 2~ Secreted lamellar bodies undergo a structural transformation to form tubular myelin, a surfactant intermediate which is thought to give rise to the monolayer of surfactant that lines the alveolar aqueous lining layer and reduces its surface tension. 2~ Surfactant phospholipids are recycled by the type II cell. 5 Another characteristic change in differentiating type II AECs is the disappearance of the large glycogen pools that are characteristic of the tall columnar undifferentiated epithelial cells in the developing lung (Fig. 4.4). 2~ It is thought that the intracellular glycogen in presumptive type II cells is broken down to provide the metabolic substrates necessary for the initial burst of surfactant phospholipid synthesis. 7~ Fatty acids used for surfactant phospholipid synthesis are probably synthesized in the alveolar type II cell. 73 Cholesterol is predominantly taken up by the type II

:

::

cell via the binding and internalization of VLDL particles from the serum. 72'74 VLDL could potentially also supply some of the fatty acids needed for phospholipid synthesis and, in fact, have been shown to increase phospholipid synthesis. 75 Premature human newborns frequently do not have adequate numbers of differentiated type II AECs and thus do not produce sufficient amounts of pulmonary surfactant, resulting in the respiratory distress syndrome ( R D S ) . 76'77 During the 1980s, supplementation with synthetic surfactants was introduced as a treatment for RDS 78 and this was followed by new generations of surfactants, many derived from natural surfactants. 79 Surfactant therapy has dramatically decreased the incidence of neonatal RDS, although some premature infants do not respond well to this therapy. 77'78

Surfactantproteins

The surfactant-associated proteins are an important component of pulmonary surfactant8~ four have been identified to date, i.e. SP-A, SP-B, SP-C and SP-D. 8~ All are expressed in type II AECs and all are developmentally regulated. 8~ SPoA: The most abundant surfactant protein is SP-A, a -35-kDa glycoprotein, sl Several reviews concerning the structure and function of this interesting protein have been published in recent years.82--86SP-A is a member of the collectin family of proteins, calcium-dependent C-type lectins that are involved in innate host defense against pathogens. 83'84 SP-A also blocks the inhibitory effects of serum proteins on surfactant surface tension lowering properties, s7'88 SP-A genedeleted mice have normal lung structure and function but are more susceptible to infection with several pulmonary pathogens than intact, wild-type mice. 89 In the human, SP-A is also expressed in submucosal glands of the conducting airways and, in smaller amounts, in the gastrointestinal tract. 90-92

.... ~~

Fig. 4.3. Electron micrographs of lamellar bodies and tubular myelin in rabbit fetal lung explants. (A) Secreted lamellar bodies and tubular myelin (arrows) observed within a lumen in a cultured explant. (B) A lamellar body observed within a differentiated type II cell. The lamellae are surrounded by a perilamellar membrane. Bars equal 0.1 l.tm. (Reproduced with permission from Snyder JM. The biology of the surfactant proteins. In: Bourbon J (ed.), Pulmonary Surfactant: Biochemical, Functional, Regulatory and Clinical Concepts, Boca Raton, FL: CRC Press, 1991, pp. 105-21.)

SP-B: SP-B is a small (-6.5 kDa), extremely hydrophobic surfactant protein that facilitates the spreading of surfactant proteins on the alveolar surface. 93 Recent studies have also implicated SP-B as being involved in lung antioxidant function. 94 SP-B is synthesized as a high molecular weight precursor that is cleaved to the active molecule within the lamellar body or its precursor. 95 SP-B gene-deleted mice die immediately after birth due to respiratory distress. 96 In the human, genetic mutations in the SP-B gene cause congenital alveolar proteinosis, a lethal condition in human newborns that can only be treated by lung transplantation. 97 The deficiency in SP-B production in these patients is also associated with a defect in the intracellular processing of the SP-C precursor protein. 97 In the human, SP-B is expressed in alveolar type II cells and in Clara cells of the conducting airways. 93 SP-C: SP-C is another low molecular weight ( - 5 kDa), extremely hydrophobic surfactant protein. 93 SP-C genedeleted mice have almost no phenotypic differences from wild-type mice other than a tendency of their surfactant to have atypical biophysical properties. 98 Interestingly, individuals with mutations in their SP-C genes and with

Development

of A l v e o l i

Fig. 4.4. Electron micrographs of rabbit fetal lung tissue. (A) Electron micrograph of a fetal lung epithelial cell at day 19 of gestation. The undifferentiated tall columnar cells contain well-defined glycogen pools. (B) Electron micrograph of a fetal lung epithelial cell at day 28 of gestation. The cells contain lamellar bodies, multivesicular bodies and no well-defined glycogen pools. Bars equal 1 l~m. (Reproduced with permission from Snyder JM, Magliato SA. An ultrastructural morphometric analysis of rabbit fetal lung type II cell differentiation in vivo. Anat. Rec. 1991; 229:73-85.)

reduced levels of SP-C protein were found to have interstitial lung fibrosis. 99 In the human, SP-C is expressed only in alveolar type II cells of the lung. 93 SP-D: SP-D is the most recently characterized surfactantassociated protein. 1~176 It is an ---43-kDa glycoprotein that is a member of the collectin protein family and, as such, is involved in host defense. 83-s5 SP-D gene-deleted mice have abnormalities in surfactant metabolism and accumulate large amounts of surfactant in their alveoli 1~ but the surface tension lowering properties of their surfactant appear to be normal. 1~ These mice go on to develop emphysema by about 1 year of age by an unknown mechanism. 1~ No human mutations in SP-D have yet been described. 97 SP-D is expressed in alveolar type II cells but is also widely distributed in mucosal surfaces throughout the body in humans. 85

DEVELOPMENT

OF THE A L V E O L A R

INTERSTITIUM

During fetal lung development, the interstitial mesenchyme situated between the branching ducts plays an important inductive role in airway and terminal sac formation. During late gestation, the mesenchyme gradually becomes more attenuated as there is a progressively closer apposition of

the epithelium of the terminal sacs and the vasculature. During alveolarization, the interstitium must assume the function of providing structural support for the gas-exchange unit, which postnatally is under the phasic mechanical stress of respiratory movements. Interstitial fibroblasts The interstitial fibroblast (IF) is the major synthetic cell in the interstitium. It is thought to produce much of the ECM in the alveolar interstitium and to provide metabolic substrates to the epithelium. During alveolarization, four functions characterize IF development: proliferation, migration, synthesis of ECM components and apoptosis. The IF is not synchronized in these functions, and since the IF population is heterogenous, some IF may be proliferating while others are migrating or involved in synthesis of ECM. Two populations of IF have been described based on the presence or absence of lipid droplets within their cytoplasm. 1~ These have been termed lipid interstitial cells (LICs) and non-lipid interstitial cells (NLICs; see below).

Fibroblast proliferation Autoradiography with 3H-thymidine has been used to characterize the proliferation of IF in postnatal rat lungs. 25'1~ IF proliferation peaks at postnatal day 4, then progressively

Th e Lungi:: bevel opm:e nti Ag::i nga nd: :the: Envir on m:e:nt::: ::: :: :::: : :::: :: ...... :: ....... ..... : : declines until day 13 with very little proliferation occurring after this time. 25 LIC proliferation declines earlier than that of NLIC so that by postnatal day 11, LIC proliferation has ceased while the labeling index of NLIC is still 2%. 104 Since LICs are frequently located at the base of the elongating secondary septa, IF proliferation is sustained for a longer period in the more distal portions of the developing lung. Under normal circumstances, IF proliferation ceases after alveolarization has been completed, but is more sustained if the developing postnatal lungs have been exposed to glucocorticoids. 1~ IF proliferation can be induced by lung injury and is an important component of the fibrotic response of the lung to injurious agents such as bleomycin and during inflammatory interstitial lung diseases.106,1~

Fibroblast migration Little is known about the migration of IF in the alveolar septa during its elongation. It is clear that cellular movement must occur as the secondary septa elongate as cellular proliferation slows before elongation is complete. Therefore, the distal septal cells must migrate and do not arise solely from cellular division at their ultimate destination. At the free (and most distal) margin of the alveolar wall, in the interstitium, is a ring of myofibroblasts, cells that contain contractile actin-myosin filament arrays arranged in parallel with the long axis of the cell. These cells differentiate late in the alveolarization process and are thought to be able to vary the dimensions of the alveolus via their contraction. 26 The septal myofibroblasts that form the alveolar septal ring are present in the attenuated secondary septa at the beginning of IF migration and maintain their position at the septal tip throughout the elongation process. 27 Cellular migration is difficult to study in tissues in vivo and most of the available information about this process has been derived from studies conducted in vitro. Embryonic and neonatal lung fibroblasts migrate towards a gradient of chemotactic molecules such as fibronectin, fibrin and elastin proteolytic fragments. 1~176 IFs that have assumed a myofibroblastic phenotype migrate in larger numbers and further in response to fibronectin than nonmyofibroblastic lung fibroblasts. 1~ Components of the elastic fiber have been shown to stimulate fibroblast migration and may also contribute to IF migration during septal elongation. 1~ Elastin degradation products may be important in the response of the lung alveolus to proteolytic injury or in the alveolar remodeling that occurs after the neonatal lung is exposed to hyperoxic conditions. 111 PDGFs and their receptors are critical for myofibroblast migration and the expression of PDGF receptors tx and are increased in myofibroblasts. 112'113 PDGF receptor ~containing myofibroblasts fail to populate the tips of the secondary septa in PDGF-~ null mice; this may result from the failure of these cells to migrate in the septa. 113 The phenotype of these mice includes a failure of alveolarization resulting in an emphysematous lung.

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Structural proteins in the alveolar wall Pulmonary IFs are the major producers of the structural proteins present in the alveolar wall interstitium. One of these, elastin, provides resilience during phasic respiration and contributes to lung recoil. It is a major component of elastic fibers, structures that also contain fibrillins, microfibrilassociated glycoproteins, emelin, lysyl oxidase and fibulins. Elastin is produced by both the LICs and the NLICs (see below), and the developmental regulation of the elastin gene has been studied in detail. 23 The structural collagens, types I, III and IX are also produced by IF and provide tensile strength to the alveolar wall. IFs produce proteoglycans which help provide a hydrated environment in which the elastic fiber retains its elastomeric nature. 114 The glycosaminoglycans hyaluronic acid, chondroitin sulfate and heparin sulfate contribute to the stiffness of the postnatal alveolar walls and changes in their contents explain agerelated differences in the mechanical properties of the distal lung. 115 The LIC accumulates lipids during late prenatal and early postnatal life that is largely dispersed by the third postnatal week. 116

Elastin The developmental regulation of elastin synthesis in the lung has been most extensively studied in mice and rats and, in these species, occurs primarily postnatally. Early morphologic studies established that extensive elastin accumulation in the alveoli occurs between postnatal days 4 and 20. 34'117'118 Amorphous elastic fbers are deposited adjacent to both LIC and NLIC in the interstitium and are crosslinked extracellularly, enzymatically by lysyl oxidase and non-enzymatically through aldol condensation. 1~ There is a close temporal correlation between the increase in elastic fiber length and the volume density of the interstitium of alveoli during postnatal days 4-20 in the rat. These morphologic findings have been corroborated by biochemical studies that demonstrate that TE production is maximal during postnatal days 7-12, and that the peak in TE production precedes the maximal desmosine and cross-linked elastin accumulation during postnatal days 10-20.120'121 Inhibition of elastin cross-linking by inhibiting lysyl oxidase (the ratelimiting enzyme) during alveolarization markedly alters alveolar structure and decreases the gas-exchange surface area of the lung. 122 Together these observations suggest that critical regulatory events occur after birth to initiate and terminate elastin synthesis. TE is synthesized during the saccular phase and surrounds the terminal sacs. 123 In mice bearing a null deletion of the elastin gene, the absence of elastin influences airway branching during the final day in utero. 124'125 The elastinnull mouse also demonstrates a decrease in the initiation of secondary crests during gestational day 18.5 to postnatal day 0.5 and a decrease in terminal sac air space units at birth. 125 The initiation of elastin synthesis in the terminal respiratory units normally results from an abrupt increase in TE transcription between days El8 and E21 in the rat, when it reaches its peak. After declining at postnatal day 2,

Development of Alveoli

TE transcription increases again at postnatal day 9.120'121 The postnatal increase in TE gene transcription is coordinated with a decrease in IF proliferation and may result from a reduction in the effects of suppressive growth factors such as FGF-2.126'127 Alveolar levels of TE mRNA reach a maximum around postnatal days 9-12 in the rat and then decline by postnatal day 15.120'128This postnatal increase in TE expression is regulated both transcriptionally and posttranscriptionally. 129'13~An increase in TE mRNA stability contributes to the postnatal increase in TE mRNA levels; the decline in TE mRNA after postnatal day 12 is solely accounted for by a decline in TE mRNA stability. 129'131The factors that are responsible for the increase in TE mRNA stability in the lung have not been identified, but studies using cultured lung fibroblasts indicate that TGF-13s can enhance TE mRNA stability by inhibiting the binding of a destabilizing protein to an element in the exon 30 region of TE mRNA. TM The destabilizing protein is more abundant in adult rat IF and an increase in the levels of this protein likely contributes to the reduction in TE mRNA stability that occurs after postnatal day 12.

The elastic fiber TE is rapidly exported from IF and associates with microfibril elements. This interaction is critically dependent on the association of basic amino acid residues in the carboxy terminus of the protein with microfibril-associated glycoproteins and with the amino terminal region of fibrillins-1 and -2.132 TE monomers also associate with one another prior to cross-linking by a process termed coacervation. Coacervation is driven by hydrophobic interactions involving TE exon 26 and is promoted by sulfated glycosaminoglycans which interact with the positively charged carboxy-terminal lysines o n T E . 133'134 These protein-protein and protein-glycosaminoglycan interactions are accompanied by an enhancement in lysyl oxidase-mediated crosslinking. 135 From postnatal days 14 to 21, alveolar septal elastin undergoes cross-linking which confers increased chemical and proteolytic stability and contributes to the extraordinary longevity of elastin in the elastic fiber. TM The pulmonary IF also contributes to the production of the microfibril, which is an essential component of the elastic fiber. Both fibrillins-1 and -2 are expressed in the adult lung with fibrillin-2 primarily present prenatally. 137'138 Postnatally, fibrillin-1 predominates and makes a significant contribution to the maintenance of the elastic fiber network. A duplication in the coding region of fibrillin-1 occurs in the tight skin mouse and this destabilizes microfibrils and leads to a reduction of elastic fibers and a decrease in the formation of alveolar septa starting 4 weeks after birth. 139

THE L I P I D I N T E R S T I T I A L CELL Based on their lipid contents, pulmonary interstitial cells (interstitial fibroblasts) are divided into two populations,

the lipid-droplet laden LICs and the NLICs which lack lipid droplets and are located more peripherally in the alveolar septum. The initial morphologic description of these cells emphasized the abundant lipid droplets, high glycogen content and localization of LIC to the central region of the alveolar septum (Fig. 4.5). 1~ Some of the lipid droplets in LIC are surrounded by glycogen deposits. 14~ Subsequent studies established that LIC contain contractile filaments similar to those observed in myofibroblasts. 119'14~ These contractile filaments are generally more dense than those present in the NLIC and are oriented with their long axis either perpendicular or oblique to the plasma membrane. 1~ Elastic fibers are found adjacent to both NLIC and LIC, and the density of the intracellular contractile filaments is greatest where cells contact elastic and collagen fibers. 26 LIC and NLIC independently undergo cell division during alveolarization, and neither cell type appears to be a precursor for the other. TM LICs are first evident in rat lungs at day El6. The triglyceride content of whole rat lung tissue increases 3-fold between days El7 and El9.141 Lung triglyceride content increases another 2.5-fold between day E21 and postnatal day 1, and then peaks during the second postnatal week. 141 The abundance of LIC in the lung follows the same timecourse. 116 The lipid droplets of LIC contain primarily neutral lipids (---86%) with phospholipids comprising the remaining 14%.116 Like adipocytes, LICs express lipoprotein lipase, fatty acid transporter and intracellular lipid binding proteins and are able to accumulate neutral lipids when purified triglycerides are added to the culture medium. 142'143 They also contain LDL and VLDL receptors whose expression increases during the postnatal period. TM LICs contain vimentin and desmin, both intermediate filaments, and m-smooth muscle actin (~SMA). The number of LIC decreases prior to weaning, a result, in part, of a decrease in cell proliferation and a decrease in plasma lipids. 1~ LICs are present at the base of the alveolar septae in adult rats and their number can be increased by the administration of retinyl palmitate. 14~ Similarities exist between the contractile filaments in neonatal LIC and those found in the contractile interstitial cell (CIC) present in the adult lung. 119 Based on the ultrastructure of the contractile filaments, several investigators have speculated that the LIC may be the neonatal equivalent of the CIC. 26 LIC lose their lipid droplets when cultured in the presence of fetal bovine serum. 143 They also assume more myofibroblastic characteristics and abundantly express ~-SMA, desmin, as well as vimentin. 146 However, they maintain their neutral lipid content if they are cultured in triglyceride-rich, neonatal rat serum or are exposed to activators of the peroxisome proliferator-activated receptors, which are involved in the induction of lipid storage by adipocytes. 146 During lung development, mesenchymal cells lie in close apposition to epithelium and play a central role in the growth and differentiation of epithelial cells into alveolar type II cells, the site of pulmonary surfactant synthesis. 16'147

The Lung: Development,

Aging and:the Environment:

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:

ALV

NLIC

Typ e

II

LIC

ALV

Fig. 4.5. Lipid-filled interstitial fibroblasts (LICs) as seen in micrographs of rat lung from late fetal developmental stage. L = lipid, Cap =capillary, ALV = alveolar lumen, NLIC = non-lipid-filled interstitial fibroblast, type II =alveolar type II cell. (Reproduced with permission from Vaccaro C, Brody JS. Ultrastructure of developing alveoli. I. The role of the interstitial fibroblast. Anat. Rec. 1978; 192:467-79.)

In the rat, LICs are first evident during the canalicular phase, with triglyceride content maximal just prior to the appearance of lamellar bodies in neighboring type II cells. 148 Triglycerides of fibroblast origin are used for surfactant phospholipid synthesis by type II cells in culture. 149 The mechanism of transfer of neutral lipids from the LIC to type II cells has not been fully characterized, but like adipocytes, the LICs contain lipase, which may de-esterify the lipids prior to their export as fatty acids. 14z It is also possible that lipid droplets are transferred directly from one cell to the other, since intercellular communications have been observed between type II cells and LIC cells in neonatal rat lung. 15~ In addition to triglycerides, the LICs accumulate retinyl esters. 151'152The retinoid content (primarily retinyl palmitate) of the fetal rat lung increases markedly after day El5, peaks at day El8 and then decreases 4-fold by day E21.153 There is a decline in retinyl ester content of LIC from day El9 to postnatal day 2 in the rat. 151 Retinol and retinoic acid (RA) in the lung increase concurrently by postnatal day 2. Retinol remains elevated until postnatal day 8 and then declines, while RA falls abruptly after day 2.151 The timing of these events has led to the hypothesis that retinoids (and in particular RA) may be important in alveolarization. 151'152'154 In whole lung tissue and in isolated LIC, the steady-state levels of RAR-mRNA appear to be the highest in the early neonatal period. 151'155 In lung tissue, the levels

of the mRNAs for RARo~, RARI3 and RARy increase significantly at birth. 155'156 In isolated LIC, RARI3 increases -4-fold while RARy increases --8-fold between day El8 and postnatal day 2.151 Endogenous retinoids increase TE expression in explant cultures of lung obtained from rats on gestational day 19 while exogenous RA increases TE expression in cultured LIC. 157 Mice that have a null deletion of RARy, and are lacking one allele of RXRt~, have diminished levels of TE mRNA in their LIC at postnatal day 10 and contain less elastin in their lungs at postnatal day 28.158 This is accompanied by a decrease in alveolar number and the gas-exchange surface area.

ALVEOLAR MYOFIBROBLASTS The term alveolar myofibroblast (AMF) has generally been applied to pulmonary IFs that express uSMA, an actin isoform that is most commonly associated with smooth muscle cells (Fig. 4.6). The AMFs have been further sub-classified based on their intermediate filament profile (vimentin and/or desmin) and the presence or absence of myosin heavy chains. 159 The AMF is of functional importance during lung development and disease. During lung development, it is present in the primary septa where secondary septal buds form. 118'125 It is responsible for producing the elastin that localizes at the tip of the elongating septum and

Development of Alveoli

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Fig. 4.6. Alveolar myofibroblasts in rat lung. (a) An alveolar myofibroblast (MF) is located at the junction of three alveolar septa. The microfilaments end in a dense body situated against the alveolar basement membrane (arrows). A, alveoli; C, capillaries. (b) Note the abundance of actin filaments parallel to the capillary basement membrane (arrows). Cytoplasmic processes of the alveolar myofibroblast extend into the thick portion of the air-blood barrier (arrowheads). cf=collagen fibers. On panel A, bar=5 ~m and on panel B, bar= 1 lLtm. (Reproduced with permission from Kapanci Y, Gabbiani G. Contractile cells in pulmonary alveolar tissue. In: Crystal R, West JB, Weibel ER etal. (eds), The Lung: Scientific Foundations. Philadelphia, PA: Lippincott-Raven, 1997, pp. 697-708.)

forms the alveolar contractile ring. In rats, the AMFs are most abundant during secondary septal formation when TE expression is at its highest; it is likely to be the major source of elastin synthesis in the pulmonary interstitium. During septal elongation, the AMF proliferates in a PDGFA-dependent process that is a requirement for secondary septal formation. 16~Mice bearing a deletion of the PDGF-A gene fail to undergo secondary septation, their primitive alveolar walls lack elastic fibers and the mice die during the first 2 weeks of life. 160 Other defects, such as a failure in AMF migration, may also contribute to failed septation in PDGF-A null mice. 113 It has also been shown that PDGFR-dependent lung myofibroblast proliferation involves signaling through the p38 MAP kinase pathway. 1~ The cellular precursor of the AMF has not been identified. In other organs, myofibroblasts arise from either primitive stem cells, pleuripotent cells such as neural crest cells or fibroblasts. 161 Based on the morphology of the subcortical cytoskeleton, some investigators have proposed that the LIC is the precursor of the AMF. 119When LICs are cultured in vitro to confluence, they express more ~SMA and also express myosin heavy chains. 146 Autocrine and paracrine factors stimulate tzSMA production by cultured lung fibroblasts. These include TGF-[3s, PDGFs, IGFs, interleukin-4, and, in inflammatory states, interleukin-1.161 The effects of TGF-[3s, secreted by myofibroblasts themselves, have been most extensively studied. 161 TGF-[31 stimulates t~SMA gene transcription through jun-kinase in lung myofibroblasts, with the effect mediated through a TGF-[3-responsive element in the proximal portion of the ~zSMA promoter. 162 The TGF-[3-responsive elements in the

~SMA promoter differ in myofibroblasts compared to smooth muscle and endothelial cells. 162 IL-I[3 stimulates inducible NO synthase in cultured lung fibroblasts, resulting in increased NO production, which reduces t~SMA mRNA and protein. 163 IL-I[3 and other inflammatory mediators may be involved in interstitial pulmonary fibrosis since in response to these mediators interstitial myofibroblasts (also termed contractile interstitial cells) proliferate and produce more ECM proteins, most notably the interstitial collagens. TM

M E C H A N I S M S OF T H I N N I N G OF THE ALVEOLAR SEPTUM Following expansion of the IF population, movement of IF into the secondary septa, and the greatest period of TE synthesis, which in the rat ends around postnatal day 13, there is a 20% loss in the number of IF; this loss results from IF apoptosis. 165 At postnatal day 16, there is an abrupt increase in IF apoptosis that is characterized by an increase in chromatin condensation and DNA strand breaks, as well as an increase in the levels of Bax, a pro-apoptotic protein, and a decrease in the expression of the anti-apoptotic protein Bcl-2.165 This apoptotic phase is transient and ends after postnatal day 19. While the number of IF decreases, the mass of the ECM does not increase and assumes a larger proportion of the volume of the interstitium. The reduction in IFs is accompanied by an increase in capillary surface area, which improves the efficiency of gas exchange. 1 During septal thinning, there is a fusion of the paired

The: Lung:Developmenti:Aging and the Environment :::

capillary loops in each septum by a process which most likely involves the formation of connecting pillars. 1 After breaching the interstitium between the two loops, these pillars then increase in diameter and decrease in height. Thus the pillars assume the shape of disks which increase capillary surface area, while reducing the need for extensive new endothelial cell proliferation. As the alveolar walls thin, the pores of Kohn form and facilitate the merging of air sacs. 166 While the lung volume of humans and rats increases 23-fold between birth and adulthood, capillary volume increases about 35-fold, a finding which is consistent with an increase in the interconnecting capillary meshwork.

DEVELOPMENTAL DEFECTS IN A L V E O L A R I Z A T I O N Studies of developmental defects in alveolar formation have taken two general approaches. The first has been to study BPD in human infants or spontaneous mutations in mice that result in defects in alveolar formation. The second approach has been to target specific genes in mice and to study the effects of their deletion or over-expression on alveolar formation. The second approach has yielded considerable information about the roles of specific regulatory factors such as PDGF-A and PDGF receptor o~, the RARs, VEGF and glucocorticoids. The phenotypes of mice with gene deletions that affect these agents will be discussed in more detail below.

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This developmental defect results from a spontaneous mutation in which a portion of the coding region of the fibrillin-1 gene is duplicated. 139 When both alleles contain this duplication, the affected mice die in utero. The heterozygotes survive, but have morphologic abnormalities: in their skin and lungs. The fibrillin-1 gene duplication results in the synthesis of a longer fibrillin protein that does not polymerize normally and results in the formation of microfibrils which are more sensitive to proteolytic degradation. 17~The abnormal microfibrils also fail to support normal elastic fiber formation and thus elastin levels are reduced in the lung parenchyma of tight-skin mice at 4 weeks. 171 In contrast, collagen deposition is increased in the lung and skin of the affected mice. The pallid mouse also spontaneously develops enlarged and fewer lung alveoli, but at 8 months of age, much later than in the tight-skin mouse. The pallid mutation maps to the same chromosome as the Tsk mutation but appears to result from a different genetic abnormality. 172 The lungs of pallid mice contain less elastin at 12 months of age and this is correlated with an increase in extracellular elastase in the pulmonary interstitium. 172 These mice may also have lower levels of circulating o~l-antiprotease, which along with the increase in elastase lead to an increase in elastin degradation. 169 Therefore, the alveolar enlargement in the pallid mouse most likely results from alveolar wall destruction rather than a defect in alveolar wall formation.

REGULATION

OF A L V E O L A R I Z A T I O N

Bronchopulmonary dysplasia BPD occurs in infants who are born prematurely and who require mechanical ventilation to treat RDS; the accompanying oxygen toxicity and pressure/volume-related lung injury was previously thought to play a major role in the development of BPD. However, following the use of surfactant replacement therapy, reduced ventilation trauma and lower O 2 levels, it has become clear that BPD is characterized by abnormal alveolarization. Infants dying of BPD have fewer and larger alveoli, with more attenuated alveolar walls and a paucity of alveolar capillaries. 7 It is hypothesized that this results from decreased alveolarization as well as injury to the existing primary septa. Multiple factors are thought to contribute to the defective alveolar formation that occurs in BPD, including prenatal or antenatal administration of glucocorticoids, nutritional deficiencies, inflammation, as well as the effects of hyperoxia and mechanical ventilation. 77 Studies in prematurely delivered baboons and lambs that received exogenous surfactant and were ventilated using conditions that minimized mechanical trauma and hyperoxia revealed that they had fewer and enlarged alveoli in their lungs, with a reduction in alveolar capillary surface area. 167'168The abnormalities in BPD have recently been reviewed in detail. 77

Tight-skin and pallid mice The tight-skin mouse is characterized by fewer and enlarged lung alveoli, apparent by 4 weeks after birth. 169

Glucocorticoids Glucocorticoids are thought to play an important role in normal lung development. 173 In many species, including the human, there is an increase in fetal serum glucocorticoid levels towards the end of gestation that correlates well with the structural and functional maturation of the fetal l u n g . 174 Liggins and H o w i e 175'176 w e r e the first to report, in the sheep and then in humans, that exogenous glucocorticoids, administered to the mother, could accelerate fetal lung development and reduce the incidence of RDS in premature infants. Transgenic mice in which the corticotrophin-releasing hormone (CRH) gene was deleted have low levels of glucocorticoids and delayed lung development. 177 Likewise, mice in which the glucocorticoid receptor gene was deleted also exhibit delayed lung development and usually die shortly after birth. 178 Since neonatal RDS is principally caused by surfactant deficiency, much attention has focused on glucocorticoid regulation of type II AEC differentiation and surfactant production. In general, glucocorticoids increase both surfactant phospholipid and surfactant protein levels. 173 The lungs of glucocorticoid-treated fetuses have thinner septal walls due to decreased connective tissue. The levels of elastin in alveolar walls are increased in glucocorticoidtreated lungs. 41'123 These structural changes may facilitate gas-exchange in the lung. However, another, well-documented

Development of Alveoli

effect of glucocorticoids on lung development is an inhibition of alveolarization. 179'18~ Septation of the terminal sac into alveoli may be impaired in glucocorticoid-treated fetuses and newborns. 179'18~Glucocorticoids may act indirectly via promoting the production of certain growth factors and their receptors which in turn stimulate premature lung differentiation at the cost of further lung growth and development. 173 The negative effect of glucocorticoids on alveolarization apparently persists into adulthood. 179 Since glucocorticoids are used prenatally to prevent RDS and are also often used postnatally to avoid the development of BPD, there is concern about the appropriateness, doses used, and duration of glucocorticoid treatment for neonatal lung disease. TM

VEGF VEGF is a growth factor that regulates endothelial cell proliferation and differentiation. 182 VEGF is present in distal epithelial cells of the human fetal lung. 183 VEGF is also expressed by alveolar II cells in adult lung tissue after oxygen injury. TM VEGF binds to at least two receptors, VEGFR1 (Fit-l) and VEGFR2 (KDR). 182 K D R is present in human fetal lung tissue. 185 The formation and growth of the capillary bed in the fetal lung parallels the formation of primary and secondary septa in the lung. 1 In premature infants and baboons that develop BPD, decreased numbers of capillaries have been observed in distal lung tissue. 186'187 Recently, two groups have reported that BPD in human infants is associated with alterations in VEGF and its receptors. 187'188 VEGF gene-deleted mice as well as those in which the VEGFR genes are deleted die early in development as a result of disrupted vasculogenesis. 182 In mice which produced excess VEGF in distal lung epithelial cells, pulmonary blood vessels were more abundant; however, alveolar development was defective. 189 The 188 amino acid isoform of VEGF (VEGF 188) is abundant in the lung and accounts for a major portion of the perinatal increase in pulmonary VEGF mRNA. 19~ Cells at the epithelial surface of the distal air sacs are responsible for much of the expression of this VEGF isoform.

PDGF The PDGFs are a family of growth factors that bind two receptors, PDGF-R~ and PDGF-R~. TM PDGF-A is expressed in the developing lung in the epithelium of the distal lung tubules as well as in the connective tissue. 192 PDGF-Rt~ is also expressed in the developing lung. 192'193 Mice in which the PDGF-A gene is deleted die soon after birth due to respiratory distress. 16~The lungs of wild type and PDGF-A null mice are indistinguishable prior to birth and during early postnatal life. 160'194 However, during the postnatal period of rapid alveolarization, lungs in the PDGF-A gene deleted mice do not form alveoli. 16~ Alveolar myofibroblasts which are PDGF-R~ positive do not proliferate in the PDGF-A null mice nor do they migrate into secondary septa. 113 In addition, elastin is not formed in the secondary septa in PDGF-A-null mice. 113 These findings confirm the

67

critical role of myofibroblasts in the formation of secondary septa in the postnatal lung. 113'16~Mice in which the PDGFR~ is deleted die before birth due to multiple abnormalities. 195 The lungs of these animals are small and up until the time of death, at about day El6, appear to undergo branching morphogenesis in a manner similar to lungs in wild type mice. TM Thus the PDGF-A-PDGF-Rt~ axis is unique in that it appears to be a specific regulator of alveolarization in the postnatal lung.

RETINOIC ACID Retinoic acid and its receptors have been shown to be involved in almost every aspect of lung development and in the maintenance of lung differentiation. 196 All-trans retinoic acid is the major, biologically active metabolite of retinol (vitamin A). 196 All-trans retinoic acid binds to RARs and to retinoid X receptors (RXRs). 197 Another metabolite of alltrans retinoic acid, 9-cis retinoic acid, binds primarily to the R X R s . 197 There are three isoforms of RARs, ct, ~ and y and, likewise, three RXR isoforms, t~, fl and Y.197 Retinol is taken up in the digestive tract and is stored in the body as retinyl esters. The fetal lung stores retinyl esters in fibroblasts during lung development. 153 Coordinate with AEC differentiation, lung stores of retinyl esters decline, suggesting that lung fibroblasts may release retinol locally, which can then be metabolized to retinoic acid in retinoid-sensitive tissues such as the adjacent lung epithelium. 198 The lung epithelium expresses all isoforms of the RARs. 156 Deletion of the RARy gene leads to an impairment of alveolarization in mice. 158 Simultaneous deletion of one RXRt~ gene (a R A R 7 - / - , RXR +/- genotype) exacerbates this effect. 158 Thus RARy is probably involved in the formation of alveoli in postnatal lung. Deletion of the RAR[3 gene has been reported to increase the number of alveoli, decrease their size and increase the rate of alveolar formation in affected mice. 199 Interestingly, there was no effect of the RAR[3 gene deletion on the gas-exchange surface area of the lung. 199 In a recent study, we found that deletion of the RAR[3 gene results in impaired alveolarization as reflected by increased alveolar size, decreased gas-exchange surface area and a decrease in respiratory function (Fig. 4.7). 200 The first evidence for a role of RA in alveolarization was presented in two studies by Massaro et al. 154'2~ In the first study, it was shown that administration of all-tram R A to newborn rats increased the number of alveoli, decreased the size of the alveoli and increased the surface area of the lungs. TM Glucocorticoids inhibit alveolarization in the newborn rat, and it was shown that RA administration could overcome this inhibition. 154'2~ In another study performed in adult rats, RA stimulated the formation of alveoli in animals previously treated with elastase to produce an emphysematous phenotype. TM Clinical evidence supports a role for retinoic acid in lung development: preterm infants are frequently deficient in retinol and low

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ACKNOWLEDGEMENTS !i~!~i!il!ii~i!!i!iii~i!i!'~i~i~:84184 i,:~i) ~i~:~:!::i~:~ i:

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The authors wish to thank Jean Gardner for preparing the manuscript. The authors' work is supported by grants from the National Institutes of Health HL62861, HL53430, DERC DK-25295, Department of Veterans Affairs Research Service, and a grant from the March of Dimes Birth Defects Foundation.

REFERENCES

k Fig. 4.7. Van Gieson elastic tissue stain of lung tissue at 28 days. Lungs from R A R - / - and wild-type mice were fixed at an inflation pressure of 20 cm and paraffin sections were prepared. Sections were stained using the van Gieson elastin stain and photographed. Lung tissues from wild-type mice were stained simultaneously so that the incubation times were equivalent for the two sources of tissue. Panel A is from a representative wild-type mouse and panel B from a representative RARy-/- mouse. Arrows point to elastic fibers in alveolar septa, airway = (a), blood vessel = (b). (Reproduced with permission from McGowan S, Jackson SK, Jenkins-Moore M e t al. Mice bearing deletions of retinoic acid receptors demonstrate reduced lung elastin and alveolar numbers. Am. J. Respir. Cell Mol. Biol. 2000; 23:162-7.)

levels of plasma retinol have been correlated with an increased risk of developing BPD in this population. 2~ Furthermore, clinical trials have shown that treatment of premature infants with retinoic acid can reduce the incidence of BPD and decrease mortality. 2~176

CONCLUSIONS The formation of alveoli in the developing lung is necessary for normal lung function. When alveolarization is impaired, as in BPD, respiratory insufficiency and chronic lung disease ensue. When alveoli are destroyed in adult lung, as in emphysema, they do not regenerate. As a result of fundamental studies such as those reviewed in this chapter, we now know more about alveolarization and its regulation than ever before. We know that the myofibroblast in the wall of the primary septa probably orchestrates the growth of the secondary septa and the deposition of elastin that result in the formation of alveolar walls. We also know that PDGF-A and its receptor as well as retinoic acid and its receptors regulate this process. Other important mediators of alveolarization will be revealed by further study. Hopefully, future insights about alveolarization will lead to new treatment approaches for lung diseases in which alveolarization is impaired. Specifically, strategies to enable reactivation of alveolarization in the adult emphysematous lung and in newborns with BPD are greatly needed.

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65. Abraham V, Chou ML, George P etal. Heterocellular gap junctional communication between alveolar epithelial cells. Am. J. Physiol. 2001; 280:L1085-93. 66. Isakson BE, Lubman RL, Seedorf GJ etal. Modulation of pulmonary alveolar type II cell phenotype and communication by extracellular matrix and KGF. Am. J. Physiol. 2001; 281 :C1291-9. 67. Newman GR, Campbell L, von Ruhland C etal. Caveolin and its cellular and subcellular immunolocalisation in lung alveolar epithelium: implications for alveolar epithelial type I cell function. Cell Tissue Res. 1999; 295:111-20. 68. Gumbleton M. Caveolae as potential macromolecule trafficking compartments within alveolar epithelium. Adv. Drug Delivery Rev. 2001; 49:281-300. 69. Crapo JD, Young SL, Fram EK etal. Morphometric characteristics of cells in the alveolar region of mammalian lungs. Am. Rev. Respir. Dis. 1983; 128:$42-6. 70. Veldhuizen R, Nag K, Orgeig S et al. The role of lipids in pulmonary surfactant. Biochim. Biophys. Acta 1998; 1408:90-108. 71. Rooney SA. Phospholipid composition, biosynthesis and secretion. In: Parent RA (ed.), Comparative Biology of the Normal Lung. Boca Raton, FL:CRC Press, 1991, pp. 511-44. 72. Orgeig S, Daniels CB. The roles of cholesterol in pulmonary surfactant: insights from comparative and evolutionary studies. Comp. Biochem. Physiol. 2001; 129:75-89. 73. Wagle S, Bui A, Ballard PL etal. Hormonal regulation and cellular localization of fatty acid synthase in human fetal lung.Am. J. Physiol. 1999; 277:L381-90. 74. Guthmann F, Harrach-Ruprecht B, Looman AC etal. Interaction of lipoproteins with type II pneumocytes in vitro: morphological studies, uptake kinetics and secretion rate of cholesterol. Fur. J. Cell Biol. 1997; 74:197-207. 75. Mallampalli RK, Salome RG, Bowen SL et al. Very low density lipoproteins stimulate surfactant lipid synthesis in vitro. J. Clin. Invest. 99:2020-9. 76. Avery ME, Mead J. Surface properties in relation to atelectasis and hyaline membrane disease.Am.J. DisabledChild 1959; 97:517-23. 77. Jobe AH, Ikegami M. Lung development and function in preterm infants in the surfactant treatment era. Ann. Rev. Physiol. 2000; 62:825-46. 78. Jobe AH. Pulmonary surfactant therapy. New Engl. J. Med. 1993; 328:861-8. 79. Halliday HL. Natural vs synthetic surfactants in neonatal respiratory distress syndrome. Drugs 1996; 51:226-37. 80. Creuwels LA, van Golde LM, Haagsman HP. The pulmonary surfactant system: biochemical and clinical aspects. Lung 1997; 175:1-39. 81. Khubchandani KR, Snyder JM. Surfactant protein A (SP-A): the alveolus and beyond. FASEBJ. 2001; 15:59-69. 82. Hawgood S, Poulain FR. The pulmonary collectins and surfactant metabolism. Annu. Rev. Physiol. 2001; 63:495-519. 83. Crouch E, Wright JR. Surfactant proteins a and d and pulmonary host defense.Annu. Rev. Physiol. 2001; 63:521-54. 84. Lawson PR, Reid KB. The roles of surfactant proteins A and D in innate immunity. Immunol. Rev. 2000; 173:66-78. 85. van Rozendaal BA, van Golde LM, Haagsman HP. Localization and functions of SP-A and SP-D at mucosal surfaces. Pediatr. Pathol. Mol. Med. 2001; 20:319-39. 86. McCormack FX. Functional mapping of surfactant protein A. Pediatr. Pathol. Mol. Med. 2001; 20:293-318. 87. Cockshutt AM, Weitz J, Possmayer F. Pulmonary surfactantassociated protein A enhances the surface activity of lipid extract surfactant and reverses inhibition by blood proteins in vitro. Biochemistry 1990; 29:8424-9. 88. Ikegami M, Elhalwagi BM, Palaniyar N et al. The collagen-like region of surfactant protein A (SP-A) is required for correction of surfactant structural and functional defects in the SP-A null mouse.J. Biol. Chem. 2001; 276:38542-8.

the:Environment

:::::::::::::::::: ::: ::

89. Korfhagen TR, LeVine AM, Whitsett JA. Surfactant protein A (SP-A) gene targeted mice. Biochim. Biophys. Acta 1998; 1408:296-302. 90. Rubio S, Lacaze-Masmonteil T, Chailley-Heu B etal. Pulmonary surfactant protein A (SP-A) is expressed by epithelial cells of small and large intestine. J. Biol. Chem. 1995; 270:12162-9. 91. Goss KL, Kumar AR, Snyder JM. SP-A2 gene expression in human fetal lung airways. Am. J. Respir. Cell Mol. Biol. 1998; 19:613-21. 92. Khubchandani KR, Goss KL, Engelhardt JF etal. In situ hybridization of SP-A mRNA in adult human conducting airways. Pediatr. Pathol. Mol. Med. 2001; 20:349-66. 93. Weaver TE, Conkright JJ. Function of surfactant proteins B and C.Annu. Rev. Physiol. 2001; 63:555-78. 94. Tokieda K, Iwamoto HS, Bachurski C etal. Surfactant protein-B-deficient mice are susceptible to hyperoxic lung injury.Am. J. Respir. Cell Mol. Biol. 1999; 21:463-72. 95. Korimilli A, Gonzales LW, Guttentag SH. Intracellular localization of processing events in human surfactant protein B biosynthesis.J. Biol. Chem. 2000; 275:8672-9. 96. Clark JC, Wert SE, Bachurski CJ et al. Targeted disruption of the surfactant protein B gene disrupts surfactant homeostasis, causing respiratory failure in newborn mice. Proc. Nat. Acad. Sci. USA 1995; 92:7794-8. 97. Cole FS, Hamvas A, Nogee LM. Genetic disorders of neonatal respiratory function. Pediatr. Res. 2001; 50:157-62. 98. Glasser SW, Burhans MS, Korfhagen TR etal. Altered stability of pulmonary surfactant in SP-C-deficient mice. Proc. Nat. Acad. Sci. USA 2001; 98:6366-71. 99. Nogee LM, Dunbar AE, III, Wert SEet al. A mutation in the surfactant protein C gene associated with familial interstitial lung disease. New Eng. J. Med. 2001; 344:573-9. 100. Crouch EC. Structure, biologic properties, and expression of surfactant protein D (SP-D). Biochim. Biophys. Acta 1998; 1408:278-89. 101. Botas C, Poulain F, Akiyama J etal. Altered surfactant homeostasis and alveolar type II cell morphology in mice lacking surfactant protein D. Proc. Nat. Acad. Sci. USA 1998; 95:11869-74. 102. Wert SE, Yoshida M, LeVine AM et al. Increased metalloproteinase activity, oxidant production, and emphysema in surfactant protein D gene-inactivated mice. Proc. Nat. Acad. Sci. USA 2000; 97:5972-7. 103. Vaccaro C, Brody JS. Ultrastructure of developing alveoli. I. The role of the interstitial fibroblast. Anat. Rec. 1978; 192:467-79. 104. Brody JS, Kaplan NB. Proliferation of alveolar interstitial cells during postnatal lung growth: evidence for two distinct populations of pulmonary fibroblasts. Am. Rev. Respir. Dis. 1983; 127:763-70. 105. Tschanz TA, Damke BM, Burri PH. Influence of postnatally administered glucocorticoids on rat lung growth. Biol. Neonate 1995; 68:229-45. 106. Adler KB, Callahan LM, Evans JN. Cellular alterations in the alveolar wall in bleomycin-induced pulmonary fibrosis in rats: an ultrastructural morphometric study. Am. Rev. Respir. Dis. 1986; 133:1043-8. 107. Lindroos PM, Wang YZ, Rice AB etal. Regulation of PDGFR-alpha in rat pulmonary myofibroblasts by staurosporine. Am. J. Physiol. 2001; 280:L354-62. 108. Kawamoto M, Matsunami T, Ertl RF et al. Selective migration of alpha-smooth muscle actin-positive myofibroblasts toward fibronectin in the Boyden's blindwell chamber. Clin. Sci. 1997; 93:355-62. 109. Senior RM, Griffin GL, Mecham RP. Chemotactic responses of fibroblasts to tropoelastin and elastin-derived peptides. J. Clin. Invest. 1982; 70:614-18.

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150. Grant MM, Cutts NR, Brody JS. Alterations in lung basement membrane during fetal growth and type 2 cell development. Dev. Biol. 1983; 97:173-83. 151. McGowan SE, Harvey CS, Jackson SK. Retinoids, retinoic acid receptors, and cytoplasmic retinoid binding proteins in perinatal rat lung fibroblasts. Am. J. Physiol. 1995; 269:L463-72. 152. Chytil F. The lungs and vitamin A. Am. J. Physiol. 1992; 262:L517-27. 153. Shenai JP, Chytil F. Vitamin A storage in lungs during perinatal development in the rat. Biol. Neonate 1990; 57: 126-32. 154. Massaro GD, Massaro D. Postnatal treatment with retinoic acid increases the number of pulmonary alveoli in rats. Am. J. Physiol. 1996; 270:L305-10. 155. Grummer MA, Zachman RD. Postnatal rat lung retinoic acid receptor (RAR) mRNA expression and effects of dexamethasone on RAR beta mRNA. Pediatr. Pulmonol. 1995; 20:234-40. 156. Grummer MA, Thet LA, Zachman RD. Expression of retinoic acid receptor genes in fetal and newborn rat lung. Pediatr. Pulmonol. 1994; 17: 234-8. 157. McGowan SE, Doro MM, Jackson SK. Endogenous retinoids increase perinatal elastin gene expression in rat lung fibroblasts and fetal explants. Am. J. Physiol. 1997; 273:L410-6. 158. McGowan S, Jackson SK, Jenkins-Moore M et al. Mice bearing deletions of retinoic acid receptors demonstrate reduced lung elastin and alveolar numbers. Am. J. Respir. Cell Mol. Biol. 2000; 23:162-7. 159. Leslie KO, Mitchell J, Low R. Lung myofibroblasts. Cell Motilility Cytoskeleton 1992; 22:92-8. 160. Bostrom H, Willetts K, Pekny M e t al. PDGF-A signaling is a critical event in lung alveolar myofibroblast development and alveogenesis. Cell 1996; 85:863-73. 161. Powell DW, Mifflin RC, Valentich JD et al. Myofibroblasts. I. Paracrine cells important in health and disease. Am. J. Physiol. 1999; 277:C1-9. 162. Roy SG, Nozaki Y, Phan SH. Regulation of alpha-smooth muscle actin gene expression in myofibroblast differentiation from rat lung fibroblasts. Int. J. Biochem. Cell Biol. 2001; 33:723-34. 163. Zhang HY, Phan SH. Inhibition of myofibroblast apoptosis by transforming growth factor beta(l). Am. J. Respir. Cell Mol. Biol. 1999; 21:658-65. 164. Hashimoto S, Gon Y, Takeshita I e t al. Transforming growth factor-betal induces phenotypic modulation of human lung fibroblasts to myofibroblast through a c-Jun-NH2terminal kinase-dependent pathway. Am. J. Respir. Crit. Care Med. 2001; 163:152-7. 165. Bruce MC, Honaker CE, Cross RJ. Lung fibroblasts undergo apoptosis following alveolarization. Am. J. Respir. Cell Mol. Biol. 1999; 20:228-36. 166. Weiss MJ, Burri PH. Formation of interalveolar pores in the rat lung. Anat. Rec. 1996; 244:481-9. 167. Coalson JJ, Winter V, deLemos RA. Decreased alveolarization in baboon survivors with bronchopulmonary dysplasia. Am. J. Respir. Crit. Care Med. 1995; 152:640-6. 168. Albertine KH, Jones GP, Starcher BC etal. Chronic lung injury in preterm lambs: disordered respiratory tract development.Am. J. Respir. Crit. Care Med. 1999; 159:945-58. 169. Martorana PA, Brand T, Gardi C et al. The pallid mouse, a model of genetic alpha 1-antitrypsin deficiency. Lab. Invest. 1993; 68:233-41. 170. Kielty CM, Raghunath M, Siracusa LD et al. The Tight skin mouse: demonstration of mutant fibrillin-1 production and assembly into abnormal microfibrils. J. Cell Biol. 1998; 140:1159-66. 171. Gardi C, Martorana PA, de Santi MM et al. A biochemical and morphological investigation of the early development

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: :::::

:

of genetic emphysema in tight-skin mice. Exp. Mol. Pathol. 1989; 50:398-410. 172. de Santi MM, Martorana PA, Cavarra E et al. Pallid mice with genetic emphysema: neutrophil elastase burden and elastin loss occur without alteration in the bronchoalveolar lavage cell population.Lab. Invest. 1995; 73:40-7. 173. Bolt RJ, van Weissenbruch MM, Lafeber HN etal. Glucocorticoids and lung development in the fetus and preterm infant. Pediatr. Pulmonol. 2001; 32:76-91. 174. Ballard PL. Hormones and Lung Maturation. Berlin; Germany: Springer-Verlag, 1986. 175. Liggins GC. Premature delivery of fetal lambs infused with glucocorticoids. J. Endocrinol. 1969; 45:515-23. 176. Liggins GC, Howie RN. A controlled trial of antepartum glucocorticoid treatment for prevention of the respiratory distress syndrome in premature infants. Pediatrics, 1972; 50:515-25. 177. Muglia LJ, Bae DS, Brown TT etal. Proliferation and differentiation defects during lung development in corticotropin-releasing hormone-defcient mice. Am. J. Respir. Cell Mol. Biol. 1995; 20:181-8. 178. Cole TJ, Blendy JA, Monaghan AP et al. Targeted disruption of the glucocorticoid receptor gene blocks adrenergic chromaffin cell development and severely retards lung maturation. Genes Dev. 1995; 9:1608-21. 179. Massaro D, Teich N, Maxwell Set al. Postnatal development of alveoli: regulation and evidence for a critical period in rats.J. Clin. Invest. 1985; 76:1297-305. 180. Massaro D, Massaro GD. Dexamethasone accelerates postnatal alveolar wall thinning and alters wall composition. Am. J. Physiol. 1986; 251 :R218-24. 181. Jobe AH, Ikegami M. Prevention of bronchopulmonary dysplasia. Curr. Opin. Pediatr. 2001; 13:124-9. 182. Ferrara N. Vascular endothelial growth factor and the regulation of angiogenesis. Recent Progr. Hormone Res. 2000; 55:15-35 (discussion 35-6). 183. Shifren JL, Doldi N, Ferrara N etal. In the human fetus, vascular endothelial growth factor is expressed in epithelial cells and myocytes, but not vascular endothelium: implications for mode of action. J. Clin. Endocrinol. Metabol. 1994; 79:316-22. 184. Maniscalco WM, Watkins RH, Finkelstein JN etal. Vascular endothelial growth factor mRNA increases in alveolar epithelial cells during recovery from oxygen injury. Am. J. Respir. Cell Mol. Biol. 1995; 13:377-86. 185. Brown KR, England KM, Goss KL etal. VEGF induces airway epithelial cell proliferation in human fetal lung in vitro.Am. J. Physiol. 2001; 281:L1001-10. 186. Coalson JJ, Winter VT, Siler-Khodr T et al. Neonatal chronic lung disease in extremely immature baboons. Am. J. Respir. Crit. Care Med. 1999; 160:1333-46. 187. Bhatt AJ, Pryhuber GS, Huyck H et al. Disrupted pulmonary vasculature and decreased vascular endothelial growth factor, Fit-l, and TIE-2 in human infants dying with bronchopulmonary dysplasia. Am. J. Respir. Crit. Care Med. 2001; 164:1971-80. 188. Lassus P, Turanlahti M, Heikkila Pet al. Pulmonary vascular endothelial growth factor and Flt-1 in fetuses, in acute and chronic lung disease, and in persistent pulmonary hypertension of the newborn. Am. J. Respir. Crit. Care Med. 164:1981-7. 189. Zeng X, Weft SE, Federici R et al. VEGF enhances pulmonary vasculogenesis and disrupts lung morphogenesis in vivo. Dev. Dyn. 1998; 211:215-27. 190. Ng YS, Rohan R, Sunday ME et al. Differential expression of VEGF isoforms in mouse during development and in the adult. Dev. Dyn. 2001; 220:112-21. 191. Betsholtz C, Karlsson L, Lindahl P. Developmental roles of platelet-derived growth factors. Bioessays 2001; 23:494-507.

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Development of the Pulmonary Basement Membrane Zone

Chapter

5

Michael J. Evans* Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, Center for Comparative Respiratory Biology and Medicine, University of California, Davis, CA, USA

Philip L. Sannes ) ~Department ofMolecular Biomedical Sciences, College of Veterinary :~ / :Medicine,~North Carolina State University, Raleigh, NC, U S A i~ii

i

i

STRUCTURE AND COMPOSITION OF THE B A S E M E N T M E M B R A N E Z O N E The basement membrane zone (BMZ) is a mat-like sheet of specialized extracellular matrix (ECM) that serves as a complex interface between epithelia, peripheral nerves or muscle cells and their surrounding tissue microenvironments. In a recent review, the morphology and composition of the BMZ and its functional significance in pulmonary development were presented. 1 In this chapter, we will discuss the structure, composition and development of the BMZ. The term basement membrane was originally derived from observations of tissues with light microscopy. In these preparations, the epithelial and endothelial basement membrane appears as a distinct layer beneath the cells. With transmission electron microscopy, the basement membrane appears as threecomponent layers: the lamina lucida, the lamina densa and the lamina reticularis. Together they make up the basal lamina. When studying the molecular structure of the basal lamina (BL) it is commonly referred to as the BMZ. In the lung, epithelial and endothelial cells, smooth muscle cells and nerves have a BMZ. In smooth muscle cells and nerves there is no basal region and the BMZ around these cells are referred to as the external lamina. However, both the BMZ and external lamina are the same functionally and molecu*To whom correspondence should be addressed. The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

larly and we will use the term BMZ to include the external lamina of nerves and smooth muscle cells. 2 The lamina lucida seen with transmission electron microscopy is a clear area between the cells and the lamina densa. This is thought to be a fixation artifact morphologically, but it appears to actually function as the region of attachment between adjacent cells and lamina densa. It contains cell adhesion molecules and anchoring filaments of laminins 5, 6 and 10. 3 The lamina densa is a sheet of connective tissue made up of type IV collagen, laminin, entactin/nidogen and proteoglycans. This region of the BMZ has been studied extensively and is commonly referred to as the basal lamina, basement membrane or true basement membrane. It is a biologically conserved structure that is virtually the same in all animals. On the ECM side of the lamina densa is the lamina reticularis. This is a region of attachment between the lamina densa and the ECM. 4-6 The lamina reticularis is variable in its distribution, thickness and composition. It is not apparent in all tissues; however, it is well developed under multilayered epithelium. The lamina reticularis is especially pronounced under the respiratory epithelium of large conducting airways, where it may be several microns thick. In the lung the lamina reticularis is thicker in the larger airways and becomes smaller as it extends into the small airways and alveoli. In alveoli, with fused BMZ, it is not physically present but still exists as a region of interaction of the lamina densa with the ECM. Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

~The L u n g - D e v e l o p m e n t , Aging and the E n v i r o n m e n t ]

Table 5.1.

.

:

Characteristics

.

:

of the basement

!/(i~i:i~ii~il~i!i 84184 Basement membrane i~i~(lightlmicroscopy)

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.

.

: ....

.

]

.

.

.

.

.

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;

membrane. ~

i

i

~

~

~

~

~ i8484 ~ ~i]i84184 i i )~i

Basal lamina ~ Basement membrane izone~ (electron microscopy) ~ ~(molecular structure)

~~

i-!,, Cellular interface

Lamina lucida

Collagen (XVll) Laminin (5, 6 and 10) Integrins (~6, IM, a, [3)

Lamina densa

Collagen (IV) Laminin (1) Entacti n/nidogen Proteoglycans (perlecan, bamacan, agrin, collagen XVlII) Stored growth factors (FGF-2)

Lamina reticularis

Collagen (I, III, V, Vl & VII) Proteoglycans (perlecan, bamacan, collagen XVlII) Stored growth factors (FGF-2)

Cellular-matrix Interface

Basement membrane

Matrix interface

The reason for the differences in the thickness of the lamina reticularis is not known. With transmission electron microscopy, the lamina reticularis is made up of numerous collagen fibrils. Immunohistochemical studies have shown that the collagen fibrils consist of types I, III, V, VI and VII collagen. 2 Collagen types I, III and V form heterogeneous fibers that account for the thickness of the lamina reticularis. Anchoring fibrils of type VII collagen loop through strands of collagen fibers in the lamina reticularis and then reattach to the lamina densa. 7 In addition proteoglycans are considered to be a structural component of the lamina reticularis as in the lamina densa. There are three proteoglycans that are considered to be an integral component of both the lamina densa and lamina reticularis. These proteoglycans (perlecan, agrin and bamacan) are specifically classified as BMZ proteoglycans. 8 In addition type XVIII collagen has recently been shown to be a BMZ heparan sulfate proteoglycan 9 and type XV collagen has been shown to be chondroitin/dermatin sulfate BMZ proteoglycan. 1~ In a study of airway whole mounts, the fiat surface of the BMZ was visualized with scanning electron microscopy and fluorescent microscopy. The lamina lucida is not visible in these preparations; however, when the epithelium is removed the lamina densa appears as a smooth, dense layer. 11 Fluorescent microscopy and scanning electron microscopy revealed that the structural proteins of the lamina reticularis are not randomly arranged, but instead appear as a mat of large fibers oriented along the longitudinal axis of the airway. Smaller fibers are cross-linked with the larger fibers to complete this structure. 12 Other small fibers are oriented around the large

fibers and an amorphous material covered individual fibers. The large fibers oriented along the longitudinal axis of the airway are consistent with prior descriptions of fibers comprised of collagen types I, III and V with some small fibers encircling the large fibers that may be collagen type VI. The cross-linking fibers are made up of elastin and probably elastin-associated microfibrils. 12'13 This study demonstrates that the structural proteins of the lamina reticularis are arranged as fibers in a highly organized manner comparable to type IV collagen and laminin in the lamina densa. A break down of the structure and composition of the BMZ is given in Table 5.1.

FUNCTIONS MEMBRANE

OF THE ZONE

BASEMENT

The BMZ has several functions. It serves as a barrier, binds growth factors, hormones and ions, and is involved with cellular adhesion, electrical charge and cell-cell and cellmatrix communication. 1'2'4'5 The lamina lucida is the interface between a layer of cells and the lamina densa. It exists as a zone of molecules attaching the layer of cells to the lamina densa. Other than a role in attachment, the molecules in this zone are important in signaling between the matrix and the adjacent layer of cells. As mentioned previously, the lamina densa is a sheet of connective tissue made up of type IV collagens, laminins, entactin/nidogen and proteoglycans. One of the main functions of the lamina densa is to provide separation between cells and the ECM. It also provides a surface for cells to migrate

Development of the Basement Membrane

and differentiate into distinct phenotypes, 1'14 which helps to determine tissue shape, stability and architecture. The lamina densa also influences important cellular activities, including adhesion spreading polarization locomotion, chemotaxis and proliferation. These functions are accomplished through binding of the cells to specific components of the lamina densa, namely collagen type IV, laminins, entactin/nidogen and proteoglycans. The interface between the lamina densa and the ECM is the lamina reticularis. It is comprised mainly of types I, III and V collagen fibers that are arranged as a mat of large fibers oriented along the longitudinal axis of the airway. Smaller fibers are cross-linked with the larger fibers to complete this structure. 12 Within and around the collagen fibrils are BMZ proteoglycans (perlecan, agrin, bamacan, type XVIII collagen and type XV collagen). Both the lamina densa and lamina reticularis store growth factors, hormones and ions, and are involved with cellular adhesion, electrical charge and cell-cell communication. The predominant BMZ proteoglycan is perlecan and the predominant stored growth factor is basic fibroblast growth factor (FGF-2). 8 FGF-2 is a ubiquitous multifunctional growth factor that plays roles during development and as a regulator of growth and differentiation in adult tissues. 15 FGF-2 is stored in the BMZ through binding with perlecan. Perlecan is a heparan sulfate proteoglycan that is an intrinsic constituent of the BMZ. FGF-2 can be released from perlecan in response to various conditions and become a signaling molecule. Thus perlecan functions as a regulator of FGF-2 transport allowing for rapid responses to local environmental conditions. 16 In the lung, FGF-2 is stored in the BMZ of airway epithelium, alveolar epithelium and endothelium of developing and adult rats. x7'18 Fibroblast growth factor receptor-1 (FGFR-1) is the primary receptor for FGF-2.17 Agrin is also a heparan sulfate proteoglycan that is most prominent in neuromuscular and glomerular basement membranes. It has also been reported in the alveolar region of the lung. 19'2~BMZs from these three tissues are similar in that they are fused. The function of agrin in the alveoli is not known. It binds to ~-dystroglycan and thus anchors the BMZ to alveolar cells and may be involved with signal transduction. It may also be involved with charge, selective filtration processes, and storage of various growth factors. It has also been related to inhibition of proteases. Agrin is not present in the BMZ of airway epithelium or smooth muscle cells. The heparan sulfate proteoglycan, type XVIII collagen, is found in alveolar epithelial and endothelial BMZs and in airway epithelial BMZs. It was not reported in smooth muscle BMZs. 21 The purpose of type XVIII in the lung is not clear. Bamacan is a chondroitin sulfate proteoglycan. Its function is also not known; however, its late appearance developmentally suggests a role in BMZ stability. 22 The recently described chondroitin sulfate proteoglycan, type XV collagen, is not found in lung BMZs. 21 The functional consequences of these collective molecules are profound by forming the basis for all cell-cell, cell growth factor interactions. Their composition defines epithelial differentiation and plasticity. 23'24

BMZ D E V E L O P M E N T The lamina densa region of the BMZ is composed of collagen IV, laminins, proteoglycans and entactin/nidogen. Collagen IV, laminin and entactin/nidogen are expressed with BMZs at early stages of lung development and persist throughout life. Entactin/nidogen is thought to play a crucial role in BMZ formation due to its ability to form complexes with type IV collagen, laminins and perlecan. Both collagen IV and laminins spontaneously polymerize into separate twodimensional networks. BMZs are thought to form by linking these two networks together with entactin/nidogen. 25 The BMZ proteoglycans are incorporated into this structure through binding with collagen IV, laminin and entactin/ nidogen. Biological diversity is maintained through different isoforms of the molecules making up the lamina densa. For example, there are six tx-chains of collagen type IV and at least three molecular forms found in various BMZs. One molecular form of collagen type IV contains the t~3, ~4 and ct5 chains and is found in the BMZ of neuromuscular, glomerular and pulmonary alveolar tissues. These three tissues are structurally similar in that they contain adjacent BMZs that appear to be fused. 26 Most other BMZs express collagen type IV ~1 and t~2 chains. 26-28 Laminins also contribute to the diversity of the lamina densa. They are a family of heterotrimeric glycoproteins containing an t~-, [3- and y-chain. Currently five t~-, three [3-, and three y-chains have been described that make up 15 laminin isoforms. Laminin-t~l and-~2 are present in fetal lungs and laminin-3, -t~, -4, t~ and -tx5 in both fetal and adult lungs. The three laminin [3-chains and laminin-y1 and -y2 are found in both fetal and adult lungs. 29-31 Recently, an isoform of entactin/nidogen was discovered and named entactin-2/nidogen-2. 32 The different BMZ proteoglycans (perlecan, agrin and bamacan, type XVIII collagen and type XV collagen) and their degree of sulfation represent diversity of the proteoglycan component of both the lamina densa and the lamina reticularis. The degree of sulfation was recently shown to be an important determinant of cell function in the lung. 33 The degree of sulfation is known to vary beneath cells lining the alveoli and different levels of the tracheo-bronchial tree. It also varies between the various regions of the BMZ. The highest levels of sulfation are found in the lamina reticularis of the trachea, bronchi and large bronchioles and the lowest levels in the lamina densa. 34 In the rat, chondroitin sulfate proteoglycans are widely distributed in the lung as early as 14 days before birth. 1 Their expression in the BMZ remains strong until day 7 after birth, after which their localization progressively becomes highly focal and limited to airways and septal regions of alveolar ducts. They are absent from the remaining BMZ regions of alveolar structures. Heparan sulfate proteoglycans are strongly expressed in these same regions from birth to adulthood. 22 Of the heparan sulfate proteoglycans, perlecan has been studied most extensively. In the

The:

Lung::Development, :

Aging

and:the::: Environment:::::

. . .: . : . :. . . . :. .

rat, perlecan mRNA is first detected in the fetus (day 19), and is abundantly expressed for the remainder of fetal and postnatal growth. It remains expressed at low levels throughout adult life. 35 Whereas the lamina densa is present at all stages of development, the lamina reticularis in rats develops postnatally. 36 Instead of collagen IV, collagens I, III and V are major components of the fibers making up the lamina reticularis. Collagen I is not expressed with BMZs during fetal lung development. Collagen III expression is light and discontinuous in epithelial BMZ. 37 Although collagens I and III are not expressed during the early stages of fetal development, collagen type V is expressed at the early stages. 37 Collagen type V is associated with determining the diameter of collagen fibrils and its early appearance indicates an important role in fiber formation. The above observations are in agreement with findings of Mariani et al. 3s who showed that, in the mouse, -- 11,000 genes are expressed throughout the morphological stages of lung development. Of these genes, they focused on a subset of ECM genes associated with development of the BMZ (collagen types III and IV) and described their pattern of expression. They found that collagen IV was expressed at early stages of lung development and collagen Ill not until much later. Cells responsible for synthesizing BMZ collagens, proteoglycans, laminins and entactin/nidogen are the fibroblasts beneath the BMZ and the epithelial and smooth muscles on the other side of the BMZ. 39-41 It has been shown that F G F signaling is required for BMZ formation. 42 Also important in development of the BMZ is tissue transglutaminase. It covalently and irreversibly cross-links ECM proteins. It is hypothesized that tissue transglutaminase prevents or delays remodeling of the BMZ and stabilizes other extracellular components during development. 43

SUMMARY In summary, development of the BMZ involves continuous growth of the lamina densa as the tissue grows. The type IV collagen and laminin are thought to spontaneously polymerize into networks that are joined together by entactin/ nidogen. Proteoglycans are then added to this structure. This is an ongoing process and the cells involved are the adjacent epithelial or smooth muscle cells and the fibroblast layer on the other side of the BMZ. The lamina reticularis is formed postnatally, probably by the same cell types as the lamina densa; however, in contrast to the lamina densa, it does not stay at the same width, but increases in width. The reason for the variable width of the lamina reticularis is not known. Understanding this process and its significance may be very important in answering questions about diseases associated with thickening of the BMZ such as asthma. However, very little research has been done on the lamina reticularis. Other important questions to be answered concern (1) the significance of the various isoforms of collagen and

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laminin in lung function, (2) the significance of proteoglycan sulfation in cellular control, (3) sequestering of growth factors in the BMZ, and (4) changes in BMZ composition during morphogenesis. Future research on the BMZs of the lung will be crucial in developing a better understanding of these processes.

ACKNOWLEDGEMENT Supported by NIH grants ES00628, ES04311, ES06700, ES05707, HL44497.

REFERENCES 1. Sannes PL, Wang J. Basement membranes and pulmonary development. Exp. Lung Res. 1997; 23:101-8. 2. Merker HJ. Morphology of the basement membrane. Microsc. Res. Tech. 1994; 28:95-124. 3. Aumailley M, Rousselle P. Laminins of the dermo-epidermal junction. Matrix Biol. 1999; 18:19-28. 4. Adachi EHI, Hayashi T. Basement-membrane stromal relationships: interactions between collagen fibrils and the lamina densa. Inter. Rev. Cytol. 1997; 173:73-156. 5. Erickson AC, Couchman JR. Still more complexity in mammalian basement membranes. J. Histochem. Cytochem. 2000; 48:1291-306. 6. Yurchenco PD, O'Rear JJ. Basal lamina assembly. Curt. Opin. Cell Biol. 1994; 6:674-81. 7. Nievers MG, Schaapveld RQ, Sonnenberg A. Biology and function ofhemidesmosomes. Matrix Biol. 1999; 18:5-17. 8. Iozzo RV. Matrix proteoglycans: from molecular design to cellular function. Annu. Rev. Biochem. 1998; 67:609-52. 9. Halfter W, Dong S, Schurer Bet al. Collagen XVIII is a basement membrane heparan sulfate proteoglycan. J. Biol. Chem. 1998; 273:25404-12. 10. Li D, Clark CC, Myers JC. Basement membrane zone type XV collagen is a disulfide-bonded chondroitin sulfate proteoglycan in human tissues and cultured cells. J. Biol. Chem. 2000; 275:22339-47. 11. Evans MJ, Burke AS, Cox RA et al. In situ preparation of rat tracheal basal cells. Tissue Cell 1993; 25:639-44. 12. Evans MJ, Van Winkle LS, Fanucchi MV et al. Three-dimensional organization of the lamina reticularis in the rat tracheal basement membrane zone. Am. J. Respir. Cell Mol. Biol. 2000; 22:393-7. 13. Kluge M, Mann K, Dziadek Met al. Characterization of a novel calcium-binding 90-kDa glycoprotein (BM-90) shared by basement membranes and serum. Eur. J. Biochem. 1990; 193:651-9. 14. Yang Y, Palmer KC, Relan N et al. Role of laminin polymerization at the epithelial-mesenchymal interface in bronchial myogenesis. Development 1998; 125:2621-9. 15. Bikfalvi A, Klein S, Pintucci G etal. Biological roles of fibroblast growth factor-2. Endocrinol. Rev. 1997; 18:26-45. 16. Dowd CJ, Cooney CL, Nugent MA. Heparan sulfate mediates bFGF transport through basement membrane by diffusion with rapid reversible binding. J. Biol. Chem. 1999; 274:5236-44. 17. Powell PP, Wang CC, Horinouchi H et al. Differential expression of fibroblast growth factor receptors 1 to 4 and ligand genes in late fetal and early postnatal rat lung. Am. J. Respir. Cell Mol. Biol. 1998; 19:563-72. 18. Sannes PL, Burch KK, Khosla J. Immunohistochemical localization of epidermal growth factor and acidic and basic

Development of the Basement Membrane

fibroblast growth factors in postnatal developing and adult rat lungs.Am.J. Respir. Cell Mol. Biol. 1992; 7:230-7. 19. Groffen AJ, Buskens CA, van Kuppevelt TH etal. Primary structure and high expression of human agrin in basement membranes of adult lung and kidney. Eur. J. Biochem. 1998; 254:123-8. 20. Groffen AJ, Ruegg MA, Dijkman H etal. Agrin is a major heparan sulfate proteoglycan in the human glomerular basement membrane.J. Histochem. Cytochem. 1998; 46:19-27. 21. Tomono Y, Naito I, Ando K. Epitope-defined monoclonal antibodies against multiplexin collagens demonstrate that type XV and XVIII collagens are expressed in specialized basement membranes. Cell Struct. Funct. 2002; 27:9-20. 22. Sannes PL, Burch KK, Khosla J e t al. Immunohistochemical localization of chondroitin sulfate, chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, entactin, and laminin in basement membranes of postnatal developing and adult rat lungs. Am. J. Respir. Cell Mol. Biol. 1993; 8:245-51. 23. Shannon JM, Nielsen LD, Gebb SA et al. Mesenchyme specifies epithelial differentiation in reciprocal recombinants of embryonic lung and trachea. Dev. Dyn. 1998; 212:482-94. 24. Demayo F, Minoo P, Plopper CG etal. Mesenchymal-epithelial interactions in lung development and repair: are modeling and remodeling the same process? Am. J. Physiol. Lung Cell Mol. Physiol. 2002; 283 :L510-17. 25. Yurchenco PD, O'Rear JJ. Basement membrane assembly. Meth. Enzymol. 1994; 245:489-518. 26. Sanes JR, Engvall E, Butkowski R etal. Molecular heterogeneity of basal laminae: isoforms oflaminin and collagen IV at the neuromuscular junction and elsewhere. J. Cell Biol. 1990; 111:1685-99. 27. Sado Y, Kagawa M, Naito I et al. Organization and expression of basement membrane collagen IV genes and their roles in human disorders. J. Biochem. (Tokyo) 1998; 123:767-76. 28. Hudson BG, Reeders ST, Tryggvason K. Type IV collagen: structure, gene organization, and role in human diseases: molecular basis of Goodpasture and Alport syndromes and diffuse leiomyomatosis. J. Biol. Chem. 1993; 268:26033-6. 29. Miner JH, Patton BL, Lentz SI etal. The laminin alpha chains: expression, developmental transitions, and chromosomal locations of alphal-5, identification of heterotrimeric laminins 8-11, and cloning of a novel alpha3 isoform. J. Cell Biol. 1997; 137:685-701. 30. Pierce RA, Griffin GL, Mudd MS et al. Expression of laminin alpha3, alpha4, and alpha5 chains by alveolar epithelial

31. 32. 33. 34. 35.

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39. 40. 41. 42.

43.

cells and fibroblasts. Am. J. Respir. Cell Mol. Biol. 1998; 19:237-44. Pierce RA, Griffin GL, Miner JH et al. Expression patterns of laminin alphal and alpha5 in human lung during development.Am. J. Respir. Cell Mol. Biol. 2000; 23:742-7. Kohfeldt E, Sasaki T, Gohring Wet al. Nidogen-2: a new basement membrane protein with diverse binding properties. J. Mol. Biol. 1998; 282:99-109. Sannes PL, Khosla J, Peters BP. Biosynthesis of sulfated extracellular matrices by alveolar type II cells increases with time in culture.Am. J. Physiol. 1997; 273:L840-7. Khosla J, Correa MT, Sannes PL. Heterogeneity of sulfated microdomains within basement membranes of pulmonary airway epithelium. Am. J. Respir. Cell Mol. Biol. 1994; 10:462-9. Belknap JK, Weiser-Evans MC, Grieshaber SS etal. Relationship between perlecan and tropoelastin gene expression and cell replication in the developing rat pulmonary vasculature. Am. J. Respir. Cell Mol. Biol. 1999; 20:24-34. Evans MJ, Fanucchi MV, Van Winkle LS etal. Fibroblast growth factor-2 during postnatal development of the tracheal basement membrane zone. Am. J. Physiol. Lung Cell Mol. Physiol. 2002; 283:L1263-70. Wright C, Strauss S, Toole K etal. Composition of the pulmonary interstitium during normal development of the human fetus. Pediatr. Dev. Pathol. 1999; 2:424-31. Mariani TJ, Reed JJ, Shapiro SD. Expression profiling of the developing mouse lung: insights into the establishment of the extracellular matrix. Am. J. Respir. Cell Mol. Biol. 2002; 26:541-8. Evans MJ, Guha SC, Cox RA etal. Attenuated fibroblast sheath around the basement membrane zone in the trachea. Am. J. Respir. Cell Mol. Biol. 1993; 8:188-92. Evans MJ, Van Winkle LS, Fanucchi MV et al. The attenuated fibroblast sheath of the respiratory tract epithelial-mesenchymal trophic unit.Am. J. Respir. Cell Mol. Biol. 1999; 21:655-7. Holgate ST, Davies DE, Lackie PM etal. Epithelialmesenchymal interactions in the pathogenesis of asthma. J. Allergy Clin. Immunol. 2000; 105:193-204. Li X, Chen Y, Scheele S etal. Fibroblast growth factor signaling and basement membrane assembly are connected during epithelial morphogenesis of the embryoid body. J. Cell Biol. 2001; 153:811-22. Schittny JC, Paulsson M, Vallan C et al. Protein cross-linking mediated by tissue transglutaminase correlates with the maturation of extracellular matrices during lung development.Am. J. Respir. Cell Mol. Biol. 1997; 17:334-43,

Chapter

Development of the Pulmonary Vasculature

6

Rosemary Jones* Harvard Medical School and Department of Anesthesia and Critical Care, Massachusetts General Hospital, Boston, MA, USA

Lynne M. Reid Department of Pathology, Harvard Medical School, Children's Hospital, Boston, MA, USA

INTRODUCTION

The vascular units of the lung form in close coordination with airways and alveoli. While many of the genes initiating lung morphogenesis, the determination of left-right asymmetry and laterality, and the regulation of airway branching have been identified, 1-s less is known about genes regulating vascularization. Emerging data indicate a role for positive and negative factors, and that the formation of pulmonary vascular units may direct epithelial morphogenesis. 2-4 As our understanding of the role of 'instructive' and 'permissive' genes in the developing lung increases (genomics), so will our need to understand the modulation of cell phenotype by correctly and incorrectly assembled proteins (proteomics). Lung vascular growth is achieved by expansion of existing structures and by change in existing templates. As vascular networks increase in size and three-dimensional complexity, growth is accompanied by regression of unneeded units until these are appropriate to the stage of lung development. In this way, vascular systems formed in utero, in the postnatal period, and in early childhood, develop, enlarge and are remodeled; in late childhood and in the young adult, they continue to enlarge and remodel until thoracic growth i s complete. Lung vessels develop by vasculogenesis and by angiogenesis, the latter including sprout formation, splitting or intusussceptive (i.e. of-itself) microvascular growth, and simple expansion. The addition of mural cells provides wall support and as the developing units increase in size, vessels form. *To whom correspondence should be addressed. The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

The growth of the small vessels from capillaries eventually gives rise to all large vessels within the lung. These mechanisms combine to form the lung's pulmonary and bronchial arterial systems, and double venous systems. Little is yet understood of the way spatial and fractal (tree-like) dimensions of developing units are determined within the confines of mesenchymal or connective tissue spaces, although familial patterns of branching in the human lung indicate that, at least for central vessels, there is a genetic component. 6 The pulmonary arteries supply capillaries in the intraacinar region and pleura (except at the hilum) and drain to pulmonary veins. While arteries run centrally, the veins are distributed at the periphery of lung units, the most distal lying at the edge of acini. Venous tributaries arise from alveolar walls, alveolar ducts, bronchial walls, pleura and connective tissue sheaths, and drain to axial vessels that increase in size towards the hilum. The wall structure of vessels proximal to the acinus reflects their role as conduits of de-oxygenated or oxygenated blood, the structure of the small thin-walled intra-acinar vessels and capillaries of the alveolar-capillary membrane their role as gas-exchanging vessels. The bronchial arteries supply the airway mucosa and peri-hilar structures; they divide with the bronchi sending one submucosal and one peri-bronchial branch along each airway wall to form communicating arcades. 7-9 When extrapulmonary, they drain to pulmonary veins and to the right side of the heart and via the azygos system; when intrapulmonary they form a network that anastomoses with the capillaries of the pulmonary arterial bed and drain via pulmonary veins to the left side of the heart. They provide nutrients to large airway and vascular structures. Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

Between the hilum and the start of the acinus, the simple endothelial channels of the lymphatics lie within connective tissue sheaths. Liquid and plasma proteins move through the matrix to the lymphatic plexus surrounding terminal airways and drain centrally through the lung's lymphatic channels to the systemic circulation. 1~ To maintain adequate function, both at rest and on exercise, each of these interconnecting systems must develop appropriately. This chapter reviews current concepts of the formation of vascular beds in the normal lung at different stages of growth and maturation, comments briefly on aberrant growth that results in a still adequately functioning lung and summarizes the little known of the lung vasculature in aging. Finally, we highlight the presence of signaling systems that, triggered by the ambient oxygen tension, change vascular density in the adult lung.

CELLULAR BASIS OF VESSEL MORPHOGENESIS Currently, a major area of study in developmental biology and in disease, particularly as it relates to new anti-angiogenic therapy in tumor growth, concerns blood vessel formation and regression. Most data relating to intercellular signaling pathways and cell assembly into capillaries and vessels arise from these studies. 12-19The cell-cell interactions and molecular mechanisms of cell survival and loss they reveal, and ones of cell proliferation, migration, recruitment and differentiation, highlight processes likely involved in normal lung vessel growth. Vessel formation first requires endothelial cells to coalesce into a tube, for their contiguous membranes to fuse, and for junctional complexes to form. 2~ Networks of large and small vessels evolve as the initial vascular webs remodel into channels. Further wall formation requires signaling between endothelial cells and mural cell precursors. As the number of wall cells increases, the formation of elastic laminae is critical for their organization into intima (endothelial cells), media (smooth muscle cells, SMCs) and adventitia (fibroblasts) to establish a fully mature vessel wall. As new growth proceeds (by cell proliferation), vascular density is adjusted by release of endothelial and mural cell contacts, and, as patterns of blood flow change, by regression (by cell apoptosis or loss) of unperfused channels (vascular pruning). Homotypic and heterotypic contacts between neighboring cells and matrix are important in determining cell phenotype, and in regulating tissue growth and organization, by interaction with growth factors and vaso-mediators 21-2s (see also Chapter 7). Adhesion receptors at the endothelial cell surface (transmembrane glycoproteins that include members of the integrin, cadherin, immunoglobulin, selectin and proteoglycan superfamilies) interact with those of adjacent cells or with matrix proteins such as collagens, fibronectins, laminins and proteoglycans forming fibrils or other macromolecular arrays. 29-31 Cytoplasmic plaque proteins further link the cell membrane receptors to the cytoskeleton, transducing

signals from the cell surface and regulating receptor function. 29-31 Nuclear DNA and its associated protein-scaffold connect to matrix components through the cytoskeleton, contributing to communication between intracellular and extracellular environments. 32

FORMATION AND GROWTH OF E N D O T H E L I A L CHANNELS

Vasculogenesis In vasculogenesis, a primitive vascular network assembles from local aggregates of progenitor cells. 13'33'34Mesenchymal cells migrate and differentiate in situ into angioblasts (i.e. cells committed to an endothelial lineage) or hemangioblasts (blood cell precursors). They form sinusoidal nests of cells and spaces (blood islands). These evolve into capillary-like structures as the angioblasts form primitive vascular networks enclosing blood cells (Fig. 6.1a). The intercellular spaces forming the lumen of the first channels arise by loss of vesicles from the apical membranes of mesenchymal cells. By thinning their processes and reforming of their apical membrane, the mesenchymal cells become endothelial-like cells (Fig. 6.1a). As these channels give rise to branches, further growth is thought to occur by angiogenesis.

Angiogenesis (sprouting) In the most widely recognized form of angiogenesis, pre-existing capillaries and small vessels form capillary-like sprouts (Fig. 6.1b). Sprout formation starts with wall destabilization. Focal degradation of the endothelial basement membrane and surrounding matrix by proteolytic enzymes is followed by the extension of endothelial pseudopodia through the gap to form a spur. Endothelial cell migration in the direction of the growth spur forms a sprout. 35'36The delicate microspikes at the leading edge of migrating endothelial cells forming the tip of the sprout lack basement membrane. Rather than migrating singly, they move as a shifting sheet of cells. 35 Typically, proliferation of cells lying behind the growing tip increases the length of the sprout. 36 Sprouts can develop in the absence of endothelial cell proliferation (e.g. as in inflammation) but an increasing cell population is needed for sustained growth. 36'37While still connected at their origin to a patent vessel or capillary, sprouts continue to elongate and to branch or fuse to an adjacent sprout at their blind-end to form a new loop. Development of a slitlike lumen followed by the entry of plasma and blood cells completes the formation of a continuous channel.

Angiogenesis (intussusceptive microvascular growth/splitting) Elegant studies by Burri and co-workers 38'39 reveal the structural basis of the division of capillary units by intussusceptive microvascular growth. Areas of contact develop between opposing endothelial cell membranes to form inter-endothelial bridges (Fig. 6.1c). These reorganize into endothelial junctions as the central region of the endothelial

Fig. 6.1. (a) Vasculogenesis: mesenchymal cells (Mc, top) differentiate to angioblasts (Ab), i.e., cells committed to an endothelial lineage, and hemangioblasts (Hb), i.e., primitive blood cells. These organize to form blood islands (center). As angioblasts assume an endothelial cell (Ec) phenotype and continue to coalesce and organize into cellular channels, they form primitive capillaries enclosing red blood cells (RBC) (bottom). (Reproduced with permission from Sadler TW. Embryonic period (third to eighth week). In: Gardner JN (ed.), Langman's Medical Embryology, 6th edition, Baltimore, MD: Williams and Wilkins. 1990, pp. 61-84.)(b) Angiogenesis (1)- sprout formation: endothelial cells and their processes, released from the constraint of a basement membrane, and in response to a signal triggering growth, invade the surrounding tissue in the direction of the growth spurt (top) to form cell-lined channels (sprouts). These remain connected to the parent channel at their origin. Sprout formation is followed or accompanied by the development of a lumen and the connection of adjacent sprouts to establish a contiguous network (bottom). (Reproduced with permission from Schoefl GI. Electron microscopic observations on the regeneration of blood vessels after injury. Ann. NY Acad. Sci. 1964; 116:789-802.) (c) Angiogenesis (2)- intussusceptive microvascular growth: diagram illustrating structural events, based on morphological data obtained from serial sections and electron microscopy. Opposing endothelial cells form a capillary inter-endothelial bridge and the area of contact is sealed and organized into interendothelial junctions (top left and right). The endothelium then thins centrally (bottom left, arrows) and gives way (bottom right, open arrows) to invading components of the interstitium (bottom right, arrow) which divide the capillary channel. (Reproduced with permission from Burri PH, Tarek MR. A novel mechanism of capillary growth in the rat pulmonary microcirculation. Anat. Rec. 1990; 228:35-45.) (d) Angiogenesis (3) - expansion: growth of an existing vascular bed occurs, by the addition of wall cells and increase in lumen diameter and segment length. (Reproduced with permission from Ref.35)

layer is perforated by an invading connective tissue post (1-2.5 ~tm in diameter). The post is subsequently stabilized by the inclusion of myofibroblast and pericyte processes and by the formation of collagen fibrils. 39 With growth, posts evolve into pillars (--2.5 l.tm in diameter) which result in the formation of a mature mesh.

Angiogenesis (expansion) Slow expansion of a formed vascular network by increase in the diameter or length of existing units is achieved by endothelial and mural cell proliferation in the absence, or in excess, of cell loss (Fig. 6.1d). 35

DEVELOPMENT OF M U R A L CELLS Origin and role of mural cells Mesenchymal cells are recruited to an endothelial channel as peri-endothelial cells (pericytes and SMCs), stabilizing the new structures, and with adventitial fibroblasts, regulating its response to vaso-mediators. 15-19'4~ In capillary and microvessel walls, particularly in post-capillary venules, peri-endothelial cells (Fig. 6.2) develop into pericytes. 43-45 In arterioles adjacent to capillaries, they develop into intermediate cells with a phenotype midway between a pericyte and SMC. 44-46 Pericytes and endothelial cells have different l i n e a g e s - pericytes develop from cells derived from the mesoectoderm, endothelial cells from the mesodermal lateral plate. 47 In vessels, where transmural pressure is higher than in capillaries, the peri-endothelial cells express a SMC phenotype. Emerging data on the origin of these cells 48-5~ indicate that embryonic endothelial cells can become mesenchymal cells that express smooth muscle proteins and so are also a possible source of these cells. 51 Adventitial fibroblasts align circumferentially to form the outermost layer of vessel walls in which SMCs develop. Their number and that of the SMCs determine oxygen diffusion, which is restricted once the perivascular tissue cuff is 100 ~tm thick. Evidence that interstitial fibroblasts are recruited as mural cells in mature vascular beds, where they differentiate into pericytes and SMCs, comes from studies of dorsal and mesenteric capillaries 44'52'53 and vascular remodeling in lung. 54-58

Fig. 6.2. Development of perivascular cells around capillary sprouts (based on intravital video recordings and electron micrographs of the mesenteric microcirculation of young rats). Small endothelial extensions or buds (1) penetrate the basal lamina (fine stippling) and evolve into cellular protrusions (2), develop into endothelial spurs and short sprouts (3) and gradually lengthen into full sprouts (4). Fibroblasts settle down on the adluminal walls of sprouts and transform into pericytes by sharing the endothelial cell basement membrane. Loss of plasma, blood cells and platelets into the interstitial tissue during sprout formation is prevented by pericytes temporarily assuming an umbrella shape. Endothelial cells can contribute to the length of forming sprouts (regardless of proliferation) by organelle streaming and re-organization of their cytoplasm into long extensions. (Reproduced with permission from Rhodin JAG, Fujita H. Capillary growth in the mesentery of normal young rats: intravital video and electron microscope analyses. J. Submicrosc. Cytol. Pathol. 1989; 21:1-34.)

Smooth muscle cells and elastic laminae Pericytes Unlike SMCs, pericytes share the endothelial basement membrane and lack extensive filament networks, dense bodies or attachment plaques. 44'45'53'59Their processes often extend between membrane leaflets to contact the endothelial cell, and rare gap junctions allow nucleotides to pass between the cells.53'59 During sprout formation, pericytes prevent plasma from escaping into interstitial tissue (Fig. 6.2). Possessing cyclic GMP-dependent kinase, actin, desmin, vimentin, u-tropomyosin and myosin, pericytes contract or relax in response to vaso-mediators. 6~Their ability to express proteins typical of contractile smooth muscle such as smooth muscle myosin heavy chain (SM-MHC), u-smooth muscle actin (u-SMA) or desmin (see below) varies greatly within a vascular bed. 61-63

The network of contractile and cytoskeletal filaments occupying the cytoplasm of differentiated SMCs (Fig. 6.3a,b) confers tensile strength and the ability to contract. 64'65 In larger vessels small bundles of collagen and collagen fibrils are present between cells. In all but the smallest venules, SMCs are surrounded by basement membrane and characterized by extensive filaments, fusiform dense bodies and attachment plaques (Fig. 6.3). To proliferate or migrate, they readily de-differentiate from a 'contractile' to a 'synthetic' phenotype by disassembling this network, and can revert to a contractile phenotype by its re-assembly. The proteins required for this (see Fig. 6.3) appear in sequence in the cells of developing vessels. 41'66-73 Thus u-SMA expression is followed by expression of calponin, h-caldesmon, u-tropomyosin and

Fig. 6.3. (a) Arrangement of the SMC contractile and cytoskeletal filament lattice (top) and organization of its structural components (bottom). Oblique, face-polar, smooth muscle (SM)-myosin filaments (14-16 nm diameter) cross-bridge to c~-SM-actin filaments (4-6 nm) and anchor to the cytoskeleton at dense bodies - ovoid structures consisting of ~-actinin and [3-actin - to form the contractile apparatus. Whether these attach to the cell membrane is not established. Longitudinal intermediate filaments (7-11 nm) composed of desmin (a SMC specific protein) or vimentin, and a cytoplasmic domain of [3-actin and filamin (an actin cross-linking protein), form the cell cytoskeleton. These filaments also anchor to the contractile apparatus at dense bodies - linking it to the cell's supporting structure to give the cell tensile strength; they also link the contractile apparatus to the plasmalemmal membrane and to elastic components of the extracellular matrix via peripherally located attachment plaques, i.e., submembranous structures (0.2-0.5 nm) containing o~-actinin, filamin, metavinculin or vinculin, which anchor at the cell membrane via proteins such as plectin. Other intermediate filaments traverse the network to provide further support by anchoring to the dense bodies and attachment plaques. Attachment plaques are separated by membrane regions rich in caveolae and characterized by transmembrane receptors or integrins that link components of the cytoskeleton to the extracellular matrix. Dense bodies and attachment plaques are considered the functional equivalent of Z-bands in striated muscle. (Adapted from Small JV, North AJ. Architecture of the smooth muscle cell. In: Schwartz SM, Mecham RP (eds), The Vascular Smooth Muscle Cell. Molecular and Biological Responses to the Extracellular Matrix. San Diego, CA: Academic Press. 1995, pp. 169-88 with permission.) (b) Example of o~-SMA immunoreactive sites identified by 10 nm gold particles decorating the filaments of a SMC developing in the wall of an alveolar vessel (31 lLtm in diameter). Unicryl section (80 nm) of rat lung after 28 days at FiO 2 0.87, using the protein A-gold technique with a monoclonal antibody to c~-SMA (1:400 dilution, Sigma) followed by uranyl acetate and lead citrate staining. The cell lies surrounded by matrix with the lumen and endothelium to the left. Filaments typically develop along the adluminal cell margin. The gold particles and filaments (at arrowhead) are shown at higher magnification in the inset. Bars = 1 and 0.1 ~tm.

metavinculin, while the SM-MHC SM1 isoform appears in immature cells during the fetal period and the SM-MHC SM2 isoform, metavinculin and ~-tropomyosin appear in differentiated cells after birth. Molecular screening techniques are beginning to identify developmentally regulated genes expressed by immature cells. 74 The number of SMCs is normally proportional to blood flow and transmural pressure. 75 The exception is in small 'resistance' vessels where wall thickness is normally high for lumen size. Their cell processes penetrate their surrounding basal lamina to

form lateral contacts and penetrate the basal lamina of adjacent endothelial cells in junctional complexes (myoendothelial junctions). 44 As vessels approach capillaries, the number of SMCs decreases, endothelial contacts increase, and it is endothelial cell processes that penetrate the basal lamina to contact SMCs. 76 Differentiated SMCs become separated from adjacent endothelial cells and from fibroblasts by internal and external laminae which form along the outer and inner margins of the SMC layer to establish the vessel media. In large vessels

additional laminae further divide the cells into multiple layers; close to capillaries, where SMCs are absent from the wall, a single lamina separates endothelium from surrounding connective tissue. The regulation of smooth muscle growth by elastin is demonstrated by obstructive intimal hyperplasia and death of mice lacking the elastin gene. 77 Each vascular cell type is elastogenic (endothelial cell, SMC and fibroblast) but their relative contributions to lamina formation is unknown. Current understanding of the process of lamina assembly derives from data for other tissue sites. 78-9~ The presence of endostatin (an inhibitor of endothelial cell proliferation) within matrix and elastic laminae of large vessels91 could restrict sprouting from the wall.

Fibroblasts Fibroblasts typically express vimentin, actin isoforms and non-muscle myosin. Under certain conditions, they express two SMC-specific proteins, ot-SMA and desmin, 92 but this is uncommon in normal vessels. Their processes are characterized by microfilaments (4--6nm) and intermediate filaments, pinocytotic vesicles and extensive rough endoplasmic reticulum:

basement membrane is absent, and collagen fibers and fibrils form along the adluminal and abluminal cell margins.

CELL-CELL INTERACTIONS AND SIGNALING

Vessel formation and wall and network remodeling are regulated by paracrine signals between the receptor tyrosine kinases (RTKs) of endothelial and peri-endothelial ceils and growth factors (ligands) (Fig. 6.4). 14-19 Close apposition to endothelial basement membrane triggers cell differentiation, likely in response to endothelial expression of TGF-13, and inhibits endothelial cell movement and proliferation. 17 The ligand-receptor signals required for vascular growth and regression (molecules that appear to modulate rather than induce the interactions required for wall formation), are still being actively analyzed. Ligands of interest include vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and the angiopoietins (Angl and Ang2). Their effect is fine-tuned

Fig. 6.4. A model for regulation of the vascular endothelium as demonstrated by the prototypical angiogenesis factor VEGF and the class of angiogenic regulators, Angl and Ang2. All three ligands bind to RTKs that have similar cytoplasmic signaling domains. Yet their downstream signals elicit distinctive cellular responses. Only VEGF binding to the VEGF-R2 (Flkl) sends a classic proliferative signal. When first activated in embryogenesis, this interaction induces the birth and proliferation of endothelial cells. In contrast, VEGF binding to VEGF-R1 (Fit1) elicits endothelial cell-cell interactions and capillary tube formation, a process that closely follows proliferation and migration of endothelial cells. Angl binding to the receptor tunica interna endothelial cell kinase-2 (Tie2) RTK recruits and likely maintains association of peri-endothelial support cells (pericytes, smooth muscle cells, myocardiocytes), thus solidifying and stabilizing a newly formed blood vessel. Ang2 although highly homologous to Angl, does not activate the Tie2 RTK; rather, it binds and blocks kinase activation in endothelial cells. The Ang2 negative signal causes vessel structures to become loosened, reducing endothelial cell contacts with matrix and disassociating peri-endothelial support cells. This loosening appears to render the endothelial cells more accessible and responsive to the angiogenic inducer VEGF (and likely to other inducers). (Reproduced with permission from Hanahan D. Signaling vascular morphogenesis and maintenance. Science 1997; 277:48-50.)

by the number and sub-type of receptors expressed by the target cell population and by availability of ligand from producer cells. Most data derive from gene inactivation studies; as most deletions are embryonic lethal, however, less is known of their role in vessel formation beyond birth. Other molecules, such as extravasated plasma proteins and inflammatory mediators such as prostaglandins, tumor necrosis factor-m, interleukins (ILs) and nitric oxide (NO), also induce angiogenesis in vir 18'19'93'94 but their role in vascular growth and remodeling in the lung is largely unknown.

VEGF to prevent endothelial cells from detaching and undergoing apoptosis, but during postnatal life endothelial cells become VEGF-independent. How endothelial cells commit to arterial or venous channels within a forming vascular bed is an intriguing question. It appears that their location within a network is determined by expression of the ligand Eprin B2 and of its receptor eph 4 (Fig. 6.5). Arterio-venous relationships are likely established and maintained by the expression of these molecules. 1~176

Endothelial channel formation- VEGF/VEGFR

and Ang/Tie

Growth factors such as the FGF family first induce angioblast and hemangioblast formation in the mesoderm. VEGF is then needed to maintain angioblast differentiation and survival. Alternative splicing produces homodimeric isoforms VEGF121, VEGF165, VEGF189 and VEGF206. Of these VEGF121 and VEGF165 act as survival factors for endothelial ceils of immature vessels. VEGF signals controlling angiogenesis come from adjacent cells, stimulating endothelial cell RTKs that include VEGF R-1 (Flt-1), VEGFR-2 (Flk-1/KDR) and VEGFR-3 (Flt-4). Assembly of angioblasts into vascular channels requires their expression of VEGFR-195-99 and after they differentiate to endothelial ceils they express VEGFR-2. VEGF binding to VEGFR-2 results in proliferation while its binding to VEGFR-1 results in tubule formation and sprouting (see Fig. 6.4). Endothelial cells develop in VEGF null mice but the vessels are malformed. VEGFR-1 null mice show similar changes while VEGFR-2 null mice fail to develop endothelial cells, possibly because hemangioblasts fail to differentiate. VEGF also stabilizes developing vessel walls by accelerating the development of peri-endothelial cells. 1~176176 Immature vessels that lack peri-endothelial ceils need

PDGF isoforms play important roles in both lung vascular and alveolar development. 1~176 While four genes are identified (A, B, C and D), most is known of PDGF-A and PDGF-B in relation to vessel formation. Dimers of two homologous polypeptide PDGF chains, a secreted A-chain and cell-associated B-chain (the c-sis homologue of the v-sis oncogene), dimerize via disulfide bonds to form functional in vivo isoforms (PDGF-AA, -AB or -BB), which in turn dimerize RTKs, PDGFR-cx and PDGFR-[3, on the cell surface. The striking absence of peri-endothelial cells, specifically pericytes, in PDGF-B gene deficient mice results in capillary dilatation, microaneurysms, and vascular leak and hemorrhage; the absence of the PDGFR-]3 produces a similar response. Absence of the PDGF-A gene, on the other hand, results in an inability to form alveolar structures, in part, because SMC progenitors fail to spread. 1~ Both isoforms promote mesenchymal cell proliferation, PDGF-B initiating progression through the cell cycle and PDGF-A (a competence factor) requiring a further signal (e.g. IL-1) to induce mitogenesis. 113 PDGF-B alone induces chemotaxis. 1~ It is suggested that SMCs use PDGF-B to enter the cell cycle,

Mural cell d e v e l o p m e n t - PDGF/PDGFR

Fig. 6.5. Presumed distribution of ephrin-B2 and Eph-B4 - which are defined as a ligand/receptor pair that identify arterial and venous endothelial cells, respectively, in a capillary plexus. 1~ These are presumed to interact between opposing arterial and venous endothelial cells in a "cis" manner (left). During remodeling of the primary plexus (right) by interdigitation, branching and differential growth of vascular segments, they remain localized to arterial and venous units ("cis" interactions) but may also interact at the interdigitating surfaces of large vessels ("trans" interactions). (Reproduced with permission from Yancopoulos GD, Klagsbrun M, Folkman J. Vasculogenesis, angiogenesis and growth factors: ephrins enter the fray at the border. Cell 1998; 93:661-4.)

and/or to suppress differentiation, and to stimulate selfreplication via synthesis of PDGF-A. 11s'116 Actin re-organization and membrane ruffling (essential for cell migration) are induced by PDGF-AB and PDGF-B, and therefore via the PDGF-[3 receptor. PDGF-B also enhances wall stabilization by inducing VEGF expression in peri-endothelial cells. 117 Tiel and Tie2 (Tek) form a second family of RTKs expressed by endothelial cells. Angl, the major physiological ligand for Tie2, is expressed by mesenchymal cells (see Fig. 6.4). As yet no ligand is identified for Tiel, which modulates transcapillary fluid exchange, and Ang2 is a negative Tie2 ligand (see Fig. 6.4). 15-17'118-123 Tie2 expression further modulates VEGF activity and is required for sprout formation. 119'120'124 While Tie2 null mice have normal numbers of endothelial cells, these assemble into immature channels that lack branching networks and the presence of large and small vessels. Angl-deficient mice die with similar vascular defects to Tie2-deficient animals. Angl together with VEGF enhances vascular density, whereas Ang2 and VEGF produce longer sprouts indicating a role for Ang2 in vessel formation in addition to one in vessel regression. TM

EMBRYONIC DEVELOPMENT

AND

FETAL

VASCULAR

Overview" In the human embryo, the formation of vascular networks starts with the onset of organogenesis in the fourth week of gestation. 34 As the lung anlage develops from the foregut, it is vascularized by ingrowth of a vascular plexus derived from the heart. The main pulmonary artery develops from migrating angioblasts. By the sixth week of gestation, the adult pattern of central vascular and airway structures consisting of lobar and segmental branches is present. The pre-acinar vessels develop hand-in-hand with airways while the intra-acinar vessels develop later within distal alveolar structures. 125-135 The precise timing of the

three recognized stages of fetal lung morphogenesis 42'136 the pseudoglandular, the canalicular and the saccular varies with species, as does the degree of distal lung maturation achieved at birth. 137 The main features of vascular beds forming in normal lung are pre-acinar and intra-acinar vessel number, vessel size and wall structure. Pre-acinar vessels run with a bronchus (Br), bronchiolus (B) or terminal bronchiolus (TB), intra-acinar ones with a respiratory bronchiolus (RB) or alveolar duct (AD), or are found within the alveolar wall (AW). The branching pattern and the size of large vessels can be assessed on angiograms lzs and additional details of development obtained from serial reconstruction of vessels in tissue. Where numerous elastic laminae envelop the SMCs, as in the main pulmonary artery and its large branches, these are 'elastic' arteries. More peripherally, as the number of laminae decreases, the arteries are 'transitional'. The arteries become 'muscular' with a wall consisting simply of SMCs between internal and external laminae. Eventually, the muscle layer thins to a few cells and forms a spiral in 'partially muscular' arteries. The wall then consists of endothelium and a single lamina as the muscle layer disappears in 'non-muscular' arteries. By the 20th week, the full number of pre-acinar pulmonary vessels is present in each segment. During fetal life there is an increase in vessel size, length and diameter but no change in the density. 12s-13~ The vessels at the hilum grow faster than at the periphery so that the gradient of diameter against length increases with fetal age.

Early vessel development Overview: Studies of early lung development in both the human and mouse reveal details of the cellular activity in vessel formation. 5~ A branching network evolves by angiogenesis from the central arterial and venous trunks and as these expand in diameter and length offshoots grow by irregular dichotomous branching (Fig. 6.6a-c). Distal

Fig. 6.6. Pulmonary arterial Mercox casts; peripheral vessels steadily increase in density in the lung within the 72-h period illustrated (a-c). (a) 12 day mouse fetus. S=systemic; R =right; L =left; PA= pulmonary artery. (b) 14-day mouse fetus; peripheral vessels steadily increase in density within the 72-h period illustrated. R=right; L=left; PA=pulmonary artery; PV=pulmonary vein; CL=cardiac lobe. (c) 15-day mouse fetus. R = right; L = left; PA = pulmonary artery; PV = pulmonary vein. (Reproduced with permission from deMello DE, Sawyer D, Galvin N etal. Early fetal development of lung vasculature. Am. J. Respir. Cell Mol. Biol. 1997; 16:568-81 .)

vessel and capillary networks develop by vasculogenesis (Fig. 6.7a,b) and then fuse with the proximal networks (see below); 139 they have also been reported to develop by sprouting from the main vascular plexus. 5~ While mesenchymal cells represent an important source of peri-endothelial precursors, 41'1~176 these cells are also reported to develop from bronchial SMCs and endothelial cells. 5~ Despite their different origin, these cells express the same smooth muscle proteins. 5~ Serial sections of human embryos show that blood lakes form first, being present in the primitive mesenchyme surrounding the lung bud at the neck (at 32-44 days). 14~As the first (5-6) airway branches form at --50.5 days, such lakes are abundant in the subpleural mesenchyme. At this stage, the pulmonary artery accompanies airways only to the third or fourth generation. The first connection between developing distal and proximal networks appears between

peripheral lakes and a thin-walled hilar vein, venous drainage thus being established before the pulmonary artery supply. At ---56.5 days, the branching of the pulmonary artery, a thick-walled blind-ended tube, lags behind airway branching by 2-3 generations. At 12-14 weeks, an extensive capillary network surrounds distal airway buds although well separated from them by the subpleural mesenchyme. By 22-23 weeks, the capillary network closely approaches the alveolar epithelium and the pulmonary artery now accompanies each airway branch. Conventional and supernumerary arteries and veins In the human fetal lung, as in the child and adult, the number of arterial and venous branches exceeds that of the airways. 12s-13~ Branches of the main pulmonary artery running alongside airways are termed 'conventional' arteries: numerous additional branches arising from the axial artery

Fig. 6.7. (a) Electron photomicrographs of a 9-day fetal mouse thorax showing (top) densely packed mesenchymal cells containing cytoplasmic vesicles and (bottom) intercellular spaces appearing between mesenchymal cells surrounding the developing lung bud. Intercellular spaces appear to result from the discharge of intra-cytoplasmic vesicles leaving a ruptured cell membrane while mesenchymal cells around intercellular spaces show membrane continuity. Bars= 10 pm (top) and 5 pm (bottom). (Reproduced with permission from deMello DE, Sawyer D, Galvin Net al. Early fetal development of lung vasculature. Am. J. Respir. Cell Mol. Biol. 1997; 16:568-81 .) (b) Electron photomicrographs of a 10-day mouse fetus showing (top) thinned mesenchymal cells which appear endothelial-like hematopoietic precursor cells are present in some spaces (bottom). Bars= 5 pro. (Reproduced with permission from Ref.139)

to the TB, i.e. pre-acinar and passing directly to adjacent respiratory tissue to supply the capillary bed are termed 'supernumerary' arteries (Fig. 6.8). For example, in the posterior basal segment of the left lung of a 19-week fetus 128 (length 16 mm), the 25 bronchial branches present to the level of the TB are accompanied by 21 conventional arteries and 58 supernumerary arteries. The number of airways and conventional artery branches is similar from 18 weeks gestation onwards. In the fetus the veins arise from the saccular respiratory region, pleura, connective tissue septae and airway walls. Conventional and supernumerary veins appear together, developing progressively from hilum to periphery. The axial veins running from the periphery to the hilum receive drainage from both conventional and supernumerary tributaries. From 20 weeks of gestation, the number of pre-acinar conventional veins is within the adult range for intrasegmental airway number. While the number of conventional veins in a segment equals that of the airways and conventional arteries, there are more supernumerary veins than supernumerary arteries. Many small arteries and veins develop in late fetal life and continue to increase with age; their density per unit area of lung increases as respiratory bronchioles and saccules appear.

Vessel wall structure Depending on gestational age and their proximal to distal location, the wall structure of fetal pulmonary arteries is either elastic, transitional, muscular, partially muscular or non-muscular. 128 ' 129 From 19 weeks of gestation, an elastic structure is present to the same level in axial and conventional arteries, and in smaller vessels than in the adult. Because of high tensile strength and the ability to maintain vessel

Fig. 6.8. Diagram of a broncho-arterial bundle illustrating conventional versus supernumerary artery branches. Conventional arteries arise at acute angles to the main axis and supply the respiratory region at the end of the axial pathway. Supernumerary arteries are short and have varying diameters. They arise at right angles to the main axis and supply adjacent air spaces (shaded areas), ultimately providing collateral circulation to a respiratory unit via the backdoor.

patency, these arteries can be regarded as supportive. Phenotypically distinct populations of SMCs in the wall of large developing pulmonary arteries raise the possibility of different lineages. TM In small vessels, when present, the SMCs are immature. The pulmonary veins are relatively free of muscle in the fetus. At 20 weeks gestation, for example, no muscle is present even in the largest veins. By 28 weeks, muscle fibers are seen within vein walls but a continuous muscle layer is present only at term. 129'138 As in arteries, the SMCs extend into veins as small as 80 ktm but unlike arteries the veins are thin-walled and no elastic laminae are present. 129'138 The walls of bronchial arteries and bronchial veins are typical of systemic vessels, being muscular and relatively thicker (for vessel size) than pulmonary arteries. 138

Distribution of intra-acinar vessels A population count of small vessels identified by size and wall structure offers a useful way to establish that the growth pattern is normal. 128 For example, in the normal human lung at term, all arteries above 150ktin in diameter are muscular: below this, they are partially muscular, the smallest being --75ktm. The largest non-muscular arteries are --90~tm in diameter, and below 60ktm all are non-muscular. This relationship between vessel size and wall structure is similar at all fetal ages. 128 While the size of the smallest muscular artery, and the size range for partially muscular and nonmuscular arteries, is the same in the fetal and adult lung, the wall thickness of an artery (of given diameter) is twice as thick in the fetus as in the adult, and muscular arteries fail to extend into the acinus. Even at birth little muscle is found in the walls of arteries beyond the terminal bronchiole (Fig. 6.9).

Fig. 6.9. The development of muscle in the wall of human intra-acinar arteries. In the fetus, no muscle is seen within the acinus. It gradually extends with age but reaches vessels in walls of alveoli only in the adult (19 years). (Reproduced with permission from Hislop A, Reid L. Pulmonary arterial development during childhood: branching pattern and structure. Thorax 1973; 28:129-35.)

Development of bronchial arteries and veins As cartilage plates form, bronchial arteries grow along the airway wall to supply the walls of vessels of the bronchopulmonary sheath. 145'146Bronchial arteries and veins gradually increase in size and number and at term are found as far into lung as bronchioli. The term 'pre-capillary anastomosis' is used to describe a channel that is pre-capillary in position and larger than a capillary in size. A small number of these connections between bronchial arteries and pulmonary arteries (35-100 ktm in diameter) are present at all fetal ages. In the late fetal stage, pre-capillary anastomoses between pulmonary and bronchial arteries are found within the bronchial wall but none are larger than 15 ktm.

POSTNATAL VASCULAR DEVELOPMENT A N D G R O W T H Overview: At 36 weeks to term of human gestation, the preacinar pattern of arteries and veins is complete. The lung now can support air breathing but structurally it is not the adult lung in miniature. It responds by a burst of vascular growth as existing units continue to expand in diameter and length, and many new intra-acinar units are added with formation of the gas-exchange surface. 42'129'130'131'136'138 In the first 4 months, as alveoli form and increase in size, the number of arteries per unit area of lung and density of capillary networks increase. 39 These vessels are thought to form and grow by angiogenesis, and the complexity of capillary networks to increase by intussusceptive microvascular growth. 39 At birth the respiratory saccules (primary septae) are supplied by small vessels and a double capillary system. Within 2 weeks, as secondary septae form and enclose the interstitial tissue between the two capillary layers, these fuse into a single network. In the first months of postnatal life, the diameter of proximal intra-acinar vessels increases more than distal ones reflecting the burst of small vessels developing at the lung periphery. After 18 months, the number of new vessels forming slows along with alveolar growth. 13~ Between 4 months and 4 years, the number of arteries (up to ---200 ~tm) per unit area of lung increases greatly. While the ratio of intra-acinar arteries to alveoli remains similar throughout childhood, the concentration of arteries per unit area of lung falls after 5 years of age as alveoli increase in size. 13~

Conventional and supernumerary arteries and veins The veins grow at the same time as airways and arteries. While the pre-acinar drainage pattern is complete half-way through fetal life, the intra-acinar pattern develops during childhood. Both conventional and supernumerary vessels continue to develop in the postnatal lung, conventional arteries accompanying new airways appearing up to 18 months and new supernumerary arteries to 8 years of age. 13~ Conventional veins, like the axial veins, run in their own connective tissue sheath. They enter the axial vein at an

acute angle, are of similar size, and lie at some distance from the capillary bed they drain. 129 Supernumerary veins drain the lung tissue immediately around the axial vein. Some have no collagen sheath but pass directly through the main vein sheath to the axial vein: others receive post-capillary tributaries and are surrounded by a collagenous sheath continuous with the sheath of the axial vein. 129 Along the axial pathway, they are equivalent in number to airway generations and the arteries accompanying them. Each type may be found along the length of the axial vein. Both conventional and supernumerary veins become more frequent towards the periphery.

Vessel wall structure During childhood, the number of large arteries with an elastic or transitional wall remains constant. Between 4 and 10 months, vessels increase in size but muscle development lags. By 10-11 years, muscle extends further distally and is present in alveolar duct vessels but not alveolar wall ones (see Fig. 6.9). The presence of a high population of thin-walled arteries within the acinus may provide children with an advantage since arteries as large as 200 ~tm in diameter that have virtually a capillary wall can contribute to oxygen transfer. The wall structure of veins is more developed in children; post-capillary veins consist only of endothelium but larger vessels have internal and external laminae and occasional SMCs. In larger veins there is a continuous muscle coat but even in the largest still no definite elastic lamina is present. 129

Distribution of intra-acinar vessels Based on the changes described above, the distribution of intra-acinar vessels shifts significantly in childhood with more partially muscular and non-muscular arteries than muscular ones present in the fetal or adult lung.

Bronchial artery to pulmonary artery connections By 10 weeks after birth pre-capillary anastomoses (formed in the fetal period) are obliterated by fibrin and muscle; pulmonary and bronchial arteries then communicate with the pulmonary veins only through their capillary bed. Pre-capillary vessels are present between the pulmonary and bronchial arteries but normally these are not functional; they have the potential, however, to open if a block occurs. In congenital heart defects, for example, these systems adapt to the altered hemodynamic state. The persistence of pre-capillary fetal anastomoses as well as selective opening of capillaries could both contribute to communication between the large vessels of different venous and arterial systems in the newborn and young lung.

VASCULAR GROWTH AND R E O R G A N I Z A T I O N IN THE A D U L T Overview" Angiograms demonstrate the branching pattern of central and peripheral pulmonary arteries and veins in

the adult lung, and density of peripheral vessels. 135 The branching pattern can be defined by counting the number of branches either as generations or by order. 143'147'148 A useful convention is to consider a segmental airway as the first g e n e r a t i o n - the trachea and lobar branches are counted separately. For example, the inferior lingular segment commonly has about 28 generations, the apical lower lobes may have as few as 15. Vessel branching parallels that of the airway in the above features. While endothelial cell turnover is extremely low in vessels of the adult lung, new vessels continue to form from pre-existing ones by angiogenesis in line with lung growth. Circulating endothelial and bone marrow derived 'stem cells' have been identified in the adult where they are thought to contribute to vessel formation. 18 The lobule represents a group of 3-5 acini clumped at the end of an airway, be it subpleural or deep in the lung. The acinus of the adult human lung is --1 cm in diameter or 1 ml in volume, each acinus consisting of many alveoli. The extensive alveolar surface of the adult lung (-~70m 2) is composed of---300 x 106 alveoli each with many small vessel and capillary segments. The density of the capillary bed increases 4-fold between birth and adulthood, when the capillary endothelial surface area is --- 126 m2. 39'149-151

Conventional and supernumerary arteries and veins The supernumerary arteries of the adult lung remain more numerous than conventional ones, both absolutely and relatively. They have a frequency ratio of 2.5-3.4:1 over the length of the pre-acinar segment and form 20-45% of the total cross-sectional area of side branches. In cattle, it has been shown that at the origin of each supernumerary artery and the parent conventional artery the wall is organized into a V-shaped musculo-elastic cushion. Beginning as a funnelshaped channel on the hilum side of each supernumerary artery it forms into a baffle that projects over the lumen and may regulate blood flow. 152 Supernumerary arteries also appear to have their own vaso-regulatory pathways; their response to vasoconstrictors is greater than for conventional arteries, and the response to NO is different. 153 Vascular wall structure The wall of the main pulmonary artery has more than 7 elastic laminae in elastic segments and 4-7 laminae in transitional o n e s . 143 Elastic arteries are >3200~tm diam., transitional ones are 3200-2000 ~m, and muscular ones are <2000 l.tm. An elastic or transitional wall structure extends approximately half-way along an axial pathway (to about the 9th airway generation); the distal region of the pathway changes to a muscular structure in arteries about 2 mm in diameter, and almost all branches from the axial pathway have a muscular wall. SMCs now extend along distal pathways and are present in alveolar wall vessels (see Fig. 6.9). Arteries as small as 30~tm in diameter may have a muscular wall and even smaller ones approaching the capillary bed can have a single SMC in their wall.

Distribution of intra-acinar vessels All arteries greater than 150 ~tm in diameter are muscular; partially muscular ones are most frequent in the 40-80 ~tm size range, and non-muscular ones appear just below 130 ktm and increase in frequency to form the majority of the vessel population below 50 ~tm.

VESSEL WALL R E O R G A N I Z A T I O N IN A G I N G In the aged lung, the large elastic and transitional arteries have less elastic fibers than in the young adult. 154'157 This 'age-related' loss in wall elasticity is associated (a) with vessel narrowing, caused by intimal and medial wall cells encroaching on the lumen, 154'155 (b) with alveolar wall thinning, (c) with the presence of large and relatively simple alveolar structures, and (d) with an overall fall in lung elastic recoil, ls6 The intima of large elastic and transitional arteries is further thickened by acellular deposits, although large muscular arteries appear free of this change. Hyaline deposits are present in the walls of small vessels less than 150ktm in diameter. 154 The medial thickness of vessels at all diameters is greater than in the normal young adult lung: most vessels are twice as thick, and the increase is nearly 3-fold in those --3000ktm in diameter. 154 The range for wall thickness, narrow in the young adult lung, is wide in the aged lung. The wall thickness of pulmonary arteries is greater than in the young adult as is the increase in the wall thickness of resistance arteries. When the number of branches arising from the axial pulmonary artery was counted on arteriograms, and the diameter of successive branches to within 0.5 cm of the pleura and the distance between them measured, no difference was found between the aged and normal young control lung. The axial pathways appear clearer in the aged lung, however, possibly because of a fall in the number of small blood vessels and capillaries. 157

FAILURE TO DEVELOP THE N O R M A L Q U O T A OF VASCULAR UNITS A N D A F U N C T I O N A L L Y ' N O R M A L ' LUNG In many conditions there is disturbance in vascular development associated with major impairment of lung function, as in cystic fibrosis, congenital heart disease and bronchopulmonary dysplasia. 142 In other conditions, adaptation to disturbance is associated with relatively normal lung function and good prognosis, although under certain circumstances, as in environmental change or in disease, reduced pulmonary reserve becomes apparent. Examples of abnormal lung development that for a time at least are consistent with satisfactory function include the solitary lung in agenesis, the lung after repair of congenital diaphragmatic hernia and the lung in certain musculo-skeletal disorders.

Single lung in agenesis The single lung in agenesis provides an example of interference in the earliest stage of the development program so that a single airway branch gives rise to a single lung that typically fills both thoracic cavities. By 3 months of age, alveolar multiplication produces the number of alveoli and associated capillaries equivalent to that of two lungs. 158 Quantitative analysis of the single lung in one infant at 3 months of age revealed that vascular, mesenchymal and epithelial components of the single lung produce twice the normal alveolar quota and associated capillaries in response to the increased space available. This solitary lung is consistent with normal lung function. Experimental work in lambs has shown that over-distension of the lung before birth or in the perinatal period stimulates alveolar multiplication. 159

Repair of congenital diaphragmatic hernia In congenital diaphragmatic hernia (CDH), lung volume and airway branches are reduced in number. Thoracic space is reduced owing to the upward movement of abdominal contents, suggesting that lung hypoplasia results predominantly from the reduced available space. The gestational age at which the hernia is produced determines the pattern of disturbance in lung and vascular growth. 160'161 The prognosis of infants delivered with CDH is strongly influenced by the deficiency in vascular development. 162'163 After CDH repair, lung volume typically increases to fill the available thoracic space but airway, alveolar and vascular multiplication do not catch up. Commonly, the most hypoplastic lobe develops the radiographic features of pan-acinar emphysema. A gradient of hypoplasia is typically apparent, the ipsilateral lung, particularly the lower lobe, being most compressed. At least during adolescence, lung function is satisfactory although physiological studies detect reduced function. 164

Musculoskeletal disorders Musculoskeletal disorders can impair lung growth metabolically or mechanically. In some of these disorders, the metabolic disturbance that impairs cartilage and bone development also affects tissues needed for lung growth: in other syndromes the resulting reduction of thoracic space also contributes to restrict lung growth and airway development. 142 For example, in osteogenesis imperfecta reduced thoracic volume leads to reduced lung volume and airway branching, while the arteries are crowded and abnormally large. In camptomelic dwarfism the thorax is small, as is lung volume, but airway and arterial size and branching are more appropriate to the volume, suggesting that a metabolic effect is less important. 142 Dissociation between the size of the thorax and lung growth in the absence of metabolic impairment is illustrated by a case of Jeune's syndrome (asphyxiating thoracic dystrophy), a rare inherited malformation characterized by disturbance in utero of bone formation, in which the thorax failed to grow, but the lungs grew to normal size

by displacing the diaphragm. 165 At the time of death from hypoxia the small arteries of the lung had remodeled structurally, and although normal in volume the lung was abnormal in shape as it conformed to the abnormal thoracic contours.

ALVEOLAR OXYGEN TENSION AND V A S C U L A R DENSITY IN A D U L T L U N G Alveolar tension as a regulator of vascular density While thoracic and vascular growth normally cease in the young adult, in pathological conditions new vascular units form from bronchial vessels, as in bronchiectasis, or from pulmonary vessels, as in pulmonary hemangiomatosis. 166-16s The mechanisms of growth and regression that remodel the alveolar vascular density in the developing lung are not generally thought to operate in the adult. Experimental studies indicate, however, that it is remodeled by the O 2 tension, such that density falls with a high FiO 2 (inspired O 2 fraction) and increases in response to a low one.58,169,170 The initial response to hyperoxia is one of alveolar epithelial and endothelial cell injury accompanied by cell loss, vascular leak and edema. Within 3-4 days, upregulation of protective systems such as antioxidant enzymes and heme-oxygenase gene expression restore the integrity of the alveolar-capillary membrane and a new structural and functional baseline is established. Despite this 'adaptation', vascular units continue to be pruned so that pulmonary hypertension results from vascular restriction if hyperoxia is maintained. If the fraction of inspired oxygen (FiO2) then falls to the level of air (relative hypoxia), it triggers a burst of vascular growth. It also seems likely that the increase in lung volume typically induced by a low FiO 2 (hypoxia) triggers vascular growth by expansion of existing segments or by development of new ones through capillary arteriolization. This may be interpreted as a response to injury and repair; or as in the retina, 117 this response may indicate the presence of sensing mechanisms that trigger cell regression or growth to match vascular density to the oxygen tension.58,169-171 In what appears an important link in this regulation of vascular density, both in the retina and lung, peri-endothelial cells are recruited to the wails of surviving segments. It is thanks to the newly recruited cells that these segments become refractory to regression signals triggered by the hyperoxia. In the lung, a significant change in FiO 2 in either direction triggers the development of peri-endothelial cells in capillaries and vessels but especially around vessels. This is demonstrated by data from the following series of studies in which we analyzed the effect on rat alveolar vessels and capillaries of breathing (a) a high 0 2 tension (90-87% 0 2 for 1-4 weeks) and (b) a high 0 2 tension (87% 0 2 for 4 weeks) followed by weaning to air (from 87% O 2 to 21% 0 2 over 1 week) and return to breathing air (for 1-8 weeks). 56-58'169-171

L o s s of vascular units by hyperoxia Hyperoxia causes loss of small arteries (Fig. 6.10, top and center). Quantitative analysis demonstrates a fall in the density per unit area of lung, in the first 7 days a reduction of about one-

third. 58'169'17~Small veins (< 150~tm) are similarly pruned. 172 Larger pre-acinar segments also remodel and are greatly narrowed by the high O 2 tension. 169'17~ Together with loss of vessel segments, extensive areas of the alveolar-capillary membrane become attenuated as capillary segments disappear (Fig. 6.11, top and center). After 4 weeks, these regions consist

Fig. 6.10. Vessel pruning and growth shown in pulmonary arteriograms of rats (original magnification: x3.6): (top) untreated control, (center) 28 days at FiO 2 0.87, (bottom) 28 days at FiO 2 0.87 + 7 days weaning (FiO 2 0.87 to FiO 2 0.21)+4 weeks air. Note the loss of background haze of small vessels filled with barium sulfate-gelatin in hyperoxia as compared to the normal lung and the increase in density of small vessels arising from larger lateral branches 4 weeks after return to air (arrow). While increased, the density of patent vessels falls short of that in normal lung. (Reproduced with permission from Jones R, Jacobson M. Angiogenesis in the hypertensive lung: response to ambient oxygen tension. Cell Tissue Res. 2000; 300:263-84.)

Fig. 6.11. Capillary pruning and growth, shown in rat alveolar-capillary membrane (1 ~m epon sections stained with toluidine blue): untreated control (top), 28 days at FiO 2 0.87 (center), 28 days at FiO 2 0.87+ 7 days weaning (FiO 2 0.87 to FiO 2 0.21)+4 weeks air (bottom). The hyperoxic lung (center) is characterized by a regionally avascular alveolar membrane (arrows). 1 and 2 weeks after return to air more segments are patent but not until 4 weeks is the lace-like structure of the normal alveolar-capillary membrane restored. Alveolus (AIv). Bar 25 ~m. (Reproduced with permission from Jones R, Jacobson M. Angiogenesis in the hypertensive lung: response to ambient oxygen tension. Cell Tissue Res. 2000; 300:263-84.)

simply of back-to-back epithelial type 1 cells and interstitial fibroblast processes with no capillary, or only a capillary remnant, in between (Fig. 6.12, top and center). In other regions, where patent capillaries persist, their lumen is narrowed by hypertrophied endothelial cells (Fig. 6.12). 58 As there is no evidence of cell necrosis in these segments where the passage of blood cells is clearly impeded, their viability is likely maintained by nutrients delivered by plasma flow.

These data confirm that vascular pruning triggered by a high FiO 2 includes effacement of capillary segments as well as functional shutdown by lumen occlusion and loss of contact with circulating erythrocytes. It also includes vessel loss due to lumen occlusion, wall disruption and rarefaction. 5s'169'17~The extent to which this loss is caused by regression signals leading to wall disassembly by cell necrosis or apoptosis, or to loss of signals for cell survival, is unknown.

Fig. 6.12. Capillary loss and restriction, shown in rat alveolar-capillary membrane (80 nm epon sections stained with uranyl acetate and lead citrate) after 28 days at FiO2 0.87. Regionally, the membrane consists of fibroblast processes with no evidence of a residual capillary segment (top). In other regions where a capillary structure persists the endothelial cells are hypertrophied, the apical processes of opposing cells partly occluding the lumen (center). In yet other regions where the membrane structure is close to normal, the processes of hypertrophied endothelial cells typically thicken the wall (bottom). Epithelial type 1 cell (Epl), endothelial cell (E), fibroblast (Fb), collagen (Col), unidentified peri-endothelial cell (*), platelet (**). Bars= 1 ~m. (Reproduced with permission from Jones R, Jacobson M. Angiogenesis in the hypertensive lung: response to ambient oxygen tension. Cell Tissue Res. 2000; 300:263-84.)

Growth of vascular units in a low alveolar oxygen tension Exposure to air for the next 2-6 weeks triggers a burst of capillary and vessel growth (Fig. 6.10, bottom). 58'17~ How closely these new segments replace those lost in terms of length, position and connections is not known. Segments expand in diameter by the addition of wall cells" in particular, endothelial cells sharply increase in number 2 weeks after return to breathing air. 174 Between 2 and 4 weeks in air, disruption of the fused apical membranes of opposing endothelial cells restores a lumen in previously closed capillary segments, and new capillaries are seen forming within avascular regions of the interstitium (Fig. 6.11, bottom). 58 This growth initially produces simple tubular capillary structures relative to the branching networks of normal lung (Fig. 6.13); by 4 weeks, vessel and capillary structures are close to normal.

Mural cells and change in oxygen tension As developing mural cells and matrix deposition significantly thicken vessel walls (to ---10 times normal), ceils expressing an SMC phenotype appear in non-muscular segments (Figs 6.14 and 6.15a,b). 46'54-58'169'17~ The majority of proliferating cells are interstitial fibroblasts. 174 Ultrastructural studies confirm that fibroblasts surrounding vessels and capillaries are temporally and spatially regulated to migrate and align as peri-endothelial cells (Fig. 6.16a,b). 54-5s This resembles the response seen in wounded mesenteric capillary networks where fibroblasts are seen migrating towards, and eventually settling onto capi11ary walls to become pericytes (see Fig. 6.2). 53 In the adult lung, fibroblasts recruited to capillary walls (Fig. 6.16b) similarly become pericytes whereas ones recruited to vessel segments become SMCs. 54-5s

Fig. 6.13. Extended tubular capillary structures, shown in rat alveolar-capillary membrane (80 nm epon sections stained with uranyl acetate and lead citrate) after 28 days at FiO 2 0.87 + 7 days weaning (FiO 2 0.87 to FiO 2 0.21)+ 2 weeks air (left) and untreated control (right). Four weeks after return to air the complexity of the membrane has increased (see also Fig. 6.11 bottom) but it remains less organized than the normal lung. Alveolus (AIv). Bars=5 ~m. (Reproduced with permission from Jones R, Jacobson M. Angiogenesis in the hypertensive lung: response to ambient oxygen tension. Cell Tissue Res. 2000; 300:263-84.)

Fig. 6.14. Alveolar wall vessel, external diameter (ED) -35 pm, in normal rat lung (80 nm epon section stained with uranyl acetate and lead citrate). Endothelial cells (E) and the processes of peri-endothelial cells (P) form a thin wall. Almost all alveolar wall vessels in the normal lung have a similar wall structure. Alveolus (AIv), capillary (Cap). Bar= 10 pm. (Reproduced with permission from Jones R, Jacobson M, Steudel W. Alpha smooth muscle actin and microvascular precursor smooth muscle cells in pulmonary hypertension. Am. J. Respir. Cell Mol. Biol. 1999; 20:582-94.)

Fig. 6.15. (a) Alveolar wall vessel (ED 20 pm) - rat lung (80 nm epon section stained with uranyl acetate and lead citrate) after 28 days at FiO 2 0.87. Endothelial cells (E) and SMC between an electron-lucent external (Eel) and internal elastic lamina (lel) now form the vessel wall. Platelet (P), leukocyte (Le). Bar = 10 pm. (Reproduced with permission from Jones R. Ultrastructural analysis of contractile cell development in lung microvessels in hyperoxic pulmonary hypertension: fibroblasts and intermediate cells selectively reorganize non-muscular segments. Am. J. Pathol. 1992; 141:1491-505.) (b) Higher magnification of SMC forming the wall of the vessel in Figure 6.15a showing the arrangement of intracellular filaments (*) and attachment plaques (arrows) characteristic of a contractile phenotype. Basement membrane surrounds the cells (arrowheads). Bar = 1 pm. (Reproduced with permission from Ref.54)

Fig. 6.16. (a) Alveolar wall vessel (ED 20 ~m) with developing peri-endothelial cells in rat lung (80 nm epon section stained with uranyl acetate and lead citrate) after 7 days at FiO 2 0.87. The wall is formed of endothelial cells (E) and fibroblasts (Fb) recruited as peri-endothelial cells. Alveolus (AIv), interstitial fibroblast (Ifb). (Reproduced with permission from Jones R. Ultrastructural analysis of contractile cell development in lung microvessels in hyperoxic pulmonary hypertension: fibroblasts and intermediate cells selectively reorganize non-muscular segments. Am. J. Pathol. 1992; 141:1491-505.) (b) Capillary with peri-endothelial cell in rat lung (80 nm epon section stained with uranyl acetate and lead citrate) after 28 days at FiO 2 0.87. The wall is formed of endothelial cells (E) and a pericyte (Pc). Note that the pericyte shares the endothelial basal lamina (arrowheads). The processes of a fibroblast (Fb) extend around the capillary (double arrows) and a basal lamina is absent from the cell. As in adjacent vessels, these fibroblasts become peri-endothelial cells. Collagen (Col), alveolus (AIv), epithelial type 1 cell (Epl). Bar = 5 ~m. (Reproduced with permission from Jones R. Role of interstitial fibroblasts and intermediate cells in microvascular wall remodelling in pulmonary hypertension. Eur. Rev. 1993; 3:569-75.) High-resolution immunogold techniques (using antibodies to detect reactive sites) confirm the evolution of interstitial fibroblasts to SMCs by their expression of SMC-specific proteins. 56'57'176 The recruited cells rapidly express a dense mesh of t~SMA-decorated filaments while SM-MHC and desmin expression is delayed and appears later as the cells organize into a well-defined medial layer. Based on immunostaining of serial sections, pericytes expressing c~SMA and SM-MHC (unpublished data) can be identified encircling the distal ends of capillary segments indicating arteriolization of capillary segments. This conversion, and vessel contracture, likely contributes to the high population of small vessels (20-25 t,tm in diameter) that develops in hyperoxia. As the recruited fibroblasts organize into a medial layer, elastin synthesis is triggered and internal and external laminae form (see Fig. 6.15a), completing the transformation of a capillarylike structure into a small thick-walled vessel. An adventitial layer also forms as those fibroblasts excluded from the inner wall by the external elastic lamina align circumferentially. Paradoxically, more SMCs develop during the early phase of return from hyperoxia to air breathing; and again interstitial fibroblasts are their source. 58 As the walls of

vessels become highly disorganized, with fragmentation and loss of elastic laminae, the processes of migrating fibroblasts penetrate into the existing cell layers. Quantitative data show loss of previously well-defined structures and elastin fragments, indicating lamina degradation. 5s Two weeks after return to breathing air, most of the peri-endothelial cells express a SMC phenotype, and well-defined laminae are again present. Many cells are then lost as vessel walls gradually thin. Four weeks after return to breathing air, as the density of vascular units within the membrane increases, the walls of most vessels consist only of attenuated endothelial and peri-endothelial cell processes and fine elastin deposits. 58 While the development of mural cells likely protects vessels from regression signals triggered by hyperoxia, 117'177 the reason for their development in response to the relative hypoxia on return to air is less clear. It may represent a response to free radicals generated by relative hypoxia, or result from signals required to promote wall support during endothelial cell proliferation and vessel growth triggered by the lower FiO2 .178 These studies raise compelling questions as to the signaling systems that produce vascular growth in the adult lung. Further studies that focus on a link between

a change in the alveolar oxygen tension and signaling pathways of cell survival or regression (e.g. the phosphatylinositol PI3k/Akt pathway) 179'1s~can be expected to identify novel mechanisms that regulate vascular density.

ACKNOWLEDGEMENTS We thank Margaretha Jacobson and Diane Capen for preparation of figures and Annalie Francis for secretarial assistance. We thank our colleagues for their generous permission to use illustrations from their publications.

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124. Asahara T, Chen D, Takahashi T et al. Tie 2 receptor ligands, angiopoietin-1 and angiopoietin-2, modulate VEGFinduced postnatal neovascularization. Circ. Res. 1998; 83:233-40. 125. Bucher U, Reid L. Development of the intrasegmental bronchial tree: the pattern of branching and development of cartilage at various stages of intra-uterine life. Thorax 1961; 16:207-18. 126. Loosli CG, Potter EL. The prenatal development of the human lung.Anat. Rec. 1951; 109:320-1. 127. Boyden EA, Tompsett DH. The changing patterns of the developing lungs of infants. Acta Anat. 1965; 61:164-92. 128. Hislop A, Reid L. Intra-pulmonary arterial development during fetal life - branching pattern and structure. J. Anat. 1972; 113:35-48. 129. Hislop A, Reid LM. Fetal and childhood development of the intrapulmonary veins in man - branching pattern and structure. Thorax 1973; 28:313-19. 130. Hislop A, Reid L. Pulmonary arterial development during childhood: branching pattern and structure. Thorax 1973; 28:129-35. 131. Hislop A, Reid L. Development of the acinus in the human lung. Thorax 1974; 29:90-4. 132. Hislop A, Reid L. Growth and development of the respiratory system. In: Davis JA, Dobbing J (eds), Scientific Foundations of Pediatrics. London: Heinemann Medical Publications. 1974, pp. 214-54. 133. Hislop A, Reid L. Formation of the pulmonary vasculature. In: Hodson WA (ed.), Development of the Lung, Lenfant C (Executive ed.), Lung Biology in Health Disease, Vol. 6. New York, NY: Marcel Dekker. 1977, pp. 37-86. 134. Reid L. The lung: its growth and remodelling in health and disease. Am. J. Roentgenol. 1977; 129:777-88. 135. Reid L. The pulmonary circulation: remodelling in growth and disease. Am. Rev. Respir. Dis. 1979; 119:531-46. 136. Burri PH. Postnatal development and growth. In: Crystal RG, West JB, Barnes PJ et al. (eds), The Lung: Scientific Foundations, Vol. 1, Chapter 4.1.2. New York, NY: Raven Press. 1991, pp. 677-87. 137. Farrell PM. Morphologic aspects of lung maturation. In: Farrell PM (ed.), Lung Development: Biological and Clinical Perspectives. Vol. I, Biochemistry and Physiology. New York, NY: Academic Press. 1982, pp. 13-25. 138. Hislop A, Reid LM. Growth and development of the respiratory system: anatomical development. In: Davis JA, Dobbing J (eds), Scientific Foundations of Pediatrics. London: Heinemann Medical Publications. 1981, pp. 390-431. 139. deMello DE, Sawyer D, Galvin N et al. Early fetal development of lung vasculature. Am. J. Respir. Cell Mol. Biol. 1997; 16:568-81. 140. deMello DE, Reid LM. Embryonic and early fetal development of human lung vasculature and its functional implications. Pediatr. Dev. Pathol. 2000; 3:439-49. 141. Schachtner SK, Wang Y, Baldwin HS. Qualitative and quantitative analysis of embryonic pulmonary vessel formation. Am. J. Respir. Cell Mol. Biol. 2000; 22:157-65. 142. deMello DE, Reid L. Pre- and postnatal development of the pulmonary circulation. In: Haddad GG, Abman SH, Chernick V (eds), Chernick-MeUins Basic Mechanisms of Pediatric Respiratory Disease, 2nd edition. Hamilton and London: Marcel Decker. 2002, pp. 77-101. 143. Elliott FM, Reid LM. Some new facts about the pulmonary artery and its branching pattern. Clin. Radiol. 1965; XVI:193-8. 144. Frid MG, Moiseeva EP, Stenmark KR. Multiple phenotypically distinct smooth muscle cell populations exist in the adult and developing bovine pulmonary arterial media in vivo. Circ. Res. 1994; 75:669-81.

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INTRODUCTION

Postnatal survival depends upon the ability of the pulmonary circulation to undergo rapid and dramatic vasodilation during the first minutes after birth. The resulting fall in pulmonary vascular resistance (PVR) allows for an 8-10-fold increase in pulmonary blood flow and is essential for the lung to assume its postnatal role in gas exchange. Although the decrease in PVR during transition of the lung at birth is abrupt, the success of this critical event follows a lengthy series of carefully orchestrated events that characterize normal growth and maturation of the fetal pulmonary circulation. Normal development of the lung circulation is determined by the precise coordination of numerous signals from multiple cell types, which include diverse transcription factors, growth factors, chemokines, cytokines, vasoactive products, matrix proteins and others. In addition to developmental changes in lung vascular growth and structure, the pulmonary circulation also undergoes maturational changes in function. While maintaining a high PVR in utero, the fetal lung must also acquire the ability to respond to vasodilator stimuli with advancing age, prior to birth. Maturational changes in endothelial and smooth muscle cell function in the lung circulation are vital for enabling the successful transition at birth. Failure of the pulmonary circulation to successfully achieve or sustain this decrease in PVR causes severe hypoxemia in cardiopulmonary disorders that constitute the syndrome, persistent pulmonary hypertension of the newborn (PPHN). PPHN is a major clinical problem, contributing substantially to morbidity and mortality in both full-term and premature neonates. Mechanisms underlying the pathogenesis of PPHN are unclear, but clinical and experimental data suggest that intrauterine events that impair vascular function can disrupt the normal maturational sequence of lung vascular The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

development and growth. Therefore, understanding basic mechanisms of normal functional and structural development of the pulmonary circulation in utero, and mechanisms that contribute to sustained pulmonary vasodilation at birth may provide insights into the syndrome of PPHN and its treatment. This chapter reviews the physiologic regulation of the developing pulmonary circulation, including: mechanisms that regulate vascular tone and reactivity in the fetal lung; mechanisms that contribute to adaptive changes at birth and the early postnatal period (see also Chapter 14); and mechanisms that may contribute to the failure of PVR to fall in neonates with severe pulmonary hypertension.

LUNG VASCULAR GROWTH AND DEVELOPMENT

Normal development of the human lung can be divided into five stages, namely the embryonic (3-7 weeks gestation), pseudoglandular (5-17 weeks), canalicular (16-26 weeks), saccular (24-38 weeks) and alveolar periods (up to 2-3 years of age). 1 Although branching morphogenesis, epithelial development and differentiation, and alveolarization have been extensively studied, relatively less is known about lung vascular growth and development (see also Chapter 6). During each stage of development, the pulmonary circulation also undergoes marked changes in growth, structural remodeling and maturation (Table 7.1). Lung vascular growth involves two basic processes: vasculogenesis, the formation of new blood vessels from endothelial cells within the immature mesenchyme, and angiogenesis, the formation of new blood vessels from sprouts of preexisting vessels. 2 Embryonic stem cells become angioblasts, which are endothelial precursor cells that have not yet been incorporated into Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

vessels. Relatively little is known concerning the exact origins of angioblasts, but endothelial differentiation occurs within the mesoderm. After the assembly of free angioblasts into cords, angioblasts differentiate into endothelial cells and form tubes (vasculogenesis). Angioblasts can migrate to distant sites and then organize into endothelial cells and capillaries. Endothelial cells were once considered homogeneous, but organ-related differences have now been found. Endothelial cell heterogeneity accounts for key functional differences in angiogenic responses and vascular functional development, and is likely to play important roles in key differences in postnatal function. 3 However, developmental mechanisms that contribute to basic functional differences between endothelial cells from different vascular beds, such as the divergent hypoxic responses of pulmonary and systemic arteries, remain uncertain. Multiple molecules contribute to angiogenesis in the developing lung (Table 7.2). 4 Several growth factors have been shown to play important autocrine and paracrine roles in vascular development, including basic fibroblast growth factor (bFGF), transforming growth factor-J3 (TGF-~3), vascular endothelial growth factor (VEGF) and plateletderived growth factor (PDGF). 2'4'5 Characterization of their precise roles and the complex interactions between these signals in the regulation of endothelial cell differentiation, migration and proliferation are unclear, but this field is rapidly expanding. 3'6 For example, bFGF induces early expression of the VEGF receptor, VEGF receptor-2 (VEGFR-2; or, KDR/flk-1), one of the earliest markers of endothelial cell lineage. 5'7 Subsequent assembly of angioblasts within the mesoderm is likely due to stimulation of VEGFR-2 by its ligand, VEGF. The importance of paracrine signaling between VEGF, produced largely from the airway epithelium, and its receptors (VEGFR-1 and VEGFR-2), as expressed by early endothelial cells, is best demonstrated by the marked

vascular disruption and embryonic lethality in VEGF or VEGFR-deficient mice. 8-1~ Signaling between airway epithelium and the immature mesenchyme provides important "cross-talk" that coordinates vascular development with airspace growth. During branching morphogenesis in the embryo and early fetus, signals from the mesenchyme are critical for normal epithelial growth and function, such as

type II cell differentiation, morphological development and surfactant protein C expression. Ix In parallel, early vascular development within the rat embryonic lung mesenchyme is dependent on epithelial-derived products, such as VEGF, bFGF, TGF-~ and others. 11-13 Once the early endothelial tubes are formed, vessel growth is extended by angiogenesis due to sprouting and non-sprouting mechanisms (such as vessel fusion and "intussusception"). 2'14Vascular development involves overlapping mechanisms that are not mutually exclusive, and are clearly dependent upon timing. For example, another role of VEGF-VEGFR-2 signaling that occurs later in vascular development is to alter endothelial cell behavior in order to promote capillary fusion, which is distinct from its mitogenic effects. 15 In addition, VEGF continues to function throughout postnatal life as an endothelial "maintenance" or "survival factor", thereby sustaining normal vascular function through the expression of such key enzymes as endothelial nitric oxide synthase (eNOS) and prostacyclin synthase. 16 For example, disruption of VEGF signaling shortly before birth in the late gestation fetus impairs lung endothelial function, blunts vasodilation and causes perinatal pulmonary hypertension, x7 Furthermore, inhibition of VEGF receptors during infancy inhibits vascular growth, causes pulmonary hypertension and impairs alveolarization, which persists into adulthood. 18 Thus, growth factors such as VEGF, which are essential during embryonic lung development, continue to play an important role of preserving lung vascular function and maintaining lung architecture later in life. Assembly and maturation of vessels requires endothelial signals to recruit mesenchymal cells around the vessel, which later differentiate into mature smooth muscle cells. 19'2~Early signaling "loops" between endothelial cells and mesenchymal cells appear to involve the endothelial production of chemotactic factors, such as PDGF or heparin-binding epidermal growth factor, which cause migration of mesenchymal cells toward the developing vessel. This response is partly dependent upon the production of angiopoietin-1 by neighboring mesenchymal cells, which activate the endothelial TIE2 receptor and stimulate the release of migratory stimuli. 21 Subsequent maturation of the vessel wall involves ongoing production of growth factors and signaling molecules, which lead to smooth muscle cell proliferation, maturation of contractile proteins and formation of extracellular matrix. As with endothelial cells, marked heterogeneity in smooth muscle cells exists within the developing vessel wall, and these may persist postnatally or with disease states. 22 In addition to numerous growth factors and transcription factors, components of the extracellular matrix are also key determinants of endothelial growth, migration and differentiation, as well as the regulation of growth factor release and activity. 23 In the embryo, early smooth muscle and fibroblast-mediated production of extracellular matrix proteins regulate vascular structure and growth. 24 Extracellular matrix proteins contribute to the initial assembly and organization of the vessel wall, cell proliferation, adventitial growth, smooth muscle cell phenotype, vascular compliance,

and controlling the activities of diverse vasoactive mediators, growth factors and cytokines. 25 Abnormal production or degradation of ECM components can markedly impair vessel growth and structure, and have been implicated in models of pulmonary hypertension. 26 Ongoing vascular growth and remodeling continues during late gestation and early postnatal life. During the period of rapid alveolarization during infancy, the lung undergoes marked vascular growth, as reflected by the 20-fold increase in alveolar and capillary surface areas from birth to early childhood. Vascular growth and remodeling during infancy and childhood is critical for the development and maintenance of normal lung architecture later in life.

P H Y S I O L O G Y OF THE FETAL PULMONARY CIRCULATION Along with the remarkable progression of lung vascular growth and structure during development, the fetal pulmonary circulation also undergoes maturational changes in function. Pulmonary vascular resistance (PVR) is high throughout fetal life, especially in comparison with the low resistance of the systemic circulation. As a result, the fetal lung receives only 3-8% of combined ventricular output, with most of the right ventricular output crossing the ductus arteriosus (DA) to the aorta. Pulmonary artery pressure and blood flow progressively increase with advancing gestational age, along with increasing lung vascular growth. 27'28 Despite this increase in vascular surface area, PVR actually increases with gestational age when adjusted for lung or body weight. Thus, pulmonary vascular tone actually increases during late gestation, especially prior to birth. Studies of the human fetus support these physiologic observations from fetal sheep. 29 Based on multiple Doppler ultrasound measurements that include assessments of human fetal left and distal pulmonary artery velocity waveforms, it has been demonstrated that pulmonary artery impedance progressively decreases during the second and early part of the third trimester. Pulmonary artery vascular impedance does not decrease further during the latter stage of the third trimester despite ongoing vascular growth. 29'3~ Several mechanisms contribute to high basal PVR in the fetus, including low oxygen tension, low basal production of vasodilator products (such as PgI 2 and NO), increased production of vasoconstrictors (including endothelin- 1 (ET- 1) or leukotrienes) and altered smooth muscle cell reactivity (such as enhanced myogenic tone) 31-4~(Table 7.3). In addition to high PVR, the fetal pulmonary circulation is characterized by progressive changes in responsiveness to vasoconstrictor and vasodilator stimuli (or "vasoreactivity"). In the ovine fetus, the pulmonary circulation is initially poorly responsive to vasoactive stimuli during the early canalicular period, and responsiveness to several stimuli increases during late gestation. For example, the pulmonary vasoconstrictor response to hypoxia, and the vasodilator response to increased fetal PO 2 and acetylcholine increase

with gestation 41-43 (Fig. 7.1). As observed in the sheep fetus, human studies demonstrate maturational changes in the fetal pulmonary vascular response to increased PaO2.29 Maternal hyperoxia did not increase fetal pulmonary blood flow between 20 and 26 weeks gestation, but increased PaO 2 causes pulmonary vasodilation in the 31-36-week fetus. These findings suggest that, in addition to structural maturation and growth of the fetal pulmonary circulation, the vessel wall also undergoes functional maturation, leading to enhanced vasoreactivity. Mechanisms that contribute to progressive changes in pulmonary vasoreactivity during development are uncertain, but are likely due to maturational changes in endothelial cell function, especially with regard to NO production 44--47 (Fig. 7.2). Lung endothelial NOS (eNOS, type III)-mRNA and protein are present in the early fetus and increase with advancing gestation in utero and during the early postnatal period in rats and sheep. 4s-5~ The timing of this increase

in lung eNOS content immediately precedes and parallels changes in the capacity to respond to endothelium-dependent vasodilators, as shown by in vivo and in vitro studies 46'51 (Fig. 7.3). The timing of this increase in lung endothelial NOS content coincides with the capacity to respond to endothelium-dependent vasodilator stimuli, such as oxygen and acetylcholine. 4~-43 In contrast, fetal pulmonary arteries are already quite responsive to exogenous NO much earlier in gestation. 46'51 These findings suggest that the ability of the endothelium to produce or sustain production of NO in response to specific stimuli during maturation lags behind the capacity of fetal pulmonary smooth muscle to relax to NO. This may account for clinical observations that extremely premature newborns are highly responsive to inhaled NO. 52 HQrmona!

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Vasodilation Fig. 7.2. Schematic diagram illustrating the NO-cGMP signaling cascade in the fetal lung.

Fig. 7.3. Maturational changes in endothelial NO synthase (type III) expression in the ovine fetal lung (upper panel: from Ref.5~ and the rat lung (lower panel: from Ref.48).

Although most studies of the perinatal lung have focused on the role of eNOS in vasoregulation, the other NOS isoforms, including neuronal NOS (nNOS; type I) and inducible NOS (iNOS; type II), have been identified by immunostaining in the rat, sheep and human fetal lung. 53-55 Lung nNOS-mRNA and protein increase in parallel with eNOS expression during development in the fetal rat. 48 Inducible (Type II) NOS has also been detected in the ovine fetal lung, and is predominantly expressed in airway epithelium and vascular smooth muscle, with little expression in the vascular endothelium. 55 Whether the "non-endothelial" (types I and II) isoforms contribute to the physiologic responses of NO-dependent modulation of fetal pulmonary vascular tone has been controversial. Treatment of pregnant rats with an iNOS-selective antagonist caused constriction of the great vessels (main pulmonary artery and thoracic aorta) and DA in fetal rats. 56 Selective iNOS and nNOS antagonists increase fetal PVR and inhibit shear stress vasodilation at doses that do not inhibit acetylcholine-induced pulmonary vasodilation. 55'57'5s These findings support the speculation that iNOS and nNOS may also modulate pulmonary vascular tone in utero and at birth (see below). 59

NOS expression and activity are affected by multiple factors including oxygen tension, hemodynamic forces, hormonal stimuli (e.g. estradiol), paracrine factors (including VEGF), substrate and cofactor availability, superoxide production (which inactivates NO), and others. 6~ Recent studies have shown that estradiol acutely releases NO and upregulates eNOS expression in fetal pulmonary artery endothelial cells. 64 Although estradiol does not cause acute pulmonary vasodilation in vivo, prolonged estradiol treatment causes late vasodilation which is sustained despite cessation of estradiol infusion. 62'63 In contrast, VEGF acutely releases NO and causes abrupt pulmonary vasodilation in vivo. 65 Chronic inhibition of VEGF receptors downregulates eNOS and induces pulmonary hypertension in the late gestation fetus. 17 These findings illustrate that diverse hormonal and paracrine factors can regulate NOS expression and activity during development. Vascular responsiveness to endogenous or exogenous NO is also dependent upon several smooth muscle cell enzymes, including soluble guanylate cyclase, cGMP-specific (type V) phosphodiesterase (PDE5) and cGMP kinase. 66-69 Several studies have shown that soluble guanylate cyclase, which produces cGMP in response to NO activation, is active before 0.7 of term gestation in the ovine fetal lung. 46'51 Similarly, PDE5, which limits cGMP-mediated vasodilation by hydrolysis and inactivation of cGMP, is also normally active in utero. 70'71 In the fetal lung, PDE5 expression has been localized to vascular smooth muscle, and PDE5 activity is high in comparison with the postnatal lung. 68 Infusions of selective PDE5 antagonists cause potent fetal pulmonary vasodilation. Thus, PDE5 activity appears to play a critical role in pulmonary vasoregulation during the perinatal period, and must be taken into account when assessing responsiveness to endogenous NO and related vasodilator stimuli. Functionally, the NO-cGMP cascade plays several important physiologic roles in vasoregulation of the fetal pulmonary circulation. 6~ These include: (1) modulation of basal PVR, 31 (2) mediating the vasodilator response to specific physiologic and pharmacologic stimuli, 31'33 and (3) opposing strong myogenic tone in the normal fetal lung. 38 Past studies in fetal sheep have demonstrated that intrapulmonary infusions of NO synthase inhibitors increase basal PVR by 35%. 31 Since inhibition of NO synthase increases basal PVR at least as early as 0.75 gestation (112 days) in fetal sheep, endogenous NOS activity appears to contribute to vasoregulation throughout late gestation. 51 NOS inhibition also selectively blocks pulmonary vasodilation to such stimuli as acetylcholine, oxygen and shear stress in the normal fetus.31,33,38,72 In addition to high PVR and altered vasoreactivity during development, the fetal lung circulation is further characterized by its ability to oppose sustained pulmonary vasodilation during prolonged exposure to vasodilator stimuli. For example, increased PaO 2 increases fetal pulmonary blood flow during the first hour of treatment; however, blood flow returns towards baseline values over time despite maintaining high PaO 2 (Fig. 7.4). 47 Similar

Fig. 7.4. (a) Effect of increased P O 2 on pulmonary blood flow in the sheep fetus. As shown, pulmonary blood flow initially increases with the rise in PO2, but subsequently falls towards baseline values. (b) Time-dependent response of pulmonary blood flow during partial compression of the ductus arteriosus. As shown, ductus compression increases pulmonary artery pressure (PAP) and blood flow, but despite constant PAP, blood flow returns to basal levels over time (from Refs44'47).

responses are observed during acute hemodynamic stress (shear stress) caused by partial compression of the DA 44 or with infusions of several pharmacologic agents. 45 These findings suggest that unique mechanisms exist in the fetal pulmonary circulation that oppose vasodilation and maintain high PVR in utero. We have speculated that this response reflects the presence of an augmented myogenic response within the fetal pulmonary circulation. Recent studies have suggested that NO release plays an additional role in modulating high intrinsic or myogenic tone in the fetal pulmonary circulation. The myogenic response is commonly defined by the presence of increased vasoconstriction caused by acute elevation of intravascular pressure or "stretch stress". 73 Past in vitro studies demonstrated the presence of a myogenic response in sheep pulmonary arteries, and that fetal pulmonary arteries have greater myogenic activity than neonatal or adult arteries. 74'75 More recent studies of intact fetal sheep have demonstrated that high myogenic tone is normally operative in the fetus, and contributes to maintaining a high PVR in utero. 44'45'47 These studies demonstrate that NOS inhibition not only blocks vasodilation to several physiologic stimuli, but also acute inhibition of NO production unmasks a potent myogenic response. 38 Thus, these findings support our hypothesis that NOS inhibition unmasks a potent myogenic response that maintains high PVR in the normal fetus. Further work suggests that downregulation of NO, as observed in experimental pulmonary hypertension, augments myogenic activity, increasing the risk for unopposed vasoconstriction in response to stretch stress at birth. Since eNOS protein is present at a stage of lung development when blood flow is absent or minimal, it has been hypothesized that NO may potentially contribute to angio-

genesis during early lung development. 49 Whether early eNOS expression implies a role in promoting vascular growth or is merely a marker of growing endothelial cells is unknown. Recent studies report conflicting data regarding the effects of eNOS activity in promoting new vessel formation in different experimental models of angiogenesis. Although NO can inhibit endothelial cell mitogenesis and proliferation, it has also been shown to mediate the angiogenic effects of substance P and vascular endothelial growth factor in vitro. 76'77 Growing bovine aortic endothelial cells in culture express greater eNOS-mRNA and protein than confluent cells, but NOS inhibition does not affect their rate of proliferation in vitro. 78 NO has also been shown to decrease smooth muscle proliferation in vitro, 79'8~but NO may have biphasic, dose-dependent effects on the growth of fetal pulmonary artery smooth muscle cells. High doses of NO donors inhibit smooth muscle cell growth, but low doses cause paradoxical stimulation in vitro. Whether NO modulates smooth muscle cell growth in vivo remains controversial; one study reported the failure of chronic NOS inhibition to alter pulmonary vascular structure during late gestation in the fetal sheep. 81 Thus, although multiple studies have examined the role of NO in vascular growth and remodeling, its effects vary between experimental settings, and the effects of NO on angiogenesis and structure of the vessel wall are controversial. Although other vasodilator products, includingprostacyclin (PgI2) , are released upon stimulation of the fetal lung (e.g. increased shear stress), basal prostaglandin release appears to play a less important role than NO in fetal pulmonary vasoregulation. For example, cyclooxygenase inhibition has a minimal effect on basal PVR and does not increase myogenic tone in the fetal lamb. 82 The physiologic roles of other dilators, including adrenomedullin, adenosine

and endothelium-derived hyperpolarizing factor (EDHF), are uncertain. EDHF is a short-lived product of cytochrome P-450 activity that is produced by vascular endothelium, and has been found to cause vasodilation through activation of calcium-activated K+-channels in vascular smooth muscle in vitro, s3 K+-channel activation appears to modulate basal PVR and vasodilator responses to shear stress and increased oxygen tension in the fetal lung, but whether this is partly related to EDHF activity is unknown.84,85 Carbon monoxide (CO) is a gaseous molecule produced by heme-oxygenase and has been shown to have several vascular effects, including vasodilation in the adult systemic and pulmonary vascular beds. 86 CO may act in part through activation of soluble guanylate cyclase, increasing cGMP content in vascular smooth muscle, and causing vasodilation, as described for NO. 87 Despite several studies that suggest an important role in vasoregulation, CO has yet to be shown to modulate vascular tone or growth in the perinatal lung. For example, inhaled CO treatment of late gestation fetal sheep had no effect on PVR, and infusions of a heme-oxygenase inhibitor did not alter basal pulmonary vascular tone. 88 Further studies are needed to demonstrate its physiologic importance in the developing lung circulation. Vasoconstrictors have long been considered as potentially maintaining high PVR in utero. Several candidates, including lipid mediators (thromboxane A2, leukotrienes C4 and D4, and platelet-activating factor) and ET-1, have been extensively studied. Thromboxane A2, a potent pulmonary vasoconstrictor that has been implicated in animal models of Group B Streptococcal sepsis, does not appear to influence PVR in the normal fetus. In contrast, inhibition of leukotriene production causes fetal pulmonary vasodilation; s9 however, questions have been raised regarding the specificity of the antagonists used in these studies. Similarly, inhibition of platelet-activating factor may influence PVR during the normal transition, but data from recent experimental studies are difficult to interpret due to extensive non-specific hemodynamic effects. ET-1, a potent vasoconstrictor and co-mitogen that is produced by vascular endothelium, has been demonstrated to play a key role in fetal pulmonary vasoregulation (Fig. 7.5). 40,90-92 PreproET-1 mRNA (the precursor to ET-1) was identified in fetal rat lung early in gestation, and high circulating ET-1 levels are present in umbilical cord blood. Although ET-1 causes an intense vasoconstrictor response in vitro, its effects in the intact pulmonary circulation are complex. Brief infusions of ET-1 cause transient vasodilation, but PVR progressively increases during prolonged treatment. 93 The biphasic pulmonary vascular effects during pharmacologic infusions of ET-1 are explained by the presence of at least two different ET receptors. The ET-B receptor, localized to the endothelium in the sheep fetus, mediates the ET-1 vasodilator response through the release of N O . 92'94'95 A second receptor, the ET-A receptor, is located on vascular smooth muscle, and

Fig. 7.5. Schematic diagram illustrating the endothelin-1 signaling cascade in the developing lung circulation. when activated, causes marked constriction. Although capable of both vasodilator and constrictor responses, ET-1 is more likely to play an important role as a pulmonary vasoconstrictor in the normal fetus. This is suggested in extensive fetal studies showing that inhibition of the ET-A receptor decreases basal PVR and augments the vasodilator response to shear stress-induced pulmonary vasodilation. 94'95 Thus, ET-1 is likely to modulate PVR through the ET-A and-B receptors, but its predominant role is as a vasoconstrictor through stimulation of the ET-A receptor.

CONTROL ARTERIOSUS

OF THE

DUCTUS

As most of the right ventricular output crosses the DA in utero, patency of the DA is absolutely vital for fetal survival and well-being. Premature DA closure in utero causes severe

pulmonary hypertension, congestive heart failure, hydrops fetalis or severe hypoxemia. In contrast, an inability of the DA to close after birth may complicate lung disease in the premature newborn with respiratory distress syndrome or cause high flow pulmonary vascular injury during postnatal life. In addition, maintaining DA patency can be critical for survival in newborns and infants with ductus-dependent cyanotic congenital heart disease. Finally, insights into the unique nature of regulation of the DA, especially in regard to smooth muscle cell tone, proliferation and synthetic functions, may provide important lessons in vascular biology. For example, changes in PO 2 have striking effects on DA smooth muscle that is unique from its neighboring smooth muscle cells in the systemic (aortic) and pulmonary circulations. Low PO 2 constricts pulmonary vessels but dilates the DA; conversely, the increase in PO 2 at birth

contributes to the fall in PVR but paradoxically constricts the DA. Past studies have shown the important role of intramural prostaglandin production on DA tone, as evidenced by the potent constrictor effects of cyclooxygenase inhibitors. However, recent studies have shown that regulation of the DA is complex, and involves multiple signals, such as ET-1, CO, the cytochrome P-450 system and K+-channel activities. 96-99 Recent in vitro data suggest that a cytochrome P-450-based monooxygenase reaction transduces the signal for DA closure, and that DA constriction is mediated through increased ET-1 production and stimulation of the ET-A receptor. Furthermore, CO can relax the DA, but its effects vary, according to ambient oxygen tension, and CO may act in part by inhibiting ET-1 production. Other studies have demonstrated that ion channels mediate early changes in DA tone, primarily through voltage-gated K+-channels. Decreased oxygen tension inhibits voltagegated K+-channels, opening CaZ+-channels and causing constriction in pulmonary artery smooth muscle cells; in fetal DA, oxygen activates a calcium-activated K+-channel and causes vasodilation. ET may act by inhibiting voltageactivated K+-channels. 9s

M E C H A N I S M S OF P U L M O N A R Y VASODILATION AT B I R T H Within minutes after delivery, pulmonary artery pressure falls and blood flow increases in response to birth-related stimuli. Mechanisms contributing to the fall in PVR at birth include establishment of an air-liquid interface, rhythmic lung distension, increased oxygen tension and altered production of vasoactive substances. 22'23 Physical stimuli, such as increased shear stress, ventilation and increased oxygen cause pulmonary vasodilation in part by increasing production of the vasodilators, NO and PgI2 .31-39 Pretreatment with the arginine analogue, nitro-L-arginine, blocks NOS activity, and attenuates the decline in PVR after delivery of near term fetal sheep (Fig. 7.6). 31 These findings suggest that about 50% of the rise in pulmonary blood flow at birth may be directly related to the acute release of NO. Specific mechanisms that cause NO release at birth include the marked rise in shear stress, increased oxygen and ventilation. 33 Increased PaO2 triggers NO release, which augments vasodilation through cGMP kinasemediated stimulation of K+-channels. s5'l~176176 Although the endothelial isoform of NO synthase (type III) has been presumed to be the major contributor of NO at birth, recent studies suggest that other isoforms (inducible (type II) and neuronal (type I)) may be important sources of NO release in utero and at birth as well. 54'56-59 Although early studies were performed in term animals, NO also contributes to the rapid decrease in PVR at birth in premature lambs, at least as early as 112-115 days (0.7 term). 51 Other vasodilator products, including PgI2, also modulate changes in pulmonary vascular tone at birth. 36'39 Rhythmic

Fig. 7.6. Effects of NO synthase inhibition on left pulmonary artery (LPA) blood flow at birth. Near term fetal lambs were delivered by cesarean section and ventilated with low (10%) and high (100%) oxygen tensions. As shown, the non-selective NOS antagonist, nitro-L-arginine (L-NA), impaired the rise in blood flow during delivery, but the effects were not different from the type II antagonists, aminoguanidine (AG) and 1400W (from Ref.59).

lung distension and shear stress stimulate both PgI 2 and NO production in the late gestation fetus, but increased PO 2 triggers NO activity and overcomes the effects of prostaglandin inhibition at birth. In addition, the vasodilator effects of exogenous PgI 2 are blocked by NO synthase inhibitors, suggesting that NO modulates PgI 2 activity in the perinatal lung. s2 Adenosine release may also contribute to the fall in PVR at birth, but its actions may be partly through enhanced production of NO. 1~ Thus, although NO does not account for the entire fall in PVR at birth, NOS activity appears important in achieving postnatal adaptation of the lung circulation. Transgenic eNOS knockout mice successfully make the transition at birth without evidence of PPHN. 1~176 This finding suggests that e N O S - / - mice may have adaptive mechanisms, such as a compensatory vasodilator mechanism (such as upregulation of other NOS isoforms or dilator prostaglandins) or less constrictor tone. Interestingly, these animals are more sensitive to the development of pulmonary hypertension at relatively mild decreases in PaO21~176 and have higher neonatal mortality when exposed to hypoxia after birth (unpublished observations). We speculate that isolated eNOS deficiency alone may not be sufficient for the failure of postnatal adaptation, but that a decreased ability to produce NO in the setting of a perinatal stress (such as hypoxia, inflammation, hypertension or upregulation of vasoconstrictors) may cause PPHN. Although these studies were performed in term animals, similar mechanisms also contribute to the rapid decrease in PVR at birth in premature lambs. 51'1~ The pulmonary vasodilator responses to ventilation with hypoxic gas mixtures (or rhythmic distension) of the lung or increased PaO 2 are partly due to stimulation of NO release in premature lambs, at least as early as 112-115 days (0.7 term). 51 Other vasodilator products, including PgI2, also modulate

changes in pulmonary vascular tone at birth. 35'36'39Rhythmic lung distension and shear stress stimulate both PgI 2 and NO production in the late gestation fetus; increased PO 2 triggers NO activity but does not appear to alter PgI 2 production in vivo. 33'35'39

M E C H A N I S M S T H A T CAUSE FAILURE OF P U L M O N A R Y V A S O D I L A T I O N AT B I R T H Some newborns fail to achieve or sustain the normal decline in PVR after birth, which constitutes the clinical syndrome known as PPHN. 1~176 As a clinical syndrome, PPHN includes diverse cardiac and pulmonary disorders, or occurs as an idiopathic disorder, in the absence of significant cardiac or pulmonary disease. Although these diverse diseases have features which are distinct from each other, they are generally included within this clinical syndrome because they share a common pathophysiologic feature: high PVR leading to right-to-left shunting of blood across the DA or foramen ovale and marked hypoxemia. Despite multiple therapeutic strategies, morbidity and mortality in neonates with severe PPHN remain high. Although cardiac and lung dysfunctions may contribute to the clinical course of PPHN, abnormalities of the pulmonary circulation are its critical features. PPHN is characterized by altered pulmonary vascular reactivity, structure, and in some cases, growth. 1~ Autopsy studies of the lungs of newborns with fatal PPHN have revealed severe hypertensive structural remodeling even in newborns who die shortly after birth, suggesting that many cases of severe disease are associated with chronic intrauterine stress. 1~176 However, the exact intrauterine events that alter pulmonary vascular reactivity and structure are poorly understood. Epidemiologic studies have demonstrated strong associations between PPHN and maternal smoking and ingestion of cold remedies that include aspirin or other non-steroidal anti-inflammatory products. 111 Since these agents can induce partial constriction of the DA, it is possible that pulmonary hypertension due to DA narrowing contributes to PPHN (see below). 112-114 Other perinatal stresses, including placenta previa and abruption, and asymmetric growth restriction, are associated with PPHN; 115 however, most neonates who are exposed to these prenatal stresses do not develop PPHN. Circulating levels of L-arginine, the substrate for NO, are decreased in some newborns with PPHN, suggesting that impaired NO production may contribute to the pathophysiology of PPHN, as observed in experimental studies. 116-118 It is possible that genetic factors increase susceptibility for pulmonary hypertension. A recent study reported strong links between PPHN and polymorphisms of the carbamoyl phosphate synthase gene; 119 however, the importance of this finding is uncertain. Studies of adults with idiopathic primary pulmonary hypertension have identified abnormalities of bone morphogenetic

protein (BMP) receptor genes; 12~whether polymorphisms of genes for the BMP or TGF-[3 receptors, other critical growth factors, vasoactive substances or other products increase the risk for some newborns to develop PPHN is unknown. Several experimental models have been used to explore the pathogenesis and pathophysiology of PPHN. 12~ Such models have included exposure to acute or chronic hypoxia after birth, chronic hypoxia in utero, placement of meconium into the airways of neonatal animals, sepsis and others. Although each model demonstrates interesting physiologic responses that may be especially relevant to particular clinical settings, most studies examine only brief changes in the pulmonary circulation, and mechanisms underlying altered lung vascular structure and function of PPHN remain poorly understood. Clinical observations that neonates with severe PPHN who die during the first days after birth already have pathologic signs of chronic pulmonary vascular disease suggest that intrauterine events may play an important role in this syndrome. 1~176 Adverse intrauterine stimuli during late gestation, such as abnormal hemodynamics, changes in substrate or hormone delivery to the lung, hypoxia, inflammation or others, may potentially alter lung vascular function and structure, contributing to abnormalities of postnatal adaptation. Several investigators have examined the effects of chronic intrauterine stresses, such as hypoxia or hypertension, in animal models in order to attempt to mimic PPHN. Whether chronic hypoxia alone can cause PPHN is controversial. A study in rats showed that maternal hypoxia increases pulmonary vascular smooth muscle thickening in newborns, 122 but this observation has not been reproduced with more extensive studies in rats or guinea pigs. 123 In contrast to hypoxia, animal studies suggest that intrauterine hypertension, due to either renal artery ligation or constriction of the DA, can cause structural and physiologic changes that resemble features of clinical PPHN. 112-114 Pulmonary hypertension induced experimentally by early closure of the DA in fetal sheep alters lung vascular reactivity and structure, causing the failure of postnatal adaptation at delivery, and providing a model of PPHN. Over days, pulmonary artery pressure and PVR progressively increase, but flow remains low and PaO 2 is unchanged. 112 Marked right ventricular hypertrophy and structural remodeling of small pulmonary arteries develop after 8 days of hypertension. After delivery, these lambs have persistent elevation of PVR despite mechanical ventilation with high oxygen concentrations. Studies with this model show that chronic hypertension without high blood flow can alter fetal lung vascular structure and function. This model is further characterized by endothelial cell dysfunction and altered smooth muscle cell reactivity and growth, including impaired NO production and activity due to downregulation of lung endothelial NO synthase mRNA and protein expression. 124-128 The fetal pulmonary hypertension also impaired soluble guanylate cyclase and upregulated cGMP-specific phosphodiesterase (type 5; PDE5) activities, suggesting further

and reactivity in an experimental model of PPHN. Further studies of the NO-cGMP cascade may provide helpful insights into new clinical strategies for more successful treatment of neonatal pulmonary vascular disease. In addition, since studies of vascular growth suggest important functions of NO in angiogenesis, we speculate that fetal NO production may contribute to normal lung vascular development. Insights into mechanisms that regulate changes in pulmonary vascular function are critical for understanding the pathogenesis and pathophysiology of neonatal respiratory diseases.

REFERENCES

Fig. 7.7. Schematic diagram illustrating the potential role of abnormalities of the NO-cGMP cascade in the pathophysiology of persistent pulmonary hypertension of the newborn (PPHN).

impairments in the NO-cGMP cascade. 129'13~Thus alterations in the NO-cGMP cascade appear to play an essential role in the pathogenesis and pathophysiology of experimental PPHN (Fig. 7.7). Abnormalities of NO production and responsiveness contribute to altered structure and function of the developing lung circulation, leading to failure of postnatal cardiorespiratory adaptation. Upregulation of ET-1 may also contribute to the pathophysiology of PPHN. Circulating levels of ET-1, a potent vasoconstrictor and co-mitogen for vascular smooth muscle cell hyperplasia, are increased in human newborns with severe PPHN. TM In the experimental model of PPHN induced by compression of the DA in fetal sheep, lung ET-1 mRNA and protein content are markedly increased, and the balance of ET receptors is altered, favoring vasoconstriction. 132 Chronic inhibition of the ET-A receptor attenuates the severity of pulmonary hypertension, decreases pulmonary artery wall thickening and improves the fall in PVR at birth in this model. 133 Thus, experimental studies have shown the important role of the NO-cGMP cascade and the ET-1 system in the regulation of vascular tone and reactivity of the fetal and transitional pulmonary circulation. 6~

CONCLUSIONS Physiologically, the fetal pulmonary circulation is characterized by the presence of high vascular resistance, and the ability to oppose vasodilation and maintain low blood flow in utero; the ability to respond to vasoactive stimuli increases with maturation. Experimental studies have clearly shown the important role of the NO-cGMP cascade in the regulation of vascular tone and reactivity of the fetal and transitional pulmonary circulation, and that abnormalities in this system contribute to abnormal pulmonary vascular tone

1. Burri PH. Structural aspects of prenatal and postnatal development and growth of the lung. In: McDonald JA (ed.), Lung Growth and Development. New York: Marcel Dekker, 1997, pp. 1-35. 2. Schachtner S, Taichman D, Baldwin HS. Mechanisms of lung vascular development. In: Haddad GG, Chernick V, Abman SH (eds), Basic Mechanisms of Pediatric Respiratory Disease. Hamilton: BC Decker, 2002, pp. 49-67. 3. Stevens T, Rosenberg R, Aird W etal. NHLBI workshop report: endothelial cell phenotypes in heart, lung and blood diseases. Am. J. Physiol. 2001; 282:C1422-33. 4. Risau W. Mechanisms ofangiogenesis. Nature 1997; 386:671-4. 5. Poole TJ, Finkelstein EB, Cox CM. The role of FGF and VEGF in angioblast induction and migration during vascular development. Dev. Dyn. 2001; 220:1-17. 6. Morrell NW, Weiser MCM, Stenmark KR. Development of the pulmonary vasculature. In: Gaultier C, Bourbon JR, Post M (eds), Lung Development. New York: Oxford University Press, 1999, pp. 152-95. 7. Gebb SA, Shannon JM. Tissue interactions mediate early events in pulmonary vasculogenesis. Dev. Dyn. 2000; 217:159-69. 8. Ferrara N, Carver-Moore K, Chen H etal. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 1996; 380:439-42. 9. Shalaby F, Rossant J, Yamaguchi TP et al. Failure of bloodisland formation and vasculogenesis inflkl deficient mice. Nature 1995; 376:62-6. 10. Carmeliet P, Ferreira V, Breier Get al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 1996; 380:435-9. 11. Shannon JM, Deterding RR. Epithelial-mesenchymal interactions in lung development. In: McDonald JA (ed.), Lung Growth and Development. New York: Marcel-Dekker, 1997, pp. 81-118. 12. Hanahan D. Signaling vascular morphogenesis and maintenance. Science 1997; 277:48-50. 13. Acarregui M, Penisten ST, Goss KL et al. Vascular endothelial growth factor gene expression in human fetal lung in vitro. Am. J. Respir. Cell Mol. Biol. 1999; 20:14-23. 14. Drake CJ, Little CD. VEGF and vascular fusion: implications for normal and pathological vessels. J. Histochem. Cytochem. 1999; 47:1351-5. 15. Drake CJ, Little CD. The de novo formation of blood vessels in the early embryo and in the developing lung. In: Weir EK, Archer S1, Reeves JT (eds), Fetal and Neonatal Pulmonary Circulation. New York: Futura, 1999, pp. 19-30. 16. Hood JD, Meininger CJ, Ziche M, Granger HJ. VEGF upregulates ecNOS message, protein and NO production in human endothelial cells. Am. J. Physiol. 1998; 274:H1054-8.

17. Grover TR, Parker TA, Zenge JP et al. Intrauterine hypertension decreases lung VEGF expression and VEGF inhibition causes pulmonary hypertension in the ovine fetus. Am. J. Physiol. Lung Cell Mol. Physiol. 2003; 284:L508-17. 18. Le Cras TD, Markham NE, Tuder RM etal. Treatment of newborn rats with a VEGF receptor inhibitor causes pulmonary hypertension and abnormal lung structure. Am. J. Physiol. Lung Cell Mol. Physiol. 2002; 283:L555-62. 19. Folkman J, D'Amore PA. Blood vessel formation: what is its molecular basis. Cell 1996; 87:1153-5. 20. Hall SM, Hislop AA, Pierce CM et al. Prenatal origins of human intrapulmonary arteries: formation and smooth muscle maturation.Am. J. Respir. Cell Mol. Biol. 2000; 23:194-203. 21. Suri C, Jones PF, Patan Set al. Requisite role of angiopoietin-1 and ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 1996; 87:1171-80. 22. Frid MG, Moiseeva EP, Stenmark KR. Multiple phenotypically distinct smooth muscle cell populations exist in the adult and developing bovine pulmonary arterial media in vivo. Circ. Res. 1994; 75:669-81. 23. Carey DJ. Control of growth and differentiation of vascular cells by extracellular matrix. Annu. Rev. Physiol. 1991; 53:161-77. 24. Roman J. Cell-cell and cell-matrix interactions in development of the lung vasculature. In: McDonald JA (ed.), Lung Growth and Development. New York: Marcel Dekker, 1997, pp. 365-400. 25. Rupp PA, Little CD. Integrins in vascular development. Circ. Res. 2001; 89:566-72. 26. Stenmark KR, Mecham RP, Durmowicz AG et al. Persistence of the fetal pattern of tropoelastin gene expression in severe neonatal pulmonary hypertension. J. Clin. Invest. 1994; 93:1234--42. 27. Heymann MA, Soifer SJ. Control of fetal and neonatal pulmonary circulation. In: Weir EK, Reeves JT (eds), Pulmonary Vascular Physiology and Pathophysiology. New York: MarcelDekker, 1989, pp. 33-50. 28. Rudolph AM. Fetal and neonatal pulmonary circulation. Ann. Rev. Physiol. 1979; 41:383-95. 29. Rasanen J, Huhta JC, Weiner S et al. Role of the pulmonary circulation in the distribution of human fetal cardiac output during the second half of pregnancy. Circulation 1996; 94:1068-73. 30. Rasanen J, Wood DC, Debbs RH et al. Reactivity of the human fetal pulmonary circulation to maternal hyperoxygenation increases during the second half of pregnancy: a randomized study. Circulation 1998; 97:257-62. 31. Abman SH, Chatfield BA, Hall SL et al. Role of endotheliumderived relaxing factor during transition of pulmonary circulation at birth.Am. J. Physiol. 1990; 259:H1921-7. 32. Cassin S. Role of prostaglandins, thromboxanes and leukotrienes in the control of the pulmonary circulation in the fetus and newborn. Semin. Perinatol. 1987; 11:53-63. 33. Cornfield DN, Chatfield BA, McQueston JA etal. Effects of birth-related stimuli on L-arginine-dependent pulmonary vasodilation in the ovine fetus. Am. J. Physiol. 1992; 262:H1474-81. 34. Cornfield DN, Reeves HL, Tolarova S etal. Oxygen causes fetal pulmonary vasodilation through activation of a calcium-dependent potassium channel. Proc. Natl. Acad. Sci. 1996; 93:8089-94. 35. Leffler CW, Hessler JR, Green RS. Mechanism of stimulation of pulmonary prostacyclin synthesis at birth. Prostaglandins 1984; 28:877-87. 36. Leffler CW, Tyler TL, Cassin S. Effect of indomethacin on pulmonary vascular response to ventilation of fetal goats. Am. J. Physiol. 1978; 234:H346-51. 37. Soifer SJ, Loitz RD, Roman C et al. Leukotriene end organ antagonists increase pulmonary blood flow in fetal lambs. Am. J. Physiol. 1985; 249:570.

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80. Thomae KR, Nakayama DK, Billiar TR et al. Effect of NO on fetal pulmonary artery smooth muscle growth. J. Surg. Res. 1996; 270:H411-15. 81. Fineman JR, Wong J, Morin FC et al. Chronic NO inhibition in utero produces persistent pulmonary hypertension in newborn lambs.J. Clin. Invest. 1994; 93:2675-83. 82. Zenge JP, Rairigh RL, Grover TR et al. NO and prostaglandins modulate the pulmonary vascular response to hemodynamic stress in the late gestation fetus. Am. J. Physiol. Lung Cell Mol. Physiol. 2001; 281 :L1157-63. 83. Campbell WB, Harder DR. Prologue: EDHF what is it? Am. J. Physiol. 2001; 280:H2413-16. 84. Storme L, Rairigh RL, Parker TP et al. Potassium channel blockade attenuates shear stress-induced pulmonary vasodilation in the ovine fetus. Am. J. Physiol. 1999; 276:L220-8. 85. Cornfield DN, Reeves HL, Tolarova S et al. Oxygen causes fetal pulmonary vasodilation through activation of a calciumdependent potassium channel. Proc. Natl. Acad. Sci. 1996; 93:8089-94. 86. Lin H, McGrath JJ. Vasodilating effects of carbon monoxide. Life Sci. 1988; 43:1813. 87. Kharitonov VG. Basis of guanylate cyclase activation of carbon monoxide. Proc. Natl. Acad. Sci. USA 1995; 92:2568. 88. Grover TR, Rairigh RL, Zenge JP etal. Inhaled carbon monoxide does not alter pulmonary vascular tone in the ovine fetus. Am. J. Physiol. Lung Cell Mol. Physiol. 2000; 278:L779-84. 89. Soifer LT, Soifer SJ, Loitz RD et al. Leukotriene end organ antagonists increase pulmonary blood flow in fetal lambs. Am. J. Physiol. 1985; 249:570. 90. Yanagisawa M, Kurihara H, Kimura Set al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988; 332:411-15. 91. Boulanger C, Luscher TF. Release of endothelin from the porcine aorta: inhibition by endothelium-derived nitric oxide.J. Clin. Invest. 1990; 85:587-90. 92. Ivy DD, Abman SH. Role of endothelin in perinatal pulmonary vasoregulation. In: Weir EK, Archer SL, Reeves JT (eds), Fetal and Neonatal Pulmonary Circulation. New York: Futura, 1999, pp. 279-302. 93. Chatfield BA, McMurtry IF, Hall SL etal. Hemodynamic effects of endothelin-1 on the ovine fetal pulmonary circulation. Am. J. Physiol. 1991; 261 :R182-7. 94. Ivy DD, Kinsella JP, Abman SH. Physiologic characterization of endothelin A and B receptor activity in the ovine fetal lung.J. Clin. Invest. 1996; 93:2141-8. 95. Ivy DD, Parker TA, Abman SH. Prolonged endothelin B receptor blockade causes pulmonary hypertension in the ovine fetus.Am. J. Physiol. 2000; 279:L758-65. 96. Coceani F, Kelsey L, Seiditz F et al. Carbon monoxide formation in the ductus arteriosus in the lamb: implications for the regulation of muscle tone. Br. J. Pharmacol. 1997; 120:599-603. 97. Tristani-Firouzi M, Reeve HL, Tolarova S etal. Oxygeninduced constriction of rabbit ductus arteriosus occurs via inhibition of 4-aminopyridine, voltage-sensitive potassium channels.J. Clin. Invest. 1996; 98:1959-65. 98. Reeve HL, Weir EK. Regulation of ion channels in the ductus arteriosus. In: Weir EK, Archer SL, Reeves JT (eds), Fetal and Neonatal Pulmonary Circulation. New York: Futura, 1999, pp. 319-30. 99. Archer SL, Huang JMC, Hampl V etal. Nitric Oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 1994; 91:7583-7. 100. Rhodes MT, Porter VA, Saqueton CB etal. Pulmonary vascular response to normoxia and Kca channel activity is developmentally regulated. Am. J. Physiol. 2001; 280:L1250-7.

101. Tristani-Firouzi M, Martin EB, Tolarova S et al. Ventilation-

induced pulmonary vasodilation at birth is modulated by potassium channel activity. Am. J. Physiol. 1996; 271:H2353-9. 102. Konduri GG, Mital S, Gervasio CT et al. Purine nucleotides contribute to pulmonary vasodilation caused by birthrelated stimuli in the ovine fetus. Am. J. Physiol. 1997; 272:H2377-84. 103. Fagan KA, Fouty BW, Tyler RC etal. The pulmonary circulation of mice with either homozygous or heterozygous disruption of endothelial NO synthase is hyper-responsive to chronic hypoxia.J. Clin. Invest. 1999; 103:291-9. 104. Steudel W, Scherrer-Crosbie M, Bloch KD etal. Sustained pulmonary hypertension and right ventricular hypertrophy after chronic hypoxia in mice with congenital deficiency ofNOS III.J. Clin. Invest. 1998; 101:2468-77. 105. Kinsella JP, McQueston JA, Rosenberg AA et al. Hemodynamic effects of exogenous nitric oxide in ovine transitional pulmonary circulation.Am. J. Physiol. 1992; 263:H875-80. 106. Levin DL, Heymann MA, Kitterman JA etal. Persistent pulmonary hypertension of the newborn. J. Pediatr. 1976; 89:626-33. 107. Kinsella JP, Abman SH. Recent developments in the pathophysiology and treatment of persistent pulmonary hypertension of the newborn.J. Pediatr. 1995; 126:853-64. 108. Geggel RL, Reid LM. The structural basis of persistent pulmonary hypertension of the newborn. Clin. Perinatol. 1984; 3:525-49. 109. Murphy JD, Rabinovitch M, Goldstein JD et al. The structural basis for PPHN infant.J. Pediatr. 1981; 98:962-7. 110. Murphy JD, Vawter G, Reid LM. Pulmonary vascular disease in fatal meconium aspiration.J. Pediatr. 1984; 104:758-62. 111. Van Marter LJ, Leviton A, Allred EN et al. PPHN and smoking and aspirin and nonsteroidal antiinflammatory drug consumption during pregnancy. Pediatrics 1996; 97:658-63. 112. Levin DL, Hyman AI, Heymann MA etal. Fetal hypertension and the development of increased pulmonary vascular smooth muscle: a possible mechanism for persistent pulmonary hypertension of the newborn infant. J. Pediatr. 1978; 92:265-9. 113. Abman SH, Shanley PF, Accurso FJ. Failure of postnatal adaptation of the pulmonary circulation after chronic intrauterine pulmonary hypertension in fetal lambs. J. Clin. Invest. 1989; 83:1849-58. 114. Morin FC. Ligating the ductus arteriosus before birth causes persistent pulmonary hypertension in the newborn lamb. Pediatr. Res. 1989; 25:245-50. 115. Williams MC, Wyble LE, O'Brien WF etal. PPHN and asymmetric growth restriction. Obstet. Gynecol. 1998; 91:336-41. 116. Castillo L, DeRojas-Walker T, Yu YM etal. Whole body arginine metabolism and NO synthesis in newborns with persistent pulmonary hypertension. Pediatr. Res. 1995; 38:17-24. 117. Dollberg S, Warner BW, Myatt L. Urinary nitrite and nitrate concentrations in patients with idiopathic PPHN and effect ofECMO. Pediatr. Res. 1994; 37:31-4.

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INTRODUCTION

Continuous secretion of liquid into the fetal lung is essential for its normal development. It is now generally accepted that the main driving force for this secretion is local osmotic gradients created across both alveolar and airway epithelia by active secretion of C1-. However, at the time of birth, the primary type of active solute transport across pulmonary epithelia switches, within minutes, from active secretion of C1- to active absorption of Na § The transepithelial osmotic gradients are reversed and luminal liquid is absorbed as the lungs fill with air.

MECHANISMS

OF

SOLUTE

TRANSPORT

In both alveolar and airway epithelia, all evidence 1-3 suggests that active Na absorption is by the mechanism first proposed by Koefoed-Johnsen and Ussing 4 (Fig. 8.1A). Net Na entry across the apical membrane occurs down both electrical and chemical gradients. Na-K-ATPase is restricted to the basolateral membrane, and actively extrudes Na entering across the apical membrane. It does this in exchange for K, which recycles via K channels in the K-selective basolateral membrane. There is currently some controversy about the nature of the apical membrane Na channels in alveolar epithelium. 5 Immunocytochemistry has shown the ubiquitous "epithelial Na channel" (ENaC) to be present in the epithelium throughout the airways and alveoli. 6-9 Furthermore, compelling evidence for the importance of this channel is provided by the finding that ENaC knockout mice die shortly after birth due to a failure to remove lung liquid. 1~ ENaC has a high selectivity to Na The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

over K, is inhibited with high affinity by amiloride, and has a unitary conductance o f - - 5 pS. However, this type of cation channel is seldom found in patch-clamp studies of isolated alveolar type II cells. 5 In fact, the most frequent of several types found is non-selective to Na vs K, and has N25pS unit conductance, though it resembles ENaC in being amiloride-sensitive. T M It must be noted though that this 25 pS channel has generally been detected in patchclamp studies on single cells shortly after their attachment to tissue culture dishes. In airway epithelium, there is evidence that the types of channel present in the upper membrane of such single cells may differ from those in the apical membrane of confluent cell sheets containing tight junctions. 13 In addition to the above channels, there is evidence for amiloride-insensitive, cyclic-nucleotide gated Na channels in alveolar epithelium, TM and amiloride-insensitive Na absorption has been demonstrated across intact sheets of adult airway epithelium by measuring transepithelial fluxes of 22Na.15 Chloride secretion across airway epithelium has been analyzed in great detail, 1 and appears to be by a mechanism widespread in vertebrate epithelia 16 (Fig. 8.1B). Though studied in less detail, all available evidence suggests that C1 secretion by alveolar epithelium is by the same mechanism. 17'18 In this model of active C1 transport, C1 entry across the basolateral membrane is by cotransport with Na and K in the ratio 1Na:IK:2C1. Basolateral Na-K-ATPase maintains intracellular [Na] at a level about one-tenth that of extracellular [Na]. Thus, the energy in the transmembrane Na gradient allows for accumulation of C1 inside the cell to levels greater than predicted for passive distribution according to the apical membrane potential difference. Net exit of C1 across the apical membrane occurs via C1 channels down Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

B

A Apical membrane

Basolateral membrane

Apical membrane

Basolateral membrane

CI

tion. 3~ Under baseline conditions, levels of H § secretion in the airways are probably too low to have much effect on transepithelial liquid transport. However, when stimulated by mucosal histamine or ATP, levels of acid secretion are comparable to those of active Na or C1 transport. 3~

I N T A C T FETAL L U N G S

N a §

i

Z~Z'Z

~? ~ZZZZZ

~

Fig. 8.1. Mechanisms of active Na § absorption (A) and active CI- secretion (B). See text for details.

the favorable electrochemical gradient. One of these channels is the cystic fibrosis transmembrane conductance regulator (CFTR) that is regulated by cAMP-dependent phosphorylation. In addition, there may be Ca-activated CI channels and cAMP-dependent C1 channels other than CFTR. 19'2~ The presence or absence of the above transport mechanisms is generally determined using specific blockers of the individual transport proteins. Sodium channels are usually blocked by amiloride, the Na-K-2CI cotransporter by bumetanide, furosemide, and other loop diuretics, Na-K-ATPase by ouabain, CFTR by diphenylamine-2carboxylic acid (DPAC), most other C1 channels by diisothiocyanatostilbene-sulfonic acid (DIDS), and most K channels by Ba 2+. It is likely that active absorption of Na and active secretion of C1 by the mechanisms outlined above are the most important solute transport processes driving liquid into and out of the lung via its epithelium. Nevertheless, other transport mechanisms may contribute. One such is Na-glucose cotransport. Thus, in adult rat lungs cross-perfused in situ with blood from a second rat, it was found that ---60% of the absorption of luminal instillate was inhibited by amiloride and the rest was inhibited by removal of glucose or by phloridzin (an inhibitor of Na-glucose cotransport). 21 Potassium secretion into the lung was detected with the same preparation and inhibited by apical Ba 2+ or basolateral ouabain. 22 Thus, K secretion may involve active uptake across the basolateral membrane on the Na-K-ATPase followed by downhill movement through apical membrane K channels. There is evidence for K secretion in airway epithelium by this mechanism. 23 However, the number of K channels in airway epithelial apical membrane is much less than that in the basolateral membrane, and active K secretion is quantitatively trivial compared to either active Na absorption or active C1 secretion. 23 Inhibition of lung liquid secretion by the carbonic anhydrase inhibitor, acetazolamide, points to a role for C1/HCO 3- exchange in lung liquid transport, 24 and C1/HCO 3- exchange has been demonstrated in isolated pneumocytes. 25 Finally, the pH of the liquids lining both the airways and alveoli is acidic. 26-28 In alveolar type II cells, acidification may be by an H§ 29 In human airway epithelium, we have recently described an apical membrane proton conductance that mediates acid secre-

The fetal lung is filled with liquid that does not appear to be derived from amniotic fluid in that the ionic compositions of lung and amniotic liquids are markedly different, with the former being relatively high in CI and low in HCO3 .31-33 Initial evidence for active secretion by the lung itself was provided by the finding in the rabbit that tracheal ligation at 19 days gestation ( t e r m - 31 days) led 9 days later to lungs abnormally distended with liquid, 34 a result confirmed in later studies. 35 Thus, the upper airways act as a partial one-way valve retarding the entry of amniotic liquid but allowing lung liquid to flow outwards. 36 Secretion of lung liquid is necessary for normal lung development; hypoplastic lungs result when lung liquid is drained out through a tracheal cannula.37, 38 The standard approach to study ion transport in intact fetal lung is to cannulate fetal tracheas in utero, and replace the lung liquid with experimental medium by repeatedly withdrawing liquid, mixing it with the contents of a reservoir, and then reinjecting the mixture. 2 Sampling of the liquid is made after a similar process of mixing. When both impermeant and permeant tracers are present in the experimental lung liquid, the concentration of the former will decline solely due to liquid secretion into the pulmonary lumen. In contrast, the concentration of permeant markers will decline both by dilution and by diffusion across the pulmonary epithelium. By comparing the rates of change in concentration of the two types of marker, transepithelial flux of the permeant marker from lumen to blood can be estimated. Flux from blood to lung lumen is measured simply by injecting tracer into the blood, and sampling pulmonary liquid at timed intervals. This approach has shown that liquid secretion into fetal sheep lung is 2-4 (ml/kg/h), and isotope studies have implicated active secretion of C1 as the driving force for liquid secretion. Thus, Ussing's flux ratio equation has been used to calculate ion fluxes into and out of the lung, after first establishing that movement of solute into the lung lumen by solvent drag was negligible compared to movement by diffusion. 39 If solvent drag is low, then the passive fluxes of any given ion into and out of the lung are given by Ussing's flux ratio equation:

YPL JLP

m

ap z E F / R T e

aL

where JPL and JLP are the unidirectional fluxes from plasma-to-lumen and lumen-to-plasma respectively, ap and

a L the ion's activities in plasma and lung luminal liquid respectively, E the transepithelial potential difference (TEP), and z, F, R and T are, respectively, the ion's valency, the Faraday constant, the Universal gas constant, and the temperature. A TEP of N4 mV (lung lumen negative) was measured and the flux ratios predicted by the flux ratio equation were compared with the measured flux ratios. It was concluded that Na was passively distributed, but that K and C1 were actively secreted. A puzzling result was that iodide (added in trace amounts to plasma) appeared to be actively secreted, though it is known that iodide cannot substitute for C1 on the active transport process illustrated in Fig. 8.1. 40

FETAL L U N G

suggesting that baseline C1 secretion via anion channels other than CFTR is sufficient for normal lung development. In addition to beta-adrenergic agents and cAMP analogues, other agents shown to stimulate C1/fluid secretion by fetal lung explants are KGF, 45 FGF-10, 45 adenosine, ATP and UTP, 46 prostaglandins, 47 and atrial natriuretic factor. 48 In mice, K G F stimulates by a mechanism independent of CFTR; it stimulates fluid secretion equally in lung cysts from wild-type or CFTR knockout mice. 49 Thus, all results on fetal lung explants show that lung cysts secrete water in response to loop-diuretic active transport of C1, and that amiloride-sensitive absorption of Na is of little or no importance in regulation of transepithelial water transport.

EXPLANTS

When pieces of fetal lung are placed in organ culture, their open ends seal and they form cysts. 41 Water transport into these cysts can be estimated by their change in size. Also, they can be pierced with a microelectrode, and the sensitivity of their TEP to pharmacological agents used to indicate the types of ion transport present. When pieces of lung from 14- or 16-day gestation rats (term = 22 days) were maintained in culture for up to 3 weeks, the cells lining the cysts contained typical lamellar bodies, and cyst volume increased N3-fold from 1 to 2 weeks in culture. 42 At 1 week in culture the [C1] inside the cysts was 42% greater than in the bathing medium, even though the cysts had a luminal negative TEP of-~3 mV. In both 1- and 2,week-old cysts, TEP was essentially abolished by ouabain or bumetanide and increased 50% by the beta-adrenergic agonist terbutaline. In contrast, amiloride m i c r o i n j e c t e d into the lumen had no effect on T E P of 2-week cysts, and only a small inhibition on 1-week cysts (-20%). All of these results are consistent with the presence of cAMPmediated C1 secretion, and with active Na absorption being of trivial magnitude compared to active secretion of C1. When isolated dispersed type II cells from 18-day gestation fetal rats were plated on collagen gels they formed cysts with the apical membranes facing the cyst lumen. 43 A T E P o f - - 2 mV, lumen negative, developed across the cyst wall. A 24-h exposure to bumetanide decreased the total number of cysts and the volume of individual cysts. Both parameters were increased by 24-h exposure to cAMP analogues. Neither bumetanide nor cAMP altered 3H-thymidine incorporation. Explant cultures of human fetal lung behaved similarly to those from rat. 44 Thus, elevation of cAMP by a variety of maneuvers caused cysts to swell more rapidly than untreated controls. Further, the lumen negative TEP (--4 mV) was doubled or trebled by these agents, an effect inhibited by loop diuretics. 44 These explants were shown to contain CFTR, and the volume and TEP of explants from fetuses with CF did not respond to cAMP elevation. 44 Thus, CFTR is presumably involved in the cAMP-dependent stimulation of C1 secretion in these alveolar cysts. This is puzzling as lung development is normal in CF. However, baseline fluid secretion was normal in the CF explants, 44

P R I M A R Y C U L T U R E S OF FETAL LUNG E P I T H E L I U M Surprisingly, several studies using primary cultures of fetal type II cells have shown amiloride-sensitive Na+absorp tion, and have often failed to detect C1 secretion. When C1 secretion has been demonstrated, it is sometimes only under permissive conditions (e.g. with Cl-free solution in the mucosal bath, or after the apical membrane has been hyperpolarized by amiloride). The significance of these findings is uncertain, given that properties of cultured cells are very dependent on growth conditions and that most of the cultures used were somewhat dedifferentiated (they had lamellar bodies, but not as many or as large as in native type II cells). 3'5~ Also, of necessity most cultures were initiated from cells derived from fetuses close to term, and it is possible that they attained the adult phenotype during the several days they were kept in culture. All studies have detected small lumen-negative TEPs (_<5mV) and transepithelial resistances (TER) of 100-1000 ~ cm 2. To determine the ion transport processes responsible for TEP, investigators have used Ussing's short-circuit current technique, 51 in which TEP is clamped to zero by passage of current in an external circuit. This current is known as the short-circuit current (Isc), and under the appropriate conditions is equal to the sum of all active ion transport processes operating across the epithelium. Short-circuit current across tracheal epithelial cultures derived from fetal rats at days 18-20 of gestation was potently inhibited by amiloride, but bumetanide and DPAC, blockers of cAMP-dependent C1 secretion, were without effect. 52 Terbutaline stimulated Isc, but sensitivity to amiloride showed that this was due to stimulation of active Na absorption rather than C1 secretion. These cultures were derived from fetuses within 2-4 days of term, and their ion transport may have switched from C1 secretion to Na absorption during the culture period of 2-6 days. Consistent with this interpretation, the inhibition of Isc by amiloride was found to be greater in cultures derived from 21- than 18-day fetuses. 53 Further, bumetanide inhibited Isc across the 18-day but not the 21-day cultures. In other words,

these rat cultures were derived from cells poised at the neonatal transition from C1 secretion to Na absorption, and may have undergone this change in culture. Also, in cultures from 18-day gestation rats, amiloride caused a 15% inhibition of lsc, and the C1 channel blocker, DPAC, caused a 50% inhibition. 54 The dose of DPAC used (3 mM), however may have been toxic and inhibition by 0.3 mM DPAC was only 10%. Later studies on midgestation human epithelial cells or on 137-142-day fetal sheep cells (term= 145 days) showed clear-cut evidence for active cAMP-dependent C1 secretion that was at least as great as the levels of amiloride-sensitive Na absorption, s5-57 Interestingly, in both rat and human cells there is evidence that CI secretion is bumetanide-insensitive. For instance, in cultures of fetal human alveolar epithelium (18-24-week gestation), amiloride reduced Is~ by 20%, and ouabain by --80%, but bumetanide was without effect. 55 However, a 26% inhibition of I~c by DPAC was consistent with the presence of basal C1 secretion. Terbutaline, ATP, and ionomycin all stimulated I~ in cultures pretreated with amiloride, consistent with the presence of both cAMP- and Ca2+-activated CI channels. 55 The low I~r ( - 2 ~tA/cm2) and comparative insensitivity to blockers of both Na absorption and C1 secretion of primary cultures of type II AECs resemble results on early airway cell cultures. 58'59 With improvement in culture conditions, baseline I~ of airway cells increased > 10-fold and the proportion of Isc inhibited by amiloride and bumetanide likewise increased. 6~ Thus, low levels of differentiation of the alveolar epithelial cultures used in the above Ussing chamber studies may have made them poor models of native epithelium. Recently, type II cells cultured on a collagen gel with an air-liquid interface have shown a considerably improved ultrastructure, 62 though the increase in I~c was disappointingly small. 63 The cultures used in Ussing chamber studies of alveolar epithelial cell transport have always been derived from type II AECs. This reflects difficulties in obtaining isolated type I cells as well as a general belief that type II cells show higher levels of vectorial ion transport than type I cells. This belief is based on immunocytochemical studies that have demonstrated both Na-K-ATPase and ENaC on type II cells, but have failed to detect either protein on type I cells. 8'64'65 Recently, however, immunocytochemistry has demonstrated alpha and beta subunits of Na-K-ATPase, and alpha, beta, and gamma subunits of ENaC on type I cells. Furthermore, experiments on freshly isolated cells showed that type I and type II cells had similar levels of both ouabain-sensitive 86Rb uptake and amiloride-sensitive 22Na uptake. 66 These findings suggest that both type I and type II AECs are capable of ion transepithelial transport of salt and water.

FETAL AIRWAYS Ion transport by fetal airways has been studied with native epithelium in Ussing chambers, with cysts in explant

culture, and in cell cultures. All studies show that the predominant solute transport process is active Na-linked secretion of C1. In tracheal epithelium from fetal dogs, Isc was entirely accounted for by net C1 secretion (measured with radioisotope), as was the case in neonates up to 21-46 days after birth; at all times, net Na movement under short-circuit conditions was zero. 67 The same was true for tracheal epithelium from sheep fetuses. 33 In contrast, the bronchial and tracheal epithelia from the mother sheep had net C1 movement of zero and net Na absorption that approximately equalled the Isc.33 Also consistent with the preponderance of active C1 transport across fetal airway epithelium, TEP of tracheal explants from fetal rats was essentially abolished by bumetanide; amiloride was without effect. 42 Compared to cultures from adult animals, cultures of fetal rabbit tracheal epithelium showed a smaller inhibitory effect of amiloride on Isc (- 2% vs - 8%), and a similar effect of furosemide: - 17% in adult a n d - 19% in fetal cells. 68

I N T A C T A D U L T LUNG Transport of ions and water has been studied in several preparations of intact adult lung: isolated perfused lungs, lungs perfused in situ, non-perfused lungs in vitro, and lungs in 7)i7.)0. 69 Saline solution containing radioactive ions, radioactive sucrose or mannitol (markers of permeation through the paracellular pathway), and an impermeant solute (e.g. dextran) can be instilled into the lung. Fluxes from lung to perfusate (or blood) can then be measured. Water transport can be calculated from the change in concentration of the impermeant marker or, if isolated, simply by weighing the lung. The health of isolated lungs declines within a couple of hours, as manifested in a generalized increase in endothelial permeability. However, even without ventilation or perfusion, active clearance of fluid from the alveoli may persist for several hours, 7~and this preparation is obviously the only one suitable for studying intact human lungs. 71 In comparison, the in situ perfused lung has the advantages of preserving the lymphatic drainage and of avoiding the trauma associated with removing the lungs from the thorax. Viability is maintained for about twice as long as in isolated lungs. Finally, saline can be introduced into lung lobes of unanesthetized animals. This preparation has the disadvantage that liquid removal can only be determined onceat the end of the experiment, when the animal is anesthetized and the lung liquid removed. Another disadvantage of all intact lung preparations is that it is not possible to distinguish between the contributions of the alveolar and distal airways epithelia. In intact lungs, liquid absorption is stimulated -2-fold by instillation of beta-adrenergic agents and by cAMP. 69 Other agents known to stimulate alveolar liquid clearance include endotoxin, 72 TNF-alpha (by a cAMP-independent mechanism),73'74and TGF-beta. Liquid absorption is inhibited

40-70% by luminal amiloride. 69 Also, in perfused lungs, addition of ouabain to the perfusate inhibits liquid clearance by >90%. 70,75 Thus, all data are consistent with Na absorption being by the mechanism indicated in Fig. 8.1A; there is no evidence for active secretion of CI in these preparations of intact adult lung. Using isolated perfused rat lung instilled with saline, the addition of ouabain to the perfusate inhibited liquid absorption by -'-60%. 21'75 The inhibition by luminal amiloride was of similar magnitude. However, amiloride in combination with either phloridzin or glucose removal arrested fluid removal completely. A feature of these experiments was that the lung was perfused with blood from a second rat. Thus, the presence or absence of Na-glucose cotransport in various preparations may depend on hormonal influences. Recently, it has been suggested that amiloride-insensitive absorption of Na may play a role in lung liquid absorption. 76 In rat lungs in vivo, terbutaline increased liquid clearance by 85%. Amiloride and 1-cis-diltiazem (an inhibitor of cyclicnucleotide gated channels) inhibited nearly equal fractions of the terbutaline-stimulated liquid absorption. Furthermore, their actions were additive. A permeable analogue of cyclic GMP stimulated liquid absorption by 36%. Compared to terbutaline, its stimulatory effects showed more sensitivity to 1-cis-diltiazem. Others, however, found no evidence for the role of cyclic-nucleotide gated channels in lung liquid absorption from fetal sheep. 77

A D U L T TYPE II CELLS In 1982, Mason eta1. 78 cultured adult rat type II cells on permeable supports, and studied them in Ussing chambers. The small TEP (N 1 mV) was stimulated by terbutaline and abolished by amiloride. These results indicate that active amiloride-sensitive absorption of Na is quantitatively the most important active ion transport process operating across adult type II cells; C1 secretion was not detectable. This basic conclusion has been confirmed in several subsequent studies. 3 Most compellingly, the use of radioisotopes has shown that Isc across rat type II cell cultures was entirely accounted for by net absorption of Na; there was no active secretion of C1 under any experimental conditions. 79 Evidence for active absorption by such cultures comes not only from Ussing chamber studies but also from the development of domes when the cultures are grown on solid supports. Such "domes" are transient blisters where absorbed liquid accumulates between the epithelium and the growth support (generally a plastic petri dish). The domes grow until ruptured by hydrostatic pressure, and then subside. Both amiloride and ouabain reduce their numbers, s~

ADULT AIRWAY EPITHELIUM The predominant form of ion transport across most shortcircuited adult airway epithelia is active absorption of Na

and this is little affected by neurohumoral agents. 23 Some airway epithelia especially those of the trachea, however, may also show active secretion of C1 when short circuited. 23 When they do, this C1 secretion can be stimulated by increases in either intracellular cAMP or Ca 2+. However, it is questionable whether these tissues secrete much water under open-circuit conditions. Thus, the presence of a luminal-negative TEP o f - - 3 0 m V causes an equivalent depolarization of the apical membrane, reducing the driving force for C1 exit or even creating a driving force for entry. In fact, most of the adult airway epithelia that show net CI secretion under short-circuit conditions show net C1 absorption or no net C1 movement under open circuit. 23 Cultures of bovine tracheal epithelium show active C1 secretion when short circuited, sl Under open-circuit conditions, however, they absorb liquid at ---5 ~l/(cm2/h). 82 Furthermore, activation of CFTR by elevation of intracellular cAMP reduces TEP and increases liquid absorption -3-fold. These results are consistent with the electrochemical potential gradient for C1 being directed inwardly across the apical membrane and with passive transcellular absorption of C1. An activated C1 channel was identified as the route by which C1 crossed the basolateral membrane. 82

SWITCH FROM L I Q U I D SECRETION TO ABSORPTION The most important factors in the switch from C1 secretion to Na absorption at birth are hormones and labor. Changes in oxygen tension and possibly also in the extracellular matrix are important in maintaining the change. Induction of amiloride-sensitive Na transport is probably the key factor in lung liquid absorption at birth; other solute absorptive processes are relatively unimportant. In addition to water following the actively transported Na +, there is evidence that oncotic and hydrostatic pressure gradients may play a role in liquid absorption. The rate of liquid secretion declines shortly before birth 83-s6 but the rate at which liquid is lost from the lungs via the trachea does not change. 84 The end result is a decline in lung liquid volume. In the sheep, for instance, secretory rates of 7.4, 16.8, and 7.1 ml/h occurred at 119, 135, and 142 days of gestation, respectively, and the corresponding volumes of lung liquid were 51,105, and 70 ml. s4 The mechanism behind the slowing of secretion is unknown. Catecholamines are strongly implicated in the removal of lung liquid after birth (see below), but in the period immediately before birth lung water content often decreases before there is any detectable rise in plasma catecholamine levels, s3,85,s7 However, the concentration of pulmonary beta-adrenergic receptors increases late in gestation 8s-9~perhaps conferring greater sensitivity to circulating catecholamines. At birth, the human lung is filled with -120 ml of liquid, and upto about 30 ml of this may be squeezed out of the mouth during passage through the birth canal. 91'92 The rest is absorbed across the pulmonary and airway epithelia.

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Hours after birth Fig. 8.2. Filling of human lungs with air after birth. The linear regression of lung volume against time is statistically significant. However, the line fitted to the data is the best least-squares exponential approach to an asymptote, with an initial increase in volume of 75 ml/h. From Milner et al. 92

How long it takes for these epithelial transport processes to remove the bulk of the lung liquid is not precisely known. However, as shown in Fig. 8.2, the lungs of human newborns fill with air at an initial rate of--75 ml/h. 92 In contrast, liquid removal from the lungs of newly born sheep is at --25 ml/h for a total lung volume of---250 ml. 93 Thus, there are clearly species differences in the rate of clearance of liquid after birth, but in general it seems that it may take several hours for complete removal of lung liquid. All studies have shown that liquid removal at birth is inhibited by amiloride. 94-96 In newborn rat lung, blockers of Na/H exchange or Na-glucose cotransport were without effect on liquid removal. 95 In fetal sheep, the removal of lung liquid immediately after birth is associated with a surge in plasma epinephrine content 93 (Fig. 8.3). Further, there is a progressive increase in sensitivity to epinephrine between 122 and 142 days of gestation. Thus, at 122 days, infusion of epinephrine merely caused a slight inhibition of liquid secretion. At 133 days, epinephrine induced a small level of absorption with the critical concentration for epinephrine being ---0.5 ng/ml. 93 Close to term, at 142 days, the magnitude of the switch from secretion to absorption was considerably greater than at 133 days, and the critical concentration of epinephrine had declined to 0.08 ng/ml. The beta-adrenergic agent, isoproterenol, had similar effects on lung liquid movement as epinephrine, and the actions of both agents were inhibited by propranolol (a beta-adrenergic blocker); norepinephrine was without effect on lung liquid secretion. 97 To respond to epinephrine it appears that the pulmonary epithelium must first be primed with triiodothyronine (T 3) and hydrocortisone (HC), circulating concentrations of which progressively increase during ovine gestation. 98'99 Thyroidectomy of fetal sheep completely abolishes the actions of epinephrine on lung liquid absorption, 1~176 and infusion of T 3 restores responsiveness. 1~ However, fetal infusion of T 3 did not change the age at which responsiveness

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Fig. 8.3. Changes in lung liquid movement and maternal plasma epinephrine levels at birth in the sheep. (Reproduced with permission from Brown MJ, Olver RE, Ramsden CA et al. Effects of adrenaline and of spontaneous labour on the secretion and absorption of lung liquid in the fetal lamb. J. Physiol. 1983; 344:137-52.) See text for details.

to epinephrine first developed (i.e. ---122 days). But, if both T 3 and HC were infused (to produce plasma levels in the upper part of the range seen just before full term), then responsiveness to epinephrine could be induced in younger fetuses (116-120 days) than normal. 1~ Work with explants of fetal rat lung or trachea indicated that the effects of hormones on lung liquid absorption were mediated predominantly by actions on alveolar rather than airway epithelium. 1~ Thus, cultures of rat lung and trachea were initiated at 14 or 16 days of gestation, respectively. They were exposed to various hormones or blocking agents for varying periods (depending on hormone). Then on day 8 of culture, wet weights (a measure of liquid secretion) and TEP were measured. A combination of T3, HC, and terbutaline reduced the weight of lung buds relative to control, but was without effect on tracheal buds. Results on both untreated tracheas and lung buds were consistent with the presence of active C1 secretion. Thus, TEP was stimulated by terbutaline and inhibited by bumetanide. Further, pretreatment with bumetanide abolished the effects of terbutaline. However, pretreatment of lung buds for 2 days with HC + T 3 produced results consistent with the presence of active Na absorption. Thus, terbutaline now increased TEP in the presence of bumetanide, and this increase was blocked by amiloride. In contrast, in tracheal buds exposed to HC + T3, the terbutaline-induced increase in TEP was blocked only by bumetanide, not amiloride. Thus, only in the lung buds did the combination of T3, HC, and beta-adrenergic agent convert C1 secretion to Na absorption. In rabbits, treatment with an irreversible beta-agonist inhibited surfactant production but had no effect on lung water content at or after birth. TM In sheep, it has also been reported that propranolol does not prevent absorption of

liquid at birth. 1~ Of course, it is possible that occupancy of only a small fraction of the total number of receptors is needed for the effects of epinephrine. Nevertheless, these results do suggest that neurohumoral agents other than betaadrenergic agents could be involved in lung liquid clearance. In fact, arginine vasopressin (AVP), prolactin, and epidermal growth factor have all been shown to influence liquid secretion in the fetal lung. AVP, for instance, at concentrations within the range seen during labor, inhibited liquid secretion in > 140-day fetal sheep by -~80%, an effect that occurred without change in circulating epinephrine levels. 1~ Unphysiologically high concentrations of prolactin stimulate lung liquid production in fetal goats. 1~ EGF slows lung liquid secretion in fetal sheep even in the presence of beta-blockade. 1~ PGE 2 and atrial natriuretic factor have also been shown to reduce tracheal liquid production in fetal lambs. 48'1~ Lung liquid absorption is stimulated by labor. In 1-7day-old human newborns, the average thoracic gas volume of babies delivered vaginally was 32.7_+1.7 ml/kg, but in those delivered by caesarian section it was significantly lower (19.7_+ 1.4 ml/kg). 92 Likewise, in rabbits delivered by caesarian, the lungs contain more water than those delivered vaginally. 11~ Thus it appears that labor in some way stimulates removal of lung liquid. It is uncertain whether this is a mechanical effect or mediated by increased levels of epinephrine or other hormones. Fetal and postnatal O 2 tensions are very different: 3 and 14%, respectively. Carbon dioxide tensions also differ: ---8% in the fetus and 5% in the adult. To determine whether these changes contributed to neonatal lung liquid absorption, lung explants from 14-, 20-, and 22-day fetal rats and 2-day neonatal rats were exposed to fetal or adult PO 2 and PCO2 .112 The wet-to-dry weight ratios of 14-day fetal or 2-day neonatal explants were independent of gas tensions. However, the water contents of 20- and 22-day explants were markedly increased by fetal as opposed to adult gas tensions. Thus, in the perinatal period, fetal gas tensions favored liquid secretion. 112 The effects of gas tension have also been investigated in primary cultures of fetal distal lung epithelium. Within 4 h of switching primary cultures of fetal lung distal epithelium from 3 to 21% O2, there was a significant decline (--30%) in TER. 113Resistance then recovered, but amiloridesensitive Isc was increased by N2-fold at 18 h and ---8-fold at 48 h after the switch. The findings suggest that the initial decline in TER was associated with an increase in H20 permeability that in the intact lung would facilitate H20 removal down oncotic and hydrostatic pressure gradients. However, water removal by active Na absorption was more important later after birth. Other studies have confirmed that the effects of changing PO 2 are quite slow, with Isc being significantly elevated 24 h but not 6 h after a switch from fetal to adult oxygen tensions. 114'115 Thus, changes in PO 2 are more important at maintaining liquid absorption in the adult than in removing lung liquid at birth. Some of the developmental changes in ion and water transport across pulmonary epithelium may be effected by changes in the extracellular matrix. Thus, when isolated

fetal pneumocytes were grown on matrix derived from mixed lung cells at the canalicular phase, they reverted to an immature phenotype with less amiloride-sensitive Na absorption and more C1 secretion than when grown on a variety of other substrates. 116

MECHANISMS SWITCH

OF THE P E R I N A T A L

Hydrostatic pressures may contribute to lung liquid absorption at birth. The first breaths are associated with an increase in permeability to non-electrolytes that could in theory correspond to a large increase in hydraulic conductivity. 117 Also, the characteristic end-expiratory pauses (expiratory grunting) that occur between the newborn's first few breaths will create positive lung pressures 118 that should also promote liquid absorption. Further enhancing hydrostatic absorption of water is a drop in interstitial hydrostatic pressure with air inflation. 119'12~ An increase in water permeability should also increase movement of water down the oncotic gradient between lung liquid and interstitium, lung liquid being essentially protein-free. 32 An increase in plasma protein concentration in the last few days before birth increases the oncotic driving force. 119'12~ Stretch of the bladder epithelium results in insertion of ENaC into the apical membrane 121'122 and a dramatic increase in active absorption of Na. This effect has not been looked for in alveolar or airway epithelium. However, the first breaths presumably stretch the lung epithelium, and it has been reported that liquid expansion of the lung of fetal goats causes a decrease in liquid secretion or induces absorption. 123 By contrast, Vejlstrup et al. TM concluded that in rabbits "pulmonary inflation renders active liquid clearance ineffective". In sheep fetuses of 135-141 days gestation, epinephrine converted a secretion of 8 ml/h to an absorption of 16 ml/h. 96 Amiloride, added in the continued presence of epinephrine, returned liquid movement to a secretion of 5ml/h. 96 Ussing's flux ratio equation was applied to Na and C1 fuxes under baseline conditions, after administration of epinephrine and after epinephrine plus amiloride. Driving forces (mV) for Na under the three conditions were 0.6, -8.6, and 0.0mV, respectively. Thus, Na was passively distributed under baseline conditions (i.e. in equilibrium with TEP), out of equilibrium (i.e. actively absorbed) after epinephrine, and passively distributed in the presence of both epinephrine and amiloride. For C1, the corresponding driving forces were 21, 2.2, and 18 mV. Thus, epinephrine not only stimulated active absorption of Na, but also inhibited active C1 secretion. This suggests that the same cells perform Na absorption and C1 secretion, and by opening Na channels in the apical membrane, epinephrine will depolarize this membrane and inhibit C1 secretion. In contrast, induction of C1 secretion by amiloride is presumably due to a hyperpolarization caused by block of Na channels. 125 Interestingly, the epithelium can still secrete C1 in the presence of epinephrine indicating

that there have been no major changes in the numbers of C1 channels or basolateral NaK2C1 exchangers; the inhibition of C1 transport by epinephrine is solely due to an unfavorable change in the apical membrane potential difference. In the longer term, however, it seems likely that the transport proteins involved in C1 secretion (apical membrane C1 channels and the NaK2C1 cotransporter) will decline in numbers, whereas numbers of ENaC will increase. The mechanisms underlying the transition from C1 secretion to Na absorption at birth have been studied with isolated dispersed type II cells from the rabbit. Levels of ouabain-sensitive 86Rb uptake in fetal, neonatal, and adult pneumocytes were in the ratio 1:3:12, respectively. 126 Studies, in which ouabain-sensitive Rb uptakes were compared to pump numbers (determined from [3H]-ouabain binding), 127 showed that during the transition from fetus to newborn there was no change in the numbers of pump sites, but that turnover of individual pumps increased -4-fold. By contrast, in moving from newborn to adult, there was no change in turnover rate, but a 5-fold increase in pump density (from - 2 5 0 to ~-1000 pumps per l.tmz ofbasolateral membrane). The ouabain-sensitive uptake of Rb is driven by entry of Na. Thus, there must be a dramatic increase in the rate of Na entry at birth that increases the rate of Na-K-ATPase turnover presumably by elevating [Na]i. Evidence, discussed below, suggests that this increase in Na entry reflects greater numbers or higher levels of activation of ENaC. The increase in pump numbers between neonatal and adult cells may reflect the effects of chronically elevated [Na]i, a condition that leads to synthesis and membrane insertion of further Na-K-ATPase units in other cell types. 12s The sensitivity of 86Rb uptake to amiloride and loop diuretics has not been studied thoroughly. Nevertheless, 86Rb uptake into adult pneumocytes is inhibited -25% by amiloride, as also is uptake of 22Na.126 Bumetanide, however, also has small effects on Rb uptake in adult pneumocytes. 126'129 Clearly, adult cells may retain the capacity for at least some active secretion of CI. Also consistent with this conclusion is the demonstration of an apical membrane CI conductance in pneumocyte cultures from adult rats. 63'13~Detailed comparison of the effects of bumetanide and amiloride on ouabain-sensitive 86Rb uptake into fetal, newborn, and adult pneumocytes could determine whether the apparatus for CI secretion is dismantled at the same time that Na transport is increased. Rats also show changes in Na-K-ATPase levels in the neonatal period, but the timing is little different from the rabbit. Thus, Na-K-ATPase alpha 1 subunit increased from fetal day 17 to fetal days 20-22 and then declined in the early postnatal period. 65 Direct measurement of Na-KATPase activity showed a 2.6-fold increase between days 17 and 20-22. Also in rat, the expression of ENaC subunits has been studied during development, fetuses being harvested at 17-22 days of gestation. TM Alpha-ENaC was first detectable at 19 days and progressively increased in utero. Beta- and gamma-ENaC were not detected until 21 and 22 days. TM Comparing the timing of expression of Na-K-ATPase and

ENaC suggested that the latter may be more important in the perinatal changes in lung liquid transport. Measurements of apical membrane Na conductance (GNa) and basolateral Na-K-ATPase activity have been made in cultures of distal lung epithelial cells from fetal rats following a switch from 3 to 14% 02 .114'115 To study GNa, the basolateral membrane was permeabilized with nystatin, and gradients in [Na] were imposed across the remaining apical membrane. Conversely, to study Na-K-ATPase activity in the basolateral membrane, the apical membrane was permeabilized and ouabain-sensitive currents across the intact basolateral membrane were measured. Short-circuit current in intact cell sheets increased 6-24 h after the change in O 2 tension, and was associated with increased Na pump capacity. Levels of GNa and of alpha-ENaC promoter activity were raised at 24 h, but increased further by 48 h. Thus, increased levels of ENaC were seemingly a consequence rather than a cause of the increased Isc seen at 6 h. Absorption of lung liquid at birth may involve changes in permeability to water as well as changes in ion transport. Thus, in the alveolar epithelium of rats, expression of mRNA for aquaporin 4 increased 8-fold during the 2 days immediately before birth. 6 In contrast, levels of AQP-4 mRNA in brains and kidneys did not change. Further, switching from 4 to 21% O 2, a change known to participate in the neonatal switch from CI secretion to Na absorption, increased AQP-4 expression.

CONCLUSION In the fetus, active secretion of CI across both airway and alveolar epithelia drives water into the lung, and the resulting elevation of intraluminal hydrostatic pressure is essential important for normal lung development. At birth, lung liquid is removed with a half-time of an hour or so. Of several factors involved in this liquid absorption, quantitatively the most important is a switch in the primary active ion transport process operating across alveolar epithelium from C1 secretion to Na absorption. This switch is effected by the interplay of a number of hormones of which the ultimate is usually epinephrine. Changes in arterial O 2 tension and in the extracellular matrix help maintain liquid absorption. The perinatal increase in Na absorption is initially due to insertion of ENaC into the apical membrane, followed later by an increase in the numbers of Na-K-ATPase on the basolateral membrane. The perinatal decrease in active C1 secretion is probably mainly due to a change in apical membrane potential difference consequent on increased Na conductance.

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In humans, the ability of the lung to exchange respiratory gases is dependent upon many unique structural, physiological and biochemical features which must have developed by the time of birth for the fetus to survive the transition to extra-uterine life. The lung must have developed an intricate tree-like airway structure to conduct air to and from the respiratory gas-exchange surface. The gas-exchange regions must, collectively, have a large surface area which is closely apposed to a rich vascular network to enhance gas diffusion between air and blood. The mechanics of the respiratory system must be sufficiently mature to readily allow lung expansion during inspiration and prevent lung collapse during expiration. Many of these features are present or have begun to develop in the lung by the time of birth, despite the lung playing no role in gas exchange before birth. Thus, at birth, the lung must immediately assume a role that it has not performed before and, in most cases, they smoothly and efficiently take over the role of gas exchange at birth. However, in some pregnancies fetal lung development is compromised, leading to respiratory insufficiency, a major cause of neonatal morbidity and mortality. Inadequate fetal lung development can result from either an insufficient period of in utero development, due to premature birth, or to inappropriate development due to disruption of developmental processes. Because appropriate lung development during fetal life is essential for the successful transition to extra-uterine life, it is important to understand the factors that control prenatal lung development. One of the major differences between fetal and neonatal lungs is that, during fetal life, the future airways of the lung are filled with liquid. This liquid is produced by the lung; it *To whom correspondence should be addressed. The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

leaves the lung by flowing out of the trachea 1 and is either swallowed or enters the amniotic sac. 2 It is now evident that fetal lung liquid is secreted across the pulmonary epithelium into the future airspace due to an osmotic gradient established by the net movement of CI- in the same direction. 3 This Cl- gradient is thought to be generated by the Na/K ATPase pump located on the basolateral surface of the epithelial cells which creates the free-energy for Cl- to enter the cell, coupled with Na +, against its electrochemical gradient. 3'4 Cl- is thought to exit the cell down its electrochemical gradient across the apical membrane into the lung lumen through specific Cl- channels (see Chapter 8). It is now recognized that fetal lung liquid plays an integral role in the development of the lung before birth by maintaining the lungs in a constantly distended state. Indeed, the lungs are not collapsed during fetal life but are maintained in a state of expansion that is greater than the end-expiratory volume of the postnatal, air-filled, lung 5'6 (see Fig. 14.1). The decrease in basal lung volume at birth results from an increase in lung recoil due to the creation of surface tension upon the entry of air into the lungs. By maintaining the fetal lungs in a distended state, fetal lung liquid is thought to be essential for fetal lung growth and maturation by acting as an internal "splint" for lung tissue. 5-7 As a result, much attention has focused on how altering the basal degree of lung expansion affects fetal lung growth and development, as discussed in detail below. Similarly, the effect of phasic alterations in lung expansion, as result of fetal breathing movements (FBM), has also been extensively studied and is detailed below. Together, the basal degree of lung expansion and FBM are considered to be important physical factors that influence fetal lung growth and development. However, it is clear that circulating Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

endocrine factors as well as paracrine growth factors can mediate, potentiate, integrate and regulate the lung growth in response to these physical factors.

ROLE OF PHYSICAL FACTORS IN R E G U L A T I N G FETAL L U N G GROWTH It is often stated that the lung is either collapsed during fetal life or is maintained at the same degree of expansion as after birth. However, it is now apparent that healthy fetal lungs are expanded to a greater degree before birth than after birth (see Chapter 14). Although there has been some controversy in the literature as to the exact volume of liquid retained in the future airways during the later stages of gestation, data from fetal sheep show that the volume is 35-45ml/kg 6'8-1~ which is considerably higher than the end-expiratory lung volume in the neonate 6 (25-30ml/kg; see Fig. 14.1); some studies have reported volumes as high as 60 ml/kg in individual ovine fetuses. 1~The discrepancies in fetal lung liquid volumes reported in the literature are probably because the fetus actively participates in maintaining its lung volume (see below) and that the physical environment of the fetus also influences the volume of lung liquid. Thus, values that have been reported from dead (e.g. histological measurements), anesthetized (and usually exteriorized) or paralysed fetuses will underestimate lung luminal volumes because lung liquid is rapidly lost following death, anesthesia and paralysis. 1~ Similarly, measurements of lung volumes in chronically catheterized fetuses (usually fetal sheep) are questionable unless it was verified that a sufficient volume of amniotic fluid was present at the time of measurement and that animals were not in labour. 8'12

following a reduction in volume is by a decrease in lung liquid efflux. 16

Role of the fetal upper airway and the trans-pulmonary pressure gradient in regulating fetal lung liquid volumes The rate of liquid efflux from the fetal lung is dependent upon the pressure gradient between the lung lumen and amniotic sac (trans-pulmonary pressure) as well as the resistance to liquid efflux through the upper airway, predominantly the glottis. 1'17 During apnea, the intra-luminal pressure within the fetal lungs is 1-2 mmHg above amniotic sac pressure is which is the driving force for liquid to leave the lungs (see Fig. 9.1). This pressure gradient is created by the inherent recoil of lung tissue and is maintained, during periods of apnea, by active adduction of the glottis which provides a high resistance to liquid movement through the tracheal7; this is analogous to constricting the neck of an inflated rubber balloon (Fig. 9.1). Thus, during apnea, liquid

ADnea

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R E G U L A T I O N OF THE BASAL DEGREE OF L U N G EXPANSION IN THE FETUS The basal degree of fetal lung expansion is determined by the volume of liquid retained within the future airways. As this profoundly influences fetal lung development, it is important to understand the factors that regulate lung liquid volumes in the fetus. In theory, lung liquid volume should be controlled by a balance between the rate at which it is secreted and the rate at which it flows out of the lungs via the trachea. In practice, however, the volume of fetal lung liquid (in the absence of labour) is controlled by its rate of efflux from the trachea, as alterations in the rate of fetal lung liquid secretion simply cause corresponding changes in the efflux of liquid, resulting in little change in lung liquid volume; this has been clearly demonstrated following reductions in fetal lung liquid secretion. 13 Similarly, although prolonged reductions in fetal lung expansion increase fetal lung liquid secretion rates in vivo, 11'14'15 the principal mechanism for restoring fetal lung liquid volume

lung liquid ............. secretion

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Fig. 9.1. Diagram showing the function of the upper airway in regulating the efflux of lung liquid during periods of apnea (upper panel) and fetal breathing movements (lower panel). During periods of apnea, the resistance to lung liquid efflux through the upper airway is increased. As a result, liquid tends to accumulate within the future airways causing the lungs to expand, generating a trans-pulmonary pressure gradient of 1-2 mmHg (intra-luminal > amniotic sac pressure). During periods of FBM, the resistance of the upper airway decreases and accumulated liquid leaves the lungs at an increased rate. As a result, the trans-pulmonary pressure gradient decreases towards 0mmHg during FBM. Reproduced from Harding and Hooper, 6 with additional data from Harding et al. 17

tends to accumulate within the fetal lungs. However, during FBM the resistance to liquid efflux through the upper airway is actively reduced, 17 due to phasic abduction of the glottis, which increases lung liquid efflux 17 (Fig. 9.1). Thus, during FBM, despite contraction of the diaphragm, there is a net loss of liquid from the lungs which is 2-3 times greater than the loss during intervening apneic periods. 1'19 Liquid can, however, enter the fetal lungs during episodes of vigorous FBM, which may be related to changes in upper airway function1 or to changes in trans-pulmonary pressure. The trans-pulmonary pressure gradient of the fetus is predominantly generated by the intrinsic recoil of lung tissue, but because the chest wall in the fetus is highly compliant, 2~ it is also influenced by external factors like abdominal pressure. Thus, increases in abdominal pressure, which could occur due to changes in fetal posture (e.g. trunk flexion), increase intra-luminal pressures, which increase the trans-pulmonary pressure gradient leading to a loss of lung liquid 12 (Fig. 9.1). These changes in fetal posture could arise from fetal movements or could be imposed on the fetus by limited intra-uterine space. 12For example, if the mechanical buffering effect of amniotic fluid is lost, as in oligohydramnios, the uterus may compress the fetus thereby increasing trunk flexion (Fig. 9.2). 12'21 This increases (-2-fold) fetal abdominal pressure, which increases the trans-pulmonary pressure gradient leading to increased lung liquid efflux (see Fig. 9.3); 12 a similar scenario may explain the loss of large quantities of lung liquid during early labour, s An increase in lung compliance late in gestation, associated with increased circulating corticosteroid levels, will greatly increase the volume change caused by even small changes in trans-pulmonary pressure. Thus, as fluid space within the intra-uterine compartment can become increasingly reduced in late gestation, particularly in multiple pregnancies, it is not surprising that different researchers have found a wide variety of lung liquid volumes late in gestation.

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Fig. 9.3. The increase in fetal tracheal pressure and lung liquid efflux associated with non-labour uterine contractions during a control period, 48h of oligohydramnios and a recovery period. These data demonstrate that, if intra-uterine volume is limited, non-labour uterine contractions increase fetal abdominal (data not shown) and tracheal pressures, resulting in an increase in liquid efflux from the fetal lungs. Data obtained from Harding et al. 12

Role of the chest wall in maintaining fetal lung liquid volumes 1.0 or) .m

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Fig. 9.2. The effect of oligohydramnios induced by drainage of amniotic fluid on the degree of spinal cord flexion (measured as a normalized spinal radius) in fetal sheep. A smaller normalized spinal radius is indicative of a greater curvature of the spine. Data obtained from Harding et al. 12

After birth the stiffness of the chest wall plays an important role in maintaining end-expiratory lung volume by opposing lung recoil and preventing lung collapse. The tendency of the lung to collapse away from the chest wall generates a negative intra-pleural pressure o f - 5 cmH20. In contrast, the fetal lung is maintained in a distended state by the retention of liquid within the future airways, resulting in a distending pressure of 1-2 mmHg during apnea. TMAs a result, at rest, the intra-pleural pressure is essentially zero. 18 This indicates that the chest wall plays little if any mechanical role in maintaining lung volume at rest, although the dome of the diaphragm extends into the chest, as in adults, indicating that its position is influenced by lung recoil and/or abdominal pressure. Consequently, it is possible that the growth and shape of the chest wall is influenced by the volume of the fetal lung. 21 For example, infants with severe pulmonary hypoplasia characteristically have bell-shaped chests which could result from a combination of the small

size of the lung as well as its reduced compliance and increased recoil. The effects of diaphragmatic contractions on fetal lung liquid volumes are discussed below.

E V I D E N C E FOR THE ROLE OF L U N G E X P A N S I O N IN FETAL L U N G GROWTH AND DEVELOPMENT Much experimental and clinical evidence has accumulated to indicate that growth and maturation of the fetal lung are critically dependent upon the degree to which it is expanded by liquid. Sustained reductions in lung expansion retard the growth and structural maturation of the fetal lung, whereas these processes are accelerated by sustained increases in lung expansion.

Clinical evidence A wide variety of disorders result in fetal pulmonary hypoplasia including oligohydramnios, congenital diaphragmatic hernias (CDH), space-occupying lesions like pulmonary cysts, tumours and pleural effusions, as well as a number of fetal muscular-skeletal deformities. Although these disorders are diverse in nature, they all share a common mechanism by which they induce fetal lung hypoplasia, namely a prolonged reduction in the degree of fetal lung expansion. 21 Oligohydramnios is a relatively common clinical problem occurring in approximately 10% of all pregnancies. It can result from the loss of amniotic fluid due to premature rupture of the membranes or from inadequate production of amniotic fluid due to urinary tract disorders, including bilateral renal agenesis, renal dysplasia as well as disruptions to urine outflow into the amniotic sac due to agenesis or stenosis to the ureters, urethra or urethral valve. 21 The severity of the lung growth deficit varies considerably between individuals depending upon a number of factors, particularly the gestational age at onset and the duration of exposure. 22 Depending upon these factors, the resulting pulmonary hypoplasia can be lethal within hours of birth in its most severe form, but can be sub-clinical during the neonatal period in less severe forms possibly contributing to neonatal respiratory distress. The mechanism by which oligohydramnios causes a reduction in fetal lung liquid volume is via an increase in the trans-pulmonary pressure gradient. 12 In the absence of amniotic fluid, the intra-uterine space is markedly reduced and the uterine wall compresses the fetus causing exaggerated flexion of the fetal trunk (Fig. 9.2). 12'21 This leads to an increase in abdominal pressure which results in an increase in the trans-pulmonary pressure gradient and the loss of lung liquid (Fig. 9.3). 12'23 The degree of compression imposed by the uterus can be so severe that it causes marked facial and limb disorders. 24 CDH is less common than oligohydramnios, but can result in very severe pulmonary hypoplasia. The mortality rates associated with this disorder vary widely between studies, arguably due to differences in the inclusion/exclusion of

subjects, but are usually reported as > 50~ 25'26 CDH is sometimes associated with other fetal malformations and occurs due to failure of the diaphragm ligament to close, thereby failing to separate the chest from the abdomen during embryonic development. 27 The hernias can either be unilateral (both left- or right-sided) or bilateral and allow abdominal contents to migrate into the thorax, thereby preventing the lung from expanding; in its most severe form, the liver can herniate into the chest. 27 It is not clear whether the abdominal contents compress the lung or simply occupy thoracic space, thereby preventing the lung from expanding and occupying that space. However, the latter mechanism is clearly consistent with the mechanism by which turnouts, cysts and intra-pleural fluid accumulation cause pulmonary hypoplasia. A variety of muscular-skeletal disorders can also result in severe pulmonary hypoplasia. 21 Although the precise mechanisms by which these disorders induce pulmonary hypoplasia will depend on the type of disorder, the mechanism likely involves a reduction in lung expansion. That is, any disorder which reduces the ability of the fetus to defend its lung volume will likely result in pulmonary hypoplasia. For instance, diaphragmatic activity and glottic adduction play important roles in maintaining lung expansion during fetal development 12 and, therefore, interfering with these activities can impact upon fetal lung liquid volumes (see

Fig. 9.4).

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Fig. 9.4. The influence of fetal muscular activity on defending the volume of liquid retained within the future airways. Compared with control fetuses (solid column), the inhibition of fetal breathing movements (FBM) by either fetal spinal cord transection TMor selective blockade of the phrenic nerves, 11 causes an -25% decrease in fetal lung liquid volume (open column), demonstrating the importance of fetal diaphragmatic activity in maintaining the volume of fetal lung liquid. If upper airway resistance (UAR) is eliminated (by by-passing the upper airway) in addition to the inhibition of FBM, the volume of lung liquid is reduced further, demonstrating the independent effect that the upper fetal airway has in maintaining fetal lung liquid volumes. The further reduction in the volume of lung liquid following the removal of the lungs from the fetus demonstrates the contribution that the fetal chest wall makes to maintaining fetal lung expansion. Diagram reproduced from Harding and Hooper. 6

Experimental evidence Most experimental evidence indicating that alterations in fetal lung expansion regulate the growth and development of the fetal lungs is derived from experiments in which the fetal trachea has been obstructed or the lungs drained of liquid. Fetal tracheal obstruction was first performed to demonstrate that fetal lung liquid is produced by the fetal lung and is not inhaled amniotic fluid. 28'29 Following tracheal ligation, the lungs accumulated liquid within the future airways, causing them to overexpand, but it was also noted that the lungs were larger and structurally more mature. 29 Subsequent experiments demonstrated that prolonged periods of tracheal ligation in fetal sheep increased fetal lung weights, whereas prolonged periods of lung deflation, caused by lung liquid drainage, reduced fetal lung weights. 7 It was also noted that reductions in fetal lung expansion increased the density of type-II cells. 7 In fetal sheep it has been demonstrated that the lung growth response to increased lung expansion is dependent on local factors within the stretched tissue, rather than circulating factors. 3~ Prolonged ligation of the left bronchus caused overexpansion and increased growth of the left lung, whereas the fight lung, which remained at a control level of expansion, remained at the same size as a control right lung. 3~ More recent studies have advanced this concept by showing that, although alteration in lung expansion at the local level is the primary determinant of lung tissue growth, circulating endocrine factors can influence the relationship between lung expansion and lung growth. For example, the lung growth response to an increase in lung expansion depends upon growth hormone both in the fetus (following tracheal obstruction) 31 and after birth (following hemipneumonectomy). 32Furthermore, exogenous cortisol enhances the fetal lung growth response to an increase in lung expansion, most probably via an increase in lung compliance. 33 Prolonged reductions in fetal lung expansion reduce lung growth rates, and the extent of the growth rate reduction appears to depend on the decrease in lung expansion. A 25% reduction in lung expansion causes a 25% reduction in lung DNA content, TM whereas total lung deflation, due to lung liquid drainage, causes lung growth to cease.7'15This cessation in growth results in severe pulmonary hypoplasia within 3-4 weeks. 7'15'34 Understanding the mechanisms by which sustained changes in fetal lung expansion alter lung growth is important because these mechanisms are likely to involve those responsible for regulating normal lung growth. In the case of increased lung expansion induced by tracheal obstruction, the growth-promoting processes are not continuously active, but show a time dependency which may depend on how the lung expands. 35 A detectable increase in lung DNA content can be measured within 2 days of tracheal obstruction in fetal sheep 36 and the increase in growth is completed within 7 days, resulting in an almost doubling (-70% increase) in DNA content. 35'37However, the rate of lung cell proliferation is not uniform throughout this period (Fig. 9.5) and appears to differ in different species and at different stages of lung

Fig. 9.5. Diagram showing how the extracellular matrix (ECM) receptors (integrins) mechanically couple the intracellular microfilaments with the ECM, to form a structural continuum. Proteins associated with the intracellular domain are associated with numerous intracellular signalling pathways 72 which may mediate the response to alterations in fetal lung expansion. Diagram reconstructed from Ingber etal. 6z and Rubin et al. 72

development. In fetal sheep, increased DNA synthesis rates peak within 2 days of obstructing the fetal trachea during the alveolar stage of lung development, 35 whereas this same period corresponds to a "stagnation in lung growth" during the pseudoglandular stage of lung development in fetal rabbits. 38 These differences most probably reflect the different stage of lung development at the time of obstruction rather than species differences. Indeed, no stagnation of lung growth was subsequently observed in older fetal rabbits following tracheal obstruction. 39 It is now known that the type and rate of lung growth induced by an increase in lung expansion differs at different stages of lung development. Although the rate of accelerated lung growth induced by tracheal obstruction is less in fetuses at the late pseudoglandular to early canalicular stages of lung development than during the alveolar stage, the eventual increase in lung size and DNA content is greater in younger fetuses. 36'4~The slower rate of accelerated growth is thought to be due to a lower rate of lung expansion in the younger fetuses, which have stiffer lungs, 36 whereas the greater final increase in lung size following tracheal obstruction in younger fetuses is thought to be due to a more compliant chest wall which allows the lungs to expand to a greater degree. 4~ However, a greater degree of lung expansion may also be responsible for the hydropic state that has been observed in fetal humans 41'42and sheep 4~when tracheal obstruction is performed during pseudoglandular/ canalicular stage of lung development; the hydrops is probably due to a restriction in venous return or the expanded lungs constraining the heart. Furthermore, the characteristics of the lung growth induced at the different stages of lung development is very different. During the alveolar stage, tracheal obstruction causes the proliferation of most major cell types in the lung, 43 but during the late pseudoglandular/ early canalicular stage it predominantly causes proliferation of mesenchymal cells. 4~ As a result, the inter-airway tissue distances markedly increase resulting in a large increase in the percentage of space occupied by tissue. 4~In contrast, these tissue distances are reduced when tracheal obstruction is performed during the alveolar stages of lung development,

resulting in marked reductions in the percentage of space occupied by tissue. 43 Thus, care should be taken when comparing the effects of increased lung expansion between species and at different stages of development. During the alveolar stage of lung development, the mechanisms responsible for the acceleration in lung growth caused by tracheal obstruction are maximally activated within 2 days. 35 Using a modified version of the left main bronchus ligation model, 3~ a recent study used a differential gene analysis technique to identify genes activated and suppressed during this maximally activated growth phase. 44 The first differentially expressed gene identified was calmodulin-2 which could be an important intracellular mediator of the proliferation induced by an increase in lung expansion. The effect of alterations in fetal lung expansion on the fetal lung are not restricted to growth. Increases in fetal lung expansion also accelerate structural maturation of the lung in a variety of species, causing a reduction in the percentage of tissue space due to a reduction in inter-alveolar tissue as well as an increase in alveolar number and surface a r e a . 7'38'39'43'45 In contrast, reductions in fetal lung expansion greatly retard structural development of the lung, resulting in marked increases in inter-alveolar tissue space and reduced alveolar development, particularly alveolar number. 7 In addition, alterations in fetal lung expansion have a profound impact on alveolar epithelial cell (AEC) differentiation. Increases in fetal lung expansion induce type-II AECs to differentiate, via an intermediate cell type, into type-I AECs such that within 10 days of obstructing the fetal trachea, <2% of AECs are of the type-II phenotype, whereas over 90% are of the type-I phenotype. 46 In contrast, reductions in fetal lung expansion promote differentiation into the type-II AEC phenotype. The most recent in vivo evidence indicates that type-I AECs, at least in the fetus, are not terminally differentiated and can trans-differentiate into type-II cells. 47 Furthermore, it is important to note that these changes are reversible and, therefore, a prolonged increase in fetal lung expansion may not cause irreversible reductions in type-II cell numbers. 47-5~ These findings are consistent with the changes in surfactant protein gene expression observed following alterations in fetal lung expansion. 47'51

EVIDENCE FOR THE ROLE OF FETAL B R E A T H I N G M O V E M E N T S IN FETAL L U N G G R O W T H A N D DEVELOPMENT Rhythmic activation of respiratory muscles begins early in fetal life and is referred to as fetal breathing. The neuronal activation of these FBM is similar to that of postnatal breathing, except that FBMs are discontinuous, particularly later in gestation. 52'53 FBM principally result from rhythmic activation of the diaphragm which causes small reductions in intra-thoracic pressure; the muscles which dilate the glottis are activated in phase with the diaphragm. 17 The fetus makes other inspiratory efforts including gasping and hiccuping, although the latter is not respiratory in origin. Fetal gasping

can be induced by severe asphyxia which causes intense activation of inspiratory muscles and is probably a primitive reflex response aimed at increasing pulmonary ventilation. 54 Much interest has focused on FBMs because they are used clinically in the assessment of fetal health and are thought to be an important determinant of fetal lung development.

In vitro evidence The in vitro evidence supporting the concept that FBMs play a critical role in fetal lung development is centred around studies in which lung cells in culture are stretched in a manner purported to simulate FBM. Although there are now a number of experimental variations to this protocol, the original studies used an electromagnet to stretch the matrix that lung cells were cultured on; the cells are usually stretched 60 times/rain with a percentage stretch of >5%. 55 These experiments have shown that simulated FBMs in vitro stimulate lung cell proliferation, causing a time-dependent increase in DNA synthesis. 56 The proliferation is thought to be mediated by increased synthesis of platelet-derived growth factor (PDGF) as the cellular proliferation can be inhibited by antisense oligonucleotides for PDGF-B. 57 PDGF is thought to act via both PDGF alpha and beta receptors to induce prenatal lung growth, 5s most probably by activating phospholipase C and PKC intracellular pathways. 56 Although these experiments have provided useful information on the transduction pathways by which mechanical stimuli initiate lung cell proliferation, the stimulus does not accurately simulate FBM in vivo. Indeed, in vivo, individual breathing movements are essentially iso-volumic and, therefore, the percentage length change experienced by a cell with each FBM is negligible. This is primarily because the fetal chest wall is very compliant, fetal lung liquid is very viscous compared with air and, due to its bulk, has a large inertia. Thus, although activation of the diaphragm causes a reduction in intra-thoracic pressure, very little liquid is inhaled because other sections of the chest wall are simultaneously drawn in; 59 indeed liquid has to be present within the pharynx before any liquid can be inhaled. 17 As a result, tidal volume in the late-gestation fetus is < 1% of resting lung volume, but is much higher immediately after birth (---20%). As the resistance to liquid movement through the upper airway during FBM is 3-4 mmHg/(ml/min) in fetal sheep, 17 the pressures required to move a volume equivalent to 5% of resting lung volume would be much greater than intratracheal pressure fluctuations during FBM. 18 In vivo evidence The in vivo evidence supporting the concept that FBMs play an important role in fetal lung development involves studies in which their thoracic components have been eliminated. Sectioning the phrenic nerves in the fetus causes a reduction in fetal lung growth, 6~ but also causes atrophy of the diaphragm resulting in its upward displacement into the thorax, resulting in a reduction in lung expansion. Paralysis of the fetal diaphragm, without causing its atrophy,

can be achieved by sectioning the spinal cord (between C1 and C2) above the level of the phrenic motoneurones. Although this procedure results in a reduction in fetal lung growth, 14'62'63 this decrease is likely due to an associated reduction in lung expansion. 14 Indeed, the reduction in lung expansion (--25%) equates to the reduction in lung DNA content (--25%) and is caused by blocking activation of the diaphragm without blocking activation of the glottis; TM the glottis is innervated by the recurrent laryngeal nerves which are not affected by spinal cord transection. The net effect is increased loss of lung liquid and a reduction in lung expansion due to continued phasic dilation of the larynx (Fig. 9.4), TMas diaphragmatic contractions inhibit the efflux of lung liquid during FBM episodes. Similarly, reversible pharmacological blockade of both phrenic nerves increases lung liquid efflux and leads to a reduction in lung liquid volume which is quickly restored after the phrenic nerve blockade is removed. 11 Thus, it is not surprising that total fetal paralysis leads to increased lung liquid efflux, a reduction in lung liquid volume 11 and a decrease in lung growth. 64 Taken together, these data provide strong evidence that fetal muscular activity, whether it is active glottic adduction during apnea, or activation of the diaphragm during FBM, plays a critical role in maintaining lung liquid volumes and hence the mean level of fetal lung expansion. However, at present there is no in vivo evidence to suggest that phasic stretch of the lung during FBM is an important determinant of fetal lung growth, other than by defending lung liquid volumes. Even the finding that bilateral thoracoplasty induces lung hypoplasia can also be explained by a decrease in fetal lung expansion. 65

MECHANO-TRANSDUCTION MECHANISMS An understanding of the transduction pathways by which mechanical forces are translated into chemical stimuli is important as all cells, tissues and whole organs of the body are subjected to mechanical forces in vivo. These forces include shear stress, strain, stretch and compression and can result from gravity, fluid flow, intracellular tensile forces (e.g. muscle contractions), ambulatory body movements, as well as from changes resulting from the expansion of organs such as the bowel, stomach, uterus, heart, bladder and lung. It has been proposed that cells exist in a state of isometric tension that is generated by the intracellular contractile filaments. 66 Thus, externally applied forces are thought to be imposed on a pre-existing force equilibrium, causing changes in cell shape and intracellular structural fibre alignment until the force equilibrium is re-established. 66'67 It has been recognized for many years that a variety of mechanical forces play an important role in cellular growth and differentiation and is a critical regulator of threedimensional tissue structure, 67 particularly in the lung. 5'68 Thus, they are an important pathway by which cells interact with and respond to their environment. 66'67'69 In general, the response of a cell to a mechanical force is to reduce the

impact of that force on the cell. For example, an attached cell exposed to a flow of fluid across it will act to reduce the shear force by orientating the longitudinal axis of the cell in the direction of flow and adopting a streamlined shape. 7~ Cells exposed to increased strain will align their intracellular structural fibres along the principal direction of strain and eventually recruit, synthesize and align new intracellular structural fibres also along this plane. 71 In addition, a cell exposed to strain may synthesize ECM components that form an extracellular framework to help resist the load. 71 Other cellular responses to strain include cellular proliferation and differentiation 67'68which can lead to marked changes in the size, structure and function of an organ.

The role of the ECM and "outside-in" cell signalling The discovery of cell-surface receptors that bind to a variety of ECM proteins has greatly advanced our understanding of mechano-transduction mechanisms. For example, the "integrin" family of trans-membrane proteins cluster at focal adhesion sites and bind to a specific sequence, arg-gly-asp (RGD), that is common in many ECM proteins. 72 The intracellular domains of these ECM receptors are mechanically linked to fibrillar-actin bundles, via a variety of cytoskeletal-associated proteins (e.g. talin, vinculin, paxillin) and are closely associated with a number of protein kinases. 72 As actin bundles form a major component of the intracellular structural scaffolding, it is clear that the intracellular and extracellular components are mechanically coupled, via ECM receptors, to form a structural continuum (Fig. 9.5). It is via these couplings that mechanical forces can be detected and translated into intracellular chemical signals. 67'72'73 The intracellular signalling pathways are less well defined, although they are thought to include stretch-activated ion channels, activation of intracellular second messenger systems and the direct activation of RNA polymerases and DNA synthetic enzymes via changes in nuclear shape. Indeed, the structural continuum between a cell and its surrounding ECM includes the nucleus, which is connected to the cell surface via intermediate filaments. Thus, mechanical forces that distort cell shape will also alter the shape of the nucleus, which can influence gene transcription and DNA synthetic machinery via pathways that are currently not understood. 67'69 Nuclear DNA is attached to the structural scaffolding of the nucleus, the spindle microtubules, via kinetochore proteins located at the centromeric regions of chromosomes. It is not difficult to envisage, therefore, that mechanical forces may influence DNA replication and gene transcription via direct manipulation of DNA and associated enzymatic pathways. The development of integrin-mediated cell adhesions also results in the relocation of pre-existing mRNA molecules and protein translation machinery to the focal adhesion to induce a rapid enhancement of protein translation at the site of cell attachment. TM

Potential cytoplasmic second messenger systems In addition to effects mediated by the cytoskeleton, focal adhesion sites also include numerous signalling molecules

that are activated or inactivated by activation of ECM receptors (e.g. integrins). The signalling molecules associated with focal adhesion sites include focal adhesion kinase (FAK), which is an intracellular tyrosine kinase that binds to a number of other signalling and structural proteins, including PI-3-kinase, Src, Grb2 and pl30Cas (Fig. 9.6). Activation of PI-3-K leads to activation of the inositol triphosphate pathway, PKA and PKC as well as enhancing calcium signalling. Activation of the Src family ofintracellular tyrosine kinases leads to the phosphorylation of a number proteins, leading to the recruitment and activation of downstream signalling molecules such as PLC-y, the Rho-like GTPases and the ERK and JNK signalling cascades (Fig. 9.6). Indeed, there is extensive cross-talk between integrin activation and the growth factor activated ERK signalling pathways that generally leads to enhanced and sustained ERK activation (Fig. 9.6). 75-77 This includes numerous mechanisms leading to the activation of Grb2 which, through its association with Sos, leads to the activation of Ras and the subsequent activation of Raf, MEK and ERK proteins and eventually to altered gene transcription. Similarly, Raf can be activated by a number of mechanisms independent of Ras. Apart from these intracellular signalling pathways, integrin clustering can also lead to the direct activation of a number of growth factor receptors in the absence of the growth factor ligand. 7s'79

Potential role of growth factors It is often assumed that any stimulus that induces cellular proliferation must be driven by growth factors, but the roles of growth factors in mediating expansion-induced fetal lung growth, at least in vivo, are unclear. Although mechanical forces are potent stimuli of cell behaviour in their own right, it is important to recognize that the application of mechanical forces in vivo (e.g. due to alterations in lung expansion) will manifest as a variety of different mechanical stimuli on different cells depending upon their location within the three dimensional structure of the lung. For instance, in response to an increase in lung expansion, alveolar epithelial cells, lying on the internal surface of the terminal gasexchange regions, will be exposed to stretch as will some capillary endothelial cells, particularly those whose basement membranes are fused with the epithelial basement membrane. On the other hand, many cells will experience compression due to terminal airway expansion. It is of interest, therefore, that an increase in fetal lung expansion induces proliferation of most major cell types within the lung and induces a coordinated growth response across the entire portion of stretched lung during the alveolar stage of lung development. 43 Thus, cells located in positions that expose them to mechanical stimuli like stretch may release growth factors which potentiate and integrate the response in adjacent non-activated cells. Similarly, remodelling of the ECM may release extracellularly bound growth factors which act to potentiate and integrate the growth response.

ROLE OF G R O W T H FACTORS IN FETAL L U N G DEVELOPMENT The role of growth factors during embryonic lung morphogenesis has been described in Chapter 1. This section wilt briefly cover the roles of the numerous growth factors and their receptors that are expressed in the lung during the later stages of lung development and that are likely to play roles in the finely coordinated development of the lung, including airway growth and branching, alveolarization, ECM remodelling, epithelial cell differentiation and angiogenesis. Particular emphasis is placed on those factors that may play a role in transducing the growth response of the lung to alterations in lung expansion/stretch. There is much evidence that the PDGF A and B and their receptors PDGFR-tx and -[3, are involved in the earlymid stages of lung development. PDGF-A and B mRNA levels increase 8~ in parallel with cell proliferation rates 82 during the pseudoglandular and canalicular stages of lung development, and cell proliferation within lung explants is inhibited by PDGF-A and B antisense oligonucleotides as well as PDGF neutralizing antibodies. 57'83 Tight regulation of PDGF-A is important as the PDGF-A knockout mouse exhibits a complete failure of alveolar septation, sl whereas overexpression of PDGF-A causes marked mesenchymal cell proliferation within the lungs, which fail to progress to the saccular stage of development, s4 Both PDGF-A s5 and B 86 have sheer stress-responsive elements (SSRE) in their promoter regions suggesting their expression levels can be influenced by exposure of cells to physical forces. This is supported by studies showing that cyclic stretch of cultured lung cells from the pseudoglandular and canalicular stages of lung development induces PDGF-B and PDGFR-[3 expression as well as cell proliferation, s7 This stretch-induced cell proliferation is abolished by PDGF-B and PDGFR-~ antisense oligonucleotides, PDGF-BB neutralizing antibodies and a PDGFR inhibitor. s7 In contrast to its role during the pseudoglandular and canalicular stages of lung development, PDGF-B expression is reduced at the time when DNA synthesis rates are maximally elevated in response to increased lung expansion during the alveolar stage of lung development (M. Wallace and S. Hooper, unpublished observations). This reduction in PDGF-B mRNA may reflect an acceleration of the normal decline in PDGF-B during the saccular and alveolar stages of development sI'82 and suggests that the factors controlling lung cell proliferation vary during the different stages of lung development. Vascular endothelial growth factor (VEGF) exerts potent mitogenic effects on endothelial cells via the VEGF receptor 2 (VEGFR2). ss VEGF and VEGFR2 mRNA increase as the lung matures, s9'9~which coincides with the increase in cross-sectional area of the pulmonary vascular bed. 9~ However, VEGF and VEGFR2 protein levels peak during the canalicular stage of lung development and decline thereafter. 92 VEGF protein is localized to the basement membrane beneath distal airway epithelial cells 93 suggesting a role in directing vascular growth to regions destined for gas exchange.

Fig. 9.6. Interactions between integrin pathways and growth factor-stimulated pathways. There are at least 18 0~ and 8 J3 integrin subunits 94 that form heterodimers between an o~ subunit and a 13subunit. These subunits consist of a large extracellular domain which binds to specific ECM molecules, a trans-membrane domain and a short intracellular domain that can interact with a large number of cytoskeletal, adapter and signalling molecules. Activation of integrins can lead to the potentiation of the effects of many growth factors via extensive interactions between signalling molecules activated by integrins and the primary growth factor-activated pathway (ERK pathway; see Fig. 9.7 and above - black arrows). Some of the pathways activated by integrins that potentiate the ERK pathway are shown in this diagram and are denoted by grey arrows, e.g. Src binds to and phosphorylates FAK generating a binding site for Grb2 which, through its association with Sos, leads to the activation of Ras. The adaptor protein Shc also binds to activated FAK and similarly binds Grb2. Src can phosphorylate p130Cas generating a binding site for Crk which, via C3G and Rap-l, also leads to activation of Raf. PI-3-K can similarly activate Raf via Rac and PAK or by the modulation of Sos. Integrins can also lead to activation of the ERK pathway in an FAK-independent manner via the association of the integrin-associated protein Caveolin and the Src family kinase Fyn which recruits and activates Shc. PKC is also activated in focal adhesions and can directly activate Raf. The p21 activated kinases (PAK) are also activated by integrin clustering via a variety of mechanisms (only one is shown above) and these kinases can phosphorylate and activate both Raf and MEK 75 and may enhance nuclear translocation of Erk.95 Integrins can also activate a number of growth factor receptors in the absence of the appropriate growth factor ligand. 79'95

Fig. 9.7. Diagram showing the ERK pathway. The extracellular binding of growth factors to their tyrosine kinase receptor (RTK) induces receptor dimerization and autophosphorylation on specific tyrosine residues in the cytoplasmic domains. The phosphorylated tyrosines (pTyr) act as binding sites for proteins containing pTyr recognition motifs (SH2 domains), which then activate a variety of intracellular signalling cascades. The pathway shown is the ERK pathway that commonly leads to cell division or differentiation (black arrows), as well as other signalling molecules that may be activated and may potentiate the ERK pathway (grey arrows). RTK autophosphorylation allows Grb2, an SH2-containing protein, to bind to the RTK, linking it to the guanine nucleotide exchange factor Sos, thus exchanging GTP for GDP on the small GTP binding protein Ras. Ras recruits Raf (a MKKK) to the cell membrane where it is activated. Raf then phosphorylates and activates MKKs (MEK1, MEK2) which phosphorylate and activate MAPKs (ERK1 = p44MAPK, ERK2 =p42MAPK). The activated MAPKs form dimers and can either directly phosphorylate and activate targets in the cell cytoplasm or they can translocate to the cell nucleus where they phosphorylate a variety of transcription factors leading to altered gene expression and subsequent cell division, differentiation or modified cell function.

Vascular development is often compromised in hypoplastic fetal lungs,92'96'97an effect which can be reversed by increased lung expansion. 96'97 As VEGF expression is upregulated by phasic stretch of lung cells,98'99 it has been suggested that VEGF was responsible for the endothelial cell proliferation induced by increased lung expansion. 43 However, a more recent study has been unable to confirm this suggestion (M. Wallace and S. Hooper, unpublished observations). The insulin-like growth factors (IGFs) I and II act as potent mitogens via the type-I IGF receptor 1~176 (IGF1R) and their bioactivity is modulated by the IGF-binding proteins 1~ (IGF-BPs). IGF1 is predominantly expressed during the saccular and alveolar stages of lung development, corresponding with a peak in IGF1R mRNA; 1~176 insufficiency of either leads to perinatal respiratory failure, possibly due to poorly formed alveoli. 1~176 IGF-II is predominantly expressed in the pseudoglandular and canalicular stages of lung development, with levels decreasing as the lung matures. 1~176 The IGF-BPs 2, 3, 4 and 5 are distributed spatially and temporally during lung development, 1~176176 suggesting that they may regulate the IGFs in a cell-specific manner. IGF-I and II mRNA levels are increased and decreased following increases and decreases in lung expansion, respectively, 14'37'1~ suggesting that they may mediate the changes in lung growth induced by altering lung expansion. However, this is unlikely, at least for IGF-II, as its expression does not increase until after the cell proliferation induced by increased lung expansion has ceased (unpublished observations). Transforming growth factor (TGF)-I3 inhibits fetal, newborn and adult type-II cell proliferation, surfactant synthesis and surfactant protein gene expression in culture. 1~ Type-II cell-specific expression of TGF-I31 has been shown to inhibit epithelial cell differentiation and halt lung development at the pseudoglandular stage of lung development in transgenic mice; 112 this was attributed to reduced lung expansion. However, although increased fetal lung expansion reduces the proportion of type-II alveolar epithelial cells, by inducing differentiation into the type-I cell phenotype, this did not correlate with increased TGF-I31 mRNA and bioactive TGF-[3 protein levels (unpublished observations). TGF-I~2 mRNA and protein are elevated after 4 weeks of increased lung expansion to reverse lung hypoplasia; 113 however, by that time it is likely that growth and structural alterations have returned to control levels. In contrast to TGF-I3, TGF-~ and epidermal growth factor (EGF) induce proliferation of isolated type-II cells from newborn rabbits 11~ and EGF treatment accelerates lung development in fetal monkeys. 114'115 The EGF receptor (EGFR) is activated by both TGF-~ and EGF, and gene deletion of this receptor prevents the attenuation of mesenchymal tissue between adjacent airways and reduces surfactant protein levels in fetal mice; these mice commonly die of respiratory failure postnatally. 116'117 The fibroblast growth factor (FGF) family consists of over 20 growth factors that bind to four FGF receptors (FGFR). 118 In fetal mice, the double knockout of FGFR3 and 4 prevents the formation of alveoli suggesting that these two receptors

act co-operatively to induce alveolarization. 119 The effects of exogenous FGF1 (acidic FGF) and FGF2 (basic FGF) on fetal lung growth differ between normal fetal mice and fetal mice with nitrofen-induced lung hypoplasia. 12~As hypoplastic lungs are also developmentally immature, this suggests that the effects of FGF1 and FGF2 differ at different stages of lung development or that prior nitrofen exposure interferes with FGF signalling pathways. The lungs of knockout mice for FGF7 (keratinocyte growth factor; KGF) appear to be normal, 121 although it has been proposed that KGF both enhances 122'123 and inhibits TM growth of the developing lung, while maturing type-II cells 123'125 and promoting this phenotype. 126 Both FGF2 and KGF mRNA are increased by cyclic stretch of postnatal lung cells. 99 Parathyroid hormone-related protein (PTHrP) and PTH/ PTHrP receptor knockout mice die at birth from respiratory failure, indicating that this protein is important for fetal lung development. Although the primary defect in the PTHrP knockout is pulmonary hypoplasia, 127 death was attributed to non-distensibility of the ribcage 128 and to reduced lung liquid clearance. 127PTHrP also induces surfactant protein and surfactant phospholipid biosynthesis 129 in lung explants from fetal mice. Furthermore, increased lung expansion 13~ and stretch of fetal lung cells in culture, TM increase PTHrP expression and receptor binding and enhance its effects on surfactant phospholipid synthesis. TM All the above growth factors act to induce cell proliferation and/or differentiation via the activation of intracellular cascades. The receptors for all of these growth factors (except TGF-13, a serine/threonine kinase receptor and PTHrP, a G-protein coupled receptor) belong to the transmembrane receptor tyrosine kinase family (RTK). The respective growth factors bind to the extracellular region, inducing receptor dimerization and autophosphorylation on specific tyrosine residues in the cytoplasmic domains. The pTyr act as binding sites for proteins containing pTyr recognition motifs (SH2 domains), which then activate a variety of intracellular signalling cascades. The most well-described pathway linking RTK activation to cell proliferation and differentiation is the extracellular signal-regulated (ERK) type of mitogenactivated protein kinase (MAPK) pathway. The core unit of this pathway consists of three successive tiers of phosphorylation (Fig 9.7). The MAPK (ERK) proteins are phosphorylated and activated by MAPK kinases (MKK also known as MEK=MAPK/ERK kinase), which themselves are phosphorylated by MKK kinases (MKKK). Although the specific components of the pathway vary depending on the stimuli and receptor activated, the most commonly described pathway involves the RTK binding of the SH2 domain-containing protein Grb2. This links the RTK to the guanine nucleotide exchange factor Sos, thus exchanging GTP for GDP on the small GTP binding protein Ras. Ras recruits Raf (a MKKK) to the cell membrane where it is activated. Raf then phosphorylates and activates MKKs (MEK1, MEK2) which phosphorylate and activate MAPKs (ERK1 = p44MAPK, ERK2 =p42MAPK). The activated MAPKs form dimers and can either directly phosphorylate

and activate targets in the cell cytoplasm or they can translocate to the cell nucleus where they phosphorylate a variety of transcription factors leading to altered gene expression (Fig. 9.7). The above schema (Fig 9.7) is greatly simplified and described in a linear format; it is more correctly viewed as a complex network of interactions, the balance of which will determine a cell's response. For example, activation of RTKs can also lead to activation of other pathways (e.g. the phosphoinositide-3 kinase pathway, PI3K), some components of which can influence the MAPK cascade. MAPK cascades can also be influenced by a variety of stimuli other than growth factors including cellular and oxidative stresses, inflammatory cytokines, G-protein stimulated cascades and integrin and ion channel activation. However, some degree of pathway specificity is determined by the MAPK family member activated; currently there are at least 12 known MAPKs, 7 MKKs and 14 MKKKs. 132 Thus, an enormous diversity of responses can be elicited by these stimuli, including changes in gene transcription, cell metabolism, proliferation, migration, differentiation, survival, apoptosis and inflammation. In mouse lung explants, the addition of an MEK inhibitor has been shown to reduce epithelial cell proliferation and to inhibit branching morphogenesis, 133suggesting that activation of the ERK pathway is necessary for normal lung development. ERK1 activity was also reduced in nitrofen-induced lung hypoplasia TM and increased following tracheal ligation. 135

ROLE OF E N D O C R I N E A N D O T H E R C I R C U L A T I N G FACTORS IN FETAL LUNG DEVELOPMENT Apart from the growth factors listed above, a number of endocrine factors have been implicated in regulating fetal lung growth and development. Much attention has focused on a variety of factors, particularly in the search for potential therapies for lung immaturity and lung hypoplasia. Corticosteroids have received much attention following the discovery that they are critical for lung maturation. However, the precise roles that most of these factors play in lung growth and development in vivo are still unclear.

The role of corticosteroids in fetal lung growth and development The pre-parturient increase in circulating corticosteroids is known to induce maturational changes in a variety of organ systems, which facilitates the transition to extra-uterine life. In the case of the lung, the maturational changes induced by corticosteroids are essential for the independent survival of the newborn, 136'137 although the precise mechanisms involved remain largely unknown. The reported effects of corticosteroids on lung development include those on (1) growth, (2) tissue remodelling, (3) type-II AECs and the surfactant system and (4) the reabsorption of lung liquid.

It is often assumed that corticosteroids induce lung maturation at the expense of lung growth, but the in vivo data are contradictory, probably a reflection of species differences as well as differences in dose, number of doses and route of administration. Most studies have used synthetic glucocorticoids (betamethasone and dexamethasone), which have a 30-40-fold greater bioactivity than cortisol. When administered to the mother, betamethasone 138'139 causes a decrease in both fetal body and lung growth. 140'141However, when administered directly to the fetus, an even greater dose of betamethasone does not affect fetal body or lung growth. 142 Similarly, physiological doses of cortisol, infused directly into the fetus to simulate the pre-parturient increase in fetal plasma cortisol levels, induce structural maturation of the lung without affecting either fetal lung or body growth. 33'143 These data suggest that the reported effect of maternally administered betamethasone on fetal lung growth may be mediated via an effect on the placenta. Furthermore, they indicate that the endogenous increase in fetal plasma cortisol concentrations does not induce lung maturation at the expense of lung growth. The principal action of corticosteroids on lung development is an effect on lung structure which greatly improves lung mechanics postnatally. TM In particular, corticosteroids markedly reduce inter-alveolar wall thickness leading to a reduction in percent tissue space and a marked increase in potential lung air-volume. Although this is arguably the major pulmonary effect of corticosteroids, the mechanisms involved remain unknown. TM Corticosteroids can also affect alveolarization, although again this affect may be dose- or species-dependent as betamethasone administered to rats has been shown to arrest alveolarization, T M whereas physiological doses of cortisol administered to fetal sheep increases alveolar number (R. Boland and S. Hooper, unpublished observations). In addition, the effect of corticosteroids on increasing lung compliance also interacts with the relationship between lung expansion and lung growth. A corticosteroidmediated increase in lung compliance is likely responsible for the high lung liquid volumes that have been observed in late gestation fetal sheep 5'6 and for the greater increase in lung expansion and lung growth in cortisol-infused fetuses following tracheal obstruction. 33 Although it was originally proposed that the beneficial effects of corticosteroids on lung maturation was due to an effect on surfactant and type-II AECs, recent studies in transgenic mice indicate that this concept needs re-evaluation. 147 Expression of the surfactant-associated proteins, SP-A, -B, -C and -D is commonly synonymously associated with type-II cell differentiation and has been used as an indicator of type-II AEC maturation. The glucocorticoid regulation of SP-B and SP-C in vitro supports a role for corticosteroid regulation of these genes and the type-II cell phenotype in vivo. 148 However, the effect of corticosteroids on surfactant and surfactant protein gene expression is complex and also appears to be dose-dependent in vitro, although the predominant in vivo effect (with supra-physiological doses) is the stimulation of surfactant components. TM As anticipated,

glucocorticoid receptor deficient mice die soon after birth with lungs that are structurally very immature. 147 However, these mice have normal surfactant protein expression levels 147 and, surprisingly, they have a greater proportion of type-II AECs than controls (T. Cole and S. Hooper, unpublished observations). These data suggest that, at least in mice, type-II cell differentiation and surfactant protein expression are independent of glucocorticoid receptor activation. An additional, often overlooked, beneficial effect of corticosteroids on the fetal lung is an increase in the lung's ability to reabsorb lung liquid. 143 Lung liquid re-absorption results from a reversal of the osmotic gradient across the pulmonary epithelium via the opening of amiloride-blockable Na +channels located on the apical surface of AECs. 4 This mechanism is thought to be activated by adrenaline during labour and is responsible for clearing significant volumes of lung liquid from the airways at birth, but only develops late in gestation. 149 Indeed, adrenaline-induced re-absorption of fetal lung liquid increases markedly near term due to the increase in endogenous corticosteroids at this time. 143'15~ Thus, the lungs of infants born preterm, who have not been exposed to prenatal corticosteroids, are likely to be incapable of re-absorbing liquid from the airways, which impairs their respiratory function.

The role of growth hormone in fetal lung growth and development Although circulating growth hormone (GH) concentrations are very high in the fetus and decrease rapidly at birth, fetal growth is thought to be independent of GH. TM Indeed, many fetal tissues contain GH receptors 152(GHR) particularly the lung, 153 but these receptor systems are thought to be immature, making fetal GH largely inactive. 153 GH receptor expression is complex; alternate leader exon usage of the 5'UTR for the GHR confers tissue-specific regulation, TM whereas alternate mRNA splicing between exons 8 and 9 can produce a short inactive form of the GHR. 155 Near term, cortisol is thought to increase GHR expression and activate the adult version of the alternately spliced version of the receptor, which may explain how postnatal growth becomes GH-dependent. However, a recent study has shown that the lung growth response to an increase in fetal lung expansion is GH-dependent. In the absence of GH (due to hypophysectomy), the initial growth response to tracheal obstruction was abolished whereas the re-infusion of GH restored the growth response. 31 These data indicate that GH may play a permissive role in regulating lung cell proliferation in response to mechanical stimuli such as alterations in lung expansion. This may not be restricted to the fetus as GH is thought to play a similar role after birth in regulating compensatory lung growth following hemipneumonectomy which is also thought to be expansion-dependent. 32

The role of retinoids in fetal lung growth and development Much interest in recent years has focused on the role of retinoids (for which vitamin A is a precursor) in fetal lung

development. Retinoids are thought to play a role in cellular proliferation and differentiation as well as airway branching and alveolarization, perhaps by altering the expression of Hox genes. 156 Retinoids exist in a variety of forms, including different isomers of retinoic acid (RA), retinyl esters as well as conjugates with specific binding proteins when circulating and within the cell. 156 Their biological action is mediated via a number of nuclear receptors (RAR) which confer on RA complex and differing responses in the lung. For example, RA administration to newborn animals induces septation and prevents glucocorticoid-induced inhibition of alveolarization in r a t s . 146'157 However, the abolition of RAR[3 by gene knockout increases septation and alveolar number in newborn mice, whereas the administration of a specific RARI3 agonist reduces alveolarization. 158 These data indicate that RAR[3 activation inhibits alveolarization and, therefore, the effect of RA on alveolarization is balanced by an interplay between RA and its receptor subtypes.

C L I N I C A L TREATMENTS FOR INFANTS WITH INAPPROPRIATE LUNG DEVELOPMENT It is clear that fetal lung development is a consequence of a complex interaction between a variety of mechanical and endocrine factors. However, the challenge is to translate this knowledge into therapeutic treatments that will improve the outcome for newborn infants that have inappropriately developed lungs. Inappropriate lung development at birth can arise due to preterm birth, which shortens in utero development time, as well as factors that compromise lung development in utero. With regard to the very preterm infant as a patient, maintaining its respiratory gas requirements is a considerable problem, yet little of what we have learnt from physiological studies in the fetus appears to have been translated into clinical practice. Indeed, many questions need to be addressed, such as the necessity to achieve blood gas tensions similar to those in the mature newborn. If these infants had remained in utero, they would have a PaO 2 of 20-30 mmHg, arterial O 2 saturations of 60-70% and a PaCO 2 of NS0mmHg. Instead, they are often ventilated with high levels of oxygen to maintain adult-like levels of oxygenation, which can damage the lungs and retard lung development (e.g. alveolarization). Although little is known about postnatal lung development in preterm infants, the mechanisms involved are probably similar to those that regulate prenatal lung development. Thus, the basal degree of lung expansion is likely to be an important factor, yet the use of ventilatory practises that specifically facilitate lung development are a minor consideration. For example, the application of a positive end expiratory pressure (PEEP), which increases functional residual capacity, is a common practice in ventilating very preterm infants and is undoubtedly beneficial. However, the primary aim is to improve oxygenation and reduce lung injury, yet appropriate lung development is arguably the key factor that will ultimately

determine the infant's long-term outcome. Thus, do the major objectives of treating preterm infants need re-evaluation in light of what we have learnt from the fetus? The finding that increased fetal lung expansion is a potent stimulus for fetal lung growth and development has prompted the suggestion that this may be used as an in utero therapeutic treatment for fetuses with severely hypoplastic lungs. 15'37'159'160 Indeed, experimental studies have shown that increases in fetal lung expansion, induced by tracheal obstruction, can rapidly reverse fetal lung growth and developmental deficits in Ut8/'O15'161-163 and improve postnatal respiratory function. 164'~65 However, clinical trials in fetal humans with severe pulmonary hypoplasia resulting from a CDH have had mixed success. 42 The principal problems relate to preterm labour, failure to stimulate fetal lung growth in some cases and, in other cases, postnatal respiratory insufficiency inspite of enhanced lung growth. 42 Thus, a significant discrepancy exists between the results obtained from clinical trials and those obtained from animal experiments. Although the reasons for these discrepancies are unknown, they may relate to the stage of lung development at which the treatment was applied, the duration of the tracheal obstruction and the severity of the fetal lung hypoplasia. Most tracheal obstructions in humans have been carried out at <28 weeks of gestation, when the lungs are at the early to mid-canalicular stage of development. 41'42'166'167 However, when tracheal obstruction is performed at the equivalent stage of development in fetal sheep, the induced lung growth is abnormal, causing marked mesenchymal cell proliferation and increased interairway distances, g~ Similarly, sustained increases in fetal lung expansion can severely reduce the numbers of type-II AECs 46 and surfactant protein gene expression; 48'5~ this reduction in type-II cell numbers 47 and surfactant protein gene expression can be reversed by releasing the obstruction. 48'168'169 Thus, inappropriate lung growth and reduced type-II cell numbers may help explain why infants subjected to prolonged TO in utero can suffer respiratory insufficiency after birth, despite having normal or increased lung growth. It is also important to recognize that the severely hypoplastic fetal lung is incompliant and therefore difficult to expand. Thus, if the internal distending pressure required to expand it is greater than the osmotic pressure (equivalent to 4-5 m m H g of hydrostatic pressure) driving lung liquid secretion, 35 the lung will not expand, despite the trachea being occluded. As increased lung expansion is the stimulus for accelerated fetal lung growth, 33'35 this observation may explain the failure of TO to induce lung growth in some infants. 42

CONCLUSIONS The growth and structural maturation of the fetal lung results from a complex interaction between mechanical and endocrine factors. The mechanical component predominantly results from the high degree of basal lung expansion, which is greater than the resting or end-expiratory lung volumes

in the air-breathing neonate. This high degree of fetal lung expansion is largely dependent upon fetal diaphragmatic and glottic activity and, therefore, factors that reduce or inhibit these muscular activities result in a reduction in fetal lung expansion. A reduction in lung expansion is now recognized as the predominant mechanism by which a variety of disorders result in pulmonary hypoplasia in human infants. However, the mechanisms by which alterations in fetal lung expansion accelerate or retard the growth and development of the lung are largely unknown, but are vital areas of research that may have substantial clinical benefits.

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124. Shiratori M, Oshika E, Ung LP etal. Keratinocyte growth factor and embryonic rat lung morphogenesis. Am. J. Respir. Cell Mol. Biol. 1996; 15:328-38. 125. Chelly N, Mouhieddine-Gueddiche OB, Barlier-Mur AM et al. Keratinocyte growth factor enhances maturation of fetal rat lung type II cells. Am. J. Respir. Cell Mol. Biol. 1999; 20:423-32. 126. Borok Z, Lubman RL, Danto SI etal. Keratinocyte growth factor modulates alveolar epithelial cell phenotype in vitro: expression of aquaporin 5. Am. J. Respir. Cell Mol. Biol. 1998; 18:554-61. 127. Ramirez MI, Chung UI, Williams MC. Aquaporin-5 expression, but not other peripheral lung marker genes, is reduced in PTH/PTHrP receptor null mutant fetal mice. Am. J. Respir. Cell Mol. Biol. 2000; 22:367-72. 128. Karaplis AC, Luz A, Glowacki J etal. Lethal skeletal dysplasia from targeted disruption of the parathyroid hormonerelated peptide gene. Genes Dev. 1994; 8:277-89. 129. Torday JS, Sun H, Wang L e t al. Leptin mediates the parathyroid hormone-related protein paracrine stimulation of fetal lung maturation. Am. J. Physiol. Lung Cell Mol. Physiol. 2002; 282:L405-10. 130. Cilley RE, Zgleszewski SE, Chinoy MR. Fetal lung development: airway pressure enhances the expression of developmental genes.J. Pediatr. Surg. 2000; 35:113-18. 131. Torday JS, Rehan VK. Stretch-stimulated surfactant synthesis is coordinated by the paracrine actions of PTHrP and leptin. Am. J. Physiol. Lung Cell Mol. Physiol. 2002; 283:L130-5. 132. Garrington TP, Johnson GL. Organization and regulation of mitogen-activated protein kinase signaling pathways. Curr. Opin. Cell Biol. 1999; 11:211-18. 133. Kling DE, Lorenzo HK, Trbovich AM et al. MEK-1/2 inhibition reduces branching morphogenesis and causes mesenchymal cell apoptosis in fetal rat lungs. Am. J. Physiol. Lung Cell Mol. Physiol. 2002; 282:L370-8. 134. Kling DE, Narra V, Islam Set al. Decreased mitogen-activated protein kinase activities in congenital diaphragmatic hernia-associated pulmonary hypoplasia. J. Pediatr. Surg. 2001; 36:1490-6. 135. Islam S, Donahoe PK, Schnitzer JJ. Tracheal ligation increases mitogen-activated protein kinase activity and attenuates surfactant protein B mRNA in fetal sheep lungs. J. Surg. Res. 1999; 84:19-23. 136. Liggins GC, Howie RN. A controlled trial of antepartum glucocorticoid treatment for prevention of the respiratory distress syndrome in premature infants. Pediatrics 1972; 50:515-25. 137. Liggins GC. The role of cortisol in preparing the fetus for birth. Reprod. Fertil. Dev. 1994; 6:141-50. 138. French NP, Hagan R, Evans SF etal. Repeated antenatal corticosteroids: size at birth and subsequent development. Am.J. Obstet. Gynecol. 1999; 180:114-21. 139. Ikegami M, Jobe AH, Newnham J etal. Repetitive prenatal glucocorticoids improve lung function and decrease growth in preterm lambs. Am. J. Respir. Crit. Care Med. 1997; 156:178-84. 140. Schellenberg J-C, Liggins GC, Stewart AW. Growth, elastin concentration, and collagen concentration of perinatal rat lung: effects of dexamethasone. Pediatr. Res. 1987; 21:603-7. 141. Adamson IY, King GM. Postnatal development of rat lung following retarded fetal lung growth. Pediatr. Pulmonol. 1988; 4:230-6. 142. Jobe AH, Newnham J, Willet K et al. Fetal versus maternal and gestational age effects of repetitive antenatal glucocorticoids. Pediatrics 1998; 102:1116-25. 143. Wallace MJ, Hooper SB, Harding R. Effects of elevated fetal cortisol concentrations on the volume, secretion

and reabsorption of lung liquid. Am. J. Physiol. 1995; 269:R881-7. 144. Jobe AH, Ikegami M. Lung development and function in preterm infants in the surfactant treatment era. Annu. Rev. Physiol. 2000; 62:825-46. 145. Johnson JW, Mitzner W, Beck JC et al. Long-term effects of betamethasone on fetal development.Am. J. Obstet. Gynecol. 1981; 141:1053-64. 146. Massaro GD, Massaro D. Retinoic acid treatment partially rescues failed septation in rats and in mice. Am. J. Physiol. Lung Cell Mol. Physiol. 2000; 278:L955-60. 147. Cole TJ, Blendy JA, Monaghan AP et al. Targeted disruption of the glucocorticoid receptor gene blocks adrenergic chromaffin cell development and severely retards lung maturation. Genes Dev. 1995; 9:1608-21. 148. Mendelson CR, Alcorn JL, Gao E. The pulmonary surfactant protein genes and their regulation in fetal lung. Semin. Perinatol. 1993; 17:223-32. 149. Waiters DV, Olver RE. The role of catecholamines in lung liquid absorption at birth. Pediatr. Res. 1978; 12:239-42. 150. Wallace MJ, Hooper SB, Harding R. Role of the adrenal glands in the maturation of lung liquid secretory mechanisms in fetal sheep. Am. J. Physiol. 1996; 270:R1-8. 151. Bassett JM, Thorburn GD, Wallace AL. The plasma growth hormone concentration of the foetal lamb. J. Endocrinol. 1970; 48:251-63. 152. Garcia-Aragon J, Lobie PE, Muscat GE et al. Prenatal expression of the growth hormone (GH) receptor/binding protein in the rat: a role for GH in embryonic and fetal development? Development 1992; 114:869-76. 153. Batchelor DC, Lewis RM, Breier BH etal. Fetal rat lung epithelium has a functional growth hormone receptor coupled to tyrosine kinase activity and insulin-like growth factor binding protein-2 production. J. Mol. Endocrinol. 1998; 21:73-84. 154. Li J, Gilmour RS, Saunders JC et al. Activation of the adult mode of ovine growth hormone receptor gene expression by cortisol during late fetal development. FASEB J. 1999; 13:545-52. 155. Finidori J. Regulators of growth hormone signaling. Vitam. Horm. 2000; 59:71-97. 156. Zachman RD, Grummer MA. Retinoids and lung development. In: Mendelson CR (ed.), Endocrinology of the Lung. Totowa: Humana Press Inc., 2000, pp. 161-79.

157. Massaro GD, Massaro D. Postnatal treatment with retinoic acid increases the number of pulmonary alveoli in rats. Am. J. Physiol. 1996; 270:L305-10. 158. Massaro GD, Massaro D, Chan WY etal. Retinoic acid receptorbeta: an endogenous inhibitor of the perinatal formation of pulmonary alveoli. Physiol. Genomics 2000; 4:51-7. 159. Wilson JM, DiFiore JW, Peters CA. Experimental fetal tracheal ligation prevents the pulmonary hypoplasia associated with fetal nephrectomy: possible application for congenital diaphragmatic hernia.J. Pediatr. Surg. 1993; 28:1433-40. 160. Hedrick MH, Estes JM, Sullivan KM etal. Plug the lung until it grows (PLUG): a new method to treat congenital diaphragmatic hernia in utero. J. Pediatr. Surg. 1994; 29:612-17. 161. Papadakis K, De Paepe ME, Tackett LD etal. Temporary tracheal occlusion causes catch-up lung maturation in a fetal model of diaphragmatic hernia. J. Pediatr. Surg. 1998; 33:1030-7. 162. Sylvester KG, Rasanen J, Kitano Y et al. Tracheal occlusion reverses the high impedance to flow in the fetal pulmonary circulation and normalizes its physiological response to oxygen at full term.J. Pediatr. Surg. 1998; 33:1071-4. 163. Kitano Y, Davies P, yon Allmen D etal. Fetal tracheal occlusion in the rat model of nitrofen-induced congenital diaphragmatic hernia.J. Appl. Physiol. 1999; 87:769-75. 164. Davey MG, Hooper SB, Tester ML et al. Respiratory function in lambs after in utero treatment of lung hypoplasia by tracheal obstruction. J. Appl. Physiol. 1999; 87:2296-304. 165. Wild YK, Piasecki GJ, De Paepe ME etal. Short-term tracheal occlusion in fetal lambs with diaphragmatic hernia improves lung function, even in the absence of lung growth.J. Pediatr. Surg. 2000; 35:775-9. 166. Harrison MR, Albanese CT, Hawgood SB etal. Fetoscopic temporary tracheal occlusion by means of detachable balloon for congenital diaphragmatic hernia. Am. J. Obstet. Gynecol. 2001; 185:730-3. 167. Vanderwall KJ, Skarsgard ED, Filly RA et al. Fetando-Clip: a fetal endoscopic tracheal clip procedure in a human fetus.J. Pediatr. Surg. 1997; 32:970-2. 168. Saddiq WB, Piedboeuf B, Laberge J-M et al. The effects of tracheal occlusion and release on type-II pneumocytes in fetal lambs.J. Pediatr. Surg. 1997; 32:834-8. 169. Lines A, Gillett AM, Phillips ID etal. Re-expression of pulmonary surfactant proteins following tracheal obstruction in fetal sheep. Exp. Physiol. 2001; 86:55-63.

The environment consists of matter in one of three physical states" solid, liquid or gas. The unequal forces of attraction between molecules in different phases result in surface tension at the boundary between the phases. The issue of phase-phase interactions is pronounced within the lung, because lungs are cyclically inflating gas-filled structures lined with fluid. In a lung lined only with an aqueous fluid, high interfacial tension would cause respiratory surfaces to adhere on expiration and impede expansion of the lung on inspiration. The primary function of the pulmonary surfactant system is to vary the surface tension at the air-liquid interface with changing volume within individual alveoli. This variation in surface tension results in very distinct lung compliance curves with a marked hysteresis, and provides the lung with alveolar stability, preventing atelectasis (alveolar collapse) at low lung volumes and overinflation at high lung volumes. In humans with surfactant deficiency diseases (e.g. respiratory distress syndrome), respiration typically demonstrates a restrictive pattern with a reduced residual lung volume often with alveolar atelectasis, greatly reduced lung compliance, high collapse pressures and a greatly reduced capacity to inspire. In this chapter, we consider the normal composition, function and development of the pulmonary surfactant system, and then discuss the effects of genetic and environmental factors on the fetal and neonatal surfactant system.

*To whom correspondence should be addressed. tPresent address: Department of Microbiology and Immunology, The University of Melbourne, Melbourne, Victoria 3010, Australia. The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

THE F O R M A T I O N OF

AND

RELEASE

SURFACTANT

Surfactant lipids and proteins are synthesized in the lung in specialized alveolar cells termed type II alveolar epithelial ceils (AECs), which are characterized by microvilli along the apical edge and lamellar bodies in the cytoplasm. Lamellar bodies, the storage organelles for surfactant, consist of a dense proteinaceous core and lipid bilayers arranged in parallel, stacked lamellae. The assembly route for phospholipids proceeds via the endoplasmic reticulum and the Golgi apparatus to lamellar bodies. The pathways for the assembly and incorporation of the surfactant proteins into the lamellar bodies are controversial, but may proceed via the endoplasmic reticulum and Golgi apparatus to multivesicular bodies before combining with the lamellar bodies. After the lamellar bodies have been released into the hypophase (the fluid lining the epithelium), they swell and unravel into another highly characteristic form of surfactant termed tubular myelin. It is from tubular myelin, that the surface film is derived, which regulates the surface tension at the air-liquid interface (reviewed by Goerke; 1 Fig. 10.1).

COMPOSITION SURFACTANT

OF

PULMONARY

In humans, pulmonary surfactant is comprised of--80% phospholipids, 12% neutral lipids and 8% protein. The components and their main functions are summarized in Table 10.1. Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

Fig. 10.1. Schematic diagram of the life cycle of pulmonary surfactant. Pulmonary surfactant components are synthesized in the endoplasmic reticulum (ER), transported to the Golgi apparatus (Golgi) and packaged into lamellar bodies (LB). Lamellar bodies are secreted into the liquid lining the alveoli (hypophase) via exocytosis across the type II cell plasma membrane. Here the lamellar bodies swell and unravel, forming a crosshatched structure termed tubular myelin (TM), which consists of lipids and proteins. This structure supplies lipids to the surface film as well as the surface-associated phase (SAP). As the mixed molecular film is compressed, lipids are squeezed out of the film into the SAP to produce a DPPCenriched film, which is capable of reducing surface tension (ST) to near 0 mN/m. It is possible that some lipids from the SAP re-enter the surface film (dashed line arrow). Lipids from the surface film and the SAP are eventually recycled and taken back up by the type II cell via endocytosis. The role of some of the surfactant proteins (SP-A, -B and -C) in regulating these processes is indicated with 1" (stimulation) or ,[, (inhibition) (modified from Goerkel).

Lipids Lipids comprise 80-90% by mass of mammalian surfactant 2 and are predominantly phospholipids (PL). Phospholipids exist in unsaturated (USP) and disaturated (DSP) forms. 3 The neutral lipid, cholesterol, is the second most abundant lipid in mammalian surfactant. 4 The most abundant phospholipid in all surfactants is phosphatidylcholine (PC) with smaller amounts of sphingomyelin (S), lysophosphatidylcholine (LPC), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylglycerol (PG). 5 The functions of most of the PL remain obscure. The disaturated form of PC, dipalmitoylphosphatidylcholine (DPPC) is largely responsible for the reduction in surface tension (see below). 6 The DPPC molecules can be compressed tightly together by virtue of their fully saturated palmitic acid chains. At low lung volumes, the lipid film at the air-liquid interface is thought to be highly enriched in DPPC, eliminating water molecules from the interface and thereby reducing surface tension markedly. 7'8 However, DPPC cannot spread by itself. Hence, upon inspiration, other lipids such as USP and cholesterol are recruited into the monolayer from the surfactant-associated phase (Fig. 10.1) and aid in spreading the lipids over the hypophase. Upon expiration, USP and cholesterol are "squeezed" out of

the surfactant film, again resulting in an enrichment of DPPC at the interface. 1 The specific role(s) of the minor PL is less clear. The acidic PL, PG and PI, may enhance the adsorption of DPPC. 9 PG may have a role in the interaction with surfactant protein B and may be preferentially squeezed out of the surfactant film to allow enrichment of DPPC. I~ The other minor phospholipid components might support the formation of structures such as tubular myelin or be involved in signaling events in surfactant metabolism. 11,12 Cholesterol accounts for 80-90% of the neutral lipid fraction, which also includes free fatty acids and mono-, di- and tri-acylglycerides. Cholesterol may function to maintain fluidity of surfactant and promote surface film respreading. 13 This is achieved by the ability of cholesterol to disrupt the cohesive forces between the PL, thereby reducing the phase transition temperature of the surface film. 14 The synthetic origin of surfactant cholesterol is not clear, as the majority of cholesterol present in lamellar bodies does not appear to be released into the alveolar hypophase. 13 Furthermore, cholesterol levels appear to be independently regulated from the surfactant phospholipids. The origin, function and regulation of surfactant cholesterol require further investigation.

Proteins Four mammalian surfactant proteins have been isolated: surfactant protein-A (SP-A), SP-B, SP-C and SP-D. SP-A accounts for 50% of the protein in purified surfactant xs and is thought to be important in the recycling of surfactant lipids, and in aiding lung immunity. 16'17 SP-A knockout mice lack tubular myelin but do not experience respiratory difficulties; TMhowever, they are more susceptible to pathogen infection. SP-B and SP-C are small hydrophobic proteins probably important in surfactant biosynthesis. 19 SP-B

promotes the "squeeze-out" of USPs from the surface film, whereas SP-C promotes PL adsorption. 2~ SP-B deficiency results in death in mice and humans 22 and therefore SP-B is the only surfactant protein which is essential for lung function. Surfactant protein D has structural and functional similarities to SP-A, and may have a role in pulmonary defence mechanisms and surfactant lipid recycling. 23'24 It is now clear that both type II AECs and non-ciliated bronchiolar (Clara) cells can produce SP-A, SP-B and SP-D. SP-A and SP-D appear to be secreted by type II cells

through routes which do not involve incorporation into lamellar bodies. 25 SP-A, SP-B and SP-D have also been located in the gut and in the Eustachian tube. 24'26-29 SP-D has been localized in the salivary, lacrimal, sweat and mammary glands, z4 In contrast, the mRNA for SP-C is found exclusively in type II cells. 3~

P H Y S I C A L PROPERTIES OF THE S U R F A C T A N T SYSTEM A N D THE L U N G Surface tension Surface tension is the force of attraction between molecules located at the surface of a liquid. It arises because the surface molecules are more attracted to each other than they are to the air above. The ability to interfere with the interaction of the surface water molecules and therefore vary the surface tension is termed surface activity. 31 Usually, surface tension is quantified as the force required to stretch the rectangular surface fluid by known length (usually 1 cm), and is expressed either as dyn/cm or mN/m (where 1 dyne/ cm= 10-5 N/cm or 1 mN/m). Pure water has a surface tension of 70dyn/cm (or 70mN/m) at 37~ Under dynamic compression in vitro, a "good" surfactant is generally able to reach a minimum surface tension (STrnin) of <5 mN/m, and an excellent surfactant < 1 mN/m. 32

Collection and analysis of pulmonary surfactant Pulmonary surfactant samples are usually obtained by rinsing the lungs with saline (broncho-alveolar lavage, BAL); this is achieved in animals by instilling saline via the trachea and filling the lungs to total capacity. 33 However, in humans and especially in infants this is more difficult, and the resultant samples are much more variable. 34 Usually BAL fluid is obtained with a bronchoscope that is wedged in a segmental or subsegmental bronchus. In some cases, tracheal aspirates and sputum samples are analysed but they contain a much greater proportion of airway material. It is therefore essential to always compare data with its own control, and that investigators provide details of the technique used. 35 Once surfactant is harvested, there are also different methods of subdividing different fractions. Cellular material can be removed by centrifugation, and surfactant can then be divided into (a) a large aggregate fraction (LA), which contains large lipid-protein complexes, including tubular myelin, and demonstrates high surface activity and (b) a small aggregate (SA) fraction, which contains small surfactant vesicles, derived from the LA fraction after repeated compression/expansion (breathing) cycles and demonstrates low surface activity. The fractions are usually separated by centrifugation; density gradient centrifugation may also be used. 35 The ratio of these subfractions is thought to be important, and a disturbance may be indicative of dysfunction. 36'37

Static lung compliance Static compliance is the retractile nature of the lung tissue at known volumes, and therefore generates pressure in the

Fig. 10.2. Typical pressure-volume curves of lungs inflated with air both before and after rinsing the lungs with saline (lavage) to remove the surfactant, and after inflation with saline. Abolition of the air-liquid interface (saline curve) abolishes the hysteresis and reduces the inflation and deflation pressures to those resulting from the elastic recoil of the lung tissue itself. The importance of the surface film to lung compliance is demonstrated by the difference in the pressurevolume curves before and after lavage.

absence of resistance due to air-flow. Static lung compliance is determined by the surface tension of the fluid lining the lung (66%) and the tone and nature of the structural units (33%). 38 The contributions are demonstrated by comparing the hysteresis curves of a normal lung with one filled with saline (which abolishes the air-liquid interface) and one, which has been lavaged with isotonic saline. If surfactant is removed and only water lines the inner lung, then compliance is dramatically decreased (Fig. 10.2).

SURFACTANT FUNCTIONS

Lung stability In the mammalian lung, an STmin of near zero at endexpiration is a necessary condition for alveolar stability. Alveoli (5-10~m in diameter) are lined with fluid of an average thickness of 0.2 l.tm.39 The surfactant film lining the interface between this fluid and the air space generates a surface tension-related elastic retractile force. According to the law of Young and Laplace, P=-2y/r, where y is the surface tension measured in mN/m and r the radius of curvature measured in m; this predicts that very small alveoli would experience a very large collapse pressure, unless the surface tension was very low. Pulmonary surfactant reduces the surface tension to near zero at very low lung volumes such that the retractile forces are equivalent both within separate regions of an alveolus as well as between respiratory units. 4~ This, together with the structural interdependence of the alveoli, provides the lung with alveolar stability and maintains a relatively large alveolar surface area necessary for efficient gas exchange at low lung volumes. 42

Alveolar interdependence and anti-adherence The elastic recoil of alveoli is responsible for about onethird of lung compliance. As alveoli are inter-connected, any alveolus tending to collapse will be held open, because it will be supported by the walls of adjoining alveoli; this interaction between alveoli is termed interdependence. A model for the structure-function relationship of the lung parenchyma, the alveoli and surface tension is shown in Fig. 10.3. 40'42'43 Furthermore, as alveoli are not perfect spheres, but rather polyhedral in shape, the walls of the alveoli may come into contact upon expiration. The work required to separate opposing alveolar walls is directly proportional to the surface tension of the fluid lining. 44 By reducing the surface tension of the alveolar hypophase, pulmonary surfactant greatly reduces the work required to initiate lung inflation. This function of surfactant is termed anti-adhesive (or occasionally "anti-glue") and is believed

Fig. 10.3. Schematic diagram of the alveolar interdependence model, illustrating the structure-function behaviour of the lung parenchyma in response to alterations in surface tension (y). The functional unit is the alveolar duct (or a set of ducts forming an acinus) embraced by peripheral connective tissue fibers. The peripheral fibers are connected to the pleura, are the main force-bearing element and are largely independent of changes in surface tension. The axial fibers are the rings of tissue forming the entrance of the alveoli and these are influenced by the surface tension of the air-liquid interface, which is continuous along the alveolar wall. The two-dimensional alveolar walls represent a negligible mechanical component. Low surface tensions allow a large alveolar surface area between slightly stretched axial fibers. However, when surface tension is abnormally high, the axial fibers become more stretched resulting in a disproportionately enlarged duct, in flattening of alveoli, and in a decreased alveolar surface area (reproduced with permission from Bachofen and Sch~rch43).

to be particularly important in non-mammals, 45 but might be significant in aiding the reinflation of a partially collapsed or atelectatic lung.

Prevention of pulmonary edema and maintenance of airway patency The alveoli and the inner surface of the small and terminal airways are lined with a fluid similar in composition to interstitial fluid. Moreover, lungs have a large surface area, high blood flow and leaky capillary endothelial cells and are therefore susceptible to fluid disturbances. Areas with a high surface tension would tend to draw fluid from the interstitium. 46 Fluid pressure is large and negative in the alveolar corners (because the radius of curvature is very small), but small in the flatter regions of the alveolus. Normally fluid probably flows from the interstitial space into the alveolar hypophase via alveolar seams, crevices and corners (Fig. 10.4). Fluid flow may occur back into the interstitium via flatter parts or type II cells. 47 In this latter case, fluid movement is aided by Na § pumps on the type II cell membrane, which set up a concentration gradient causing fluid to flow passively down an osmotic gradient. The pumping of Na § from the hypophaseinto the interstitium

Fig. 10.4. Schematic diagram of an alveolar wall illustrating the movement of fluid (arrows) between the fluid lining the air spaces (hypophase) and the interstitial space. The small radius of curvature of corners and crevices leads to a large negative fluid pressure in the alveolus, which tends to draw fluid into the alveolus from the interstitium. Furthermore, under hydrostatic pressure, net fluid movement occurs out of the capillaries into the surrounding tissue and the alveolus. Sodium pumps in the alveolar membrane of type II cells remove sodium from the hypophase and transport it into the interstitium, causing a net passive fluid movement out of the alveolus, thereby preventing fluid build-up. Excess fluid in the interstitium is removed by the lymphatic circulation. BM, basement membrane; LB, lamellar bodies; N, nucleus.

may also promote the reabsorption of fluid from the small airways. Fluid build-up within the alveolus (pulmonary edema) increases the gas diffusion distance and compromises breathing. By lowering surface tension, surfactant reduces the amount of fluid entering the alveolar space. Moreover, surfactant decreases surface tension to values lower than the surrounding interstitium, and thereby encourages the movement of fluid from the alveolus into the tissue (Fig. 10.4). 48 Pulmonary surfactant aids in airway patency. As the terminal airways leading to alveoli lack cartilage, they participate in volume changes during ventilation. Therefore, during expiration, the airways would narrow further and fluid would accumulate in the narrowest section. 49 Since narrow portions of the airway experience greater pressures than wide portions, this could potentially result in fluid extravasation into the narrower regions. By lowering surface tension during expiration, pulmonary surfactant lowers the pressure in the narrow sections to less than that of the wide portions, and therefore maintains airway patency. 49

Immune functions The lung is susceptible to invasion by air-borne bacteria, fungi, viruses and other foreign material. Surfactant contributes to lung immunity by reducing the work required for the airway cilia to beat, 5~ increasing beat frequency 51

and improving cilia-mucus coupling. 52 In doing so, particles are moved to the upper respiratory tract and removed by swallowing. SP-A and SP-D also play a role in lung defence; binding sites on the proteins complement those found on the surface of bacterial cells zl and both proteins enhance the ability of alveolar macrophages to destroy bacteria. 8'I7'53 SP-A knockout mice are especially susceptible to bacterial, viral and fungal attack, yet the processing of the surfactant lipids remains the same. TM SP-D knockout mice show increased numbers of macrophages. 54

REGULATION SECRETION

OF

SURFACTANT

The release of surfactant from alveolar type II cells is mediated by breathing, 55 signals from the autonomic nervous system, 56 and by other biochemical factors. 1 In the intact lung, paracrine factors or physical forces may also influence surfactant secretion. Some of the factors and signaling pathways which stimulate surfactant secretion are summarized in Fig. 10.5.

Autonomic agonists Regulation of surfactant secretion by [3-adrenergic agonists occurs in vivo and in isolated type II cells. The order of

Fig. 10.5. Schematic diagram summarizing the major factors and signaling pathways that stimulate surfactant secretion from type II alveolar epithelial cells. Several [3-adrenergic agonists, including isoproterenol, adrenaline and noradrenaline stimulate the 13-2 adreno-receptor. The receptor is coupled to adenylate cyclase (AC), which produces cyclic AMP (cAMP) via a trimeric GTP-binding protein (G), in order to stimulate cAMP-dependent protein kinase (protein kinase A, PK-A). Another pathway involves the direct or indirect stimulation of protein kinase C (PK-C). The synthetic surfactant secretagogue tetra-decanoylphorbol acetate (TPA) and cell-permeable diacylglycerols (DAGs) are potent direct stimulators of PK-C. ATP and UTP bind to the purinergic receptor (P2Y2) which is coupled to phospholipase C (PLC) via another G protein. Activation of PLC leads to the formation of inositol triphosphate (IP3) and DAG. The latter stimulates PK-C, while IP3 feeds into the third secretory mechanism, which involves the elevation of intracellular Ca 2+ levels by IPg or calcium ionophores. Calcium in turn stimulates calmodulin-dependent protein kinase (PK-CA2§ The stimulatory effect of stretch may be mediated via the P2Y2 receptor, and therefore the PK-C and/or PK-Ca2§ signaling pathways, although physiological stimulation of surfactant secretion by ventilation or labour may be mediated via the 13-2 receptor. The exact subsequent mechanisms leading to lamellar body exocytosis are not well understood, but are thought to involve protein kinase-stimulated protein phosphorylations, which presumably activate contractile proteins to move lamellar bodies to the apical surface to fuse with the plasma membrane (figure modified from Mason and Voelker, 25 and Rooney57).

potency on isolated type II cells is isoproterenol >adrenaline > noradrenaline, indicating that the ~2 receptor mediates the response. 25 Adrenergic agonists bind to ~2 receptors located on the type II cells, causing an increase in intracellular cAMP and activation of a cAMP-dependent protein kinase. 55'58 Adrenergic agonists decrease lamellar body volume density within 0.5 h after injection, 59 demonstrating that the response is rapid. While type II AECs possess muscarinic cholinergic receptors, 6~ cholinergic agonists fail to stimulate surfactant secretion from isolated type II cells of rats 58'61 or humans. 62 Cholinergic agonists enhance PC secretion in the isolated perfused rat 56'63 and rabbit 64 lung. In this case, cholinergic agonists may cause contraction of smooth muscle cells in the intrapulmonary tissue, resulting in the distortion of type II cells. 56 However, cholinergic agonists do act directly on isolated type II cells to release surfactant lipids in the heterothermic marsupial, Sminthopsis crassicaudata. 65'66

Ventilation Hyperventilation results in a stimulation of surfactant secretion within minutes, 67 and a single deep breath increases the amount of alveolar PL, possibly through increased secretion. 68 Ventilation-induced increases in surfactant secretion are likely to occur via direct mechanical stimulation of type II cells, which are preferentially located in corners where they are exposed to maximum distortion. 69 Physical stretch of isolated type II cells results in an increase in surfactant secretion equivalent to a combination of agonists. 7~ This effect is likely to be mediated by an increase in cytosolic calcium 7~ or, in vivo, by leakage of surfactant components from the alveolar compartment. 72

Towards the end of gestation, the content and saturation of the PL increase. For example, total PC is low in fetal sheep 3-4 weeks prior to birth, but reaches adult levels 3-4 days prior to parturition. 84 Likewise, there is a 10-20-fold increase in the amount of phospholipid in lung homogenates of fetal rats between days El9 and E21. s5 The disaturated phospholipids appear late in gestation, with the relative saturation of PC increasing 4-5-fold in the lavage of the fetal rabbit between days 24 and 25 of gestation, s6 The acidic PL also change during development; there is an increase in the relative proportion of PG concomitant with a decrease in the proportion of PI, leading to an increase in the PG/PI ratio. This ratio can be used as an index of fetal lung maturity. 87 The surfactant proteins appear late in gestation. SP-A is not detectable in human fetal lung fluid until the 30th week of gestation, ss SP-B and SP-C are expressed earlier in gestation, with the pre-proteins present in the human lung by the 15th week of pregnancy. 89 SP-D mRNA in humans is first detected in the second trimester, 9~ whereas in rats, SP-D appears in very low quantities at day 18 (82%) of gestation. 91

Regulation of surfactant development Mechanisms controlling the maturation of the mammalian surfactant system are still poorly understood. While numerous neuro-hormonal factors are known to influence the development of the surfactant system including prolactin, 92 prostaglandins, 93 insulin, 94,95 estrogen, 96 theophylline, 97 leukotrienes 75 and growth factors, particularly keratinocyte growth factor (KGF), 9s'99 we outline only the major controllers here.

Other factors Many biochemical factors influence the secretion of surfactant. ATP is a powerful stimulant for surfactant release, mediated though P~ and P2 purinergic receptors. 73'74 Metabolites of arachidonic acid, 75 calcium ionophores, 76 endothelin-1, 77 vasopressin, 7s lipoproteins 79 and phorbol esters s~ all stimulate surfactant secretion. The secreted components of surfactant themselves regulate further secretion. At low lung volumes, when USP and the proteins (especially SP-A) are squeezed out of the surfactant monolayer, these components act to inhibit further secretion. 25 Purified SP-A inhibits all forms of agonist-induced secretion in vitro. 81'82 However, this effect only occurs when SP-A is delipidated. 81 Below the phase transition temperature, saturated PC also inhibits surfactant secretion in vitro. 83

D E V E L O P M E N T OF THE P U L M O N A R Y S U R F A c T A N T SYSTEM The pulmonary surfactant system is one of the last systems to develop before birth, and in humans, it matures between the 29th and 32nd week of gestation. The amount and composition of the surfactant lipids change during gestation.

Glucocorticoids Glucocorticoids are increasingly produced by the fetal adrenal cortex during late gestation. 1~176176 Administration of glucocorticoids to the fetus decreases respiratory distress probably by increasing surfactant phospholipid synthesis and the appearance of PL in the alveolar compartment. In vivo, long-term exposure to betamethasone increases the saturated PC in lavage in the fetal sheep 1~ and total PL in lavage of the fetal rabbit. 1~ Similarly, long-term exposure to dexamethasone results in an increase in saturated PC in lavage in the fetal rat ~~ and in the tracheal fluid of the fetal sheep. 1~ Dexamethasone increases the choline incorporation into PC in the lungs of the fetal rat, 1~ rabbit 1~ and human l~ in vitro. Glucocorticoids appear to increase the amounts 1~ and activity 11~ of CTP phosphocholine cytidylyltransferase, an enzyme which catalyses the regulatory step in PC synthesis. However, the mechanism by which this occurs remains unclear. As glucocorticoids have a minimal effect on isolated type II cells, 111-113 they may act through interstitial fibroblasts. A factor produced from fibroblasts (termed fibroblast-pneumocyte factor, FPF) has been isolated that increases PC production in fetal type II cells. ~12 The release

of FPF from interstitial fibroblasts may be mediated by glucocorticoids.112 Glucocorticoids also appear to regulate the surfactant proteins. Treatment of fetal lambs with betamethasone results in an increase in the levels of SP-A, SP-B and SP-C mRNA 24h following injection. 114 Dexamethasone also increases the appearance of SP-B, 115 S P - C 116 and S P - D 117 in humans. Similarly, the transcription rate of genes for SP-B and SP-C is increased by dexamethasone in organ culture. 118 However, dexamethasone decreases the levels of SP-A mRNA at high doses. 119-121 This may, in part, be explained by the differential regulation of the two genes for SP-A by glucocorticoids. 121 Glucocorticoids have a number of effects other than changing the composition or amount of surfactant. The activity of pulmonary antioxidant enzymes is increased under the influence of glucocorticoids. 122'123The rate of clearance of fetal lung fluid is also increased. 124'125 Dexamethasone increases the density of 13-adrenergic receptors in the fetal rabbit 126 and rat 127 lung and also increases ]3-receptor gene expression in the rat. 128 In addition, glucocorticoids enhance the stimulatory effect of secretagogues on surfactant secretion. 129 Given that this effect occurs in the presence of a number of secretagogues, it is likely that glucocorticoids influence the signaling pathways downstream from the cellular receptors. 129 However, many fetuses do not respond to antenatal glucocorticoids. 130 Furthermore, animals that are deficient in corticotropin-releasing hormone (CRH - / - ) have normal lungs if born to mothers that are CRH (+/_).131 Therefore, although glucocorticoids may affect surfactant production experimentally, their effects are not well understood in vivo. 13~ Thyroid hormones The thyroid hormones, triiodothyronine (T3) and thyroxine (T4), enhance surfactant development. An increase in the levels of T 3 in the late gestation fetus is accompanied by a surge in pulmonary surfactant content. 13z T 3 is the most biologically active of the thyroid hormones and generally only acts in the final stages of gestation. The thyroid gland produces only a small amount of T 3, the majority being produced in peripheral tissues (predominantly in the liver) by deiodination of T 4. Thyrotropin-releasing hormone (TRH) is released from the hypothalamus and acts on the anterior pituitary to release thyroid-stimulating hormone (TSH). The products of the thyroid gland then act by negative feedback to decrease the production of TRH and/or TSH. However, at birth there is an increase in the thyroid hormones concomitant with an increase in TSH. In the preterm lamb, the processes of thyroid function are altered. There is significantly less plasma T 3 and significantly more T4, indicating that the conversion of T 4 to T 3 may be impaired. 133The thyroid of premature lambs is also insensitive to a rise in TRH and does not respond by releasing thyroid hormones. 133 Therefore, these factors may also contribute to the dysfunction of the surfactant system in preterm infants (see below).

Since thyroid hormones do not readily cross the placenta, TRH has been maternally administered to increase fetal T3 .134 TRH increases the alveolar concentrations of PC in fetal rabbits TM and sheep. 135Directly administered thyroxine increases the number and size of the lamellar bodies, 136 increases the amount of disaturated PC in the lavage of fetal rabbits 137 and increases the total lung PL and disaturated PC content in fetal rats. 138 In lung slices from the fetal rabbit 1~ and human, 1~ T 3 increases choline incorporation into PC, indicating that the thyroid hormones increase surfactant synthesis. However, thyroid hormones administered alone may not increase surfactant synthesis; 139 rather, glucocorticoids and thyroid hormones act synergistically to enhance surfactant PC synthesis. 92'1~176176 The effects of thyroid hormones are mediated via receptors in the nuclei of type II cells, TM but the exact maturation mechanism remains unclear. 142The activities of the enzymes that control the synthesis of DPPC are increased by administration o f T3 .109'143 The thyroid hormones may also act to enhance surfactant lipid metabolism. 136 Fetal administration of thyroid hormones also increases the number of [3-adreno-receptors in the lungs of rabbits 137 and rats. TM However, thyroid hormones do not appear to affect the surfactant protein content. 145'146 Autonomic neurotransminers Adrenergic and cholinergic agonists also contribute to surfactant maturation, presumably by the same regulatory mechanisms as in the adult. 147'148 Administration of the adrenergic agonist, terbutaline, to fetal rabbits leads to an increase in lung compliance, total PL in lavage and surface activity. 149 Similarly, the adrenergic agonist, isoxsuprine, increases synthesis, storage and release of surfactant in the rabbit fetus. 150 Furthermore, if the synthesis of adrenaline is experimentally inhibited during the latter part of gestation, there is a 40% reduction in alveolar PC at birth and a 23% reduction in alveolar PC at 1 h of age in the newborn rabbit. TM In mammals, adrenergic influences are the most important in stimulating surfactant secretion, 132 although adrenergic agonists increase choline incorporation in PC 152'153 and therefore may also increase surfactant synthesis. Adrenergic agonists also increase the amounts of SP-A in explants of human fetal lung. 154 During birth, there is a massive surge in circulating catecholamine levels, 155 which might provide the physiological source of adrenergic stimulation. However, sympathetic nerve activity may also contribute. 156 There is a developmental increase in response to 13-adrenergic agonists towards the end of gestation, believed to reflect maturation of the receptor pathway. 148 Furthermore, the 13-receptors mature more rapidly in females than males. 157 The influence of cholinergic agonists on surfactant development during the perinatal period is more obscure. Pilocarpine stimulates PC release in fetal lung explants in the rat, 158 but does not stimulate PC surfactant release in newborn rabbit lung slices. 93 Furthermore, carbachol does not stimulate PC secretion from fetal hamster lung slices. 159

Stretch~Distension Surfactant secretion is stimulated by air-breathing and ventilation in newborn mammals. 16~ Before birth, expansion of the fetal lung by lung fluid may stimulate the production of surfactant lipids during the latter part of gestation, x64 Prostaglandin E 2, released by type II cells in response to stretch, mediates the release of surfactant lipids, x65 However, artificially induced fetal lung expansion by tracheal obstruction induces differentiation of type II cells into type I A E C s 166'167 and results in a decrease in the levels of mRNA for SP-A, SP-B and S P - C . 168-170 However, mechanical strain of organotypic cultures of type II cells increases the expression of SP-C mRNA. 171

The surfactant system at birth At birth, the activity of AEC Na+-K § ATPase pumps establishes an osmotic gradient between the lung fluid and the interstitium leading to fluid reabsorption (see Chapter 8). Both the entry of air into the lungs coupled with tidal breathing excursions stimulate surfactant release. The surfactant lipid, which quickly moves to the surface of the lung liquid, rapidly reduces the surface tension, an activity that further promotes the reabsorption of the fetal lung fluid. The surfactant lipids coat the thin layer of fluid remaining, and balance surface tension to ensure alveolar stability and reduce the work of breathing. If the surfactant system is not fully developed, as occurs when an infant is born prematurely, the respiratory distress syndrome (RDS) ensues (see below). However, surfactant deficiency (or a lack of one or more components) can occur as a result of genetic or environmental factors, and will also result in respiratory distress in the infant.

GENETIC CAUSES OF SURFACTANT DEFICIENCIES D U R I N G DEVELOPMENT Is there a genetic basis to respiratory distress in infants? While the frequency of certain SP-A alleles correlates with increased likelihood for respiratory distress, 172'173and family clusters of affected infants and of ethnic- and gender-based respiratory phenotypes point to the contribution of inheritance, 174 only one classically inherited surfactant deficiency disease (SP-B deficiency) is known. The consequences of this disease for the surfactant system and the lung are described below.

Surfactant protein-B deficiency Inherited deficiency of SP-B is an autosomal recessxve disorder that leads to lethal respiratory failure immediately after birth. 175 The most common null mutation is due to a 2 base pair insertion in codon 121 of the SP-B gene, resulting in a frameshift mutation. 176'177 Affected infants can be supported temporarily with assisted ventilation, supplemental oxygen and exogenous surfactant replacement, but without a lung transplant all infants have died within the

first year of life. 178'179 Surfactant replacement has been ineffective in SP-B-deficient infants. 18~ The SP-B gene targeted mouse also suffers from lethal respiratory failure and all homozygous null mutants ( S P - B - / - ) die within 30min of birth, TM evidence of the crucial role of this protein in the successful transition to air breathing. Type II AECs of S P - B - / - mice lack lamellar bodies and secreted tubular myelin, demonstrating that SP-B is crucial for these complexes. 182 Occasionally, a few loosely arranged lamellae are observed in their lamellar bodies; it is likely that these represent disorganized lamellar bodies, and that SP-B is responsible for the packaging of surfactant PL into concentric lamellae. 182 This abnormal cellular processing also appears to affect other surfactant components, for example, SP-C processing is abnormal in SP-B-deficient humans and mice, with type II cells exhibiting an accumulation of intermediate pro-SP-C peptides. 178'182'183 This suggests that there is a block in normal SP-C processing as a result of SP-B deficiency. As the latter stages of normal SP-C processing have been localized to the lamellar bodies, 184'185 this disruption is readily understood. Impaired lamellar body processing could also result in abnormal PL secretion, which may lead to delayed accumulation of PL in amniotic fluid. 18~ Both lavage and lung tissue of SP-B-deficient infants demonstrated a decreased phospholipid to protein ratio, possibly a result of increased protein, as surfactant PL content is normal in SP-B - / mice. TM Furthermore, both lung tissue and lavage exhibited elevated PI and decreased PG concentrations, whereas disaturated PC was elevated in lung tissue. 178 However, phospholipid synthetic rates were normal. 178'181 In SP-B - / - mice, both residual and total lung volumes and compliance were markedly decreased. Pressure-volume curves demonstrated no hysteresis and significant atelectasis was evident in the lungs. In heterozygous mice (SP-B +/-), lung compliance decreased slightly. TM As surfactant replacement therapy in SP-B-deficient infants is rarely successful, perfluorocarbon ventilation has been trialled and has prolonged survival by improving lung expansion, lung compliance and oxygenation. TM However, lung function is not completely restored, as lungs virtually collapse at end expiration, presumably because the perfluorocarbons only reduce surface tension to ---15 raN/m, 186 and not to < 1 mN/m required for alveolar stability. 32 Hence, the use of perfluorocarbons which has also been used in RDS 187 may also be used for the treatment of hereditary SP-B deficiency. However, perfluorocarbon also causes structural damage to epithelial, vascular and alveolar structures in SP-B - / - and SP-B +/- mice, and may not be successful if used long term. TM

Variations of surfactant protein B deficiency Over the past few years, several other cases of inherited surfactant protein B and other deficiencies have been reported, which do not match either the genetic or pathological pattern of inherited SP-B deficiency. These include partial SP-B deficiency, xss transient SP-B deficiency, is9 milder

SP-B deficiency leading to prolonged survival, 19~other novel mutations TM or other phenotype abnormalities involving lamellar bodies or other surfactant components. 192-194 These findings suggest that the level of genetically induced lung disease may be much higher than previously thought. It is possible that many cases previously attributed to environmental factors may in fact have a genetic basis.

E N V I R O N M E N T A L EFFECTS ON THE DEVELOPING P U L M O N A R Y SURFACTANT SYSTEM Fetal hypoxia and intrauterine growth retardation Intrauterine growth retardation (IUGR) is a major cause of low birthweight and increases the risk of respiratory distress and death in both term and preterm infants. 195'196 Causes of IUGR include maternal undernutrition, hypertension, anemia, placental infarction and tobacco smoking 197 (see Chapter 17). Growth retarded fetuses suffer from fetal hypoxemia and hypoglycemia, and elevated levels of circulating catecholamines and cortiso1198'199 and decreased levels of insulin-like growth factors and their binding proteins. 2~176 During late gestation, activation of the hypothalamopituitary axis (HPA) leads to an increase in the basal levels of adrenocorticotropic hormone (ACTH) and circulating cortisol, facilitating lung maturation. TM Furthermore, the administration of exogenous glucocorticoids improves postnatal lung function and stimulates fetal growth. 2~176 Cortisol is also increased during periods of physiological stress such as fetal hypoxemia, which may be expected to stimulate lung maturation by increasing surfactant protein expression. The hypothesis that IUGR leads to enhanced lung maturation stems from the observation that the lecithin to sphingomyelin ratio (PC/SM) increases in amniotic fluid of IUGR fetuses. 2~ However, this finding was not supported in fetal sheep in which IUGR was induced by carunclectomy; 2~ here a decrease in the total PL concentration of luminal liquid occurred. Similar findings were also made in IUGR induced by maternal undernutrition in neonatal guinea pigs, 2~176 where substantial reductions in total surfactant PL (including disaturated PC) in lavage and lung tissue occurred, although PL composition, the number of lamellar bodies per type II cell and lung compliance remained normal. In the fetal sheep, prolonged hypoxemia induced by maternal hypoxia (48 h at gestational days 126-130 or days 134-136) leads to an elevated plasma cortisol level, which is more pronounced later in gestation 2~ and correlates with elevations in lung tissue SP-A and SP-B mRNA levels. SP-C mRNA level was unaffected. The alterations are dependent on the age of the fetus, as only older fetuses (134-136 days) responded to hypoxemia. 2~ Hypoxemiainduced increases in SP-A a n d - B mRNA occurred in an ovine model of chronic placental insufficiency and IUGR. 2~ Chronic fetal hypoxemia, maintained for 21 days

during late gestation (--109-130 days) decreased fetal growth and lung growth proportionately, and decreased lung DNA content. Fetal cortisol levels increased, and correlated significantly with increases in SP-A and -B mRNA (but not SP-C mRNA). Although lung morphology and function were not assessed, the decrease in lung DNA content and concentration, with an increase in SP mRNA synthesis, suggests that there was a switch from lung cell proliferation to fetal lung cell maturation. 2~ Hence, fetal hypoxemia and/or nutrient restriction may affect fetal lung and surfactant maturation via fetal glucocorticoids. However, as hypoxia can also stimulate other factors such as catecholamines, prostaglandins and cAMP levels, cortisol may not be solely responsible for the increase in SP mRNA. Although catecholamines and cAMP analogues stimulate SP-A mRNA transcription, ss there was no correlation between prostaglandin E z plasma levels and SP mRNA levels in chronically hypoxemic sheep lungs. 2~ In direct contrast, chronic placental insufficiency during late gestation (120-140 days) did not change the SP-A, -B or -C mRNA or SP-A protein levels in the lung tissue of fetal sheep. 21~There was also no correlation between SP mRNA or SP-A protein levels and cortisol levels. Furthermore, although DNA content decreased, relative to lung weight, the DNA concentration was higher in the growth-retarded sheep. As DNA concentration decreases during normal gestation, TM the increase found here suggests that the lungs of the growth-retarded fetuses were structurally immature. There is still a relative paucity of information on the effect of fetal hypoxia and growth retardation on the surfactant system and the data on the levels of SP mRNA expression are inconsistent. The primary difference between the study of Gagnon et al. 2~ and that of Cock et al. 21~lies in the timing of sampling relative to gestation. Cock etal. 21~ induced IUGR between days 120 and 140 days of gestation compared to ---109-130 days in the study by Gagnon et al. 2~ It is possible that the levels of SP mRNA in the older fetuses had already reached their maximal expression and could not be stimulated further by cortisol. Age exerted a specific effect on SP mRNA expression and cortisol levels after 48 h of mild hypoxia, 2~ and so it appears that there is a very narrow window in which surfactant maturation can be perturbed by environmental factors. While this robustness may represent an adaptive advantage during normal development, it may also explain the variable outcomes described 13~ in therapeutic interventions such as glucocorticoid administration. Therefore, in order to optimize treatment strategies of fetuses and infants at risk of IUGR-related postnatal respiratory complications, it is essential that the mechanisms and timing of lung and surfactant maturation during late gestation are understood, especially in relation to environmental factors which lead to IUGR.

Alterations in fetal lung fluid The alveolar epithelium consists of type I and type II AECs, which cover approximately 95 and 5% of the alveolar surface area, respectively. The type II cell is the progenitor of both

epithelial cell types because it proliferates into new type II cells and differentiates into type I cells. The tightly coupled regulation of these two processes is important during restoration of the alveolar epithelium after injury and also during normal intrauterine development and postnatal lung remodelling. The number and proportions of the two cell types are influenced by fetal lung expansion (i.e. by accumulation of fetal lung fluid). 167 The transition of a type II AEC into a differentiated type I cell phenotype is reversible 212 and occurs via an intermediate cell type, which combines features of both cells. 167 Both the transition between, and maintenance of, the type II/type I AEC phenotypes are modulated by mechanical signals recognizable both in vitro and in vivo (see Chapter 9). Hence, the application of stretch to isolated type II AECs, or expansion of the fetal lung results in changes to AEC phenotype, which are also associated with alterations in surfactant proteins, their mRNA as well as the P L . 213'214 Hence, the level of surfactant protein gene expression is regulated by the degree and pattern of lung expansion. The biochemical mechanisms are not understood, but probably involve paracrine mediators (e.g. cytokines) released by neighbouring cells such as fibroblasts. 164 The interactions between the mechanical forces resulting from the volume of fetal lung fluid and the development of the alveolar epithelium have profound implications for the normal development of the lung and the surfactant system. For example, if the lung is physically prevented from expanding (e.g. if a diaphragmatic hernia is present), there is a lack of lung fluid, which leads to pulmonary hypoplasia and structural immaturity.

and reduced lung volume. In the adult, the stiffness and natural tendency of the chest wall to spring outwards reduces the extent of the lung collapse. However, in the neonate, the ribs are highly compliant and cannot oppose the decreased lung compliance and signifcant atelectasis will result. If the surfactant system is not activated soon after birth in very premature infants, fibroblasts may invade the alveolar interstitium and form large quantities of hyaline cartilage and elastin, which thicken and rigidify the membrane. This alteration in the membranes can exacerbate respiratory distress and can lead to death. Oxygen therapy and ventilation are only partially successful in treating the syndrome, because they do not address the primary cause (a lack of surfactant). Administration of artificial surfactants, dispensed via the airways, is a remarkably effective treatment, and if accompanied by continued corticosteroid therapy, has a very high success rate. 218 In spite of advances in management, complications leading to chronic lung injury are still common. In fact, due to the greater rate of survival of premature babies, the incidence of chronic lung injury has increased, because these babies are most at risk of developing bronchopulmonary dysplasia. 219

Surfactant abnormalities associated with R D S In general, surfactant from infants with RDS demonstrates reduced levels of total phospholipid, T M especially PC 222-224 and PG. 225-228 Furthermore, SP-A is greatly r e d u c e d . 22~ These changes result in a poorly surface-active material. 228'229 Pre- and postnatal treatment with dexamethasone reverses many of these symptoms, leading to improved surface activity and reducing the amount of inhibitory proteins and increasing the level of SP-A and S P - D . 230'231

Premature birth and the respiratory distress syndrome

Chronic lung disease/bronchopulmonary dysplasia

Much of the interest in surfactant research has focussed on the consequences of the failure of the system, particularly in the newborn infant. Von Neergaard 38 first suggested that the alveolar collapse, observed in some newborns, may be a result of high surface tension in the lungs. Avery and Mead 215 performed autopsies on infants who had died from hyaline membrane disease (HMD). They defined the condition as infiltration of the alveoli by cellular and fibrotic material and concluded that lack of a surface-active film was partly responsible for the regions of alveolar collapse seen in the RDS. The link between pulmonary surfactant deficiency and RDS was later confirmed. 216'217 If birth occurs prior to the complete maturation of the surfactant system, then both the reabsorption of the fetal lung fluid and the subsequent maintenance of a low work of breathing can be compromised. Initially, alveolar fluid may remain, and the increased diffusion distance may cause hypoxemia, with or without hypercapnia. The low PO 2 reduces the extent of the arteriolar dilation and pulmonary hypertension may remain. In addition, the high work of inspiration and elevated elastic recoil of the lung results in a very high frequency, low volume breathing pattern, accompanied by a collapsed sternum, elevated diaphragm

Premature birth and RDS may necessitate prolonged mechanical ventilation with supplemental oxygen. This treatment effect can lead to chronic lung injury and often produces bronchopulmonary dysplasia (BPD), a major cause of morbidity and mortality in premature infants. 232'233 (see Chapter 16). However, the relationship between ventilation, hyperoxia and the pathology is not clearly understood; recent evidence suggests a possible involvement of pulmonary surfactant dysfunction. Histological analysis of the presence and distribution of SP-D in postmortem lungs from infants who died of BPD has demonstrated that only open terminal airways were lined with SP-D. Injured areas, or alveoli filled with blood, infection, or edema, in which type II cell function is likely to be impaired, were characterized by low levels of SP-D staining on the alveolar surface, and protein was not detected in bronchioles and bronchi from these infants. TM In a neonatal rat model of chronic lung injury, the SP-A mRNA level of lung tissue, specifically in the terminal bronchioles and in type II cells, was greatly elevated. SP-B and SP-C mRNA levels were elevated only in inflamed areas. These changes may reflect a natural protective response to the inflammation. A reduction in FIO 2 resulted in a reversal

of the mRNA changes, possibly indicating recovery of normal type II cell metabolism. 235 However, in a primate model of premature birth, the induction of SP-A mRNA at birth significantly lagged behind those of SP-B and SP-C mRNA. This defect in the response of the SP-A gene may be a contributing factor in the development of BPD after premature birth. 236 Furthermore, an analysis of both tissue and lavage SP-A and -D levels in primates with BPD indicated that, although the tissue levels of SP-A and SP-D were greatly enhanced following 10 days of ventilation with 100% oxygen, the level of SP-A in the BAL was greatly reduced. Hence secretion was reduced, suggesting an impairment of normal type II cell function due to the inflammation. 237 In animals that developed infections naturally during long-term ventilation, the lavage SP-A level was further reduced. 238 Because of their important immune functions, the reduction in either SP-A or SP-D in the airways of infants with BPD may render the lung susceptible to secondary infection and inflammation, or may inhibit the resolution of infection and thereby contribute further to the developing injury. Very little information is available on the surfactant PL or surfactant function in the lead-up to or during BPD. A study of preterm infants with RDS, some of whom developed BPD, demonstrated a reduced surfactant PC content and impaired surface activity in non-survivors; however, there was no association between surfactant phospholipid composition or function and the risk for BPD development. 239 In a baboon model of mild to moderate BPD, the phospholipid composition of pulmonary surfactant lavage including disaturated PC and total PG, was similar between animals with BPD and a control-ventilated group after 21 days. 24~ Although the early administration of glucocorticoids to preterm infants with RDS did not reduce the incidence of BPD, TM it is apparent that early or prophylactic treatment of RDS with exogenous surfactant can reduce neonatal mortality and the incidence of BPD. However, there is a lack of consistency in the treatment outcome for different drugs. This is likely to be related to the complex interaction of many factors (e.g. surfactant, antioxidant and inflammation), which leads to the heterogeneous pathologies of this disease. 219 Furthermore, in preterm RDS infants, an initial high level of polyunsaturated fatty acids (PUFA) and plasmalogens (i.e. phospholipid that contains an aldehyde of a fatty acid) in the tracheal effluent correlated with a reduced risk of developing BPD. 242 These data suggest that PUFA may protect against oxidative injury and highlight the need to examine and trial therapeutic agents with an antioxidant capacity. 187

The effect of pathogens/toxins on the pulmonary surfactant system The immature lung is very susceptible to respiratory infections. The prolonged artificial ventilation required by premature infants leads to an increased risk of pulmonary infection. Other infants particularly at risk are those with

cystic fibrosis and human immunodeficiency virus. Common infectious agents include Pneumonia, Staphylococcus, tuberculosis and respiratory syncytial virus (RSV). All these agents affect the pulmonary surfactant system. 35

Respirawry syncytial virus (RS V) This is presently one of the most prevalent respiratory infections in infants and children, occurring as an annual epidemic in winter. It causes viral bronchiolitis, which if severe requires hospitalization; 1-2% of infants are affected and RSV is the most common cause of intensive paediatric care. 243 The predominant pathology is an inflammation of the bronchioles, which leads to a mucous build-up and narrowing of the airways, and the clinical manifestations can vary from cold-like symptoms to death of respiratory failure. 244 The mortality rate in industrialized countries is 0.5-1% of hospitalized patients. 245'246 In addition to the inflammation, surfactant abnormalities occur, which may further increase the severity. Tracheal aspirates of infants with viral bronchiolitis have shown a reduction in the concentration of PC and SP-A, 247'248 as well as an impairment of surface activity. 248 SP-B content was unchanged between healthy controls and ill patients. 247 Patients with bronchiolitis had a lower concentration of DPPC (PC16:0/16:0), but normal levels of P G . 249 However, others have reported a lack of PG in some infants, which correlated exactly with impaired surface activity. 25~Moreover, in patients with severe RSV infection, the pattern of expression of specific genetic variants of SP-A suggests that there may be a genetic association between SP-A gene locus and severe RSV infection, as certain alleles dominated in RSV-infected infants, and others were greatly reduced. TM An association between certain SP-A genes and an increased risk for ARDS has also been proposed. 251'252 The standard treatment of acute bronchiolitis includes mechanical ventilation, supplemental oxygen and bronchodilators. However, as bronchodilators and glucocorticoid treatment have not been very successful, 253 exogenous surfactant supplementation has recently been trialled. 249 This treatment was able to arrest the deterioration of the phospholipid composition observed in the placebo group and affected an increase in lung compliance. However, there were no changes in various indices of gas exchange. This study therefore presents some evidence that exogenous surfactant treatment may be a useful therapeutic strategy in infants with severe bronchiolitis.

Conclusions The pulmonary surfactant system is complex in terms of its composition and function, and neither the specific functions nor the exact biophysical interactions of its different components are fully understood. During development, numerous regulatory elements interact to affect surfactant function and maturity. The surfactant system develops in utero during late gestation, and it is crucial that this system is established and operational at birth. Dysfunction of the surfactant system at this time, either through preterm birth

or other environmental or genetic causes, may lead to RDS. Although the use of antenatal glucocorticoids and the administration of exogenous surfactant to the neonate have been remarkably successful in the treatment of RDS, a significant number of affected infants develop chronic lung injury (BPD). The search for the causes of this condition together with the development of suitable animal models and treatment strategies represent major areas of research. There is a growing awareness that the intrauterine environment may have profound effects on the physiology of the infant and also in later adult life. Lung and surfactant functions also appear to be profoundly affected in the infant by intrauterine influences such as severe growth retardation. Whether there are long-term influences, which reach into adult life, is as yet unknown. Furthermore, it is becoming apparent that there is a genetic basis to many cases of RDS. The recent identification of a number of specific gene defects relating to the surfactant system suggests that the search for clinically useful genetic markers of risk for respiratory distress in infancy will become an important area of future research. It is also now recognized that the developing surfactant system is susceptible to pathogens and toxins, and surfactant dysfunction represents a major complication in respiratory illnesses of neonates. Here too, genetic risks appear to play a role, and the search for genetic markers, as well as the development of suitable therapies, including exogenous surfactant replacement, are major targets for research effort.

ACKNOWLEDGEMENTS This work was supported by the Australian Research Council.

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INTROD

UCTIO N

It is well established that newborn infants have a limited ability to mount an effective immune response against microbes. It is likely that the cellular basis for the immunological restrictions of the fetus and neonate are multifaceted; functional immaturity of immune cell types in conjunction with developmental immaturity of organ systems (lack of immune cell 'seeding') may both play a role in susceptibility. Based on data obtained from human and laboratory animal studies, it is apparent that both aspects contribute to limitations of the maturing immune system. This chapter will discuss current knowledge within the field of developmental immunology, with an emphasis on the pulmonary system. This area of investigation suffers from too little information in the literature, particularly in human/non-human primate species. It is clear that postnatal development of rodent airways and immunity is quite different from that in humans; further, there are species differences in the release of cytokines and other mediators by leukocytes. Because of these important distinctions between human and rodent pulmonary/ immune system development, it is imperative to expand our understanding of the maturing mucosal immune system in the lung within the context of primates (either human or monkey). As such, this chapter will focus on data obtained from human/non-human primate studies, with supportive data from other species included where appropriate.

MATURATION OF IMMUNE SYSTEM

THE

POSTNATAL

Human neonatal blood lymphocytes differ from their adult counterparts both in expression of cell surface markers and overall number within the circulation, emphasizing the The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

need to evaluate lymphocyte subsets both by frequency and absolute counts. 1 In 1935, it was reported that total leukocyte and lymphocyte counts in humans are highest at birth and decline with maturation; 2 these findings have been confirmed over the past 10 years by several other laboratories. 1'3'4 In comparison with values obtained from cord blood samples, total blood lymphocyte numbers are elevated at 1 week of age.3 Total blood lymphocyte counts and frequency remain high, relative to adult blood values, until approximately 6 years of age; these values begin to decline within the age group of 7-17 years of age. 1'3 A peak of blood T-lymphocyte counts and frequency occurs at 1 week of age, followed by a peak of blood B-lymphocyte counts and frequency at 2-3 months of age (Fig. 11.1). 3 Following the early rise in circulating numbers after birth, T-lymphocyte populations show a modest, but progressive reduction in frequency and number through the first year of life. In contrast, B-lymphocyte populations remain relatively stable both in frequency and number until 2 years of age. 3'5 For both T and B peripheral blood lymphocytes, a dramatic drop in absolute counts occurs between the ages of 7 and 17 years. 1 Although B-lymphocyte frequency falls with the onset of adulthood, the frequency of T-lymphocytes appears to slightly increase at this stage. 1'4 Both absolute numbers and frequency of circulating NK cells are reduced almost immediately following birth and continue to fall during the first year of life, suggesting that this population is functionally more important during gestation. 3 Based on these findings alone, it is clear that the first 6 years of life are an important period of immune system growth and maturation. The particularly pronounced changes in T- and B-lymphocyte populations observed during the first 2 months of life suggest that this is a particularly significant developmental window within which immune cell populations expand as a result of environmental stimuli. Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

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Fig. 11.1. Numbers of T-lymphocytes, B-lymphocytes, and NK cells in blood (cells x 109/litre) from birth to 1 year of age. Each graph represents one individual infant (infants 1-10). Reproduced from de Vries et al. 3

Differences in the relative frequencies of CD4+ and CD8+ cells during childhood and into adulthood are somewhat controversial due to conflicting reports, likely due to technical issues; however, it is apparent from a longitudinal analysis during the first year of life that both of these T-lymphocyte populations demonstrate a pronounced fall in frequency with increased age. 3 Activated CD3+ T-lymphocytes, as measured by CD25 (IL-2 receptor) expression, peak in number at 1 week of age. An additional marker of activation, HLA-DR, shows little change during the first year of life and HLA-DR+ T-cells are reduced in number in comparison with adult values. The relative frequencies of both CD25 and HLA-DR subpopulations of CD3+ T-lymphocytes are considerably higher in adults versus infants, likely reflecting the status of the immune system for both age groups. Infants encounter a variety of environmental antigens throughout childhood, yet functional immaturity may limit the types of responses that the developing immune system can produce; this is particularly evident for the production of cytokines (see below). At birth, the majority of peripheral blood T-lymphocytes are CD45RA+ and do not express CD45RO (the low molecular weight isoform of CD45R); this cell population likely represents a 'naive' or antigen-inexperienced phenotype. Because CD45RA+ T-cells are reduced and CD45RO+ T-cells are increased in frequency during maturation from infancy to adulthood, it has also been assumed that cell surface expression of CD45RO is representative of an antigen-experienced or 'memory' phenotype. Although these findings would suggest that the humans are immunologically naive at birth, numerous lymphoproliferation and cytokine studies indicate that a newborn does come in contact with antigens during gestation, most likely from transplacental transfer. 6-12 Such antigens include common environmental allergens such as house dust mite and milk protein, as well as parasites. At present, it is not known how this apparent development of immune responses to antigens in utero can affect the maturation of immunity during postnatal development.

There is a higher frequency and cell count for CDlc+, CD5+, and to a lesser extent, CD38+ circulating B-lymphocytes within the first year of life, as compared with adult values. 3 Absolute counts of CDlc+ B-lymphocytes continue to rise up until 3 months of age, yet the frequency of this population declines following the first week of life. In contrast, both absolute counts and frequency of CD5+ cells peak at 6 weeks of age. Because of this expression profile, it has been suggested that CDlc and CD5 are markers of untriggered B-lymphocytes during the neonatal period of development. 3 However, the apparently different expansion profile of CD5+ cells during the first year of life suggests additional developmental roles. It is now well established that CD5+ B-lymphocytes are B-1B-cells, a distinct, self-renewing subset of IgM secreting B-lymphocytes that exhibit a restricted V-region repertoire. The immune functions of B-1B-cells are not well defined; the secretion of highly cross-reactive IgM by this B-lymphocyte subset may be an important defense mechanism for the immature infant immune system. At birth, production of significant levels of immunoglobulin isotypes other than IgM in response to antigenic challenge is poor. 13 Neonatal B-lymphocytes can secrete IgE in vitro provided there are high levels of exogenous IL-4. This suggests that the lack of appropriate cytokines in the environment may play a role in this apparent deficiency of immunoglobulin production. 14 In addition, it has been reported by several groups that cord blood mononuclear cells express very low levels of CD40 ligand (CD40L). 15-17 As the interaction of CD40L on activated T-lymphocytes with CD40 on B-lymphocytes is required for isotype switching, the apparent lack of sufficient CD40L on newborn mononuclear cells suggests a potential mechanism whereby secretion of IgA, IgG and IgE is suppressed. Under in vitro priming conditions, cord blood mononuclear cells can be stimulated to express CD40L at adult levels, demonstrating that the newborn T-lymphocyte repertoire does have the capacity to respond to antigenic stimulation.16,18

Resident immune/inflammatory cells of developing airways Airways of newborn infants without pulmonary lesions contain few leukocytes. In contrast, histological examination of lungs from stillborn infants diagnosed with congenital pneumonia show the presence of alveolar macrophages as early as 20 weeks' gestation. 19 Alveolar macrophages are observed histologically within alveoli of healthy preterm Macaca nemestrina monkeys at 140 days, albeit at very low numbers. 2~ Macrophages have also been identified within the airway compartment by lavage of healthy fetal monkeys at 162-165 days of gestation (term = 168 days). 21 In addition to pulmonary pathology during gestation, alveolar macrophage number within airways correlates with duration of postnatal life for both monkey and man. Alveolar macrophages have been found within airways of preterm human infants that had no apparent pulmonary lesions, but survived for at least 48 h. 19 Alveolar macrophages in lavage of healthy newborn monkeys are increased 33-fold at 2 days of age (versus fetal) and 4-fold at 3-4 weeks of age (versus 2 days). 21 Based on these data, it has been suggested that in healthy infants, 'seeding' of leukocytes within the airways must be initiated following birth and exposure to environmental stimuli; this process likely continues during the first 2 years of life. 22 A multi-center study with standardized methods has reported that bronchoalveolar lavage from healthy adults contains an average of 85.2% alveolar macrophages, 11.8% lymphocytes, 1.6% neutrophils, and 0.2% eosinophils. 23 Reference values for bronchoalveolar lavage from healthy children do exist and are generally similar to those reported for adults, yet the range in ages sampled were wide (4 months to 16 years), thus limiting the ability to determine age-related effects. 24-27 There are no significant changes in lavage cell profiles of children between the age groups of 3-8 and 8-14 years, suggesting that accumulation of resident immune/ inflammatory cells within airways is essentially complete after the age of three. 26 It is important to note that methods for obtaining airway lavage samples can produce varying results. For example, in preterm infants, leukocyte populations from tracheal aspirates versus deep pulmonary lavage yielded pronounced differences in cellular phenotype. 28 Although this study had too few subjects to draw significant conclusions with regard to correlation of cell profiles with airway levels, the findings suggest that the region of sampling should be taken into consideration for interpretation of results. In a small study of 18 healthy children (3 months to 10 years), it has been reported that age significantly correlates with the frequency of lymphocytes within lavage, however there was no other correlation with other lavage cell phenotypes. 24 A direct comparison of airway lavage from children less than 2 years versus 2-6 years of age does show a significant decrease in frequency of alveolar macrophages with age, with a concomitant increase in lymphocyte frequency. 29 Available data on the immunophenotype of lavage lymphocyte subsets in normal children are limited. It has been shown that the overall frequency of B-lymphocytes, T-lymphocytes and NK cells in lavage from children

(3-16 years of age) is similar to that obtained from adults, although the limited number of subjects and wide range of age groups in this study may mask significant changes that could occur in younger subjects. 3~ Several groups have reported striking differences in the CD4/CD8 ratio of lavage lymphocytes from children (3 months to 16 years of age); CD4/CD8 ratios ranged from 0.6 to 0.8 for children, in contrast with 1.8-2.7 for a d u l t s . 23'24'30-33 The reduction in CD4/CD8 values for bronchoalveolar lavage from children appears to be due to an overall increase in CD8 cell number. 3~ In a recent histological analysis of human fetal airway tissues (with no apparent lung abnormalities), it has been reported that T-lymphocytes, mast cells, and macrophages were observed within lung interstitium as early as the pseudoglandular stage of development (9-18 weeks). 34 From the pseudoglandular to saccular/alveolar stage, numbers of T-lymphocytes and mast cells progressively increased in number within lung interstitium, whereas numbers of tissue macrophages decreased with time in utero. In this same study, neutrophils and B-lymphocytes were rarely observed within lungparenchyma up until the saccular/alveolar stage (27-41 weeks); a few B-lymphocytes were localized within lymphoid structures of the trachea and small bronchi. In contrast, another study reported finding no lymphoid follicular structures or plasma cells within the bronchus at birth. 35 The investigators observed small lymphoid nodules within the airway wall as early as 12 h after birth; the structures continued to increase in number after 8 weeks of age. A few B-cells with surface IgM were also found in bronchial tissue immediately following birth, with IgA and IgG plasma cells appearing at 4 and 13 weeks, respectively. In addition, secretory component (receptor for IgM and dimeric IgA) was observed to be expressed by airway epithelial cells at birth. The ability for plasma cells to undergo isotype switching is an important indicator of developmental immune maturation, as this step is dependent upon T- and B-lymphocyte interactions via CD40/C40L. As with lymphoid nodules within the conducting airways, peripheral lymphzreticular aggregates have also been evaluated within alveolar walls of the newborn infant lung. 36 Peripheral lymphoreticular aggregates are not found in the normal lung at birth; aggregates are observed following the first week of life, and like bronchial lymphoid follicles, progressively increase in number during the first year of life. Bronchus-associated lymphoid tissue (BALT) was first formally described in rabbit lung in the early 1970s. 37'38 Structurally, BALT consists of a follicular aggregation of lymphocytes within the airway lamina propria, containing a central, germinal center-like region of B-lymphocytes surrounded by T-lymphocytes (reviewed in Ref.39). There are no afferent venules; high endothelial venules within the peripheral regions of BALT allow for trafficking of lymphocytes into the BALT structure. The most distinguishing feature of BALT is a dome-like protrusion into the bronchial epithelium. In the human adult, BALT is not normally found in the lung but can be found under disease conditions. 4~ In contrast, BALT is often observed in children and adolescents

under apparently healthy conditions, likely indicating the antigenic stimulation and expansion of memory lymphocyte populations that this age group undergoes. In the fetal lung, BALT has been observed as early as 20 weeks and most often correlates with chorioamnionitis or intrauterine pneumonia; BALT is only found in 10% of'normal' fetal lungs. 42 BALT has also been observed within infant airways, with the caveat that post-mortem specimens were obtained from infants that had died from the sudden infant death syndrome. 43 No evidence exists to indicate that sudden infant death syndrome is related to the presence of BALT, but because of the limited ability to obtain information from healthy human infants, this area of investigation is still speculative.

DEVELOPMENTAL EXPRESSION OF P U L M O N A R Y CYTOKINES AND CHEMOKINES Very little data are available on the expression of cytokines during infancy, particularly with respect to in situ production in the lungs. In peripheral blood, expression of IL-12 by mononuclear phagocytes and dendritic cells is much lower in cells from human neonates compared with adult cells. 44'45Correspondingly, because IL-12 plays a key role in the differentiation of the Thl cell phenotype, interferon gamma expression is reduced in neonatal lymphocytes. 46 These findings would indicate that the newborn is deficient in Thl responses and therefore carries a Th2 'bias'. However, there is additional evidence that IL-4, a key cytokine in the differentiation of the Th2 cytokine profile is also much reduced in the neonate. 47 The deficiency of IL-4, interferon gamma, and IL-12 in the neonate does not extend to other cytokine family members; expression of IL-1, IL-2, IL-6, IL-13, TNF-alpha and interferon-alpha in neonatal cells is comparable to that of adults. 46'48-51 Therefore, a deficiency of cytokines that is central to the differentiation of memory/ effector T-helper cells at birth may represent a key regulatory step in the subsequent structuring of a unique immune cell repertoire that is personalized to the newborn environment. In the fetal lung, protein and mRNA expression of colony stimulating factor-1 (CSF-1), a hematopoietic growth factor that regulates the development of tissue macrophages, is positively correlated with gestational age in the human fetal lung and is present as early as 10 weeks of age. 52 In addition, IL-6, IL-8, and IL-10 protein expression is detectable throughout the pseudoglandular, canalicular and alveolar stages of development by standard immunostaining methods. 34 Expression of IL-6, IL-8, and IL-10 in normal fetal lung is primarily localized to epithelial cells, and observed as early as 9-18 weeks of gestation. 34 In contrast to an epithelial cell expression profile under 'healthy' conditions, a study of 22 neonates (average postnatal or gestational age, 28 weeks) with hyaline membrane disease showed elevated IL-8 protein expression within airway neutrophils and connective tissue, in addition to epithelium. 53 Messenger RNA for IL-1 alpha, IL-1 beta,

IL-6, IL-8, and TNF-alpha is detectable in cells obtained by tracheal aspirates and deep pulmonary lavage from premature infants at 1, 7, and 28 days of age. 28 Infants that eventually develop chronic lung disease show elevated levels of IL-1 beta, IL-6, and IL-8 in lavage at different time points within the first month of life, correlating with inflammatory cell influx into the airways. 54'55 Bronchopulmonary dysplasia (BPD) is a major cause of the morbidity associated with preterm birth. In animal models, exposure to hyperoxic conditions mimics many of the features of BPD. 56'57 Interestingly, adult animals are more susceptible to the toxic effects of hyperoxia than newborn animals, suggesting that supplementary mechanisms exist in the young that are no longer present in the adult. 56'58 In newborn rats, CINC-1 and MIP-2 (rat homologues for IL-8 and GRO-beta) expression is detectable in whole lung homogenates; expression of these chemokines is increased in response to hyperoxia and occurs in parallel with neutrophil accumulation within airways. 59 In comparison with adult mice, exposure of newborn mice to hyperoxic conditions results in elevated expression of TNF-alpha, IL-1 beta, and IL-6. 6~ As an extension of this approach, chemokine expression was evaluated in lung homogenates of mice; newborn mice exposed to hyperoxic conditions showed higher elevations of MCP-1, MIP-2, IP-10 mRNA within airways as compared with adult counterparts. 61 Further, chemokine induction in the hyperoxic newborn mouse occurred prior to the onset of significant airway inflammation, whereas in the adult, chemokine expression was concurrent with ongoing airway inflammation. Although cytokines and chemokines have a primary role in the induction and regulation of airways inflammation, it has been suggested that elevation of these proteins in newborn airways in response to hyperoxic conditions also functions as a protective mechanism. 61

DEVELOPMENTAL REGULATION OF P U L M O N A R Y I M M U N E / I N F L A M M A T O R Y CELL TRAFFICKING In the adult, lymphocytes may accumulate within the lung via two different mechanisms: (1) expansion of precursors that are already present within the lung parenchyma, or (2) expansion of lymphocytes within regional lymph nodes, followed by trafficking via the circulation into the lung. 62 Because there are limited numbers of precursor or 'memory' lymphocytes within neonatal airways, it is likely that the infant is most dependent upon immune cell expansion in regional lymph nodes to respond to environmental exposure to antigens and infectious agents. Trafficking patterns of circulating lymphocytes have been best described in the sheep, whereby cannulation of lymph nodes allows for access to lymphocytes that are draining from specific sites; radioactive labeling and subsequent reinfusion into animals enables one to follow recirculation patterns of lymphocytes obtained from regional lymph notes. A direct comparison of lymphocyte

migration patterns in fetal versus adult sheep suggests a significant incapacity of fetal lymphocytes to be transported to mucosal sites such as the small intestine or lung. 63 A related finding in a rodent model of infectious disease supports the notion that trafficking of lymphocytes to mucosal organs during the fetal/neonatal period is somewhat restricted. It has been shown that, unlike adult mice, neonatal mice exhibit a 3-week delay in the initiation of a pulmonary inflammatory response to Pneumocystis carinii infection. 64 Adoptive transfer of normal splenocytes from adult mice into neonatal SCID mice does not eliminate the delay in pulmonary inflammatory response to P. carinii infection. In reverse, adoptive transfer of splenocytes from neonatal mice into adult SCID mice does permit resolution of P. carinii infection. 65 These findings clearly demonstrate that newborn lymphocytes are functional and capable of responding to infectious agents such as P. carinii. Importantly, the data suggest that the neonatal lung environment is the limiting factor in resolving infection. Further analysis showed fewer antigen presenting cells and reduced efficiency of phagocytic activity within neonatal mouse airways; both factors could contribute to the apparent ineffectiveness of the newborn lung to promote robust lymphocyte recruitment into the airways in response to a challenge with an infectious organism. 64

P U L M O N A R Y DEFENSE M E C H A N I S M S DURING INFANCY

Alveolar macrophage As discussed above, the alveolar macrophage is the predominant leukocyte phenotype in the lung for all age groups (greater than 89%). It has been reported that the frequency of alveolar macrophages in bronchoalveolar lavage (BAL) is significantly higher in children less than 2 years of age (98% frequency), as compared with children and teenagers up to 17 years of age (91-92% frequency). 29 The predominance of macrophages within airways of infants is likely to be representative of the first level of innate immunity established for the 'naive' lung; as the infant matures and comes in contact with environmental antigens, cells from the adaptive arm of the immune system will contribute to the resident leukocyte population of the airways. Functionally, the alveolar macrophage of the infant lung is not identical to that of the adult. As compared with their more mature counterparts, alveolar macrophages from children under 2 years of age express less HLDA-DR, are impaired with regards to respiratory burst (as measured by the NBT assay), and produce less IL-1 and TNF-alpha in response to LPS stimulation. 29 In the newborn monkey, the ability of alveolar macrophages to migrate in response to endotoxin-activated plasma is significantly inhibited as compared with adults; chemotactic ability is increased to nearly adult levels as early as 6 days of age. 66 In a similar fashion, intracellular killing of Candida albicans by newborn monkey alveolar macrophages is much reduced, but functions at nearly adult capacity by 6 days of age. The ability to

phagocytose C. albicans is also much reduced in the newborn monkey alveolar macrophage, however this function does not reach adult capacity until after 6 months of age. In contrast with the monkey, newborn rat macrophages have greater phagocytic capacity for certain gram-negative and positive bacteria as compared with adults and may represent a compensatory mechanism for the immature neonatal immune system. 67 These findings emphasize the distinctive immunological differences between species.

Neutrophils Cord blood-derived neutrophils from healthy newborns exhibit an attenuated chemotactic response to stimuli such as leukotriene B4 in vitro, as compared with peripheral blood neutrophils from adults. 68 Interestingly, the chemotactic ability of newborn neutrophils to leukotriene B4 is as enhanced by a larger pore size chemotaxis chamber, suggesting that limited deformability plays a role in attenuation of chemotaxis. In a related study, it was shown that newborn neutrophils have a diminished capacity to undergo cellular orientation in response to a chemotactic gradient; this correlated with a reduced capacity for cytoplasmic microtubule complexes. 69In vivo, chemotaxis of neutrophils from 4-week-old rabbits into inflamed airways was impaired in comparison with adult rabbit neutrophils, demonstrating a fundamental difference in the migratory behavior of neutrophils from very young animals. 7~ Impairment of migratory behavior has been attributed to a number of factors, including decreased receptor binding sites for formyl-methionyl-leucyl-phenylalanine, reduced cell surface expression of Mac-1 (CDllb/CD18), and diminished actin polymerization. 71-73

Antigen presenting cells Dendritic cells are the primary antigen-presenting cells of the lung and are critical for the immunosurveillance of inhaled antigens. By the 13th week of gestation, HLA-DR positive dendritic-like cells are observed in the human fetal lung. TM HLA-DR positive cells within the fetal lung are dispersed as single cells within the interstitium, do not stain for markers of macrophage/monocyte lineage, and increase in frequency with gestational age (to 21 weeks). Studies performed in the rat demonstrate that MHC Class II positive dendritic cells are very few in number within neonatal airways. 75'76 Isolated MHC Class II positive dendritic cells from the fetal rat lung are not as effective as adult-derived cells in stimulating cell proliferation within the context of an autologous mixed lymphocyte reaction, yet, at birth, the dendritic cells of the newborn rat function nearly as well as in the adult. 76 In contrast, another study has shown that MHC class II positive dendritic cells obtained from airways of very young rats respond poorly to maturation signals via GM-CSF and provide an attenuated response to microbial challenge. 77 Cumulatively, these studies suggest that lung-derived dendritic cells from the newborn have limited functional competency, as compared with their adult counterparts. Lastly, MHC class II expression

by airway epithelial cells has been reported in fetuses greater than 21 weeks of age, primarily in conjunction with lung inflammation. 7s Most infants at birth express M H C class II on airway epithelium regardless of the presence of an inflammatory response. At present, the capacity for airway epithelial cells to function as effective antigen presenting cells is not well defined.

CONCLUSIONS Epidemiological studies suggest that early childhood immunerelated events can have a profound impact on the development of airways disease later in life (reviewed by Holt79). Experimental data to support this notion in the very young h u m a n population does not exist and cannot be obtained due to ethical reasons. Although descriptive findings from post-mortem h u m a n specimens and peripheral blood have provided important insights into the dramatic changes that occur within the pulmonary/systemic i m m u n e system during the first year of life, these data provide a restricted view of the developmental dynamics that occur within the airways during this important period of development. In addition, data obtained from adult populations are not comparable to infants owing to i m m u n e changes associated with introduction of antigens from the environment. If, in fact, the infant i m m u n e system (pulmonary or otherwise) is plastic and can be 'molded', then the introduction of therapeutics or avoidance of antigenic stimuli must occur at this time. T h e establishment of animal models to address mechanistic questions with regard to i m m u n e events during postnatal development would substantially enrich our knowledge base to support this approach in humans.

ACKNOWLEDGEMENT Supported by N I E H S P01 ES00628, N C R R RR00169, and American L u n g Association Research Grant RG-070.

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27. Khan TZ, Wagener J S, Bost T et al. Early pulmonary inflammation in infants with cystic fibrosis. Am. J. Respir. Crit. Care Med. 1995; 151:1075-82. 28. LoMonaco MB, Barber CM, Sinkin RA. Differential cytokine mRNA expression by neonatal pulmonary cells. Pediatr. Res. 1996; 39:248-52. 29. Grigg J, Riedler J, Robertson CF et al. Alveolar macrophage immaturity in infants and young children. Eur. Respir. J. 1999; 14:1198-1205. 30. Ratjen F, Bredendiek M, Zheng L et al. Lymphocyte subsets in bronchoalveolar lavage fluid of children without bronchopulmonary disease. Am. J. Respir. Crit. Care Med. 1995; 152:174-8. 31. Clement A, Chadelat K, Masliah J et al. A controlled study of oxygen metabolite release by alveolar macrophages from children with interstitial lung disease. Am. Rev. Respir. Dis. 1987; 136:1424-8. 32. Costabel U, Bross KJ, Ruhle KH etal. La-like antigens on T-cells and their subpopulations in pulmonary sarcoidosis and in hypersensitivity pneumonitis. Analysis of bronchoalveolar and blood lymphocytes.Am. Rev. Respir. Dis. 1985; 131:337-42. 33. Hunninghake GW, Crystal RG. Pulmonary sarcoidosis: a disorder mediated by excess helper T-lymphocyte activity at sites of disease activity. N. Engl. J. Med. 1981; 305:429-34. 34. Hubeau C, Puchelle E, Gaillard D. Distinct pattern of immune cell population in the lung of human fetuses with cystic fibrosis. J. Allergy Clin. Immunol. 2001; 108:524-9. 35. E1 Kaissouni J, Bene MC, Thionnois Set al. Maturation of B cells in the lamina propria of human gut and bronchi in the first months of human life. Dev. Immunol. 1998; 5:153-9. 36. Emery JL, Dinsdale F. The postnatal development of lymphoreticular aggregates and lymph nodes in infants' lungs. J. Clin. Pathol. 1973; 26:539-45. 37. Bienenstock J, Johnston N, Perey DYE. Bronchial lymphoid tissue. I. Morphologic characteristics. Lab. Invest. 1973; 28:686-92. 38. Bienenstock J, Johnston N, Perey DYE. Bronchial lymphoid tissue. II. Functional characteristics. Lab. Invest. 1973; 28:693-8. 39. Tschernig T, Pabst R. Bronchus-associated lymphoid tissue (BALT) is not present in the normal adult lung but in different diseases. Pathobiology 2000; 68:1-8. 40. Hiller AS, Tschernig T, Kleemann WJ etal. Bronchusassociated lymphoid tissue (BALT) and larynx-associated lymphoid tissue (LALT) are found at different frequencies in children, adolescents and adults. Scand. J. Immunol. 1998; 47:159-62. 41. Pabst R, Gehrke I. Is the bronchus-associated lymphoid tissue (BALT) an integral structure of the lung in normal mammals, including humans? Am. J. Respir. Cell Mol. Biol. 1990; 3:131-5. 42. Gould SJ, Isaacson PG. Bronchus-associated lymphoid tissue (BALT) in human fetal and infant lung. J. Pathol. 1993; 169:229-34. 43. Tschernig T, Kleemann WJ, Pabst R. Bronchus-associated lymphoid tissue (BALT) in the lungs of children who had died from sudden infant death syndrome and other causes. Thorax. 1995; 50:658-60. 44. Lee SM, Suen Y, Chang L etal. Decreased interleukin-12 (IL-12) from activated cord versus adult peripheral blood mononuclear cells and upregulation of interferon-gamma, natural killer, and lymphokine-activated killer activity by IL-12 in cord blood mononuclear cells. Blood 1996; 88:945-54. 45. Langrish CL, Buddle JC, Thrasher AJ et al. Neonatal dendritic cells are intrinsically biased against Th-1 immune responses. Clin. Exp. Immunol. 2002; 128:118-23.

46. Wilson CB, Westall J, Johnston L et al. Decreased production of interferon-gamma by human neonatal cells. Intrinsic and regulatory deficiencies.J. Clin. Invest. 1986; 77:860-7. 47. Tang ML, Kemp AS. Ontogeny of IL-4 production. Pediatr. Allergy Immunol. 1995; 6:11-19. 48. Weatherstone KB, Rich EA. Tumor necrosis factor/cachectin and interleukin-1 secretion by cord blood monocytes from premature and term neonates. Pediatr. Res. 1989; 25:342-6. 49. Yachie A, Takano N, Yokoi T et al. The capability of neonatal leukocytes to produce IL-6 on stimulation assessed by whole blood culture. Pediatr. Res. 1990; 27:227-33. 50. Cederblad B, Riesenfeld T, Aim GV. Deficient herpes simplex virus-induced interferon-alpha production by blood leukocytes of preterm and term newborn infants. Pediatr. Res. 1990; 27:7-10. 51. Williams TJ, Jones CA, Miles EA etal. Fetal and neonatal IL-13 production during pregnancy and at birth and subsequent development of atopic symptoms. J. Allergy Clin. Immunol. 2000; 105:951-9. 52. Roth P, Stanley ER. Colony-stimulating factor-1 expression in the human fetus and newborn.J. Leukoc. Biol. 1995; 58:432-7. 53. G~ihler A, Stallmach T, Schwaller Jet al. Interleukin-8 expression by fetal and neonatal pulmonary cells in hyaline membrane disease and amniotic infection. Pediatr. Res. 2000; 48:299-303. 54. Kotecha S, Wilson L, Wangoo A et al. Increase in interleukin (IL)-I beta and IL-6 in bronchoalveolar lavage fluid obtained from infants with chronic lung disease of prematurity. Pediatr. Res. 1996; 40:250-6. 55. Munshi UK, Niu JO, Siddiq MM etal. Elevation of interleukin-8 and interleukin-6 precedes the influx of neutrophils in tracheal aspirates from preterm infants who develop bronchopulmonary dysplasia. Pediatr. Pulmonol. 1997; 24:331-6. 56. Frank L, Bucher JR, Roberts RJ. Oxygen toxicity in neonatal and adult animals of various species. J. Appl. Physiol. 1978; 45:699-704. 57. Holm BA, Matalon S, Finkelstein JN et al. Type II pneumocyte changes during hyperoxic lung injury and recovery. J. Appl. Physiol. 1988; 65:2672-8. 58. Bonikos DS, Bensch KG, Northway WH Jr. Oxygen toxicity in the newborn. The effect of chronic continuous 100 percent oxygen exposure on the lungs of newborn mice. Am. J. Pathol. 1976; 85:623-50. 59. Deng H, Mason SN, Auten RL. Lung Inflammation in Hyperoxia Can Be Prevented By Antichemokine Treatment in Newborn Rats. Am. J. Respir. Crit. Care Med. 2000; 162:2316-23. 60. Johnston CJ, Wright TW, Reed CK et al. Comparison of adult and newborn pulmonary cytokine mRNA expression after hyperoxia. Exp. Lung Res. 1997; 23:537-52. 61. D'Angio CT, Johnston CJ, Wright TW etal. Chemokine mRNA alterations in newborn and adult mouse lung during acute hyperoxia. Exp. Lung Res. 1998; 24:685-702. 62. Joel DD, Chanana AD. Comparison of pulmonary and intestinal lymphocyte migrational patterns in sheep. Ann. N.Y. Acad. Sci. 1985; 459:56-66. 63. Cahill RN, Poskitt DC, Hay JB et al. The migration of lymphocytes in the fetal lamb. Eur. J. Immunol. 1979; 9:251-3. 64. Garvy BA, Qureshi MH. Delayed inflammatory response to Pneumocystis carinii infection in neonatal mice is due to an inadequate lung environment.J. Immunol. 2000; 165:6480-6. 65. Qureshi MH, Garvy BA. Neonatal T cells in an adult lung environment are competent to resolve Pneumocystis carinii pneumonia. J. Immunol. 2001; 166:5704-11. 66. Kurland G, Cheung ATW, Miller ME et al. The Ontogeny of Pulmonary Defenses: Alveolar Macrophage Function in Neonatal and Juvenile Rhesus Monkeys. Pediatr. Res. 1988; 23:293-7.

67. Lee PT, Holt PG, McWilliam AS. Role of alveolar macrophages in innate immunity in neonates: evidence for selective lipopolysaccharide binding protein production by rat neonatal alveolar macrophages. Am. J. Respir. Cell Mol. Biol. 2000; 23:652-61. 68. Dos Santos C, Davidson D. Neutrophil chemotaxis to leukotriene B4 in vitro is decreased for the human neonate. Pediatr. Res. 1993; 33:242-6. 69. Anderson DC, Hughes BJ, Wible LJ et al. Impaired motility of neonatal PMN leukocytes: Relationship of abnormalities of cell orientation and assembly of microtubules in chemotactic gradients.J. Leukoc. Biol. 1984; 36:1-15. 70. Hyde DM, Downey GP, Tablin F etal. Age-dependent neutrophil and blood flow responsiveness in acute pulmonary inflammation in rabbits. Am. J. Physiol. 1997; 272:L471-8. 71. Bortolussi R, Howlett S, Rajaraman K et al. Deficient priming activity of newborn cord blood-derived polymorphonuclear neutrophilic granulocytes with lipoppolysaccharide and tumor necrosis factor-alpha triggered with formyl-methionylleucyl-phenylalanine. Pediatr. Res. 1993; 34:243-8. 72. Harris MC, Shalit M, Southwick FS. Diminished actin polymerization by neutrophils from newborn infants. Pediatr. Res. 1993; 33:27-31. 73. Anderson DC, Freeman KL, Heerdt B etal. Abnormal stimulated adherence of neonatal granulocytes: impaired

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induction of surface Mac-1 by chemotactic factors or secretagogues. Blood 1987; 70:740-50. Hofman FM, Danilovs JA, Taylor CR. HLA-DR (La)-positive dendritic-like cells in human fetal nonlymphoid tissues. Transplantation 1984; 37:590-4. Nelson DJ, McMenamin C, McWilliam AS et al. Development of the airway intraepithelial dendritic cell network in the rat from class II major histocompatibility (La)-negative precursors: differential regulation of La expression at different levels of the respiratory tract. J. Exp. Med. 1994; 179:203-12. McCarthy KM, Gong JL, Telford JR. Ontogeny of La+ accessory cells in fetal and newborn rat lung. Am. J. Respir. Cell MoL Biol. 1992; 6:349-56. Nelson DJ, Holt PG. Defective regional immunity in the respiratory tract of neonates is attributable to hyporesponsiveness of local dendritic cells to activation signals. J. lmmunol. 1995; 155:3517-24. Peters U, Papadopoulos T, Muller-Hermelink HK. MHC class II antigens on lung epithelial of human fetuses and neonates. Ontogeny and expression in lungs with histologic evidence of infection. Lab. Invest. 1990; 63:38--43. Holt PG. Programming for responsiveness to environmental antigens that trigger allergic respiratory disease in adulthood is initiated during the perinatal period. Environ. Health Perspect. 1998; 106(Suppl. 3): 795-800.

IMPORTANCE OF AND XENOBIOTIC ENZYMES

ANTIOXIDANTS METABOLIZING

The cellular composition and architectural organization of the adult distal respiratory system (trachea, bronchi, and lungs) is highly complex. The respiratory system is comprised of a large number of morphologically distinct cell phenotypes organized into a highly branched series of tissues surrounding air passages (see Breeze and Wheeldon I for a complete review of cell types). This very complex structure begins as an evagination of the undifferentiated epithelium from the foregut into a surrounding mesenchymal bundle. All of the developmental stages (embryonic, pseudoglandular, canalicular, and saccular) associated with the transformation from this simple tubular structure into a highly complex organ system are potentially susceptible to modification by toxic agents. These stages have been reviewed in more detail in earlier chapters. The susceptibility of the lung as a target organ for specific toxicants may be altered by the cellular processes involved in each of these stages of development. When considering the potential pulmonary toxicity of a compound, a backdrop of developmental issues should be kept in mind. These issues have been addressed in more detail previously 2 and will be only briefly mentioned here. First, one must remember that lung development is a multi-event process and occurs during both prenatal and postnatal periods. The initial evagination of the tracheobronchial bud occurs early in gestation and a significant amount of lung growth and development continues after birth. Only a few maturational events need to be complete at birth for survival. Second, overall growth, branching morphogenesis, and cellular differentiation are events The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

common to all stages of lung development. Third, all of these developmental events occur in combination with a steadily increasing total cell mass. The interplay and balance between the xenobiotic activating enzymes and detoxifying enzyme systems have been shown to be critical factors in dictating the toxic response of the respiratory system to bioactivated compounds in adults. 3-6 This interplay is also important in perinatal animals during pulmonary morphogenetic and differentiation processes. An obvious role of antioxidants in the lung is protecting the lung from oxidative stress at birth, when the lung switches from a relatively hypoxic state to a relatively hyperoxic state. However, the antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase), nonenzymatic antioxidants (glutathione), as well as the xenobiotic metabolizing enzymes (cytochrome P450 monooxygenases, glutathione S-transferases, epoxide hydrolases, and glucuronyl transferases), all have important roles in modulating cellular interaction with environmental toxicants. Antioxidants and antioxidant enzymes protect the lung from oxidant pollutants such as ozone and nitrogen dioxide. Xenobiotic metabolizing enzymes act on compounds to make them more water-soluble and increase their rate of elimination. Many lung-targeted toxicants, such as furans, 7 chlorinated hydrocarbons, 8'9 aromatic hydrocarbons, 4'10 indoles, T M and pyrrolizdine alkaloids 13 require bioactivation in order to produce their toxicity. The reactive metabolites of these bioactivated compounds are then detoxified by a number of pathways. Other compounds, such as organometallic 14 and amphiphilic agents, 15 are toxic when introduced to the body and also require metabolic detoxification. This section defines the pattern of differentiation for antioxidant enzyme pathways as well as bioactivation Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

and detoxification enzyme systems during pre- and postnatal lung development.

DEVELOPMENT OF ENZYME SYSTEMS

ANTIOXIDANT

During late gestation, changes in the fetal lung include the development of the antioxidant enzymes. This group of enzymes includes superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx). These enzymes play an important role in the detoxification of highly reactive oxygen metabolites that are produced during normal aerobic cellular respiration as well as during oxidant injury. In general, the pulmonary antioxidant enzyme system develops during the last 10-15% of gestation in humans 16 as well as in laboratory animals such as rats, hamsters, guinea pigs, rabbits, and lambs. 17-25

Superoxide dismutase The antioxidant enzyme SOD rapidly catalyzes the conversion of superoxide anion to hydrogen peroxide and oxygen. Manganese SOD (MnSOD), an inducible form of superoxide dismutase, is predominantly located in the mitochondria, 26'27 while the non-inducible copper-zinc SOD (Cu-ZnSOD) is located in the cytosol. 28 There is also an extracellular form of SOD (EC-SOD) localized in extracellular matrix and extracellular fluid. 29'3~EC-SOD is thought to play a role in modulating nitric oxide concentrations by controlling the amount of superoxide anion available to react with it, 31 but very little is known about this form in the developing lung. 32 In most experimental animals studied, SOD activity increases throughout the pre- and postnatal periods, however, there are some species differences. A comparison of the timecourse of expression of pulmonary antioxidant enzymes during lung development is represented in Fig. 12.1.

In rats, pulmonary Cu-ZnSOD activity and total enzyme content peak during the late gestational period (last 10-15% of gestation) and activity peaks again early in the postnatal period, finally reaching adult levels at 4 weeks. 20'23'33-35 Temporal expression of mRNA does not correlate with the activity levels. 35'36 In contrast, pulmonary MnSOD activity and mRNA content remain steady throughout late gestation and the early postnatal period in the rat, 17'33'36 even though total enzyme content increases 6.9-fold. 2~ In the mouse, Cu-ZnSOD mRNA expression peaks at 15 days gestation (early pseudoglandular stage of lung development), is low during the later stages of fetal lung development, and peaks again at birth. 36'37 In rabbits, pulmonary Cu-ZnSOD and MnSOD activities also peak in late gestation and in the postnatal period. 22 The only species in which pulmonary EC-SOD protein expression has been evaluated is the rabbit. Expression is low and contained within the intracellular compartment during the fetal period. Expression increases and shifts to the extracellular compartment with increasing age.32 In contrast to experimental animals, human Cu-ZnSOD and MnSOD activity remains constant during fetal and neonatal lung development, and no surge in expression is seen. 16'38

Glutathione peroxidase Glutathione peroxidase (GPx) is a cytosolic enzyme that catalyzes the reduction of hydrogen peroxide to water and oxygen as well as catalyzing the reduction of peroxide radicals to alcohols and oxygen. 19 As with SOD, GPx activity and total enzyme content increase during late gestation in two of the experimental animals studied: guinea pig and rat (Fig. 12.1). 20,22,23,33,36 However, mRNA expression steadily decreases during fetal development and does not correlate with activity levels. 17'36 In the mouse, however, both GPx activity and mRNA levels peak in late gestation and then activity steadily decreases throughout the postnatal period. 37'39 In humans, GPx activity is lowest at 10-20 weeks gestation,

Fig. 12.1. Species-dependent expression of antioxidant enzyme protein and/or activity during stages of lung development. Gray portion of bars represent transitional expression, black portions of bars represents mature expression for human,16'38guinea pig, 22 rat 18'20'23'34'35'101 and mouse.39

highest during the early postnatal period (41-50 weeks post conception) and drops at 3-5 months of age. 16'40

in higher abundance in Clara cells than in type II cells. 47 Pulmonary glutathione synthetic activity is affected by several factors. One factor, maternal nutrition, has been shown to be very important in regulating the perinatal activity of glutathione cycle enzymes. Pulmonary y-GCS activity is lower in rat pups from dams that were fed low protein diets than in pups from dams fed diets supplemented with casein. 48 Surgical procedures in newborn guinea pigs also affect glutathione synthetic activity and result in increased pulmonary glutathione content. 49

Catalase Like GPx, catalase accelerates the combination of two hydrogen peroxide molecules to produce water and oxygen. Catalase activity steadily increases during both the fetal and postnatal periods of lung development in humans 16 and in all experimental animals studied, including guinea pigs, rats, and mice (Fig. 12.1). 20,23,33,36,39 In contrast to GPx, though, mRNA expression during this time is consistent with the increase in activity. 36 In humans, catalase activity increases Miscellaneous antioxidants 3.5-fold between 10 weeks of gestation and 3 months postThere is little information regarding the expression of other natal age, unlike SOD and GPx, which remain constant antioxidants during lung development. Ceruloplasmin, throughout human lung development. 16 The increase in .....a free radical scavenger, is a major extracellular antioxidant catalase activity during the late gestational period has been in human lung epithelial fluid. 5~Ceruloplasmin mRNA has suggested by McElroy and colleagues to be linked to been detected in fetal lung of species as varied as mouse and maturation of the surfactant system and has been correlated baboon. 51 The mRNA first appears in baboon lung during temporally to increases in lung dipalmitoylphosphatidylthe pseudoglandular stage (60d gestation) in bronchial choline (DPPC) content. 16 epithelium. By the saccular stage of development (140d gestation), ceruloplasmin mRNA is detected in all airway Glutathione epithelium. In mice, ceruloplasmin mRNA is detected Glutathione acts as an antioxidant by directly scavenging during the saccular stage and is expressed in epithelium of free radicals through the donation of a hydrogen atom. all airways. Peroxiredoxin, an antioxidant which reduces The synthesis of glutathione is homeostatically controlled, hydrogen peroxide to molecular oxygen has also been both inside the cell and outside. 41-44 Glutathione is not described in lungs of newborn baboons. 52 Both pulmonary taken up by pulmonary cells in its intact form. It is syntheperoxiredoxin mRNA and activity are expressed at low sized and utilized through the 7-glutamyl cycle, a multistep, levels in fetal baboons, and are upregulated by oxygen ATP-dependent process. Very simplistically, it begins with treatment. Urate and ascorbate (vitamin C) levels in bronthe cleavage of extracellular glutathione and other glutamatechoalveolar lavage fluid have been described in preterm containing peptides by y-glutamyl transpeptidase, a human infants. 53 Urate levels drop during the first two membrane-bound enzyme. The resulting amino acids can weeks of life, while ascorbate levels drop during the first then be moved by specific transporters into the intracellular week then increase during the second week of life. environment. 45 The rate-limiting step in the synthesis of glutathione, however, is the formation of y-glutamylcysteine by y-glutamylcysteine synthetase (y-GCS). The formation of DEVELOPMENT OF X E N O B I O T I C glutathione is complete when glutathione synthetase M E T A B O L I Z I N G ENZYME SYSTEMS. catalyzes the addition of glycine to y-glutamylcysteine to form 7-glutamylcysteinylglycine (glutathione). 44 Even though Cytochrome P450 monooxygenases glutathione plays an important role in protection against The cytochrome P450 (CYP) monooxygenases are very both oxidant and reactive metabolite injury, there is very important enzymes in both bioactivating and detoxifying little information available on glutathione levels or y-glutamyl compounds. In adults, immunoreactive CYP enzymes have cycle enzyme activity during fetal and postnatal lung been detected in four pulmonary cell types: the nonciliated development. Evidence from the few rodent studies availbronchiolar (Clara) cell, the type II epithelial cell, the able suggests that neonates have similar glutathione levels endothelial cell and the alveolar macrophage. The developas adults. In rats, pulmonary glutathione levels are at mental expression of CYP monooxygenases is the most mature levels right before birth, decrease 30% at birth and extensively studied of all the pulmonary xenobiotic metabthen increase back to mature levels between 10 and 15 days. 46 olizing enzymes. Even so, the expression of pulmonary The activities of two important enzymes of the y-glutamyl CYP monooxygenases in the differentiating lung has been cycle, y-GCS and glutathione synthetase, are similar in the evaluated in only five species: rabbit, hamster, mouse, rat lungs of 1-2-week old mice and adult mice. 39 7-Glutamyl and goat. 54-63 Some of the CYP isozymes (and representative transpeptidase mRNA is detectable by polymerase chain substrates) that have been evaluated during lung developreaction in late fetal and early postnatal rat lung, and is ment include CYPIA1 (ethoxyresorufin), CYP2B (O,O,Slocated in the type II cell. 47 However, pulmonary 7-glutamyl trimethylphosphorothioate), CYP2F (naphthalene), and CYP4B transpeptidase activity is very low in newborn rat, and only (2-aminofluorene). In addition, NADPH CYP reductase, gradually increases to mature levels. 47'48 In the late postimportant for electron transport during the catalytic cycle natal and adult rat lung, 7-glutamyl transpeptidase is found of CYP monooxygenases, has also been evaluated. In general,

the first detectable expression of CYP monooxygenases occurs after the development of the smooth endoplasmic reticulum (SER). Protein for NADPH CYP reductase is detected before the monooxygenase isozymes in all species evaluated (Fig. 12.2). The youngest age at which the intracellular protein can be detected immunohistochemically varies substantially within the five species evaluated. Isozymes CYP2B and CYP2F are expressed earliest: they are detectable in the late fetal stage in mice and hamsters, 56'61 in the early postnatal period of rabbits and rats, 54'58'64'65 and around 6 weeks of age in goats. 62'63 In goats, CYP4B is detected 2-3 days of age later. Activity for these proteins is first detected approximately 2-3 days after the protein is immunologically detectable. The temporal and spatial distribution of immunoreactive pulmonary CYP protein has been described in detail for the rabbit. 54 Initially, immunoreactive protein is detected only in the most apical border of a small percentage of the nonciliated cell population. During the period of time in which the amount of detectable protein increases, the distribution changes in two ways. First, an immunologically detectable protein is found in an increasing proportion of the nonciliated cells. Second, the distribution of detectable protein within an individual cell moves from the apex to the base until it is evenly distributed throughout the cell. The timeframe between when the protein is first detectable and when it reaches mature distribution and intensity also varies among species. Mature expression of CYP proteins can take up to

four weeks in rabbits and mice,54'61 three weeks in hamsters, 56 and as little as one week in rats. 58'65 Mature expression of the immunoreactive protein, however, does not necessarily indicate mature enzyme activity, especially in rats and rabbits. 58'65 This suggests that the activity of these enzymes continues to increase after the protein density and organelle composition have reached adult levels. It is critical to understand the temporal and spatial development of CYP monooxygenase activity in order to be able to extrapolate among species.

Glutathione S-transferase It is essential to understand the temporal and spatial development of the detoxifying enzymes, including glutathione S-transferase, in order to appreciate the role they play in protecting the lung during development. Glutathione S-transferases (GSTs) are a group of dimeric cytosolic enzymes that catalyze the conjugation of GSH to electrophilic compounds. In human tissues, GSTs are grouped into four classes based on isoelectric points: alpha, mu, pi, and theta. 66-68 All four classes have been described in adult lung, although only the expression of GST alpha, mu, and pi has been evaluated in developing lung of mice, 69'7~ rabbits, 71 goats, 62 guinea pigs, 71 and humans. 72-75 Unlike CYP monooxygenases, which are expressed in only four cell types in the lung, pulmonary GSTs are expressed in multiple cell types including both ciliated and nonciliated bronchiolar epithelial cells, alveolar epithelial cells, endothelial cells, and smooth muscle cells. The ubiquitous distribution of

Fig. 12.2. Species-dependent expression of CYP monooxygenase protein and/or activity during stages of lung development. Gray portion of bars represent transitional expression, black portions of bars represents mature expression for goat, 63 rabbit,54 hamster,56 rat, 58'65 and mouse. 61 Question marks (?) indicate a lack of available information during the fetal period.

the transferase enzymes suggests a role in protecting cells from reactive intermediates from both exogenous and endogenous metabolism. In goats, rabbits and guinea pigs, information regarding the expression of GSTs is available for only the postnatal period of lung development (Fig. 12.3). 62'71 Rabbits and mice at birth have pulmonary GST protein expression and activity levels similar to adults, while GST expression is not mature in goats until later in the postnatal period. In mice, the only laboratory animal in which GST expression has been described during fetal lung development, GST isozymes are present very early in development, however, the isozymes do not reach mature expression levels until early in the postnatal period. 7~ In contrast to mice, human fetal lung has been shown to contain more overall GST activity than adult lung. 74 During human lung development, the total activity of pulmonary GST decreases 5-fold between 13 weeks gestation and birth and then remains constant. 75 Pulmonary GST activity is due to a combination of all the isozymes, and the contribution of each isozyme changes during development. In humans, 72'76'77 GST1 (mu) increases as a percentage of total GST activity over development, from 5% at 10 weeks gestation (pseudoglandular stage of lung development) to 25% at birth and 60% in adult tissue. GST2 (alpha) consistently contributes 50% of the total activity. GST3 (pi) decreases as a percentage of total activity over development, from 50% of the total GST activity at 10 weeks gestation to 15% at birth, and is almost undetectable in adult tissue. These activity data are supported by immunohistochemical data. 73 GST2 is expressed consistently throughout development: strongly in proximal airway epithelium and weakly in more distal airways in fetus, newborns and adults. GST3 decreases in expression during development. It is expressed strongly in all epithelial cells in early gestation, and then expression is lost in the distal airways by 24 weeks of gestation. 73'74'78

Epoxide hydrolase There are three general forms of epoxide hydrolase (EH) which all create 1,2-dihydrodiols from epoxides, although each form has different substrate specificities and tissue distribution. 79 The first form, microsomal EH (mEH), is involved in the conversion of cyclic epoxides and is found in high levels in the smooth endoplasrnic reticulum (SER) and in lower levels in other membranous organelles. The second form, cytosolic EH (cEH), hydrates aliphatic epoxides and is found in the cytosol and peroxisomes. The third form, cholesterol EH, catalyzes the hydration of cholesterol epoxides and is located in the microsomal fraction. Substrate specificity may also differ immensely among species and tissues (reviewed in Wixtrom and Hammock79). As with most metabolic enzymes, the highest levels of both mEH and cEH activity are found in the liver, and this is true in adults of a wide variety of species: rabbits, s~ humans, 7s rats, sl and mice. 7~ The ratio of cytosolic to microsomal EH activity, though, varies by species and by organ. In humans, cEH activity is higher than mEH activity in both the liver and the lung. 78'83-85 In mice, while cEH activity is higher than mEH activity in the liver, the reverse is true in the lung. 86 EH activity also varies within the lung. EH activity is reported to be present in microdissected airways of dogs with the highest activity in distal airway generations and lower activities in proximal airway generations. 87 There are few studies on the development of epoxide hydrolase (Fig. 12.3). In mice, the predominant pulmonary form of epoxide hydrolase is microsomal, with the cytosolic form found only in vascular smooth muscle. 7~ Immunoreactive mEH protein is detectable in airway epithelial cells soon after birth, and reaches mature density and distribution near the age of weaning. However, specific activity for mEH in mouse lung is detectable at mature levels at 7 days

Fig. 12.3. Species-dependent expression of glutathione S-transferase (alpha, mu, and pi combined) and epoxide hydrolase (mEH and cEH combined) protein and/or activity during stages of lung development. Gray portion of bars represent transitional expression, black portions of bars represents mature expression for goat, 62 rabbit, 71'1~176 guinea pig, 71 mouse, 7~ and human. 72-75'83-85Question marks (?) indicate a lack of available information during the fetal period.

postnatal age. 7~ In contrast to the mouse, the predominant pulmonary form of epoxide hydrolase in humans is cytosolic and EH mRNA expression gradually increases to adult levels by 65 days postnatal. 55 Increases in fetal human pulmonary EH activity do not correlate with increases in mRNA expression. 85

Glucuronyl transferase Glucuronidation is another major pathway for the metabolic elimination of parent compounds (such as 4-ipomeanol) or primary metabolites (such as 1-naphthol) and as such can provide important means of protecting extrahepatic tissues from toxicants. 88 As with the other Phase II enzymes discussed in this chapter, glucuronyl transferases are widely distributed throughout tissues, with the highest activity found in the liver. These enzymes have been described in the lungs of adult rabbits, 89 dogs,87'90humans, 84 and sheep. 91 Similar to CYP monooxygenases, the distribution of UDPglucuronyl transferase is restricted to bronchial epithelial cells, Clara cells and type II pneumocytes in rat lung. There are reported species-specific differences in activity levels. UDP-glucuronyl-transferase activity has been shown to be evenly distributed throughout the respiratory tract of the dog and is similar to activity levels found in the liver. 87 In the human, 92 pulmonary UDP-glucuronyl transferase activity is considerably lower than hepatic activity. In contrast to the detailed studies of the development of hepatic glucuronyl transferases, there is only one report on the developmental expression of this enzyme in the lung. In goats, pulmonary UDP-glucuronyl-transferase activity has been reported to be lowest at birth and highest in adults. However, the pattern of enzyme expression also varies with the substrate used. 62

Miscellaneous enzymes In addition to the four enzyme systems already mentioned, there is a small amount of information available concerning the expression of two other enzymes during lung development: flavin-containing monooxygenases and sulfotransferases. 64'93 Flavin-containing monooxygenases are important oxidative metabolizing enzymes and have much in common with the cytochrome P450 monooxygenases. They have similar molecular weights, are localized in SER, have the highest expression in liver, require NADPH, and have multiple isozymes. 94'95 To date, flavin-containing monooxygenases have only been studied during lung development in rabbits. 64 Activity, protein and mRNA are all expressed as early as 25 d gestation (canalicular stage). Flavin-containing monooxygenase expression is high prenatally (except for an unexplained decrease at 28 d gestation), drops immediately after birth, and then steadily increases throughout postnatal lung development. This expression pattern matches that of CYP2B4 and CYP4B1 in the rabbit lung. 64 Sulfation is a major detoxification pathway, resulting in a highly watersoluble product. 96'97 There are two major subfamilies: phenol sulfotransferase and hydroxysteroid sulfotransferase,

each of which has a different substrate specificity. 98 Human pulmonary hydroxysteroid sulfotransferase, which sulfates steroids and cholesterol, is found in low levels early in gestation (around 56d gestation) and expression peaks at one year after birth. 93 Hydroxysteroid sulfotransferase is expressed in most ciliated, nonciliated, and basal airway cells, but not in mucus-secreting cells. Human phenol sulfotransferase is highly expressed and widely distributed in fetal lung. 93'99 After birth, expression decreases and the distribution is restricted to the proximal airways.

CONCLUSIONS Humans are exposed to multiple compounds early in life, yet most toxicological studies focus on the effects in adults. In addition, decisions regarding acceptable levels of environmental contaminants are based on adult data, possibly missing the most susceptible portion of the population, our children. Indeed there is growing evidence that exposure to bioactivated pollutants produces much higher pulmonary toxicity in neonates than in adults. Although the human lung needs to be sufficiently formed at birth in order to perform its primary function of gas-exchange, lung development continues for approximately 8-12 years. 1~176 During both the pre- and postnatal period of lung development, there are rapid rates of cellular differentiation, cell division and alveolarization occurring, making the early postnatal lung uniquely susceptible to injury by environmental oxidant and toxic air pollutants. 2 The enzyme systems responsible for protection from oxidant injury and toxicantinduced injury differentiate during the perinatal period, with the majority of differentiation activity occurring for an extended period of time after birth. Each of these enzyme systems is expressed in a different pattern during pre- and postnatal lung development. Postnatal patterns for expression of these enzyme systems suggest that their distribution and activity in postnatal lung are not reflective of the situation in adults. In the absence of clear epidemiologic evidence suggesting an association between chemical or oxidant exposure and an adverse human health consequence, we still do not, in many instances, understand how results obtained in animal models extrapolate to the human and whether or not there is an age-related susceptibility. As shown in Fig. 12.1, the pulmonary antioxidant enzyme system in humans appears to mature earlier than the corresponding systems in laboratory animals, although most species follow a similar timecourse of expression. Variability between species becomes much greater when evaluating the Phase I and II enzyme systems (Figs 12.2 and 12.3). The few data available indicate that Phase II enzymes are expressed early in lung development, suggesting a role in protecting the lung during the period of rapid cellular differentiation and cell division. CYP monooxygenases are expressed later during lung development, suggestive of a role in protecting the lung from exogenous, rather than endogenous, compounds.

It is clear that there is a lack of information regarding the expression of antioxidants and Phase I and Phase II enzyme systems during lung development in laboratory animals as well as in humans. It is imperative that we obtain more information regarding the temporal and spatial expression of these enzymes in order to understand the mechanisms of susceptibility to environmental insults and how we may extrapolate among species.

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INTRODUCTION

How does the lung grow? The answer to this question remains fragmentary, despite the recent explosion of information concerning the cellular and molecular events that influence normal and compensatory lung growth. In particular, we lack an adequate understanding of how the escalating number of known molecular signals and mediators acting on individual components of the lung are coordinated to form the complex three-dimensional architecture that fully meets but does not exceed the functional requirement of the organism. We understand even less about the altered coordination of growth and remodeling events in adaptation to loss of functioning lung units by injury or disease. The objective of this chapter is not to exhaustively review the known pathways of lung growth, but rather to illustrate the guiding principles of growth regulation by comparing the response to a specific signal, namely mechanical lung strain, during normal and compensatory lung growth, and to interpret the growth response in the context of structure-function relationships with respect to the whole organ.

*To whom correspondence should be addressed. The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

MECHANICAL DEVELOPMENT

FORCES

AND

LUNG

All organic and inorganic branching structures arise as the result of persistent physical forces acting on one medium against the resistance offered by another medium. Branching morphogenesis is characterized by self-similarity, i.e. the same basic unit is repeated at different scales in a manner that can be described by fractal mathematics. Examples of fractal structures abound in nature. In the lung, iterative dichotomous branching of the broncho-vasculo-alveolar complex serves the purpose of maximizing gas exchange surface area within a confined thoracic cavity defined by the rib cage and diaphragm. Since form follows function and vice versa, physiologic variables such as ventilation, perfusion and diffusion must also follow fractal principles, 1 and lung growth and function are intrinsically linked to that of the thorax. During development, the enlarging rib cage exerts a negative intrathoracic pressure resulting in chronic traction on the lung (Fig. 13.1). The mechanical stress and strain activates a cascade of cellular events leading to cell proliferation and growth of lung tissue in addition to a host of metabolic alterations. Lung growth, in turn, reduces septal stress and strain, creating a negative feedback loop that continues until somatic maturity is reached, at which Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

Fig. 13.1. Mechanical interactions between the lung and the thorax. Thick arrows indicate directions of strain. See text for discussion.

time the epiphyses close and the thorax reaches its maximum size and final shape. The lungs, which must conform to the size and shape of its container, also stop growing as the mechanical signals diminish. During the period of growth, recoil of the lung exerts traction on the thorax and modulates thoracic growth in a reciprocal manner. Any process that interferes with mechanical lung-thorax interdependence can alter lung development. For example, congenital diaphragmatic hernia (CDH) is associated with reduced mechanical lung strain and lung hypoplasia. 2 After restoration of strain from the thorax by CDH repair, 'catch-up' alveolar growth occurs associated with vascular remodeling and amelioration of pulmonary arterial hypertension. 3 Long-term pulmonary function in survivors is surprisingly well preserved, 4'5 highlighting the potential for accelerated postnatal alveolar development once normal lung-thorax mechanical interactions are restored. Severe childhood kyphoscoliosis is also associated with blunted or delayed alveolar development, 6-9 resulting in fewer alveoli and larger alveolar airspaces. Conversely, selective inhibition of lung growth may deform the growing rib cage. 1~

Mechanical stretch of alveolar septa in vitro stimulates alveolar epithelial cell proliferation, 11'12 and induces apoptosis, 13 signal transduction pathways, 12'14ion channel flux, 15'16 turnover of matrix proteins, 15'17 cytoskeletal proteins, 18 cytokine growth factors, 19'2~as well as gene expression of surfactant-associated proteins, zl Increased intratracheal pressure following tracheal ligation in utero accelerates lung growth in fetal lamb; the resulting lung hypertrophy can be inhibited by replacing tracheal fluid with saline, 22 suggesting distention-induced accumulation of growth factors in the lung. Fetal breathing movements and chest expansion are critical for prenatal lung development. 23 Even the lack of in utero skeletal muscle movements, as in transgenic mice without the muscle-specific transcription factor myogenin, secondarily impairs lung organogenesis. 24 Postnatal lung stretch induced in vivo by continuous positive airway pressure in weanling ferrets increases total lung capacity, lung weight, protein and DNA content without changing lung recoil. 25 Sustained segmental lung distension with perfluorocarbon in neonatal lambs also accelerates lung growth in an age-dependent fashion. 26 Growth activity is

most pronounced at the lung periphery 27'28 where mechanical forces are largely borne by the septa due to a relative absence of physical support from the more rigid airway and vascular scaffold.

MECHANICAL PNEUMONECTOMY

FORCES

AFTER

The anatomical effects of removing one lung by pneumonectomy (PNX) are illustrated in Fig. 13.2 by magnetic resonance imaging in a human subject (top panels) and by computerized tomographic (CT) scan in a dog (lower panels) in comparison with their normal counterparts. Normally the right and left lungs comprise 55-58% and 42-45% of total volume and diffusing capacity respectively,

and receive a similar proportion of total ventilation and blood flow. 29'3~Gross mediastinal shift is observed after right PNX in dog or man. The remaining lung expands across the midline and its volume increases to --90% of two normal lungs. Blood flow to the remaining lung at a given cardiac output increases by a factor of 1/(fraction of remaining lung tissue) as the entire cardiac output is directed through one lung. The ipsilateral rib cage and hemidiaphragm are distorted. The mediastinal structures are displaced and rotated. The cardiac fossa, a potential space in which the heart resides, loses its compliance as the heart becomes surrounded by the rigid rib cage, elevated right hemidiaphragm and a stiffer remaining lung. Three-dimensional reconstruction of conducting airways in the left lower lobe by CT scan, shown for a dog ten months after right PNX, illustrates the magnitude of post-PNX lung strain (Fig. 13.3).

Fig. 13.2. Upper panels: Magnetic resonance images from a normal human subject (left) and a subject after right PNX (right). Lower panels: CT images from a normal dog (left) and a dog after right PNX (right). See text for details.

Fig. 13.3. Three-dimensional reconstructions from CT scan of airways from the trachea to conducting airways in the left lower lobe within the thorax of a normal dog (left panel) and a dog 10 months after right pneumonectomy (right panel). (Reproduced with permission from Dane DM, Johnson RL Jr, Hsia CCW. Dysanaptic growth of conducting airways after pneumonectomy assessedby CT scan. J. Appl. Physiol. 2002; 93:1235-42.)

After right PNX, the trachea deviates to the right side, and lobar airways are displaced, rotated and splayed. Each airway generation becomes elongated and dilated as a result of chronic traction. 31 In the early post-operative period, acute microcirculatory hyperperfusion may increase endothelial permeability and cause pulmonary edema. 32 Acute mediastinal shift can cause hemodynamic instability as a result of cardiac herniation and/or torsion, kinking of major blood vessels and leakage of lymph. 33-3s Sustained mechanical strain of intrathoracic structures can cause the 'post-pneumonectomy syndrome', particularly after right PNX and in pediatric patients, where a distorted main stem or lobar bronchus gradually becomes obstructed, necessitating the insertion of space-occupying prosthesis to reposition the mediastinum. 36'37 In addition, disrupting the normal mechanical coupling between lung and thorax may cause asymmetric thoracic deformities. 3s Massive dilatation of the esophagus has been reported in dogs after extensive lung resection. 39 Measures that had been used clinically to prevent mediastinal shift after PNX include thoracoplasty, pneumothorax, oleothorax, and plastic sponge plombage on the side of PNX. 4~ Such intervention may relieve symptomatic airway obstruction, but does not always confer long-term benefit. 41'42

POST-PNEUMONECTOMY COMPENSATORY RESPONSE In general, the remaining lung can adapt to the loss of functioning tissue in disease through: (a) greater utilization or recruitment of remaining physiological reserves; (b) remodeling of remaining lung structure; and (c) compensatory growth of new lung tissue. All three mechanisms are invoked in response to PNX, detailed in the following sections. In this regard PNX is a useful model for understanding the consequences and adaptive principles in response to a known and reproducible severity of alveolar destruction irrespective of the specific etiology of disease. Early studies of PNX, summarized by Schilling in 196529 generally regarded post-PNX lung expansion as detrimental, causing emphysema-like changes of the remaining parenchyma leading to gas exchange and mechanical dysfunction. However, recent studies suggest that sustained mechanical strain on the remaining lung is in fact a major signal initiating beneficial adaptation 31 '43 '44 and long-term compensation, evidenced by the maintenance of a near normal exercise capacity 43-4s in adult dogs after removal of 55-58% of lung by right PNX. 4s

R e c r u i t m e n t of physiologic reserves In any biological transport system, 'load' is reflected by flux through the system and 'capacity' is the maximum flux that can be handled. Normally, capacity far exceeds flux by many fold; the difference between load and capacity has been termed 'safety factor' in engineering or 'physiologic reserves' in biology. 46 The preservation of adequate reserves that are not utilized at the basal state but can be readily recruited under conditions of metabolic stress is a fundamental principle for survival and adaptation. Physiologic reserves for pulmonary gas exchange are huge, attributed to both structural and non-structural variables. Structural determinants of diffusive oxygen transport include lung volume, effective alveolar-capillary surface area at the air-tissue interface, and effective diffusion path length from the air-tissue interface to the capillary erythrocytes. Non-structural determinants of diffusive oxygen transport include ventilation, pulmonary blood flow and erythrocyte mass (hematocrit) as well as their distributions. At rest, erythrocytes are unevenly distributed within the lung; many capillaries particularly at the apex are perfused only with plasma but devoid of erythrocytes. 47 Studies using in vivo videomicroscopy in dog lung have shown that as blood flow or pressure increases, the number of subpleural capillary segments perfused with red cells progressively increases, 48 i.e. effective surface area for gas exchange increases without intrinsic structural change. The result of alveolar-capillary recruitment is a linear increase in diffusive gas transport, measured as lung diffusing capacity (DL) for carbon monoxide (DLco), oxygen (DL02) or nitric oxide (DLNo), as cardiac output increases. 49-51 From rest to peak exercise, DL can increase 2-3 fold without evidence of an upper limit as a result of recruitment of microvascular reserves. 49'52'53 The ability to increase DL with respect to cardiac output is critical for maintaining adequate oxygenation of end-capillary blood leaving the lung. If the ratio of DL to blood flow (DL/(~c) falls below a critical region, endcapillary 0 2 saturation will fall precipitously and arterial hypoxemia will develop. 54 Although measurement of DL has been performed for many decades, its importance as a source of physiologic compensation was not generally understood until recently. After PNX, as in all destructive lung diseases, the entire ventilation and blood flow are directed to the remaining functioning lung units, causing the remaining alveolar surface to unfold, and the remaining capillaries to open and distend. Effective surface for gas exchange and therefore DL per unit of lung is higher than that expected based on anatomical lung destruction. Through numerous studies in animals and human subjects from rest up to heavy exercise, we have validated a conceptual framework for interpreting structural and functional compensation in diffusive gas exchange from the DL vs blood flow (DL-()c) relationship (Fig. 13.4). 54 Normally DL increases linearly with t)c. When some alveolar-capillary units are destroyed, DL is reduced at a given blood flow, but as long as the remaining units can be recruited DL should continue to increase

Fig. 13.4. Compensation in gas exchange achieved by microvascular recruitment (left panel) or septal regrowth (right panel). Alveolar destruction reduces diffusing capacity (DL) at a given pulmonary blood flow. Blood flow and ventilation, redirected to the remaining functioning units, cause those units to distend, thereby recruiting gas exchange surface area and DL along a parallel lower relationship. As a result, apparent DL is higher than expected from anatomical alveolar destruction. Growth of new alveolar-capillary tissue elevates the entire relationship between DL and pulmonary blood flow back to normal. Arrows indicate direction of change. (Adapted from Hsia CCW. Recruitment of lung diffusing capacity: update of concept and application. Chest 2002; 122:1774-83.)

along a lower but parallel relationship (i.e. lower intercept and normal slope of the D L / 0 c curve). If alveolar-capillary destruction is diffuse and the remaining lung units cannot be recruited, the slope of the (DL/Q c) relationship will also be reduced. On the other hand, any re-growth of new gas exchange tissue would increase DL at a given blood flow and return the entire relationship to normal. Accordingly, the slope of the DL/Q c relationship provides an index of the integrity of microvascular recruitment, while the absolute magnitude of DL at a given (~c provides an index of the effective gas exchange surface area and/or diffusion path. An example of compensation by recruitment is seen in dogs after 42-45% lung resection, where DL at a given workload declines by only 25% because the remaining capillary bed is normal and diffusive reserves can be recruited. 55 Similarly, in patients after PNX where the remaining lung is relatively normal, recruitment of DL is preserved and the decline in DL at a given workload is considerably less than expected from the amount of lung removed. 56'57 For the same resting DL, patients with chronic lung disease who are unable to recruit microvascular reserves demonstrate more severe reductions in DL and arterial hypoxemia upon exercise. 56,58

Septal remodeling Considerable enhancement of gas exchange can be achieved through remodeling of septal architecture to maximize the efficiency of oxygen uptake in the remaining lung without the addition of new septal tissue. Burri et al. 59'6~ proposed that initially after PNX, alveolar duct airspace expands more than alveolar airspace followed by rearrangement of existing septal tissue that ultimately restores the original volume proportions between alveolar ducts and alveoli.

The net result is lengthening of interalveolar septa and increased gas exchange surface, even in the absence of alveolar multiplication. In adult dogs studied about 2 years after 42-45% lung resection by left PNX, 43 there is no evidence of new septal tissue growth. Rather, the remaining alveolar surfaces unfold, the alveolar airspaces enlarge and the septum becomes thinner in response to lung expansion. Chronic unfolding of the septum together with an enlargement of the capillary network increases the effective surface area for gas exchange, while septal thinning reduces the effective barrier distance (mean harmonic thickness) for diffusion. Along with microvascular recruitment, these mechanisms are sufficient to mitigate the post-PNX reduction in DL and to maintain a nearnormal exercise capacity in the dog, despite an earlier decline in arterial oxygen saturation during exercise. 61 In progressive lung disease, alveolar-capillary recruitment and remodeling in remaining normal areas are responsible for the clinical observation that overt arterial hypoxemia and symptomatic escalation such as exertional dyspnea often do not develop until lung destruction is extensive (exceeding 60% of total). 62 Both alveolar-capillary recruitment and remodeling are ubiquitous adaptive mechanisms that are invoked regardless of whether septal growth is initiated (see below).

Re-initiation of septal growth In contrast to the response after 42-45% lung resection by left PNX, the response to 55-58% lung resection by right PNX in adult dogs is characterized by limited new septal tissue growth in addition to microvascular recruitment and remodeling. 44 Evidence suggests that the loss of functional lung tissue can be tolerated up to a certain threshold beyond which the remaining alveolar-capillary reserves are inadequate and additional mechanisms of compensation are elicited in the remaining lung. Chronic volume expansion and increased perfusion to the remaining lung increase alveolar strain and endothelial distention and shear, respectively; presumably when these signals exceed a critical threshold septal cell proliferation and synthesis of matrix proteins are stimulated. 17'63-65 The ability to induce compensatory alveolar growth is clearly dependent on the species, maturity and ability of the remaining lung to expand. There is an extensive body of literature detailing the vigorous post-PNX lung growth in mice, 66-7~ rats, 71-s~ ferrets, sl-83 and rabbits, sg-s6 In rodents, PNX activates a host of early response genes 66'78 leading to rapid induction of septal tissue growth, which restores lung volume, weight, DNA and protein content to that of two lungs within two weeks. 68'77 Perhaps because somatic growth continues throughout life, or because lung structure is simpler and alveolar-capillary reserves are limited, postnatal alveolar growth is more easily stimulated in rodents than in larger mammals, such as after resection of only one or two lobes. 59'71'87'88 Although mice normally lack a bronchial circulation, a functioning bronchial circulation can be generated de novo within one week

after unilateral pulmonary artery ligation. 89 In addition, since the lungs and thorax of rodents are more compliant than those of larger animals, a given mechanical force results in greater tissue strain and hence a more vigorous growth response. The brisk proliferative activities in pneumonectomized rodents are associated with increased susceptibility to experimental carcinogenesis and tumor metastasis. 68,69 In contrast to rodents, compensatory lung growth in large adult animals such as dogs follows a protracted course in which functional compensation lags behind tissue growth. 44'9~ During the first few months after right PNX, when cell proliferation and/or hypertrophy is most pronounced, total septal tissue volume increases more than 2-fold compared to the same lung in sham controls. 44 However, aerobic capacity remains markedly reduced; both the magnitude of DL and the slope of DL recruitment with respect to blood flow are impaired. 9~At 5 mo after right PNX, the alveolar septa are thickened due to a disproportionate increase in volume of the interstitial cells and matrix by more than 3-fold above that in the control lung; this leads to a longer mean alveolar epithelial-to-erythrocyte barrier distance and increased resistance to gas diffusion that offsets the benefit of septal tissue growth. Between 5 and 16 months after PNX, tissue remodeling occurs consisting of thinning of the septum and increased complexity of the gas exchange surfaces, resulting in normalization of the mean barrier distance for diffusion and a greater effective alveolarcapillary surface area. As a result, DL at a given blood flow and the slope of DL recruitment improve significantly, although this relationship never completely normalizes. When comparing the response in adult dogs one year after right PNX with that one year after left PNX, 52'55 we were surprised to find that DLco and arterial O z saturation, at a given blood flow per unit of remaining lung during heavy exercise, was significantly higher in dogs after right PNX while DLco and arterial 0 2 saturation in their respective control groups were similar, i.e. intensity of compensatory response is directly related to the intensity of lung destruction (Fig. 13.5). Thus, diffusion impairment is the major limitation after 45-55% of lung resection; within this range compensation occurs by recruitment and remodeling of the remaining lung, until a threshold is exceeded when septal tissue growth is also initiated. However, active septal tissue growth does not translate immediately into functional enhancement of DL until after normal alveolar morphology is reconstituted. Determinants of the threshold remain unclear, but this is an important concept to understand. In both large and small animals, compensatory lung growth is most marked during the period of somatic maturation but diminishes with age; 44'53'91'92 ultimately, restoration of functional capacity is more complete in growing animals than in adult animals. In immature dogs undergoing PNX, resting DL and in vivo estimates of lung air and tissue volume return to control values for two lungs within 8 weeks. 93'94 At full maturity one year later, aerobic capacity

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Pulmonary Blood Flow (ml/min) Fig. 13.5. At a given blood flow, diffusing capacity of the lung for carbon monoxide (DLco) is higher in adult dogs after right PNX (55-58% resection) than after left PNX (42-45% resection, right panel). In comparison, DLco at a given blood flow is similar in the respective control groups (pre-L-PNX and sham R-PNX, left panel). (Adapted from Hsia CCW, Herazo LF, Ramanathan M et al. Cardiopulmonary adaptations to pneumonectomy in dogs. IV. Membrane diffusing capacity and capillary blood volume. J. Appl. Physiol. 1994; 77:998-1005.)

and DL are normal at rest and up to peak exercise, compared to simultaneously raised SHAM animals. 53 Alveolar architecture returned to normal, such that DL estimated from morphometric estimates of surface area and harmonic mean diffusion path in the postmortem fixed lung is also normal compared to both lungs of control animals. 53 The relative volume increase of most septal cell volumes and surface areas is greater in dogs undergoing right PNX as immature puppies than as mature adults 53 with the exception that type II epithelial cells increase to a similar extent in the growing remaining lung of both immature and mature dogs, suggesting the type II cell to be a major compartment that proliferates to repopulate and extend the alveolar septa. The comparison between mature and immature dogs suggests that stimuli for developmental and compensatory lung growth may be synergistic. Somatic growth is not affected by PNX; hence normal progressive enlargement of the thorax further intensifies the already accelerated growth of the remaining lung. In patients followed for more than 30 years after PNX, there is an age-related gradual decrease in ventilatory capacity and reserve depending on whether PNX was performed during infancy, early childhood, adolescence or after, although adaptation is evident at all ages. 95'57 The above analysis emphasizes the importance of effective surface area, barrier distance and physical features of erythrocytes as key determinants of the functional usefulness of alveolar septal growth. These variables are independent of anatomical alveolar distortion that occurs after PNX or in disease. Estimates of DL based on these variables correlate strongly with physiologic measurements of DL during heavy exercise in the same animals; 53 an indication that these structural variables accurately index physiologic reserves of gas exchange. Another commonly used parameter, the

number of alveoli, describes the airspace units surrounded partially by gas exchange tissue. In spite of its appeal, however, it is an insufficient descriptor of adaptive events because assessment of alveolar number does not take into account surface complexities at the air-tissue or tissuecapillary interface and may not accurately reflect gas exchange capacity.

I M P O R T A N C E OF L U N G S T R A I N AS A S I G N A L FOR SEPTAL G R O W T H Experimental models to examine the effect of lung strain on post-PNX adaptation date back to the 1930s when Cohn 87 reported that wax plombage packed into the chest of rats after left upper lobectomy prevents expansion of the remaining lung, an observation replicated in adult ferrets when the right lung was replaced by an oil-filled silicone balloon. 96 Other investigators 7~ found that plombage only delays but does not eliminate the post-PNX increase in mitotic index, 97 DNA synthesis 7~ or in vivo lung volume. 98 These studies were of uniformly short duration and corresponding structural or functional changes were not examined. To avoid the unintended adverse effects caused by the weight, rigidity and unnatural shape of plombage material used in previous studies, we reconstructed a physical model of the normal right lung of an adult dog using magnetic resonance imaging. 99 From this model an inflatable silicone prosthesis was manufactured. At the time of right PNX, the prosthesis was placed into the empty hemithorax and inflated via a subcutaneous injection port buried at the back of the neck. In half of the animals the prosthesis was kept inflated with an SF6-air mixture to a volume ---20% above the dog's resting functional residual capacity, sufficient to

keep the mediastinum in the midline. In the other half of the animals the prosthesis was deflated, containing a minimum amount of air to prevent pleating and allowing marked mediastinal shift to occur. Volume of the prosthesis was checked by helium dilution and refilled, and a normal mediastinal position was verified by chest X-rays at regular intervals. After surgical recovery, animals were trained to exercise on a treadmill. Exercise studies and thoracic CT scan were performed 5-10 months after surgery, followed by postmortem studies at 12-15 months. Results were compared to adult dogs after right PNX without prosthesis and adult sham dogs. The presence of a silicone prosthesis, even when deflated, significantly reduced maximal oxygen uptake compared to pneumonectomized animals without a prosthesis. Inflation of the prosthesis, however, reduced maximal oxygen uptake further by --30%, and arterial hypoxemia developed earlier during increasing exercise than in animals with deflated prosthesis, particularly at low inspired oxygen tensions. Ventilation-perfusion distributions and maximal cardiac output were not different between groups with inflated and deflated prosthesis. The most pronounced difference was a marked parallel reduction in DLco 99 and DLo2 (unpublished observation) at a given cardiac output in animals with inflated prosthesis compared to those with deflated prosthesis (Fig. 13.6). The lower DL with inflated prosthesis was entirely due to a low membrane diffusing capacity, that is, lower conductance by diffusion across the tissue-plasma barrier, at a given blood flow. These results are consistent with the interpretation that reducing lung strain impairs structural adaptation of gas exchange tissue rather than functional recruitment, as confirmed by postmortem morphometric analysis. 1~176 The remaining lung from animals with an inflated prosthesis showed septal crowding with an elevated capillary blood volume. Septal tissue volume, volume of septal cell components, and alveolar and capillary

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surface areas were significantly lower than in animals with a deflated prosthesis or in both lungs of sham controls, but significantly greater than in the left lung of sham animals. 1~176 Hence, compensatory lung growth was impaired but not completely eliminated by the inflated prosthesis. CT scans in these animals 99 extended previous observations in rabbits 98 by showing that when lung expansion across the midline was prevented by the inflated prosthesis, the remaining lung changed shape and continued to increase 20-30% in air and tissue volume via caudal displacement of the diaphragm and outward expansion of the thorax. This observation is somewhat counter-intuitive and suggests that the remaining lung after PNX does not simply expand passively to fill an empty space, but rather there are intrinsic signals that induce the lung to grow and expand even when space is not readily available. Effects of preventing lung expansion with the inflated prosthesis are reversible. In pneumonectomized ferrets when the resected lung was replaced with an oil-filled silicone balloon, there was a reversible increase in lung volume following deflation of the balloon 3-13 weeks later. 96 In our study, the inflated prosthesis in one dog developed a leak 9-10 mo after PNX. Subsequent to the unintentional deflation, lung volume, DLco and exercise capacity progressively increased. 99 Lung structure in this animal examined at 13 mo after PNX showed higher septal cell volumes and surface areas more in keeping with those in animals that had continuously deflated prosthesis for the same duration than in animals with continuously inflated prosthesis. In particular, volume of type II epithelial cells in this animal was the highest of all animals studied, l~176 suggesting vigorous septal cell growth following delayed re-institution of lung strain. These data collectively show that in vivo septal strain can account for the majority (about 70%) of the observed functional and structural compensation after PNX. However, neither plombage nor the prosthesis eliminated tidal stretch of the remaining lung, endothelial distention or shear forces after PNX. These additional mechanical factors could have contributed to the partial compensation. Lung growth may also be modulated by non-mechanical signals, such as exercise-induced alveolar hypoxia, 77 endocrine or paracrine growth mediators such as adrenocorticosteroid hormone, 1~ epidermal growth factor (EGF), s~ hepatocyte growth factor, 1~ platelet-derived growth factor, 1~ retinoic acid 79 and nitric oxide, TM among others. Such modulating influences may interact with mechanical signals in ways that are presently not known.

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A I R W A Y G R O W T H , REMODELING A N D F U N C T I O N AFTER P N E U M O N E C T O M Y

(L/min)/kg Fig. 13.6. dogs with shift after a deflated

At a given blood flow, DLco was markedly lower in adult an inflated intrathoracic prosthesis to prevent mediastinal right PNX compared to pneumonectomized animals with prosthesis. Adapted from Wu et al. 99

From the foregoing it is clear that mechanical lung strain constitutes the most potent inducer of alveolar tissue growth, acting in a dose-dependent fashion during normal maturation and after PNX. During compensatory lung growth

bronchial epithelial cells, mesothelial cells as well as alveolar septal cells multiply, 1~ and peripheral alveolar tissue proliferates more vigorously than central alveolar tissue. 28 Peripheral alveoli can grow by adding new septa, increasing the complexity of existing septa, and perhaps branching to form another generation of alveolar ducts. Respiratory bronchioles also increase in number after PNX 1~ presumably via (a) transforming the first generation of alveolar ducts into respiratory bronchioles, or (b) alveolarizing the terminal bronchiole. In contrast to intraacinar structures, the potential for growth and remodeling of proximal conducting airway is limited. Airway branching patterns are set in utero; essentially lengthening and dilation are the only options for postnatal adaptation. In addition, the rigid cartilaginous rings reduce the strain experienced by airway cells at a given airway stress or lung stretch; hence strain-induced response is also blunted. After PNX, airway flow resistance (Raw) in the remaining lung increases precipitously due to: (a) reduction of total airway cross-sectional area, (b) increased volume flow through the remaining lung at a given ventilation, and (c) anatomical airway distortion as a result of asymmetric lung expansion. In response to the chronic flow and traction forces, the remaining airways lengthen 81's4'1~ and dilate. 81 However, airway lengthening increases Raw while airway dilatation decreases Raw; hence the net functional compensation depends on a balance of the two processes. Since Raw is inversely proportional to the 4th power of airway diameter and directly related to airway length, only a minimal increase in airway diameter is needed to offset the effect of airway lengthening. To determine the time course of airway structural changes and their contribution to flow resistance, we measured in vivo airway dimensions by CT scan under physiologic conditions at normal distending pressures in immature dogs 4 and 10 months after right PNX. 31 By 4 months after PNX the remaining airway lengthened with minimal dilation without any compensatory reduction of Raw. PostPNX airway dilatation became apparent subsequently. By 10 months after PNX, the average cross-sectional area of a given lobar airway was 24% greater than in the control airway, which can be expected to attenuate lobar Raw by 40-50% compared to that of a normal lobe at the same volume flow. However, since total airway cross-sectional area still remains below that in two normal lungs the net reduction of estimated work of breathing against Raw in the whole animal is only ---30%. Thus, airway remodeling after PNX progresses slowly; the delayed increase in total airway volume and cross-sectional area lags behind compensatory septal growth, 94 and can only partially mitigate the increased work of breathing in the whole animal. Structural analysis from CT scan predicts a 3-fold higher work of breathing against Raw 10mo after PNX, 31 consistent with the 2.5-fold actual increase in work of breathing done against the whole lung measured at rest and during exercise in pneumonectomized immature dogs raised to maturity. 1~ Post-PNX airway remodeling amplifies the normal pattern

of declining Raw during postnatal maturation, and requires the addition of new airway tissue and/or a reduction in airway compliance. Long-term elevation in work of breathing is greater after right PNX than after left PNX, 1~ suggesting that the intensity of airway growth and remodeling does not increase with more extensive lung resection, as is the case with alveolar-capillary response. The increase in work of breathing is also similar regardless of the maturity of the animal at the time of lung resection, 1~ in contrast to the maturity-dependence of compensation in gas exchange function. Thus, parallel physiologic and structural analysis reached similar conclusions, that compensatory growth and remodeling of the parenchyma is more vigorous and rapid than that of conducting airways. After PNX, the rate and magnitude of increase in airway volume or cross-sectional area at all generations was less than expected from the increase in lung air or septal tissue volume. This concept of unequal potential for compensatory growth among different structural components of the lung, termed 'dysanaptic growth', was first used to explain the highly variable expiratory flow rates with respect to lung volume observed in normal subjects, 11~ and it was later used to explain the relatively low maximal airflow rates with respect to the larger increase in lung volume observed in natives of high altitude, 111 in animals after P N X 112 and in children after lobectomy. 113 A similar 'dysanaptic' adaptive pattern is observed in the response of pulmonary blood vessels to PNX. In immature dogs raised to maturity after PNX, pulmonary arterial pressure and total pulmonary resistance during exercise are persistently elevated even after normal alveolar oxygen transport has been restored. 53'1~ Exercise-induced pulmonary arterial hypertension is even more pronounced in dogs pneumonectomized as adults. 1~ Long-term structural changes in pulmonary arteries are shown to be more prominent in dogs pneumonectomized as adults than as puppies, but there is evidence of a chronically elevated pulmonary vascular resistance in both groups. 114 An age-related increase in pulmonary arterial pressure has also been observed in patients after PNX at exercise and sometimes at rest. 115 In adult dogs, long-term mechanical and hemodynamic abnormalities become further and disproportionately accentuated after even more extensive bilateral resection removing up to 68% of lung; 116'117these abnormalities impose major ventilatory and cardiac limitations on exercise capacity. While dysfunction of alveolar-capillary gas exchange is the functional bottleneck after resection of up to 55% of lung, with more extensive resection, adaptive responses of the highly plastic alveolar septum outstrip those of the less plastic conducting structures, and the bottleneck shifts to airway and hemodynamic dysfunction. Consequently, during post-PNX adaptation the extent of concurrent growth and remodeling of conducting airway and blood vessels sets the upper limit of functional compensation that can be achieved, regardless of the strength and completeness of alveolar septal regeneration.

REGULATORY PATTERNS D U R I N G DEVELOPMENTAL A N D COMPENSATORY GROWTH It has been assumed that compensatory lung growth represents reactivation of normal developmental pathways. 1~ If true, changes in key regulators known to modulate developmental lung growth should parallel those seen after PNX. We tested this hypothesis directly in dogs undergoing right PNX at two months of age by examining the protein expression of EGF axis, as well as surfactant protein (SP) system, using lung tissue obtained three weeks and ten months after PNX compared to simultaneous age-matched controls. 2s In normal growing lungs, cell proliferation measured by proliferating cell nuclear antigen (PCNA) level was nearly 24-fold that in the adult lung; the vigorous proliferative activity in growing animals was further enhanced by 80% at three weeks after PNX followed by a decline to normal by ten months. Whereas EGF and its receptor (EGFR) were modestly but significantly elevated in the proliferating immature lung compared to the adult lung, expression of both proteins decreased modestly but significantly after PNX compared to age-matched sham controls. SP expression also differed during postnatal and compensatory lung growth. In the normal growing lung, SP-A and surfactant proprotein-C (pro SP-C) levels were 60-80% lower while SP-D and pro SP-B levels were slightly higher compared to the adult lung. In contrast, in the growing lung three weeks after PNX, SP-A level rose nearly 5-fold and SP-D rose modestly compared to sham controls, while pro SP-B and pro SP-C levels did not change. These divergent directions of response between normal growing lungs and post-PNX lungs suggest that compensatory lung growth involves different regulatory mechanisms from postnatal maturation. The selective prominent upregulation in SP-A after PNX suggests a large differentiated and highly active population of type II epithelial cells and/or Clara cells. Selective regulation of SPs may also play a role in modulating post-PNX lung growth. What factors might contribute to the differential expression during postnatal and compensatory lung growth in the growing animal? Obviously the metabolic and hormonal milieu in the post-PNX lung differs from that in the normal lung. Mechanical strain on the remaining lung after PNX is imposed suddenly at a larger magnitude than that experienced by the normal growing lung. During development, the increase in tidal volume matches the ventilatory requirement of the animal. After PNX, minute ventilation is unchanged but volumetric flow through the remaining lung with each breath is doubled. It is possible that a mismatch between static strain due to lung expansion and cyclic lung strain due to tidal breathing elicits a different set of responses than either alone. PNX markedly alters the distribution and uniformity of mechanical stress and strain experienced by the thorax and remaining lung, and may lead to differential regional biochemical response not seen

during development. In developing lungs the increase in pulmonary blood flow matches growth of the heart and microvascular bed. After PNX, the rapid and disproportionate increase in perfusion to the remaining lung might exaggerate any endothelium-derived response to normal growth signals. These possibilities remain speculative.

CONCLUSIONS Any assessment of the functional significance of lung growth must take into account structural as well as non-structural sources of adaptation. Recent evidence directly supports mechanical lung strain as the major signal underlying postnatal and post-PNX lung growth, although non-mechanical signals must also play a role in modulating the growth response. Reducing lung strain markedly impairs normal lung development as well as post-PNX growth and compensation, and compensatory lung growth is clearly not a recapitulation of normal developmental events. As the magnitude of post-PNX lung strain increases, a disparate pattern of adaptation between lung parenchyma and conducting structures emerges, reflecting differences in the plasticity of the respective components. The extent of disparity or 'mismatch' between the adaptive potential of these components effectively limits the functional compensation that can be derived from alveolar septal growth and regeneration.

ACKNOWLEDGEMENTS Supported by National Heart Lung and Blood Institute Grants RO1-HL40700, HL54060, HL45716 and HL62873, Training Grant TL-07362, and the Swiss National Science Foundation.

REFERENCES 1. Robertson HT, Altemeier WA, Glenny RW. Physiological implications of the fractal distribution of ventilation and perfusion in the lung.Ann. Biomed. Eng. 2000; 28:1028-31. 2. Nagaya M, Akatsuka H, Kato J etal. Development in lung function of the affected side after repair of congenital diaphragmatic hernia.J. Pediatr. Surg. 1996; 31:349-56. 3. Beals DA, Schloo BL, Vacanti JP et al. Pulmonary growth and remodeling in infants with high-risk congenital diaphragmatic hernia. J. Pediatr. Surg. 1992; 27:997-1001; discussion 1001-2. 4. Ijsselstijn H, Tibboel D, Hop WJ et al. Long-term pulmonary sequelae in children with congenital diaphragmatic hernia. Am.J. Respir. Crit. Care Med. 1997; 155:174-80. 5. Marven SS, Smith CM, Claxton D etal. Pulmonary function, exercise performance, and growth in survivors of congenital diaphragmatic hernia.Arch. Dis. Child 1998; 78:137-42. 6. Berend N, Marlin GE. Arrest of alveolar multiplication in kyphoscoliosis. Pathology 1979; 11:485-91.

7. Davies G, Reid L. Effect of scoliosis on growth of alveoli and pulmonary arteries and on right ventricle. Arch. Dis. Child 1971; 46:623-32. 8. Olgiati R, Levine D, Smith JP etal. Diffusing capacity in idiopathic scoliosis and its interpretation regarding alveolar development.Am. Rev. Respir. Dis. 1982; 126:229-34. 9. Boffa P, Stovin P, Shneerson J. Lung developmental abnormalities in severe scoliosis. Thorax 1984; 39:681-2. 10. Lund DP, Mitchell J, Kharasch V e t al. Congenital diaphragmatic hernia: the hidden morbidity. J. Pediatr. Surg. 1994; 29:258-62; discussion 254-62. 11. Liu M, Skinner SJ, Xu J et al. Stimulation of fetal rat lung cell proliferation in vitro by mechanical stretch. Am. J. Physiol. 1992; 263:L376-83. 12. Chess PR, Toia L, Finkelstein JN. Mechanical strain-induced proliferation and signaling in pulmonary epithelial H441 cells.Am. J. Physiol. Lung Cell Mol. Physiol. 2000; 279:L43-51. 13. Sanchez-Esteban J, Wang Y, Cicchiello LA etal. Cyclic mechanical stretch inhibits cell proliferation and induces apoptosis in fetal rat lung fibroblasts. Am. J. Physiol. Lung Cell Mol. Physiol. 2002; 282:L448-56. 14. Liu M, Qin Y, Liu J et al. Mechanical strain induces pp60src activation and translocation to cytoskeleton in fetal rat lung ce|ls.J. Biol. Chem. 1996; 271:7066-71. 15. Xu J, Liu M, Liu J et al. Mechanical strain induces constitutive and regulated secretion of glycosaminoglycans and proteoglycans in fetal lung cells. J. Cell Sci. 109:1605-13. 16. Liu M, Xu J, Tanswell AK etal. Inhibition of mechanical strain-induced fetal rat lung cell proliferation by gadolinium, a stretch-activated channel blocker. J. Cell. Physiol. 1994; 161:501-7. 17. Breen EC. Mechanical strain increases type I collagen expression in pulmonary fibroblasts in vitro. J. Appl. Physiol. 2000; 88:203-9. 18. Smith PG, Moreno R, Ikebe M. Strain increases airway smooth muscle contractile and cytoskeletal proteins in vitro. Am. J. Physiol. 1997; 272:L20-7. 19. Liu M, Liu J, Buch S etal. Antisense oligonucleotides for PDGF-B and its receptor inhibit mechanical strain-induced fetal lung cell growth.Am. J. Physiol. 1995; 269:L178-84. 20. Waters CM, Chang JY, Glucksberg MR etal. Mechanical forces alter growth factor release by pleural mesothelial cells.Am. J. Physiol. 1997; 272:L552-7. 21. Sanchez-Esteban J, Tsai SW, Sang J et al. Effects of mechanical forces on lung-specific gene expression. Am. J. Med. Sci. 1998; 316:200-4. 22. Papadakis K, Luks FI, De Paepe ME et al. Fetal lung growth after tracheal ligation is not solely a pressure phenomenon. J. Pediatr. Surg. 1997; 32:347-51. 23. Harding R, Hooper SB, Han VK. Abolition of fetal breathing movements by spinal cord transection leads to reductions in fetal lung liquid volume, lung growth, and IGF-II gene expression. Pediatr. Res. 1993; 34:148-53. 24. Tseng BS, Cavin ST, Booth FW et al. Pulmonary hypoplasia in the myogenin null mouse embryo. Am. J. Respir. Cell Mol. Biol. 2000; 22:304-15. 25. Zhang S, Garbutt V, Mcbride JT. Strain-induced growth of the immature lung.J. Appl. Physiol. 1996; 81:1471-6. 26. Nobuhara KK, Fauza DO, Difiore JW etal. Continuous intrapulmonary distension with perfluorocarbon accelerates neonatal (but not adult) lung growth.J. Pediatr. Surg. 1998; 33:292-8. 27. Massaro GD, Massaro D. Postnatal lung growth: evidence that the gas-exchange region grows fastest at the periphery. Am. J. Physiol. 1993; 265:L319-22. 28. Foster DJ, Yan X, Bellotto DJ et al. Expression of epidermal growth factor and surfactant proteins during postnatal and compensatory lung growth. Am. J. Physiol. Lung Cell Mol. Physiol. 2002; 283:L981-90.

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72. Rannels DE, Stockstill B, Mercer RR et al. Cellular changes in the lungs of adrenalectomized rats following left pneumonectomy.Am. J. Respir. Cell Mol. Biol. 1991; 5:351-62. 73. Thet LA, Law DJ. Changes in cell number and lung morphology during early postpneumonectomy lung growth. J. Appt. Physiol.: Respir. Environ. Exercise Physiol. 1984; 56:975-8. 74. Nattie EE, Wiley CW, Bartlett D Jr. Adaptive growth of the lung following pneumonectomy in rats. J. Appl. Physiol. 1974; 37:491-5. 75. Mcanulty RJ, Guerreiro D, Cambrey AD, Laurent GJ. Growth factor activity in the lung during compensatory growth after pneumonectomy: evidence of a role for IGF-1. Eur. Respir. J. 1992; 5:739-47. 76. Faridy EE, Sanii MR, Thliveris JA. Influence of maternal pneumonectomy on fetal lung growth. Respir. Physiol. 1988; 72:195-209. 77. Sekhon HS, Smith C, Thurlbeck WM. Effect of hypoxia and hyperoxia on postpneumonectomy compensatory lung growth. Exp. Lung Res. 1993; 19:519-32. 78. Gilbert KA, Rannels DE. Increased lung inflation induces gene expression after pneumonectomy. Am. J. Physiol. 1998; 275:L21-9. 79. Kaza AK, Kron IL, Kern JA et al. Retinoic acid enhances lung growth after pneumonectomy. Ann. Thorac. Surg. 2001; 71:1645-50. 80. Kaza AK, Laubach VE, Kern JA et al. Epidermal growth factor augments postpneumonectomy lung growth. J. Thorac. Cardiovasc. Surg. 2000; 120:916-21. 81. Mcbride JT. Postpneumonectomy airway growth in the ferret. J. Appl. Physiol. 1985; 58:1010-14. 82. Kirchner KK, Mcbride JT. Changes in airway length after unilateral pneumonectomy in weanling ferrets. J. Appl. Physiol. 1990; 68:187-92. 83. Mcbride JT, Kirchner KK, Russ G e t al. Role of pulmonary blood flow in postpneumonectomy lung growth. J. Appl. Physiol. 1992; 73:2448-51. 84. Boatman ES. A morphometric and morphological study of the lungs of rabbits after unilateral pneumonectomy. Thorax 1977; 32:406-17. 85. Das RM, Thurlbeck WM. The events in the contralateral lung following pneumonectomy in the rabbit. Lung 1979; 156:165-72. 86. Langston C, Sachdeva P, Cowan MJ et al. Alveolar multiplication in the contralateral lung after unilateral pneumonectomy in the rabbit.Am. Rev. Respir. Dis. 1977; 115:7-13. 87. Cohn R. Factors affecting the postnatal growth of the lung. Anat. Rec. 1939; 75:195-205. 88. Burri PH, Sehovic S. The adaptive response of the rat lung after bilobectomy. Am. Rev. Respir. Dis. 1979; 119:769-77. 89. Mitzner W, Lee W, Georgakopoulos D et al. Angiogenesis in the mouse lung.Am. J. Pathol. 2000; 157:93-101. 90. Hsia CCW, Herazo LF, Ramanathan M et al. Cardiopulmonary adaptations to pneumonectomy in dogs. II. Ventilationperfusion relationships and microvascular recruitment. J. Appl. Physiol. 1993; 74:1299-1309. 91. Johnson RL Jr, Cassidy SS, Grover R et al. Effect of pneumonectomy on the remaining lung in dogs. J. Appl. Physiol. 1991; 70:849-58. 92. Holmes C, Thurlbeck WM. Normal lung growth and response after pneumonectomy in rats at various ages. Am. Rev. Respir. Dis. 1979; 120:1125-36. 93. Takeda S, Ramanathan M, Wu EY et al. Temporal course of gas exchange and mechanical compensation after right pneumonectomy in immature dogs. J. Appl. Physiol. 1996; 80:1304-12. 94. Takeda S, Wu EY, Epstein RH etal. In vivo assessment of changes in air and tissue volumes after pneumonectomy. J. Appl. Physiol. 1997; 82:1340-8.

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INTRODUCTION

The smooth transition from fetal to extrauterine life is largely dependent upon the ability of the lung to take over the role of respiratory gas exchange, a function that is performed by the placenta during fetal life. To perform this task the lungs must have grown and matured appropriately during fetal life; in particular, they must have developed a large internal surface area, which is closely apposed to a large vascular bed to facilitate postnatal gas exchange (see Chapter 9). For pulmonary gas exchange to commence, a number of important adaptive events must occur at the time of birth. The pulmonary vascular bed, which receives only a small fraction of cardiac output in the fetus, must be able to accept the entire output of the right side of the heart; the ductus arteriosus (DA) that in the fetus shunts blood away from the lungs must close and remain closed. The liquid that occupies the lung lumen throughout gestation must be rapidly cleared after birth to allow the entry of air into the lungs, although a thin film of liquid must remain to protect the epithelium of the air-filled lung. Importantly, continuous rhythmic breathing must begin. The entry of air into the lungs at birth creates an air-liquid interface across the large internal surface of the lung, which is not present in the liquid-filled fetal lung. Thus, unless the type-II alveolar epithelial cells (AECs) are sufficiently mature to produce and release surfactant into the alveolar lumen, the surface tension created at the air-liquid interface will greatly increase the recoil pressure of the lungs. This will oppose expansion of the lungs during inspiration and favour collapse of the peripheral airspaces during expiration, thereby increasing the work of breathing. Many of the changes in lung physiology that occur late in gestation to facilitate the transition to air-breathing are closely linked to the processes that initiate labour. Indeed, early evidence that the increase in fetal circulating corticosteroid concentrations preceding birth plays an important *To whom correspondence should be addressed. The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

role in maturing the lungs 1 has gained universal acceptance and has led to the widespread use of antenatal corticosteroids in women at risk of delivering before term. 2'3 Although the success of antenatal corticosteroids in reducing respiratory insufficiency in the preterm infant is incontrovertible, 4 the mechanisms by which corticosteroids act on the immature lung to facilitate gas exchange after birth remain poorly understood. 5 The aim of this chapter is to review the physiological changes that take place in the lungs around the time of birth, and how these changes are regulated.

FETAL LUNG MATURATION, GLUCOCORTICOIDS AND

BIRTH

In most mammalian species, particularly those giving birth to precocial offspring, birth is preceded by an exponentiallike increase in circulating corticosteroid concentrations. In some species (e.g. sheep), the increase in circulating cortisol levels in the fetus plays a vital role in the initiation of parturition 6 and, at the same time, stimulates the maturation of a variety of fetal organ systems, including the lung. 7-9 From a teleological standpoint, it is appropriate that the same processes that initiate parturition also contribute to the maturation of fetal organ systems that, at birth, must rapidly assume a role that is vital for postnatal survival. Preterm birth represents an example of the failure of this link between parturition and maturation, resulting in the birth of an infant before vital organs such as the lungs have been able to mature to the point that they can sustain independent life. To determine the mechanisms by which fetal corticosteroids accelerate lung maturation, most attention has focussed on their effects on surfactant production and type-II AEC maturation; the role of corticosteroids in fetal lung development has been the subject of a number of reviews. 5'1~ As described in Chapter 10, the phospholipid component of surfactant plays a vital role in lung function by forming a stable monolayer at the air-liquid interface, thereby reducing surface tension. In particular, the recruitment and expulsion of Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

phospholipids from this monolayer during the respiratory cycle help to stabilize the lung, particularly at end-expiration. It has been well established that exogenous corticosteroids increase surfactant synthesis by inducing many of the synthetic enzymes and by inducing surfactant protein (SP) expression both in vivo and in vitro. 5'1~ However, some discrepancies persist in the literature, particularly relating to the induction of SP gene expression, which may reflect differences in the dose and duration of corticosteroid exposure and differences between in vitro and in vivo studies. 1~ Indeed, most studies have used high doses of synthetic glucocorticoids (e.g. dexamethasone or betamethasone), which have a -~30-fold greater bioactivity than natural cortisol. Thus, to define the precise role of endogenous cortisol on type-II AEC maturation and surfactant, studies are required that use physiological doses of cortisol that mimic the preparturient increase in fetal cortisol concentrations. The development of a glucocorticoid receptor (GR) deficient mouse has provided insights as to the role of endogenous corticosteroids in fetal lung maturation. 11 Homozygous GR (-/-) deficient mice die of respiratory insufficiency at birth, confirming the vital role that endogenous corticosteroids play in fetal lung maturation. ~ However, the major abnormality appears to be a marked immaturity of the tissue architecture rather than type-II AEC dysfunction or surfactant deficiency. Indeed, recent data from these GR deficient mice indicate that type-II AEC numbers are increased, and type-I AEC numbers are reduced (T. Cole & S. Hooper, unpublished observations), and SP gene expression is unaltered. 11 These findings support the concept that the principal effect of corticosteroids on the developing lung is to mature its architecture so as to create a more compliant structure with a greater surface area for gas exchange and with less tissue between airspaces and capillaries. 5 In preterm infants, respiratory problems arise because their lungs (1) have had insufficient time to grow in utero (see also Chapter 16) and (2) have not been exposed to the pre-parturient increase in circulating corticosteroids. These two consequences of preterm birth are not necessarily analogous. Based on available evidence, it appears that the immature lungs of preterm infants have a reduced gas exchange surface area with few mature type-I and type-II epithelial cells due to insufficient time for in utero development; these lungs are mechanically and structurally immature with a thick air-blood gas barrier and a reduced ability to reabsorb lung liquid (see below) due to a lack of exposure to glucocorticoids.

LUNG

LIQUID

CLEARANCE

BEFORE B I R T H In the fetus, the lungs contain a liquid that is secreted by the pulmonary epithelium (see Chapter 8) and, during the latter part of gestation, the volume of this liquid is greater than the equivalent volume of air in the neonate (Fig. 14.1).

19 50E

363847~~~ ~

r

40-

E o 30-

14

e.-

~ ~

t-

n,

20100

'"

~ 120

~ 130

Gestational age (days)

~ 140

/"2 / ~

/ Birth

~ 10

~ 20

30

40

Postnatal age (days)

Fig. 14.1. The volume (ml/kg) of luminal liquid within the lungs of fetal sheep during the last third of gestation (open circles) is greater than the end-expiratory volume (functional residual capacity; FRC) of the air-filled lung in postnatal lambs (closed circles). Fetal lung liquid volumes were measured by an indicator dilution technique and FRC was measured in postnatal lambs by helium dilution. Note the fall in lung volume after birth. The numbers above each data point represent the number of animals in which measurements were made. Data are taken from Refs21'39'44.

Before effective air breathing can be established at birth, the airways must be cleared of this luminal liquid. Although the mechanisms by which this process occurs have been the subject of much investigation, the timing and relative contributions of the different mechanisms that influence liquid clearance from the airways remain unclear. 12 Initially, it was proposed that the clearance of lung liquid begins days, even as long as a week, before the onset of labour. This concept originated from studies in which reductions in lung liquid volume were observed to occur over the last week of gestation in fetal sheep. 13'14 However, in making these measurements, many factors that influence the volume of fetal lung liquid are often overlooked (e.g. the trans-pulmonary pressure gradient). As lung compliance increases late in gestation, due to increased structural maturity and the effects of corticosteroids, 5'15 even small changes in trans-pulmonary pressure can cause large changes in lung liquid volumes. Thus, one would expect considerable natural variability in lung liquid volumes late in gestation, depending upon the factors influencing the trans-pulmonary pressure gradient. For example, changes in fetal posture, imposed by reductions in intrauterine volume (perhaps due to amniotlc fluid loss), leading to increased flexion of the fetal trunk, increase the abdominal pressure, elevate the diaphragm and increase the transpulmonary pressure gradient, leading to the loss of lung liquid 16 (see Chapter 9, Figs 9.2 and 9.3). Consequently, unless the volume of amniotic fluid is reduced (e.g. reduced fetal urine production, or membrane rupture) and the fetus is unable to defend its lung volume, there is presently no basis for reduced lung liquid volume before the onset of

labour. Indeed, studies in sheep have failed to show a reduction in lung liquid volume in healthy fetuses near term. 17 Furthermore, it is unlikely that reduced lung liquid production rates, perhaps due to an increasing sensitivity of the lung epithelium to stimuli that inhibit lung liquid secretion and activate lung liquid absorption (e.g. adrenaline, see below), leads to a reduction in lung liquid volume late in gestation. In fetal sheep, a reduction in fetal lung liquid secretion rate simply leads to a simultaneous reduction in tracheal fluid efflux, resulting in no net change in the volume of lung liquid, ls'19

doses to fetal sheep in utero, cortis01 has no acute effect (within hours) on fetal lung liquid secretion rates. 29 When infused over a number of days, cortisol paradoxically increased secretion rates, 29 which was attributed to an increase in the intracellular machinery (e.g. Na+-K § ATPase) responsible for fetal lung liquid secretion in 7.)i7)0. 29 Similarly, fetal mice with a mineralocorticoid receptor knockout appear to have normal lung liquid secretion and reabsorption activities as their lungs develop normally and they survive the transition to extrauterine life but die ---10 days after birth due to a severe loss of Na § and water. 31

The active reabsorption of lung liquid at birth

Other mechanisms of liquid clearance from the airways

The clearance of liquid from the airways at birth undoubtedly involves a reversal of the osmotic gradient across the pulmonary epithelium that normally drives fetal lung liquid secretion. A detailed account of the ionic basis for fetal lung liquid reabsorption is presented in Chapter 8. In brief, it is apparent that the osmotic gradient that promotes liquid secretion into the lung lumen during fetal life is reversed at birth due to the activation of amiloride-inhibitable Na § channels located on the apical surface of pulmonary epithelial cells. 2~ It is proposed that fetal endocrine and metabolic changes caused by active labour result in a large increase in fetal circulating concentrations of adrenaline (epinephrine) and arginine vasopressin (AVP) which act via ]3-adrenergic and V2 receptors, respectively, to induce a cAMP-mediated activation of apical surface Na + channels. 21 This, in turn, leads to an increase in Na § and Na+-linked flux of chloride ions across the pulmonary epithelium, resulting in a reversal of the osmotic gradient and a reversal in the direction of liquid movement. 2~ The ability of adrenaline and AVP to inhibit fetal lung liquid secretion and initiate liquid reabsorption matures late in gestation, increasing in an exponential-like manner close to term. 23-25 This maturational increase is thought to be dependent upon the actions of both cortisol and triiodothyronine (T3), as thyroidectomy26 and adrenalectomy 27 abolish the gestational age-related increase in lung liquid reabsorption in response to adrenaline. On the other hand, infusions of either cortisol alone or cortisol and T3 together (T3 alone had no effect) precociously mature the pulmonary reabsorptive response to adrenaline. 28'29 Following preterm birth, this mechanism for lung liquid reabsorption may be inactive and hence preterm infants can suffer from significant liquid retention within their airways. The mechanisms by which cortisol and T3 induce and enhance the potential of the fetal lung to reabsorb liquid undoubtedly include the processes that regulate Na § and Na+-linked C1- ion flux across the epithelium. Indeed, glucocorticoids increase the expression of the amiloride-inhibitable Na § channels, both in vivo and in vitro, which contributes to the increase in Na § flux across the epithelium during liquid reabsorption. 22 Studies using excised lungs from fetal guinea pigs have implicated cortisol and aldosterone in the inhibition of fetal lung liquid secretion and the reabsorption of lung liquid late in gestation. 30 However, when infused in physiological

Although active liquid reabsorption plays a major role in liquid clearance from the airways, 22 there are a variety of other potential mechanisms that may contribute to the clearance of lung liquid at birth. For example, changes in fetal oncotic pressure, interstitial hydrostatic pressure associated with the formation of an air-liquid interface (see below) and the mechanical effects of labour are likely to have profound effects on the volume of liquid retained within the airways. As indicated above and in Chapter 9, late in gestation the fetal lungs and chest wall are very compliant 32 and, therefore, factors (such as changes in fetal posture) that influence the trans-pulmonary pressure gradient can have profound effects on the volume of liquid retained within the future airways. 16 It is often assumed, probably incorrectly as previously discussed, 12 that the passage of the fetus through the birth canal 'squeezes' the majority of liquid from the fetal lung. Instead, a large loss of liquid can occur much earlier during labour, probably in response to a reduction in amniotic fluid volume and/or to the contractions and shortening of the myometrium. Both of these factors have the potential to impose marked changes in fetal posture in both sheep 16 and humans. 33 This would cause an increase in the trans-pulmonary pressure gradient leading to the loss of lung liquid 16 (see Chapter 9). Indeed, in the absence of amniotic fluid, even mild non-labour uterine contractions cause phasic compression of the fetus which increases the trans-pulmonary pressure gradient and the loss of lung liquid. 16 Consequently, the strong coordinated uterine contractions associated with labour are likely to strongly enhance liquid efflux from the fetal lungs, particularly after the membranes have ruptured. Marked reductions in the volume of lung liquid have been observed within hours of the first signs of labour in sheep (as indicated by uterine EMGs), well before the second stage of labour and the expected increase in the release of stress-related hormones.17,34 A recent report has suggested that increased lung liquid efflux may occur with each breath during fetal breathing movements (FBM) late in gestation, due to a change in breathing activity, leading to a reduction in lung liquid volume. 35 Although this is consistent with a n increasing degree of compression of the fetus, such data are difficult to

evaluate as much of the efflux of lung liquid occurs at slow flow rates 36 (e.g. ml/h), even during uterine contractions 16and it is unlikely that the measurement technique used is capable of accurately measuring these slow rates of flow. Thus, only rapid rates of flow will be detected which may bias the measurements to those only associated with fetal muscle activity. The available evidence indicates that the mechanisms leading to the clearance of lung liquid at birth are multifactorial and vary between species and individuals. The contribution of the mechanical effects of a reduction in intrauterine space and the impact of uterine contractions on the loss of fetal lung liquid at birth are unknown, but they may account for an initial substantial reduction in lung liquid volume, based on studies in sheep. 17'34'37 Similarly, it is difficult to assess the relative contribution of active liquid reabsorption resulting from Na § channel activation in the clearance of lung liquid from the airways. Although blockade of epithelial Na § channels using amiloride and gene knockout of the epithelial Na § channel, ct-rENaC, can restrict lung liquid clearance at birth, 22 this does not preclude the involvement of other mechanisms. Indeed, as maximal reabsorption rates of-~30ml/h have been reported in fetal sheep in response to the infusion of high doses of adrenaline, 38 the clearance of---200ml of liquid from the fetal airways 39 via this mechanism is likely to take many hours. Although circulating catecholamine levels remain elevated after birth, 4~ it is unlikely that they remain sufficiently elevated to drive liquid reabsorption at these rates.

THE CHANGE F R O M A L I Q U I D - F I L L E D T O A N A I R - F I L L E D L U N G AT B I R T H : WHAT ARE THE PHYSIOLOGICAL CONSEQUENCES? The physiological consequences of changing the luminal contents of the lung from liquid to air at birth have long been overlooked as an important contributor to the changes in lung physiology that occur at this time. In particular, the decrease in the basal degree of lung expansion associated with this change in the lung's internal environment is not widely acknowledged or understood. Our studies using healthy fetal sheep in utero have shown that, before birth, the volume of liquid retained within the future airways is considerably greater than the end-expiratory volume of the air-filled lung after birth 21'39 (Fig. 14.1). Although the degree of difference in resting lung volume between the fetus and new-born is debatable due to differences in fetal lung liquid volume measurements between studies (see Chapter 9), the reduction in end-expiratory lung volume upon air entering the lung is dictated by fundamental laws of physics. Before birth, the fetal lung is maintained in an expanded state due to the retention of liquid, which maintains an internal hydrostatic distending pressure; this retention of fluid is due to the active participation of fetal skeletal muscles in conjunction with a background of fluid secretion by the lung (see Chapter 9). During fetal apnea,

active adduction of the glottis maintains an internal hydrostatic distending pressure of 1-2 mmHg and, consequently, resting intra-pleural pressures within the fetus are close tO zero. 41'42 During episodes of FBM (see below), active dilation of the glottis reduces its resistance to lung liquid efflux, 43 resulting in an increased loss of lung liquid. However, the loss of luminal fluid during FBM is opposed by repeated contraction of the diaphragm. 21'39 After birth, the replacement of fetal lung liquid with air causes the generation of an air-liquid interface across the large surface area of the lung which greatly increases lung recoil, despite the presence of surfactant; as air is compressible it is less able to oppose lung recoil. In combination, an increase in lung recoil and the absence of the distending influence of lung liquid lead to the reduction in lung expansion at birth shown in Fig. 14.1. Recognising that the basal degree of lung expansion is reduced when air first enters the lungs helps to explain a number of physiological changes that occur in the respira, tory system at birth. For instance, the increase in lung recoil and the partial collapse of the lung away from the chest wall explains why intra-pleural pressure becomes more 'negative' after birth. 41 Before birth, the lungs are expanded with liquid and intra-pleural pressure is similar to ambient (amniotic sac) pressure, 41'42 but within hours of birth, the tendency of the lung to pull away from the chest wall reduces the intra-pleural pressure to --2cmH20 below ambient (atmospheric). 41 This has two further consequences: (1) at birth the mechanical load experienced by the chest wall must increase with the increase in lung recoil, which may play an important role in the stiffening of the chest wall after birth, 44 and (2) the pulmonary interstitial hydrostatic pressure must decrease in selected areas and approach intra-pleural pressure at rest; this may contribute to a reduction in capillary pressure and a reduction in pulmonary vascular resistance (PVR) (see below). Indeed, an increase in recoil pressure at birth due to the creation of surface tension within the lungs would be expected to promote the tendency for alveoli to pull away from adjacent alveoli, thereby lowering interstitial pressure between them. Such a reduction in pressure may also contribute to the clearance of liquid from the airways by providing a hydrostatic pressure gradient for liquid to move from the airways into the tissue and leave the lungs via either the lymphatics or circulation. 45 A predictable, yet until recently undocumented consequence of the decrease in lung expansion associated with birth and the onset of gaseous ventilation is its effect on the alveolar epithelium. It has been shown both in vivo and in vitro that the differentiated state of AECs is strongly influenced by the degree of mechanical strain experienced by these cells. In culture, increasing the degree of strain imposed on AECs stimulates type-II to type-I AEC differentiation. 46-48 Similarly, increased fetal lung expansion in vivo increases the number and relative proportions of type-I AECs, 49'5~ most probably due to type-I to type-II cell transdifferentiation via an intermediate cell type. 5~ As a result, within 10 days of increased lung expansion in fetal sheep,

only --2% of the AECs remain in the type-II cell phenotype. 5~ A more recent study has shown that a sustained reduction in fetal lung expansion, following a prolonged increase in lung expansion, results in the re-appearance of type-II AECs, most probably due to type-I to type-II cell trans-differentiation; 51 similar results have been obtained from in vitro studies. 46'47 These studies provide compelling evidence to indicate that (1) the basal degree of lung expansion is an important determinant of AEC phenotypes and (2) that type-I cells are not terminally differentiated but can trans-differentiate between phenotypes depending upon the degree of lung expansion. Based on these relationships it is interesting to consider what happens at birth. It may be expected that the proportion of type-I AECs would decrease whereas the proportion of type-II cells would increase after birth in response to the decrease in the basal level of lung expansion at this time (Fig. 14.1). A recent study has confirmed this by showing that the proportion of type-I AECs decreased from 60 to 65% in the late gestation fetus to ---30% after birth whereas the proportion of type-II cells increased from ---30% in the fetus to 50-55% after birth 52 (Fig. 14.2). This increase in type-II cell proportions after birth corresponded with an increase in the expression of SP-A, but not SP-B and -C. In summary, it is clear that end-expiratory lung volume decreases after birth, most likely due to an increase in lung recoil resulting from the creation of an air-liquid interface and the removal of the distending influence of lung liquid. This reduction in lung volume helps explain many of the changes in lung physiology that occur at birth which enable it to successfully adopt the role of gas exchange. As indicated

above, and in Chapter 9, skeletal muscle activity plays a critical role in maintaining high lung liquid volumes in the fetus. Intrauterine space and fetal posture are important determinants of the trans-pulmonary pressure gradient and, therefore, also affect lung liquid volume. Consequently, studies reporting normal lung liquid volumes in the fetus must ensure that fetuses are alive and unanaesthetised and that appropriate levels of amniotic fluid are present at the time of measurement.

THE C H A N G E S IN PULMONARY B L O O D F L O W AT B I R T H The development of the pulmonary vascular bed during fetal development is described in detail in Chapter 6. Chapter 7 provides a comprehensive review of the cellular processes involved with maintaining a high PVR in the fetus, how PVR is reduced at birth, and pharmacological treatments for persistent pulmonary hypertension of the newborn. In this section, we focus on the role of physical factors in the decrease in PVR at birth, in particular the effects induced by the reduction in end-expiratory lung volume shown in Fig. 14.1. Previous studies have shown that PVR in the fetus is closely associated with the degree of lung expansion 53'54 and alveolar pressure, as in the newborn 55'56 and adult. 57 Indeed, it has been shown that, in the fetus, pulmonary vascular conductance (the reciprocal of PVR) is inversely proportional to the degree of lung expansion, from very low lung volumes (residual lung volume) to total lung

Fig. 14.2. The proportions of type-I (filled bars) and type-II (hatched bars) alveolar epithelial cells (AECs), expressed as a percentage of the total number of AECs counted, before and after birth. Note that type-I AECs predominate before birth whereas soon after birth type-II cells predominate. The decrease in type-I cells and increase in type-II cells may result from the reduction in the basal degree of lung expansion that occurs at birth (see Fig. 14.1) due to the increase in lung recoil associated with the formation of an air-liquid interface. Data from Flecknoe etal. 52

capacity. 53 Expansion of the fetal lung to total lung capacity was found to cause complete cessation of flow, which was due to compression and closure of blood vessels of <300 ~tm diameter. 53 It is interesting to speculate, therefore, as to the contribution that the normal high degree of lung expansion, as well as a positive luminal pressure, makes to the maintenance of a high PVR in the fetus. To address this question and to assess the relative contribution that a reduction in lung expansion at birth may make to the decrease in PVR at this time, the effect of reducing the volume of fetal lung liquid to a volume equivalent to end-expiratory volumes in the air-filled lung after birth has been examined. 54 In fetal sheep, a 60% reduction in lung liquid volume caused a 4-fold increase in PBF, a corresponding 4-fold reduction in PVR and the abolition of the pressure gradient between the pulmonary and systemic arterial circulations. These findings indicate that the maintenance of a high degree of lung expansion in the fetus may contribute to the high PVR and help explain why the fetal pulmonary circulation is relatively resistant to prolonged vasodilatory stimuli (see Chapter 7). In the adult, control of the pulmonary circulation and PVR has few similarities with the control of the systemic circulation. In particular, the pulmonary circulation in the adult is characterised as a high flow, low pressure circuit with low mean arterial pressures (---15 mmHg) and a high flow that equals cardiac output. As a result PVR is low (--10-fold lower than in the systemic circulation), but has the capacity to fall even further in response to increases in pulmonary arterial pressure. 5s As most of the resistance within the adult pulmonary circulation resides within the capillaries, this decrease in resistance has been attributed to recruitment and distension of capillary beds. 5s In contrast, the pulmonary circulation of the fetus is characterised as a low flow, high pressure circuit with mean arterial pressures that are ---5mmHg greater than systemic arterial pressures. 54'59 Furthermore, it has been shown that only - 4 5 % of the fetal lung is perfused at any one time, 6~with the areas being perfused cycling apparently randomly across the lung at intervals of---35 min. 61 This raises the possibility that a large component of the decrease in PVR at birth results from the recruitment of capillary beds. 6~ It is pertinent to ask, therefore, why these capillary beds are closed and at what stage after birth does the pulmonary circulation develop adult-like characteristics and how? In the adult, the pulmonary capillaries are unique in that many of them lie immediately adjacent to relatively large air-filled sacs (alveoli). Fusion of the capillary endothelial cell and AEC basement membranes plays a critical role in facilitating respiratory gas exchange as it leads to a very thin air-blood diffusion barrier (N0.2ktm); this process begins before and continues after birth (see Fig. 14.3). As a result, the capillary wall is mechanically coupled to both the alveolar wall and interstitial tissue, which means that forces exerted in both compartments must be transferred onto capillaries. Indeed, transmural pressure across the alveolar-capillary wall is known to be a key factor regulating

capillary patency and PVR in the adult as capillary blood flow occurs only when capillary pressure exceeds alveolar pressure. 58 In adults, changes in the capillary-alveolar transmural pressure, leading to changes in PVR, are mainly determined by changes in capillary pressure. 58 Increases in capillary pressure, which can be caused by changes in cardiac output, account for the decrease in PVR with increasing pulmonary arterial pressure. Similarly, differences in capillary pressure account for the gravity-induced zonal differences in pulmonary blood flow through the apical and basal regions of the lung in adults at rest. 5s In the context of comparing the pulmonary circulations of the adult and mature fetus near term, both the alveolarcapillary wall transmural pressure and the pressure in interstitial tissue (i.e. between adjacent alveoli) are likely to be important. In the air-filled lung at rest, the high level of recoil (relative to that of the liquid-filled fetal lung) is likely to reduce the interstitial hydrostatic tissue pressure below alveolar pressure, which will help to maintain capillary pressures higher than interstitial tissue pressures. In contrast, a lower lung recoil in the fetus, due to the absence of surface tension, in combination with an intra-alveolar distending pressure of 1-2 mmHg, will reduce the transmural pressure across both the capillary-alveoli and capillary-interstitial tissue walls compared with the air-filled lung. Although few studies have focussed on the role of intrapulmonary pressures in regulating PVR in the fetus, some experimental evidence exists to support this concept. For example, a recent study (unpublished) has shown that during FBM in fetal sheep, mean PBF can increase ---3-fold, compared with apneic periods, due to a decrease in PVR; this only occurs when the changes in intra-luminal pressure caused by FBM exceed ---3 mmHg (Fig. 14.4). Although this finding may be explained by the release of vasodilators in response to the vascular shear stress induced by accentuated FBM (see Chapter 7), the role of capillary transmural pressure changes cannot be ignored. Indeed, the reduction in PVR also occurred in isolated brief periods of vigorous FBM and were tightly constrained to these periods (see Fig. 14.4), which would not be expected if vasodilators were involved. Similarly, changes in intra-pulmonary tissue pressures may provide an alternative explanation for the contribution that the 'onset of air breathing' makes to the decrease in PVR at birth. 62 Previous studies have examined the independent effect of the onset of gaseous ventilation on the decrease in PVR at birth by ventilating fetal sheep while they remained in utero. 62'63 It was found that the onset of gaseous ventilation per se, without an associated increase in oxygenation, could account for a large proportion of the decrease in PVR observed. 62 However, before these lambs were ventilated in utero the lungs were drained of liquid; 62'63 thus, the lungs were first deflated and then lung recoil was increased by the generation of an airliquid interface. In summary, the role of an increase in lung recoil, due to the creation of an air-liquid interface and the generation of surface tension across the lung epithelium, has been largely overlooked as a contributor to the decrease in PVR at birth.

Fig. 14.3. Electron micrograph of an alveolus and an adjacent capillary, demonstrating the very thin barrier that separates the airspace from the capillary lumen (air-blood barrier). Note that this barrier consists of the attenuated cytoplasm of an alveolar epithelial cell and a capillary endothelial cell, which are separated by their respective basement membranes that have fused and is -0.2 Ftm thick (see enlarged inset). In this micrograph, the attenuated cytoplasmic extension of a single type-I cell (nucleus indicated) extends around the entire alveolus.

However, the associated changes in interstitial tissue-capillary and alveolar-capillary transmural pressures resulting from the increase in lung recoil at birth are likely to be important additional factors.

CLOSURE

OF

THE

DUCTUS

A R T E R I O S U S AT B I R T H The DA is a large vascular shunt interposed between the main pulmonary artery and descending aorta, allowing the majority of the output from the right heart of the fetus to be directed into the systemic circulation (Fig. 14.5). Soon after birth, the DA must close to enable the lungs to receive the entire output of the right heart to facilitate pulmonary gas exchange; failure of ductal closure (i.e. patent DA) results

in reduced pulmonary blood flow and hence impaired gas exchange in the lungs. The mechanisms underlying closure of the DA at birth are complex and involve both chemical and physical factors; the chemical control of ductal closure at birth has been the focus of much attention and is discussed in Chapter 7. In this section we focus on the physical relationship between PVR and blood flows through the pulmonary circulation and DA after birth. During fetal life, N88% of right ventricular output by-passes the lungs by flowing through the DA, from the main pulmonary artery into the descending aorta. 59 This flow not only occurs during systole, but also during diastole due to back flow from the lungs (Fig. 14.6). That is, during the later part of diastole, because of the very high PVR, blood flows retrogradely along the left and right pulmonary arteries and through the DA into the descending aorta. Thus, the DA acts as a shunt during fetal life, which promotes the flow of

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0 .........i;i:~:,g .......~;i::g;00........~i~:,3:20ii;,;:40 .......1ii.~i00-.... I~:S:3;20..... Fig. 14.4. Pulmonary arterial blood flow (ml/min), tracheal pressure (mmHg) and mean pulmonary arterial blood flow (ml/min)in a fetal sheep during an episode of vigorous breathing movements (FBM; upper panel) and during apneic periods (lower panel). During vigorous FBM (amplitude > 3 mmHg, shown in the upper panel), mean pulmonary arterial blood flow increases 3-4-fold compared with apneic periods in the same fetus shown by bars in the lower panel. Note that, in the lower panel, a brief period of vigorous FBM was associated with a transient increase in the mean pulmonary arterial blood flow. Clock time is given at the bottom of the panels.

Fig. 14.5. The ductus arteriosus (DA) in a sheep fetus showing its connections to the main pulmonary artery (MPA) and descending aorta. Note the oblique angle at which the DA joins the thoracic aorta, which facilitates flow from the pulmonary circulation, via the DA into the descending aorta. Thus, a reversal of the pressure gradient across the DA (i.e. when systemic arterial pressure > pulmonary arterial pressure), leading to the reversal of blood through the DA, would cause substantial turbulence within it. The left atrium (LA) is retracted to reveal the left pulmonary artery - LPA; pd Aorta, preductal aorta. (See Color plate 5.)

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blood from the pulmonary into the systemic circulation due to the pressure gradient (of 4-5 mmHg) between these circulations (Fig. 14.6). After birth, despite the large increase in PBF, pulmonary arterial pressure gradually decreases due to the very large decrease in PVR. As a result, backflow along the left and right pulmonary arteries quickly diminishes (within hours of birth) resulting in only forward flow through the pulmonary arteries, even during diastole (Fig. 14.6). If the DA remains open, blood will pass between the pulmonary and systemic circulations, depending upon the pressure gradient across this vessel. Thus, as pulmonary arterial pressures decrease after birth, unless the DA closes, blood will flow retrogradely from the systemic into the pulmonary circulation. Due to the anatomic relationship between the pulmonary artery, DA and the descending aorta (Fig. 14.5), considerable turbulence must occur at this site if blood

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Fig. 14.6. Instantaneous pulmonary blood flow through the left pulmonary artery before birth in a near-term sheep fetus (left panel) and in the same animal 1 hour after birth (right panel). These flow profiles demonstrate the beat-by-beat changes in pulmonary arterial blood flow. Note the large degree of backflow (- 100 ml/min) during diastole before birth, whereas after birth, this backflow is abolished.

flow through the DA is reversed. Such turbulence would likely elicit the release of endothelial-derived factors (see Chapter 7) that would contribute to the constriction of the DA.

FETAL B R E A T H I N G A N D THE O N S E T OF C O N T I N U O U S BREATHING AT BIRTH Although it is often stated that an infant takes its first breath at birth, it is now clear that respiratory movements begin during fetal life, long before birth. Like postnatal breathing, FBM involve rhythmical contractions of the diaphragm and other inspiratory muscles, such as dilator muscles of the pharynx and larynx, which are driven by brainstem centres that are stimulated by CO2 .64 The major differences between fetal breathing and postnatal breathing are that in the fetus (1) the lungs are liquid-filled and hence tidal volume is very small (see Chapter 9), (2) breathing movements are episodic, (3) breathing movements are inhibited, rather than stimulated, by hypoxia and (4) the breathing movements play no role in gas exchange, but rather result in a net oxygen consumption. Breathing movements in healthy human and ovine fetuses can first be detected during the first half of gestation and continue until the onset of labour. 64 Characteristically, they occur in episodes which become more organised later in gestation as fetal behavioural states become organised. During late gestation, FBM in humans and sheep occur 40-50% of the time 64 and are primarily associated with a state resembling rapid eye movement sleep; during episodes of 'quiet sleep' fetuses are largely apneic. 65 The incidence of FBM is reduced during active labour, but the mechanisms underlying this inhibition are not presently understood. 64 Although it is well established that prostaglandins can inhibit FBM, 66 it is thought that prostaglandins such as PGE 2 are not involved in the labour-related reduction of FBM. 67 Adenosine, which can be released into the fetal circulation from the placenta and fetal liver may play a role, as it is known to inhibit FBM via cerebral adenosine receptors. 68 During parturition and when the umbilical cord is cut at birth, the fetus-neonate may become profoundly hypoxemic, hypercapnic and acidemic, as well as being exposed to a lower environmental temperature with increased heat loss. It will also be exposed to a greatly increased degree of external sensory stimuli; in addition, its behavioural state may change to one of arousal. 69 What triggers continuous breathing at the time of birth is presently the subject of debate, although it is apparent that many factors, such as those listed above, are likely to be involved. A major factor is thought to be increased fetal CO 2 production, perhaps resulting from an increased rate of metabolism at birth, and/or an increased sensitivity to CO 2. Circulating levels of catecholamines are elevated at birth, 4~ which would be expected to stimulate metabolic activity and hence CO 2 production. It has also been shown that a reduction at birth in fetal circulating concentrations of adenosine, which is produced by the placenta, can stimulate thermogenesis TM leading to an increase in CO 2 production. A decrease in the concentrations of one or more circulating factors of placental origin (e.g. PGE 2, adenosine, progesterone metabolites)

that are known to inhibit FBM may facilitate continuous breathing after birth. 66'71'72 Removal of the placenta from the fetal circulation results in an increased ventilatory sensitivity to CO 2 suggesting that an inhibitory factor may act at the level of the fetal brainstem. 69 Central and peripheral chemoreceptors are active in the f e t u s , 73 although it has been suggested that their sensitivity may be tonically suppressed by factors released from the placenta into the fetal circulation such as PGE2, 72 receptors for which have been identified in the fetal brainstem. TM Studies of fetal sheep maintained e x u t e r o by extracorporeal oxygenation with the umbilical cord occluded, thereby eliminating potential effects of placental factors, have shown that continuous breathing is dependent upon blood CO 2 levels, 75 supporting the notion that CO 2 plays a crucial role in the maintenance of continuous breathing after birth. The integrity of the vagus nerves has been shown to be essential for the onset of adequate breathing at birth. 76 Although the critical pathways have not yet been identified, it is likely that volume receptive feedback from the lungs is involved. Studies in unanaesthetized neonatal lambs have shown that the application of negative airway pressures or creation of a tracheostomy, both of which would reduce end-expiratory lung volume (FRC) and the amount of vagal neural traffic from lung volume receptors (pulmonary stretch receptors), result in profound hypoventilation, periodic breathing and active glottic adduction during periods of apnea. 77'78 This indicates that volume receptive vagal feedback at end-expiration, which is normally maintained by an adequate FRC, is essential for continuous breathing in the newborn, and explains, at least in part, the benefits of positive end-expiratory pressure (PEEP) in the treatment of infantile apnea.

CONCLUSIONS During normal gestation, with labour and delivery occurring at term, the fetal lung is well prepared for its critical role of gas exchange after birth. Both endocrine and physical factors play a major role in preparing the lung for extrauterine function. However, if gestation is shortened as a result of preterm birth, maturation of the lung may not have occurred to a sufficient degree, resulting in respiratory compromise; in particular, the lung may not have developed structurally and epithelial type-II cells may not be able to produce sufficient quantities of surfactant, resulting in respiratory distress. If birth occurs in the absence of labour, as a result of caesarian section, this may result in a delay in the clearance of lung liquid after birth, which can result in transient tachypnea and a delay in establishing independent respiratory function. Although much is already known, there remain many unanswered questions relating to physiological and molecular mechanisms underlying the preparation of the lung for birth and its postnatal adaptation to airbreathing.

REFERENCES 1. Liggins GC. Premature delivery of foetal lambs infused with glucocorticoids. J. Endocrinol. 1969; 45:515-23. 2. Liggins GC, Howie RN. A controlled trial of antepartum glucocorticoid treatment for prevention of the respiratory distress syndrome in premature infants. Pediatrics 1972; 50:515-25. 3. Crowley P, Chalmers I, Keirse MJNC. The effects of corticosteroid administration before preterm delivery: an overview of the evidence from controlled trials. Br. J. Obstet. Gynaecol. 1990; 97:11-25. 4. Crowley P. Prophylactic corticosteroids for preterm birth. Cochrane Database Syst. Rev. 2000; CD000065. 5. Jobe AH, Ikegami M. Lung development and function in preterm infants in the surfactant treatment era. Annu. Rev. Physiol. 2000; 62:825-46. 6. Liggins GC. The foetal role in the initiation of parturition in the ewe. In: Wolstenholme GEW, O'Connor M (eds) Foetal Autonomy (Ciba Foundation Symposium). Churchill, London. 1990; 218. 7. Crone RK, Davies P, Liggins GC et al. The effects of hypophysectomy, thyroidectomy, and postoperative infusion of cortisol or adrenocorticotrophin on the structure of the ovine fetal lung.J. Dev. Physiol. 1983; 5:281-8. 8. Kitterman JA, Liggins GC, Campos GA etal. Prepartum maturation of the lung in fetal sheep: relation to cortisol. J. Appl. Physiol. 1981; 51:384-90. 9. Liggins GC, Schellenberg JC, Finberg K etal. The effects of ACTH1-24 or cortisol on pulmonary maturation in the adrenalectomized ovine fetus. J. Dev. Physiol. 1985; 7:105-11. 10. Ballard PL. The glucocorticoid domain in the lung and mechanisms of action. In: Mendelson CR (ed.) Endocrinology of the Lung. Humana Press, Inc., Totowa. 2000; 1-44. 11. Cole TJ, Blendy JA, Monaghan AP et al. Targeted disruption of the glucocorticoid receptor gene blocks adrenergic chromaffin cell development and severely retards lung maturation. Genes Dev. 1995; 9:1608-21. 12. Bland RD. Loss of liquid from the lung lumen in labor: more than a simple "squeeze". Am. J. Physiol. Lung Cell Mol. Physiol. 2001; 280:L602-5. 13. Kitterman JA, Ballard PL, Clements JA et al. Tracheal fluid in fetal lambs: spontaneous decrease prior to birth. J. Appl. Physiol. 1979; 47:985-9. 14. Dickson KA, Maloney JE, Berger PJ. Decline in lung liquid volume before labor in fetal lambs. J. AppL Physiol. 1986; 61:2266-72. 15. Mitzner W, Johnson JWC, Scott R et al. Effect of betamethasone on pressure-volume relationship of fetal rhesus monkey lung.J. Appl. Physiol. 1979; 47:377-82. 16. Harding R, Hooper SB, Dickson KA. A mechanism leading to reduced lung expansion and lung hypoplasia in fetal sheep during oligohydramnios. Am. J. Obstet. Gynecol. 1990; 163:1904-13. 17. Lines A, Hooper SB, Harding R. Lung liquid production rates and volumes do not decrease before labor in healthy fetal sheep.J. Appl. Physiol. 1997; 82:927-32. 18. Dickson KA, Harding R. Restoration of lung liquid volume following its acute alteration in fetal sheep. J. Physiol. 1987; 385:531-43. 19. Hooper SB, Dickson KA, Harding R. Lung liquid secretion, flow and volume in response to moderate asphyxia in fetal sheep.J. Dev. Physiol. 1988; 10:473-85. 20. Olver RE, Ramsden CA, Strang LB et al. The role of amiloride-blockable sodium transport in adrenaline-induced lung liquid reabsorption in the fetal lamb. J. Physiol. 1986; 376:321-40.

21. Hooper SB, Harding R. Fetal lung liquid: a major determinant of the growth and functional development of the fetal lung. Clin. Exp. Pharmacol. Physiol. 1995; 22:235-47. 22. Matalon S, O'Brodovich H. Sodium channels in alveolar epithelial cells: molecular characterization, biophysical properties, and physiological significance. Annu. Rev. Physiol. 1999; 61:627-61. 23. Brown MJ, Olver RE, Ramsden CA et al. Effects of adrenaline and of spontaneous labour on the secretion and absorption of lung liquid in the fetal lamb.J. Physiol. 1983; 344:137-52. 24. Hooper SB, Harding R. Effects of 13-adrenergic blockade on lung liquid secretion during fetal asphyxia. Am. J. Physiol. 1989; 257:R705-10. 25. Wallace MJ, Hooper SB, Harding R. Regulation of lung liquid secretion by arginine vasopressin in fetal sheep. Am. J. Physiol. 1990; 258:R104-11. 26. Barker PM, Brown MJ, Ramsden CA et al. The effect of thyroidectomy in the fetal sheep on lung liquid reabsorption induced by adrenaline or cyclic AMP.J. Physiol. 1988; 407:373-83. 27. Wallace MJ, Hooper SB, Harding R. Role of the adrenal glands in the maturation of lung liquid secretory mechanisms in fetal sheep. Am. J. Physiol. 1996; 270:R1-8. 28. Barker PM, Markiewicz M, Parker KA et al. Synergistic action of triiodothyronine and hydrocortisone on epinephrineinduced reabsorption of fetal lung liquid. Pediatr. Res. 1990; 27:588-91. 29. Wallace MJ, Hooper SB, Harding R. Effects of elevated fetal cortisol concentrations on the volume, secretion and reabsorption of lung liquid.Am. J. Physiol. 1995; 269:R881-7. 30. Kindler PM, Chuang DC, Perks AM. Fluid production by in vitro lungs from near-term fetal guinea pigs: effects of cortisol and aldosterone. Acta Endocrinol. 1993; 129:169-77. 31. Berger S, Bleich M, Schmid W e t al. Mineralocorticoid receptor knockout mice: pathophysiology of Na § metabolism. Proc. Natl. Acad. Sci. USA 1998; 95:9424-9. 32. Dickson KA, Harding R. Compliances of the liquid-filled lungs and chest wall during development in fetal sheep. J. Dev. Physiol. 1991; 16:105-13. 33. Albuquerque CA, Smith KR, Saywers TE etal. Relation between oligohydramnios and spinal flexion in the human fetus. Early Hum. Dev. 2002; 68:119-26. 34. Berger PJ, Kyriakides MA, Smolich JJ et al. Massive decline in lung liquid before vaginal delivery at term in the fetal lamb. Am.J. Obstet. Gynecol. 1998; 178:223-7. 35. Kalache KD, Chaoui R, Marks Bet al. Does fetal tracheal fluid flow during fetal breathing movements change before the onset of labour? Br. J. Obstet. Gynaecol. 2002; 109:514-9. 36. Harding R, Sigger JN, Wickham PJD et al. The regulation of flow of pulmonary fluid in fetal sheep. Respir. Physiol. 1984; 57:47-59. 37. Pfister RE, Ramsden CA, Neil HL et al. Volume and secretion rate of lung liquid in the final days of gestation and labour in the fetal sheep.J. Physiol. 2001; 535:889-99. 38. Waiters DV, Olver RE. The role of catecholamines in lung liquid absorption at birth. Pediatr. Res. 1978; 12:239-42. 39. Harding R, Hooper SB. Regulation of lung expansion and lung growth before birth.J. Appl. Physiol. 1996; 81:209-24. 40. Hagnevik K, Faxelius G, Irestedt Let al. Catecholamine surge and metabolic adaptation in the newborn after vaginal delivery and caesarean section. Acta Paediatr. Scand. 1984; 73:602-9. 41. Avery ME, Cook CD. Volume-pressure relationships of lungs and thorax in fetal, newborn, and adult goats.J. AppL Physiol. 1961; 16:1034-8. 42. Vilos GA, Liggins GC. Intrathoracic pressures in fetal sheep. J. Dev. Physiol. 1982; 4:247-56. 43. Harding R, Bocking AD, Sigger JN. Upper airway resistances in fetal sheep: the influence of breathing activity. J. Appl. Physiol. 1986; 60:160-5.

44. Davey MG, Johns DP, Harding R. Postnatal development of respiratory function in lambs studied serially between birth and 8 weeks. Respir. Physiol. 1998; 113:83-93. 45. Bland RD, Hansen TN, Haberkern CM etal. Lung fluid balance in lambs before and after birth. J. Appl. Physiol. 1982; 53:992-1004. 46. Shannon JM, Jennings SD, Nielsen LD. Modulation of alveolar type II cell differentiated function in vitro. Am. J. Physiol. 1992; 262:L427-36. 47. Danto SI, Shannon JM, Borok Z et al. Reversible transdifferentiation of alveolar epithelial cells. Am. J. Respir. Cell Mol. Biol. 1995; 12:497-502. 48. Gutierrez JA, Gonzalez RF, Dobbs LG. Mechanical distension modulates pulmonary alveolar epithelial phenotypic expression in vitro.Am. J. Physiol. 1998; 274:L196-202. 49. Alcorn D, Adamson TM, Lambert TF et al. Morphological effects of chronic tracheal ligation and drainage in the fetal lamb lung.J. Anat. 1977; 123:649-60. 50. Flecknoe S, Harding R, Maritz G et al. Increased lung expansion alters the proportions of type I and type II alveolar epithelial cells in fetal sheep. Am. J. Physiol. 2000; 278:L1180-5. 51. Flecknoe SJ, Wallace MJ, Harding R et al. Determination of alveolar epithelial cell phenotypes in fetal sheep: evidence for the involvement of basal lung expansion. J. Physiol. 2002; 542:245-53. 52. Flecknoe SJ, Wallace MJ, Harding R et al. Changes in alveolar epithelial cell proportions before and after birth. Am. J. Respir. Crit. Care Med. 2002; 165:A643. 53. Walker AM, Ritchie BC, Adamson TM etal. Effect of changing lung liquid volume on the pulmonary circulation of fetal lambs.J. Appl. Physiol. 1988; 64:61-7. 54. Hooper SB. Role of luminal volume changes in the increase in pulmonary blood flow at birth in sheep. Exp. Physiol. 1998; 83:833-42. 55. Fuhrman BP, Smith-Wright DL, Kulik TJ etal. Effects of static and fluctuating airway pressure on intact pulmonary circulation.J. Appl. Physiol. 1986; 60:114-22. 56. Fuhrman BP, Smith-Wright DL, Venkataraman S etal. Pulmonary vascular resistance after cessation of positive end-expiratory pressure. J. Appl. Physiol. 1989; 66:660-8. 57. Roos A, Thomas LJ, Nagel EL et al. Pulmonary vascular resistance as determined by lung inflation and vascular pressures. J. Appl. Physiol. 1961; 16:77-84. 58. West JB. Pulmonary blood flow and metabolism. In: West JB (ed.) Physiological Basis of Medical Practice, 12th edn. Williams & Wilkins, Baltimore. 1989; 529-536. 59. Heymann MA. Control of the pulmonary circulation in the perinatal period. J. Dev. Physiol. 1984; 6:281-90. 60. Lipsett J, Hunt K, Carati C et al. Changes in the spatial distribution of pulmonary blood flow during the fetal/neonatal transition: an in vivo study in the rabbit. Pediatr. Pulmonol. 1989; 6:213-22.

61. Lipsett J, Gannon B. Regional cycles of perfusion and nonperfusion in the lung of the term fetal rabbit. Pediatr. Pulmonol. 1991; 11:153-60. 62. Teitel DF, Iwamoto HS, Rudolph AM. Changes in the pulmonary circulation during birth-related events. Pediatr. Res. 1990; 27:372-8. 63. Iwamoto HS, Teitel DF, Rudolph AM. Effects of lung distension and spontaneous fetal breathing on hemodynamics in sheep. Pediatr. Res. 1993; 33:639-44. 64. Harding, R. Fetal breathing movements. In: Crystal RG, West JB, Weibel ER etal. (eds) The Lung: Scientific Foundations, 2nd edn. Raven Lippincott, New York. 1997; 1655-63. 65. Dawes GS, Fox HE, Leduc BM et al. Respiratory movements and rapid eye movement sleep in the foetal lamb. J. Physiol. 1972; 220:119-43. 66. Kitterman JA. Arachidonic acid metabolites and control of breathing in the fetus and newborn. Semin. Perinatol. 1987; 11(1):43-52. 67. Wallen LD, Murai DT, Clyman RI et al. Effects of meclofenamate on breathing movements in fetal sheep before delivery. J. Appl. Physiol. 1988; 64(2):759-66. 68. Koos BJ, Maeda T, Jan C. Adenosine A(1) and A(2A) receptors modulate sleep state and breathing in fetal sheep. J. AppL Physiol. 2001; 91:343-50. 69. Adamson SL. Regulation of breathing at birth. J. Dev. Physiol. 1991; 15:45-52. 70. Sawa R, Asakura H, Power GG. Changes in plasma adenosine during simulated birth of fetal sheep. J. Appl. Physiol. 1991; 70(4):1524-8. 71. Crossley KJ, Nicol MB, Hirst JJ etal. Suppression of arousal by progesterone in fetal sheep. Reprod. Fertil. Dev. 1997; 9:767-73. 72. Thorburn GD. The placenta and the control of fetal breathing movements. Reprod. Fertil. Dev. 1995; 7:577-94. 73. Jansen AH, Chernick V. Onset of breathing and control of respiration. Semin. Perinatol. 1988; 12(2):104-12. 74. Tai TC, MacLusky NJ, Adamson SL. Ontogenesis of prostaglandin E2 binding sites in the brainstem of the sheep. Brain Res. 1994; 652:28-39. 75. Kuipers IM, Maertzdorf WJ, De Jong DS et al. Initiation and maintenance of continuous breathing at birth. Pediatr. Res. 1997; 42:163-8. 76. Wong KA, Bano A, Rigaux A et al. Pulmonary vagal innerration is required to establish adequate alveolar ventilation in the newborn lamb. J. Appl. Physiol. 1998; 85:849-59. 77. Johnson P. Physiological aspects of regular, periodic and irregular breathing in adults and in the perinatal period. In: Von Euler C, Lagercrantz H (eds) Central Nervous Control Mechanisms in Breathing. Pergamon Press, Oxford. 1979; 337-51. 78. Harding R. State-related and developmental changes in laryngeal function. Sleep 1980; 3:307-22.

INTRODUCTION

The process of lung aging begins at birth. Development of the respiratory system for a variety of mammalian species has been analyzed both anatomically, as well as physiologically, during early postnatal life. 1-3 However, few studies have quantitatively examined structural changes that occur with aging. Descriptions of age-related alterations in the respiratory system that do exist 4-6 have focused almost exclusively on the gas exchange portions of the lungs. Less information is available for detailed description of cellular and structural changes in the aging process of the tracheobronchial tree. The majority of data related to lung aging is typically confined to brief descriptive observations of control animals through lifetime toxicity studies. 7'8 This chapter will cover aspects of normal aging in a number of mammalian species, focusing primarily on the mouse, rat, and dog. Aging of the human lung will be covered in Chapter 28. Lifespan characteristics for each of these species is different, with the mouse having a median lifespan of 29-30 months, the rat, 30-34 months, the dog, 12-14 years, and the human, 72-78 years. These striking differences in lifespan are likely to have a significant impact on the resultant aging process within the respiratory system of each species. However, there also exists a multitude of morphologic features in aging that cross boundaries for all species. These include postnatal alveolarization of the lungs during early childhood development that is complete in small laboratory species within four to six weeks of birth, in dogs within the first year of life, and in humans within the first eight years of life. Thinning of alveolar septa within the gas exchange portions of the lung is a common characteristic across all species. Pores or fenestrations within alveolar The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

walls are also present in all species. These anatomical structures are thought to serve in cross-collateral ventilation between adjacent alveoli. They may also serve to facilitate the migration of cells such as macrophages within the airspaces, in a short circuit path from one alveolus to another. These pores appear to enlarge during the aging process and may play an important role in the development of an emphysematous condition in the lungs with larger and greater numbers of fenestrations and loss of alveolar septal walls with age. These characteristics have been studied in detail in the mouse, dog, and human. Airspace enlargement is a characteristic feature of aging in all species; however, it is unclear whether such enlargement is always accompanied by the destruction or partial loss of alveolar wall structures. Nevertheless, these general characteristics present in each species described in this chapter can serve to better understand the process of lung aging over the normal lifespan. The most vital function of the lungs is gas exchange. To enable the most efficient exchange of 0 2 and CO 2 air must be brought into close proximity to blood as it passes through the lungs. That portion of the lungs involved in gas exchange represents 80-90% of the total lung volume. Billions of cells in the lungs are arranged to form a delicate, yet sturdy and highly vascular air-tissue interface creating a surface area 25 times greater than that of the external surface of the body. This tissue barrier separates the inspired air from the blood by less than one micrometer. It is easily deformed by the passage of blood through the underlying capillary bed, causing bulging of the walls into the airspace with the outline of red blood cells easily visible through the thin alveolar partitions (Fig. 15.1). This highly efficient surface design for gas exchange is limited to the small space enclosed by the bony thorax and muscular, Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

Fig. 15.1. Alveolar septum from the lungs of a 5-month-old Fischer 344 rat. A fibroblast (F) can be seen at the junction of the septa. A pocket (*) of interstitial matrix is present, but most of the alveolar septum is composed of a thin air-to-blood tissue barrier (bar, 3 l.tm). (Reproduced with permission from Pinkerton KE, Barry BE, O'Neil JJ et al. Morphologic changes in the lung during the life span of Fischer 344 rats. Am. J. Anat. 1982; 164:155-74.)

dome-shaped diaphragm. A highly ordered airway branching beginning at the carina of the trachea gives rise to millions of alveoli lining several generations of alveolar ducts and sacs to bring about this exponential increase in area within a tightly packaged volume. The process of alveolarization during early life leads to the formation of approximately 80% of all alveoli postnatally. However, in later life the number of alveoli may be reduced through a destructive process, with loss of alveolar septal wall surface area and/or the rearrangement of essential extracellular matrix components such as collagen and elastin; these processes allow the alveoli to become stretched and shallow leading to distention of alveolar ducts, referred to as alveolar ductasia. These phenomena are discussed in this chapter as well as in Chapter 28.

AGING, BODY IN MAMMALS

MASS

AND

THE

LUNGS

For the mammalian lung, a strong allometric relationship exists between body mass and the following features: lung volume, lung capillary volume, alveolar surface area, and pulmonary diffusing capacity. 9'1~ Postnatal development is associated with significant increases in body mass and lung volume. For the mouse, the most dramatic increase in body mass occurs during the first month of life. After 2 months, weight increases slowly, but a slight increase occurs up to 19 months of age, usually followed by a decrease after 28 months of age (Table 15.1). For rats, the two most commonly used strains for lifetime studies are SpragueDawley and Fischer 344 rats. The popularity of this species

for lifetime studies is attributed to their small body size, simple housing requirements, as well as a relatively short lifespan of 30-36 months. The Fischer 344 rat has been used extensively because of a slow increase in body mass with age and resistance to pulmonary disease (Fig. 15.2). Dogs cover a vast span in body size and therefore have pulmonary size characteristics that cover a wide range of values compared with mice and rats. Lung characteristics such as airspace volume or alveolar surface area may span at least two orders of magnitude in dogs. However, the lungs of dogs possess many characteristics similar to the human lung, including the presence of respiratory bronchioles, anatomical structures lacking in the lungs of mice and rats. Lung physiology and tissue structure in dogs also change in a similar fashion to humans during the process of lung growth and development. 12 For the human, lung development continues beyond birth, with over 80% of alveoli formed postnatally. This process of alveolarization is thought to be complete by eight years of age. However, it has also been stated that alveolarization may end as early as two years of age. During adolescence, the lungs continue to expand to the dimensions of the thoracic cavity and increase physiological capacity. By the age of 18, lung growth is considered to be complete. The following sections will cover normal aging in the mouse, rat, and dog, with an emphasis on lung anatomy.

THE

MOUSE

Lifespan characteristics of the mouse The white-footed mouse maintained under barrier conditions can survive for up to 8 years, 13'14 the median lifespan being 4.5-6 years. 15 Mouse strains most commonly used in

research, including BALB/c, Swiss-Webster, and C57BL/6, have a shorter median lifespan of 29-30 months. The life span of laboratory mice depends on the strain, nutritional status, and environment in which they are maintained. Special mouse strains have been established, including the senescence-accelerated mouse (SAM), a murine model of rapid aging. 16 Both prone (SAM-P/I) and resistant (SAM-R/I) strains have been developed and have median lifespans of 11.9 and 17.5 months, respectively. 16

The aging mouse lung The majority of studies using mice begin with animals at 1-2 months of age. Studies on the process of pulmonary aging rarely extend beyond 12 months of age. Therefore, the amount of information available on the effects of aging on the structure and function of the lungs in mice, including age-related changes of the airways, vasculature, nerves, lymph vessels, or immune system is limited. Selected changes in the pulmonary parenchyma of the mouse from 1 to 28 months of age have been established. 2'17'1s Studies with SAM have prompted a growing interest in this strain as a potential model of the aging process. Senescence in this mouse strain is marked by behavioral changes, hair lOSS,19 senile amyloidosis, 2~ cataracts, 19 and osteoporosis. 21 Pulmonary studies in SAM have noted accelerated changes in the degree of pulmonary hyperinflation similar to that noted in other mouse strains at older ages. 22'23 Therefore, SAM may provide insights into the aging process in mouse lungs over a shorter period of time, as compared with other conventional mouse strains.

Changes in lung volume of mice during aging Changes in total lung volume with increasing age in mice of differing strains are given in Table 15.2. The volume of the lungs continues to increase in a significant age-dependent

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AGE (months)

Fig. 15.2. Changes in body weight with increasing age in male and female Fischer 344 rats. Each datum point represents the mean +SD of 4 animals. Asterisk on line connecting different age groups denotes significant change in body weight (p< 0.05). (Reproduced with permission from Pinkerton KE, Barry BE, O'Neil JJ et al. Morphologic changes in the lung during the life span of Fischer 344 rats. Am. J. Anat. 1982; 164:155-74.)

manner to age 25-28 months. 17 These age-related increases occur in an approximately linear manner throughout the lifespan of the mouse (Table 15.2). Measurement of lung volume changes is based on a simple technique that utilizes intratracheal instillation of fixative at a standard pressure. For the measurements in Table 15.2, the lungs were removed from the thoracic cavity and degassed before instillation of the fixative at 25-30 cm I-I20; once fixed, lung volume was measured by liquid displacement. 24 Fixed lung volume can serve as a simple reference for measurements of size, volume, and surface area of those structural components present in the lungs by applying morphometric techniques. Intratracheal instillation of fixative at a standard pressure is one of the most accurate and reproducible means of inflating excised lungs. However, in the absence of the chest wall, the increased compliance of pulmonary tissues in excised old mouse lungs could allow distention beyond those volumes permitted within the thoracic limits of the animal. However, for the measurements in Table 15.2, special care was taken to ensure that fixation pressures did not exceed 15 cm H20 after an initial inflating pressure of 25cm H20. Therefore, subsequent measures of parenchymal airspace volume and surface area, as determined using morphometric techniques on these tissues, can be considered as a true reflection of changes due to aging in the lungs of each mouse strain.

in the mouse. Only studies of mice aged 2-3 months have reported the number of epithelial cells per mm of basal lamina in the trachea to be approximately 215. 25 The epithelial cell types in the trachea at this age consist of 10% basal cells, 39% ciliated cells, 49% Clara cells, and 2% unknown cells. The density of epithelial cells does not change in second and third airway generations compared with the trachea. The proportion of epithelial cell types also remains constant. Within the mainstem bronchi of the mouse, basal cells constitute 4% of the total population, ciliated cells 47%, Clara cells 46%, and unknown cells 3%. 25 Epithelial cell density in airway generations 4-6 in the mouse is slightly reduced, with 199 cells per mm of basal lamina. The proportion of epithelial cell types shifts, with basal cells accounting for 1%, ciliated cells 36%, Clara cells 61%, and unknown cells 2%. Epithelial cells of the bronchioles, including the terminal bronchiole, consist of a simple cuboidal to columnar epithelial layer formed by ciliated cells and Clara cells. The relative proportion of Clara cells in the bronchiole is as high as 80% and ciliated cells constitute the remaining cells of the epithelial airway lining. Clara cells within the terminal bronchiole of the mouse have dome-shaped apical surfaces protruding into the lumen of the airway and numerous secretory granules and mitochondria found within the apical cytoplasm. Little is known about the effects of aging on the structure or function of Clara cells in the mouse.

Tracheobronchial airways of aging mice

Lung parenchymal structure in aging mice

In the trachea and proximal bronchi of the mouse, the epithelial cells form a pseudostratified layer, with a staggered arrangement of nuclei located above the basal lamina. The airway epithelium is composed of three primary cell types: basal cells, ciliated cells, and secretory cells. In the mouse, secretory cells present in the trachea form the nonciliated bronchiolar epithelial cells, or Clara cells. In the mouse, the location of these cells within the trachea is unique because Clara cells are typically found only in more distal bronchioles of the airways in most species. Little information is available on epithelial cell populations of the tracheobronchial tree during the aging process

The parenchyma of the lungs is composed of alveoli, alveolar ducts, and alveolar sacs. These structures form - 90% of the total lung volume in the mouse. 17 The alveoli represent the smallest anatomical unit involved in gas exchange and are composed of the airspace bounded by the alveolar wall and its opening into the alveolar duct. The alveolar duct is formed by the airspace shared in common with alveoli opening along a common channel created by the tissue ridges forming the mouth opening of individual alveoli. The branching of alveolar ducts to form discrete alveolar duct generations begins at the bronchiole-alveolar duct junction (BADJ) and ends 3-5 generations away as a blind

alveolar sac formed by several alveoli. The total alveolar surface area formed by these structures in the aging mouse is depicted in Fig. 15.3 for BALB/c mice and two strains of senescent mice (SAM). The proportion of air volume found in ducts and alveoli is given in Table 15.3. Total volume of alveolar air is approximately 0.5 ml at 1 month and steadily increases until 28 months of age. The age-related increase in air volume is more dramatic in young animals compared with older animals. The total volume of air within alveolar ducts and sacs increases in a consistent and significant degree with increasing age in mice (Table 15.3). A 3-fold increase in the air volume of the ducts and sacs occurred from age 1 to 28 months. The total volume of the alveolar wall (Table 15.3) was not significantly changed from 1 to 9 months. In older animals (19 months), alveolar wall volume was significantly greater than in younger animals. An increase in total alveolar surface area (Fig. 15.3) in combination with a continual increase in the total air volumes of the alveoli, ducts, and sacs (Table 15.3) strongly

suggest that the aging process in the mouse lung results in a hyperinflated lung with significant airspace distention. Although there is some evidence for the loss of alveolar surface area in SAM mice with increasing age, there is no loss in alveolar surface area in the BALB/c mouse during the aging process. Interalveolar pores form a communication between adjacent alveoli. Changes in the size and frequency of these pores are best detected using scanning electron microscopy on critical point dried lung tissues. In mice, alveolar pores or wall fenestrations increase in frequency with age. At 1 month, these pores are relatively sparse but increase in size with age. The frequency of interalveolar pores per alveolus varies, depending on location within the lung parenchyma. TM Subpleural and peribronchiolar regions appear to have alveoli that contain higher numbers of interalveolar pores compared with parenchymal tissues in other regions of the lungs. The number of pores per alveolus more than doubles between 1 month and 28 months of age (Fig. 15.4). The total area of interalveolar pores in the aging BALB/cNNia

Fig. 15.3. Alveolar surface area in aging strains of mice. (Reproduced with permission from Pinkerton KE, Cowin LL, Witschi H. Development, growth, and aging of the lungs. In: Mohr U, Dungworth DL, Capen CC et al. (eds) Pathobiology of the Aging Mouse (Volume 1), 1996, pp. 261-72. Washington, DC: ILSI.)

Fig. 15.4. Changes in the number of interalveolar pores per alveolus with aging in the BALB/cNNia mouse. (Reproduced with permission from Pinkerton KE, Cowin LL, Witschi H. Development, growth, and aging of the lungs. In: Mohr U, Dungworth DL, Capen CC et al. (eds) Pathobiology of the Aging Mouse (Volume 1), 1996, pp. 261-72. Washington, DC: ILSI.)

mouse increases throughout life. From I month to 28 months of age, the total area of alveolar pores increases more than 4-fold (Table 15.4). A rapid increase in the number of interalveolar pores early in life has been described in both mice is and dogs. 26 Interalveolar pores are extremely rare during the first 10 days of life in mice, but rapidly increase in number and size after day 14. 27 The increase in the total area of interalveolar pores, as well as the number of interalveolar pores per alveolus, corresponds to the increase in total alveolar surface area during the lifespan of the mouse. Rapid enlargement of the lungs after birth may account in part for the formation of some pores; however, degenerative processes during aging may also be responsible for an increase in the total interalveolar pore surface area as well as the number of interalveolar pores per alveolus. Pore enlargement may occur as a result of the rupture of tissue strands between adjacent pores, especially in the senescent animal. A process of fenestration in the lungs has been described that would allow

these tissues to attenuate and rupture between intervening capillaries. 28 This process could also occur in the aging mouse lungs. The proportion of alveolar wall formed by pores is about 3.5% at 1 month, 5.9% at 2 months, and 8.5-9% in mice older than 9 months. A decrease in pulmonary elasticity with aging has been verified physiologically. The morphological and chemical basis for changes with age in elastic tissue and the organization of the elastic fiber network throughout the lungs and alveoli is not well understood. A number of investigators have examined elastin content in aging mouse lungs by morphology through measurements of total fiber length. 5'17'22 They found aging occurs in the absence of changes in total elastic fiber length, despite an increase in pulmonary volume (Tables 15.2 and 15.3). However, if elastic fiber length is normalized to pulmonary volume, an age-related decrease in elastic fibers was noted in BALB/c mice. 17 This decrease in elastic fibers was associated with an increase in the static compliance of excised lungs of aged mice, due to a progressive loss of elastic recoil pressure. If an increase in pulmonary compliance could occur without the destruction of elastic fibers, an increase in the total elastic fiber length would be expected) However, biochemical analysis of elastic tissue content demonstrated a loss of elastic fibers in aging lungs. During biochemical analysis, special care must be taken to separate pseudoelastin, a form of elastin in human lungs that increases with age, and elastin, or the actual amount of elastin will be overestimated. Pseudoelastin has not been found in mice. Therefore, it has been suggested that this absence of pseudoelastin fibers accounts for the decrease in elastin content in the aging BALB/c mouse lung. 5 Studies of histological changes in lung elastic fibers in SAM mice have found no evidence of destruction of the alveolar wall or elastic fibers during aging. Physiological studies have also demonstrated lung compliance in these mice at age 10 months to be significantly greater than that at 2 months of age. 22 Therefore, age-related changes in lung distension in SAM occur in a manner similar to that noted for other strains of mice. In contrast, for mice of the SAMP/I strain, changes in lung hyperdistention occur in an accelerated manner compared with the resistant strain (SAM-R/l) or other mouse strains. Therefore, SAM-P/I strain of mice could prove useful in the study of senilerelated lung hyperinflation.

Macrophages and lung aging (mouse) Aging is thought to be associated with a decline in immune function. Changes in macrophage function can be an important component in compromised lung defense. Phagocytic cells collected by bronchoalveolar lavage are useful to study the characteristics of these cells. In comparing C57BL/6 mice of differing ages, the number of cells recovered by bronchoalveolar lavage was found to be greater at 26 months than at 1.5 months (Table 15.5). 29 The cell differentials at both ages were similar, with 95-96% of macrophages recovered from the lungs. Neutrophils represented a small fraction (0.1%) in the youngest animals and a slightly higher fraction in older animals (1.8%). Lymphocytes in both young and old animals were approximately 2%. This slight increase in the number of neutrophils and lymphocytes within the lungs of aged animals compared with young animals may reflect a subtle change in the immune system of these animals or the need for greater numbers of cells in old versus young animals to maintain the proper sterility of the lungs. The difference between young and old mice in the numbers of cells recovered by bronchoalveolar lavage may also be a simple reflection of the aging process, with greater numbers of phagocytic cells present in the lungs of old mice. The ability of cells to phagocytize particles has been tested, and has shown that a greater proportion of cells from 26-month-old mice were unable to phagocytize latex spheres than cells from lungs of 1.5-month-old mice. 29

Antioxidant enzyme activity in the aging mouse Antioxidant enzymes are an important means of protecting cells from damage due to gases and particles that have the oxidizing capacity to alter vital cellular components. Superoxide dismutase (SOD), catalase (Cat), and the glutathione (GSH) system all serve to protect against the toxic effects of oxidants. GSH in sufficient concentrations can adequately detoxify oxidants through conjugation. However, if glutathione concentrations become depleted, toxic intermediates can form and injure pulmonary cells. Pulmonary glutathione

concentrations have been determined over the lifespan of male C57BL/6 mice, at ages of 3, 6, 12, 26, and 31 months. GSH concentrations decreased by 30% in the lungs of aged mice, whereas GSSG (oxidized glutathione) cysteine and cystine concentrations remained unchanged. Depletion of pulmonary GSH by injection of acetaminophen demonstrated that young (3--6 months) and mature (12 months) mice recovered hepatic GSH levels more efficiently than senescent mice (31 months), but no differences were noted in the lungs. 3~ These findings suggest that detoxification capacity decreases as age increases in the mouse. However, no information is available regarding cell numbers or cell types responsible for maintaining GSH concentrations in the lungs of mice during the aging process.

Conclusions for lung aging in the mouse Little information is available to adequately describe the effects of aging in mouse lungs. Although changes in air space and tissue volumes and alveolar surface area with aging are known, less is known for cell populations lining the lung airways or forming the alveolar structures of the pulmonary parenchyma over the lifespan of the mouse. A number of studies have implied that lung aging in mice is associated with decreases in specific functional and structural parameters. These include increases in the phagocytic cell populations present in the lung airspaces, but decreased ability to engulf foreign particles. Decreases in antioxidant defense systems have also been noted in the lungs of aging mice. From a structural perspective, hyperinflation of the lungs and increases in interalveolar pore size and number are key features of the lung aging process in mice. Future knowledge of changes in pulmonary cell number, type, distribution, and function with aging would greatly increase our understanding of their impact on pulmonary physiology, metabolism, and immunity in the mouse. Age-related changes could significantly affect the normal function of the lungs as well as greatly increase host susceptibility to injury.

TH E RAT Lifespan characteristics of the rat As previously mentioned, aging has been examined in a number of rat strains, but the two most commonly used for lifetime studies are Sprague-Dawley and Fischer 344 rats. The lifespan characteristics of the Fischer 344 rat have been well-documented. 4'31-38 The majority of rats and mice maintained under barrier conditions show no gross pathological changes of the lungs. The pathology of nasal, laryngeal, tracheal, and respiratory systems that may be associated with aging in the rat has been reviewed. 39

The aging rat lung Development of the respiratory system in the rat has been analyzed morphometrically during early postnatal life 1-3 but few studies have quantitatively examined structural changes related to aging. Descriptions of age-related alterations in

the respiratory system which do exist 4 have focused almost exclusively on the gas exchange portions of the lungs. Less information is available on cellular and structural changes in the tracheobronchial airway tree with age with the exception of the postnatal changes in the nonciliated bronchiolar epithelial (Clara) cell. 2'3 The majority of data related to lung aging in the rat are confined to brief descriptive observations of control animals in lifetime toxicity studies. 7'8

The tracheobronchial tree and epithelium of the aging rat The tracheobronchial epithelium of adult rats varies in terms of the types of cells present and the relative proportions of specific cell types throughout the conducting airway tree. In the trachea, four cell types have been identified: basal cells, serous cells, ciliated cells, and mucous goblet cells. 4~ In contrast, the epithelium of bronchi and bronchioles consists of ciliated cells and nonciliated bronchiolar epithelial (Clara) cells. 4~ The composition of epithelial cells also varies from proximal to distal airway generations with postnatal development. 4~ The transformation of tracheal epithelial cells in the rat from the perinatal period through to adulthood appears to be continual. At birth, the rat trachea contains some mature ciliated cells, obvious secretory cells, and the beginning of basal cell differentiation. 4~ Ciliogenesis begins at about 80% gestation in the rat. Nonciliated secretory cells are obvious in the tracheal epithelium at about 90-95% gestation. We have recently evaluated the general characteristics of the airway epithelium in the aging rat. Epithelial cells were examined in three different airway generations of the left lung of male Fischer 344 rats at 5 and 22 months. 45 Fig. 15.5 shows the 3 airway levels selected (A, B, and C) which are designated as cranial, central, and caudal bronchi, respectively. At each site, ciliated, non-ciliated and basal cells were identified, and the average volume density (cell volume/basal lamina surface area) of each cell type determined (Table 15.6). No significant differences in the

Fig. 15.5. Location of tissue samples taken from the rat lung. (A) Silicone cast of the tracheobronchial airway tree. Lung casts served to standardize sampling. Samples of terminal bronchiole-alveolar duct junctions were taken from the three regions (cranial, central, and caudal) indicated with letters and arrows within the figure. (B) Mediastinal half of a fixed, microdissected rat lung. Note how closely the pathways and sampling regions match the silicone cast shown above. The same level of airway in each figure is designated by A, B and C. (Reproduced with permission from Pinkerton KE, M~nache MG, Plopper CG. Consequences of prolonged inhalation of ozone on F344/N rats: collaborative studies, Part IX. Changes in the tracheobronchial epithelium, pulmonary acinus, and lung antioxidant enzyme activity. Health Effects Institute Research Report No. 65, 1995, pp. 41-98.)

abundance of each cell type were noted between 5 and 22 months; total epithelial cell volume also remained constant. No appreciable differences were noted between each of the three bronchial regions examined. These findings suggest that few changes occur during aging, with a lack of significant shifts in the proportion of epithelial cell types in the tracheobronchial tree of the aging rat. However, far less is known regarding changes in the molecular and biochemical functions of cells with aging.

Lung parenchymal structure in the aging rat Total alveolar airspace volume of the lungs progressively increases over a 2-year period of growth in Fischer 344 rats (Fig. 15.6). Airspace volume was measured in lungs fixed following airway instillation of 2% glutaraldehyde at 20 cm H20; 4'48 prior physiological tests showed that the lungs were fixed at 75% of total lung capacity. 4'49'5~The marked increase in air space volume from I week to 5 months of age

Fig. 15;.6. Total airspace volume (cm3) in glutaraldehyde-fixed lungs of male and female Fischer 344 rats. Each point represents the mean (+ SD) of four animals. Asterisks indicate significant changes between consecutive age groups (p < 0.05). (Reproduced with permission from Pinkerton KE, Vincent R, Plopper CG et al. Normal development, growth, and aging of the lung. In: Mohr U, Dungworth DL, Capen CC (eds) Pathobiology of the Aging Rat (Volume 1), 1992, pp. 97-109. Washington, DC: ILSI.)

was associated with a significant increase in alveolar number. The peak rate of formation of alveoli in the neonatal rat has been estimated to be 1000 per minute. 51 Light microscopic examination of large airways, terminal bronchioles, large blood vessels, alveoli, and alveolar septa demonstrated no detectable differences from 5 to 26 months of age. Rats aged 26 months appeared to have slightly enlarged alveolar ducts compared with younger animals, but otherwise were indistinguishable from the younger animals (Fig. 15.7). The proportion of the lungs forming the lung parenchyma was 0.81 to 0.82 in young and old animals, respectively. 1'4 Epithelial and capillary surface areas of the gas exchange regions of the lungs are given in Table 15.7. Between 1 and 6 weeks of age the surface area formed by epithelial type I cells increased 6-fold, while epithelial type II cell surface area increased 3-fold. The squamous type I cell covered more than 95% of the total alveolar surface, while the cuboidal type II cell covered the remaining 5%. The alveolar type III cell is a rather rare epithelial cell type within the alveoli and contributes very little to the total alveolar surface area; they are found most frequently within alveolar regions near bronchiole-alveolar duct junctions. 42 From 6 weeks to 5 months of age alveolar surface area almost doubled. From 5 to 26 months, total alveolar surface area remained unchanged. Since airspace size increased by 50% from 5 to 26 months in the absence of any significant change in alveolar surface area, alveolar duct enlargement in older animals could explain, in part, how airspace volume increased without a loss of surface area within the lung parenchyma. Enlargement of alveolar ducts was not quantitatively confirmed, but emphysematous changes (alveolar wall destruction) appeared to be absent in the aging Fischer 344 rat. 35'52'53 Changes in capillary surface area with age were proportional to changes in alveolar surface area (Table 15.7). The capillaries within the alveolar septa of the neonatal rat undergo a fascinating transformation from a double capillary system to a single capillary system associated with the growth of secondary alveolar septa to form new alveoli. This reorganization of the pulmonary vasculature is complete by 3 weeks of age. 1'4 From 5 to 26 months of age the

Fig. 15.7. Photomicrographs of the lung parenchyma from Fischer 344 rats aged (A) 5 months and (B) 26 months. A slight increase in alveolar size can be noted in the lungs of 26-month-old rats compared with 5-month-old rats. Scale bar is 100 ~tm.

total surface area of the capillary bed remained unchanged in both male and female rat lungs (Table 15.7).

Alveolar tissue compartments in the aging rat During the first months of life in the rat, alveolar tissue volumes increase dramatically. However, little change is noted in tissue volumes from 5 to 26 months, with the exception of the noncellular component of the interstitium (Table 15.8). The volume of the capillary bed also increases dramatically during the first 5 months of life, but remains relatively unchanged from 5 to 26 months. Alveolar tissue volumes for epithelial, interstitial, and endothelial compartments of the lung parenchyma are presented in Table 15.8. Cell number for each major alveolar cell type is given in Table 15.9 and morphometric characteristics are presented in Table 15.10. The lung parenchyma consists of three tissue compartments, the epithelium, the interstitium, and the endothelium. Alveolar macrophages form a unique tissue compartment of individual cells which freely migrate along the surfaces of alveoli and airways and which are also present within the pulmonary connective tissues as interstitial macrophages. The composition of the alveolar epithelium in the rat changes dramatically during postnatal development (Table 15.8). The volume of type I epithelium increased more than 6-fold from 1 week to 6 weeks, while the type II epithelium displayed a more modest 3-fold increase during the same period. The total surface area of type I and type II cells also increased more than 5-fold from 1 to 6 weeks (Table 15.8). The ratio of type II to type I cells in the lungs at 6 weeks was 1.8, but it decreased to 1.0 by 26 months 4 (Fig. 15.8). The significance of the reduction in type II to type I cell ratio is unknown, but it may contribute to an

altered secretory response in type II cells of old rats since a greater surface area must be served per type II cell to form the surfactant lining layer of the lungs compared with that in younger animals. The type II cell undergoes dramatic changes during the perinatal period. Just prior to birth, the rat type II cell is heavily laden with glycogen and first acquires its characteristic lamellar bodies 48-72 h prior to parturition. From the last day of gestation to a few hours later, the cellular content of glycogen drops from about 10% to zero. On the day of birth the cells have polarized their secretory granules (lamellar bodies) towards the basal pole. The putative immediate precursor of the lamellar body contains eccentrically placed vesicles in addition to slips of phospholipid lamellae and is termed a composite body. These are polarized toward the basal side of type II cells throughout the life span of the animal, but the mature lamellar bodies become randomly distributed between 2 and 6 weeks. 54 In contrast, the Clara cell has its secretory granules polarized toward the apical region like many other secretory cells in the body. Another interesting polarization of type II cell intracellular organelles is that of the light and dark multivesicular bodies. Light multivesicular bodies are not rich in lysosomal enzyme and accumulate endocytosed membrane markers most quickly, like endosomes do in other cells. Dark multivesicular bodies are rich in lysosomal enzymes and are basally polarized like composite bodies. There could be functional differences between the two multivesicular body types, but more direct experimental evidence is needed to define them. Type II cellular composition between birth and adulthood differs mainly by a doubling of the volume density of the lamellar bodies and a 50% increase in mitochondrial

Fig. 15.8. Changes in the ratio of alveolar type II cell number to alveolar type I cell number in the lungs of aging male and female Fischer 344 rats. Each datum point represents the mean + SEM of four animals. Asterisk on the line connecting two age groups denotes a significant change in this ratio (p < 0.05). (Reproduced with permission from Pinkerton KE, Barry BE, O'Neil JJ et al. Morphologic changes in the lung during the life span of Fischer 344 rats. Am. J. Anat. 1982; 164:155-74.)

volume density. Neither the appearance nor the size distribution of lamellar bodies changes during postnatal life, but dramatic changes do occur in mitochondria. They shift from an isolated spheroid form to a highly branched interconnected web in the adult cell (Fig. 15.9). The significance of such a shift is unknown but very similar changes occur in other phyla (such as insect flight muscle) and may accompany cell division. Although the notion is untested, it is easy to see how division of the mitochondria would be simplified by the fetal disconnected form. The adult mitochondria have only a small surface area-volume ratio advantage over the spheroid form and it seems unlikely to be the entire advantage of that shape. It seems possible that the extended shape would allow an entire mitochondrion to respond to very focal cellular changes in high energy phosphates.

Fig. 15;.9. Computer-assisted three-dimensional reconstruction of the mitochondria from an alveolar type II cell within the lungs of a 1-day-old neonate (A) and an adult (B) Sprague-Dawley rat. The globular appearance of individual mitochondria in the neonate is in striking contrast to the filamentous branching mitochondria of the adult. (Reproduced with permission from Pinkerton KE, Vincent R, Plopper CG et al. Normal development, growth, and aging of the lung. In: Mohr U, Dungworth DL, Capen CC (eds) Pathobiology of the Aging Rat (Volume 1), 1992, pp. 97-109. Washington, DC: ILSI.)

One more key change occurs in type II cell morphology during postnatal development. At birth, each type II pneumocyte has as many as 200 cytoplasmic extensions, which perforate the basement membrane, and these are termed foot processes. A fraction of these have close apposition with interstitial lipofibroblasts. Although there is an electron-dense cytoplasmic condensation, there is no intra-gap substructure on tilted sections to suggest a true gap junction. Occasional profiles from serial sections show cytoplasmic bridges between the epithelial type II cell and the mesenchymal lipofibroblast. The function of these three structures is unknown but is widely speculated to be related to epithelial mesenchymal interactions. They can be induced with corticosteroid administration in the perinatal period, and decrease by 20-fold in number with maturation of the lung. In response to injury resulting in interstitial pneumonitis in humans, the foot processes proliferate. Over the lifespan of the Fischer 344 rat, the absolute volume of the cellular interstitium of the lung parenchyma does not change (Table 15.8). Interstitial cell number (Table 15.9) and cell size (Table 15.10) were similar at 1 week and 26 months, although the types and ratios of interstitial cell types forming the parenchyma cell pool were markedly different in neonatal pups compared with adult rats (Fig. 15.10). 55 In contrast, the noncellular components of the interstitium demonstrated dramatic volume changes from 1 week to 5 months of age, increasing approximately 10-fold in total volume. From 5 to 26 months of age interstitial volume continued to change at a lower, but significant rate (Table 15.8). Compared to 5-month-old rats, the interstitial matrix increased 18% by 14 months of age and 39% by 26 months (Fig. 15.11). Females also demonstrated a 36% increase in interstitial matrix volume

Fig. 15.10. Upper micrograph: An alveolar septum from the lungs of a 1-week-old Fischer 344 rat. The alveolar surface is covered by type I (I) and type II (11)epithelial cells. Note the presence of capillaries (C) on both sides of the septum and numerous lipid-containing cells (arrow) in the interstitium (bar is equal to 3 lam). Lower micrograph: An alveolar septum from the lungs of a 6-week-old Fischer 344 rat (bar is equal to 3 ~m). (Reproduced with permission from Pinkerton KE, Barry BE, O'Neil JJ et al. Morphologic changes in the lung during the life span of Fischer 344 rats. Am. J. Anat. 1982; 164:155-74.)

from 81 ml at 5 months; a further 89% increase in matrix volume occurred by 26 months. These changes could be a result of two different conditions: (a) pulmonary edema and (b) an increase in the noncellular matrix components of the lung such as collagen or elastin. Changes in the constituents of the interstitial matrix at the level of the proximal alveolar region (PAR) have been examined in Fischer 344 rats. 56 The PAR consists of alveolar tissue sampled in a perpendicular orientation relative to the axis of the terminal bronchiole, approximately 300-400 ~tm down the alveolar ducts. The overall characteristics of the alveolar septum in this region were similar to those of the more distal parenchyma. Morphometric analysis of the PAR revealed that the increase in interstitial matrix volume in the parenchyma could be attributed almost entirely to a thickening of basement membranes and the deposition of collagen fibers. Basement membrane thickness went from

40-45 nm at 4-6 months to 75-80nm at 20-24 months. Similarly, collagen fiber volume, normalized to the surface of the alveolar epithelium, increased by more than 100% during this same period. Basement membrane volume and collagen fiber volume were similar in the proximal alveolar regions and accounted for 50% of the noncellular matrix at 4-6 months and 80% at 20-24 months. By comparison, no change was noted in the relative volume of elastin and remaining acellular space. The volume ratio of collagen fibers to elastin fibers shifted from 3 to 5 in young adults (4-6 months) to 10 in the older animals (20-24 months). The same collagen to elastin fiber ratio was found in Sprague-Dawley rats as in young Fischer 344 rats. 57 It appears that the volume of the ground substance, relative to epithelial surface, is not significantly modified in the lungs of the aging Fischer 344 rats. Therefore, it is unlikely that edema contributes significantly to the increase

Fig. 15.11. The alveolar septum from the lungs of male (upper micrograph) and female (lower micrograph) Fischer 344 rats 26 months of age. A prominent interstitial matrix space (*) is present in the septum. Some cytoplasmic extensions of interstitial cells are also present. Collagen is present within the matrix. In spite of the increase in the interstitial matrix, a thin air-to-blood tissue barrier is still maintained. Magnification is the same as in Fig. 15;.10. (Reproduced with permission from Pinkerton KE, Barry BE, O'Neil JJ et al. Morphologic changes in the lung during the life span of Fischer 344 rats. Am. J. Anat. 1982; 164:155-74.)

in interstitial matrix volume with age. The absence of detectable changes in the volume of elastin is consistent with its low rate of synthesis and slow turnover in adult animals. 58'59 A progressive thickening of basement membranes may be explained by epithelial and endothelial cell turnover if the cells lay down basement membrane material during each cell cycle, and possibly also during interphase. The increase in the volume of collagen fibers documented morphometrically in adult rats confirms our previous morphologic observations 4 and is in agreement with biochemical studies of collagen in the lungs of Lewis rats 6~ and Fischer 344 rats. 62 Morphologic and biochemical evidence supports the notion that there is a continuous deposition of mature cross-linked extracellular collagen in the lung parenchyma of aging rats which can be interpreted as an age-related excess of collagen (or fibrosis). However, it should be noted that qualitative examination revealed no visible change in the overall gross architecture of the matrix in older animals

compared to younger rats. Although speculative, it is conceivable that the dominant stimulus for collagen fiber deposition, through life, is the stress and strain exerted on fibers and translated to fibroblasts. Following this concept, fibroblasts may add fibrils to the existing network while maintaining the general spatial relationship between collagen fibers and other tissue components, resulting in a net enrichment in collagen. It is not clear how this increase in collagen mass and the shift in the ratio of collagen to elastin with age impact on the micro mechanical behavior of the lungs. A logical assumption would be stiffer lungs, but physiologic measurements suggest otherwise. 4'53 Threedimensional reconstructions in concert with physiologic measurements 57'63 could specifically address this issue in the senescent rat lung. An interesting feature of senescent lungs in the Lewis rat 61 is an abrupt 4-fold decrease in the measured rate of gross collagen synthesis between 15 months and 24 months. More than 80% of newly synthesized collagen is apparently

degraded intracellularly in 15-month-old animals and 60% degraded intracellularly at 24 months. The net result is only a small fraction of product being deposited as extracellular cross-linked collagen. It was suggested that maintenance of a high rate of collagen synthesis provides an adaptive capacity that allows the fibroblast to rapidly redirect the procollagen from intracellular degradation pathways towards secretory pathways in response to injury or pathologic insult. 61 In the Fischer 344 rat there was no decline in fibroblast populations in aging lungs. 4 If these observations 61 can be extended to other strains of rats, then a fall in the rate of collagen synthesis may constitute an intrinsic characteristic of the senescent lung fibroblast in vivo, resulting in a compromised ability to repair collagen fibers. If these assumed dynamics of the lung interstitium prove to be true, there are specific and profound metabolic changes that accompany aging which need to be understood to more fully appreciate the nature of the morphologic and functional alterations seen in normal aging and as a consequence of exposure to toxicants in the aged lung. 64 Acute toxicity studies are seldom performed in senescent animals, while chronic studies are usually not extended through to the last third of the lifespan due to low survival rates. Therefore, our present understanding of age-related interstitial changes is severely limited. The endothelium forms the third major tissue compartment of the alveolar septum. The volume of the endothelium in the lungs of Fischer 344 rats increased more than 3-fold from 1 to 6 weeks of age. Beyond 6 weeks of age, total endothelial cell volume did not increase significantly through to 26 months (Table 15.8). The total surface area of the capillary endothelium, like the epithelial surface, demonstrated nearly a 10-fold increase from 1 week to 6 weeks. From 6 weeks to 5 months, capillary surface area increased by an additional 20%. From 5 months to 26 months, the surface area of the capillary endothelium did not change significantly. Endothelial cell volume and surface area were relatively unchanged in male and female rats from 5 months to 26 months of age (Tables 15.7 and 15.8).

Alveolar macrophages in the aging rat Alveolar macrophages form an important defense against inhaled particulates and pathogens in the lungs. In 1-weekold rats, there were approximately 2 million macrophages, and these increased to 9 million by 6 weeks; numbers increased to 20 million by 5 months of age. Although no further increases in alveolar macrophage number were evident at 26 months (Table 15.10), this population of cells is highly dynamic and numbers can change rapidly by recruitment of monocytes and macrophages from the interstitium and the blood 65 and by the in situ proliferation of cells within the lung airspaces. 66

Conclusions for lung aging in the rat The dynamics of lung growth, development, and aging in the rat is a continuous process that involves every tissue compartment of the lungs. Significant changes in aging

adult rats are primarily within alveolar type II cells and the noncellular portions of the interstitium. Because such changes may influence the response of the lungs to inhaled chemical agents and dusts, the age of the rat should be considered in the evaluation of any experimental study. Different responses of the lungs to inhaled pollutants have been noted in young versus old r a t s . 64'67 Such differences may be due to changes within target cell populations and/or alterations in the functional status of cells through the aging process. Although cellular changes are most prominent during postnatal growth and development, modifications in cells continue up to advanced age. Changes in cell number, size, and function associated with aging are likely to impact on lung physiology, metabolism, and immunity. Such changes could significantly alter the normal functions of the lung and its susceptibility to injury. Therefore, an understanding of the aging process in the rat is essential to the evaluation and interpretation of chronic (lifetime) exposures to a variety of substances that present a potential health risk to all mammalian species.

TH E D O G General characteristics of the lungs in aging dogs Morphological changes in the lungs during aging have been studied most extensively in the dog. As discussed above, a strong allometric relationship exists between body mass and several features of the respiratory tract. Table 15.11 illustrates a number of these morphometric characteristics of the lungs for dogs of differing body mass and age. Changes in alveolar size, enlargement of respiratory bronchioles and alveolar ducts, and the accumulation of anthracotic pigment are all common features of the aging process in the dog lung. In a study of the lungs of 20 beagle dogs, aged < 1 year to 10 years, it was found that the primary lesion in older dogs was large accumulations of pulmonary macrophages containing dust and pigment in the walls of respiratory bronchioles and at the mouth openings of alveoli into alveolar ducts. 68 It was observed that these dustladen macrophages became more prominent with increasing age. Focal pneumonitis was also frequently associated with the accumulation of macrophages. It was further noted that the volume of the lungs occupied by alveolar ducts increased. 68 In the airways, aging was associated with larger submucosal glands and a greater degree of calcification of bronchial cartilage. Due to the typical outdoor habitat of dogs, the accumulation of macrophages loaded with dusts and anthracotic pigment is to be expected with age. Although not studied, impaired clearance of particles may also occur with increasing age, leading to greater retention of pigment with age. The relationship of each of these lung features with age were further analyzed using linear regression to determine which parameters were significantly associated with the aging process. 68 Tissue sections taken from a total of 33 blocks from both lungs of each animal were examined without

knowledge of the dog's identity or age. Scores were based on the degree of change relative to the youngest animals observed. Significant age-related changes were identified as increases in pigment surrounding respiratory bronchioles and alveolar duct mouth openings, enlargement of lumenal size of respiratory bronchioles and alveolar ducts, increased abundance of airway submucosal glands, and bronchial calcification of cartilage with age. Two features not changed with age were subpleural airspace size and the degree of focal pneumonitis. 68

The tracheobronchial tree of the aging dog Little information is available to describe or quantify the aging process of the trachea or conducting airways in the lungs of dogs. It is known that body mass is proportional to the dimensions of the trachea in terms of its length to diameter ratio. In most mammalian species, this ratio is approximately 8:1. Compared to other species, the trachea and central airways of the dog are slightly larger due to their need of dissipating body heat by panting. The relatively large central airways also minimize impedance produced by rapid shallow breathing. Age-related alterations of the bronchi and the non-respiratory bronchiole are unknown with the exception of age-related calcification of cartilaginous plates within the bronchi as well as hypertrophic changes of the submucosal glands. 68 No changes have been described for the non-respiratory bronchioles of dogs with aging. The most peripheral airways in the lungs of dogs are formed by respiratory bronchioles and alveolar ducts. In a study of mucus velocity in the trachea of dogs aged from 1 to 15 years,73 it was found that the velocity increased during the first few years of life, followed by a gradual age-related

reduction beginning around 4 years of age (Fig. 15.12). With extrapolation of equivalent dog years to human years, a close correlation has been found between dogs and humans, with similar patterns of decline in tracheal mucus velocity rates with increasing years during adult life. No information is available to correlate changes in flow rates with cellular and/or functional parameters in epithelial cells lining the trachea. Although extensive studies have examined the branching pattern of the tracheobronchial tree in dogs, 74'75 only one study focused on postnatal growth. TMIn that study, resin casts were made of the bronchial tree of 4 Labrador dogs weighing 0.5, 3.4, 7.5, and 30 kg; TM the smallest dog was one week of age, while the ages of the other dogs were not given. The branching pattern of the lungs was ordered by number, mean diameter, and mean length of branches in each generation; these were plotted by order and expressed according to diameter and length. It was found that the diameter and length of similar airway orders ran in a parallel fashion for dogs of varying size. If these measurements were normalized to body weight, they were found to be identical. This correction factor was equal to the cube root of body weight. These observations provide strong evidence that airway branching morphogenesis of the bronchial tree of the dog is complete at birth and postnatal growth reflects a simple expansion of the length and diameter of each airway generation in the absence of any further airway branching. Lung parenchymal structure in the aging dog To define morphological changes of the aging canine lung, the parenchymal characteristics of the lungs of 14 beagle

Fig. 15.12. Tracheal mucus velocity (mean and SEM) for beagle dogs versus age. The fitted function that describes this relation is V(t)= 11 [1/2 exp (-0.9t)] - 0.6t. The available data for humans are also shown after transforming for age. (Reproduced with permission from Pinkerton KE, Murphy KM, Hyde DM. Morphology and morphometry of the lung. In: Capen CC, Benjamin SA, Hahn FF et al. (eds) Pathobiology of the Aging Dog (Volume 1), 2001; 5:43-56. Iowa State University Press, Ames.)

dogs have been analyzed morphometrically. 6 These dogs, which were also used in another study, 68 ranged from 1 to 10 years of age. The volume and surface densities of tissues and air, the volumes of alveoli, alveolar ducts, and alveolar sacs (i.e. endings of the alveolar duct), as well as the number of alveoli per unit volume of lung parenchyma were measured. It was found that the volume density of alveoli decreased with age, while the volume density of alveolar ducts and sacs increased with age. The number of alveoli per unit volume also decreased with age. There was no correlation between the numerical density of alveolar ducts and alveolar sacs with age. Alveolar tissue density also decreased with age. Multiple regression analysis was also used to determine the relationship between stereological parameters of the lung parenchyma, body weight, age, and diffusion capacity in the dog. 6 Analyses were repeated on males and females, as well as the total population. There were no significant multiple correlations among stereological lung parameters, diffusing capacity, body weight, and age. No sex-related differences in the slope of the regression lines relating to morphometric parenchymal lung values and age were noted. In the earlier study of these dogs 68 a significant agerelated increase in alveolar duct profile area was observed; minimal emphysema was also seen, with only occasional signs of fibrosis associated with focal pneumonitis. These observations were confirmed by the later morphometric measurements, 6 showing an increased volumetric density of alveolar ducts associated with decreases in the volumetric density of alveoli and parenchymal tissue, as well as decreases in the numerical density of alveoli, and the surface density of parenchymal tissues. These observations strongly correlate with a prevalence of lung hyperdistention, or ductasia, in the aging process for dogs. Similar findings have also been noted in the human lung, without a significant decrease in the number of alveoli. In ductasia, those alveoli adjacent to enlarged alveolar ducts in respiratory bronchioles decreased in depth, thus maintaining a constant number of alveoli within the lung. 6 The observed decrease in surface density in the lungs of dogs with age can be explained by an increase in lung volume, a loss of interalveolar septa, a rearrangement of the geometry of the lung by alveolar flattening and duct enlargement, or a combination of any of these anatomical changes. Similar changes have been observed in aged human lungs, with a geometric rearrangement of alveoli in alveolar ducts, resulting in an increase in the average interalveolar septal distance. The total alveolar surface area for the lungs decreased as a result of an increase in the average interalveolar septal distance rather than an increase in lung volume. In these dogs, no significant change in total lung capacity with age was observed. 6s The reduction in volume density of alveolar parenchymal tissue could be interpreted as a loss of interalveolar septa. This type of reduction has also been observed in human lungs. Cross collateral ventilation of the lungs within the parenchyma is accomplished in part through communications formed by alveolar pores, as discussed above. A number of studies have examined the alveolar

Fig. 15.13. Average diameter of alveolar pores plotted against age. Each point represents one dog. Closed circles are males, and open circles are females. (Reproduced with permission from Pinkerton KE, Murphy KM, Hyde DM. Morphology and morphometry of the lung. In: Capen CC, Benjamin SA, Hahn FF et al. (eds) Pathobiology of the Aging Dog (Volume 1), 2001; 5:43-56. Iowa State University Press,Ames.)

pores during aging in dogs. 6'26 These pores appear to be absent at birth, but become prominent within the first year of life. 26 The average number and size of these pores remain constant through the first 10 years of life (Fig. 15.13). Prolonged exposure to a number of environmental air pollutants has been shown to dramatically increase the number and size of these pores 6 with extensive fenestration of alveolar walls.

Conclusions for the aging dog lung Aging of canine lungs has many similarities to that seen in human lungs, but the aging process in dogs occurs over a shorter time frame than in humans. The accumulation of dust-laden macrophages as well as lumenal enlargement of alveolar ducts are hallmarks of this aging process. Although some tissue thinning and loss of alveolar septal tissues may occur, the increase in lumenal size of alveolar ducts is in large measure due to the stretching and shallowing of alveolar outpocketings within these regions of the lung parenchyma. Changes in the relative velocity of mucus flow in the trachea with aging also parallels changes observed in the aging human trachea. Little is known regarding the canine aging process at the cellular level for either the airways or gas exchange portions of the lungs. However, a significant advantage of studying dog lungs is their similarity to the human lung in both composition and structure. These similarities may offer unique opportunities to better understand the aging process of the lungs for both dogs and humans.

OVERALL

CONCLUSIONS

Aging is a natural process in the respiratory system. A number of similarities have been noted in all species

associated with aging. An increase in the total alveolar airspace volume is a natural consequence of aging with enlargement of alveolar ducts immediately beyond the terminal and respiratory bronchioles. This enlargement is typically seen in the form of ductasia, with stretching and shallowing of alveoli in affected regions. Although some destruction of alveolar tissue walls may be evident in the aging lung, such changes appear to be due to an increase in the size and frequency of alveolar pores connecting adjacent alveoli. An increase in the n u m b e r of phagocytic cells in the lung airspace is also a c o m m o n finding, with less efficient phagocytic properties. Increased collagen deposition with focal increases in the interstitium also appears to be a consequence of the aging process. Metabolic functions of cells are also likely to be compromised in the lungs as aging advances. A n u m b e r of environmental factors are likely to accentuate all of these changes during the aging process with the greatest consequences likely to be asthma, emphysema, and chronic obstructive pulmonary disease. These topics are more fully covered in Chapter 28.

REFERENCES 1. Burri PH, Dbaly J, Weibel ER. The postnatal growth of the rat lung: I. Morphometry.Anat. Rec. 1974; 178:711-30. 2. Massaro GD, Davis L, Massaro D. Postnatal development of the bronchiolar Clara cell in rats. Am. J. Physiol. 1984; 247:C197-203. 3. Massaro GD, Massaro D. Development of bronchiolar epithelium in rats. Am. J. Physiol. 1986; 250:R783-8. 4. Pinkerton KE, Barry BE, O'Neil JJ et al. Morphologic changes in the lung during the life span of Fischer 344 rats. Am. J. Anat. 1982; 164:155-74. 5. Ranga V, Kleinerman J, Sorensen J. Age-related changes in elastic fibers and elastin of lung. Am. Rev. Respir. Dis. 1979; 119:369-76. 6. Hyde D, Orthoefer J, Dungworth D et al. Morphometric and morphologic evaluation of pulmonary lesions in beagle dogs chronically exposed to high ambient levels of air pollutants. Lab. Invest. 1978; 4:455-69. 7. Coleman GL, Barthold SW, Osbaldistan GW etal. Pathological changes during aging in barrier-reared Fischer 344 rats.J. Gerontol. 1977; 32:258-78. 8. Goodman DG, Ward JM, Squire RA etal. Neoplastic and non-neoplastic lesions in aging F344 rats. Toxicol. Appl. Pharmacol. 1979; 48:237-48. 9. Pinkerton KE, Gehr P, Crapo JD. Architecture and cellular composition of the air-blood barrier. In: Parent RA (ed.) Treatise on Pulmonary Toxicology, Comparative Biology of the Normal Lung (Volume I), 1992; pp. 121-8. CRC Press, Boca Raton LA. 10. Gehr P, Mwangi DK, Ammann A etal. Design of the mammalian respiratory system. V. Scaling morphometric pulmonary diffusing capacity to body mass: wild and domestic mammals. Respir. Physiol. 1981; 44:61-86. 11. Pinkerton KE, Cowin LL, Witschi H. Development, growth, and aging of the lungs. In: Mohr U, Dungworth DL, Capen CC et al. (eds) Pathobiology of the Aging Mouse (Volume 1), 1996, pp. 261-72. Washington, DC: ILSI. 12. Boyden EA, Thompsett DH. The postnatal growth of the lung in the dog. Acta Anat. 1961; 47:185-215. 13. Sacher GA, Hart RW. Longevity, aging and comparative cellular and molecular biology of the house mouse (Mus musculus)

and the white-footed mouse (Peromyscus leucepus). Birth defects: Original article series 1978; 14:71-96, March of Dimes Foundation, New York. 14. Banfield AWF. The Mammals of Canada, 1974; p. 438. University of Toronto Press, Toronto. 15. Burger J, Gochfield M. Survival and reproduction in Peromyscus leucopus in the laboratory: viable model for aging studies. Growth Dev. Aging 1992; 56:17-22. 16. Takeda T, Hosokawa M, Takeshita S etal. A new murine model of accelerated senescence. Mech. Ageing Dev. 1981; 17:183-94. 17. Kawakami M, Paul JL, Thurlbeck WM. The effect of age on lung structure in male BALB/cNNia inbred mice. Am. J. Anat. 1984; 170-1. 18. Ranga V, Kleinerman J. Interalveolar pores in mouse lungs. Am. Rev. Respir. Dis. 1980; 122:477-81. 19. Hosokawa M, Takeshita S, Higuchi K et al. Cataract and other ophthalmic lesions in senescence accelerated mouse (SAM): morphology and incidence of senescence accelerated ophthalmic changes in mice. Exp. Eye Res. 1984; 38:105-14. 20. Takeshita S, Hosokawa M, Irino M e t a l . Spontaneous ageassociated amyloidosis in senescence accelerated mouse (SAM). Mech. Ageing Dev. 1982; 26:91-102. 21. Matsushita M, Tsuboyama T, Kasai R etal. Age-related changes in bone mass in the senescence accelerated mouse (SAM): SAM-R/3 and SAM-P/6 as new murine models for senile osteoporosis. Am. J. Pathol. 1986; 125:276-83. 22. Kurozumi M, Matsushita T, Hosokawa Metal. Age-related changes in lung structure and function in the senescenceaccelerated mouse (SAM): SAMP/1 as a new murine model of senile hyperinflation of lung. Am. J. Respir. Crit. Care Med. 1994; 149:776-82. 23. Uejima Y, Fukuchi Y, Nagase T et al. A new murine model of aging lung: the senescence accelerated mouse (SAM)-P. Mech. Ageing Dev. 1991; 61:223-36. 24. Scherle WA. A simple method for volumetry of organs in quantitative stereology. Mikroskopie 1970; 26:57-60. 25. Pack RJ, A1-Ugaily LH, Morris G. The cells of the tracheobronchial epithelium of the mouse: quantitative light and electron microscope study. J. Anat. 1981; 132:71. 26. Martin H. The effect of aging on the alveolar pores of Kohn in the dog.Am. Rev. Respir. Dis. 1963; 88:773-8. 27. Amy RWM, Bowes D, Burri PH et al. Postnatal growth of the mouse lung.J. Anat. 1977; 124:131-51. 28. Pump K. Emphysema and its relation to age. Am. Rev. Respir. Dis. 1976; 114:5-13. 29. Higashimoto Y, Fukuchi Y, Shimada Y e t al. The effects of aging on the function of alveolar macrophages in mice. Mech. Aging Dev. 1991; 69:207-17. 30. Chen TS, Richie JP Jr, Lang CA. Life span profiles of glutathione and acetaminophen detoxification. Drug Metab. Dispos. Biol. Fate Chem. 1990; 18:882-7. 31. Boorman GA, Eustis SL. Lung. In: Boorman GA, Eustis SL, Elwell MR etal. (eds) Pathology of the Fischer Rat, 1990; pp. 339-367. Academic Press, New York. 32. Chesky JA, Rockstein M. Life span characteristics in the male Fischer rat. Exp. Aging Res. 1976; 2:399-407. 33. Jacobs BB, Huseby RA. Neoplasms occurring in aged Fischer rats with special reference to testicular, uterine, and thyroid tumors.J. Natl. Cancer Inst. 1967; 39:303-9. 34. Massaro EJ. Mortality and growth characteristics of rat strains commonly used in aging research. Exp. Aging Res. 1980; 6:219-33. 35. Mauderly JL, Likens SA. Relationships of age and sex to function of Fischer 344 rats. Fed. Proc. 1980; 39:10-91. 36. Rockstein M, Chesky JA, Sussman ML. Comparative biology and evolution of aging. In: Finch CE, Hayflick L (eds) Handbook of the Biology of Aging, 1977; pp. 3-34. Van Nostrand Reinhold, New York.

37. Sass B, Rabstein LS, Madison R et al. Incidence of spontaneous neoplasms in F344 rats throughout the natural life-span. J. Natl. Cancer Inst. 1975; 54:1449-56. 38. Snell KC. Spontaneous lesions of the rat. In: Ribelin WE, McCoy JR. (eds) The Pathology of Laboratory Animals, 1965; pp. 211-302. Thomas, Springfield. 39. Boorman GA, Morgan KT, Uriah LC. Nose, larynx and trachea. In: Boorman GA, Eustis SL, Elwell MR et al. (eds) Pathology of the Fischer Rat, 1990, pp. 315-37. Academic Press, New York. 40. Jeffery PK, Reid LM. Ultrastructure of airway epithelium and submucosal glands during development. In: Hodson (ed.) Development of the Lung, 1977, pp. 87-134. Dekker, New York. 41. Plopper CG, Mariassy AT, Wilson DW et al. Comparison of nonciliated tracheal epithelial cells in six mammalian species: Ultrastructure and population densities. Exp. Lung Res. 1983; 5:281-94. 42. Chang L, Mercer RR, Crapo JD. Differential distribution of brush cells in rat lung.Anat. Rec. 1986; 216:49-54. 43. Cireli E. Elektronenmikroskopische anaivse der priiund postnatalen differenzierung des epithels der oberen luftwege der ratte. Z. Mikrosk. Anat. Forsch. 1966; 41:132-78. 44. Kober HJ. Die lumenseitige oberflache der rattentrachea wiihrend der ontogenese. Z. Mikrosk. Anat. Forsch. 1975; 89:399--409. 45. Pinkerton KE, Weller BL, M6nache MG et al. Consequences of prolonged inhalation of ozone on F344/N rats: collaborative studies. Part XIII. A comparison of changes in the tracheobronchial epithelium, and pulmonary acinus in male rats at 3 and 20 months. Health Effects Institute Research Report No. 85, 1998, pp. 1-32. 46. Pinkerton KE, M6nache MG, Plopper CG. Consequences of prolonged inhalation of ozone on F344/N rats: collaborative studies, Part IX. Changes in the tracheobronchial epithelium, pulmonary acinus, and lung antioxidant enzyme activity. Health Effects Institute Research Report No. 65, 1995, pp. 41-98. 47. Pinkerton KE, Vincent R, Plopper CG et al. Normal development, growth, and aging of the lung. In: Mohr U, Dungworth DL, Capen CC (eds) Pathobiology of the Aging Rat (Volume 1), 1992, pp. 97-109. Washington, DC: ILSI. 48. Crapo JD, Peters-Golden M, Marsh-Salin J etal. Pathologic changes in the lungs of oxygen-adapted rats. A morphometric analysis. Lab. Invest. 1978; 39:640-53. 49. Takezawa J, Miller FJ, O'Neil JJ. Single-breath diffusing capacity and lung volumes in small laboratory mammals. J. Appl. Physiol. 1980; 48:1052-9. 50. Hayatdavoudi G, Crapo JD, Miller FJ et al. Factors determining degree of inflation in intratracheally fixed rat lungs. J. Appl. Physiol. 1980; 48:389-93. 51. Randell SH, Mercer RR, Young SL. Postnatal growth of pulmonary acini and alveoli in normal and oxygen-exposed rats studied by serial section reconstructions. Am. J. Anat. 1989; 186:55-68. 52. Liebow AA. Summary: biochemical and structural changes in the aging lung. In: Cander L, Moyer JH (eds)Aging of the lung, 1964; pp. 97-104. Grune and Stratton, New York. 53. Mauderly JL. Effect of age on pulmonary structure and function of immature and adult animals and man. Fed. Proc. 1979; 38:173-7. 54. Young SL, Spain CL, Fram EK et al. Development of type II pneumocytes in the rat lung. Am. J. Physiol. Lung Cell Mol. Physiol. 1991; 260:L113-22.

55. Brody JS, Kaplan NB. Proliferation of alveolar intersitial cells during postnatal lung growth. Evidence for two distinct populations of pulmonary fibroblasts. Am. Rev. Respir. Dis. 1983; 127:763-70. 56. Vincent R, Mercer RR, Chang LY et al. Morphometric study of interstitial matrix in the lungs of the aging rat. FASEB J. 1990; 4:A1915 (Abstract). 57. Mercer RR, Crapo JD. Spatial distribution of collagen and elastin fibers in the lungs.J. Appl. Physiol. 1990; 69:756-65. 58. Rucker RB, Dubick MA. Elastin metabolism and chemistry: potential roles in lung development and structure. Environ. Health Perspect. 1984; 55:179-91. 59. Foster JA, Curtiss SW. The regulation of lung elastin synthesis.Am. J. Physiol. 1990; 259:L13-23. 60. Mays PK, Bishop JE, Laurent GJ. Age-related changes in the proportion of types I and III collagen. Mech. Aging Dev. 1988; 45:203-12. 61. Mays PK, McAnulty RJ, Laurent GJ. Age-related changes in lung collagen metabolism. A role for degradation in regulating lung collagen production. Am. Rev. Respir. Dis. 1989; 140:410-16. 62. Sahebjami H. Lung tissue elasticity during the lifespan of Fischer 344 rats. Exp. Lung Res. 1991; 17:887-902. 63. Mercer RR, Crapo JD. Three-dimensional reconstruction of alveoli in the rat lung for pressure-volume relationships. J. Appl. Physiol. 1987; 62:1480-7. 64. Stiles J, Tyler WS. Age-related morphometric differences in responses of rat lungs to ozone. Toxicol. Appl. Pharmacol. 1988; 92:274-85. 65. Brain JD, Sorokin S, Godieski IJ. Quantification, origin, and fate of pulmonary macrophages. In: Brain JD, Proctor DF, Reid LM (eds) Lung Biology in Health and Disease: Respiratory Defense Mechanisms, part 11 (Volume 5), 1977, p. 849. Dekker, New York. 66. SheUito J, Esparza C, Armstrong C. Maintenance of the normal rat alveolar macrophage cell population. The roles of monocyte influx and alveolar macrophage proliferation in situ. Am. Rev. Respir. Dis. 1987; 135:78-82. 67. Tyler WS, Tyler NK, Last JA et al. Effects of ozone on lung and somatic growth. Pair fed rats after ozone exposure and recovery periods. Toxicology 1987; 46:1-20. 68. Robinson NE, Gillespie JR. Morphologic features of the lungs of aging beagle dogs.Am. Rev. Respir. Dis. 1973; 108:1192-9. 69. Bartlett D Jr, Areson JG. Quantitative lung morphology in newborn mammals. Respir. Physiol. 1977; 2:193-200. 70. Siegwart B, Gehr P, Gil J et al. Morphometric estimation of pulmonary diffusion capacity. IV. The normal dog. Respir. Physiol. 1971; 13:141-59. 71. Crapo JD, Young SL, Fram EK et al. Morphometric characteristics of cells in alveolar region of mammalian lungs. Am. Rev. Respir. Dis. 1983; 128:$42-6. 72. Pinkerton KE, Murphy KM, Hyde DM. Morphology and morphometry of the lung. In: Capen CC, Benjamin SA, Hahn FF et al. (eds) Pathobiology of the Aging Dog (Volume 1), 2001; 5:43-56. Iowa State University Press, Ames. 73. Mauderly JL, Hahn FF. The effects of age on lung function and structure of adult animals. Adv. Vet. Sci. Comp. Med. 1982; 26:35-77. 74. Horsefield K, Cumming G. Morphology of the bronchial tree in the dog. Respir. Physiol. 1976; 26:173-82. 75. Raabe OG, Yeh HC, Schum GM et al. Tracheobronchialgeometry: human, dog, rat, hamster. 1976; Lovelace Foundation, Albuquerque, NM.

Environmental Influences on Lung Development and Aging

ISBN 0 12 324751 9

Part 2

Copyright © 2004 Elsevier

INTRODUCTION Today, infants who are born as early as 22 weeks postconceptional age (term is 40 weeks) may survive if supported by antenatal steroids and postnatal surfactant replacement, mechanical ventilation, supplemental oxygen, antibiotics, and appropriate nutrition. These, and other supports are required because of the sudden event of preterm birth, an abrupt change in environment that profoundly impacts the lung. Two questions are addressed in this chapter. First, what are the causes of preterm birth and, second, how does the abrupt change in environment influence lung development?

Evaluation of birth certificates for most of the country showed an incidence of preterm birth of 9% in 1981, which rose to 11% in 1989. 2 The second source, a multicenter trial, which evaluated preterm birth weight and gestational age also showed a 10% incidence of prematurity. 3 The extensive use of tocolytic agents to arrest preterm labor has not reduced the incidence of preterm birth in the United States or Canada. 4-6 The preterm birth incidence among non-Hispanic African-American women is about twice that of non-Hispanic Caucasian women. 7 Other factors such as maternal smoking, s illicit-drug u s e , 9 previous reproductive history, 1~ incompetence of the cervix, and uterine anomalies are also associated with preterm birth. Mothers who bear their first child before 20 years of age or after 35 years of age are also at high risk, with mothers 35 years or older being at higher risk. 11

CAUSES OF PRETERM B I R T H Factors leading to preterm birth Definition of preterm birth Preterm birth is any delivery, regardless of birth weight, that occurs before 37 completed weeks from the first day of the menstrual cycle. 1 Pregnancies that end before 20-22 completed weeks of gestation are termed abortions. Thus, a reasonable definition of preterm birth is any delivery that occurs between 20 and 37 weeks of gestation.

Incidence of preterm birth In the United States, the overall rate of prematurity is approximately 10%. This estimate derives from two sources. *To whom correspondence should be addressed. The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

Maternal factors dominate the factors that lead to preterm birth. 12 Maternal infection is clinically identified in 30 to 40% of pregnancies complicated by preterm labor. 13 Infection in the maternal cervix or vagina may involve extra-embryonic fetal tissue (i.e. infection of the fetal membranes, called chorioamnionitis) or uterine decidua. Alternatively, low-grade systemic infection in the mother may cross the placenta and inflame the fetus and/or placental villi (called villitis) (Fig. 16.1). On the other hand, at least 35 to 40% of pregnancies ending in preterm labor have no documented infection. TM Because such a large percentage of pregnancies end in preterm labor without clinical evidence of infection, uncertainty persists regarding the role of intrauterine infection Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

100 -

J Premature

birth Inflammation ,~

J

Initiation of • ~ mechanical IRDSI ventilation /

Pre- or postnatal

"~ Nutrition

infection

Prolonged

~ - " / ~ ' ~

Inflammation

]Recovery]

80-

Supplemental O=

~ Postnatal ~ infection

Ic,DI

mechanical ventilation

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60-

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(%)

40-

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Fig. 16.1. Factors contributing to the development of respiratory distress syndrome (RDS) and its evolution to chronic lung disease (CLD) following preterm birth.

in initiating preterm labor. Although preterm labor has also been associated with preterm rupture of membranes, pregnancy-induced hypertension, abruption of the placenta, or intrauterine fetal growth retardation, such associations have not been consistently found. 15

Preterm birth and perinatal mortality Deaths of very-low-birth-weight (VLBW) preterm infants (defined as weighing 500-1500gm at birth) have declined since 1988.16 Death before hospital discharge was 26% in 1988 and 20% in 1991. A recent prospective, 14-center survey (the National Institute of Child Health and Human Development Neonatal Research Network) showed that 16% of 4438 VLBW preterm infants died prior to discharge from hospital or long-term care facility in 1995-1996.16 Death prior to discharge, however, was inversely related to birth weight (Fig. 16.2). The incidence of death prior to discharge ranged from 89% for 195 preterm infants who weighed 401-500gm at birth (defined as extremely lowbirth-weight, ELBW) to 3% for 1299 preterm infants who weighed 1251-1500 gm at birth. Perinatal mortality associated with preterm birth is also related to sex of the preterm infant, plurality (twins, triplets, etc.), ethnicity and socioeconomic status, intrauterine growth rate, late-onset sepsis, and neonatal respiratory failure. 16-18 Specific examples include the mortality rate in male preterm infants, which is approximately twice that of female preterm infants when birth occurs before 29 completed weeks of gestation. Twin preterm infants have 3 to 4 times the mortality of singleton preterm infants. Mortality risk in the presence of late-onset sepsis was 18% compared to 7% without infection. 18 Preterm birth and morbidities and adverse outcomes Among the survivors of preterm birth, morbidities such as poor in-hospital growth, patent ductus arteriosus, intracranial hemorrhage, late-onset sepsis, neonatal respiratory failure, necrotizing enterocolitis, and neurological impairments are frequent. Poor in-hospital growth, the most frequent morbidity in a recent study, 16was present in 97% of VLBW infants. Overall morbidity increased from 27% in 1991 to 30% in

20-

0500-750

m

751-1000

1001-1250

1251-1500

Birth Weight Categories (gm)

Fig. 16.2. Histogram summarizing the results of a clinical study that assessed mortality among very-low-birth-weight prematurely born infants, stratified for birth weight. Death prior to discharge from hospital is inversely related to birth weight. Adapted from Lemons and coworkers. 16

1996, primarily because the incidence of chronic lung disease (CLD) of prematurity (also known as bronchopulmonary dysplasia or BPD) in all survivors increased from 19% in 1991 to 23% in 1996.16 The incidence of major morbidity was greatest (63%) among the smallest preterm infants (501-750 gm at birth). Adverse outcomes later in life, such as asthma and neurological impairments, continue to affect a high percentage of prematurely born infants. Because the incidence of morbidities has not changed significantly, the absolute number of surviving preterm infants with morbidities has increased. 16-18 Some VLBW infant survivors of newborn intensive care are now 20 years old. 19 Studies of these infants are providing both good and bad news about adverse outcomes later in life. Encouraging observations are that 51% of the survivors had IQ scores within the normal range, 74% had completed high school, and 41% were continuing their education beyond high school. In addition, alcohol use, illicit-drug use and criminal behavior occurred at a frequency that was not different from peers who were born at normal birth weight (>2500 gm). On the other hand, these infants had more chronic health problems, such as cerebral palsy, blindness and deafness. 19 As a group, they were also shorter, had lower IQ scores and lower scores on academic achievement tests compared to peer young adults who had normal birth weight. The VLBW infants were also less likely to have graduated from high school or enrolled in postsecondary education compared to their young adult peers who had normal birth weight. Whether these differences will diminish as VLBW survivors reach maturity remains an important question.

In summary, preterm birth is an abrupt environmental stress. The incidence of preterm birth is increasing, the size of preterm infants that can be supported is declining, and the incidence of chronic lung disease among the smallest preterm infants remains high. How does the abrupt change in environment influence lung development? This question will be addressed in the next section of this chapter.

PRETERM B I R T H AS AN E N V I R O N M E N T A L INFLUENCE ON LUNG DEVELOPMENT Before term birth, gas exchange is among the functions of the placenta, which is interposed between the fetal and maternal circulations. Because the fetal lung is not required to function as a gas-exchange organ before 37 weeks of gestation, the lung architecture does not develop to facilitate gas exchange, particularly oxygen. In the mature lung, exchange of oxygen and carbon dioxide occurs in relatively large units that are referred to as terminal respiratory units. Terminal respiratory units are all alveolar ducts, together with their accompanying alveoli, that stem from the most proximal respiratory bronchiole. 2~ In the adult human lung, such units contain approximately 100 alveolar ducts and 2000 anatomic alveoli. A terminal respiratory unit is approximately 5 mm in diameter and has a volume of about 0.02ml at functional residual capacity. Together, the two lungs of the adult human contain about 150,000 terminal respiratory units. 21 The function of the terminal respiratory unit is such that diffusion of oxygen and carbon dioxide is so rapid that the partial pressures of each gas are uniform throughout the unit. From each inspired breath, oxygen that reaches the alveolar duct gas diffuses into the alveoli because the incom-

ing air has a higher oxygen concentration than the alveolar gas. Oxygen subsequently diffuses through the air-blood barrier into the red blood cells, where oxygen combines with hemoglobin as the red blood cells flow along the capillaries. Carbon dioxide diffuses in the opposite direction. In the normal adult human lung, the air-blood barrier is exceedingly thin, which facilitates gas diffusion. The average width of this barrier is about 1.5 ktm.21'22 For oxygen, the air-blood barrier's anatomic elements consist, in order, of alveolar epithelium and its subjacent basal lamina, alveolar wall interstitium, basal lamina of the capillary endothelium, the capillary endothelium, plasma, and the membrane of red blood cells. For carbon dioxide, those anatomic elements are traversed in the opposite direction. The terminal respiratory units of the prematurely born infant are incompletely developed so that the air-blood barrier is too thick to allow efficient exchange of oxygen and carbon dioxide. This structural problem is greatest with more immature preterm infants because the development of the terminal respiratory units occurs during the second half of gestation, with the thickness of the gas-exchange barrier inversely related to gestational age. A brief review of the stages of lung development for humans illustrates these features (Table 16.1 and Fig. 16.3). 23 Branching of the bronchial tree is complete by 16 weeks gestation; prior to 17 weeks of gestation, the future bronchi and bronchioles branch into relatively undifferentiated mesenchyme that contains no terminal respiratory units or blood vessels (Fig. 16.3a). This period of lung development is known as the pseudoglandular stage. From weeks 17 to 28 of gestation, the human fetal lung is at the canalicular stage. Lung architecture at this stage is characterized by development of capillaries within the thick partitions of mesenchyme that are delimited by rudimentary air canals. These immature air canals are lined by cuboidal or even columnar epithelial

Fig. 16.3. Normal lung development in humans. Panel a: Although the lung of this infant stillborn at 12-14 weeks of gestation shows considerable autolysis (post-mortem degeneration), a smooth muscle wall (arrow) is visible around airways (AW) that are lined by (largely desquamated) columnar epithelium. There is no development in the intervening mesenchyme (M), which is devoid of blood vessels (BV), except at the periphery of the developing lung tissue. Panel b: By 20 weeks of gestation, smooth-walled respiratory bronchioles and alveolar ducts lined by low cuboidal epithelium (arrow) are visible beyond the larger Y-shaped terminal bronchiole (TB). Numerous capillaries (arrowhead) are visible deep within the developing interstitium (mesenchyme). Panel c: At 30 weeks of gestation, the distal airspaces are subdivided, and many capillaries are present in the thick primary septa (arrow). A terminal bronchiole (TB) and neighboring pulmonary artery (PA) are visible in the center of the panel. Panel d: By 40 weeks of gestation, alveolar development has progressed to a point where the primary septa are thinner (arrow) and secondary septa (crests; arrowhead) protrude into the developing alveoli (A). The alveolar walls are replete with capillaries. Thus, at term, the infant lung clearly resembles its adult form, although considerable growth and development have yet to occur. All four panels show tissue sections that were stained with hematoxylin and eosin, and are the same magnification. (See Color plate 6.)

cells. Although capillary proliferation is robust during this stage of lung development, the developing capillaries are far removed from the epithelium that lines the air canals (Fig. 16.3b). From about week 24 to week 36 of gestation, lung development is at the saccular stage. This stage is characterized structurally by thinning of the mesenchyme, continued proliferation of capillaries, proliferation and expansion of the distal air sacs, and thinning of their lining epithelial ceils (Fig. 16.3c). Combination of these structural transformations creates a thinner connective tissue compartment and decreases the distance between the developing air sacs and the proliferating capillaries. However, intimate structural association between the capillaries and air sac epithelial cells is not reached during this stage. From about 32 weeks of gestation and continuing through the first 18 to

24 months of postnatal life, alveoli are formed by progressive expansion and subdivision (secondary septation, also called secondary crest formation) of the terminal air sacs (Fig. 16.3d). During the alveolarization stage of lung development, the air-blood barrier attains its adult thinness by reduction of the connective tissue compartment in the developing alveolar walls. At the same time, the epithelial cells that line the developing alveoli differentiate into two cell populations. 24 One population, known as alveolar type II epithelial cells, remains cuboidal and develops the cellular organelles that synthesize and secrete surface-active material (surfactant and its apoproteins). Also arising from this population of epithelial cells are alveolar type I (squamous) epithelial cells. These terminally differentiated cells, although fewer in number than their cuboidal counterpart,

cover about 90 to 95% of the alveolar surface area of the peripheral lung. 25 Both structural attributes of alveolar type I epithelial cells provide a large, thin cellular barrier specialized for gas exchange. Before birth, the fetal lungs are filled with liquid that flows from the pulmonary vascular compartment, through the interstitium to the potential airspaces. This liquid then percolates centrally along the conducting airways to the oropharynx, where it is either swallowed or expelled into the amniotic sac. As alveolar type II epithelial cells develop, they secrete surfactant lipids and apoproteins into this fluid. Detection of these secretory products in the amniotic fluid provides a means of assessing lung development prior to birth. 26 Liquid flows from the interstitium into the fetal air spaces because chloride is secreted across the fetal respiratory epithelium into the potential airspaces, 27 causing a concentration gradient that promotes flow of water into the potential airspaces. Near the time of term birth, the secretory activity of the respiratory epithelium switches from a predominantly chloride-secreting membrane to a predominantly sodium-absorbing membrane. 28-3~ The presence of fluid in the potential airspaces, as well as in the mesenchyme surrounding those potential airspaces, is an impediment to gas exchange in the preterm infant. Fluid is an impediment because diffusion is much faster in the gas phase than in water. For example, the solubility of oxygen in water is low (0.03 ml/L x mmHg PaO2) compared to carbon dioxide, the solubility of which is about 20 times more than oxygen (0.7 ml/L • mmHg PaCO2).The advantage for carbon dioxide persists even though the driving pressure for carbon dioxide diffusion is only one tenth that for oxygen entering the blood. Therefore, oxygen diffusion is much slower in the relatively over-hydrated environment of the preterm lung. Balance between adequate production of lung luminal liquid and its drainage is required for normal intrauterine lung growth. 31 When drainage exceeds production, the fetal lung is not exposed to continuous liquid distending pressure and lung growth is inhibited. This occurs in fetuses with prolonged oligohydramnios due to rupture of amniotic membranes. 32 Other conditions that disturb lung growth include diaphragmatic hernia 33 or pulmonary artery occlusion. 34 Structural and functional characteristics of the airways may also be affected by preterm birth. During the canalicular and saccular stages of lung development, the airways have little smooth muscle in their wall, the epithelium is immature, and cell-cell adhesion is weak. Physiological studies of human tracheobronchial segments ex vivo indicate that pressure-volume relations are affected by maturity, 35'36 such that airway compliance was inversely related to maturity. The greater compliance of the airways of preterm infants may contribute to the need for higher airway pressures, and the associated increase in lung volume needed to inflate the collapsed gas exchange regions compared to the airways of term infants. 37 The dearth of smooth muscle and weak

adhesion between cells of the immature airway may make the preterm infant's airways susceptible to injury by the increased airway pressures and lung volumes that are required to effect adequate ventilation. Evidence of injury is manifest as sloughed airway epithelial cells in tracheal aspirates and lung lavage fluid, as well as development of air leaks (interstitial emphysema and pneumothorax). Other structural and functional attributes of the developing lung important to normal lung function are alveolar type II epithelial cells and the surfactant phospholipids and apoproteins that these cells synthesize, secrete and recycle. In the human, type II cells containing lamellar bodies are histologically recognizable at the transition from the canalicular to the saccular stages of lung development (weeks 20 to 24 of gestation). Surfactant synthesis begins later, during the saccular stage (about 30 weeks of gestation). 38 From the saccular stage to the end of term gestation, the concentrations of dipalmitoyl phosphatidylcholine and other surfactant phospholipids increase in lung tissue, lung lavage fluid and amniotic fluid. Surfactant apoproteins (SP) A, B and C, expressed only in lung tissue, are also developmentally regulated but not concordantly. Human SP-A mRNA and protein in lung tissue are not detectable until the saccular stage of lung development (about 30 weeks of gestation). 38-41 Human SP-B and SP-C are detectable in lung tissue at very low levels earlier than SP-A, during the canalicular stage (about 24 weeks of gestation). 42 Detection of SP-B and SP-C proteins, however, is later in development, during the saccular stage (about 30 weeks of gestation). 41 To our knowledge, human SP-D protein has only been detected in amniotic fluid during the saccular stage of lung development (about 24 weeks of gestation). 43'44 Thus, the cellular and biochemical machinery to reduce surface tension at air-liquid interfaces in the future airspaces develops during the third trimester in humans. Many preterm infants are born before the third trimester, and are at risk for respiratory failure because their lungs are deficient in surfactant (Fig. 16.1).

Respiratory distress syndrome When an infant is born prematurely, if its lungs are structurally immature and deficient in surfactant, surface tension forces are high and the alveoli are unstable. 45 Alveolar instability results in alveolar collapse (atelectasis). This atelectasis results in ventilation-perfusion mismatch. Such mismatch results, in turn, in intrapulmonary shunting and thus contributes to poor oxygenation. 46 Opening the collapsed air spaces requires high ventilatory pressure that is transmitted to the immature distal airways and gas exchange regions of the lung. In effect, the extreme effort required to expand the lungs with the first breath must be repeated with each breath because surfactant is not present to prevent collapse of the distal airspaces. Prematurely born infants who have these characteristics usually develop respiratory distress syndrome (RDS; also known as acute lung injury) (Fig. 16.1).

Respiratory distress syndrome affects about 30,000 infants annually in the USA. The incidence is about 50 to 60% of infants who are born before 30 weeks of gestation. 47 The incidence increases with decreasing gestational age.16,18 RDS is more prevalent and severe in male compared to female preterm infants, for reasons that remain unclear. Respiratory distress syndrome is characterized by tachypnea, chest retractions, and cyanosis (hypoxemia) soon after birth. Chest radiographs demonstrate low lung volumes, air bronchograms, and opacities (i.e. reticulargranular infiltrates). Lung function studies have shown increased airway resistance 4s and decreased pulmonary compliance. 49 Pulmonary artery pressure and pulmonary vascular resistance are elevated, particularly in infants with severe RDS. 5~ The pathologic findings in RDS are similar to adults with acute respiratory distress syndrome (ARDS), 51 superimposed on the immature lung. 52 Mechanical ventilation is instituted very early in the course of this disease, and therefore lesions observed reflect both the disease and its treatment. Within the first 3 to 4 h, the lungs may only show uneven ventilation of distal airspaces, resulting from surfactant deficiency, interstitial edema, resulting from incomplete removal of fetal lung liquid or acute injury to microvascular endothelial cells, and congestion of capillaries (Fig. 16.4a). By 12 to 24 h, necrosis of alveolar and bronchiolar epithelial cells develops and the denuded walls become coated by characteristic hyaline membranes, which are brightly eosinophilic transudates of plasma proteins admixed with necrotic epithelial cells and fibrin (Fig. 16.4b). 53 Hyaline membranes accumulate especially at branch points. These pathologic features are similar to the exudative phase of ARDS in older patients, but the alveolar type II epithelial cell hyperplasia that constitutes the proliferative phase of ARDS is not as prominent in autopsy slides of hyaline membrane disease in preterm infants. Mechanical ventilation of preterm infants is frequently associated with ventilator-induced lung injury 54 (Fig. 16.1), even following repeated doses of exogenous surfactant. The injurious effects of mechanical ventilation depend on a number of factors, among which are the magnitude of airway pressure (barotrauma) and lung volume (volutrauma), 55'56 and the concentration of inspired oxygen. Several experimental animal studies have compared indices of ventilator-induced lung injury between conditions that raised airway pressure and increased lung volume. One study distinguished between the effects of pressure and volume by subjecting rats to identical peak airway pressure and either large or small tidal volume ventilation. 57 Small tidal volume ventilation with a high peak airway pressure was accomplished by strapping the thorax and abdomen. Rats subjected to a high airway pressure and large tidal volume ventilation developed increased permeability pulmonary edema and ultrastructural evidence of cellular damage. Strikingly, the rats that were subjected to high peak airway pressure, by strapping the abdomen, and small

tidal volume had no edema or ultrastructural evidence of cellular damage. This and other 58'59 studies, which used normal animals, suggest that large lung volumes, rather than high peak airway pressures per se, are important in the pathogenesis of ventilator-induced lung injury. For this reason, volutrauma is used to describe the injury that is associated with mechanical ventilation. How does volutrauma induce lung injury, especially following preterm birth? Evidence suggests that some of the circumstances that have already been described are exacerbated. For example, regional overinflation increases microvascular permeability, either directly or indirectly (through release of inflammatory mediators from sequestered leukocytes in the lung), which leads to alveolar flooding. Alveolar flooding, in turn, is associated with inactivation of surfactant, which in turn leads to atelectasis and less compliant lungs. If these cycles are not broken, lung injury recurs, necessitating more supplemental oxygen, higher airway pressure and a larger tidal volume. As the extent and duration of mechanical ventilation with supplemental oxygen increases, and lung volutrauma persists or increases, mild RDS may become BPD. Conventional mechanical ventilation, most frequently delivered by a time-cycled, pressure-limited ventilator, is regarded as an important contributing factor in inducing acute lung injury and chronic lung injury (Fig. 16.1). For this reason, other approaches to ventilation continue to be explored, such as high-frequency oscillatory ventilation, high-frequency jet ventilation, ECMO, and liquid perfluorocarbon ventilation. 6~ The aim of these alternative ventilation strategies is to decrease the peak inspiratory pressure and mean airway pressure, thereby resting the lung and reducing ventilator-induced lung injury. Another approach is to avoid mechanical ventilation altogether by use of continuous positive airway pressure delivered through nasal prongs (nasal CPAP). These alternatives offer benefits but each also carries unwanted side effects that limit utility. 61-64 The pathogenesis of increased permeability pulmonary edema during RDS is also associated with accumulation of leukocytes (Fig. 16.1), particularly neutrophils, in the vascular, interstitial and airspace compartments of the lung. 65-67 A recent study that ventilated preterm lambs for 8 h with 100% oxygen showed that the number of circulating neutrophils decreased in the first 30 to 90min of life and that this decrease correlated with an increase in the number of neutrophils that accumulated in the lung's air spaces at the end of the 8 h study. 65 A complimentary, retrospective chart review study in human preterm infants subsequently showed that a low concentration of mature neutrophils in the systemic circulation within 24 h of birth (ascribed to egress of neutrophils from the circulation coupled with an inability of the immature bone marrow to replenish the circulating pool) is associated with more severe respiratory distress during the first postnatal week of life. 68 Such preterm infants were more likely to need mechanical ventilation with supplemental oxygen.

Fig. 16.4. Hyaline membrane disease (HMD; panels a and b) and chronic lung disease of prematurity (CLD; panels c and d) in humans. Panel a: HMD, birth at 25 weeks of gestation followed by 6 h of mechanical ventilation. In the first few hours of life, the preterm, mechanically ventilated lung shows uneven expansion of the distal airspaces, vascular congestion, and interstitial edema. Some patchy hemorrhage also may be present. Panel b: HMD, birth at 29 weeks of gestation followed by 2 days of mechanical ventilation. Hyaline membranes (arrow head) are uniformly present by 12 to 24 h, but may appear within 3 to 4 h of preterm birth. Hyaline membranes are typically most evident at the branch points of distal airways (AW). The distribution of ventilation is uneven, with centriacinar expansion and peripheral collapse. Panels c and d: Infants who progress to CLD show marked simplification of the distal airspaces (compare panels c and d with the expected appearance of the lung at 30 and 40 weeks of gestation in Fig. 16.3, panels c and d, respectively; all four panels are at the same magnification). Panel c: CLD, birth at 22 weeks of gestation followed by 44 days of mechanical ventilation (28-29 weeks post-conceptual age). The distal airspaces appear as distended circles that are filled, in this specimen, with cellular debris (*). Thick, cellular mesenchyme (M) separates the adjacent airspaces. Panel d: CLD, birth at 24 weeks of gestation followed by 151 days of mechanical ventilation (45 weeks post-conceptual age). Simplification and overdistension of the distal airspaces (DAS) is clearly evident. The primary septa (arrow) are thick and cellular. Secondary septa (crests; arrowhead) are infrequently visible; those that are visible are short, thick and devoid of capillaries near their tip. Panels a and b show tissue sections that were stained with hematoxylin and eosin. Panels c and d show tissue sections that were stained with Masson's trichrome. All four panels are the same magnification. (See Color plate 7.)

Because lung immaturity is a consistent feature of RDS, strategies have been developed to accelerate lung development before preterm delivery. One strategy is administration of hormones, such as glucocorticoids. 69'7~The seminal studies of Liggins demonstrated that glucocorticoids accelerated lung maturation of fetal sheep and decreased the incidence of RDS in human infants after antenatal corticosteroid therapy. 71'72 The maturational effect of corticosteroid treatment on lung structure was first described by Kikkawa and colleagues. 73 Among the structural effects, corticosteroids (betamethasone or dexamethasone) accelerate the maturation of alveolar type II epithelial cells, which are the source of

pulmonary surfactant. For these reasons, mothers who are in preterm labor, and at risk of giving birth to a preterm infant between 24 and 35 weeks, of gestation, are treated antenatally with glucocorticoids. 74 Evidence has been demonstrated that maternal treatment with thyroid-releasing hormone enhances the beneficial effects of antenatal glucocorticoids by further promoting lung maturation from structural, biochemical and functional standpoints, including surfactant production. 75'76 For example, combination treatment increased the content of saturated phosphatidylcholine, stability of alveoli, and clearance of lung liquid. 77-82 However, a recent follow-up

study of children two years after receiving antenatal combination therapy of thyroid-releasing hormone and corticosteroids showed delayed mental development as well as more respiratory problems, ventilation days, and chronic lung disease. 83 These results, in combination with meta-analysis,84 have led to the recommendation that antenatal thyroidreleasing hormone treatment should not be given to women at risk of preterm birth. A treatment strategy to reduce lung stiffness (i.e. increase lung compliance) after preterm birth is to instill surfactant into the airways. 85 Surfactant replacement therapy is beneficial because once it becomes widely and thinly distributed in the lung, the exogenous surfactant reduces surface tension at air-liquid interfaces, thereby stabilizing alveoli when they are deflated. After surfactant replacement, oxygenation improves swiftly, followed for several hours by progressive improvement in gas exchange and lung mechanics.86'87 Other improvements, at least in preterm lambs, are reduced vascular injury and edema. 88 Thus, surfactant replacement therapy reduces the incidence and severity of RDS following preterm birth.

100 Yipp and Tan, 1991 Kraybill et al., 1987

80

Preterm Infants Who Developed BPD

(%)

60m

40-

20-

I I i

0 500-750

751-1000

1001-1250

1251-1500

Birth Weight C a t e g o r i e s ( g m )

Fig. 16.5. Histogram summarizing the results from two clinical studies that assessed the incidence of bronchopulmonary dysplasia (BPD) among birth weight categories for prematurely born infants. The incidence of BPD is inversely related to birth weight. Adapted from Yipp and Tan16Sand Kraybill and colleagues. 166

Chronic lung disease of prematurity Survival of prematurely born infants with RDS has significantly increased since the introduction of antenatal steroids and postnatal surfactant replacement therapies, and gentler ventilation strategies. 16'6~176 As described earlier, 84% of preterm infants weighing 500-1500gin at birth now survive. Unfortunately, about 8000-10,000 preterm infants who develop RDS in the United States annually, and who receive antenatal glucocorticoids and postnatal surfactant replacement, go on to develop CLD of prematurity (also called BPD) (Fig. 16.1). The reported incidence of CLD ranges between 15 and 60% of preterm infants. 91-93 As expected, smaller infants were at greater risk for CLD than larger preterm infants (Fig. 16.5). Other risk factors for CLD include male gender, white race, greater severity of RDS, and a low Apgar score 1 rain after birth. After adjusting for these risk factors, development of CLD is associated most with a need for high inspired oxygen 96 h after birth, high peak inspiratory pressure, and high fluid intake. The original description of BPD was made by Northway and colleagues (none of whom were neonatologistsI). 94 These authors defined BPD based upon clinical, radiological and pathological criteria. The principal clinical criteria were the requirement for supplemental oxygen for at least 28 days after birth (a duration selected because that is a month; W. Northway, personal communication). The major radiological criteria were cyst formation and hyperexpansion mixed with atelectasis. The characteristic pathological findings at autopsy depended upon the duration of disease prior to death. Classic hyaline membrane disease was seen in the first 2-3 days. A regenerative phase was seen over the next week that consisted of necrosis and regeneration ofbronchiolar and alveolar epithelium, in addition to hyaline membranes.

Over the next 10 days, hyperplasia of bronchiolar and arteriolar smooth muscle, fibrosis, and dilated or collapsed distal air sacs ensued. In the final stage, these alternating areas of overdistention and atelectasis became fixed. The term 'bronchopulmonary' was originally chosen because it identified airway and blood vessel involvement; "dysplasia" was chosen because it described the failure of the distal air sacs to develop into normal anatomic alveoli (W. Northway; personal communication). Today, CLD is usually clinically defined as either oxygen dependency at 28 days of life with appropriate radiological findings or oxygen dependency at 36 weeks' postconceptional age. 95'96 As originally described by Northway etal., 94 BPD was diagnosed in preterm infants who did not recover from RDS. However, treatment of respiratory failure from other causes, including meconium aspiration pneumonia, 97 neonatal pneumonia, 98 congestive heart failure, 99 or congenital diaphragmatic hernia, 1~176 may be associated with CLD. Prematurely born infants who develop CLD today, compared to those who developed BPD over 30 years ago, are much younger (23-26 weeks of gestation compared to 31-34 weeks of gestation) and smaller (<1000gm versus N2000gm at birth), and the radiological and pathological features of their lung disease are less severe. The radiological findings include lung hyperinflation, emphysema, and interstitial densities. The principal pathological findings, at autopsy, are dilated and simplified distal air sacs 101'102 (Figs 16.4e,d). In fact Husain et al. found that the number of alveoli in the lungs of infants dying of BPD was not significantly different than the number that would have been expected to have been present when those infants were born. 1~ Generally missing are the pathological changes in

the central airways. Recognition of these distinctions between CLD that is seen today and the BPD described by Northway etal. 94 more than 30 years ago has prompted clinicians and investigators to call today's CLD the 'new' BPD. Whether the disease that is seen today is 'new' or a less severe variant of BPD is debatable. Prematurely born baboons and lambs that are ventilated for 3 weeks or more also have simplified distal air sacs. 104'105 Simplification is related to failure of alveolar secondary septa (crests) to sprout into the distal air sacs. A consequence of failed septation is a reduced surface area for gas exchange, particularly the capillary surface area. 1~ However, the latter observation in experimental animals with evolving CLD has not been confirmed in autopsy tissue from infants who died with established CLD. 107 The mechanisms that participate in normal or abnormal secondary septation of alveoli are not known. Studies using knockout mice for PDGF-AA 1~ or tropoelastin 1~ suggest that elastin synthesis and secretion at the site of initial outgrowth may be involved. Our studies using preterm lambs with evolving CLD indicate that the tropoelastin gene is excessively and continuously up-regulated. 11~ Although the results from the knockout mice and preterm lambs seem contradictory, they actually suggest that tropoelastin gene expression has to be tightly regulated for normal septation. Too little or too much tropoelastin gene expression is detrimental to normal septation. Recent studies using preterm baboons 112 or preterm lambs 113 suggest that appropriate temporal and spatial expression of vascular endothelial growth factor (VEGF) and related growth factors and receptors may also guide septation. Pulmonary arteries are also structurally and functionally affected in infants who develop CLD. Autopsy studies have demonstrated structural remodeling of arterial vessels. The most characteristic changes are greater muscularization of small arteries and extension of smooth muscle into the smallest arterioles that normally are partially muscular or are nonmuscular. 114'115 Studies of lung tissue from preterm lambs that are ventilated for 3 weeks showed that vascular smooth muscle does not regress during the evolution of C L D . 106 Postmortem histology and angiography showed that infants whom had CLD for 1 to 7 months also have decreased arterial density, compared to infants who had CLD for <1 month. 116 Disruption of pulmonary blood vessel formation in rats results in lung histopathological changes that look like CLD. 117'118Diminished blood vessel formation may contribute to the elevated pulmonary arterial pressure (pulmonary hypertension) and pulmonary vascular resistance that typically accompany C L D . 106'119'120 In this regard, studies using preterm lambs that are mechanically ventilated for 3 weeks have provided some new insights into the pathogenesis of CLD. For example, endothelial nitric oxide synthase (eNOS), the enzyme that catalyzes the endogenous generation of nitric oxide by vascular endothelial cells, 121 is reduced in quantity in the endothelial cells of large and small pulmonary arterial vessels during the evolution of CLD. 122

An additional predisposing factor for the development of CLD may be oxygen toxicity and overwhelmed endogenous antioxidants. Oxygen toxicity occurs when prolonged increased concentration of inspired oxygen is needed for prematurely born infants with R D S . 55'94'123'124'125 Endogenous antioxidant enzymes, such as catalase, copper-zinc superoxide dismutase or manganese superoxide dismutase, may be overwhelmed during the evolution of RDS to CLD. Evidence of oxidant stress in prematurely born infants is detected as shifts in the plasma concentrations of reduced glutathione and oxidized glutathionine, lz6 Oxidant stress is exacerbated by endogenous release of toxic metabolites of oxygen (e.g. superoxide anion, hydroxyl radical) from activated neutrophils and macrophages that accumulate in the lung during the acute inflammatory response characteristic of RDS.65,67,125,127

Nutrition is important in the pathogenesis of CLD from several standpoints. One standpoint is energy expenditure related to rapid growth in the face of limited nutritional reserves, both of which apply to preterm infants. To that energy expenditure is compounded the additional energy need associated with the superimposed lung disease (RDS and CLD). Much of the added energy expenditure is related to the added work of breathing. If these compounded energy needs are not met, a catabolic state ensues, which is thought to contribute to the pathogenesis of CLD. 128 In addition, most antioxidant enzymes require trace amounts of metals, such as copper, zinc, and selenium, for optimal activity. 129'13~Deficiencies in such trace metals, therefore, may contribute to the imbalance between oxidants and antioxidants and participate in the pathogenesis of CLD. TM Vitamin E (tocopherol) is an antioxidant that prevents peroxidation of lipid membranes. However, a number of studies have shown that vitamin E supplementation does not alter the pathogenesis of C L D . 132-136

Vitamin A (retinol) levels may be low in the plasma of some preterm infants who are less than 36 weeks of gestation; 137-14~this observation has prompted vitamin A supplementation trials. 141'142 Experimental animal studies have provided some exciting results in this regard. In one study, pregnant rats were repeatedly exposed to dexamethasone to inhibit lung development in utero; newborn pups were then treated with all-trans-retinoic acid postnatally. 143 Alveolar formation in the treated rat pups was greater than that in the untreated control rat pups. Our studies using preterm lambs with evolving CLD of prematurity have shown that daily treatment with vitamin A (Aquasol) is associated with greater formation of alveoli, alveolar capillaries and increased expression of VEGF and its natural ligand, fetal liver kinase-1 (FLK- 1). 113 Imbalances in proteinases (elastase and collagenase) and anti-proteinases (Ctl-anti-proteinase) may also play a role in evolution of RDS to CLD. Part of the imbalance may result from inactivation of Ctl-anti-proteinase by toxic metabolites of oxygen. 144 Such imbalances may result in destruction of

elastin and collagen, two prominent extracellular matrix molecules in the l u n g . 145-147 Mechanical ventilation also contributes to the evolution of CLD from RDS. As the extent and duration of mechanical ventilation with supplemental oxygen increases, and lung volutrauma persists or increases, recovery from RDS does not occur and the acute lung injury evolves into CLD. Inhomogeneous distribution of ventilation, causing regional overdistension, and the potential need to use high inflation pressures and large lung volume, set the stage for ongoing ventilator-induced lung injury. The detrimental effects of prolonged mechanical ventilation on the lung have prompted the development of alternative treatment approaches and modes of ventilation. An alternative treatment approach that has proven unfavorable is postnatal administration of dexamethasone. Initial clinical trials looking at short-term respiratory outcome suggest that postnatal administration of dexamethasone to ELBW infants within 24-48 h after preterm birth may reduce the risk of CLD. 14s-lsl These studies used a high initial dose of dexamethasone (_>0.5mg/kg/day) and many of the treated preterm infants developed hypertension or hyperglycemia. A recent study examined the effect of a moderate dose of dexamethasone (0.15 mg/kg/day for 3 days) on other outcome variables in ELBW infants. 152 Although the relative risk of death or CLD in the dexamethasone-treated preterm infants was not different from the placebo group of infants, several adverse effects were observed. The rate of spontaneous gastrointestinal perforation within 14 days of birth was >3-fold higher in the dexamethasone-treated infants compared to the placebo-treated infants. The trial was stopped for this reason. More importantly, among the other adverse effects identified in other studies is long-term cognitive deficits associated with dexamethasone use in preterm infants. 153These important adverse effects have led to a marked decrease in the use of postnatally administered steroids to ELBW infants to treat anticipated or evolving CLD. The outcome of children and young adults who had BPD as originally described by Northway and colleagues indicate persistent pulmonary dysfunction. 154 At 10 years of age, BPD survivors had increased airways resistance, air trapping, blood gas abnormalities and persistent reactive airways disease, lss Such children also had increased lung volumes, airway obstruction, and increased transcutaneous carbon dioxide tensions. 156 These abnormalities did not seem to disappear in later years of life, even though symptoms were infrequently evident. TM For example, of 25 young adults about 18 years of age, 19 had some degree of pulmonary dysfunction. Six of the young adults had severe pulmonary dysfunction, including increased airway resistance, air trapping, and reactivity to methacholine. Only six of the 25 young adults who had 'old' BPD had respiratory symptoms, however. Thus, some persistent pulmonary dysfunction is to be expected for many children and young adults who had BPD. Persistent pulmonary dysfunction in children and young adults who had CLD raises questions about persistent

structural abnormalities in their lungs. No published information is available beyond 34 months of life. 157 Little published histopathological information is available for such infants and children during their first months of life; however, Margraf and associates 1~ reported morphometric results obtained for the lungs of eight infants who died with persistent BPD. The oldest infant died at 28 months of age. The principal findings were decreased numbers of alveoli and alveolar surface area compared to controls. Bronchiolar smooth muscle and glands were increased. One might predict less severe histologic changes in young adults who had the less severe, contemporary variant of BPD, but the long-term sequelae of the apparent 'simplification' of lung architecture in infancy remains speculative. Chest imaging results suggest that the radiographic appearance of the lungs of children who had BPD as originally described by Northway and colleagues may return to normal by 2-3 years of age, lss although this is not always the case. 154'159 When radiographic changes persist, they are evident as peribronchial cuffing, diffuse and focal linear densities, hyperexpansion, pleural scarring, and skeletal changes of the chest. Computed tomographic imaging further reveals septal thickening, focal areas of lucency, and vascular remodeling. While it is tempting to ascribe such chest imaging changes to BPD, the changes could develop in response to the recurrent respiratory infections that typically affect survivors of BPD. Again, we do not know if the radiographic appearance of the lungs will be different for children who had the less severe, contemporary variant of BPD.

CONCLUSIONS In conclusion, preterm birth is an environmental stress that abruptly affects lung development. The structural and functional immaturity of the preterm infant's lungs necessitates use of antenatal steroids, perinatal surfactant replacement therapy, ventilatory and oxygen support, and other supportive measures (Fig. 16.1). Acute lung injury often occurs as a result of lung immaturity and the therapy that it necessitates. If recovery does not ensue, the affected preterm infants progress to CLD. While CLD continues to be one of the most serious of pediatric public health issues, important progress has been made. For example, CLD of prematurity occurs infrequently today in the larger (_>2kg) and more mature (_>32 weeks) preterm infants, the population that Northway and colleagues described in the original paper on BPD. 94 Moreover, the severity of CLD is less today than it was 30 years ago. These encouraging changes reflect a better understanding of lung developmental biology with resultant improvements in clinical management of prematurely born infants. Nonetheless, challenges remain because the incidence of preterm birth is increasing, the size of the preterm infants that can be supported continues to decline, and the incidence of CLD among the smallest preterm infants remains very high. Therefore, much more has to be

learned to better understand lung developmental biology and alveolar formation, and how both processes are impacted by mechanical ventilation, supplemental oxygen, infection and inflammation, nutrition, and volutrauma, etc., if respiratory failure in the preterm infant is to be more successfully treated or prevented.

ACKNOWLEDGEMENT Portions of this work were supported by N I H grants HL62875 (KHA), HL56401 (KHA; P L Ballard, P.I.), and AHA Grant-in-Aid 96014370 (KHA).

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163. Emery JL, Wilcock PF. The postnatal development of the lung.Acta Anat. 1966; 65:10-29. 164. Langston C, Kida K, Reed M et al. Human lung growth in late gestation and in the neonate. Am. Rev. Respir. Dis. 1984; 129:607-13. 165. Yipp Y, Tan K. Bronchopulmonary dysplasia in very low birth weight infants. J. Pediatr. Child Health 1991; 27:34-8. 166. Kraybill E, Bose C, D'Ercole A. Chronic lung disease in infants with very low birth weight. A population-based study.Am. J. Dis. Child. 1987; 141:784-8.

INTRODUCTION

An increasing body of evidence from epidemiological and experimental studies indicates that nutritional and oxygen status during development can induce both immediate and long-term alterations in the structure and function of many organs including the lung. Associations between nutrition and lung development, lung function and respiratory symptoms or illnesses have long been recognized, and have been the subject of several recent reviews. 1-6 Sustained alteration in respiratory function following compromises during development has been referred to as 'tracking', 7 which is likely a consequence of persistent alterations in lung structure or function caused by adverse environmental conditions during early life, either before or after birth. Evidence is accruing that a number of common respiratory illnesses of childhood and adulthood may have their origins in early life, or that predisposing conditions may be laid down at that time? Although epidemiological and clinical studies have indicated that low birth weight is associated with later respiratory illness, 9'1~ it is not yet clear which of the many factors that may be associated with low birthweight and early postnatal growth are responsible for altered respiratory development. In this chapter, we present current understanding of how the early nutritional environment impacts upon lung development and respiratory function; hypoxia is included, as we consider oxygen to be an important nutrient for the developing *To whom correspondence should be addressed. The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

organism. As well as influencing lung development, it is also likely that nutritional impairments during development can influence the rate of 'pulmonary aging'. Indeed, it is becoming apparent that the early nutritional environment may be important for the entire life-cycle of the lung.

CAUSES OF NUTRITION

RESTRICTED FETAL AND GROWTH

The term 'fetal growth restriction' (FGR) implies that fetal weight falls below the 10th percentile for gestational age; normally growth has been restricted by factors affecting the intrauterine environment or the delivery of nutrients to the fetus. 11 The underlying etiology is varied and can include maternal, fetal or placental causes (Table 17.1); a common underlying cause is reduced placental transport of substrates including oxygen from mother to fetus. Blood samples taken from the umbilical cord show that FGR arising from a range of etiologies is associated with fetal hypoxemia, hypoglycemia, hyperlactinemia, acidemia, and altered endocrine status indicative of intrauterine stress. 12 Maternal causes

These include chronic disease states of the mother, drug use (e.g. cigarette smoking) and undernutrition. Maternal vascular disease, whether chronic hypertension, pre-eclampsia or diabetes, accounts for 25-30% of FGR cases due to impairments of utero-placental perfusion in non-anomalous fetuses. 11 The Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

incidence of FGR is increased in the presence of chronic maternal hypertension and is directly correlated with the severity of the hypertension. 13 Maternal diabetes is a risk factor for FGR due to damage of the placental microcirculation. Maternal renal insufl]ciency may also be accompanied by FG1L14 Maternal hypoxemia has multiple causes, including cyanotic heart disease, severe chronic anemia, sickle cell anemia and high altitude. Chronic pulmonary disease, such as severe asthma, also may be associated with impaired fetal growth. 15Each of these conditions may be expected to reduce the delivery of oxygen, and possibly nutrients, to the placenta and hence to the fetus. The single most common cause of restricted fetal growth in Western societies is maternal cigarette smoking, and a dose-response relationship has been demonstrated. 16 The smoking-related impairment of fetal growth likely results from a combination of carbon monoxide exposure, which decreases the O 2 carrying capacity of fetal hemoglobin, and nicotine, which induces the release of maternal catecholamines. The repeated release of maternal catecholamines by cigarette smoking may reduce maternal perfusion of the placenta, restricting nutrient transfer to the fetus. Recent evidence suggests that activation of ct-2 nicotinic receptors can have a direct inhibitory effect on cell division and therefore may play a role in restricting fetal growth. 17 Excessive maternal alcohol (ethanol) intake can cause FGR and the fetal alcohol syndrome, is Reduced birthweight can result from maternal alcohol ingestion of only 1-2 standard drinks per day. 19 Maternal use of drugs such as steroids, dilantin, coumadin, cocaine and heroin has also been implicated in causing FGR. Maternal infectious diseases account for 5-10% of FGR cases. Rubella and cytomegalovirus infections are associated with FGR; varicella zoster, human immunodeficiency and a first episode of herpes simplex virus infection may also be associated with impaired fetal growth. 2~

Recent evidence suggests that thrombophilia, congenital or acquired, can cause FGR. 21 Antiphospholipid antibody syndrome, anticardiolipin antibody syndrome, and the presence of lupus anticoagulant can all lead to FGR. 11

Placental causes Placental dysfunction is a major cause of FGR. A decrease in placental mass or impaired umbilical cord development can restrict the rate of substrate delivery to the fetus and thus can contribute to FGR. 2z In particular, placental anomalies such as circumvallate placenta, chorioangioma of the placenta have been associated with restricted fetal growth. 23 Placental abruption and placental previa can chronically restrict placental function, and occur more frequently among cigarette smokers. 24 Fetal causes Fetal anomalies arising from chromosomal disorders (e.g. trisomy 18, 13, 21 and sex chromosome disorders) and congenital malformations (both chromosomal and nonchromosomal) account for 20% of FGR cases. 11 Multiple gestation is another cause of fetal growth restriction. FGR can occur in any multiple gestation pregnancy, but is severe if there is a shared fetal circulation. Twinning results in a decreased placental mass in relation to fetal mass, thereby restricting fetal nutrient supply. The incidence of FGR in twins is 15-30% and the figure is increased with greater numbers of fetuses. 25 Fetal viral infections such as parvovirus, rubella, cytomegalovirus can cause up to 5% of FGR cases particularly if they occur in early gestation. 2~ Fetal growth restriction and organ development It is now recognized that FGR can affect the development of multiple organs. Usually the organ weight or dimensions are measured as an index of growth, but these may not

"~

associated with adverse outcomes, including alterations of respiratory function at all stages of postnatal life. As low birth weight can also be caused by preterm birth, birth weight per se may not be a reliable indicator of fetal nutrition; therefore, in this chapter we have made the distinction between low birth weight (LBW) and FGR. As outlined above, FGR can be caused by many factors, and this has led to difficulties, in human studies, in identifying factors responsible for altered lung development.

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gestational age (days) Fig. 17.1. Body weight trajectories of control (--) and placentally restricted (PR) (---) fetuses over the last third of ovine gestation (term, 147 days). Placental restriction was induced by pre-pregnancy, surgical removal of implantation sites. The growth curve of the control fetuses (r= 0.94, n= 74) was significantly steeper than that of the placentally restricted fetuses (r=0.72, n=64), resulting in fetal growth restriction and low birth weight at term. The data show that restricted placental function exerts its most significant effect upon fetal growth during late gestation. Data from Edwards et al. 155 based on fetal weights at delivery.

provide a true indication of how organ development has been affected by the factors that have restricted fetal growth. For example, brain weight is often spared in FGR (leading to an increase in brain-body weight ratio) yet brain structure can be altered. 26 Apart from the brain and adrenal glands, most organs including the lungs are smaller in individuals affected by FGR. In a sheep model of chronic, late-gestational FGR, it is apparent that lung weight may be spared, 27 yet it has been established that lung structure and function are detrimentally altered. 28'29 Thus, organ weight is an unreliable indicator of the effects of early nutrient restriction, and structure and/or function must be examined. Developmental alterations in organ structure may not only affect organ function immediately after birth but may have persistent effects on function and could lead to increased vulnerability to later disease or effects of aging. The FGR caused by placental dysfunction and nutrient restriction has its greatest effects during the later stages of gestation, when the fetus is growing most rapidly (grams/day). Measurements of fetal growth throughout gestation indicate that the greatest divergence from normal growth profiles occurs during the second half of gestation (Fig. 17.1) at a time when organs are growing in complexity. Hence it is likely that FGR primarily affects developmental processes that occur during the late stages of in utero development.

EFFECTS

OF

RESTRICTION FUNCTION"

PRENATAL

GROWTH

ON POSTNATAL HUMAN DATA

LUNG

A number of studies have shown that low birth weight, which can be a marker of impaired fetal nutrition, is

Effects during infancy It was once considered that infants whose growth had been restricted in utero were at a lower risk of respiratory compromise in the immediate neonatal period than normally grown infants. However, recent studies controlling for gestational age have shown that FGR increases the risk of adverse metabolic and respiratory complications in the early postnatal period. 3~ In both term and preterm neonates, FGR increases the risk of respiratory insufficiency and the need for respiratory support after birth. 32'33 The reasons for impaired gas exchange in FGR infants are unclear at present, but they could relate to delayed clearance of lung liquid from the airways, impaired pulmonary perfusion or pulmonary structural abnormalities, but are unlikely to involve surfactant deficiency. 34 Owing to technical barriers, few studies of airway function have been conducted in infants, but it has been recently shown that FGR infants (approx. 6 weeks after term birth) of non-smoking mothers have impaired lung function, as indicated by reduced FVC and FEF75. 35

Effects during childhood A study of more than 2000 children showed that FGR was associated with a reduction in FEVI; importantly, this effect was demonstrated in FGR children born both at term and preterm. 36 Similarly, a study of twins aged 7-15 years has indicated that the smaller twins have lower forced expiratory flow rates than their larger siblings. 37 Together, these studies suggest that restricted fetal growth impairs airway development, although it is possible that alterations in lung parenchyma and impaired skeletal muscle function contribute to the reduction in forced expiratory flows. In a recent study of airway reactivity in 7-15-year old children from multiple pregnancies, it was found that FGR was not associated with altered bronchoreactivity38; however, respiratory infections in the post-neonatal period were associated with later hyper-responsiveness.

Effects in the adult Three studies of different adult populations have provided evidence that fetal undernutrition, as indicated by low birth weight, can affect airway function in adult life. As the gestational age at birth of the subjects was unknown in these studies, it is possible that low birth weight could have been due, at least in part, to preterm birth; however, given that individuals in the study group were born at a time when few severely preterm infants survived the neonatal period, it is likely that low birth weight was mainly

attributable to FGR. A large study of British men aged 59-70 years showed that low birth weight was associated with reduced FEV 1 (after adjustment for height, age and smoking status), and an increased risk of death from r e s p i r a t o r y c a u s e s . 39 A similar study of men and women living in southern India also showed that FEV 1 was associated with positively correlated with birth weight. 4~ More recently, an analysis of lung function in adults who, as fetuses, experienced the Dutch winter famine of the World War II showed that they had a tendency towards impaired lung function. 41 Together, these studies suggest that restricted nutrition during fetal life can alter lung development such that lung function is impaired through to adult life; specifically, it is likely that airway or alveolar development may have been affected.

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Respiratory effects of FGR following preterm birth Improvements in neonatal care over the last two decades have resulted in the survival of most preterm infants (>25 weeks of gestation) and thus it is likely that, in the future, the delayed, postnatal effects of preterm birth may compound those of FGR. In particular, preterm infants who develop chronic lung disease as a result of bronchopulmonary dysplaia (BPD) may be at increased risk of later lung illnesses, particularly if they were also subjected to FGR. A recent study of Canadian births has shown that a large proportion of preterm infants are indeed growth restricted, 42 and the same is likely to be true of other centres in developed countries. Hence, in the future, with increasing survival rates for the youngest preterm infants, the incidence of respiratory illnesses may also increase. Preterm birth in normally grown infants has been shown to convey respiratory deficiencies after birth, even in the absence of lung disease. 43 That is, preterm birth itself, even in the absence of iatrogenic factors, apparently leads to abnormal lung development. Hence it is likely that, with the added insult contributed by FGR, respiratory illness will be exacerbated and possibly prolonged. In the neonatal period, FGR infants who are born prematurely have higher risks of morbidity and mortality 44 and a greater requirement for ventilatory support. 45 A recent study of preterm lambs following FGR has indicated how respiratory function may be additionally compromised. 46 This study showed that the level of hypoxemia in the first 2 weeks of postnatal life was more severe if FGR was confounded by preterm birth (Fig. 17.2a). The ability of the lung to exchange gas, measured by diffusing capacity for carbon monoxide (DLco), was reduced in FGR lambs, and further reduced if born preterm (Fig. 17.2b). A lower DLco indicates impaired pulmonary gas exchange and it is likely that the lower DLco measured during the first 2-4 postnatal weeks contributed to their relative hypoxemia observed during this period. The site at which pulmonary gas exchange occurs (the alveolar blood-air barrier), was thicker following FGR in near term fetuses and postnatal lambs, 28 and it may be even thicker in preterm lambs. While preterm birth in FGR lambs had an

Fig. 17.2. (a) Arterial PO2 (mmHg) and (b) pulmonary diffusing capacity (DLco; ml/min/mmHg/100 ml FRC) measured serially in 3 groups of postnatal lambs between birth and 8 weeks after birth. The lambs were either previously subjected to fetal growth restriction (FGR) by placental embolisation from 120 days of gestation (term, 147 days) and born either prematurely (..O.., n=6) or at term (-O-, n=8), or control lambs (-@-, n=8). In the first 2-4 postnatal weeks PaO 2 and DLco were lower (p < 0.05) in both groups of FGR lambs compared to controls; PaO 2 and DLco in the FGR lambs born preterm were lower than in FGR lambs born at term. Reproduced with permission from Cock et al. 46

additive detrimental affect on gas exchange, it did not further affect the decrease in lung compliance observed following FGR alone.

RESTRICTED

GROWTH

NUTRITION

AFTER

HUMAN

AND

BIRTH:

DATA

Animal studies indicate that impaired nutrition after birth, like prenatal nutrient restriction, can contribute to altered lung development; however, little data on effects in humans are available. During infancy and childhood, there are numerous potential causes of inadequate nutrition. Severe protein deficiency may occur in infancy and childhood in some Third World countries in situations of poverty and during times of famine. Nutrition may also be impaired during disease states, such as respiratory or other infections, and in association with congenital anomalies such as cystic fibrosis. Altered nutrition associated with illnesses during early life may contribute to the later effects of these illnesses on respiratory function. 47'48 This is supported by the finding that size at one year was a significant predictor of later deaths from respiratory c a u s e s . 39 A group that is particularly vulnerable to inadequate nutrition during early development is preterm infants, who represent approximately 10% of all births. Impaired nutrition can occur in these infants as a result of limited fat deposits, parenteral feeding, poor feeding ability, gut immaturity or unavailability of breast milk. It is now thought likely that

undernutrition, both before and after birth, may contribute to the etiology of neonatal chronic lung disease or BPD; 49 hence it has been suggested that preterm infants require particular nutritional management. 49 Owing to ongoing lung development in the preterm infant, undernutrition, or a lack of necessary minerals or micronutrients, could have detrimental effects on lung antioxidant and defence mechanisms, surfactant production, and the process of alveolar formation. Little is known of the effects of postnatal undernutrition after birth on human lung development, although there is a large body of data from animal models supporting such an effect (see below). Effects of malnutrition on lung function have been documented in undernourished children. In Indian children aged 6-12 years, evidence of current malnutrition and body wasting, indicated by low weight for height, was associated with lower than expected peak expiratory flow rates. 5~ In a more recent study, wasted and stunted children were found to have lower lung volumes and reduced forced inspiratory flow rates, 51 indicating that nutrition influences aspects of lung development in children other than lung size. 52 In undernutrition, whether during development or adulthood, it is likely that respiratory skeletal muscle function may be impaired, contributing to reduced flow rates. A number of studies in humans and other mammalian species have shown that undernutrition can impair the function and structure of major respiratory muscles. It is considered possible that nutritional factors resulting in weight loss may contribute to respiratory illnesses such as chronic obstructive lung disease, 53'54 although it is recognized that increased energy expenditure due to airway obstruction may be involved. A study in the rat found that prolonged undernutrition (40% of estimated requirements) led to reduced oxidative capacity in the diaphragm, secondary to a reduced production of NADH in the Krebs cycle. 55 Biopsies of adult human intercostal muscles indicate that nutritional status, as well as gender, was related to altered fibre morphometry, although no relation was found between muscle structure and lung function or respiratory muscle strength. 56 Undernutrition may also affect other aspects of the lungs, such as defence mechanisms. As in animal studies, data from humans indicate that malnutrition causes a reduction in macrophage function, mucociliary clearance and specific B- and T-cell responses to infection. 57 Such effects may explain the elevated incidence of respiratory infections in undernourished infants and children.

EFFECTS OF I M P A I R E D N U T R I T I O N ON THE D E V E L O P I N G LUNGS" EXPERIMENTAL EVIDENCE Numerous experimental studies have explored the relation between early impairment of nutrition or oxygen availability and lung development. This complex but important topic 14 has been the subject of recent comprehensive reviews.-

As oxygen is an essential nutrient, we will consider the impact of chronic hypoxia, as well as restriction of other nutrients, on lung development (effects of chronic hypoxia are also considered in Chapter 18). Concisely reviewing the effects of nutrient restriction on lung development is difficult as results vary according to species, the developmental stage at which nutrition was restricted, and the nature and severity of the dietary restriction (e.g. protein or caloric restriction, acute or chronic). A complicating factor that must be considered is that different species of animals reach term at differing stages of lung development, and hence care must be taken to make comparisons taking these stages into account; this is important, for example, when comparing rodent models, in which alveolar development is largely a postnatal event, s8 with long gestation species such as larger mammals including man, in which alveolar formation begins before birth. 59 Another major problem with studies on the effects of nutrient and oxygen restriction on the fetus is that this intervention can induce a range of endocrine changes that could in themselves affect lung development; such changes include elevated circulating levels of corticosteroids, catecholamines and prostaglandins. 60'61 Even maternal undernutrition can alter the endocrine status of both mother and fetus. 62

Expansion of the fetal lung Throughout much of lung development, the future airways and airspaces are not collapsed but contain a unique liquid (fetal lung liquid/fluid) secreted by the epithelial cells lining the 'airways' (see Chapter 8). This liquid plays an important role in lung development as it maintains the fetal lungs in an expanded state, an essential factor for normal growth and structural development of the lungs (see Chapter 9). As lung liquid secretion depends upon the metabolic activity of the airway epithelial cells, it is likely that this process is affected by restricted nutrient and oxygen availability. Indeed it has been shown that both acute 63'64 and chronic hypoxia and hypoglycemia 65'66 induced in fetal sheep by the restriction of placental blood flow, inhibit the secretion of lung liquid, and alter its composition. 67 However, most critical to lung growth is the volume of luminal liquid retained within the lungs, 68 and it is apparent that this volume is not significantly altered, when adjusted for lung or body weight, as a result of prolonged hypoxemia and hypoglycemia. 66 The reason for this lack of effect is that lung liquid volume is determined principally by physical factors, the most important of which are the pressure gradient across the fetal lungs and the resistance of the upper respiratory tract. 68

Fetal breathing and F G R Fetal breathing movements (FBM) play an important role in lung development as they retard the efflux of liquid from the future airspaces of the lungs, thereby maintaining an adequate level of lung expansion. 6s They may also stimulate lung development via the small phasic alterations in lung dimensions. 69'7~ Surprisingly few studies, however, have

examined FBM in growth restricted fetuses. One such study in sheep examined the effects of FGR induced by restriction of placental mass71; it was found that the proportion of time spent 'apneic' was increased in FGR fetuses. Although it is well established that acute hypoxia of the fetus inhibits FBM, in the presence of chronic hypoxia, FBM return to a frequency and amplitude that are similar to those of a normoxic fetus. 72'73 In the human growth restricted fetus, FBM are reduced in incidence, and this is associated with elevated adenosine concentrations in fetal blood. 74 Thus it appears that FGR, perhaps due to the associated chronic hypoxemia and hypoglycemia, may have an inhibitory effect on FBM; this in turn may affect lung development, although not via a reduction in lung liquid volume. 66

Fetal lung growth Studies in several species have shown that restriction of nutrient supply during fetal life can alter lung development, but the effects vary according to the form and gestational timing of nutritional intervention. FGR induced in sheep by pre-pregnancy removal of placental attachment sites has been shown to either reduce, 71 or have no effect 7s on fetal lung weights, relative to fetal body weight, near to term. In the sheep, 10 days of maternal undernutrition during late gestation reduced lung weight (wet), although fetal body weight was unaffected. 76 More prolonged periods of fetal undernutrition, induced by chronic placental insufficiency, restrict fetal growth, but lung growth relative to body growth is not impaired. 27'66 Similarly, in the rat, 4 days of late gestational undernutrition did not affect fetal lung weight or DNA content. 77

there was a reduction in the number of alveoli per respiratory unit, an increase in the mean diameter of alveoli and a thickening of the inter-alveolar septa (Fig. 17.3).28 This reduction in alveolar number was still evident in 2-year old adult sheep. 81 Thus it appears that adequate nutrition is necessary for normal alveolar formation and that the effects may persist into adulthood. Similar effects on alveolar formation have been made in the undernourished postnatal rat 78 and in the guinea pig following undernutrition late in gestation and during the neonatal period. 82 The causes of impaired alveolar formation in the presence of undernutrition are poorly understood. In the case of fetal growth restriction, numerous metabolic and endocrine changes occur in the fetus which have the potential to affect lung development, for example hypoxia, hypoglycemia and increased circulating glucocorticoid concentrations. 6~It has been shown that glucose transport to the lungs of fetal rats subjected to FGR was reduced, which could contribute to reduced metabolic activity of lung cells. 83

Pulmonary blood-air barrier The tissue separating alveolar gas from pulmonary capillary blood, the blood-air barrier, is essential for normal lung function, as its properties, as well as the total alveolar 8

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Alveolar formation Studies in a number of species have shown that undernutrition during early development can interfere with alveolar formation. Most studies on environmental effects on alveolar formation have been performed in the rat, in which alveoli are formed after birth. 5s In this species, intermittent starvation during the first week after birth (induced by removing the mothers on days 1 and 5) resulted in enlarged alveoli, thicker septa, with an apparent reduction in elastin deposition; these effects were seen at 2 weeks of age but not at one week. 7s In the rat, it has been found that protein deficiency during postnatal development led to changes in lung morphometry which were mostly attributable to growth restriction and smaller lung volume, with normal parenchymal maturity being apparent at weaning. 79 Thus it appears that the degree and type of nutrient restriction during development may determine the form of structural alteration in the lungs. In sheep, the effects of fetal hypoxemia and nutritional restriction during late gestation, a time of rapid fetal growth coinciding with pulmonary saccular and alveolar formation, have been studied in the near-term fetus, 8-week old lamb and 2-year old sheep. In these studies, fetal growth was restricted by repeated umbilical-placental embolisation. 28'29'8~ At 8 weeks after term-birth, but not in near-term fetuses,

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Fig. 17.3. Effects of fetal growth restriction on (a) the thickness of the alveolar walls (Tsept) and (b) number of alveoli per respiratory unit in lungs of fetal and postnatal lambs. Following late gestational growth restriction (placental embolisation from 120 days of gestation), Tsept was not different to controls in near-term fetuses, but was significantly thicker in 8-week old postnatal lambs subjected to FGR. At 8 weeks after birth, but not at 140 days of gestation, there were significantly fewer alveoli per respiratory unit in FOR lambs than in controls. Open bars show data from control animals; filled bars, FGR animals. Taken with permission from Maritz et al. 28

surface area, determine the rate of gas exchange between blood and respired air (pulmonary diffusing capacity). The blood-air barrier is comprised of three components, the endothelial cell, alveolar epithelial type I cell and the interposed basement membrane. It is now apparent that undernutrition during early life can retard the normal thinning of the blood-air barrier that occurs during development, possibly owing to a reduction in the rate of tissue remodelling in the alveolar walls. In a study of fetal rats whose mothers had been undernourished during the latter half of pregnancy, 84 the alveolar epithelial cells appeared less mature with glycogen retention. In fetal sheep that had been subjected to FGR in late gestation, the blood-air barrier was increased in thickness, and this increase was also observed at 8 weeks 28 and 2 years after birth following FGR 81 (Fig. 17.4). This effect of FGR was associated with a reduction in pulmonary diffusing capacity for carbon monoxide during the first 8 weeks after birth. 29 The diffusing capacity of the lung can also be reduced by a reduction in alveolar surface area, which is likely to occur in growth restricted individuals. For example, in guinea pig offspring subjected to maternal undernutrition during gestation the alveolar surface area was reduced, leading to a reduced diffusing capacity, but this was related to the smaller body size. 82 However, a smaller surface area was still apparent at 126 days after birth in spite of catch up growth in body weight and lung volume.

ExtraeeUular matrix The extracellular matrix (ECM) of the lung plays a vital role not only in providing the framework of alveoli but by

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Fig. 17.4. Effects of fetal growth restriction induced by placental embolisation from 120 days of gestation on the harmonic mean thickness of the blood-air barrier (ThBAB) and its components in (a) near-term fetuses and (b) 8-week old postnatal lambs. In both fetuses and lambs, the blood-air barrier and the basement membrane (ThBM) were significantly thicker in animals exposed to fetal growth restriction. The thickness of the endothelium (Then) was increased in FGR fetuses, whereas the thickness of the alveolar epithelial cells (Thep) was not affected in either FGR fetuses or lambs. Open bars show data from control animals; filled bars, FGR animals. Taken with permission from Maritz et al. 28

having a profound influence on the mechanical properties, and hence the function, of the lung. Major groups of ECM components in the lungs are elastic fibres, collagens, proteoglycans and basement membrane proteins. Elastic fibres are involved in the organization of tissue architecture and in tissue elasticity; collagens are also involved in tissue architecture development, provide tensile strength to the lungs and are a major component of basement membranes; proteoglycans exert a large effect on lung compliance and fluid balance due to their large hydrodynamic volumes. 85 These and other structural proteins are laid down during lung development and have a profound effect on the mechanical properties of the lungs after birth. There is considerable evidence that nutritional status during development can have a persistent effect on the pulmonary ECM such that lung function is affected. Effects of major components of the ECM are discussed below.

Elastin Elastin is the principal structural protein of the terminal airspaces, and is encoded by a single gene, the expression of which can be affected by a range of factors including TGF-[3, TNF-~, corticosteroids, vitamin D, basic FGF, IGF-1 and retinoic acid. 86'87 As demonstrated by elastin-null mice, elastin expression is necessary for normal branching and development of the terminal airways. 8s During development, increased lung compliance is related to the presence of elastin, s9 In the lung, the tropoelastin gene is expressed principally by fibroblasts during early development, particularly during the period of alveolar formation. 9~ As the half-life of elastin matches the lifespan of the species, 86 physiological factors affecting the development of elastin have the potential to exert a persistent effect upon elastin content and mechanical properties of distensible organs such as the lung. It is now apparent that a range of intrauterine and early postnatal factors, such as hypoxia, nutritional restriction and FGR, can affect elastin deposition. In isolated rat pulmonary fibroblasts, hypoxia (2%) reversibly downregulates tropoelastin gene expression. 93 A similar inhibitory effect on tropoelastin synthesis has been observed in vitro in pulmonary artery smooth muscle cells from neonatal calves. 94 Hypoxia (12-13% O2) causes an increase in pulmonary lysyl oxidase activity, the extracellular enzyme responsible for crosslinking of elastin and collagen. 95'96 Hypoxia also reversibly inhibits amino acid uptake into human lung fibroblasts. 93 In vivo, an inhibitory effect of hypoxia on elastin synthesis was not observed in growing rats. 97 Protein deficiency in growing rats had little effect on elastin (or collagen), which was supported by the finding that adjusted, saline-filled compliance was not significantly altered. 98 A similar study in growing rats subjected to protein restriction, sufficient to restrict body growth, found a loss of lung desmosine and recoil, when adjusted for lung size, with enlarged alveoli. 99 In contrast, severe starvation of growing postnatal rats, but not adult rats, led to a reduction in pulmonary elastin content and altered lung compliance. 1~176

In humans, elastin accumulates in the lungs between 25 weeks of gestation and 15 weeks after birth, and it is apparent that this accumulation is not significantly affected by FGR. 1~ Similarly, in fetal sheep affected by late gestational FGR, pulmonary tropoelastin expression and elastin content were not significantly different from controls. 1~ During fetal and postnatal life, both hypoxia and fetal undernutrition may be associated with increased levels of corticosteroids; 62 these challenges may exert an effect on elastin synthesis as it has been shown that exogenous corticosteroids can both increase 92 and decrease 1~ elastin formation in the fetal lungs.

Collagen Collagens, which are produced within the lung by fibroblasts and other cell types, are among the strongest soft tissues of the body, providing the lung with mechanical strength and hence protection from damage during inflation. s5 In contrast to elastin, lung collagen is synthesized and degraded throughout life. s5 It is well established that collagen metabolism is affected by nutritional status. TM Lung collagen content was signifcantly reduced in growing and adult rats fed on a low protein diet. 98'99 Collagen IV is a major component of basement membranes, and therefore it affects the strength and function of the blood-gas barrier. 1~ With advancing maturity the blood-gas barrier thins, allowing an increased diffusing capacity. In the sheep, FGR has been found to impair thinning of the blood-air barrier, due to a thickened basement membrane; this was seen in the near-term fetus, and at 8 weeks after birth. 2s More recent evidence suggests that this effect persists for 2 years after birth, which in sheep is early adulthood, sl It is of interest that procollagen gene expression in lung fibroblasts is increased by hypoxia 1~ which is present in fetuses exposed to FGR.

Proteoglycans No data apparently exist on the influence of impaired nutrition or oxygenation on proteoglycan synthesis in the developing lung. In vascular tissue, hypoxia has been shown to decrease proteoglycan production by bovine pulmonary artery endothelial cells 1~ and by human aortic smooth muscle cells, l~

Airway development Data from s o m e 35'37'109 but n o t all 38'47 studies on infants and children suggest that low birth weight due to prenatal growth restriction, resulting from factors such as maternal undernutrition or inadequate placental function, can alter airway function after birth, and it has been suggested that such changes may be long-lasting, potentially accounting for alterations in adult lung function. 41 These studies have used standard tests of airway function, such as peak flow rates or airway responsiveness to challenge by bronchoconstrictors, and their relevance to airway development may be questionable. Very few studies have been performed examining the effects of restricted fetal growth on airway

development, although some evidence exists that airway wall structure can be altered. In a lamb model of FGR induced by placental restriction, tracheal wall structure was affected in the near-term fetus, such that there was less cartilage, less mucosal folding, impaired development of submucosal glands and evidence of reduced ciliation of epithelial cells. 7s In a more recent study, FGR was induced for 20 days during late ovine gestation also affected the structure of cartilaginous airways near to term; the walls were thinner and more folded and the submucosal glands were less well developed, s~ In this study, however, there was postnatal recovery of airway wall thickness (by 8 weeks), but changes in mucus elements were still evident. In rats, early postnatal undernutrition was shown to affect bronchiolar epithelium, such that cell division was reduced, conversion of Clara cells to ciliated cells was reduced and there was a persistent abnormality of bronchiolar epithelial cells. 11~ Such changes could underlie longterm effects of early postnatal undernutrition on lung function. There is clearly a need for more data on the effects of early nutritional compromises on airway function and structure later in life. Effects on surfactant s y s t e m It has been known for some time that maternal underfeeding can affect the surfactant system of fetuses. Underfeeding maternal rats has been shown to impair surface tension lowering properties of fetal lung extracts, 111 and similar effects have been seen when neonates were underfed. 112 Maternal underfeeding of rats during pregnancy slows the prenatal and postnatal maturation of alveolar type II cells of offspring, as indicated by increased glycogen content and decreased volume density of lamellar bodies and rough endoplasmic reticulum; 84'113in this species, type II cells are immature at birth. Similar changes related to undernutrition were seen in bronchiolar Clara cells of rats. 114 There is some evidence that these changes are transient and recovery occurs after birth. 84 In the prenatally undernourished guinea pig, it was found that numbers of alveolar type II cells and their lamellar bodies were not different to controls. In fetal sheep, prolonged mild hypoxemia (48 hours), similar to that associated with placental insufficiency, led to increases in both fetal blood cortisol levels and SP-A mRNA. 115 More prolonged periods of placental restriction during late ovine gestation have been found to have stimulated SP-A and SP-B expression at 0.88 of gestation 34 but not close to term. 66 This suggests that FGR or nutrient restriction may have beneficial effects on lung maturity in preterm infants, but not in those born at term. The effects of undernutrition and hypoxia on surfactant producing cells are likely to differ according to gestational timing and alterations in circulating cortisol levels. However, at present, it is not known whether the effects are long lasting, but recent evidence indicates that the proportion of type II cells in the alveolar epithelium depends upon the prevailing local physical environment.

Lung defence There is now evidence that nutrition can affect the four major defence systems of the lungs, namely antioxidant defence, surfactant production, mucous secretion and transport, and immunological competence. 1 In the developing lung, particularly that of the preterm infant, each of these is particularly important for protection of the lungs and the organism. Further information is provided in Chapters 10-12. It is apparent that malnutrition can impair pulmonary immunological defence including non-specific functions (phagocytic cell functions and inflammation) and specific functions, such as B- and T-cells. 57 For example, in fetal rats from underfed mothers, alveolar macrophages were less abundant. 84 Similarly, maternal underfeeding has been shown to alter the resistance of neonatal rats to oxidant injury. 116 Airway mucus development has been shown to be altered in postnatal lambs following FGR. 8~ Together, the experimental data suggest that ability to resist pulmonary infection in the neonatal period may be impaired in infants subjected to intrauterine nutritional compromises.

EFFECTS OF H Y P O X I A O N THE LUNGS D U R I N G DEVELOPMENT Fetal hypoxemia is commonly associated with FGR and may have different effects on the developing lung to those of nutrient restriction (see also Chapter 18). Prolonged hypoxemia in the absence of hypoglycemia, induced in the ovine fetus by restriction of utero-placental blood flow, led to reduced DNA synthesis in the lungs. 117 In the growth restricted fetal rat, following maternal and hence fetal hypoxia during the latter two-thirds of gestation, the lungs near term were small relative to body weight. 118 However, hypoxia starting from later stages of gestation did not appear to affect fetal lung growth relative to body growth. 118'~19In sheep exposed to hypobaric hypoxia throughout much of gestation, there was no effect on fetal relative lung weight, nor on protein or DNA concentrations. 12~In fetal sheep, acute episodes of hypoxia induced by repeated umbilical cord occlusion have been found to reduced surfactant protein expression. 121 Postnatal hypoxia (hypobaric and normobaric) in growing rats increases lung growth by hypertrophy and hyperplasia, resulting in increased connective tissue. 97 Postnatal hypoxia increases gene expression of extracellular matrix proteins (alpha I, III, IV procollagens, fibronectin) in adult rats. This effect may be due to the induction of pulmonary hypertension and vascular remodeling by alveolar hypoxia. 122

ROLE OF M I C R O N U T R I E N T S IN L U N G D E V E L O P M E N T Micronutrients, the collective term for vitamins and minerals, are now recognized as being of considerable importance in maternal health during pregnancy and in the develop-

ment of the fetus and newborn infant. 123 Of particular relevance to lung development are Vitamins A, D and E, and selenium.

Retinoic acid Vitamin A (retinol) and retinoic acid have long been known to play an important role in lung development during the stages of embryogenesis and organogenesis, and retinoic acid appears to be the most active of the retinoids in terms of interactions with the genome. TM Indeed retinoic acid has been shown to affect many aspects of lung development ranging from early airway branching, structural remodelling during development leading to alveolar formation and the synthesis of surfactant and surfactant proteins. 125 Vitamin A deficiency during gestation has been shown to cause reduced expression of surfactant proteins A, B and C in fetal rat lungs. 126 In recent years, considerable interest has been directed towards the effects of retinoic acid on alveolar formation (see Chapter 4) and surfactant synthesis. The spatio-temporal expression pattern of retinoic acid receptors in the fetal lung supports a role in the maturation of the peripheral airspaces. 127 These effects may involve increased elastin synthesis, as inhibitors of retinoid metabolism decrease tropoelastin expression in fetal rat lung explants, 128 and Vitamin A deficiency during pregnancy in rats reduces elastin staining in fetal lungs. 129 T h e r e is some evidence that a reduction in alveolar formation during early development may be reversible, as treatment with retinoic acid is able to increase alveolar numbers, at least in rats and mice. 13~ In a number of trials on preterm infants, it has been shown that Vitamin A supplementation allows a reduction in oxygen requirements, although the mechanisms are as yet unknown. 132 Vitamin D Vitamin D deficiency is normally associated with impaired calcium metabolism and bone disorders; these may impact upon respiratory function via metabolic bone disease affecting the rib cage. 133 Vitamin D can also affect the developing lung directly. In contrast to the mature lung, the developing lung possesses specific binding sites for Vitamin D, specifically on type II alveolar epithelial cells. TM In fetal rat lung explants, Vitamin D has the effect of stimulating maturational changes in type II cells; in particular, the amount of glycogen was reduced and there was evidence of increased surfactant synthesis by these cells. 135 Vitamin E At birth, the lung is exposed to high levels of oxygen, with the result that reactive oxygen species are increased. Antioxidative agents include Vitamins E, C, A and B2.136 Among these, Vitamin E is the most important lipophilic, radical-scavenging vitamin, and a number of recent reports have implicated it with fetal and neonatal lung development; in particular, there has been interest in the possible role of Vitamin E in the treatment of lung injury and respiratory

distress in preterm infants. As Vitamin E accumulates in the fetus, along with fat accumulation, during late gestation, it is likely that preterm infants are Vitamin E deficient. 137 Vitamin E is taken up by type II cells and may therefore play a role in respiratory distress and chronic lung disease of prematurity. However, Vitamin E supplementation of preterm infants with bronchopulmonary dysplasia had no beneficial effect. 138

Selenium Selenium is important to lung development as it is necessary for the activity of glutathione peroxidase, an important antioxidant defence enzyme which reduces both organic and inorganic hydroperoxidases. Experimental selenium deficiency in rats led to altered lung development, in particular attenuated alveolar septation, whether they were raised in air or high oxygen levels. 139 Low selenium levels develop in preterm infants that are fed parenterally, but less so in those fed breast milk; however, even healthy preterm infants had lower levels than term infant at 6 weeks after birth. ~4~ In preterm infants, low plasma selenium levels are associated with increased respiratory morbidity; TM however, selenium supplementation of very preterm infants failed to alter neonatal oxygen dependency. 142

NUTRITIONAL MATURITY

RESTRICTION

(ADULT

AFTER STARVATION)

It has been recognized for some time that nutritional deprivation during adulthood, for example during starvation or severe protein restriction, can adversely affect lung function and structure in otherwise healthy individuals. 1 Undernutrition during maturity can also affect pulmonary surfactant, respiratory control and muscle function. 143 The pulmonary effects of impaired nutrition may in part be due to the breakdown of structural proteins in the lungs ~44 which may exacerbate chronic lung diseases such as COPD. 54 Nutrition is known to affect all aspects of host defence, and it is now well established that sub-optimal nutrition can impair the immune functions of the lungs. 57

Nutritional emphysema In rats, severe postnatal starvation (3 weeks of food deprivation) reduces both lung and body weights, the effects being more marked in younger, rapidly growing animals than in older (non-growing) animals. 1~176 In the younger animals connective tissue including elastin and collagen was lost from the lungs. Less severe starvation caused similar effects on the lung ECM, resulting in a reduction in tissue elasticity 145 and increases in mean linear intercept, an index of alveolar size. 146 It seems unlikely that the loss of elastin and collagen was due to protein restriction; in young adult rats, it was found that protein restriction caused less severe emphysema than did caloric restriction, and the effects of protein restriction appeared to be due to a loss of growth in the lungs. 147

While the processes underlying nutritional emphysema remain unclear, it is apparent that the structural component of the lung most affected by impaired nutrition is the interalveolar septum, or alveolar capillary membrane. 144 It is believed that nutritional emphysema is largely due to the breakdown of structural proteins of the septum, in particular, elastin and collagen; however, the undernutrition must be severe to induce these changes. In the rat, it has been shown that starvation caused a reversible reduction in the activity of lysyl oxidase, an enzyme involved in post-transational cross-linking of elastin and collagen (Fig. 17.5). 148 Elastase treatment of the lungs of rats has been shown to induce a form of emphysema that more closely resembles the condition in humans than undernutrition, suggesting that elastin breakdown may be involved in the humandisease. 149 Of considerable interest, a recent study has shown that nutritional emphysema in adult animals is reversible; caloric restriction in mice reduced the number of alveoli and alveolar surface area, as occurs in human emphysema, but refeeding fully reversed these changes. 15~This suggests that alveolar walls are able to regenerate following their destruction due to undernutrition. The effect of undernutrition may be species-dependent; although starvation of hamsters caused an enlargement of airspace dimensions and a reduction in surface area, it was not associated with a reduction of pulmonary collagen, elastin or glycosaminoglycan content, nor was there evidence of alveolar wall destruction. 151 Such differences may be due to differences in somatic growth at the time of the nutritional insult. In general, however, it appears that restricted substrate availability, protein degradation, impaired crosslinking of pulmonary structural proteins and an imbalance between synthetic and catabolic processes, all contribute to the remodeling that results in emphysematous changes in adult lung structure. 2

20

[ ~'-

t

10

o

. 0

.

. 24

.

,//t~ 48

72

144

Hours Fig. 17.5. Effects of starvation on the specific activity of lysyl oxidase in the lungs of postnatal rats. Lysyl oxidase activity, which is essential for cross-linking in the formation of elastin fibres, was profoundly reduced throughout 144 hours of starvation (-O-, n= 5) compared to normally fed controls (-@-, n= 5). In a separate group of rats (--7-1--, n= 5), lysyl oxidase activity was reduced after 48 hours of starvation but returned to control values following 3 hours of refeeding. Data taken from Madia et al. 148

Pulmonary surfactant As during early development 111'152 nutritional status can affect pulmonary surfactant during maturity. A nutritional effect in adults can be induced rapidly; for example, after 72 hours of fasting, the pulmonary surfactant content of adult rat lungs was reduced, as was lung compliance and the volume density of lamellar bodies in type II cells. 153 These effects may be due to a reduced availability of substrates necessary for the synthesis of surfactant or surfactant proteins. In adult rats, caloric restriction or complete fasting caused a reduction in pool size of pulmonary surfactant, but within 4-8 days, these pools had recovered to normal sizes, 154 suggesting that the lung is able to adapt rapidly to reduced nutrient availability.

CONCLUSIONS It is now well established that impaired nutrition (including hypoxia) can affect the respiratory system at all stages of life; as well as the lung, nutritional status can affect the neurochemical control of breathing and respiratory muscles. Owing to the diverse models employed to investigate the impact of nutritional restrictions on the lungs, it is extremely difficult, and perhaps dangerous, to make generalizations as to the consequences of individual classes of nutrients. It can be concluded, however, that as the lung is an organ whose architecture is laid down early in life, nutritional compromises during development, as well as other environmental challenges, have the potential to induce long-lasting alterations in lung structure and function; these alterations form the basis of 'tracking' of lung function. Multiple aspects of lung structure and function can be affected by nutritional factors during development, but those that are most likely to have persistent effects on lung function are alterations in structural proteins such as elastin and basement m e m b r a n e proteins, formation of alveoli and airway structure. Although it is suggested by studies of human subjects that early nutrition and growth, both before and soon after birth, can lead to later alterations in lung function and respiratory health, there is a need for controlled studies using animal models to define the specific alterations in the lung.

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36. Rona RJ, Gulliford MC, Chinn S. Effects of prematurity and intrauterine growth on respiratory health and lung function in childhood. Br. Med. 1993; 306:817-20. 37. Nikolajev K, Heinonen K, Hakulinen A et al. Effects of intrauterine growth retardation and prematurity on spirometric flow values and lung Volumes at school age in twin pairs. Pediatr. Pulmonol. 1998; 25:367-70. 38. Nikolajev K, Korppi M, Remes K etal. Determinants of bronchial responsiveness to methacholine at school age in twin pairs. Pediatr. Pulmonol. 2002; 33:167-73. 39. Barker DJP, Godfrey KM, Fall C et al. Relation of birth weight and childhood respiratory infection to adult lung function and death from chronic obstructive airways disease. Br. Med. 1991; 303:671-5. 40. Stein CE, Kumaran K, Fall CH et al. Relation of fetal growth to adult lung function in south India. Thorax 1997; 52:895-9. 41. Lopuha~i CE, Roseboom TJ, Osmond C etal. Atopy, lung function, and obstructive airways disease after prenatal exposure to famine. Thorax 2000; 55:555-61. 42. Lackman F, Capewell V, Richardson B etal. The risks of spontaneous preterm delivery and perinatal mortality in relation to size at birth according to fetal versus neonatal growth standards. Am. J. Obstet. Gyncol. 2001; 184:946-53. 43. Hjalmarson O, Sandberg K. Abnormal lung function in healthy preterm infants. Am. J. Respir. Crit. Care Med. 2002; 165:83-7. 44. Piper JM, Xenakis EM, McFarland M etal. Do growthretarded preterm infants have different rates of perinatal morbidity and mortality than appropriately grown premature infants? Obstet. Gynecol. 1996; 87:169-74. 45. Morley R, Brooke OG, Cole TJ etal. Birthweight ratio and outcome in preterm infants.Arch. Dis. Child. 1990; 65:30-4. 46. Cock ML, Camm EJ, Louey S et al. Postnatal outcomes in term and preterm lambs following fetal growth restriction. Clin. Exp. Pharmacol. Physiol. 2001; 28:931-7. 47. Shaheen SO, Sterne JA, Tucker JS etal. Birth weight, childhood lower respiratory tract infection, and adult lung function. Thorax 1998; 53:549-53. 48. Pullan CR, Hey EN. Wheezing, asthma, and pulmonary dysfunction 10 years after infection with respiratory syncitial virus in infancy. Br. Med. 1982; 284:1665-9. 49. Ryan S. Nutrition in neonatal chronic lung disease. Eur. J. Pediatr. 1998; 157(Suppl. 1):S19-22. 50. Primhak R, Coates FS. Malnutrition and peak expiratory flow rate. Eur. Respir. J. 1988; 1:801-3.

51. Nair RH, Kesavachandran C, Shashidhar S. Spirometric impairments in undernourished children. Indian J. Physiol. Pharmacol. 1999; 43:467-73. 52. Ong TJ, Mehta A, Ogston S etal. Prediction of lung function in the inadequately nourished. Arch. Dis. Child. 1998; 79:18-21. 53. Lewis MI, Belman MJ. Nutrition and the respiratory muscles. Clin. Chest Med. 1988; 9:337-48. 54. Schols AM. Nutrition in chronic obstructive pulmonary disease. Curr. Opin. Pulmonol. Med. 2000; 6:110-5. 55. Matecki S, Py G, Peyreigne C et al. Effect of prolonged undernutrition on rat diaphragm mitochondrial respiration. Am. J. Respir. Cell Mol. Biol. 2002; 26:239-45. 56. Hards JM, Reid WD, Pardy RL et al. Respiratory muscle fiber morphometry. Correlation with pulmonary function and nutrition. Chest 1990; 97:1037-44. 57. Bellanti JA, Zeligs BJ, Kulszycki LL. Nutrition and development of pulmonary defense mechanisms. Pediatr. Pulmonol. 1997; 16:170-1. 58. Massaro D, Teich N, Maxwell Set al. Postnatal development of alveoli. Regulation and evidence for a critical period in rats. Anat. Rec. 1974; 180:77-98. 59. Burri PH. Development and growth of the human lung. In: Handbook of Physiology, Fishman AP, Cherniack NS, Widdicombe JG et al. (eds). American Physiological Society, Bethesda, 1986, pp. 1-46. 60. Gagnon R, Challis J, Johnston Let al. Fetal endocrine responses to chronic placental embolization in the late-gestation ovine fetus.Am. J. Obstet. Gynecol. 1994; 170:929-38. 61. Gagnon R, Murotsuki J, Challis JRG etal. Fetal sheep endocrine responses to sustained hypoxemic stress after chronic fetal placental embolization. Am. J. Physiol. 1997; 272:E817-23. 62. Dwyer CM, Stickland NC. The effects of maternal undernutrition on maternal and fetal serum insulin-like growth factors, thyroid hormones and cortisol in the guinea pig. J. Dev. Physiol. 1992; 18:303-13. 63. Hooper SB, Dickson KA, Harding R. Lung liquid secretion, flow and volume in response to moderate asphyxia in fetal sheep.J. Dev. Physiol. 1988; 10:473-85. 64. Wallace MJ, Hooper SB, McCrabb GJ etal. Acidaemia enhances the inhibitory effect of hypoxia on fetal lung liquid secretion in sheep. Reprod. Fertil. Dev. 1996; 8:327-33. 65. Hooper SB, Harding R. Changes in lung liquid dynamics induced by prolonged fetal hypoxemia. J. Appl. Physiol. 1990; 69:127-35. 66. Cock ML, Albuquerque C, Joyce BJ etal. Effects of intrauterine growth restriction on lung liquid dynamics and lung development in fetal sheep. Am. J. Obstet. Gynecol. 2001; 184:209-16. 67. Albuquerque C, Boland R, Cock ML etal. Lung fluid composition in the chronically hypoxemic ovine fetus. Am. J. Obstet. Gynecol. 1998; 178:$37. 68. Harding R, Hooper SB. Regulation of lung expansion and lung growth before birth.J. Appl. Physiol. 1996; 81:209-24. 69. Harding R, Liggins GC. Changes in thoracic dimensions induced by breathing movements in fetal sheep. Reprod. Fertil. Dev. 1996; 8:117-24. 70. Liu M, Skinner SJM, Xu J e t al. Stimulation of fetal rat lung cell proliferation in vitro by mechanical stretch. Am. J. Physiol. 1992; 263:L376-83. 71. Maloney JE, Bowes G, Brodecky Vet al. Function of the future respiratory system in the growth retarded fetal sheep. J. Dev. Physiol. 1982; 4:279-97. 72. Koos BJ, Kitanaka T, Matsuda K et al. Fetal breathing adaptation to prolonged hypoxaemia in sheep. J. Dev. Physiol. 1988; 10:161-6. 73. Bocking AD, Gagnon R, Milne KM et al. Behavioural activity during prolonged hypoxemia in fetal sheep. J. Appl. Physiol. 1988; 65:2420-6.

74. Yoneyama Y, Shin S, Iwasaki T etal. Relationship between plasma adenosine concentration and breathing movements in growth-retarded fetuses. Am. J. Obstet. Gynecol. 1994; 171:701-6. 75. Rees S, Ng J, Dickson K etal. Growth retardation and the development of the respiratory system in fetal sheep. Early Hum. Dev. 1991; 26:13-27. 76. Harding JE, Johnston BM. Nutrition and fetal growth. Reprod. Fertil. Dev. 1995; 7:539-47. 77. Rhoades RA, Ryder DA. Fetal lung metabolism response to maternal fasting. Biochim. Biophys. Acta 1981; 663:621-9. 78. Das RM. The effects of intermittent starvation on lung development in suckling rats. Am. J. Pathol. 1984; 117:326-32. 79. Kalenga M, Tschanz SA, Burri PH. Protein deficiency and the growing rat lung. II. Morphometric analysis and morphology. Pediatr. Res. 1995; 37: 789-95. 80. Wignarajah D, Cock ML, Pinkerton KE etal. Influence of intra-uterine growth restriction on airway development in fetal and postnatal sheep. Pediatr. Res. 2002; 51:681-8. 81. Maritz GS, Cock ML, Louey S etal. Altered lung structure persists until maturity following fetal growth restriction. Am. J. Respir. Crit. Care Med. 2003; 167:A379. 82. Lechner AJ 1985 Perinatal age determines the severity of retarded lung development induced by starvation. Am. Rev. Respir. Dis. 2002; 131:638-43. 83. Simmons RA, Gounis AS, Bangalore SA etal. Intrauterine growth retardation: fetal glucose transport is diminished in lung but spared in brain. Pediatr. Res. 1992; 31:59-63. 84. Curie DC, Adamson IY. Retarded development of neonatal rat lung by maternal malnutrition. J. Histochem. Cytochem. 1978; 26:401-8. 85. Chambers RC, Laurent GJ. The lung. In: Extracellular Matrix. Comper WD (ed.). Hardwood. Academic Publishers, Amsterdam, 1996, pp. 378-409. 86. Mecham RP. Elastic Fibers. In: The Lung RG Crystal, West JB, Weibel ER, Barnes PJ (eds). Lippincott-Raven, Philadelphia, 1997, pp. 729-36. 87. Foster JA, Curtiss SW. The regulation of lung elastin synthesis. Am. J. Physiol. 1990; 259:L13-23. 88. Wendel DP, Taylor DG, lbertine KH et al. Impaired distal airway development in mice lacking elastin. Am. J. Respir. Cell Mol. Biol. 2000; 23:320-6. 89. Nardell EA, Brody JS. Determinants of mechanical properties of rat lung during postnatal development. J. Appl. Physiol. 1982; 53:140-8. 90. Shibahara S, Davidson JM, Smith K etal. Modulation of tropoelastin production and elastin messenger ribonucleic acid activity in developing sheep lung. Biochemistry 1981; 20:6577-84. 91. Pierce RA, Mariani TJ, Senior RM. Elastin in lung development and disease. In: The Molecular Biology and Pathology of Elastic Tissues. Chadwick DJ, Goode JA (eds). J Wiley, New York, 1995, pp. 199-214. 92. Pierce RA, Mariencheck WI, Sandefur Set al. Glucocorticiods upregulate tropoelastin expression during late stages of fetal lung development.Am. J. Physiol. 1995; 268:L491-500. 93. Berk JL, Massoomi N, Hatch C et al. Hypoxia downregulates tropoelastin gene expression in rat lung fibroblasts by pretranslational mechanisms. Am. J. Physiol. 1999; 277:L566-72. 94. Stenmark KR, Aldashev AA et al. Cellular adaptation during chronic neonatal hypoxic pulmonary hypertension. Am. J. Physiol. 1991; 261:97-104. 95. Brody JS, Vaccaro C. Postnatal formation of alveoli: interstitial events and physiologic consequences. Fed. Proc. 1979; 38:215-23. 96. Brody JS, Kagan H, Manalo A. Lung lysyl oxidase activity: relation to lung growth. Am. Rev. Respir. Dis. 1979; 120:L1289-95.

97. Sekhon HS, Thurlbeck WM. Lung growth in hypobaric normoxia, normobaric hypoxia, and hypobaric hypoxia in growing rats.J. Appl. Physiol. 1995; 78:L124-31. 98. Myers BA, Dubick MA, Gerreits J etal. Protein deficiency effects on lung mechanics and the accumulation of collagen and elastin in rat lung.J. Nutr. 1983; 113:2308-15. 99. Matsui R, Thurlbeck WM, Fujita Y et al. Connective tissue, mechanical, and morphometric changes in the lungs of weanling rats fed a low protein diet. Pediatr. Pul. 1989; 7:L159-66. 100. Sahebjami H, MacGee J. Effects of starvation on lung mechanics and biochemistry in young and old rats. J. Appl. Physiol. 1985; 58:778-84. 101. Desai R, Wigglesworth JS, Aber V. Assessment of elastin maturation by radioimmunoassay of desmosine in the developing human lung. Early Hum. Dev. 1988; 16:61-71. 102. Joyce BJ, Cock ML, Maritz GS et al. Tropoelastin expression and elastin content in fetal and postnatal lung following intra-uterine growth restriction. Am. J. Respir. Crit. Care Med. 2002; 164:A642. 103. Willet KE, McMenamin P, Pinkerton KE etal. Lung morphometry and collagen and elastin content: changes during normal development and after prenatal hormone exposure in sheep. Pediatr. Res. 1999; 45:615-25. 104. Berg RA, Kerr JS. Nutritional aspects of collagen metabolism. Ann. Rev. Nutr. 1992; 12:369-90. 105. West JB, Mathieu-Costello O. Structure, strength, failure, and remodeling of the pulmonary blood-gas barrier. Annu. Rev. Physiol. 1999; 61:543-72. 106. Zhao L. Yang H, Guo X etal. Effect ofhypoxia on proliferation and alpha 1 (I) procollagen gene expression by human fetal lung fibroblasts. Chung-Kuo i Hsueh Ko Yuan Hsueh Pao Acta Academiae Medicinae Sinicae 1998; 20:109-13. 107. Humphries DE, Lee SL, Fanburg B etal. Effects of hypoxia and hyperoxia on proteoglycan by bovine pulmonary artery endothelial cells. J. Cell Physiol. 1986; 126:249-53. 108. Figueroa JE, Tao Z, Arphie TG et al. Effect of hypoxia and hypoxia/reoxygenation on proteoglycan metabolism by vascular smooth muscle cells. Atherosclerosis 1999; 143:135-44. 109. Dezateux C, Stocks J. Lung development and early origins of childhood respiratory illness. Br. Med. 1997; 53:40-57. 110. Massaro GD, McCoy L, Massaro D. Postnatal undernutrition slows development of bronchiolar epithelium in rats. Am. J. Physiol. 1988; 255:R521-6. 111. Faridy EE. Effect of maternal malnutrition on surface activity of fetal lungs in rats.J. Appl. Physiol. 1975; 39:535-40. 112. Guarner V, Tordet C, Bourbon JR. Effects of maternal protein-caloric malnutrition on the phospholipid composition of surfactant isolated from fetal and neonatal rat lungs. Compensation by inositol and lipid supplementation. Pediatr. Res. 1992; 31:629-35. 113. Massaro GD, Clerch L, Massaro D. Perinatal anatomic development of alveolar type II cells in rats. Am. J. Physiol. 1986; 251 :R470-5. 114. Massaro GD, Davis L, Massaro D. Postnatal development of the bronchiolar Clara cell in rats. Am. J. Physiol. 1984; 247:C197-203. 115. Braems GA, Yao L, Inchley K et al. Ovine surfactant protein cDNAs: use in studies on fetal lung growth and maturation after prolonged hypoxemia. Am. J. Physiol. Lung Cell Mol. Physiol. 2000; 278: 754-64. 116. Langley-Evans SC, Phillips GJ, Jackson AA. Fetal exposure to low protein maternal diet alters the susceptability of young adult rats to sulfur dioxide-induced lung injury. J. Nutr. 1997; 127:202-9. 117. Hooper SB, Bocking AD, White S etal. DNA synthesis is reduced in selected fetal tissues during prolonged hypoxemia. Am. J. Physiol. 2002; 261:508-14.

118. Faridy EE, Sanii MR, Thliveris JA. Fetal lung growth: influence of maternal hypoxia and hyperoxia in rats. Respir. Physiol. 1988; 73:225-42. 119. Larson JE, Thurlbeck WM. The effect of experimental maternal hypoxia on fetal lung growth. Pediatr. Res. 1988; 24:156-9. 120. Jacobs R, Robinson JS, Owens JA etal. The effect of prolonged hypobaric hypoxia on growth of fetal sheep. J. Dev. Physiol. 1988; 10:97-112. 121. Nardo L, Zhao L, Possmayer F et al. The effects of repeated umbilical cord occlusions (rUCO) on surfactant protein (SP) mRNA levels in the ovine fetal lung. J. Soc. Gynecol. Invest. 2002; 9:267A. 122. Berg JT, Breen EC, Fu Z, Mathieu-Costello O et al. Alveolar hypoxia increases gene expression of extracelluar matrix proteins and platelet-derived growth factor-B in lung parenchyma. Am. J. Respir. Crit. Care Med. 1998; 158:1920-80. 123. Black RE. Micronutrients in pregnancy. Br. J. Nutr. 2001; 85:S193-7. 124. Chytil F. Retinoids in lung development. FASEB J. 1996; 10:986-92. 125. Zachman RD. Role of Vitamin A in lung development. J. Nutr. 1995; 125:1634S-8S. 126. Chailley-Heu B, Chelly N, Lelievre-Pegorier M e t a l . Mild vitamin A deficiency delays fetal lung maturation in the rat.Am. J. Respir. Cell Mol. Biol. 1999; 21:89-96. 127. Grummer MA, Thet LA, Zachman RD. Expression of retinoic acid receptor genes in fetal and newborn rat lung. Pediatr. Pulmonol. 1994; 17: 234-8. 128. McGowan SE, Doro MM, Jackson SK. Endogenous retinoids increase perinatal elastin gene expression in rat lung fibroblasts and fetal explants. Am. J. Physiol. 1997; 273:410-6. 129. Antipatis C, Ashworth CJ, Grant Get al. Effects of maternal vitamin A status on fetal heart and lung: changes in expression of key developmental genes. Am. J. Physiol. 1998; 275:L1184-91. 130. Massaro GD, Massaro D. Postnatal treatment with retinoic acid increases the number of pulmonary alveoli in rats. Am. J. Physiol. 1996; 270:L305-10. 131. Massaro GD, Massaro D. Retinoic acid treatment partially rescues failed septation in rats and mice. Am. J. Physiol. 2000; 278:L955-60. 132. Darlow BA, Graham PJ. Vitamin A supplementation for preventing morbidity and mortality in very low birthweight infants. Cochrane Database Syst. Rev. 2000; 2: CD000501. 133. Glasgow JF, Thomas PS. Rachitic respiratory distress in small preterm infants.Arch. Dis. Child. 1977; 52:268-73. 134. Marin L, Dufour ME, Tordet C et al. 1,25 (OH) 2D3 stimulates phospholipid biosynthesis and surfactant release in fetal rat lung explants. Biol. Neonate 1990; 57:257-60. 135. Marin L, Dufour ME, Nguyen TM etal. Maturational changes induced by 1 alpha, 25-dihydroxyvitamin D3 in type II cells from fetal rat lung explants. Am. J. Physiol. 1993; 265:L45-52. 136. Bohles H. Antioxidative vitamins in prematurely and maturely born infants. Int.J. Vitam. Nutr. Res. 1997; 67:321-8.

137. Chan DK, Lim MS, Choo SH etal. Vitamin E status of infants at birth.J. Perinat. Med. 1999; 27:395-8. 138. Watts JL, Milner R, Zipursky A et al. Failure of supplementation with vitamin E to prevent bronchopulmonary dysplasia in infants less than 1,500 g birth weight. Eur. Respir. J. 1991; 4:188-90. 139. Kim HY, Picciano MF, Wallig MA et al. The role of selenium nutrition in the development of neonatal rat lung. Pediatr. Res. 1991; 29:440-5. 140. Daniels L, Gibson R, Simmer K. Selenium status of preterm infants: the effect of postnatal age and method of feeding. Acta Paediatr. 1997; 86:281-8. 141. Darlow BA, Inder TE, Graham PJ et al. The relationship of selenium status to respiratory outcome in the very low birth weight infant. Pediatrics 1995; 96:314-9. 142. Darlow BA, Winterbourn CC, Inder TE et al. The effect of selenium supplementation on outcome in very low birth weight infants: a randomized controlled trial. J. Pediatr. 2000; 136:473-80. 143. Chin R, Haponik EF. Nutrition, respiratory function, and disease. In: Modern Nutrition in Health and Disease. Shils ME, Olson JA, Shike M (eds). Lea & Febiger, Philadelphia, 1994, pp. 1374-90. 144. Riley DJ, Thakker-Varia S. Effect of diet on lung structure, connective tissue metabolism and gene expression. Exp. Biol. 1994; 125:1657S-60S. 145. Sahebjami H, Vassallo CL, Wirman JA. Lung mechanics and ultrastructure in prolonged starvation. Am. Rev. Respir. DIS. 1978; 117:77-83. 146. Sahebjami H, Vassallo CL. Effects of starvation and refeeding on lung mechanics and morphometry. Am. Rev. Respir. DIS. 1979; 119:443-51. 147. Kerr JS, Riley DJ, Lanza-Jacoby S etal. Nutritional emphysema in the rat Influence of protein depletion and impaired lung growth. Am. Rev. Respir. DIS. 1985; 131:644-50. 148. Madia AM, Rozovski SJ, Kagan HM. Changes in lung lysyl oxidase activity in streptozotocin-diabetes and in starvation. Biochem. Biophys. Acta 1979; 585:481-7. 149. Harkema JR, Mauderly JL, Gregory RE et al. A comparison of starvation and elastase models of emphysema in the rat. Am. Rev. Respir. Dis. 1984; 129:584-91. 150. Massaro GD, Radaeva S, Clerch LB etal. Lung alveoli: endogenous programmed destruction and regeneration. Am. J. Physiol. Lung Cell Mol. Physiol. 2002; L305-9. 151. Karlinsky JB, Goldstein RH, Ojserkis Bet al. Lung mechanics and connective tissue levels in starvation-induced emphysema in hamsters. Am. J. Physiol. 1986; 251 :R282-8. 152. Lin Y, Lechner AJ. Surfactant content and type II cell development in fetal guinea pig lungs during prenatal starvation. Pediatr. Res. 1991; 29:288-91. 153. Gail DB, Massaro GD, Massaro D. Influence of fasting on the lung.J. Appl. Physiol. 1977; 42:88-92. 154. Brown LA, Bliss AS, Longmore WJ. Effect of nutritional status on the lung surfactant system: food deprivation and caloric restriction. Exp. Lung Res. 1984; 6:133-47. 155. Edwards LJ, Simonetta G, Owens JA et al. Restriction of placental and fetal growth in sheep alters fetal blood pressure responses to angiotensin II and captopril. J. Physiol. 2001; 515:897-904.

INTRODUCTION

It has been estimated that 139 million people reside at altitudes of > 2500m (> 8200 feet). 1 Environmental stressors at altitude are cold and hypoxia, and the capacity of humans to thrive under such adverse conditions attests to their adaptability as a species. Acclimatisation and adaptation to altitude represent a continuum in the process of biological and genetic change which, at its best, enables an animal to survive, tolerate and thrive when they ascend to and live at altitude. Acclimatisation occurs relatively quickly (hours to weeks), whereas adaptation occurs more slowly (decades or generations). The degree of adaptation between individuals and populations varies considerably as a consequence of genetic factors and pressures of natural selection. Many studies have been published on the acute effects of altitude on the respiratory system. This chapter reviews the impact of hypoxia on lung growth and development in the fetus, as well as on the lungs of the adolescent and adult, largely by examining evidence from studies performed in populations who have lived at altitude for decades and/or generations. We will also briefly comment on the evidence for cellular alterations at altitude.

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PO 2

ALTITUDE

The concentration of oxygen in the atmosphere is about 21% and this remains constant as altitude increases. At sea *To whom correspondence should be addressed. The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

level, the barometric pressure (Pb) is 7 6 0 m m H g but this falls with ascent to altitude due to the diminishing 'weight' of the overlying atmosphere. Ambient inspired PO 2 (PIO2) is the product of Pb and oxygen concentration and PIO 2 therefore falls in direct proportion to the fall in Pb as altitude increases. At 3000m (9843 feet) the atmospheric pressure is 537mmHg, and the PIO 2 will be 103mmHg compared to 150 m m H g at sea level (see Table 18.1). An important determinant of arterial PO 2 (PaO2) is the partial pressure of oxygen in alveolar gas (PAO2). PAO 2 can be estimated using the alveolar air equation (PAO 2- PIO 2[PACO2/0.8], where PIO 2 is the partial pressure of inspired air, given by 0.21(Pb-47), 0.8 is the respiratory exchange ratio, and 47 is the partial pressure of water vapour in the lung at 37 ~ Note that the PAO 2 is critically dependent on the partial pressure of alveolar carbon dioxide (PACO2). Assuming a constant PACO2 of 40 mmHg, the PAO 2 will be 100 m m H g at sea level but at 3000 m (i.e. P b - 537 mmHg) it falls to only 53 m m H g (see Table 18.1). At altitude, however, alveolar ventilation increases due to the hypoxic drive to ventilation. This reduces the PACO 2 with a concomitant increase in PAO 2 and represents an important compensatory mechanism that maintains PAO 2 and hence PaO 2 levels at altitude. A doubling of alveolar ventilation (VA) at 3000 m for a given rate of CO 2 production (VCO2) effectively reduces the PACO 2 from its normal sea level value of 40 m m H g to 20 m m H g (since PACO 2 ~ VCOz/VA), resulting in an increase in PAO 2 from 53 m m H g to approximately 78 mmHg. This reduction of PACO 2 through increased ventilation is one of the key mechanisms which allows some mountaineers to ascend to the summit of Mount Everest without supplementary oxygen. Indeed, at such extremes of Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

altitudes hyperventilation can force the PACO 2 down to levels as low as 7.5 mmHg.

ACCLIMATISATION AND ADAPTATION TO ALTITUDE Human biological responses to living at altitude are complex and it is not easy to differentiate adaptive changes due to hypoxia from those due to the effects of genetics, nutritional status, cultural practices and socioeconomic differences. It is also difficult to differentiate between the influence of genetic (i.e. inheritable) factors from long-term biological adaptations which may similarly affect lung development. Evidence for adaptation of the respiratory system to altitude comes from studies which have compared populations resident at, or near, sea level with long-term residents at altitudes greater than 2500m. Many of the physiological differences described between such populations can be rather difficult to interpret as apparent biological adaptations to altitude are confounded by the fact that the populations are often not identical in terms of race and body size. Further, the duration of residency at altitude may range from only a few years to many generations making direct comparisons between the many published studies complex. However, comparing populations who have migrated to altitude relatively recently with those who have dwelt there for many generations does provide some useful information about the variable contributions of genetic and physiological adaptation.

PREGNANCY, THE FETUS A N D POSTNATAL G R O W T H Residents at high altitude have fewer children than their lowland counterparts. This is due to increased mortality during fetal and neonatal life and a shorter reproductive period rather than reduced fertility per se or to increased

rates of prematurity, z'3 At altitude, placental weight and blood flow in relation to the size of the fetus are increased and serve to facilitate the transport of oxygen to the fetus. 4'5 The hypoxic ventilatory drive in highland natives is blunted relative to lowlanders (see below). However, during pregnancy at altitude the mothers' hypoxic ventilatory drive increases, thereby facilitating fetal oxygenation. 6-8 Postpartum, the mothers ventilatory drive diminishes and is again blunted relative to sea level values. 6 These strategies for maximising oxygen transport to the fetus are critically important because birth weight is directly related to fetal blood oxygen levels. The effect of altitude on intrauterine growth is to reduce birth weight by approximately 100 gm for each 1000 m increase in altitude (Fig. 18.1). 1 This fetal growth restriction is related to the reduced supply of oxygen from placental blood at altitude. 1 Intrauterine growth restriction is not associated with reduced gestational age and is believed to be less severe in residents who have spent many generations at altitude which suggests a degree of adaptation or perhaps genetic advantage. 3'9'1~ The restricted early growth appears to continue into infancy with continued exposure to altitude 11 although the effect of other factors such as nutritional and socioeconomic status probably play a role and make interpretation purely in terms of the hypoxic stress difficult. 12'13 Growth restriction appears to continue into adulthood and adolescent females resident at high altitude are not as tall and weigh less than their lowland counterparts. 13 Interestingly, ventilatory response to the effects of hypoxia in the fetus and adult human are quite different. The net outcome of hypoxia depends on the balance between the stimulating peripheral and inhibitory central chemoreceptors. TM In the adult the peripheral chemoreceptors dominate and they stimulate ventilation in response to hypoxia. However, in the infant the central chemoreceptors play the major role and they effectively suppress ventilation as PaCO 2 falls due to hypoxic hyperventilation. 15 Based on

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Fig. 18.1. Mean birth weights of high altitude residents of North America, South America and Tibet (labelled). The dotted lines show the 90% confidence limits. (Reprinted from Moore LG, Zamudio S, Curran-Everett L et al. Genetic adaptation to high altitude. In: Wood SC, Roach RC (eds). Sports and Exercise Medicine. Marcel Dekker, New York, 1994, pp. 225-62 with permission of the authors and publishers.)

this mechanism, Eden and Hanson proposed that chronic exposure to hypoxia early in life may blunt the response to acute hypoxia by resetting or altering the maturation of the central, inhibitory chemoreceptors. 17 Their results suggest that chronic hypoxia from birth may prevent or inhibit the development of the adult response to acute hypoxia, thus leaving the central chemoreceptors set at a potentially dangerous and inhibitory level. Theoretically, this could severely diminish the infant's capacity to mount an appropriate and sustained ventilatory response when hypoxic and this mechanism may play a role in sudden infant death syndrome at low altitudes. 17 At birth, the sudden increase in PIO 2 and mechanical effects of lung expansion play a crucial role in increasing pulmonary blood flow by stimulating vasodilation of the pulmonary circulation, and thus decreasing pulmonary artery pressure and closing intracardiac shunts (see Chapters 7 and 14). However, at altitude, the low PIO 2 is less effective, resulting in a slower decrease in both pulmonary artery pressure and flow, presumably due to continued vasoconstriction. 18 This probably accounts for the higher prevalence of atrial septal defects reported in some high altitude residents 19 and may contribute to the increased neonatal mortality rate. Despite intrauterine growth restriction and delayed maturation overall, it has been repeatedly reported that children resident at altitude show evidence of accelerated lung growth in relation to body size. 20-22 Thus, they demonstrate phenotypic adaptation to altitude with improved oxygen uptake due to increased ventilation, increased lung compliance and higher pulmonary diffusing capacity relative to lowlanders. Infants at altitude also have an increased red cell mass (Hb concentration), greater blood viscosity, larger

lung volumes and chest wall dimensions, increased respiratory compliance and a decrease in PaO 2 associated with increased pulmonary artery pressure. 23'24 Whether these changes represent adaptations to the environment or genetic differences remains unknown. However, laboratory animals reared in a hypoxic atmosphere develop larger lungs, and consequently higher diffusing capacities, compared with those reared at a normal sea level PO 2 provided the exposure occurred during the animal's active growth period. 25'26 This suggests that the dominant mechanism is adaptation. Despite these observations, genetic factors probably play an important role as chest size is genetically determined. Lung development that occurs as the infant grows to adulthood includes a substantial increase in the number of alveoli as the lung expands towards adult values. If they reside at altitude during this growth phase they appear to develop larger and heavier lungs with more alveoli than their lowland counterparts. 27 Larger lungs and greater number of alveoli confer an advantage in terms of gas exchange by increasing the alveolar surface area available for gas exchange. These adaptive changes are primarily acquired during growth and development as they do not appear to be observed in adults who move to altitude. 28 Thus it appears that prolonged exposure to the hypoxia of altitude results in accelerated lung growth towards values typical of normal adults living at sea level.

ADOLESCENCE

AND ADULT

Relative to acute acclimatisation, long-term adaptation appears to add considerably to one's ability to reside at altitude.

It is unclear, however, what proportion is due to an individuals capacity to respond to the stresses of altitude as opposed to inherent ancestral genetic effects. 29 Adaptive lung growth resulting in larger lungs with greater surface area has been reported in young rats experimentally exposed to the hypoxia of altitude during their growth phase for periods as short as 3 weeks. 26 Similarly, guinea pigs experimentally exposed to a PIO 2 of 72-87 mmHg (equivalent to 5100m) for periods up to 14 weeks also demonstrated significantly increased lung volumes (32%) and alveolar surface area (27%) compared to weight-matched controls exposed to a PIO 2 of 133 mmHg (Figs 18.2 and 18.3). 30 However, the differences between the groups raised at a high altitude PIO 2 and at sea-level disappeared as the animals approached adulthood, indicating an accelerated rate of lung growth. Similarly no difference was found in lung volume or alveolar surface area of adult sheep and guinea pigs raised for generations at about 4500m compared with sea level animals. 31 These data contrast with many studies of humans which have found that highlanders have increased in chest dimensions, vital capacity (VC) and carbon monoxide diffusing capacity, the latter probably being associated with the increased lung volume and quantity of parenchyma. 21'32-35 In a study which compared three healthy Central Asian populations resident at 3200, 2100 and 900m, no differences in VC or forced expiratory

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volume in one second (FEV1) were found between groups; 36 this suggests that the residents at the highest altitude were not genetically different. The larger lung volumes reported in other studies may suggest a genetic rather than environmental adaptation. Therefore, it appears that there is a limit to the hypoxia-induced acceleration of lung growth; 3~ lung growth will also be limited by the chest wall dimensions, which are genetically determined. This is likely as the larger the thoracic cavity the greater the capacity for lung growth. 35 Overall, these data suggest a genetic contribution, and alterations in the dimensions of the chest wall may be possible over many generations, but there remains a great deal of variability between species and indeed between human populations. 3~ Additionally, such data are often difficult to interpret due to the differences in the body dimensions of highland natives compared to their larger lowland counterparts. Tibetans who have resided at altitude for many more generations than Andean or Rocky Mountain residents, have less intrauterine growth restriction, higher levels of resting minute ventilation, a stronger ventilatory response to hypoxia, less pulmonary vasocontriction, lower Hb concentrations and are less susceptible to mountain sickness. These observations suggest Tibetans have a biological advantage compared relative to newcomers as a consequence of their longer generational period of time at high altitude. 1

In a study of five population samples from urban and rural areas of La Paz, Bolivia, representing long-term natives and recent newcomers, Frisancho etal. found that RV and VC were higher in all five groups compared with values predicted for matched lowland residents. 38 Of particular interest, individuals who were native to high altitude had larger vital capacities (corrected for body size) than subjects who, although not natives, had lived at altitude from birth. This conclusion is supported by others; Brutsarert et al. compared highland natives of Bolivia with two lowland groups (one raised at 3600 m) and showed that the high-altitude natives had higher arterial oxygen saturations and physical work capacity suggesting there may be a genetic component to the adaptations of native highlanders. 39 However, Frisancho argues that the attainment of altitude adaptation to aerobic work is due to growth and development in a hypoxic atmosphere. 4~ In a sample of 369 Andean highlanders of Aymara ancestry, Grekas concluded that they had enhanced lung growth and that this was influenced by both genetic and environmental factors. 41 To try to delineate these issues further, skin reflectance has been used to assess the influence of genetic factors on the development of lung volumes because skin colour is largely genetically determined. 42 Frisancho et al. found an inverse relationship between skin reflectance and RV in urban natives and estimated that 25-29% of the variability in RV was explained by genetically determined factors whereas VC was not. 38 There was also a positive relationship between the level of occupational activity and VC but not RV which demonstrates the complexity of such data. TarazonaSantos etal. found ventilatory function (FEV 1, FVC and peak expired flow) to decrease significantly with age but not Hb concentration or hematocrit. 43 They found a negative age-independent association between lung function and Hb and hematocrit in a population of Quechua natives of the Peruvian Central Andes indicating two adaptive responses conferring different physiological work capacities. The hematopoietic response to altitude shows regional differences, with miners from South America showing higher haemoglobin concentrations than residents at altitude in the Himalayas. Fiori etal. provide evidence that the increased haemoglobin, red blood cell count and hematocrit and higher oxygen content in higher altitude residents was achieved through physiological adaptation. 36 The carotid body and, to a lesser extent, the aortic bodies are the main peripheral chemoreceptors responding to arterial PO 2. Acute exposure to hypoxia results in an increase in ventilation which is hyperbolic in shape with a progressively more rapid increase in ventilation as PaO 2 falls below 50mmHg. The adult carotid body weighs only 10mg but relative to its mass is highly vascularised and therefore it does not remove much of the oxygen contained in the blood perfusing it. Thus, the carotid body produces neural signals in response to the PaO 2 of arterial blood rather than to its oxygen content. Chronic exposure to hypoxia has been shown to increase the mass of the carotid body due to increased blood volume and angiogenesis. 44

At both sea level and altitude the effect of hypoxia on ventilation is very variable between individuals and is also directly influenced by PaCO 2. During short-term visits to high altitude there is a strong ventilatory response but as exposure continues over several years this response becomes blunted. However, this observation is by no means uniform and may depend to some extent on the population studied. A recent study comparing Tibetans (3800-4000m) and Andean (3900-4000m) natives over a wide age range showed significantly higher resting ventilation and hypoxic ventilatory responses in the Tibetan population. 45 On average the Tibetans' resting ventilation was 50% higher due to increased tidal volume than the Andeans' and their ventilatory response to hypoxia was about double that of Andeans. The results of this extensive study suggests quite different phenotypes of populations who have successfully adapted to living at altitude. A wide range of hypoxic ventilatory responses have been reported ranging from very little response to responses very similar to residents at sea level. 46 Thus high altitude residents have a higher PaCO 2 than fully acclimatised newcomers to altitude. 47 As mentioned above, the blunted ventilatory response observed in high altitude Peruvian women disappears during pregnancy thus allowing them to mount a heightened ventilatory response during gestation. 6 This suggests that hypoxic ventilatory drive can be rapidly reversed. Remodelling of the smaller vessels of the pulmonary circulation in response to chronic hypoxia has been described consisting principally of increased vascular smooth muscle in the intima of the pulmonary arterioles. 48 The increased muscularisation leads to a higher than normal pulmonary vascular resistance and the common finding of modest pulmonary hypertension. 49 For example, the mean pulmonary artery pressure of adult natives of Peru (4800m) has been reported at 28 mmHg, 5~ compared to typical sea-level values of 9 mmHg. Pulmonary hypertension only appears in native populations resident at elevations above about 2000m where the alveolar PO 2 is less than about 75 mmHg; 51 at greater altitudes the degree of pulmonary hypertension varies between populations being less severe in those who have lived longest at altitude. This indicates that the development of a reduced hypoxic vasoconstrictive response occurs only after generations at altitude and that genetics may play an important role. Williams has pointed out that this slight rise in pulmonary artery pressure is little more than a marker of chronic hypoxia as it has little effect on the well-being of the high altitude native or their capacity to engage in sustained work. 52

U L T R A - S T R U C T u R A L A N D CELLULAR CONSIDERATIONS As previously discussed, there are considerable data suggesting that the lungs of long-term residents at altitude are morphometrically different from their lowland counterparts. 22'27'53 The lungs of the highland native are larger,

heavier and contain significantly greater numbers of alveoli. 27 These morphological changes serve to increase the surface area available for oxygen diffusion and clearly confer a survival advantage. Whether the increase in alveolar numbers occurs as a direct result of genetic adaptation or as an adaptive response to hypoxaemia in utero and postpartum remains unknown and difficult to ascertain at present. However, it is probable that the majority of the increased alveolar budding and growth takes place in utero although it may continue to a lesser degree in the first years of life. There is, in fact, little data on lung structure in infants or adults resident at high altitude and we can only speculate on potential changes in the pulmonary vasculature, airways and parenchyma by examination of limited post-mortem and animal data. However, potentially useful information may be inferred from an understanding of the processes involved in the development of cor pulmonale in chronically hypoxic patients at sea level and from post-mortem studies of high altitude pulmonary oedema (HAPE or mountain sickness). In emphysema, chronic hypoxia as a result of parenchymal destruction and loss of alveolar units results in the development of pulmonary hypertension. The pulmonary arterial circulation undergoes a variety of functional and anatomic changes resulting in vasoconstriction and an increase in the thickness of the pulmonary arteriolar wall as a consequence of smooth muscle hypertrophy and elastin deposition. Histological studies of the lungs of Aymaran Indians demonstrate vascular remodelling that is very similar to those seen in COPD (i.e. muscularisation of the pulmonary arterial tree and the presence of longitudinal muscle within the vessel intima). 53'55 These anatomical alterations are thought to be responsible for the very mild increases in pulmonary artery pressure observed in this population. In contrast, Mestizos Indians do not appear to develop vascular remodelling or pulmonary hypertension despite living at similar altitudes, s4 Interestingly, Aymara Indians with pulmonary artery remodelling have increased numbers of airway mast cells whereas Mestizos appear to have normal numbers of resident mast cells. 55 The mast cell is implicated in vascular remodelling in asthma and is a source of proinflammatory mediators and growth factors, such as interleukin (IL)-8 and vascular endothelial growth factor (VEGF), that are involved in the formation of new vessels (angiogenesis). 56 The mast cell is also implicated in the pathogenesis of HAPE and post-mortem studies demonstrate an increase in pulmonary surfactant and mast cells within the alveoli. 57 Additionally, there is gross congestion of pulmonary vessels, formation of alveolar hyaline membranes, haemorrhage and multi-thrombi and fibrin clots within the pulmonary and bronchial circulations. These observations are intriguing and suggest the mast cell is involved in the pathogenesis of both chronic and acute pulmonary hypertension at altitude. Additionally, in COPD and in highland natives there appears to be an alteration in the morphology of cells

immediately adjacent to pulmonary venules such that they develop characteristics of arachnoid villi. 58 In the central nervous system, these collections of cells or 'granulations' serve to transfer excess cerebrospinal fluid to the dural venous sinus and it is possible that in COPD and in residents at altitude they may contribute to the avoidance of pulmonary oedema by facilitating the return of fluid into pulmonary venules. The yak has successfully adapted to residence at altitudes in excess of 4500 m despite belonging to the same genus as the domestic cow, which is well known for its exaggerated pulmonary vasoconstrictor response. The yak has smaller pulmonary arteries and attenuated endothelial cells than the cow and has a blunted vasoconstrictor response, all of which result in a reduction in the blood-gas barrier and improved oxygenation. 59'6~ Whether similar adaptations occur in man has not been established but there is some evidence to suggest that the vasoconstrictor response of the pulmonary artery may be altered in highland natives. 61-63 An increased resistance to the pulmonary vasoconstriction that normally occurs in response to hypoxia appears to be another survival advantage of residents at altitude and there are some suggestions from animal and human studies that this response may be genetically determined. 61'64'65 Differences in pulmonary haemodynamics have been demonstrated in high altitude populations resident in the Himalayas, Andes and North America, supporting the possibility that there may be an evolutionary, genetic influence. Structural changes in the pulmonary artery probably underlie the majority of these differences but there is some evidence to suggest that there may be functional alterations in the way in which the pulmonary vasculature responds at a cellular level to mediators of pulmonary vasoconstriction. Potentially important mediators that may be involved in these differing responses to hypoxia include nitric oxide (NO), endothelin-1 (ET-1) and prostacyclin (PGI2). Evidence to suggest a role for each in the pathogenesis of pulmonary hypertension at altitude will be examined and potential mechanisms of adaptation considered. Nitric oxide is a ubiquitous molecule with potent vasodilator properties and data suggest it is an important determinant of pulmonary vascular tone. 66-68 Increased production of NO in the setting of hypoxia might therefore be expected to maintain pulmonary arteriolar patency and thus reduce pulmonary artery pressures. Interestingly, limited data from Aymara Indians suggest that an increase in NO production does indeed occur as a direct consequence of upregulated expression of the enzyme endothelial nitric oxide synthase ( e N O S ) w i t h i n pulmonary endothelial cells. 69 Furthermore, mountaineers who are susceptible to the development of HAPE exhibit reduced levels of nasal NO that are significantly and negatively related to the rise in pulmonary artery systolic pressure seen during hypoxic challenge. In contrast, nasal NO levels and pulmonary artery pressures do not alter in HAPE-resistant controls during acute hypoxia. 7~ This suggests that decreased pulmonary NO production during hypoxia contributes to

the development of HAPE. Further evidence to support a role for NO comes from a rat model of primary pulmonary hypertension. In this study, rats deficient in eNOS have abnormal pulmonary vascular growth during the fetal, neonatal and postnatal periods and develop pulmonary hypertension in adulthood. 71'72 Additionally, these abnormalities are exacerbated by altitude and exposing rats to sea-level PiO 2 during the perinatal period improves lung eNOS expression and reduces abnormal vascular growth, further suggesting a potential beneficial role for eNOS in lung vascular development. 72 ET-1 is a peptide vasoconstrictor that is secreted predominantly by endothelial and vascular smooth muscle cells within the lung. 73 Abnormalities of endothelin-1 have been implicated in both primary and secondary pulmonary hypertension and it is therefore appropriate to consider its potential role in modifications of the function and structure of the pulmonary vascular system at altitude. 74-76 ET-1 has been implicated in the development of vascular remodelling associated with hypoxic pulmonary hypertension through its mitogenic effects on vascular smooth muscle cells. 77-81 However, at high altitude the role of ET-1 in the development of acute pulmonary hypertension is unclear and data are conflicting. Despite the apparent contradictions, the majority of studies suggest that ET-1 production is increased within the lung during hypoxia and airway and peripheral blood ET-1 levels are positively correlated with rises in pulmonary arterial pressure. 82-84 Interestingly, NO has been shown to down-regulate ET-1 expression at the transcriptional level and it would seem biologically plausible to assume that at least some of the potential protective effects of NO at altitude are mediated through inhibition of ET-1 production. PGI 2 is a potent vasodilator that also attenuates the proliferation of cultured vascular smooth muscle cells. 8s-s7 Nitric oxide and PGI 2 are both involved in the reduction of pulmonary vascular resistance that occurs at birth and a reduction in PGI 2 receptor expression on pulmonary arterial smooth muscle has been associated with severe pulmonary hypertension, s5,88 Knockout mice that lack PGI 2 smooth muscle receptors (PGI-R KO) develop greater degrees of pulmonary hypertension than wild type mice following exposure to chronic hypocarbic hypoxia, s9 Additionally, animal models demonstrate that an ability to up-regulate PGI 2 synthesis may be an adaptive response that protects against the stress of chronic hypoxia. 9~ In conclusion, the extent to which ultra-structural alterations in the pulmonary vasculature and changes in endothelial or vascular smooth muscle cell function are involved in determining long-term adaptation of humans to life at altitude remains unknown. Although animal studies suggest genetic factors such as alterations in eNOS, ET-1 or PGI 2 gene expression may play a role in determining survival advantage at altitude, whether these putative mechanisms are relevant to man has not been established. However, recent advances in medical technology should make this a fertile field for future research.

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81. Aguirre JI, Morrell NW, Long L e t al. Vascular remodeling and ET-1 expression in rat strains with different responses to chronic hypoxia. Am. J. Physiol. Lung Cell Mol. Physiol. 2000; 278:L981-7. 82. Cruden NL, Newby DE, Ross JA et al. Effect of cold exposure, exercise and high altitude on plasma endothelin-1 and endothelial cell markers in man. Scott. Med. J. 1999; 44:143-6. 83. Droma Y, Hayano T, Takabayashi Y e t al. Endothelin-1 and interleukin-8 in high altitude pulmonary oedema. Eur. Respir. J. 1996; 9:1947-9. 84. Goerre S, Wenk M, Bartsch Pet al. Endothelin-1 in pulmonary hypertension associated with high-altitude exposure. Circulation 1995; 91: 359-64. 85. Shaul PW. Regulation of vasodilator synthesis during lung development. Early Hum. Dev. 1999; 54:271-94. 86. Shirotani M, Yui Y, Hattori R, Kawai C. U-61,431F, a stable prostacyclin analogue, inhibits the proliferation of bovine vascular smooth muscle cells with little antiproliferative effect on endothelial cells. Prostaglandins 1991; 41:97-110. 87. Olschewski H, Rose F, Grunig E et al. Cellular pathophysiology and therapy of pulmonary hypertension. J. Lab. Clin. Med. 2001; 138:367-77. 88. Ghanayem NS, Gordon JB. Modulation of pulmonary vasomotor tone in the fetus and neonate. Respir. Res. 2001; 2:139-44. 89. Hoshikawa Y, Voelkel NF, Gesell TL et al. Prostacyclin receptor-dependent modulation of pulmonary vascular remodeling.Am. J. Respir. Crit. Care Med. 2001; 164:314-8. 90. Pshennikova MG, Kuznetsova VA, Kopylov Iu et al. The role of the prostaglandin system in the cardioprotective effect of adaptation to hypoxia in stress. Kardiologiia 1992; 32:61-4.

INTRODUCTION

It has become increasingly clear that many diseases are complex disorders that involve multiple intrinsic (internal) and extrinsic (external) components. External factors that contribute to disease pathogenesis may include physical factors (e.g. temperature), socioeconomic status, and exposure to environmental stimuli or triggers (e.g. allergens, molds, air pollutants, tobacco smoke). A n important role for environmental factors in disease is supported by numerous epidemiological studies that demonstrate strong associations between disease morbidity and mortality and exposure, including pulmonary diseases such as asthma, chronic obstructive pulmonary disease (COPD), and lung cancer. For example, asthma morbidity has been associated with the potent oxidant ozone (03) TM as well as particulate matter (PM) 5-8 in numerous industrial cities throughout the world. A large body of literature also supports a number of indoor allergens as important environmental factors in allergy and asthma. These include house dust mite, molds associated with indoor dampness problems, and cockroach allergen. 9-11 However, not everyone responds similarly to these environmental stimuli, and this implicates intrinsic factors as important inter-individual determinants of response. Important internal factors include gender, age, diet, and genetic background. An individual's genetic make-up has become increasingly recognized as a critical internal or host factor in environmental and occupational disease predisposition. Numerous family studies have argued for a genetic basis for multiple lung diseases, ranging in design Present Address: Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, NIH, 111, T.W. Alexander Dr. Building 101, Rm. D240 Research Triangle Park, NC 27709, USA. The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

from twin studies to segregation analysis of extended pedigrees. 12 Investigators have found evidence of linkage between genetic markers and unobserved susceptibility loci for many lung diseases and their associated phenotypes, although such evidence is seldom definitive within any one study and often is conflicting across studies. 12 The lists of candidate genes for these unobserved susceptibility loci are long, and sometimes vary from one study to another. It is also generally agreed that the diseases are highly complex, since multiple genes (each with modest effects) are likely to be operating through interactions with multiple environmental factors. Furthermore, the roles of both these genes and environments vary across populations, and without explicit consideration of both simultaneously it is not possible to accurately identify the critical actions of either. An important genetic component has been well established for a number of lung diseases, including COPD (Table 19.1), idiopathic pulmonary fibrosis (Table 19.2), and sarcoidosis (Table 19.3). Asthma has perhaps been the most intensely studied pulmonary disease, and excellent reviews for the genetic basis of asthma susceptibility to each have been published recently. 12,38-41 The reader is directed to these reviews for greater in-depth discussion of the contribution of genetic background to this disease. A major challenge in understanding how specific susceptibility genes and environmental exposures interact in pulmonary disease pathogenesis is to determine which environmental factors might be relevant to which genetic markers in the etiology of the disease. 42 Environmental exposures and genetic factors associated with disease risk interact in a complex fashion that varies from one population to another. The relationships between the independent variables and disease risk and severity can only be evaluated through multi-variate analysis of large samples to determine Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

their relative contributions to disease risk and the interactions among them. 42'43Well-designed case-control studies of risk factors (i.e. environmental exposures and genetic background) are necessary to guide investigators in the search for mechanisms of disease outcome and risk. Well-known examples of gene-environment interaction and disease pathogenesis include increased incidence of bladder and lung cancer, craniofacial malformations, coronary atherosclerosis, beryllium disease, and rheumatoid arthritisY 47

A study by Bell and colleagues 44 is an excellent illustration of the power of the gene-environment approach to study the pathogenesis of disease in human populations. These investigators evaluated the role of a common genetic

polymorphism and carcinogen exposure in determining risk to bladder cancer. They found that among individuals with similar exposure to cigarette smoke, the risk of developing bladder cancer was significantly elevated in those with a defect in the glutathione S-transferase (GSTM1) gene. That is, the risk of bladder cancer was almost 2-fold greater in individuals homozygous for the null allele compared to those with the wild-type alleles. Individuals with the null genotype and no exposure were at no greater risk than those with no exposure and the normal genotype. This study clearly demonstrates how an understanding of complex environmental disease pathogenesis depends not only on the genetic background and degree of exposure experienced by an individual, but also on the interaction of the two. A thorough discussion of the genetic factors involved in susceptibility to all lung diseases is, of course, beyond the limitations of single book chapter. I have accordingly narrowed this topic to the genetic factors that may contribute to environmental and occupational lung diseases as follows. Initially, I present a brief introduction to experimental approaches used to identify lung disease genes, and to convey some of the challenges that must be overcome in study design and application. Next, studies are presented that have identified a genetic basis for susceptibility to environmental stimuli and acute lung injury in animal models and human subjects. Lastly, the potential role of genetic susceptibility to occupational lung diseases is discussed, and candidate susceptibility genes are highlighted.

RESEARCH STRATEGIES EMPLOYED TO I D E N T I F Y C A N D I D A T E DISEASE SUSCEPTIBILITY GENES Before describing the process of identifying candidate disease susceptibility genes, it is necessary to define a few concepts. The first of these is a quantitative trait: this can be considered as a phenotype that varies in a quantitative manner when measured among different individuals. The variable phenotype or expression is due to a combination of genetic and environmental factors, as well as by chance, and is often controlled by the cumulative action of alleles at multiple loci. 48 While many single gene (Mendelian) disorders have been identified, 49 it has become clear that most diseases are quantitative or complex, the result of an interaction between multiple genes and environmental exposures or stimuli. A quantitative trait locus (QTL) is a region on a chromosome that has been identified by linkage or linkage disequilibrium analyses or association studies (see below) to contain a gene or genes that contribute to the phenotype of interest. Each QTL may be as large as 10-20cM (centi-Morgan) and include hundreds of genes. Therefore, a major task facing an investigator is to reduce the size of a QTL to a size (ideally < 1 cM) that is more 'manageable' for physical mapping or other approaches (including the use of single nucleotide polymorphisms or SNPs) that will lead to the identification of a candidate gene that determines

the phenotype of interest. The combined contribution of each locus (QTL) with environmental influences (stimuli) determines an individual's response phenotype. It is well known that many complex diseases cluster in families, and the challenge to investigators is to understand whether clustering can be explained by genetic background or shared environment, or a combination of the two. Two broad research strategies have been utilized to identify genes (or QTLs) that determine disease susceptibility. The first is meiotic mapping and positional cloning. Generally, two kinds of meiotic mapping exist. The first is linkage mapping, which exploits within-family associations between marker alleles and putative trait-influencing alleles that arise within families and are may be followed by methods of co-segregation analyses. 5~ This approach is designed to identify association of a chromosomal interval(s) within the entire genome that may contain genes that are polymorphic and may account for the differential response phenotype under study. This approach is ideally suited for genetically well-controlled models, particularly inbred mice. These strategies, alone or in combination, have been utilized in inbred mice to identify genetic mechanisms of a number of diseases. These include models of Huntington's disease, 51'52 Duchenne muscular dystrophy, 53 amyotrophic lateral sclerosis, 54 insulin-dependent diabetes mellitus, 55 Alzheimer's disease, 56 yon WiUebrand disease, 57 chronic granulomatous disease, 58 and Niemann-Pick C1 disease. 59 One of the unifying concepts for these studies is the linkage relationship of homologous loci from human and mouse. That is, highly significant homologies in gene order and chromosomal structure have been maintained since the divergence of the human and mouse. 6~ Therefore, identification of the chromosomal location of a susceptibility gene in the mouse provides the basis for potentially localizing a homologous gene in the human. 61'62 The considerable resources and energy applied to mapping of the mouse genome as an integral component of the Human Genome Project underlie the importance of this animal model for human diseases. 63 Linkage mapping is applicable to human populations as well, and has had considerable success in Mendelian (single gene) diseases. Unfortunately, genome-wide linkage scans have yielded few significant findings in complex human diseases (multigenic). 5~ The reasons for this general lack of success are numerous, but the primary reason is that, for most complex traits, no single locus confers a high degree of risk. Current technologies and statistical methods are not sufficient to permit fine mapping of the location of multiple genes that influence a complex disorder in genetically heterogeneous species like humans, particularly when environmental exposure may vary from one study population to the next. Because of the limitations of linkage analysis in complex human disease, the candidate gene approach or association study in which loci are chosen a priori as likely mechanisms that determine the phenotype of interest, have been increasingly accepted as the optimum approach for fine-mapping susceptibility loci. 65'66 With association studies, linkage is

assessed between the phenotype of interest and markers flanking the candidate genes or the candidate genes themselves, and evaluates across-family associations. The principle underlying the association of genetic polymorphisms not directly involved in disease pathogenesis is that of linkage disequilibrium, which arises from the co-inheritance of alleles at loci that are in close physical proximity on an individual chromosome. 65 This approach requires a dense map of chromosomal markers because the chromosomal regions that co-segregate with a disease across different families may be very small. 5~The emergence of finely mapped SNPs has facilitated this approach for mapping susceptibility loci. Study designs with individual functional/nonfunctional SNPs, or multiple SNPs in a small chromosomal region (haplotype analyses), have come to provide researchers with the potential means to dissect genetic contributions to complex human diseases. Central to these studies is, of course, the identification and application of SNPs. The advantages, limitations, and application of SNP technologies have been discussed thoroughly elsewhere. 5~ It is important to note that employing association studies alone could implicate certain genes in the expressed phenotype of interest, but other important loci that determine a quantitative trait, as well as the interaction between them, may be missed. Clearly, neither the genetic linkage approach nor association study is sufficient to reach a clear understanding of the genetic basis of complex diseases. While SNP technologies are, at present, not practical for use in genome-wide strategies, they are quite useful when applied to limited chromosomal regions, such as those already defined by genome-wide screens for genetic linkage. 5~ Therefore, when practical, the most productive strategy to understand complex diseases will likely be a combination of the linkage and association approaches.

GENETIC SUSCEPTIBILITY TO E N V I R O N M E N T A L STIMULI A N D ACUTE LUNG INJURY Inter-individual differences in drug metabolism and sensitivity due to polymorphisms in genes that encode N-acetyltransferase and G6PD, respectively, are examples of genetic contribution to human susceptibility to environmental agents. 68-7~ Genetic background contributes to the responses to other environmental agents including inhaled pollutants and pesticides (e.g. t~l-antitrypsin deficiency, aryl hydrocarbon hydroxylase inducibility, plasma paraoxonase activity), and infectious agents and autoimmune disorders (e.g. Duffy blood groups, predisposition associated with histocompatibility phenotypes). 6s'7~Identification of genetic polymorphisms that predispose individuals to the toxic effects of drugs and environmental stimuli is the basis for the burgeoning fields of pharmacogenetics and ecogenetics. 71 The remainder of this section is focused on recent studies that have been designed to identify genes that determine pulmonary susceptibility to environmental stimuli and acute lung injury.

Genetic modeling and candidate genes for susceptibility to pulmonary effects of environmental stimuli Air pollution continues to be an important public health and economic concern in industrialized cities throughout the world. Numerous epidemiological associations of adverse health outcomes with air pollution episodes support these concerns. Inter-individual variation in human responses to air pollutants indicates that not all individuals exposed to pollutants respond similarly. That is, some subpopulations are at increased risk to the detrimental effects of pollutant exposure. Inter-individual variation in biological responses to environmental stimuli is a consequence of internal and external factors. External factors include physical forces (e.g. temperature, altitude), socioeconomic status, and previous exposure. Internal factors include gender, age, diet, and predisposing disease (e.g. asthma). Genetic background has become increasingly recognized as another important internal or host factor in environmental disease predisposition. In human populations, gene polymorphisms have been associated with susceptibility to environmental agents including pesticides and infectious agents. 69 It is also clear that multiple internal and external factors contribute to individual responses to air pollutants and occupational stimuli. A number of populations have been identified that are particularly susceptible to the toxic effects of airborne oxidants and particulates, including the elderly and individuals with cardiopulmonary disease.72, 73 Few linkage or association studies have been attempted in human subjects. However, a number of linkage analyses with genetically standardized animal models have been done, and they provide useful tools to identify genetic factors that contribute to host responses to environmental pollutants. In particular, using segregant populations derived from inbred mice, QTLs have been identified which contain genes that control susceptibility to pulmonary responses induced by exposure to a number of occupational and environmental stimuli. Identification of a QTL is a multi-step process that may be broadly grouped into three research objectives. The first objective is to determine whether the response phenotype is genetically determined and quantitative. After determining that the quantitative phenotype has a genetic basis, then the susceptibility QTL(s) are sought. Finally, significant informative QTLs are searched for candidate genes that may explain differential susceptibility/responsiveness in the model. It is beyond the scope of this chapter to explain all of the details of linkage mapping; therefore the reader is referred to excellent reviews of this topic by Broman, TM Moore and Nagle, 75 and Silver. 48 Below I describe selected investigations that have led to the identification of a number of pulmonary susceptibility QTLs. This discussion is not exhaustive, and is meant only to illustrate the approach to identify the genetic basis of susceptibility to pulmonary toxicants, and candidate genes that may be important determinants of susceptibility.

Genetic determinants of susceptibility to O3-induced lung inflammation and injury Ozone exposure induces multiple pulmonary and extrapulmonary responses in humans and animal models. Ozone elicits inflammation, hyperreactivity, and epithelial damage of the airways, as well as altered ventilation and decrements in pulmonary function. 72'76 Ozone has also been demonstrated to either suppress or enhance immune responsiveness, depending upon the exposure regimen, genetic background (inbred strains), pre-existing diseases, etc. 77 Significant inter-strain variation in the magnitude of these responses has also been demonstrated in rats and mice, and has thus provided strong evidence of a genetic component to 0 3 responsiveness. 78-8~ The inter-strain variation in susceptibility among inbred mice led us to conduct studies to identify the chromosomal location of the 0 3 susceptibility genes using susceptible C57BL/6J (B6) and resistant C3H/HeJ (C3) strains. A genomewide search for linkage of the inflammation (polymorphonuclear leukocytes, PMNs) phenotype was performed with informative simple sequence length polymorphisms (SSLPs) distributed at approximately 10cM intervals throughout the genome. 81 The number and spacing of markers yielded complete coverage of the mouse genome with 95% confidence. Linkage was carried out with individual intercross animals derived from B6 and C3 progenitors (B6C3F2). The phenotyped F 2 progeny were genotyped for each of the SSLPs, and linkage of susceptibility to 0 3 was evaluated using Map Manager QT and MAPMAKER-QTL software packages. Interval mapping by simple linear regression in the entire F 2 cohort determined the presence of a susceptibility locus on chromosome 17 in the interval b e t w e e n approximately 16-22 cM. 81 An additional QTL was detected on chromosome 11 between DllMit20 and DllMitl2. Within the chromosome 17 QTL are a number of candidate genes, including the pro-inflammatory cytokine Tnf (tumor necrosis factor-t~, TNF-ct). Because Tnf may be postulated to have a role in the inflammatory response to oxidantrelated lung injury, we made preliminary evaluations of this candidate gene for determination of differential O3-induced inflammation in B6 and C3 mice. Pre-treatment of susceptible B6 mice with a monoclonal antibody to TNF-t~ significantly attenuated the inflammatory response to 0 3 relative to control B6 mice, providing support of TNF-t~ as a candidate susceptibility gene in this model. 81 To further understand the mechanisms of O3-induced lung injury, we performed a genome-wide linkage analysis for susceptibility QTLs to explain interstrain differences in hyperpermeability induced by 72 h exposure to 0.3 ppm 0 3. Because an apparent dissociation exists between inflammatory cell infiltration and lung hyperpermeability induced by 03, 82 we hypothesized that different loci control the hyperpermeability response. To determine the susceptibility QTLs, we performed a genome screen using recombinant inbred (RI) strains of mice derived from B6 and C3 progenitors (see Ref. 48 for explanation of the use of RIs for QTL mapping). A significant QTL was identified on

chromosome 4, and suggestive QTLs were identified on chromosomes 3 and 11.83 The chromosome 4 QTL contains a candidate gene, toll-like receptor 4 (Tlr4), that recently has been implicated in innate immunity and endotoxin susceptibility, s4-s6 As a 'proof of concept' that Tlr4 has an important functional role in susceptibility, we compared the hyperpermeability responses to 0 3 in C3H/HeOuJ (OuJ) and C3 mice. These strains differ only at a polymorphism in the coding region of Tlr4 and the polymorphism confers resistance to endotoxin-induced injury in the C3 mouse compared to wild type OuJ. Significantly greater protein concentrations were found in OuJ mice compared with C3 mice after exposure to 03 .83 Furthermore, RT-PCR analysis demonstrated Tlr4 message levels in the lungs of C3 mice were markedly downregulated, while levels increased in the OuJ strain after 0 3 exposure, s3 Together, results indicate that a QTL on chromosome 4 explains a significant portion of the genetic variance in O3-induced hyperpermeability, and support a role for Tlr4 as a strong candidate susceptibility gene. This is the first demonstration that innate immune mechanisms modulated by Tlr4 are involved in the pulmonary response to oxidant exposure. Prows et al. s~ have performed a linkage analysis of susceptibility to death induced by exposure to high concentrations of 0 3. Using susceptible A/J and resistant B6 mice, these investigators identified a significant QTL on chromosome 11, and suggestive QTLs on chromosomes 13 and 17. Interestingly, the QTLs on chromosomes 11 and 17 are similar to those described by us for susceptibility to inflammation induced by exposure to 0.3 p p m 03 .81 Evidence also exists for genetic determinants of susceptibility to 0 3 in human subjects. A number of laboratories have reported inter-individual variation in pulmonary function responses to 0 3 in otherwise normal, healthy human subjects. 87-89Inter-individual variation in the inflammatory response to 0 3 has also been described. 9~ A second line of evidence is the demonstration that specific gene polymorphisms associate with response phenotypes in exposed human subjects. It has been found that polymorphisms in genes for quinone-metabolizing enzymes may have an important role in the pulmonary function and epithelial permeability responses to 0 3 in nonsmoking exercising subjects. 95

Genetic determinants of susceptibility to particle-induced lung inflammation and injury Considerable attention has been focused on the adverse respiratory effects of particle inhalation. Epidemiological studies have reported significant association of acute and chronic respiratory and non-respiratory health effects with increases in particulate exposure throughout the industrialized world. 72'96'97 Susceptible subpopulations include the aged (>65 year of age) and patients with cardiopulmonary disease such as chronic heart disease, chronic obstructive pulmonary disease, and asthma. 8'72'98'99To determine whether genetic background is an important determinant of pulmonary responses to particulates, we studied the inter-strain

variance of lung responses to acid sulfate-coated particles (ACP) in inbred strains of mice. 1~176 Although the 4h challenge to ACP did not elicit a detectable inflammatory response, significant inter-strain differences were found in Fc receptor-mediated phagocytosis of alveolar macrophages (an indicator of innate immune defense). A genome scan similar to that described for 0 3 susceptibility (see above) was then performed with susceptible B6 and resistant C3 mice. 1~ Interestingly, linkage analyses identified a susceptible QTL on chromosome 17 and a suggestive QTL on chromosome 11 that nearly overlapped similar QTLs identified for 0 3 susceptibility. The common linkages suggest that similar genetic mechanisms may control pulmonary responses to O3-induced inflammation and macrophage phagocytic dysfunction induced by ACP, but further genetic analyses are required to confirm this hypothesis. Tolerance to the pulmonary inflammatory and hyperpermeability effects of air pollutants has been demonstrated repeatedly in animal models and human subjects. 1~176 To investigate the potential genetic contribution to tolerance, the inter-strain variation in the ability of inbred mice to 'become tolerant' to the toxic effects of repeated exposure to zinc oxide (ZnO) has been evaluated. TM Interestingly, significant inter-strain variation was found in the inflammatory cell and hyperpermeability responses to single and multiple ZnO exposures. TM The investigators then performed a genome-scan for susceptibility QTLs for the development of pulmonary tolerance to ZnO in an intercross cohort from DBA/2J and Balb/cByJ mice. 1~ A significant QTL was identified on chromosome 1, and toll-like receptor 5 (Tlr5) was identified as a candidate susceptibility gene. To our knowledge, this is the first attempt to identify the genes responsible for development of tolerance and may have important implications for understanding susceptibility and resistance to repeated exposures to pulmonary toxicants.

Genetic determinants of susceptibility to acute lung injury Because of its interface with the environment, the lung is a major target organ for injury by exogenous oxidants such as environmental pollutants, cigarette smoke, drugs, chemotherapeutic agents and hyperoxia, 1~176 as well as by endogenous reactive oxygen species (ROS) generated by inflammatory cells. In addition, many pulmonary diseases (e.g. adult respiratory distress syndrome, emphysema) require supplemental oxygen therapy to maintain lung function that further increases the oxidant burden of the lung. It is believed that the damaging effects of oxygen are mediated by superoxide and hydroxyl radicals and H202, products formed by the incomplete reduction of oxygen. There is ample evidence showing increased cellular formation of these ROS during hyperoxia and the deleterious effects on cellular constituents such as nucleic acid, proteins, and lipids. Because of the potential impact that oxidants may have on lung function, identification of those factors that may influence individual susceptibility remains an important issue. An understanding of susceptibility factors

could lead to better interventive strategies and, potentially, a means to identify individuals at risk for the development of oxidative stress injury. Our laboratory has begun to develop a model of genetic mechanisms that regulate lung injury induced by exposure to hyperoxia. In this model, susceptible (after the highly susceptible B6 mouse) and resistant (after the minimally susceptible C3 strain) phenotypes are easily distinguished on the basis of lung permeability and cytotoxic responses to 72 h exposure to 100% O 2. In B6 and C3 mice, and progeny of crosses between them, we have estimated by segregation analysis and analyses of variance that two genes account for a significant amount of the variance in susceptibility to hyperoxia induced hyperpermeability, l~ A genome wide linkage analysis of intercross (F2) and recombinant inbred (RI) cohorts identified significant and suggestive QTLs on chromosomes 2 (hyperoxia susceptibility locus 1 [Hsll]) and 3 (Hsl2), respectively. 1~ Comparative mapping of Hsll identified a strong candidate gene, Nfe212 (nuclear factor, erythroid derived 2, like 2 or Nrf2) that encodes a transcription factor NRF2 that regulates antioxidant and phase 2 gene expression. Strain-specific variation in lung Nrf2 messenger RNA expression and a T ~ A substitution in the B6 Nrf2 promoter that cosegregated with susceptibility phenotypes in F2 animals supported Nrf2 as a candidate gene in this model. 1~ To provide proof of concept for the role of NRF2 in susceptibility to hyperoxic lung injury, we compared pulmonary responses to 100% oxygen exposure between mice with site-directed mutation ofNrf2 (Nrf2 -/-) and wild type mice (Nrff2+/+).11~ Because NRF2 is an important regulator of several important antioxidant and phase 2 detoxifying enzymes, we hypothesized that deletion of the gene for this transcription factor would enhance susceptibility to oxidant-induced lung injury. As predicted, pulmonary response phenotypes (hyperpermeability, macrophage inflammation, and epithelial injury) were significantly elevated in Nrf2 -/- mice compared to Nrf2 +/+ mice. 11~Consistent with a role for NRF2 in susceptibility to oxidant-induced lung injury, hyperoxia-induced mRNA levels of NQO1, GST-Ya and -Yc, UGT, GPx2, and HO-1 were significantly lower in Nrf2 -/- mice compared to Nrf2 +/+ mice. These experiments therefore strongly suggest that Nrf2 has a significant protective role against pulmonary hyperoxic lung injury in mice, and provide confirmation of a candidate gene identified by linkage analyses. Verification of Nrf2 as the candidate susceptibility gene is required by physical mapping strategies. Another model of acute lung injury is exposure of mice to nickel sulfate aerosol. 111 Continuous exposure to 150mcg/m 3 nickel sulfate causes death in a strain-dependent manner, with A/J mice succumbing to exposure significantly faster than B6 mice. 112 A QTL analysis with backcross mice derived from these two strains identified significant linkage to chromosome 6, and suggestive linkage to chromosomes 1 and 12. Interestingly, these QTLs are distinct from those identified for acute lung injury induced by hyperoxia,

but are similar to those identified for death induced by continuous exposure to high concentrations of 0 3. Although no candidate genes have been tested for this model, these studies suggest relatively few loci determine susceptibility to irritant-induced lung injury and subsequent survival. 112 Lung injury and fibrosis may result from irradiation and chemotherapy for the treatment of cancer, and lead to significant morbidity and mortality in a subset of cancer patients. 113 A series of important investigations to identify the genes that confer -susceptibility to bleomycin (a chemotherapeutic agent) and radiation in inbred mice has recently been initiated. In a cohort derived from fibrosisresistant C3Hf/Kam and -susceptible B6 mice it has been demonstrated that heritability of fibrosis induced by bleomycin is approximately 53% in males and 54% in females. TM A role for the MHC complex in this model was demonstrated using various MHC congenic mouse strains. A genome-wide scan for susceptibility loci identified a QTL on chromosome 17 that was highly significant in males and females, and a second QTL on chromosome 11 that was significant in males only. 113Bleomycin phenotypes in reduced congenic mice narrowed the chromosome 17 QTL to a 2.7 cM region that includes the MHC complex. The chromosome 11 QTL was confirmed in a chromosome substitution (consomic) mouse strain, where B6 mice with chromosome 11 transferred from the C3Hf/Kam strain were found to be resistant to bleomycin-induced fibrosis (i.e. the susceptibility phenotype was reduced compared to 'wild type' B6 controls). Further, the gene for bleomycin hydroxylase is located within the chromosome 11 QTL, and functional studies of bleomycin hydrolase activity were consistent with this being a candidate gene in this model. However, further studies are required to confirm this hypothesis. The C3Hf/Kam and B6 mice have also been found to be similarly differentially responsive to lung fibrosis induced by radiation treatment. 115 A subsequent genome scan using an F2 cohort derived from these strains identified significant fibrosis susceptibility QTLs on chromosomes 1, 6, and 17, and a potential QTL on chromosome 18.116The chromosome 17 QTL was confirmed using a battery of mice congenic for the MHC region, further supporting this chromosomal region as an important genetic determinant of lung injury and fibrosis sequelae in inbred mice. Chromosomal position and length of all susceptibility QTLs discussed above, as well as key candidate genes, are summarized in Table 19.4. Although many chromosomes harbor susceptibility loci for pollution effects and acute lung injury (see also Fig. 19.1), it is readily apparent that QTLs on chromosomes 11 and 17 account for genetic variance in multiple susceptibility models. A cursory examination of the mouse genome informatics database (http:// www.jax.org) indicates that these two regions of the mouse genome (and by comparative mapping, the corresponding regions in the human genome) are rich with candidate genes for lung injury, inflammation, and immune function.

While it is tempting to speculate that polymorphisms in one or two key genes in both chromosomal regions may account for susceptibility to multiple environmental stimuli, 1~ the presence of multiple candidates indicates that much further work is necessary to confirm this idea.

GENETIC SUSCEPTIBILITY TO O C C U P A T I O N A L LUNG DISEASE As discussed above, inter-individual differences in genetic background (polymorphisms) havebeen demonstrated to influence susceptibility to pulmonary disease pathogenesis and responses to environmental pollutants. Considerable evidence is also accumulating for genetic susceptibility to the detrimental pulmonary effects of occupational exposures. While genome-wide scans for susceptibility loci have not been attempted to date, a number of recent association studies have been done for candidate susceptibility genes in chronic beryllium disease (CBD), coal worker's pneumoconiosis, silicosis, and occupational asthma that have provided strong evidence for a genetic contribution to some occupational lung diseases (Table 19.4). Perhaps the most well-known study was done by Richeldi et al. 46 who investigated the role of MCH class II genes in CBD, a lung disorder related to beryllium exposure and characterized by accumulation in the lung of beryllium-specific CD4 + MHC class II-restricted T-lymphocytes. Using a case-control design, it was found that 97% of the CBD cases expressed the HLA-DPB1 x0201-associated glutamic acid at residue 69, a position that has been associated with susceptibility to autoimmune disorders. 46 Subsequent studies have extended this observation, lz7'128 and have led to the possibility that residue 69 of HLA-DPB1 may be used as a diagnostic indicator of risk for CBD. Silicosis is a disease characterized by fibrosing nodular lesions that may eventually develop into progressive massive fibrosis, 129 and is prevalent in individuals such as coal and gold miners exposed chronically to dusts and other irritants. The gene for the proinflammatory cytokine tumor necrosis factor alpha (TNF) has been investigated in South African gold miners, and a functional polymorphism in the T N F promoter (at t h e - 3 0 8 position) has been associated with disease severity, rather than disease frequency. 12~ An SNP in the regulatory element of the gene for another proinflammatory cytokine (interleukin-1 alpha receptor antagonist [IL1RA]) was shown to associate with the incidence of silicosis in coal miners. 123 As demonstrated for a number of occupational lung diseases (Table 19.5), polymorphisms in the HLA class II alleles may also have an important role in disease pathogenesis involved with silica exposure. 122 The common link to all of these studies is inflammation, and suggests that those individuals with polymorphisms that promote inflammation and exposed chronically to silica, may be predisposed to the development of disease. Pneumoconiosis is a pulmonary disease also found in coal miners, and is characterized by inflammation that

commonly leads to fibrosis due to irritation caused by inhalation of dust. The mechanisms of susceptibility to onset and disease progression are not clear, but a role for genetic background has been proposed. 13~ It has been demonstrated that t h e - 3 0 8 promoter polymorphism in TNF, found to be related to the development of silicosis (see above), was also associated with coal workers' pneumoconiosis compared with miners without pneumoconiosis and controls. 119 Further support for a genetic component of susceptibility to pneumoconiosis was provided by Nadif etal. 11s They evaluated the role of the T N F - 3 0 8 polymorphism and a functional polymorphism (NcoI RFLP) in the gene for the proinflammatory cytokine lymphotoxin alpha (LTA) in a prospective epidemiological study in 253 coal miners differentially exposed to coal dust and cigarette smoke. They found an interaction of T N F - 3 0 8 genotype with coal dust exposure on erythrocyte GSH-Px activity (an intermediate response phenotype), with a significant association in those with high exposure whereas no associa-

tion was found among those with low exposure. Further, a significant association of pneumoconiosis prevalence with the LTA NcoI polymorphism was found in miners with low blood catalase activity, whereas no association was observed in those with high (a priori protective) activity. These experiments thus provide well-documented support of interactions of genetic background with environmental exposure and intermediate response phenotype in the pathogenesis of coal workers pneumoconiosis. Occupational asthma has been described in some individuals exposed to isocyanates and sawmill byproducts. However, only a small proportion of those who are exposed to these agents develop occupational asthma, suggesting that intrinsic factors such as genetic background may be important disease determinants. MCH class II genes have been associated with isocyanate- and western red cedarinduced asthma, 124'126'131 indicating that specific genetic immune mechanisms may reduce or increase the risk of developing these occupational diseases in exposed individuals.

Fig. 19.1. Distribution of acute lung injury and pollutant-induced inflammation and immune dysfunction quantitative trait loci (QTLs) in the mouse genome. Numbers above chromosomes are the chromosome numbers. Numbers beside the chromosomes indicate the approximate center of the QTL on the chromosome (in centiMorgans, cM). Patterned bars represent the approximate length of the QTLs. (See Color plate 8.)

Recent evidence also suggests that glutathione-S-transferase genotypes may be important determinants of occupational asthma induced by exposure to isocyanates. 125'132

CONTRIBUTION OF AGE GENETIC SUSCEPTIBILITY AIRBORNE POLLUTANTS

AND TO

To date, few studies have examined the influence of age on genetic susceptibility to pulmonary diseases, and none have examined the interaction of these host factors on pollutant susceptibility. A segregation analysis suggested that the effect of genotype on lung cancer varies by age, such that age-specific relative risks being greatest in young and declining thereafter. 133 Though no specific genes were identified, this study suggests strongly that age and genetic background may be important co-determinants of pulmonary disease. Both factors should be considered in future proposals/studies to understand susceptibility to air pollutant-induced lung disease.

SUMMARY

Pulmonary morbidity and mortality due to exposure to environmental and occupational stimuli continue to be important public health concerns worldwide, and identification of susceptible sub-populations is of critical importance. It is clear that numerous factors may contribute to inter-

individual susceptibility to the detrimental effects of these stimuli, including genetic background. Linkage analyses with inbred mice, and association studies with h u m a n subjects, have led to the identification of candidate susceptibility genes for p u l m o n a r y responses to air pollutants, including 0 3 and particles. Animal modeling of acute lung injury induced by hyperoxia, pulmonary irritants and chemotherapeutic agents (bleomycin, radiation) has also confirmed a role of candidate susceptibility genes. Casecontrol association studies in humans have also demonstrated that genetic background is an important intrinsic susceptibility factor in subsets of occupationally exposed individuals. An understanding of the biology of candidate genes will lead to an understanding of the genetic mechanisms of differential responses to environmental exposures. Further, characterization of a polymorphism in a pollutant susceptibility gene(s) may thus provide the means to identify individuals who are genetically susceptible to the development of injury and improve methods of risk assessment.

ACKNOWLEDGEMENTS Dr. Kleeberger was supported by grants from the National Institutes of Health (HL57142, ES09606, and P30 ES00002) and the Environmental Protection Agency (R826724).

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INTRODUCTION In the United States, 43% of children aged 2 months to 11 years of age live in a home with at least one tobacco smoker. 1 There is increasing evidence that this exposure to cigarette smoke has substantial adverse effects on the respiratory health of children. This chapter will address the effects of exposure to cigarette smoke during prenatal and postnatal life on the developing lungs.

EXPOSURE T O SMOKE The developing lung can be exposed to the constituents of smoke in utero from transplacental transfer from the pregnant woman, postnatally from ingestion of breast milk from the mother, and postnataUy from inhalation of environmental tobacco smoke (ETS). Environmental tobacco smoke is produced primarily from the smoldering end of a cigarette, with a small contribution from exhaled mainstream smoke and differs from mainstream smoke in important ways. ETS is aged rather than fresh and has a smaller particle size. Nicotine is in the gas phase of ETS but in the particulate phase of mainstream smoke. The uptake of various chemical constituents of cigarette smoke in a mainstream smoker vs a passive smoker has been estimated by Scherer et al. 2 (Table 20.1). Exposure to ETS for 8 hours and smoking 20 cigarettes per day result in similar uptakes of gas phase constituents. In contrast, exposure to ETS results in a much smaller uptake of particulate phase constituents than does mainstream smoking. 2 The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

Cotinine is a metabolite of nicotine which can be found in measurable quantities in plasma, urine and saliva of smoke-exposed individuals. Because of the ease with which cotinine can be measured, it is the most commonly used qualitative and quantitative marker of cigarette smoke exposure, and is considered an indirect marker of exposure to the numerous other constituents of cigarette smoke. If a pregnant woman smokes or is exposed to ETS, cotinine and presumably some of the other smoke constituents can be transferred to the developing fetus. In fetal life, the concentrations of cotinine in the serum of fetuses 21-36 weeks gestational age are about 90% of maternal levels, regardless of gestational age or number of cigarettes smoked. 3 Newborn infants of mothers who smoked have higher concentrations of cotinine in their hair than infants whose mothers did not smoke but were exposed to ETS in the home, who in turn had greater concentrations of cotinine in their hair than infants not exposed to smoke at all during pregnancy. 4 Thus, a fetus can be significantly exposed to smoke even when the pregnant mother's exposure is only environmental. Cotinine is passed via breast milk. Indeed, exclusively breast fed infants whose mothers are mainstream smokers have urinary cotinine concentrations that are in the same range as those of active smokers. 5 In general, the serum cotinine concentrations of children living with ETS are about 100-fold less than that of active smokers. 1'6 Cotinine concentrations of children are greater if the mother smokes than if the father smokes the same number of cigarettes per day, 6'7 suggesting greater uptake from the primary caretaker. As would be expected, cotinine concentrations increase with the number of cigarettes smoked in the home and in the same room as the child? Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

RESPIRATORY EFFECTS OF IN UTERO EXPOSURE T O S M O K E A number of studies have documented that exposing the fetus to smoke during gestation leads to decreased lung function, airway obstruction, and airway hyperresponsiveness in the newborn period. Lung function was measured by Stick et al. in 461 term infants at about 58 hours of life 9 and by Lodrup Carlsen etal. i~ in 804 infants at about 65 hours of life. Both groups showed that the time to peak tidal expiratory flow (tPTEF) as a proportion of total expiratory time (tE) was lower in infants of smoking mothers in a dose-dependent fashion, tPTEF/tE has been shown to correlate with indices of airway obstruction and with the development of wheezing lower-respiratory-tract illness. 1~ In the study by Lodrup Carlsen et al., 1~ compliance of the respiratory system was also lower in female babies. Similar findings of airway obstruction were found in one monthold infants of smoking mothers using VmaxFRc (maximum flow rate at functional residual capacity) as a measure of airway obstruction. 11'12 In a study by Hanrahan etal., 11 the VmaXFRC was about half that in control unexposed infants. In premature neonates, both tPTEF/tE and VmaxFRc were reduced by 14-18% in 40 infants of smoking mothers compared with 68 infants of nonsmoking mothers when studied at about 3 weeks of age. Since the infants were on average 7 weeks premature, the data suggest that at least some of the effect of smoke exposure on lung function occurs before 33 weeks of gestation. Newborn infants of smoking mothers also have greater airway responsiveness, a hallmark of asthma. In month-old healthy infants, the dose of inhaled histamine required to

reduce VmaxFRC by 40% was 5-fold less in infants with at least one parent who smoked as compared with infants whose parents did not smoke. 13 Interestingly, although maternal smoking is associated with premature delivery, it is also associated with enhanced lung maturity of the fetus as measured by lecithin/sphyngomyelin ratio (L/S). L/S ratio was one week advanced in fetuses of smoking mothers whose amniotic fluid was sampled between 28 and 36 weeks of gestation. The elevated L/S ratio correlated with the cotinine concentration in the amniotic fluid and was associated with increased concentrations of free, conjugated and total cortisol. TM This may explain why premature infants of smokers are at lower risk for developing infant respiratory distress syndrome at a given gestational age. Animal studies confirm the adverse effects of maternal smoke exposure on the developing fetus and also demonstrate histopathologic abnormalities. When pregnant rats were exposed to smoke (mainstream or ETS), their fetuses had reduced lung volume, reduced number of enlarged alveoli, decreased parenchymal elastic tissue, 15 increased density of interstitium, poorly developed elastin and collagen, 16 and increased Clara cell secretory protein. 17 Thus there is ample evidence from both human and animal studies that mainstream smoking by the pregnant mother changes fetal lung development. These include changes in airflow, airway responsiveness and lung maturity in human studies and changes in morphology and enzyme function in animal studies. In humans, at least some of these effects may occur even if the mother herself does not smoke but is exposed to ETS in the home.

RESPIRATORY S Y M P T O M S P R O D U C E D BY P O S T N A T A L EXPOSURE TO ETS Many studies have used questionnaires to determine if children raised in the homes of smokers have more respiratory symptoms than those raised in the absence of smoke exposure. These studies provide a large body of data from around the world showing that smoke exposure is associated with cough, wheeze and respiratory illness. When studies are performed in children beyond the neonatal period, it is difficult to differentiate the relative contribution of the effects of smoke exposure on lung symptoms produced by fetal exposure from the subsequent exposure of the child to ETS. In general, it has been shown that the mother's smoking is more deleterious to the respiratory health of her children than is the father's smoking. There are two possible explanations for this: one is that the lung damage occurs prenatally due to transplacental exposure to the mother's mainstream smoke; the other is that the mother is usually the primary caretaker and thus provides the infant and child with a greater exposure to ETS postnatally than do other smokers in the household. The effects of prenatal exposure to mainstream smoke have been reviewed above. There is, however, also good evidence that postnatal exposure to ETS plays a role in the adverse effects of smoking on children's respiratory health. The most useful types of studies which examine the effects of postnatal ETS exposure separate from prenatal smoke exposure are those in which (1) the mother is not a smoker but other household members are smokers, (2) the influence of smoking by other household members is reported, (3) there is a dose-response relationship between current ETS exposure and the effect reported, (4) statistical methods are used to separate in utero from postnatal effects, and (5) animals are used to explicitly study known exposures. With increasing numbers of smokers and cigarettes smoked in the home, children aged 8-11 years have more wheeze with colds, persistent wheeze, shortness of breath with wheeze and emergency room visits for wheeze; TM the odds ratio was 1.5-1.8 for 30 or more cigarettes smoked per day in the home. At this age, the association of total exposure to ETS was greater than the association with maternal smoking. In a Chinese study, smoking was very rare in young women in Shanghai and there were no mothers who smoked. 19 A dose-response relationship for ETS exposure was demonstrated whereby the odds ratio for hospitalization for respiratory illnesses in the first 2 years of life of 160 low birth weight infants was 2.9 if they were exposed to light ETS (1-19 cigarettes/day) and 4.5 if they were exposed to heavy ETS (20 or more cigarettes/day). In a study of the effect of day care on children's health, it was found that after adjusting for the smoking status of the mother, the effect of placing a 3-year old child in a day care setting where the caregiver smokes increases the risk of a wheezing lower respiratory illness by more than 3-fold. 2~ In a study of 1501 Malaysian 12-year old children, only 1.9% of the mothers were

smokers; 21 this study showed a significant 1.7-fold risk of the child having chest illness if he/she slept in the same room as a smoker. A meta-analysis of 50 publications published prior to 1997 showed that, for children less than 3 years of age, the odds ratio for lower respiratory illness was 1.72 for children whose mothers smoked. For children whose mothers did not smoke but other household members did, the odds ratio was still greater than 1.0 at 1.29.22 In a separate meta-analysis of 17 publications, ETS exposure in infancy or early childhood was associated with 1.93-fold increased incidence of lower respiratory infection requiring hospitalization. 23 Since cough and wheeze are major symptoms that are increased in children raised in the homes of smokers, we studied the activity of the sensory nerves of the lungs which are responsible for initiating cough and airway narrowing in a guinea pig model of pediatric ETS exposure. We exposed guinea pigs to sidestream smoke ( l m g total suspended particulates/m, 3 the equivalent of a smoky bar) for 6 hours/day, 5 days/week from age 1 week to age 6 weeks of life, the equivalent o f human childhood. We found that both their rapidly adapting receptors 24 and C-fibers 25 were more excitable when stimulated. Furthermore, the increased neural exitability was relayed to the second order neurons in the brainstem (Fig. 20.1). 26 Thus, using a model

Fig. 20.1. Effect of chronic sidestream smoke (SS) exposure on the response of neurons in the nucleus tractus solitarius (NTS) to stimulation of bronchial C-fibers. Guinea pigs were exposed to SS (1 mg/m 3 total suspended particulates) or to filtered air (FA) from 1 to 6 weeks of life and studied 16 hours after the last exposure. The top trace is an example of a recording from a FA-exposed guinea pig and the trace below it is an example of a recording from a SS-exposed guinea pig. The graph below shows the grouped data for all guinea pigs. After injection of capsaicin into the left atrium (marked by the triangles), the frequency and duration of action potentials of the NTS neurons increased more in the SS-exposed animals than in the FA-exposed animals. (Reproduced with permission from Mutoh T, Joad JP, Bonham AC. Chronic passive cigarette smoke exposure augments bronchopulmonary C-fibre inputs to nucleus tractus solitarii neurones and reflex output in young guinea-pigs. J. Physiol. 2000; 523(Part 1):223-33.)

in which animals were exposed to ETS only in the postnatal period, we showed that a clinically relevant concentration of ETS enhanced the excitability of the nerves responsible for cough and wheeze.

L U N G F U N C T I O N C H A N G E S IN C H I L D R E N EXPOSED P O S T N A T A L L Y TO ETS In addition to symptoms of airway obstruction such as wheeze, children beyond the neonatal period raised in the homes of smokers demonstrate small but measurable changes in baseline lung function, consistent with airway obstruction. In a study of 2765 Australian children 8-11 years of age, FEF25_75 was decreased by 2.4% if more than 20 cigarettes were smoked in the home daily. 27 A similar small decrease in FEV1/VC of 1.5% in male children of smoking parents was noted in a study of 635 New Zealand children 9-15 years old. z8 As with respiratory symptoms, the effect of the mother's smoking on the lung function of the child is greater than that of other household members. In a longitudinal study of 1196 children 5-19 years of age, smoking of the mother but not the father was associated with a 7% decrease in the expected increase in FEV x over a 5 year period. 29 Thus, as with respiratory symptoms, it is important to examine data from studies which focus on the effects of postnatal ETS exposure separate from prenatal smoke exposure. Statistical techniques have been used to separate the effects of smoke exposure in early life (in utero and the first 5 years of life) from current maternal smoke exposure in 6-10-year old children; 3~ it was found (after adjusting for early smoke exposure) that current exposure to maternal smoking decreased FEF25_75 by 2.3%. In a study of 317 children 12-15 years of age who did not live with smokers or smoke themselves, and hence experienced only occasional exposure to ETS, there was a measurable effect of ETS exposure on lung function; 31 the children with greater exposures to ETS as documented by urinary cotinine had lower lung function as measured by FEF25_75. Even in the absence of clinical asthma, children exposed to smoke may have increased airway responsiveness. Airway responsiveness has been determined by measuring spontaneous changes in airflow with peak flow variability or by using drug, exercise, or cold air challenges. A doseresponse effect of ETS on peak flow variability has been shown in boys 6-9 years of age; 32 as the urinary cotinine excretion increased, so did the ratio of the amplitude over the mean of the diurnal peak flow rates. In this study there was no similar effect in girls. Another study showed that the father smoking in a crowded household increased the odds ratio for hyperresponsiveness to methacholine to 2.7 in their 7-10-year old daughters; 33 in this study, the effects were less prominent in their sons. Thus it is not clear whether one gender is more susceptible to ETS than the other. Interestingly, using exercise challenge as a measure of res-

ponsiveness of airways, it has been showed that exposure to ETS during the first year of life but not during pregnancy or currently was associated with a 1.8-fold increased risk of airway hyperresponsiveness. 34 A recent meta-analysis suggests a small but real increase in airway responsiveness in school aged children exposed to ETS. 35 In summary, the data suggest that exposure to ETS is associated with a decrease in airway caliber and increase in airway responsiveness in children exposed to ETS.

EFFECTS OF PRENATAL A N D P O S T N A T A L SMOKE EXPOSURE ON THE D E V E L O P M E N T OF A S T H M A Children affected by wheezing have been grouped according to temporal criteria into (1) 'transient early wheezers' who wheeze before 3 years of age but not at 6 years of age, (2) 'late onset wheezers' who do not wheeze in the first 3 years but do wheeze at 6 years, and (3) 'persistent wheezers' who wheeze both in the first 3 years and at 6 years. 36 Although many variables such as rhinitis, a family history of asthma, and maternal smoking were associated with persistent wheeze, only maternal smoking was associated with transient early wheeze. Interestingly, only the transient early wheezers had airflow obstruction (a lower Vmaxvg c) in infancy. By 6 years of age, airflow obstruction was still found in the transient early wheezers, but it was also found in the persistent wheezers. Late onset wheezing was not associated with maternal smoking or airway obstruction in infancy or at 6 years of life. These data suggest that maternal smoking plays a primary role in causing children to develop repeated wheezing illnesses in early life but that later in life other factors also become important in the maintenance of wheezing respiratory illness. Other studies indicate that smoke exposure, both prenatally and postnatally, is associated with the development of asthma in infants and young children. Data from the Child Health Supplement to the 1981 National Health Interview Survey of 4331 children 0-5 years of age showed a 2.1-fold risk for having asthma, and a 2.6-fold risk for developing asthma early (in the first year of life) among children whose mothers smoked at least one-half pack per day. 37 In another study, the 2.5-fold increased risk of developing asthma before 12 years of age due to maternal smoking occurred only in children whose mothers had less than 12 years of formal education. Possible reasons for this interaction with socioeconomic status include crowding, exposure to unique aeroallergens, nutrition and infections. 38 In a case-controlled study, children 3-14 years of age attending an emergency department or an asthma clinic were compared with children without asthma attending the emergency department. 39 It was found that smoking was more prevalent in the maternal caregiver (odds ratio 2.0) and that the urinary cotinine concentration (cotinine to creatinine ratio) was 65% higher in the asthmatic children. In a separate case control study of children 7-9 years

old, 4~ both maternal smoking in pregnancy (odds ratio 1.9) and each additional household smoker (odds ratio 1.15) were independent predictors of asthma/wheeze. Furthermore, as the child's urinary cotinine/creatinine ratio increased, the odds ratio for asthma/wheeze increased. Using the recent Third National Health and Nutrition Examination Survey 1988-1994, the odds ratio for asthma in young children 2 months to 5 years of age was 2.1 for household exposure to more than one pack per day of cigarette smoke and 1.8 for prenatal exposure. 41 It was estimated that for young children 2 months to 2 years of age, 40-60% of the cases of asthma, chronic bronchitis, and three or more episodes of wheezing were attributable to ETS exposure. A recent meta-analysis suggested that maternal smoking was associated with developing wheeze/asthma in the first 5-7 years of life (odds ratio 1.31) but not in later childhood (odds ratio 1.13). 42 In epidemiological studies, it is difficult to determine the risk of developing asthma due to smoke exposure separated from other confounding factors such as household crowding, infections, nutrition, or exposure to other pollutants or allergens. Thus we used an animal model to determine if exposure to sidestream smoke would result in hyperreactive airways as occurs in asthma. 43 We exposed rats to sidestream smoke (1 mg total suspended particulates/m 3) or to filtered air for 4 hours/day, 7 days/week from day 3 of gestation until birth. At birth the pups were randomly assigned to receive either sidestream smoke or filtered air until the equivalent of adolescence (7-10 weeks of age). We found that rats exposed to sidestream smoke only during gestation or only during postnatal life did not develop hyperreactive airways. However, if they received it both during gestation and in postnatal life, their airways were hyperreactive to methacholine. In the sidestream smoke-exposed group, the increase in pulmonary resistance (a measure of airway obstruction) at the highest doses of methacholine was more than 2-fold that in the filtered air-exposed group (Fig. 20.2). This increase in airway responsiveness was associated with an increase in the number of pulmonary neuroendocrine cells, suggesting that their bronchoconstrictor mediators may play a mechanistic role (Fig. 20.3). In a subsequent study, we showed that ETS exposure need only occur during gestation and in the first 3 weeks of life. Despite 5 subsequent weeks in filtered air, their airways were still hyperresponsive to methacholine in adolescence (8 weeks of age). 44

EFFECT OF E X P O S I N G C H I L D R E N T O ETS O N SEVERITY OF A S T H M A Environmental tobacco smoke exposure also appears to increase the severity of asthma once it has become established. A number of studies have shown that asthma is worse in children of smoking mothers. 4s A study of 94 children with asthma found that children whose mothers smoked had 47% more symptoms, a 4-fold increase responsiveness to histamine, and 13% decrease in FEV1/FVC; 4s it was found that the effect was greater in boys than in girls and

Fig. 20.2. Methacholine-induced changes in pulmonary resistance (RL) in isolated lungs from rats exposed in utero to filtered air (FA) followed by 8-10 weeks postnatal FA (FA/FA), in utero FA followed by postnatal sidestream smoke (SS, 1 mg/m 3 total suspended particulates, FA/SS), in utero SS followed by postnatal FA (SS/FA), and in utero SS followed by postnatal SS (SS/SS). SS/SS exposure resulted in hyperresponsiveness to methacholine. (Reproduced with permission from Joad JP, Ji C, Kott KS et al. In utero and postnatal effects of sidestream cigarette smoke exposure on lung function, hyperresponsiveness, and neuroendocrine cells in rats. Toxicol. Appl. Pharmacol. 1995; 132:63-71 .)

Fig. 20.3. Number of neuroendocrine cells (identified by neuronspecific enolase staining) per centimeter basal lamina in lungs from rats exposed in utero to filtered air (FA) followed by 8-10 weeks postnatal FA (FA/FA), in utero FA followed by postnatal sidestream smoke (SS, 1 mg/m 3 total suspended particulates, FA/SS), in utero SS followed by postnatal FA (SS/FA), and in utero SS followed by postnatal SS (SS/SS). SS/SS exposure resulted in more pulmonary neuroendocrine cells. (Reproduced with permission from Joad JP, Ji C, Kott KS et al. In utero and postnatal effects of sidestream cigarette smoke exposure on lung function, hyperresponsiveness, and neuroendocrine cells in rats. Toxicol. Appl. Pharmacol. 1995; 132:63-71 .)

in older boys (with presumably longer exposure to ETS) compared with younger boys. 46 An increased responsiveness to cold dry air was also seen in asthmatic children whose mothers smoked. 47 In a study of 276 children 4-17 years old with asthma, 48 it was found that passive smoking was associated with a 63% increase in emergency department visits but not with hospitalizations. A similar study of 199 asthmatic children 8 months to 13 years old showed that

the children with the highest urinary cotinine values had a 1.8-fold increase in the risk of acute exacerbations of asthma. 49 More recently, a study of 74 children 7-12 years old with asthma showed that ETS exposure decreased peak expiratory flow rates, and increased bronchodilator use, cough, and phlegm production 8-12-fold. 5~ In contrast, data from the Third National Health and Nutrition Examination Survey 41 suggested that household exposure to cigarettes was not associated with increased use of asthma medications as would be expected if asthma severity was worse.

EFFECTS OF ACUTELY EXPOSING A S T H M A T I C C H I L D R E N TO ETS Only a few studies have acutely exposed children with asthma to ETS under an experimental protocol. In one such study, 11 children 8-13 years of age with asthma who were receiving controller asthma therapy were exposed to 2.7 mg/m 3 total suspended particulates ETS for 1 hour or to a sham exposure to air. sl Although the subjects reported increased eye irritation with the ETS exposure there was no reduction in their FEV 1, SRaw, and no increase in airway responsiveness to histamine. In a later study by the same group, 52 13 children with mild asthma who were not using antiinflammatory controller therapy were exposed to 3.1 mg/m 3 total suspended particulates ETS for 1 hour. ETS exposure caused a significant drop in their FEV 1 (7.2% vs 3.2% with air exposure) at rest but did not affect the decrease in FEV 1 with exercise. The resting drop in FEV 1 occurred within 5 minutes and then remained stable for the next hour. There was a significant interaction between subjects and exposures suggesting that the mean changes due to ETS were mainly caused by 3 of the 13 children. A similar reproducible sensitivity to ETS has been shown in adult asthmatics. 53

EFFECTS OF PRENATAL A N D POSTNATAL SMOKE EXPOSURE ON THE D E V E L O P M E N T OF ALLERGY One of the greatest risk factors for the development of asthma is atopy as measured by IgE-mediated skin test positivity or allergen-specific IgE serum concentrations. Several studies have shown that children of smoking parents have increased atopy. In one study, maternal cigarette smoking was found to be associated with atopy in 5-9-year old children as evaluated by skin tests to 4 environmental allergens. 54 In another study, male but not female children of smoking parents had both higher IgE levels but also a higher concentration of eosinophils in the blood, a hallmark of the immune changes associated with allergy. The effect of smoke exposure on eosinophils was even more pronounced than that on IgE, since for a given IgE concentration, the eosinophils were at least 3-fold higher in the boys exposed to smoke. Furthermore there was a dose-response relation-

ship between the number of cigarettes to which the boys were exposed and the magnitude of their eosinophilia. 55 As with the other manifestations of smoke exposure, it may not be known how much of the effect is due to in utero exposure as compared with postnatal exposure. Newborn infants of nonallergic parents whose mothers smoked had a 3-fold increase in the risk of having an elevated IgE and a 4-fold increased risk of having allergic symptoms such as eczema, urticaria, asthma, or food allergy before 18 months. 56 In a separate study of children 1-4 years of age, the odds ratio for allergy to cat increased from 3 when the children were exposed to an indoor cat only to 42 when they were exposed to ETS in addition to a damp environment with an indoor cat. 57 A study in mice confirms that exposure to smoke enhances the transformation of the immune response to a T helper-2 type allergic response, s8 T-cells involved in allergic responses are predominantly of the T helper-2 phenotype and produce interleukin-4 (IL-4), IL-5, IL-6, IL-9, IL-10, and IL-13 rather than interferon-),, a T helper-1 cytokine. 59 Six- to eight-week old mice were made allergic to ovalbumin by injection with adjuvant followed later by an exposure to aerosolized ovalbumin. They were exposed either to filtered air or to ETS (1 mg/m 3 total suspended particulates) for 6 hours/day, 5 days/week for 6 weeks. ETS exposure increased total IgE, ovalbumin-specific IgG1, and eosinophils in the blood and IL-4 and IL-10 in the lung. Thus, both human and animal studies suggest that smoke exposure results in allergic changes in the immune system. This is important since most infants and young children who will go on to have persistent wheezing and asthma show allergic inflammation such as high IgE production and esosinphilic immune responses both in the airways and in circulation. 59

EFFECTS OF PRENATAL A N D POSTNATAL SMOKE EXPOSURE ON S U D D E N I N F A N T DEATH A N D OBSTRUCTIVE APNEA Exposure to smoke appears to be one of the main preventable causes of sudden infant death syndrome (SIDS). In a nationwide case-control study of 485 SIDS deaths from New Zealand 6~ it was shown that the risk of SIDS was 4-fold greater in infants of mothers who smoked during pregnancy; the risk increased as the number of cigarettes smoked by the mother increased. Furthermore, smoking by other household members increased the risk of SIDS 2.4-fold but only if the mother smoked, suggesting that a prenatal effect was probably important as well as a postnatal effect. This was confirmed in another case-control study of 435 SIDS deaths reported in the National Maternal and Infant Health Survey (USA); this study showed that the increased risk for SIDS death was 3-fold if the mother smoked both during and after pregnancy and 2-fold if she smoked only after the birth of the child. 61 Interestingly, although breast feeding is protective in preventing SIDS for infants of nonsmoking

mothers (odds ratio-0.37), it is not protective in smoking mothers (odds ratio= 1.38) suggesting that the beneficial properties of breast milk are counteracted by the deleterious smoke constituents. 62 Although it is not known what causes SIDS, several possible mechanisms are related to smoke exposure: narrower airways and obstructive apneas. It was recently shown that 19 infants who died of SIDS and whose mothers smoked both during pregnancy and thereafter had thicker inner airway wall thickness than 29 infants who died of SIDS whose mothers did not smoke. 63 This thicker inner airway wall could contribute to airway narrowing. In a polysomnographic study of 509 infants (115 newborn and the others 5-29 weeks of age) it was found that infants whose mothers smoked 10 or more cigarettes per day during pregnancy had a 2.76-fold increased risk of having obstructive apneas during sleep. 64 Father's smoking enhanced the risk only if the mother also smoked suggesting that the major effect occurred prenatally. Finally, the risk of laryngospasm is greater in young children exposed to ETS. In a retrospective study of 310 children (mean age 2 years) undergoing anesthesia with halothane, 9.4% developed laryngospasm if they were exposed to ETS in the home whereas only 0.9% developed laryngospasm if they were not exposed to ETS in the home. 65 This finding is consistent with data from an animal model showing that volatile anesthetics stimulate capsaicinsensitive laryngeal neurons 66 and we found that sidestream smoke exposure enhances the capsaicin-stimulated activity of C-fibers. 25

CONCLUSIONS Many children are exposed to smoke both prenatally and postnatally. Prenatal exposure to mainstream smoke from the mother and even to ETS from the mother in utero has been shown to affect fetal lung development. Soon after birth, the lungs of infants exposed prenatally to smoke show evidence of airway obstruction, airway hyperresponsiveness, and changes in maturation. Children exposed to ETS postnatally have more symptoms of cough, wheeze, and respiratory illnesses. They have small decreases in lung function and increases in airway responsiveness. Smoke exposure is associated with the early development of asthma and may increase the severity of asthma once it develops. Furthermore, smoke exposure is associated with the development of atopy and T helper-2 i m m u n e responses which may further worsen asthma. Finally smoke exposure is associated with sudden infant death, obstructive apnea and anesthesia-induced laryngospasm. In general, these effects are most apparent in infancy and early childhood. These effects are potentially preventable, and public health efforts should be directed at reducing the prenatal and antenatal exposure of children to cigarette smoke. The greatest reduction in adverse respiratory health effects in children would result from encouraging women to stop smoking during pregnancy and in the first years of their children's lives.

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20. Holberg CJ, Wright AL, Martinez FD et al. Child day care, smoking by caregivers, and lower respiratory tract illness in the first 3 years of life. Pediatrics 1993; 91:885-92. 21. Azizi BHO, Henry RL. The effects of indoor environmental factors on respiratory illness in primary school children in Kuala Lumpur. Int. J. Epidemiol. 1991; 20:144-50. 22. Strachan DP, Cook DG. Health effects of passive smoking. Parental smoking and lower respiratory illness in infancy and early childhood. Thorax 1997; 52:905-14. 23. Li JS, Peat JK, Xuan W et al. Meta-analysis on the association between environmental tobacco smoke (ETS) exposure and the prevalence of lower respiratory tract infection in early childhood. Pediatr. Pulmonol. 1999; 27:5-13. 24. Bonham AC, Kott KS, Joad JP. Sidestream smoke exposure enhances rapidly adapting receptor responses to substance P in young guinea pigs.J. Appl. Physiol. 1996; 81:1715-22. 25. Mutoh T, Bonham AC, Kott KS et al. Chronic exposure to sidestream tobacco smoke augments lung C-fiber responsiveness in young guinea pigs.J. Appl. Physiol. 1999; 87:757-68. 26. Mutoh T, Joad JP, Bonham AC. Chronic passive cigarette smoke exposure augments bronchopulmonary C-fibre inputs to nucleus tractus solitarii neurones and reflex output in young guinea-pigs. J. Physiol. 2000; 523(Part 1):223-33. 27. Haby MM, Peat JK, Woolcock AJ. Effect of passive smoking, asthma, and respiratory infection on lung function in Australian children. Pediatr. Pulmonol. 1994; 18:323-9. 28. Sherrill DL, Martinez FD, Lebowitz MD et al. Longitudinal effects of passive smoking on pulmonary function in New Zealand children.Am. Rev. Respir. Dis. 1992; 145:1136-41. 29. Tager IB, Weiss ST, Munoz A et al. Longitudinal study of the effects of maternal smoking on pulmonary function in children. N. Engl. J. Medical 1983; 309:699-703. 30. Wang X, Wypij D, Gold DR etal. A longitudinal study of the effects of parental smoking on pulmonary function in children 6-18 years. Am. J. Respir. Crit. Care Med. 1994; 149:1420-5. 31. Corbo GM, Agabiti N, Forastiere F e t a l . Lung function in children and adolescents with occasional exposure to environmental tobacco smoke. Am. J. Respir. Crit. Care Med. 1996; 154:695-700. 32. Kuehr J, Frischer T, Karmaus W et al. Cotinine excretion as a predictor of peak flow variability. Am. J. Resp. Crit. Care Med. 1998; 158:60-4. 33. Forastiere F, Agabiti N, Corbo GM et al. Passive smoking as a determinant of bronchial responsiveness in children. Am.J. Respir. Crit. Care Med. 1994; 149:365-70. 34. Frischer T, Kuehr J, Meinert R et al. Maternal smoking in early childhood: a risk factor for bronchial responsiveness to exercise in primary-school children. J. Pediatr. 1992; 121:17-22. 35. Cook DG, Strachan DP. Parental smoking, bronchial reactivity and peak flow variability in children. Thorax 1998; 53:295-301. 36. Martinez FD, Wright AL, Taussig LM etal. Asthma and wheezing in the first six years of life. N. Engl. J. Medical 1995; 332:133-8. 37. Weitzman M, Gortmaker S, Walker DK et al. Maternal smoking and childhood asthma. Pediatrics 1990; 85:505-11. 38. Martinez FD, Cline M, Burrows B. Increased incidence of asthma in children of smoking mothers. Pediatrics 1992; 89:21-6. 39. Ehrlich R, Kattan M, Godbold J e t al. Childhood asthma and passive smoking: urinary cotinine as a biomarker of exposure. Am. Rev. Respir. Dis. 1992; 145:594-9. 40. Ehrlich RI, Du Toit D, Jordaan E et al. Risk factors for childhood asthma and wheezing: importance of maternal and household smoking.Am.J. Respir. Crit. Care Med. 1996; 154:681-8. 41. Gergen PJ, Fowler JA, Maurer KR et al. The burden of environmental tobacco smoke exposure on the respiratory health of children 2 months through 5 years of age in the United States. Third National Health and Nutrition Examination Survey, 1988-94. Pediatrics 1998; 101:E81-6.

42. Strachan DP, Cook DG. Parental smoking and childhood asthma, longitudinal and case-control studies. Thorax 1998; 53:204-12. 43. Joad JP, Ji C, Kott KS et al. In utero and postnatal effects of sidestream cigarette smoke exposure on lung function, hyperresponsiveness, and neuroendocrine cells in rats. Toxicol. Appl. Pharmacol. 1995; 132:63-71. 44. Joad JP, Bric JM, Peake JL etal. Perinatal exposure to aged and diluted sidestream cigarette smoke produces airway hyperresponsiveness in older rats. Toxicol. Appl. Pharmacol. 1999; 155:253-60. 45. Murray AB, Morrison BJ. The effect of cigarette smoke from the mother on bronchial responsiveness and severity of symptoms in children with asthma. J. Allergy Clin. Immunol. 1986; 77:575-81. 46. Murray AB, Morrison BJ. Passive smoking by asthmatics: its greater effect on boys than on girls and on older than on younger children. Pediatrics 1989; 84:451-9. 47. O'Connor GT, Weiss ST, Tager IB et al. The effect of passive smoking on pulmonary function and nonspecific bronchial responsiveness in a population-based sample of children and young adults.Am. Rev. Respir. Dis. 1987; 135:800--4. 48. Evans D, Levison J, Feldman CH, et al. The impact of passive smoking on emergency room visits of urban children with asthma.Am. Rev. Respir. Dis. 1987; 135:567-72. 49. Chilmonczyk BA, Salmun LM, Megathlin KN, et al. Association between exposure to environmental tobacco smoke and exacerbations of asthma in children. N. Engl. J. Medical 1993; 328:1665-9. 50. Schwartz J, Timonen KL, Pekkanen J. Respiratory effects of environmental tobacco smoke in a panel study of asthmatic and symptomatic children. Am. J. Respir. Crit. Care Med. 2000; 161:802-6. 51. Oldigs M, Jorres R, Magnussen H. Acute effect of passive smoking on lung function and airway responsiveness in asthmatic children. Pediatr. Pulmonol. 1991; 10:123-31. 52. Magnussen H, Lehnigk B, Oldigs M etal. Effects of acute passive smoking on exercise-induced bronchoconstriction in asthmatic children.J. Appl. Physiol. 1993; 75:553-8. 53. Menon PK, Stankus RP, Rando RJ et al. Asthmatic responses to passive cigarette smoke: persistence of reactivity and effect of medications.J. Allergy Clin. Immunol. 1991; 88:861-9. 54. Weiss ST, Tager IB, Munoz A et al. The relationship of respiratory infections in early childhood to the occurrence of increased levels of bronchial responsiveness and atopy. Am. Rev. Respir. Dis. 1985; 131:573-8. 55. Ronchetti R, Macri F, Ciofetta Get al. Increased serum IgE and increased prevalence of eosinophilia in 9-year-old children of smoking parents.J. Allergy Clin. Immunol. 1990; 86:400-7. 56. Magnusson CGM. Maternal smoking influences cord serum IgE and IgD levels and increases the risk for subsequent infant allergy.J. Allergy Clin. Immunol. 1986; 78:898-904. 57. Lindfors A, Hage-Hamsten M, Rietz H etal. Influence of interaction of environmental risk factors and sensitization in young asthmatic children. J. Allergy Clin. Immunol. 1999; 104:755-62. 58. Seymour BWP, Pinkerton KE, Friebertshauser KE etal. Second-hand smoke is an adjuvant for T helper-2 responses in a murine model of allergy. J. Immunol. 1997; 159:6169-75. 59. Martinez FD. Maturation of immune responses at the beginning of asthma.J. Allergy Clin. Immunol. 1999; 103:355-61. 60. Mitchell EA, Ford RPK, Stewart AW, etal. Smoking and Sudden Infant Death Syndome. Pediatrics 1993; 92:893-6. 61. Schoendorf KC, Kiely JL. Relationship of sudden infant death syndrome to maternal smoking during and after pregnancy. Pediatrics 1992; 90:905-8. 62. Klonoff-Coher HS, Edelstein SL, Lefkowitz ES, et al. The effect of passive smoking and tobacco exposure through breast milk on Sudden Infant Death Syndrome. JAMA 1995; 273: 795-8.

63. Elliot J, Vullermin P, Robinson P. Maternal cigarette smoking is associated with increased inner airway wall thickness in children who die from sudden infant death syndrome. Am. J. Respir. Crit. Care Med. 1998; 158:802-6. 64. Kahn A, Groswasser J, Sottiaux M et al. Prenatal exposure to cigarettes in infants with obstructive sleep apneas. Pediatrics 1994; 93:778-83.

65. Lakshmipathy N, Bokesch PM, Cowan DE et al. Environmental tobacco smoke: a risk factor for pediatric laryngospasm. Anesth. Analg. 1996; 82:724-7. 66. Mutoh T, Tsubone H, Nishimura R etal. Responses of laryngeal capsaicin-sensitive receptors to volatile anesthetics in anesthetized dogs. Respir. Physiol. 1998; 111:113-25.

INTRODUCTION

DALLY

Maternal tobacco smoking during pregnancy has been associated with many adverse outcomes such as increased incidences of pneumonia and bronchitis, 1-3 impaired lung function, and general respiratory disorders have been observed in children of smoking parents. 4'5 In some epidemiological studies a relationship has been drawn between diseases of the distal airways of children of smoking parents and chronic bronchitis and emphysema of the same individuals in adulthood. 6 It is therefore clear that certain components of cigarette smoke interfere with normal lung cell function and development. Nicotine, a major component of tobacco smoke, is implicated in the adverse effects of tobacco smoke on the metabolic 7 and structural development of the lung. 8 Indeed, several studies have shown that maternal nicotine exposure during gestation and lactation adversely affect lung development. 9'1~

In regular smokers of cigarettes, the daily intake of nicotine varies widely between 10.5 mg and 78.6 mg, with an average intake of 35rag. 13 The nicotine intake per cigarette averages- 1 mg. 13 The absorption of nicotine by the lungs from tobacco smoke is rapid and arterial blood concentrations reach a peak of 49.2+9.7 ng/ml after 5 min. When a cigarette is smoked, the maximum concentration of nicotine in the jugular vein peaks at 22.4+3.9ng/ml at about 7 min; TM the nicotine concentration then levels off at 4-6 h. Because its half-life in adults is about 120min, nicotine persists in potentially biologically active concentrations throughout the night, despite nocturnal cessation of smoking. 15 Although the mean daily intake is not different between men and women, blood nicotine concentrations may differ as men metabolize nicotine faster than women. 16 Under experimental conditions, animals are usually treated with 1 mg nicotine/kg body weight/day, a dose equivalent to that of heavy smokers. 13 In pregnant rats, this dose results in an average nicotine level in amniotic fluid of 15.4+3.9ng/ml, equivalent to the levels of nicotine found in amniotic fluid of pregnant women who smoke tobacco. 17 By comparison, light smokers would typically receive one-half of this dose, and transdermal nicotine and nicotine gum would deliver one-fourth and one-eighth of this dose, respectively. TM

UPTAKE

OF NICOTINE

Nicotine is typically absorbed into the body by means of inhalation, transdermal patches, gums, nasal sprays, snuff and chewing tobacco. 11 When inhaled in tobacco smoke, nicotine is directly absorbed via the pulmonary capillaries into the pulmonary venous circulation; it then enters the arterial supply to tissues which take it up in variable amounts. When taken intranasally, nicotine is absorbed into a rich submucosal venous plexus that drains into the facial, sphenopalatine, and ophthalmic veins, from where it enters the left side of the heart and appears in arterial blood. 12

The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

NICOTINE

INTAKE

ABSORPTION, DISTRIBUTION AND METABOLISM OF NICOTINE IN PREGNANCY Nicotine crosses the placenta rapidly from mother to fetus, reaching higher concentrations in the fetal circulation than

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in the mother's; that is, the disappearance of nicotine from fetal blood is slower than from the maternal circulation. It is important to note that fetal skin, due to its low keratinisation during the second trimester, is readily permeable to substances such as nicotine in amniotic fluid. 17Nicotine is even absorbed by adult skin. 19 Elevated levels of nicotine occur in the amniotic fluid and placental tissue of pregnant women who smoke. 2~ Considerable amounts of nicotine enter fetal blood and nicotine reaches high concentrations in the fetal adrenal glands, heart, kidneys, stomach wall, spleen 22 and lung. 23 During the first half of pregnancy, concentrations of cotinine, the major metabolite of nicotine, are greater in amniotic fluid and fetal serum than in maternal serum in both active and passive smokers. 21 The relatively large area available for nicotine absorption by the fetus, together with the slower elimination of nicotine by fetal tissues, explains why the fetus is exposed to higher concentrations of nicotine than the mother. This could enhance the toxicity of nicotine to the fetus. 24 After birth, elevated levels of nicotine occur in the maternal milk 17 and nicotine is rapidly absorbed by the infant, as determined by its presence in the saliva (166ng/ml) of breast-fed infants. 25

EFFECTS OF MATERNAL N I C O T I N E EXPOSURE D U R I N G GESTATION A N D L A C T A T I O N ON THE PLACENTA A N D FETAL G R O W T H Extensive epidemiological studies have demonstrated a positive correlation between the concentration of nicotine in the maternal blood and fetal growth restriction. 26 Smokinginduced alterations in the morphology of the umbilical arterial wall and a reduction in umbilico-placental blood flow are thought to contribute to fetal growth restriction and low birth weight of infants of mothers who smoke during pregnancy. 27 As nicotine and its metabolites readily cross the fetal membranes and accumulate in amniotic fluid, 17 it is reasonable to assume that they will also affect placental morphology and function. It has been demonstrated that placental tissue from smoking mothers has both a reduced uptake and transfer of amino acids. 28 Addition of high levels of nicotine (10-100 times greater than serum concentrations measured 5 min after smoking one cigarette) to cultures of placental slices from smoking mothers results in an inhibition of placental amino acid transport. 29 This suggests that the decrease in placental amino acid uptake induced by nicotine, as demonstrated in vitro, may contribute to the growth restriction observed in fetuses of smoking mothers. 28 Trophoblast differentiation during the first trimester of pregnancy is required for implantation and subsequent formation of the placenta, a clear prerequisite for normal fetal development. It has been suggested that the effects of maternal smoking on the placenta may depend on the stage

of pregnancy at which exposure occurs. 2s The early (first trimester) period is critical because that is when differentiation of the speciaiised epithelial cells of the placenta (trophoblasts) occurs.

EFFECTS OF N I C O T I N E EXPOSURE ON L U N G DEVELOPMENT Energy metabolism Glucose uptake and metabolism are essential for the proliferation and survival of cells, and may be enhanced in actively proliferating cell systems such as embryonic tissue. Glucose is considered to be an essential source of energy in lung tissue 3~ and is necessary for the functional development of t h e lung. 31'32'33 Glucose is also the main source of o~-glycerophosphate for surfactant synthesis in adult lung 34 while in fetal lung, loss of cellular glycogen from alveolar type II cells just before birth is associated with increased surfactant synthesis. 3s'36 During the alveolar phase of lung development, lung tissue is more dependent on glycogen as an energy substrate than adult lung. This is illustrated by the fact that, during fasting, the activity of phosphorylase in adult lung tissue decreases to conserve glycogen while the activity of phosphorylase in fetal and neonatal lung increases, thereby increasing the utilization of glycogen. This means that the control of glycogen metabolism during the alveolar phase of lung development is different from that of adult animals. 37 Although glucose and glycogen are the primary energy substrates of the adult and developing lung, fatty acids can also be important. For example, during fasting, when blood fatty acid levels are elevated, fatty acids replace glucose as primary energy substrate. Under these circumstances, glucose is conserved by the lung for ot-glycerophosphate synthesis and eventual surfactant formation by alveolar type II cells. 38 Maternal nicotine exposure during gestation results in sustained suppression of glycogenolysis and glycolysis in lung tissue of the rat fetus. 39 The lower glycogenolytic activity is due to a lower phosphorylase activity in the lungs of nicotine exposed offspring. 39 The ratio of inactive to active phosphorylase of lung tissue of nicotine exposed offspring is the same as for animals that were not exposed to nicotine during gestation and lactation. However, the tissue levels of both the phosphorylase fractions are lower than in the lungs of control animals (Fig. 21.1), which implies that the total phosphorylase content of the lungs of the nicotine exposed animals was lower than that of the control animals. This means that nicotine exposure suppressed the synthesis of phosphorylase in the lungs. 39 It also implies that nicotine had no direct inhibitory effect on phosphorylase, but that the slower rate of glycogenolysis in the lungs of these animals is rather due to a decrease in the level of phosphorylase available to catalyse glycogenolysis. This implies that the developing fetal and neonatal lung, of animals exposed to nicotine during gestation and lactation, is more dependent

Fig. 21.1. The influence of maternal nicotine exposure during gestation and lactation in rats on the phosphorylase activity of the lungs of offspring. (Active = active phosphorylase; Total = total phosphorylase).

on exogenous glucose for utilization via the glycolytic pathway and the hexose monophosphate shunt than on glucose from pulmonary glycogen stores. The uptake of exogenous glucose is usually carried out by glucose transporters. Glucose transporter isoforms 1 (GLUT 1) and 4 (GLUT 4) are not present in adult lung, but are present in developing lungs. Over-expression of these GLUT isoforms can enhance glucose uptake into fetal lung cells to support active cell proliferation, which is a common characteristic of developing lung epithelium. 4~The decrease in the flux of glucose through the glycolytic pathway (Fig. 21.2) of lungs of nicotine exposed rat pups is, however, not due to a compromised glucose transporter system because the total glucose turnover of lung tissue of these pups is higher than in control pups. This is because flux of glucose through the hexose monophosphate shunt (HMP) of lung tissue of nicotine exposed rat pups exceeds that of control animals. The flux of glucose through the glycolytic pathway remains suppressed after nicotine withdrawal (Fig. 21.2), while the flux through the HMP shunt returns to normal; 7 thus nicotine has no inhibitory effect on the detoxification function of the lung. This is supported by the observation that nicotine exposure induces the synthesis of the microsomal monooxygenases such as NADH-cytochrome bs-reductase , NADPH-cytochrome c-reductase, NADPHcytochrome P450-reductase and upregulates the expression of cytochrome P450 in lung tissue of neonatal and adult rats.40,41 Hexokinase catalyses the phosphorylation of glucose before it can be metabolized by the various metabolic pathways in the cell. In rats, maternal nicotine exposure during pregnancy and lactation 7 had no influence on the hexokinase activity of offspring and thus on the phosphorylation of glucose. Maternal nicotine exposure during pregnancy and lactation also had no effect on lactate dehydrogenase and pyruvate kinase activity of the lungs of the offspring which means that the site of action of nicotine is between hexokinase and lactate dehydrogenase. 42 The decrease in the glycolytic activity, and thus the flux of glucose through this pathway, can be attributed to an inhibition of phosphofruc-

Fig. 21.2. The influence of maternal nicotine exposure during gestation and lactation on glucose utilisation and lactate production by the lungs of offspring. Glucose utilisation and lactate production were determined during lactation in each of 3 groups: a control group that received no nicotine, a nicotine treated group, and another group killed following a 4-week withdrawal period during which the offspring received no nicotine.

tokinase, the rate limiting enzyme of the glycolytic pathway. 43 It appears that the lower phosphofructokinase activity, like the reduced phosphorylase activity, is due to interference of phosphofructokinase synthesis at either the pre- or post-translational level, 44 resulting in a lower concentration of phosphofructokinase and thus a slower flux of glucose via glycolysis. In a recent study it was shown that the activity of hexokinase is highest during the phase of rapid alveolarisation. 43 This implies that the glycolytic pathway plays an important role in supplying energy and precursors to the developing lung during this phase of lung development. 43 The lower phosphofructokinase activity in the lungs of nicotine exposed animals will therefore impact adversely on this phase of lung development. In the offspring of maternal rats exposed to nicotine, surfactant production by alveolar type II cells appears to be unaffected by the sustained suppression of glycogenolysis and glycolysis; this suggests that fatty acids contribute to the supply of precursors for surfactant synthesis when the flux of glucose through the glycolytic pathway is reduced as a result of a lower glucose supply or an inhibition of this pathway. 42 This could explain the increase in the surfactant content of the type II cells of the lungs of nicotine exposed neonatal rats. 45 In addition, the adenine nucleotide content of the lungs of nicotine exposed neonatal rats is increased due to a decrease in the rate of ATP hydrolysis. 46 This inhibition of ATP hydrolysis gives rise to an increase in the ATP/ADP ratio in the lungs of the offspring. The reduced rate of ATP hydrolysis can be partly attributed to an inhibition of Na+-K + ATPase. 47 This inhibition of Na+-K + ATPase may result in swelling and bleb formation of the alveolar type I cells and death of these cells.

Nicotine exposure during pregnancy and lactation indirectly stimulates the hexose monophosphate shunt in the lungs of offspring. 41 This implies that nicotine had no inhibitory effect on the supply of precursors for synthetic processes, for example, synthesis of surfactant in the fetal and neonatal lung, as well as in the detoxification of foreign substances. Lung s t r u c t u r e Fibroblasts play a critical role in the transition from the saccular to the alveolar stage of lung development, during which there is a 4-fold increase in the number of interstitial fibroblasts in the neonatal rat lung. 48 Perturbations such as hyperoxia, barotrauma and steroid therapy have been shown to interfere with alveolar development in the rat, 49 baboon 5~ and human infant, 51 the net result of which is a significant, often permanent, decrease in the number of alveoli. Although the control of the alveolarisation process is poorly understood, a substantial body of information exists regarding events that coincide with alveolar septation, many of which may influence fibroblast proliferation. Lung elastic fibres are also thought to be involved in septation by providing structural support for newly emerging secondary septa. Inhibition of elastic fibre assembly has been linked to impaired septation. 52 In neonatal rat lung fibroblasts, elastin expression peaks during the second postnatal week and declines rapidly thereafter. 53 Cigarette smoke inhibits fibroblast proliferation and migration by increasing cell cycle transit time, thereby reducing the rate of alveolarisation. 54 Consequently, the surface area available for gas exchange is reduced. Cigarette smoke exposure will also compromise fibroblast-induced repair responses, and may be one of the factors that contributes to the development of smoke-induced lung disease. 54 Accumulation of nicotine in fibroblasts can affect their metabolism and function. In vitro studies, however, have shown that nicotine has no effect on fibroblasts from human fetal lungs. 54 These in vitro studies were, however, performed on cells that were not metabolically permanently compromised as opposed to the fibroblasts of lung cells of neonatal rats that had been exposed to nicotine during gestation and lactation. Therefore, since maternal nicotine exposure during gestation and lactation interferes with glucose metabolism and apoptosis in the fetal and neonatal lung, and since it may cause disruption of the interaction between lung fibroblast glucose metabolism and fibroblast function, it is plausible that nicotine will interfere with the structural and thus functional development of the fetal and neonatal lung. Many agents that induce injury in lung tissue may do so by modifying key metabolic events for various cell populations in the lung. The type I alveolar epithelial cell for example, which covers more than 90% of the alveolar surface, 55 depends on glycolysis for energy. 56 Glycolysis also supplies the ATP required to maintain the membranelinked Na+--K§ ATPase. 57 The Na+-K + ATPase pump plays

a vital role in maintaining cell volume; reducing its activity by the inhibition of glycolysis will therefore result in swelling of these cells and the formation of membrane blebs. 58 Inhibition of glycolysis will therefore interfere with the ability of the type I cell to adapt to changes in the environment and to maintain cell volume. Since glycolysis is irreversibly suppressed in the lungs of nicotine-exposed rat pups, the activity of this pump will be permanently lower and this could result in rupturing of the cell membranes. The type I epithelial cells are the most vulnerable to injury in the lung 59 and the permanent inhibition of glycolysis will therefore make them more susceptible to damage, especially when exposed to blood and airborne toxic substances. An analysis of the broncho-alveolar lavage fluid of rat pups exposed to nicotine via the placenta and mother's milk has shown an increase in levels of alkaline phosphatase; 6~ such an increase is considered to be a marker of alveolar type I epithelial damage. 61 The glucose 6-phosphate dehydrogenase activity of the bronchoalveolar lavage is also increased, and is a marker of type II cell proliferation. 61 Scanning and transmission electron micrographs indeed reveal blebbing as well as more comprehensive damage of alveolar type I cells in these animals. Alveolar wall fenestrations also o c c u r 63 and are considered to be an indication of the early onset of emphysema. 63 Many of these changes only become apparent after the withdrawal of nicotine. 9 Alveolar type II cell numbers have been found to be increased in the lungs of nicotine exposed animals, 65 which is thought to be a response to type I cell damage and death. As a consequence of the proliferation of alveolar type II cells, the type I to type II cell ratio decreased in the lungs of these animals. 65 Pulmonary fibroblasts are thought to be positive modulators of this process through the synthesis of keratinocyte and hepatocyte growth factors, both known to be potent mitogens for type II cells. 66 It appears that the negative impact of maternal nicotine exposure during gestation and lactation on the growth, development and repair processes of the lungs of the offspring is of such a nature that lung structure will gradually deteriorate with age. In pregnant rats, cigarette exposure at days 5-20 of gestation causes a reduction in lung volume, number of saccules and septal crests, and elastin content in fetal lungs. 67 Maternal nicotine exposure during pregnancy and lactation produces emphysema-like changes in the lungs of the r a t pups 62'68 as well as an accumulation of lamellar bodies in type II alveolar epithelial cells. 45 The elastic tissue framework of the lungs of the offspring is also compromised. 69 These structural changes that are induced during pregnancy as a result of maternal nicotine exposure are irreversible and render the lungs of the offspring more susceptible to damage. 9 Exposure of fetal monkeys to nicotine via the placenta during the late saccular/early alveolar phase of lung development results in an increase in the size and volume density of the primitive alveoli and the surface area for gas exchange decreases. 67 These findings are similar to those from two other models of the effects of smoking on lung development. The lungs of fetal rats exposed to cigarette

smoke during pregnancy also have enlarged and fewer saccules; 67 similarly, pre- and postnatal exposure of developing rats to nicotine resulted in a decreased alveolar number (Fig. 21.3), an increased alveolar volume (Fig. 21.4) and a decrease in the surface area available for gas exchange. 8'9 In addition it was found that the alveolar septa became shorter and also incomplete (Figs 21.5 and 21.6), thereby reducing the alveolar wall surface area. 7~ The finding that nicotine alters alveolar development in both monkeys and rodents makes it highly likely that prenatal exposure to nicotine will similarly affect human lung development. It has been shown that much of the effect of maternal smoking on the developing lung may be mediated by the interaction of nicotine with nicotinic receptors expressed in the developing lung. 1~ It is interesting to note that hospital admissions are seen less frequently for children whose mothers smoked only after pregnancy, arguing for a prenatal effect. 71'72

Fig. 21.3. Effects of nicotine exposure during gestation and lactation on the number of alveoli (Na) in the lungs of postnatal rats. Nicotine exposure results in an age-related decrease in Na, relative to controls. 7~

Fig. 21.5. Scanning electron micrographs of the lung parenchyma of (A) 14-day-old control and (B) nicotine-exposed rat pups. The surface morphology of the lungs of the nicotine-exposed rat pups shows normal alveoli as well as focal flattened alveoli (encircled) with incomplete alveolar walls (arrow). The parenchyma of 42-day-old control animals (C) showed normal intact alveoli, while enlarged alveoli, likely due to alveolar destruction (inside circle), occur in the lungs of 42-day-old nicotine-exposed rats (D) fenestrations are also visible (arrows) (E) Flattened alveoli (long arrows) as well as more pronounced alveolar damage (short arrows) are apparent. (Bar =290 Bm). 7~

NICOTINE APOPTOSIS

Fig. 21.4. Effects of nicotine exposure during gestation and lactation on the volume of alveoli (Valv) in the lungs of postnatal rats. Nicotine exposure results in an increase in Valv in the lungs of offspring after the completion of alveolarisation. 7~

AND AND

CELL SIGNALLING: LUNG DEVELOPMENT

Programmed cell death or apoptosis is an energy-dependent and genetically controlled process 73 that can be induced by a number of molecular tools. 74 Apoptosis occurs in the mesenchyme as early as day 14 of gestation in fetal rat lung, the embryonic phase of lung development, during which branching of conducting airways is the predominant feature. The percentage of cells undergoing apoptosis increases dramatically between 18 and 22 days of gestation and remains elevated in the first day of postnatal life. This marked increase at birth may be initiated by a number of

Fig. 21.6. Scanning electron micrographs of 42-day-old rat lung showing the alveoli of lungs of (A) control rats, and (B) rats that were exposed to nicotine during gestation and lactation. The alveoli of the lungs of the nicotine exposed rats are larger than those of the control rats. The arrows indicate short incomplete alveolar septa. (Bar = 380 l.tm)

factors such as air breathing, hormonal changes due to labour and delivery, and/or expansion of the lungs with changes in cell shape and cell-cell relationships. Most ceils require attachment to the extracellular matrix for proper growth and function. Lung epithelial cell adhesion to the extracellular matrix is mediated by cell surface receptors known as integrins 75 which trigger a number of intracellular signalling pathways. Some of the pathways that have been shown to be involved in apoptosis include the Ras-Raf-MAP kinase pathway and the phosphatidylinositol 3-kinase pathway. 76,77 During the phase of rapid alveolarisation between postnatal days 4 and 13 in rats, interstitial fibroblasts undergo rapid proliferation. Few new alveoli are formed after the phase of rapid alveolarisation. Between postnatal days 13 and 21, the number of fibroblasts and type II cells decrease. This decrease in fibroblasts and type II cells occurs by means of programmed cell death or apoptosis, which peaks between postnatal days 17 and 19. Apoptosis therefore plays a key role in the thinning of the alveolar septa that occurs after alveolarisation. 78'79 Although apoptosis is an ongoing process in the immature lung, the rate of apoptosis after alveolarisation increases owing to a decrease in bcl-2 mRNA and an increase in BAX mRNA in the fibroblasts on postnatal day 16. The gene products of bcl-2 and BAX interact to form homodimers and heterodimers. Although bcl-2 and BAX heterodimers are inactive, when BAX is in excess and BAX homodimers predominate, cells are likely to undergo apoptosis. 8~ This explains the decrease in the total numbers of type II epithelial ceils and fibroblasts in rat lungs during the third postnatal week. 81 The reduction, due to apoptosis, in the number of fibroblasts in the interstitium of the developing lung is likely to play a critical role in lung maturation, the final process of which is the transition of the alveolar wall from a double to a single capillary network layer. 79 Interference with the apoptotic process would be expected to have an adverse effect on lung maturation.

Cigarette smoke inhibits the proliferation and migration of human lung fibroblasts and fibroblast-mediated repair responses, and therefore may play a role in the development of emphysema, s4 Nicotine and cotinine inhibit apoptosis in fibroblasts, s2 but the mechanism by which they suppress apoptosis is not known. Nicotine is known to exert its effects on many cell types by binding to nicotinic cholinergic receptors. It has been suggested that paediatric, smokingassociated pulmonary diseases and small cell lung carcinoma may be caused by the direct chronic stimulation of an ix7 nicotinic acetylcholine receptor-initiated autocrine loop by nicotine and 4-(methylnitrosoamino)-(3-pyridyl)-lbutanone (NNK), where NNK is formed from nicotine by nitrosation in the mammalian organism and during the curing of tobacco, s3'84 It is also possible that certain effects of nicotine are not receptor mediated or may operate through unconventional nicotine receptors. 82 There is evidence that nicotine: (a) activates the mitogen-activated protein (MAP) kinase signalling pathway and extracellular signal-regulated kinase (ERK2), resulting in increased expression of the bcl-2 protein and inhibition of apoptosis, and (b) blocks the inhibition of protein kinase C (PKC) activity in lung cells. Nicotine appears to have no effect on the activities of c-jun NHz-terminal protein kinase (JNK), c-myc or p38 MAP kinases, which are also involved in apoptosis. While exposure to nicotine can result in the activation of two major signalling pathways, (MAP-kinase and PKC) that are known to inhibit apoptosis, nicotine regulation of MAP (ERK) kinase activity is not dependent on PKC. These effects of nicotine occur at concentrations that are generally found in the blood of smokers, and could lead to disruption of the critical balance between cell death and proliferation. 85 The inhibition of apoptosis by nicotine may contribute to the slower thinning of the alveolar septa of the lungs of the nicotine exposed rat pups. 64 It has been suggested 86 that in utero exposure of fetal pulmonary neuro-endocrine cells to nicotine or NNK in cigarette smoke may contribute to the development of

paediatric lung disorders such as bronchitis and lower respiratory illnesses, 4'5 along with altered pulmonary mechanics in infants and children. 87 T h e nicotine-induced alterations in lung function of monkeys parallel those observed in infants of mothers who smoke during pregnancy. 4'87 These alterations in lung function could be induced via two different mechanisms. The first is a direct effect of released 5-hydroxytryptamine (5-HT) in response to t~7 nicotinic receptor stimulation on bronchial and vascular smooth muscles and fibroblast growth; the second is an indirect effect of 5-HT on p u l m o n a r y neuro-endocrine cell numbers, via activation of a Raf-1/MAP kinase pathway, resulting in yet more cells that can synthesize and release 5-HT. Chronic exposure to nicotine and N N K of pregnant mothers may therefore upregulate the t~7 nicotinic receptor as well as components of its associated mitogenic signal transduction pathway, thereby increasing the vulnerability of infants to the development of paediatric lung disorders 4'5 mentioned above.

CONCLUSIONS Research on animals has shown that prenatal nicotine exposure produces alterations in lung function that parallel those observed in infants of mothers who smoked during pregnancy. Nicotine and products of nicotine metabolism can affect lung growth, development and function via several mechanisms. Understanding the mechanisms whereby prenatal tobacco smoke exposure alters lung development and eventually lung function, may lead to therapeutic interventions to block its effects as well as help to further discourage smoking or the use of nicotine replacement therapies during pregnancy.

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54. Nakamura Y, Romberger DJ, Tate L etal. Cigarette smoke inhibits lung fibroblast proliferation and chemotaxis. Am. J. Respir. Crit. Care Med. 1995; 151:1497-503. 55. Naimark A. Non-ventilatory functions of the lung. Am. Rev. Respir. Dis. 1977; 115:93-8. 56. Massaro GD, Gail DB, Massaro D. Lung oxygen consumed,,.. and mitochondria of alveolar epithelial and endothelial cells.J. Appl. Physiol. 1975; 38:588-92. 57. Paul RJ. Functional compartmentalization of oxidative glycolytic metabolism in vascular smooth muscle. Am. J. Physiol. 1983; 224:C399-C409. 58. Contran RS, Kumar V, Robbins SL. Cellular injury and adaptation. In: Pathologic Basis of Disease, 4th edn. WB Suanders Co, Philadelphia, 1989; pp. 16-38. 59. Witschi H. Proliferation of alveolar type II cells: a review of common responses in toxic lung injury. Toxicol. 1976; 5:267-77. 60. Maritz GS, Najaar K. Biomedical response of neonatal rat lung to maternal nicotine exposure. Pathophysiology 1995; 2:47-54. 61. Henderson RF. Use of broncho-alveolar lavage to detect lung damage. Environ. Health Perspect. 1984; 56:115-29. 62. Maritz GS. Maternal nicotine exposure induces microscopic emphysema in neonatal rat lung. Pathophysiology 1997; 4:135-41. 63. Linhartova A. Fenestrations of the pulmonary septa as a sign of early destruction in emphysema. J. Cesk. Patol. 1983; 19:211-21. 64. Maritz GS, Matthews HL, Aalbers J. Maternal copper supplementation protects the neonatal rat lung against the adverse effects of maternal nicotine exposure. Reprod. Fertil. Dev. 2000; 12:97-103. 65. Maritz GS, Thomas R-A 1994 The influence of maternal nicotine exposure on the interalveolar septal status of neonatal rat lung. Cell Biol. Int. 2000; 18:747-57. 66. Panos RJ, Rubin JS, Csaky KG etal. Keratinocyte growth factor and hepatocyte growth factor/scatter factor are heparin-binding growth factors for alveolar type II cells in fibroblast-conditioned medium. J. Clin. Invest. 1993; 92:969-77. 67. Collins MH, Moessinger AL, Klinerman J. Fetal lung hypoplasia associated with maternal smoking: a morphometric analysis. Pediatr. Res. 1985; 19:408-12. 68. Maritz GS, Woolward K, du Toit G. Maternal nicotine exposure during pregnancy and development of emphysema-like damage in the offspring. S. Afr. Med. J. 1993; 83:195-8. 69. Maritz GS, Dolley L. The influence of maternal nicotine exposure on the status of the connective tissue framework of developing rat lung. Pathophysiology 1996; 3:212-20. 70. Maritz GS. Maternal nicotine exposure during gestation and lactation of rats induce microscopic emphysema in the offspring. Exp. Lung Res. 2002; 28:391-403. 71. Sekhon HS, Jia Y, Raab R etal. Prenatal nicotine increases pulmonary o~7 nicotinic receptor expression and alters fetal lung development in monkeys. J. Clin. Invest. 1999; 103:637-47. 72. Tager IB, Hanrahan JP, Tosteson TD etal. Lung function, pre- and postnatal smoke exposure, and wheezing in the first year of life. Am. Rev. Respir. Dis. 1992; 147:811-7. 73. White E. Life, death and the pursuit of apoptosis. Genes Dev. 1996; 10:1-15. 74. Wertz IE, Hanley MR. Diverse molecular provocation of programmed cell death. Trends Biochem. Sci. 1996; 21:359-64. 75. Pilewski JM, Albelda SM. Adhesion molecules in the lung. An overview. Am. Rev. Respir. Dis. 1993; 148(Suppl.): 532-7. 76. Ichijo H, Nishida E, Irie K et al. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/ JNK and p. 38 signalling pathways. Science 1997; 275:90-4.

77. Yao R, Cooper GM. Requirements for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science 1997; 267:2003-06. 78. Schnittny JC, Djonov V, Fine Aet al. Programmed cell death contributes to postnatal lung development. Am. T. Respir. Cell Mol. Biol. 1998; 18:786-93. 79. Bruce MC, Honaker CE, Cross RJ. Lung fibroblasts undergo apoptosis following alveolarisation. Am. J. Respir. Cell Mol. Biol. 1999; 20:228-36. 80. Yang A, Zha J, Jockel J e t al. Bad, a heterodimeric partner for Bcl-xl and Bcl-2, displaces Bax and promotes cell death. Cell 1995; 80:285-91. 81. Randell SH, Silbajoris R, Young SL. Ontogeny of rat lung type II cells correlated with surfactant apoprotein expression. Am. J. Physiol. 1991; 260:L562-70. 82. Wright SC, Zhong J, Zheng H etal. Nicotine inhibition of apoptosis suggests a role in turnout promotion. FASEB J. 1993; 7:1045-51.

83. Fischer S, Spiegelhalder B, Eisenbarth Jet al. Investigation on the origin of tobacco-specific nitrosamines in mainstream smoke of cigarettes. Carcinogenesis 1990; 11:723-30. 84. Hecht SS, Hoffmann D. Tobacco-specific nitrosamines, an important group of carcinogens in tobacco and tobaccosmoke. Carcinogenesis 1990; 9:875-84. 85. Heusch WL, Maneckjee R. Signaling pathways involved in nicotine regulation of apoptosis of human lung cancer cells. Carcinogenesis 1998; 19:551-6. 86. Schuller HM, Jul BA, Sheppard BJ etal. Interaction of tobacco-specific toxicants with the neuronal l.t7 nicotinic acetylcholine receptor and its associated mitogenic signal transduction pathway: potential role in lung carcinogenesis and pediatric lung disorders. Eur. J. Pharmacol. 2000; 393:265-77. 87. Sekhon HS, Keller JA, Benowitz NL et al. Prenatal nicotine exposure alters pulmonary function in newborn Rhesus monkeys. Am. J. Respir. Crit. Care Med. 2001; 164:989-94.

INTRODUCTION In this chapter the influence of allergen exposure during development will be examined. Exposure to allergen can occur during fetal, neonatal, and juvenile years, which are critical times for immunological and structural development of the lung. The increasing incidence of allergic asthma in the human population is most noticeable in children and adolescents. The greatest increases in asthma incidence have occurred among children between the ages of birth to 4 years of age, particularly among Latino and African American populations. 1 Maternal factors as well as environmental influences have been shown to enhance sensitization to allergens. Moreover, the cytokine environment that favors maintenance of a successful pregnancy is also compatible with allergic sensitization. Thus, this chapter will discuss what is known about these interactions and how exposure to allergen at a critical time in development may modulate the immune system towards an allergic phenotype.

Principles of allergic sensitization: induction of type 1 hypersensitivity An understanding of the principles of allergic sensitization is paramount to understanding how exposure to allergens during development can induce chronic pulmonary allergy. The response to allergen is a hypersensitivity response, i.e. a response that is exaggerated and in fact, one that the normal individual would not be expected to make. Close to half of the human population is atopic, which means that they have a genetic tendency to develop IgE antibodies specific for a variety of environmental proteins, called allergens. Upon contact with an allergen, usually by inhalation (also by ingestion, injection, or skin contact) an atopic individual will begin an immunological response that The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

results in production of IgE antibodies. The specific events that precede the production of IgE antibodies will be addressed below. However, once formed, the IgE binds very tightly to surface receptors on mast cells that exist in tissues that are in close proximity to mucosal surfaces. At this point the mast cell (and hence the individual) is sensitized. Subsequent exposures to allergen cause degranulation of mast cells with release of mediators, which cause the clinical signs that we associate with allergy. Thus, release of histamine causes increased capillary permeability and smooth muscle contraction. Stimulation of eicosanoid production stimulates not only smooth muscle contraction, but also chemotaxis of leukocytes. The later acting mediators produced by activation of the arachidonic acid pathway include leukotrienes, formerly called slow reacting substance of anaphylaxis. These reactions, when initiated in the lung, set the stage for allergic asthma. 2

Immune modulation by helper T-cell subsets The process of exposure to allergen alone is insufficient to initiate the type I hypersensitivity response described above. It is critical that the allergen gains access to appropriately situated cells called dendritic cells, which can present the allergen to T-lymphocytes called type 2 helper cells (Th2). These cells must secrete cytokines, called interleukins, that direct the immune system to ultimately make IgE antibodies that can bind to the allergen. Specifically, interleukins (IL)-4 and-13 stimulate B-lymphocytes to develop into IgE producing plasma cells. These cytokines work in opposition to another set of cytokines produced by lymphocytes called T-helper 1 (Thl) cells. The T h l immune response favors development of a cell-mediated immune response. 2 The Thl cells make IL-2, IL-12, and ),-interferon, cytokines which stimulate T-cells, NK cells, and macrophages- cytokines which Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

are important in control of infectious diseases. Thus, high levels of ?-interferon down-regulate production of IL-4 and IL-13.

INFLUENCE OF I N UTERO EXPOSURE TO ALLERGENS O N D E V E L O P M E N T OF THE A T O P I C PHENOTYPE M a t e r n a l i n f l u e n c e s on the i m m u n e status of the fetus The human fetus spends nine months within the womb of its mother. In humans and non-human primates there is a very close interaction between maternal and fetal circulations during this period of in utero development. These species have the fewest layers of cells dividing the maternal and fetal circulations due to the hemochorial nature of the placenta. This type of placentation allows for transfer of immunoglobulin and other small molecules from maternal to fetal circulation. Moreover, it is now well accepted that during pregnancy there is a change in the local environment of the placenta towards a T-helper type 2 cytokine profile. This environment is thought to assist in survival of the fetal allograft from implantation in the uterus up to parturition. 3 This is because a strong cellular immune response might facilitate maternal rejection of the fetus, which shares major histocompatiblity antigens with its father. The Th2 cytokine profile is less likely to facilitate such 'graft' rejection. When the production of the cytokine IL-13 by cells at the materno-fetal interface was examined, it was found that normal pregnancy was associated with production of IL-13 by the placenta and subsequently by the fetus. 4 The recognition that the human fetus is capable of developing an antigen-specific IgE response in utero was made as a result of examination of cord blood. In one epidemiological study, cord blood IgE concentrations were higher among infants whose mothers had a history of atopic disease and allergen immunotherapy compared with infants whose mothers were not atopic, s During pregnancy, the maternal influence is exerted on the fetus. In one study, correlations were found between house dust mite and ovalbumin specific IgG subclass levels in cord blood, maternal atopy, and the magnitude of perinatal lymphoproliferative responses to allergens in human infants at birth. An inverse relationship was found between levels of interferon gamma in the fetus/ neonate and a maternal history of atopy. 6 However, it was concluded that allergen-specific antibody transferred through the placenta is less likely to have an impact on the future development of allergy in the infant than the maternal influence on interferon gamma production by T-cells of the fetus. These findings support the importance of the uterine cytokine environment to subsequent allergic predisposition of the fetus. Exposure of the fetus to allergens inhaled by the mother during pregnancy can result in its sensitization to those allergens. Studies performed on cord blood lymphocytes have revealed that sensitization to environmental allergens during intrauterine life does occur. For example, in one

study neonatal peripheral blood T-cells were obtained from cord blood and stimulated in vitro with several antigens: tetanus toxoid, streptokinase, purified protein derivative of Mycobacterium (PPD), or common inhaled allergens, Dermatophygoides pteronyssinus (Der pI) and Lolium perenn~ (LolpI). It was expected that maternally derived T-cells would respond to the commonly encountered antigens, such as tetanus toxoid, and that fetal cells would respond only to the inhaled allergens, thereby allowing discrimination between maternal and fetal cells to be accomplished. In fact, the neonatal T-cells responded strongly to PPD and to Der pI, but not to Tetanus toxoid or streptokinase. The response to the seasonal allergen Lol pI allergen was variable. The authors of this study concluded that aeroallergen sensitization can occur in utero. 7 Several studies have shown that exposure to cockroach antigen during the first few years of life increases the risk of wheezing in children from atopic parents. These same children had T-cell proliferative reactivity in response to cockroach antigen. 8 Based on this data and other studies suggesting that prenatal antigen exposure can lead to sensitization of the newborn, Miller et al. performed a study on a cohort of 167 pregnant women, living in the inner-city, all either African-American or Dominican. 1 Dust samples were taken from a subset of their homes for analysis of allergen concentrations. At delivery cord blood was collected as well as maternal blood within a day of childbirth. Analysis of mononuclear cells in response to mouse protein, cockroach, house dust mite, and tetanus toxoid was performed by lymphocyte proliferation and compared with a mitogen (PHA) control. The most significant finding from T-cell stimulation was the response to the house dust mite (D. farinae). There was no significant correlation between cord blood or maternal IgE and proliferation in response to any antigen. Interestingly, the occurrence of maternal allergen-specific T-cell proliferation was not a prerequisite for cord blood mononuclear cell proliferation. T-cell reactivity was measured to mouse and cockroach antigen as well as to dust mite. There was no correlation between the T-cell proliferation and allergen-specific IgE levels. Thus, while stating that cord blood cells readily proliferate in response to allergens in the environment of the pregnant mother, the exact link with induction of allergic asthma in the fetus/neonate was not determined. 1 A recent study sought to determine if maternal avoidance of allergen could influence the development of allergic reactivity in the fetus. To accomplish this goal, concentrations of house dust mites were measured from beds of mothers at the 36th week of pregnancy. At birth the cord blood mononuclear cells were stimulated with either mitogen or dust mite allergen, then subjected to measurement of cytokine synthesis. Levels of IL-4, IL-5, IL-10, y-interferon were determined and compared with allergen data. The results showed that the cytokine levels did not correlate with the house dust mite levels in the beds of the mothers at week 36 of gestation. Thus, maternal avoidance of house dust does not appear to be an important factor in determination of a child's future allergy status. 9 However, maternal immuno-

logical factors were not examined, i.e. maternal IgG and IgE specific for house dust mite allergen. In another study two seasonal allergens were used to study the importance of gestational age on intrauterine priming to allergens inhaled by the mother. Birch and timothy grass allergens were used to stimulate cord blood T-cells from neonates born at different times during the year. From the data obtained it was concluded that the susceptibility of fetal lymphocytes for priming with birch and timothy grass allergens decreased when exposure was initiated towards the end of the pregnancy. The authors proposed that this observation may be the result of either decreased access of the allergen to the fetus due to decreased permeability of the placenta, or possibly to enhanced sensitivity of lymphocytes to priming during the earlier phase of pregnancy. Indeed, most of the positive proliferative responses seen to the allergens were obtained from cord blood taken from neonates whose mothers would have been exposed to the allergens during the first 6 months of pregnancy. 1~ Another study took these findings further to demonstrate that not only does transplacental priming of the human immune system occur to environmental allergens, but it can also be attributed to a skewed cytokine profile. This study attempted to address concerns that what was attributed to intrauterine priming might actually be either reactivity of maternal cells that were in the cord blood or the stimulatory effect of lipopolysaccharide (LPS), a contaminant of the allergen preparations, on the neonatal T-cells. The experiments performed showed that neither of these alternate explanations of the results were valid. It has been hypothesized that adult Th cell cytokine patterns are determined during infancy; ~1 although stimulation of neonatal T-cells with allergens produced cytokine profiles, as demonstrated by RT-PCR, consistent with a Th2 response, 11 studies over time showed that the response patterns of atopic and non-atopic children were different. Thus, the authors studied both normal and atopic families and found that although normal newborns were born with a Th2 bias, they rapidly altered their cytokine pattern in response to allergens and began producing some T h l cytokines. In contrast, the atopic individuals displayed an age-associated up-regulation of the Th2 immune response. ~2 From this study it is possible to suggest that exposure to allergen during development may be a critical factor for the development of the allergic phenotype in infants and children who are genetically predisposed to allergy. A recent review of multiple studies addressing the effect of the in utero environment on subsequent development of an allergic phenotype developed several well-supported conclusions. One was that a fetus is able to mount a proliferative response to common allergens as early as 22 weeks of pregnancy. Another was that maternal exposure to allergens can result in a protective IgG response that may decrease the likelihood of fetal sensitization, but that atopic mothers produce a more Th2 skewed immune environment than non-atopic mothers. Proliferation of umbilical cord blood cells in one study was inversely proportional to levels of

cord blood dust mite specific IgG. These results tend to suggest that if the mother has made an IgG response to the allergen, then the fetus is less likely to develop an IgE response to that same allergen. Thus, manipulation of the maternal environment may prevent development of an allergic phenotype in infants. 13 Studies on the role of maternal IgG in modulating the fetal IgE response have shown that increased levels of maternal anti-Bet vl IgG at birth correlated with reduced prevalence of allergic disease at 18 months of age. 14 Thus intrauterine IgG may play a modulatory role in the fetus. The route by which a fetus is exposed to environmental allergens has not been definitively proven. It has been hypothesized that exposure of the fetus to allergen is transplacental, perhaps as part of a complex with IgG. IgG crosses the placenta beginning early in gestation and reaching a maximum by 32 weeks. 15 IgE does not cross the placenta. However, IgE has been detected in the amniotic fluid as early as 16-17 weeks of gestation. An alternative explanation has been proposed to explain the exposure of the fetus to allergen: x5 allergen crosses from the maternal circulation to the fetal side where these tissues are in intimate contact. In support of this theory the authors state that house dust mite allergen has been detected in the amniotic fluid at 16-17 weeks of gestation. 15 There are multiple determinants of cord blood IgE concentrations, as shown in a study on 6401 German neonates. 16 Since the levels of cord blood IgE have been considered to be a good means of determining the risk of allergic disease for the neonate, a multicenter study was performed. 16 There was a significant difference in IgE concentrations between sexes, with boys having greater amounts of IgE in cord blood. If parents smoked during pregnancy, there was an increase in cord blood IgE. This study did not show any significant difference between full term babies and those of shorter gestational age. There were significant differences based on nationality (i.e. genetics), with non-German mothers from Far Eastern countries showing the highest values. Food allergens have also been examined as potential intrauterine immune stimulants. Indeed, cow's milk proteins were used to stimulate cord blood lymphocytes and found to have stimulatory effects on cells from both atopy prone and non-atopy prone individuals. The degree of production of gamma interferon by stimulated T-cells was thought to have a stronger likelihood of predicting the future development of atopic disease. 17 Proliferation of cord blood T-cells has also been reported in response to bovine serum albumen, is Since the predominance of a Th2 phenotype during pregnancy may favor the prenatal development of allergy, several studies have attempted to address this hypothesis using murine models. In one study, adult BALB/c mice were sensitized to ovalbumin (OVA) prior to mating followed by allergen exposures during pregnancy. The cytokine profile of the OVA-sensitized mice had enhanced Th2 reactivity when compared to offspring of non-sensitized mothers. 19 Recent studies have demonstrated that fetal life is a critical time for development of the allergic phenotype. Multiple

epidemiological studies have shown a strong correlation between maternal atopy and subsequent development of atopy in the infant. In fact, exposure of the atopic mother to allergens while pregnant appears to have a distinct effect on modulation of cytokine profiles and IgE production by the fetal immune system. Thus interaction of environment and genetic background during in utero life of a human infant may determine the future allergic status of the child and adult.

The influence of genetics on responses to allergen exposure during development The influence of genetic background on development of allergic diseases, including asthma, has been recognized for many years. 2~ Recently, however, a number of genetic loci have been associated with either development of high IgE production and/or asthma. The multigene control of allergy and asthma ultimately determines whether exposure to allergen results in development of allergic disease. For example, a gene on chromosome 1 lql 3 is linked to maternal inheritance of asthma; it involves polymorphisms in the beta subunit of the high affinity IgE receptor. Another polymorphism of importance occurs in the gene coding for interleukin-13. 21 It is generally thought that atopy is inherited and that predisposition for asthma is a multi-gene effect. According to this hypothesis it follows that only a fetus of the appropriate genetic make-up would be likely to develop in utero sensitization. This area of research will undoubtedly yield more information as the information discovered by the human genome project becomes linked with functional data.

N E O N A T A L EXPOSURE TO ALLERGENS The T-Helper cell type 2 phenotype in the neonate As previously discussed the intrauterine environment is prejudiced for a Th2 cytokine profile. While this type of immune environment is likely to facilitate survival of the fetus in utero, it results in a neonate that is born with a prejudice towards the Th2 (allergic) phenotype. The exposures that occur in the first months of life are very likely crucial to phenotypic development of the child and subsequently the adult. Thus, exposures to allergens, such as pollens, may facilitate future development of allergic disease, especially in the atopy prone genotype. However, exposure to certain other antigens during this critical time of development can influence or modulate the immune response. For example, as discussed below, endotoxin from gram negative bacteria has an allergy-sparing effect by shifting the Th2/Thl ratio in favor of T h l cytokines.

Role of dietary factors in allergen sensitization Dietary exposure to allergen may be an important factor in mucosal sensitization of the neonate to allergen. For example cross-reactive antigens have been identified between cereal proteins and grass pollen. 22 In support of these ideas, a cross-sectional study was performed in Spain to evaluate

the relationship between grass-pollen asthma and sensitization to cereals in the diet of the child. A relationship was shown between cereal allergy and pollen sensitivity. It was found that early introduction of cereals into the diet of children was a risk factor for grass-pollen allergy.22 Thus, avoidance of cereal by infants from an atopic family may reduce pollen sensitivity in future years. A controlled study was performed to determine if prophylactic dietary control could modulate induction of allergy in children. 23 The prophylactic group of 58 children were either breast fed from mothers whose diets excluded highly antigenic foods, or fed an extensively hydrolyzed formula. The control group of 62 did not have any specific prophylaxis. Results of the study showed that after one year there was significantly less total allergy, including asthma in the prophylactic group. After four years the difference between these groups was still significant, with more control children having positive skin tests to allergens. 23 Thus, it seems that another important source of exposure of the child to allergen during development can be in the neonatal period through breast milk.

The role of infectious diseases in modulating the effects of inhaled allergen during the neonatal period Several infectious diseases are thought to have a role in either facilitating development of allergic sensitization to inhaled allergen during the neonatal period or subverting the allergic response. The 'hygiene hypothesis' (discussed later in this chapter) focuses on early exposures to bacterial products that appear to have an allergy-sparing effect. In contrast, certain viral respiratory infections have been implicated in promotion of allergen sensitization and development of asthma. Viral infections are frequently associated with wheezing in small children. The fact that many of these children progress to become asthmatic has led to the supposition that the early viral infection may either facilitate sensitization or damage airways leading to an increased likelihood of developing airways hyperreactivity. 24 One of the major respiratory viruses that appears to have a link to asthma is respiratory syncytial virus (RSV). RSV is one of the most important neonatal pathogens currently recognized. Yearly epidemics of RSV cause high morbidity and some mortality in young infants and children under two years of age. The disease is much less severe in older children and healthy adults. However, in older people and those immunocompromised, RSV can be equally devastating. In the severe form, RSV causes wheezing and severe bronchiolitis and often interstitial pneumonia. It was recognized in the 1980s that children with most severe RSV accompanied by wheezing were often subsequently diagnosed with childhood asthma. 25 Indeed, IgE specific for viral proteins as well as elevated histamine concentrations in respiratory fluids were found in severely affected children. 25 More recently it has been found that RSV preferentially induces a Th2 cytokine environment in atopic children and in some animal models. Studies have shown that infection of balb/c mice with human RSV induces a T-helper cell type 2

response. 26 Work in the author's laboratory has demonstrated that some calves infected with bovine respiratory syncytial virus (a closely related bovine pathogen) develop a Th2 cytokine response when infected with the virus. 27 Indeed production of IgE is also associated with this model. Further work with the bovine model has demonstrated that disease is exacerbated when allergen is inhaled during the virus infection; 2s and that sensitization can be enhanced by exposure to allergen during the viral infection. 29 In contrast to these observations, a recent study has demonstrated that, in severe cases of respiratory disease with wheezing, y-interferon is present in elevated amounts. However, in this study the identity of the virus causing the infection and wheezing was not elucidated. 3~ Following up on these observations, Garofalo et al. 31 examined the role of T h l and Th2 cytokines as well as several chemokines in RSV disease. They found that macrophage inflammatory protein-la and was associated with severe RSV bronchiolitis. Thus, the role of cytokines in induction of clinical disease may be different to that previously thought to stimulate subsequent development of asthma. Using a murine model of RSV infection, it has been found that the production of IL-13 during a primary RSV infection has an important effect on exacerbation of cockroach allergen-induced disease. 26 When mice were depleted of IL-13, RSV infection did not exacerbate airway hyperactivity. The production of IL-13 during acute RSV infection is thought to also facilitate allergic sensitization. 26 In another murine model the effects of infection with influenza A on induction of tolerance to aerosolized allergen was examined. These studies were performed in adult mice, in which induction of tolerance is the most usual sequel to inhalation of allergen. However, infection of mice with influenza changed the response to intranasal ovalbumin such that Th2 cytokines, IgE, and airway hyperactivity developed in response to O V A . 32

of life had a protective effect; additional protection was conferred by exposure until the age of five years. 35 This protective effect of exposure to farms during early life has been attributed to bacterial components, such as LPS and unmethylated CpG motifs. The raw milk consumed by farm children has a large content of gram negative bacteria, which are a primary source of endotoxin (LPS). Other studies have focused on childhood environmental factors other than farm life as potential influences on development of allergic disease. For example, one retrospective study obtained information from 13,932 adults between the ages of 20 and 44 from 36 areas in Europe, New Zealand, and the United States. The results of this study showed that growing up in a large family, with sharing of bedrooms, and the presence of a dog in the household appeared to offer protection from development of atopy in later life. The authors of this study point out that if the subject has strong genetic predisposition for allergic reactivity, the environmental factors are probably less important. 36 Bacteria endotoxin, a component of gram negative bacteria commonly associated with intestinal flora, is capable of inducing the synthesis of the T h l cytokines y-interferon and IL-12. As discussed above, exposure to bacterial endotoxin could be expected to decrease the allergic response. House dust is another source of bacterial endotoxin, containing varying levels of endotoxin. To examine the effect of endotoxin in house dust on development of allergy in infants, a study was performed on infants aged 9-24 months, all of whom had at least three documented wheezing episodes. 37 Levels of house dust endotoxin were compared with allergic sensitization of these infants. It was found that the homes that had the lowest levels of endotoxin were associated with increased numbers of allergen-sensitized infants. 37 This study further supports the hygiene hypothesis.

Hygiene hypothesis

EXPOSURE TO ALLERGENS D U R I N G THE JUVENILE P E R I O D

Several epidemiological studies have shown that growing up on a farm has a protective effect against development of allergic disease as a child. 33 Exposure to stable dust and farm animals is thought to be the essential component in this 'protective' environment. Thus, endotoxin, from gram negative bacteria, may modulate the immune response. When environmental endotoxin exposure was measured in farming and non-farming families, it was found that endotoxin concentrations were highest in stables of farming families and also high in dust from the farmhouse f l o o r s ; 34 these concentrations were significantly different from nonfarming families. This study supports the hypothesis that environmental exposure to endotoxin from bacterial cell walls is an important protective determinant in prevention of atopic disease in children. In another study the effect of timing of the exposure was examined by comparing 812 children younger than one year with those from 1 to 5 years of age. 35 Results of this study showed that prenatal exposure and exposure to stables and farm milk during the first year

The age group of 7-8 years was targeted in a study to examine the incidence of allergic respiratory disease in Swedish children. 38 In this study sensitization to allergens was evaluated with skin-prick tests and a questionnaire was used to evaluate the incidence of asthma and rhinoconjunctivitis, as well as lifestyle factors. It was found that while skin-prick test reactivity was not significantly different between the children of farmers and non-farmers, there was a reduced risk among children of farmers for having allergic respiratory disease. It would be of interest to know whether the children with less respiratory disease had similar or dissimilar serum IgE and IgG concentrations. The presence of house dust mite (HDM) allergen in the environment of children and its relationship to development of asthma was addressed in an Australian study. 39 School children with a clinical history of H D M allergy, including wheeze, were followed for one year. During this time, they

reported their peak expiratory flow rates and allergen concentration was periodically measured in their homes. Results showed that there was a significant association between decreased peak expiratory flow values and HDM allergen concentration. Thus, in children already sensitized to H D M allergen, development of clinical signs of asthma was exacerbated by increased exposure to the HDM allergen. Another study addressed the relationship between allergen exposure in the environment and atopic sensitization. 4~ When HDM allergen reactivity was examined in school children living in the Alps versus children living at sea level, it was found that in the Alps where HDM is less common, skin-test reactivity to H D M allergen was less than in the lowlands. However, when grass pollens were used as the test allergen, positive skin tests were significantly higher in the Alps where grass pollens are prevalent, as were clinical signs of allergic reactivity. Thus, exposure to inhaled allergen during the school-age period leads to specific sensitization. 4~ Development of allergic reactivity during early childhood was demonstrated in a study that compared cytokine responses of T-cells, and skin-test reactivity to the oral antigen ovalbumin with the response to the inhaled allergens of house dust mite in 2-5-year old children. It was found that the inhaled allergen-specific cytokine response was associated with a positive skin-prick test for children five years of age or less. However, this association was not true for the oral allergen, ovalbumin. The authors suggest that a mechanism exists for deletion of the allergic response to food antigens during early childhood. 41 Indeed, the children with negative skin tests had cytokine profiles that showed higher levels of gamma interferon, indicative of a non-allergic T-helper cell type I phenotype (Thl). In another study, skin-prick tests were found to correlate with early exposure to animal danders, such as dog and cat, while the development of clinical allergic asthma was not. 42 In yet another study that involved a variety of allergens, a positive correlation was not found between early childhood exposure to allergens and development of allergic asthma. 43 The influence of a variety of environmental exposures of children on development of atopy as adults was examined in a study of 13,932 subjects in Europe, New Zealand, USA, and Australia. This study (European Community Respiratory Health Survey) examined a variety of childhood environmental factors and correlated these with the presence of atopy in adults. The most significant findings were that an association between large family, bedroom sharing, and a dog in the home was associated with a lower prevalence of atopy in families without a genetic predisposition to atopy. However in subjects that had a strong genetic predisposition for allergy, these environmental factors were less important. 36 Overall, we can conclude from this and other studies that the overbearing influence of genetics on induction of allergic response to allergen cannot be ignored. Allergy-inducing environmental factors appear to have their greatest effect on those individuals that have a familial history of atopy.

I N T E R A C T I O N OF ALLERGENS WITH E N V I R O N M E N T A L FACTORS D U R I N G DEVELOPMENT Epidemiological data in humans as well as experimental studies in mice have suggested a correlation between exposure to air pollutants and the incidence of asthma. This observation is not unexpected considering the dramatic increase that has occurred in the incidence of asthma in industrialized countries, where air pollution has also been increasing. In fact, studies have shown that there is a significant association between ambient levels of pollutants and the increased incidence of asthma. 44 Animal studies have been performed to demonstrate more specifically a link between exposure to environmental pollutants and enhancement of allergic sensitization. Initial animal studies were performed with adult mice. Swiss webster mice exposed to either 0.8 ppm ozone (O3), 0.5 ppm 03, or 03 and sulfuric acid mist in an episodic schedule were anaphylactically sensitized to aerosolized ovalbumin and had higher numbers of IgE producing cells in the lung than did similarly OVA-aerosol exposed mice exposed to filtered air instead of pollutant. 45 Later studies in the author's laboratory using adult BALB/c mice exposed to environmental tobacco smoke (ETS) showed a similar phenomenon, i.e. enhanced sensitization to OVA in pollutant exposed animals. 46 In these latter studies, production of Th2 cytokines by lung lymphocytes and IgE production was significantly greater in the ETS-exposed mice than in OVA-aerosol sensitized ambient air controls. The mouse serves as an excellent model with which to predict the effects of in utero and neonatal exposure to pollutants on the development of allergic lung disease in humans. In an experiment performed in the author's laboratory, pregnant female mice were exposed to either ETS or ambient air (control) during pregnancy. The progeny were born into and raised in the environment of the mother. At the age of 6 weeks all mice were primed with intraperitoneal ovalbumin and alum. Subsequently they received a series of aerosol OVA challenges. It was found that mice born into and raised in the ETS environment had increased levels of IgE and Th2 cytokines as compared to those born and raised in ambient air. Thus, ETS exposure facilitates development of the allergic phenotype. 47 Others have demonstrated that exposure to diesel exhaust (DE) enhances the IgE response to inhaled allergen in adult mice. 48 Diesel exhaust had an adjuvant effect on total and allergen-specific IgE production. This resulted in production of chemokines and cytokines that are important in sensitization to inhaled allergens. In a study using rats, the effect of intrauterine diesel exhaust exposure on development of an IgE response to inhaled pollen was examined. Developing rats were exposed to DE either during the fetal period (via the mother), the suckling period, or the weaning period. The study demonstrated that inhalation of DE during the differentiation periods of the immune system accelerated the increase in IgE in response to exposure to

Japanese cedar pollen. 49 The environmental pollutant pyrene is a component of DE, which has been found to induce IL-4 production. Thus, the mechanism by which the allergic enhancement occurs most likely involves the production of IL-4. 5~ Epidemiological evidence obtained from surveys in Japan indicate that allergic rhinoconjunctivitis was found to be more prevalent in individuals living near motorways than in cedar forests. Children were found to be more likely to have severe asthma than mild asthma when they were living in polluted areas, thus demonstrating the impact of pollution on allergic disease. 51 It has been previously thought that cord blood IgE concentrations could be a useful predictor for development of atopic disease in later life. Although correlations have been found between cord blood IgE, atopic history of the mother and smoking history of the parents during pregnancy, more recent data indicates that intrauterine exposure to allergens may, in fact, be the most important factor in development of sensitization to inhaled allergens. In one study of 7609 neonates in Germany most of the samples of cord blood assayed for IgE showed very low levels. This study confirmed the previous findings that there is an association

between higher IgE levels in children and parental smoking, thus confirming that passive smoke exposure is stimulatory for IgE production. 16 In another study it was found that maternal smoking was correlated with development of allergy in the child, whereas paternal smoking did not. 52 While these and other studies point to explicit causal factors for enhanced induction of allergic responses by environmental pollutants, the influence of these factors in sensitization of children is less well understood. It has been proposed that the strong association between ETS exposure and asthma in young children may relate to both prenatal and post natal effects on physical and functional parameters such as airway diameter and bronchial responsiveness. 53

SUMMARY

Epidemiological and experimental evidence suggests that the increasing incidence of asthma in the human population has been facilitated by sensitization of infants and children as early as during gestation. Maternal factors such as a

Allergen Maternal Factors: [ m

Smoking Genetic- atopic vs nonatopic Allergen exposure Air pollution Cleanliness of environment

~1

"m

''

Nature of immune response - IgG?

Fetal Factors: Genetic Atopy- parental Sex Season of the year

Neonatal and Infant Factors: Environment- allergen, pollution, bacteria. Infections- RSV Vaccinations Pets in the home Parental smoking Siblings Farm vs city

Fig. 22.1. Inhalation of allergen during pregnancy can result in intrauterine exposure of the fetus to allergen through the maternal and fetal circulation. Maternal, fetal and environmental factors influence the consequences of pre-natal allergen exposure, as shown in the boxes.

genotype that favors development of an atopic phenotype, smoking habits, and exposure to allergens during pregnancy are all contributing factors. Additional fetal factors include sex and time of the year in relation to gestational age. Finally, the e n v i r o n m e n t in which the neonate, infant, and finally the child lives and breathes interacts with the genotype to facilitate or suppress development of the allergic phenotype (Fig. 22.1).

REFERENCES 1. Miller RL, Chew GL, Bell CA etal. Prenatal exposure, maternal sensitization, and sensitization in utero to indoor allergens in an inner-city cohort. Am. J. Respir. Crit. Care Med. 2001; 164:995. 2. Janeway CA, Travers P, Walport M et al. Immunobiology, 5th edn. Garland Publishing, New York, NY, 2001. 3. Dealtry G, O'Farrell MK, Fernandez N. The Th2 cytokine environment of the placenta, lnt. Arch. Allergy lmmunol. 2000; 123:109. 4. Williams TJ, Jones CA, Miles EA etal. Fetal and neonatal IL-13 production during pregnancy and birth and subsequent development of atopic symptoms. J. Allergy. Clin. lmmunol. 2000; 105:951. 5. Johnson C, Ownby DR, Peterson EL. Parental history of atopic disease and concentration of cord blood IgE. Clin. Exp. Allergy 1996; 26:624. 6. Prescott S, Holt PG, Jenmalm M e t a l . Effects of maternal allergen-specific IgG in cord blood on early postnatal development of allergen-specific T cell immunity. Allergy 2000; 55:470. 7. Piccinni M, Mecacci F, Sampognaro S etal. Aeroallergen sensitization can occur in fetal life. Int. Arch. Allergy Immunol. 1993; 102:301. 8. Finn PW, Boudreau JO, He H etal. Children at risk for asthma: home allergen levels, lymphocyte proliferation, and wheeze.J. Allergy Clin. lmmunol. 2000; 105:933. 9. Marks GB, Zhou J, Yang HS et al. Cord blood mononuclear cell cytokine responses in relation to maternal house dusts mite allergen exposure. Clin. Exp. Allergy 2002; 32:355. 10. Van Duren-Schmidt K, Pichler J, Ebner C etal. Prenatal contact with inhalent allergens. Pediatr. Res. 1997; 41:128. 11. Prescott S, Macaubas C, Holt BJ et al. Transplacental priming of the human immune system to environmental allergens. universal skewing of initial T cell responses toward the Th2 cytokine profile.J. Immunol. 1998; 160:4730. 12. Prescott SL, Macaubas C, Smallacombe T etal. Reciprocal age-related patterns of allergen-specific T-cell immunity in normal vs. atopic infants. Clin. Exp. Allergy 1998; 28 (Suppl. 5):39. 13. Warner JA. Primary sensitization in infants. Ann. Allergy Asthma lmmunol. 1999; 83:426. 14. Warner JA, Jones CA, Jones AC etal. Immune responses during pregnancy and the development of allergic disease. Pediatr. Allergy Immunol. 1997; 8:5. 15. Jones CA, Holloway JA, Warner JO. Does atopic disease start in foetal life? Allergy 2001; 55:2. 16. Bergmann RI, Schylz J, Gunther S etal. Determinants of cord-blood IgE concentrations in 6401 German neonates. Allergy 1995; 50:65. 17. Szepfalusi Z, Nentwich M, Gerstmayr E et al. Prenatal allergen contact with milk proteins. Clin. Exp. Allergy 1997; 27:28. 18. Kondo N, Kobayashi Y, Shinoda S. Cord blood lymphocyte responses to food antigens for the prediction of allergic disorders. Arch. Dis. Child. 1992; 67:1003.

19. Herz U, Joachim R, Ahrens B eta1. Allergic sensitization and allergen exposure during pregnancy favor the development of atopy in the neonate. Int. Arch. Allergy Immunol. 2001; 124:193. 20. Cookson WO, Young RP, Sandford AJ et al. Maternal inheritance of atopic IgE responsiveness on chromosome 1l q. Lancet 1992; 340:381. 21. Yang K. Childhood asthma. Aspects of global environment, genetics, and management. Changgeng Yi Xue Za Zhi 2000; 23:641. 22. Armentia A, Banuelos C, De Arranz MLI et al. Early introduction of cereals into children's diets as a risk-factor for grass pollen asthma. Clin. Exp. Allergy 2001; 31" 1250. 23. Hide DW, Matthews S, Tariq S etal. Allergen avoidance in infancy and allergy at 4 years of age.Allergy 1996; 51:89. 24. Gershwin LJ. Asthma, infection, and environment. In: Bronchial Asthma Principles of Diagnosis and Treatment (ed. M E G A T E Albertson), Vol. 1, 4th edn., Humana Press, Totowa, 2001, p. 279. 25. Welliver RCOP. RSV, IgE, and wheezing. J. Pediatr. 2001; 139:903. 26. Lukacs NW, Tekkanat Berlin KK, Hogaboam A etal. Respiratory syncytial virus predisposes mice to augmented allergic airway responses via IL-13-mediated mechanisms. J. lmmunol. 2001; 167:1060. 27. Gershwin LJGRA, Anderson ML, Woolums AR, etal. Virus specific IgE is associated with IL-2, IL-4, and i-Interferon expression in pulmonary lymph of calves during experimental bovine respiratory syncytial virus infection. Am. J. Vet. Res. 2000; 61:291. 28. Gershwin LJ, Dungworth DL, Himes SR etal. Immunoglobulin E responses and lung pathology resulting from aerosol exposure of calves to respiratory syncytial virus and Micropolyspora faeni. Int. Arch. Allergy Appl. lmmunol. 1990; 92:293. 29. Gershwin LJ, Himes SR, Dungworth DL etal. Camacho: effect of bovine respiratory syncytial virus infection on hypersensitivity to inhaled Micropolyspora faeni, lnt. Arch. Allergy Appl. Immunol. 1994; 104:79. 30. van Schaik SM, Tristram DA, Nagpal IS et al. Increased production of IFN-gamma and cysteinyl leukotrienes in virusinduced wheezing. J. Allergy Clin. lmmunol. 1999; 103:630. 31. Garofalo RP, Patti J, Hintz KA et al. Macrophage inflammatory protein-lalpha (not T helper type 2 cytokines) is associated with severe forms of respiratory syncytial virus bronchiolitis. J. Infect. Dis. 2001; 184:393. 32. Tsitoura DKS, Dabbagh K, Berry G etal. Respiratory infection with influenza A virus interferes with the induction of tolerance to aeroallergens. J. lmmunol. 2000; 165:3484. 33. Von Ehrenstein OS, Von Mutius E, Illi S et al. Reduced risk of hay fever and asthma among children of farmers. Clin. Exp. Allergy 2000; 30:187. 34. Von Mutius E, Braun-Fahrlander C, Schierl R et al. Exposure to endotoxin or other bacterial components might protedt against the development of atopy. Clin. Exp. Allergy 2000; 30:1230. 35. Riedler J, Braun-Fahrlander C, Eder W etal. Exposure to farming in early life and development of asthma and allergy: a cross-sectional study. Lancet 2001; 358:1129. 36. Svanes C, Jarvis D, Chinn Set al. Childhood environment and adult atopy: results from the European Community Respiratory Health Survey. J. Allergy Clin. Immunol. 1999; 103(3 Part 1):415. 37. Gereda JE. E.Y.L., Thatayatikom A et al. Relation between house-dust endotoxin exposure, type I T-cell development, and allergen sensitisataion in infants at high risk of asthma. Lancet 2000; 355:1680. 38. Klintberg B, Berglund N, Lilja G etal. Fewer allergic respiratory disorders among farmer's children in a closed birth cohort from Sweden. Eur. Respir. J. 2001; 17:1151.

39. Jalaludin B, Xuan W, Mahimic A et al. Association between Der p l concentration and peak expiratory flow rate in children with wheeze: a longitudinal analysis. J. Allergy Clin. Immunol. 1998; 102:382. 40. Charpin D, Birnbaum J, Haddi E et al. Altitude and allergy to house-dust mites. A paradigm of the influence of environmental exposure on allergic sensitization. Am. Rev. Respir. Dis. 1991; 143:983. 41. Yabuhara A, Macaubas C, Prescott SL etal. TH2-polarized immunological memory to inhalent allergens in artopics is established during infancy and early childhood. Clin. Exp. Allergy 1997; 27:1261. 42. Arshad SH, Hide DW. Effect of environmental factors on the development of allergic disorders in infancy. J. Allergy Clin. Immunol. 1992; 90:235. 43. Lau S, Illi S, Sommerfeld C et al. Early exposure to house-dust mite and cat allergens and development of childhood asthma: a cohort study. Lancet. 2000; 356:392-7. 44. Salvi S. Pollution and allergic airways disease. Curr. Opin. Allergy Clin. Immunol. 2000; 1:35. 45. Gershwin LJ, Osebold JW, Zee YC. Immunoglobulin E-containing cells in mouse lung following allergen inhalation and ozone exposure. Int. Arch. Allergy Appl. Immunol. 1981; 65:266.

46. Seymour BWP, Pinkerton KE, Friebertshauser KE etal. Second hand smoke is an adjuvant for T helper-2 responses in a murine model of allergy.J. Immunol. 1997; 159:6169. 47. Seymour BWP, Friebertshauser KE, Peake JL etal. Gender differences in the allergic response of mice neonatally exposed to environmental tobacco smoke. Dev. Immunol. 2002; 9:47-54. 48. Devouassoux G, Saxon A, Metcalfe DD et al. 2002 Chemical constituents of diesel exhaust particles induce IL-4 production and histamine release by human basophils. J. Allergy Clin. Immunol. 2002; 109:847. 49. Watanabe N, Ohsawa M. Elevated serum immunoglobulin E to Cryptomeria japonica pollen in rats exposed to diesel exhaust during fetal and neonatal periods. BMC Pregnancy Childbirth 2002; 2:1. 50. Bommel H, Li-Weber M, Serfling E et al. The environmental pollutant pyrene induces the production of IL-4. J. Allergy Clin. Immunol. 2000; 105:796. 51. Devalia JL, Rusznak C, Davies RJ. Allergen/irritant interactionits role in sensitization and allergic disease.A//er~ 1998; 53:335. 52. Tariq S, Hakim EA, Matthews SM et al. Influence of smoking on asthmatic symptoms and allergen sensitisation in early childhood. Postgrad. Med. J. 2000; 76:694. 53. Gold D. Environmental tobacco smoke, indoor allergens, and childhood asthma. Environ. Health Perspect. 2000; 108:643.

Atopy generally refers to development of a specific immune response to non-self antigens characterized by the production of antigen specific IgE. This process is associated with the production of a number of cytokines by T-lymphocytes, generally referred to as "Th2" cytokines, which promote the production of antigen specific- IgE and support growth and differentiation of cells such as eosinophils and mast cells. These cytokines include IL-4, IL-5, and IL-13. In addition to increased production of Th2 cytokines, there has been a reported decrease in production of so-called Thl cytokines, such as 33-interferon, associated with development of atopy. Diseases associated with atopy include eczema, food allergy, stinging insect anaphylaxis, allergic rhinitis and extrinsic asthma. 1'2 Various studies suggest that 7-20% of the general population have some clinical manifestation ofatopic disease. Expression of atopic disease, including food allergy, eczema and asthma, is common in the first few years of life. There are a number of possible risk factors associated with development of atopy, including family history of atopy, increased IgE in early life, decreased monocyte production of ),-interferon, no (or short duration of) breast feeding, early allergen exposure, exposure to environmental tobacco smoke, certain viral infections (such as respiratory syncytial virus) and decreased occurrence of bacterial infections. These risk factors demonstrate the importance of gene-environment interactions in the development of atopy. This chapter will focus on development of allergic disease in childhood and the proposed environmental and genetic factors that likely play an important role in this process.

cells. Certain cytokines promote a "Thl" non-allergic immune response to specific antigens (characterized by antigen specific IgG and neutrophilic inflammation), whereas others promote a "Th2" allergic response (characterized by antigen specific IgE, eosinophils and mast cell responses). Both genetic and environmental factors may influence the cytokine profile of a given individual. Thus, to more fully appreciate the significance of these factors in expression of atopy, it is necessary to review the actions of some of these cytokines. 1'2 Cytokines shown to play a role in developing a TH1 response include y-interferon and IL-12. y-Interferon is associated with induction of expression of Fc Y receptors, MHC class I and MHC class I! molecules on the surface of macrophages (facilitating their actions as antigen presenting cells), promoting B-cells to switch from secretion of IgM to IgG and to not generate IgE. IL-12 is primarily secreted by macrophages (an antigen presenting cell) and acts on T-lymphocytes to induce secretion of a TH1 rather than TH2 cytokine profile. 3 So-called "Th2" cytokines include IL-4, IL-5, IL-10, and IL-13. Interleukin-4 acts on B-lymphocytes to induce a switch from IgM secretion to IgE and IgG4 secretion and also contributes to expression of VCAM-1 on endothelial cells in postcapillary venules, allowing for migration of eosinophils from the bloodstream to end-organ tissues. Interleukin-5 promotes eosinophil maturation and survival. IL-10 acts on macrophages to inhibit expression of MHC class II molecules and inhibits TH1 responses by blunting production of y-interferon. This cytokine also promotes T-cells to exhibit a TH2 phenotype by enhancing the action of IL-4. IL-13 is very homologous with IL-4 and, like IL-4, induces B-cell switching from IgM to IgE. 2

CYTOKINES OF ATOPY

GENETIC FACTORS OF ATOPY

INTRODUCTION

AND

DEVELOPMENT

Development of atopy is significantly influenced by the action of cytokines produced by monocytes, T-lymphocytes and other The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

IN

DEVELOPMENT

The earliest support for the hypothesis that genetic factors played a role in allergic diseases came from studies focused Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

on development of asthma in which disease phenotypes in monozygotic (MZ) and dizygotic (DZ) twin pairs were examined. 4-6 In one study, monozygotic twins were 19.8% concordant for asthma compared to 4.8% in DZ twins. A second study involving 2902 twin pairs revealed 30% vs 12% concordance in MZ vs DZ twins. In studies using atopy, rather than asthma, as the primary endpoint, 50-60% concordance has been reported in twin pairs. These studies indicate that a genetic component for asthma and/or atopy exists. However, even monozygotic twins, despite having identical genomes, do not have concordance of asthma or atopy of 100%. This observation suggests that environmental, as well as genetic, influences are important in eliciting the expression of an atopic or asthma phenotype. Gene mapping studies carried out in a number of laboratories indicate that chromosome 5q31-33 may be important in asthma and atopy, with genes for IL-3, IL-4, IL-5, IL-13 and GM-CSF proving to be clustered on the 5q locus. This technique also indicates that the [3 subunit of the high affinity IgE receptor is located on chromosome 1l q. Taken together, candidate gene studies have suggested linkages of potentially important genes with regions 5q ([52 AR and those listed above), 6p (HLA-DR), 1lq, 12q (y-interferon), 13q, and 14q (TCR). Positional cloning techniques suggest linkages of asthma or atopy with regions 2q, 5p, lip, 17p, 19q, and 21 q.4,6-9 Recent understanding of the role of innate immunity in influencing development of atopy will also lead to identification of other genes that might be important in influencing asthma or atopy development. An example of this is the appreciation borne from observations in many laboratories that the T-allele for the -159 position of the CD14 gene promoter on chromosome 5 protects against the development of TH2 responses, whereas the C-allele is a risk factor for atopy development. 1~ One may anticipate that as genomic and proteomic approaches are developed, new gene products associated with atopy may be identified that were previously unknown or not thought to play a role in development of an atopic phenotype.

PRENATAL I M M U N O L O G I C A L FACTORS IN DEVELOPMENT OF ATOPY It has been suggested that Th2 cytokines, best known for their role in immune response to allergens and parasites, also play a role in maintaining pregnancy. Indeed, traditional allergens, parasites and mammalian antigens (such as fetal antigens from a maternal perspective and maternal antigens from a fetal perspective), all derive from eukaryotic organisms, and all seem to elicit TH2 responses. Conversely, traditional TH1 responses are primarily directed against microbial (prokaryotic) organisms. 2 Some TH1 molecules appear to have a negative impact on pregnancy, y-Interferon has been reported to be an abortifactant. This may be through activation of maternal CD8

cytotoxic cells and NK cells which may injure or destroy fetal tissue. IL-2 may also negatively affect pregnancy through actions on uterine large granular lymphocytes which evolve into lymphokine-activated killer cells, which in turn may injure fetal tissues. 17-2~Conversely, TH2 cytokines may have a positive effect on maintenance of pregnancy. IL-1 is thought to signal initiation of parturition. IL-4, which has been identified in amniotic fluid, fetal and maternal endothelial cells may counter the effects oflL-1, preventing premature labor. Interleukin-10 has been observed in cells recovered from villous sampling throughout pregnancy. IL-10 is known to interfere with the action of IL-12, which is important in antigen presentation and induces y-interferon. Thus, it is possible that IL-10 promotes maternal immune tolerance of the fetus, allowing pregnancy to be established and continue until term. 21'22 Apart from the pro-Th2 milieu reported during pregnancy, it has been suggested that prenatal events may promote development of atopy in childhood. Of interest are reports that specific allergens, including Der p 1 from house dust mite and ovalbumin are present in amniotic fluid. As the fetus both swallows and aspirates amniotic fluid, amniotic allergens may induce active sensitization to these allergens in the fetus. Indeed, support for the hypothesis that antigen specific TH2 immune responses occur in utero can be found in observations that antigen-specific IgE are present in cord blood. Given that maternal IgE does not cross the placenta (though it can be found in amniotic fluid), IgE in cord blood would suggest an active response to an allergen by the fetal immune system. 17 Also consistent with development of active Th2 responses in fetal life are observations that mononuclear cells recovered from cord blood have robust proliferative responses to stimulation with allergens (including house dust mite antigen, rye grass pollen extract, Fel d I [cat allergen], ovalbumin and beta lactoglobulin). 21'23 Stimulated cord blood mononuclear cells also secrete TH2 cytokines, including IL-4, IL-5, IL-9, IL-10 and IL-13. Interestingly, TH2-type responses in cord blood mononuclear cells occur both in infants who were thought to be at low risk for development of atopy (based on family history) as well as those thought to be at higher risk for atopy. 2'17 Decreased levels of y-interferon also influence development of atopy at a young age. As outlined above, y-interferon is associated with TH1 responses, antagonizes the action of IL-4 and blunts production of IL-4. Studies in many laboratories demonstrate that both cord and peripheral blood mononuclear cells from neonates have diminished ability to produce y-interferon when compared to cells from healthy adult subjects following stimulation with mitogens. This blunting generally resolves by 5 years of age. However, a trend appears to exist in which y-interferon responses in cells obtained from children who develop atopic disease are even more blunted than those from non-atopic children. Likewise, some have argued that production of adult levels of y-interferon by mononuclear cells occurs later in atopic children than in non-atopic children. Thus, blunting of

TH1 responses may be important in maintaining TH2 responses. 2'2~ There is also evidence that antigen specific T-lymphocyte responses occur during fetal life. In studies of adults, CD3 positive cells (T-lymphocytes), the cell surface marker CD45RA is associated with naive cells whereas the marker CD45RO is associated with recall responses to antigen. Studies of fetal lymphocytes obtained at various stages of gestation suggest that the proportion of CD3+cells that express the CD45RO marker increases with increasing gestational age. Stimulation of neonatal cord blood mononuclear cells with specific allergens demonstrates that a significant proportion of T-lymphocytes responding to allergen challenge express the CD45RO marker, suggesting that these neonates had prior immunological experience with these specific allergens. 25 Consistent with these ideas are observations that infants can have positive skin tests to food allergens, presumably due to maternal ingestion of food allergens. However, attempts at decreasing maternal exposure to food allergens have not convincingly demonstrated a decrease in fetal levels of IgE or the likelihood of development of atopic disease in childhood. 22 Nonetheless, several studies have shown that maternal factors outweigh paternal factors in development of atopy or asthma in children. 26'27 These observations support the hypothesis that maternal influences may play a role in development of atopy or asthma.

P O S T N A T A L ALLERGEN EXPOSURE A N D D E V E L O P M E N T OF A T O P Y It has been argued that early exposure to allergens by persons who are genetically susceptible to development of atopy may be important in development of an atopic phenotype (eczema, rhinitis or asthma) during childhood. Season of birth has been reported to be an important event in development of atopy, with relationships between early exposure to seasonal airborne allergens and development of TH2 responses to those allergens at an early age being observed. 22 Likewise, seasonal allergens present in ambient air during the neonatal period correlate with development of airway allergy to those allergens later in childhood. It has also been argued that children living in environments with increased levels of mite allergen during the first year of life are more likely to develop asthma. 28 Studies that examined efforts to reduce the incidence of atopic airway disease by decreasing allergen exposure during the first year of life are at very early stages. Interestingly, somewhat different observations have been made regarding the relationship of exposure to mammalian pet antigens and development of allergic responses to those pets. It is clear that persons with well-established animal allergy may have significant exacerbation of disease when exposed to animal allergens. However, early exposure to animal allergens has been reported to protect against the development of allergy. 29 Whether this represents a process

of development of immune tolerance is unclear. It is also known that domestic endotoxin levels are increased in homes with mammalian pets. As endotoxin is thought to promote the development of TH1 responses in preference to TH2 responses, it could be argued that pet-related endotoxin might be outweighing pet-related allergen exposure. 30-35 Nonetheless, early exposure to animal allergens may be protective, rather than being a risk factor for development of animal allergy. The relationship between food allergen exposure and development of food allergy and eczema in children is better understood. Evidence of development of allergen specific IgE responses has been reported in nearly 30% of children born to atopic parents within the neonatal period. In infants with food allergy, the most common atopic phenotype is eczema. Between 80 and 90% of infants with eczema will develop antigen specific responses to airborne allergens as well. It has been argued that both maternal and neonatal exposure to food allergens contributes to development of specific allergen responses. Milk and egg allergies are perhaps the most common of the food allergy states in infants. 22 Infants who are exclusively breast-fed are less prone to develop food allergy than infants who are exposed to other food antigens. One mechanism for this may simply be decreased foreign food exposure. However, the presence of maternal IgA in breast milk may also have a protective effect, perhaps binding food allergens in breast milk so that they cannot interact with the neonatal mucosa. While support can be found on either side of the breast feeding argument regarding its role in protection against food allergy, the weight of evidence indicates that food allergen avoidance in at risk infants is protective against development of atopy. 22'36-38

Hygiene hypothesis As described above, there is evidence to suggest that fetal immune function is skewed towards a TH2 expression relative to adult immune function. As children get older, they experience more exposure to bacteria and bacterial products. 30-35'39-43 The argument can be made that the resulting innate response to these stressors likely redirects the acquired immune response towards a TH1 character. With these ideas in mind, a "hygiene hypothesis" to explain the development of atopy in modernized western society developed. This hypothesis suggested that decreased incidence of bacterial infection or decreasing bacterial colonization of the GI tract in infancy 44-47 contributes to the development of atopy by reducing pro-TH1 responses in the infant, thus allowing fetal TH2 immune character to be maintained, and promoting the expression of an atopic phenotype. This hypothesis is supported by observations that antibiotic use in early life is associated with subsequent development of atopy. Likewise, in societies in which antibiotic use is decreased and natural infections are more frequent, atopy occurs less frequently. In this same society, foods containing lactobacillus are also more commonly consumed. However, despite these data, it seems unlikely that children at very

high risk for asthma (e.g. inner-city African Americans) have increased exposure to antibiotics when compared to more affluent populations at lesser risk. 16 Perhaps one of the most interesting and controversial observations on the role of TH1 stimuli on TH2 expression is that examining the effect of the anti-tuberculosis vaccine BCG on development of atopy. Japanese school children, who routinely undergo BCG vaccination, were reported to have a significant inverse relationship between delayed hypersensitivity to M. tuberculosis and incidence of asthma and elevation of IgE. 48 As with many studies of environmental influences on atopic disease, there are confounding data. Studies in Britain fail to show a relationship between response to BCG vaccination and atopy. 49 However, supporting the idea that BCG vaccine is a potent TH1 stimulus are experiments in mice which demonstrate that immunization with BCG blunts development of allergen specific IgE and eosinophilic responses to allergen following allergen challenge. 2 Taken together, these observations support the notion that BCG stimulates increased TH1 function and is associated with decreased TH2 immune responsiveness. In addition to antigen specific immune responses, it has been suggested that accessory molecules expressed by bacteria (classic TH1 stimuli) may contribute to immune maturation such that TH1 responses are emphasized, z Lipopolysaccharide (LPS) is a molecule expressed on all gram-negative bacteria that interacts with antigen presenting cells and other immune effector cells via the CD 14 receptor. Antigen presenting cells (APCs) include dendritic cells and macrophages which process foreign particles and express a digested portion of that on their surface for interaction, or presentation, to T-lymphocytes for development of a specific immune response. Treatment of antigen presenting cells with LPS results in secretion of IL-12, which in turn blunts TH2 responses and stimulates 7-interferon secretion, promotes development of a TH1 immune response. It has been argued that mucosal colonization with bacteria, including LPS bearing organisms, allows for non-specific deviation toward the TH1 phenotype. Recently, it was found that the gene for CD14 co-localizes with genes for IL-3, IL-4 and GM-CSF on the chromosome region 5q31.1. Furthermore, a specific polymorphism has been identified (a cytosine [C] to thymidine [T] transition at base p a i r - 1 5 9 ) in which those children homozygous for the T-allele have significantly higher levels of soluble CD14 than do heterozygotes or those who are homozygous for the C allele. In turn, serum levels of sCD14 (which could mediate LPS interaction with APCs) have a significant positive correlation with y-interferon and a negative correlation with IL-4. This observation supports the hypothesis that an imbalance between TH1 and TH2 influences the development of atopic disease. 1~176

LIFESTYLE Many biological agents that contribute to airway inflammation in asthma include allergens from house dust mite and mold.

Non-specific irritants derived from microbes such as endotoxin from gram-negative bacteria and 1,3 beta-glucans from molds may also impact upon airway inflammation in both atopic and non-atopic subjects. Additionally, volatile organic compounds produced by microorganisms may have an impact on human health. 51 House dust mites, molds and environmentally encountered bacteria all thrive in humid environments, suggesting that high relative humidity in indoor environments is linked to increased adverse health effects. Several European studies demonstrate that asthma is increased in association with high indoor humidity or in homes with water damage. For example, both asthma and respiratory tract infections were increased in schools with increased humidity and mold spore counts; 52 furthermore, 53 measures of airway obstruction, variability in peak flow rates and peripheral blood eosinophils and mold but not mite allergy were increased in children living in homes with increased humidity. 54 A large cross-sectional study of fourth grade school children in Munich, Germany identified 234 children with active asthma (5% of the total cohort). 5s Three years later, 155 of these children underwent measurement of lung function and non-specific airway reactivity. Dampness was associated with increased night-time wheeze and shortness of breath but not with persisting asthma. Risk factors for bronchial hyperreactivity in adolescence included allergen exposure and damp housing conditions. Mite antigen levels were examined from homes of 70% of the asthma cohort and found to significantly correlate with dampness and bronchial hyperreactivity. However, the effect of dampness was not due to mite allergen alone as bronchial hyperreactivity remained significantly correlated with humidity even when adjusting for mite allergen levels. Studies in the United States, Australia and New Zealand also report increased indoor humidity as a risk factor in asthma and wheezing disease. 56-59 It has recently been reported that use of mechanical ventilation systems can minimize indoor humidity. While it has been reported that such systems do not modify mite allergen levels, other studies suggest air-handling systems which impact on humidity may mitigate increased levels of mite allergen and airway dysfunction. It has been reported that homes that have mechanical ventilation systems are significantly less humid, with decreased numbers of mites and Der p 1 concentrations in bedroom carpets when compared to homes without such systems. 58 Non-specific responses to histamine were improved in a nearly significant number of patients (p=0.085) living in homes with mechanical ventilation systems. In school buildings randomly selected to have air-exchange rate increased, relative humidity and concentrations of several airborne pollutants were reduced compared with classrooms in control buildings; reports of asthma symptoms were less in the students who attended schools with increased air exchanges. 6~ A number of epidemiological studies have pointed to the potential role of lifestyle as a factor that modulates expression

of atopy in susceptible individuals. One of the most intriguing examples of the effect of lifestyle is provided by an examination of the prevalence of asthma and atopy in children from eastern and western Germany at times following political reunification of that state. Shortly after reunification in 1990, studies of the prevalence of atopy and asthma in children from East and West Germany revealed that children from the East, while more likely to be diagnosed with bronchitis, were less likely to have atopy, had fewer positive skin tests and were less likely to have asthma than their Western counterparts. 61 While it was unclear which lifestyle factors were influencing atopy development, there were some candidate influences. Children in the East were more likely to be placed in day care than those in the West. Also, potential differences in diet, especially fat intake, were suggested as a possible influence. In the early 1990s, particulate pollution was higher in the East whereas private automobile use and ozone exposure were more common in the West. Allergen exposure was not thought to be substantially different in the East than in the West. 62 A few years later, rates of atopy in the East have increased and are approaching those found in Western Germany. This has been associated with development of a more "westernized" lifestyle in the East, including decreased use of coal in industry, increased automobile use, and increased availability of high fat foods. Decreased exercise and changes in architectural style have also been associated with development of atopy and asthma. Comparison of heating styles in rural vs urban Western Germany in which wood and coal burning furnaces in homes are used in rural settings is associated with decreased asthma and atopy in the rural setting. It was thought that bedroom and indoor temperatures were lower in these homes than in urban homes and that this might contribute to decreased expression of atopy. Dampness and water damage have also been associated with increased expression of allergic disease. 54'55'63 Similar observations have been made between other previously Eastern bloc and Western countries as well as comparison of asthma in rural vs urban areas in Africa, Europe, New Zealand and Australia. This effect of a greater degree of "westernization" of lifestyle in previously undeveloped locations mirrors development of asthma in already developed locations. Likewise, the problem of asthma in inner city minority populations suggests a role for urbanization in expression of atopy. Therefore, based on current evidence, it seems quite likely that urbanization is a key feature in the development of asthma and atopy. This likely represents alterations in the environment, which allow for expression of important genes that result in an atopic phenotype. Delineation of the specific features of urban lifestyle, which allow the atopic phenotype to be expressed, is incomplete.

Environmental tobacco smoke Maternal tobacco smoking has also been linked to increased rates of wheezing and asthma in exposed children, increased bronchial reactivity and increased total and antigen-specific

IgE. 2 Exposure to environmental tobacco smoke (ETS) may act to enhance atopy by a number of mechanisms. These include increased airway mucosal permeability or a direct effect on immune function. Environmental tobacco smoke (side stream smoke from the burning end of the cigarette and exhaled mainstream smoke from the smoker) is a major source of gases and respirable particles in indoor environments. 64'65 The most extensive literature on the impact of ETS in airway health and asthma is in children. 66-6s Environmental tobacco smoke exposure has been associated with increased occurrence of otitis media, upper and lower respiratory tract infections, wheezing and even increased occurrence of cancer in adults living with active smokers. 66'69 ETS is clearly a significant factor in exacerbating airway illnesses affecting airway mucosa. Overall particle levels in homes without smokers is roughly equivalent to that found in ambient air, whereas levels found in homes with smokers are often several-fold higher than those found in ambient air. Thus, the effect of particulate air pollution is magnified in homes with smokers compared to those in which smok, ing does not occur. Numerous case reports and studies suggested that ETS is an important factor in asthma exacerbation. 69 Furthermore, a recent meta analysis of 37 studies indicated an increased risk for asthma and wheezing disease in children exposed to ETS. 7~ Maternal tobacco smoking is associated with increased incidence of wheezing and asthma, increased bronchial reactivity and increased total and antigen-specific IgE in exposed children. 71'72 The link between ETS and asthma appears very clear with regard to exacerbation of pre-existing disease. While there is still debate on the role of ETS in development of asthma or atopy, there are numerous reports that support the hypothesis that ETS enhances atopy development in susceptible individuals. 71'72 However, there is a growing body of evidence in animals that tobacco smoke may enhance development of TH2 responses. Increased levels of the TH2 cytokines IL-4 and IL-10 as well as eosinophils are found in the airway after allergen challenge when such a challenge is preceded by ETS exposure. 73 Tobacco smoke extracts have been reported to alter monocyte function in mice, 65 including suppression of phagocytosis, decreased MHC class II molecule expression, blunting of oxidative burst and NO synthesis, all of which are activities that are mediated by ),-interferon, a TH1 cytokine. Compared with control mice, mice exposed to ETS during primary sensitization with ovalbumin (OVA) have exaggerated TH2 responses to recall challenge with O V A . TM Taken together, these studies provide biological plausibility for the many epidemiological reports indicating that ETS exposure enhances development of atopy and asthma.

Ozone exposure There are a number of ambient air pollutants that are regulated by the US Environmental Protection Agency. Of these, the two pollutants thought to have the biggest impact on allergic diseases are ozone and elements of particulate air

pollution, particularly diesel exhaust particles. As outlined below, ozone and diesel exhaust have been reported to both augment allergic response and to potentially promote the development of primary TH2 responses to antigens. Exposure to ambient ozone is related to increased asthma morbidity. Various studies which collectively examined admissions to 79 hospitals in southern Ontario reveal a significant association between ozone and admissions for respiratory symptoms. 75-77 Of note is a 24-48 h time lag between the ozone exposure and occurrence of hospital admission. Also, an association has been found between ER visits for asthma and ozone levels >0.11 ppm (but not <0.11 ppm) in school children. 78 Similar observations have been made in Mexico City, 79-81 and more recently in Los Angeles. 82 In general, ozone exposure is strongly associated with increased asthma morbidity, and may have been a major trigger for asthma exacerbation in summer months. It is notable that, in concert with 1996 Olympic Games held in Atlanta, there was a significant attempt by the local government to decrease ozone generation by vehicle exhaust. Not only were ozone levels improved, but there was also a significant decrease in asthma morbidity during this time. 83 In addition to its impact on exacerbation of disease, recent studies suggest that ozone may promote development of asthma. A cohort of 3535 children with no history of asthma from schools in 12 southern California was studied for up to 5 years; 84 during this period, 265 children developed a newly recognized diagnosis of asthma. It was observed that participation in outdoor sports (presumably associated with increased minute ventilation) in areas of increased ozone concentration was a risk factor for asthma development relative to similar exercise in areas with ozone exposures w e r e lOW.67'84 A number of studies have examined the effect of ozone on lung function in asthmatics. For example, challenge with 0.25 ppm ozone for 2 h causes decreased pulmonary function in asthmatics. 85 Furthermore, exposure to 0.4ppm ozone for 2 h with rigorous exercise has a greater effect in asthmatics than non-asthmatics as reflected in measurements of airway resistance and non-specific airway reactivity. 86 These studies are supported by other studies that demonstrate an ozone-induced bronchospasm in asthmatics. 87'88 Taken together, most studies suggest that ozone generally appears to worsen airflow in asthmatics to a greater extent than in healthy subjects, although there is disagreement regarding the increased sensitivity of asthmatics to ozone. 89 Indeed, ozone levels as high as 0.4 ppm do not appear to have an adverse effect on exercise-induced bronchospasm (EIB). 9~ In the nasal airways of persons with allergic rhinitis, ozone has been observed to induce both neutrophil and eosinophil influx. 91'92 A number of studies have also examined the effect of ozone exposure on lower airway inflammation. One study did not reveal a marked difference in response to ozone between asthmatics and non-asthmatics, nor was there an eosinophil response to ozone in the asthmatics. 93 However, other studies do indicate that asthmatics have a different inflammatory response to ozone when compared

to non-asthmatics. Two studies reported increased numbers of neutrophils in BAL fluid from asthmatics exposed to ozone than in normal subjects, with no effect on eosinophil numbers. 94'95 Three other studies, one employing induced BAL and two examining sputum, report increased eosinophil numbers or evidence of eosinophil activation after ozone exposure. 96-98 It is also important to note that in a large study involving 877 children, ozone exposure was significantly associated with increased levels of ECP (eosinophil cationic protein) as reflected in urine samples. 99 Overall, the available evidence indicates that asthmatics have a greater inflammatory response to ozone inhalation, including influx of eosinophils to the airway. Ozone also has an effect on responses to allergen challenge. Following an ozone exposure of 0.12ppm for 1 h, some investigators have observed increased immediate response to inhaled allergen whereas others have not. 1~176176 However, levels of 0.16 and 0.25 ppm ozone have clearly demonstrated increased response to inhaled allergen. 1~176 Nasal studies of allergic asthmatics do not demonstrate enhanced immediate phase mast cell responses to allergen following exposure to 0.4 ppm ozone. TM However, increased late phase response to house dust mite allergen 4 h after challenge following 0.4 ppm ozone exposure have been observed; measures of late phase response, which were enhanced, included eosinophil influx, ECP levels and IL-8 levels. 92 Taken together, these studies suggest that the effect of ozone on inhaled allergen challenge is likely dose-related, may be mediated by mechanisms other than increased sensitivity of mast cells to allergen after ozone exposure, and can exacerbate allergen-induced inflammation in asthmatics. Given the strong epidemiological associations between ozone exposure and asthma exacerbation coupled with the observed effect of ozone on responses to allergen, it has been suggested that asthmatics with more severe disease may be more sensitive to the effect of ozone. However, a comparison of ozone response of mild episodic asthmatics to those asthmatics with disease significant enough to require inhaled corticosteroids, revealed increased inflammatory response in the milder group. These data would suggest that more severe asthmatics are less responsive to ozone than milder subjects. However, the mild persistent asthmatics were also using inhaled steroids. It is possible that this study really demonstrates an effect of inhaled corticosteroids on ozoneinduced inflammation in asthmatics that is not observed in non-asthmatic volunteers. 1~

Diesel exhaust As with gaseous pollutants, respirable particulate matter (less than 10 ktm in size) is also associated with episodes of increased asthma exacerbation. 1~ Increased ambient air particulate levels were linked to a need for asthma medication in a cohort of asthmatics in Utah. 1~176 Hospitalization due to increased asthma severity in Seattle was found to occur in conjunction with increases in airborne particulate matter. 1~ Similar observations have been made in other locations as well, including Germany, the Czech Republic

and Mexico City, and other locations, s~176 These data demonstrate the important role that particulates could play in asthma morbidity. There are also studies that demonstrate that acute increases in ambient air particulates are associated with increased mortality, especially in subjects with pre-existing cardiopulmonary disease. 112'113 A study performed in the Utah Valley offered a unique opportunity to link the effect of particulates to the activity of a local steel mill. 114 That study focused on occurrence of respiratory disease episodes (asthma exacerbation, hospital admission for respiratory complaints, etc.) that occurred during a year in which the mill was not operational due to a labor dispute, as well as the years that preceded and followed the work stoppage. Compared to the previous and following years, respiratory disease markers were markedly decreased during the strike year. Also decreased during the strike was the level of ambient air particulates. Thus, there is a strong suggestion that ambient air particulate levels do correlate with increased occurrence of disease exacerbation in disorders such as asthma. However, identification of specific agents that may mediate asthma exacerbation is difficult, as there is a vast array of particles that have been identified and shown to have biological effects in animal and in vitro studies. Active agents in particulate matter include silica, metal ions (such as iron, vanadium, nickel and copper), organic residues (polyaromatic hydrocarbons found on diesel exhaust particles), acid aerosols and biological contaminants such as endotoxin, ss Human challenge studies have recently been undertaken employing chamber exposures to diluted diesel exhaust (DDE). This is different to what is observed with challenge by diesel exhaust particles (DEP) as the DEPs are freshly generated and exposure conditions contain all components of diesel exhaust, not just those retained on particles alone. Exposure to DDE for 1 h results in increased number of neutrophils, B-lymphocytes, histamine and fibronectin in BAL. In bronchial biopsy specimens, increased numbers of mast cells, neutrophils, CD4 and CD8 positive T lymphocytes, as well as increased ICAM-1 and VCAM-1, were found 6h after challenge; 115'116 increased polymorphonuclear leukocytes and platelet numbers were also observed in peripheral blood after challenge. 116 The same investigators also found induced expression of IL-8 and GRO-ct at the message and protein level in airway samples collected 6 h after DDE exposure. It has also been reported that airway inflammation is increased 4 h after exposure to resuspended diesel particles (polymorphonuclear leukocytes and myeloperoxidase). 117 Thus, diesel exhaust can induce non-specific, TH1 type inflammation in humans. Diesel exhaust particles also have actions that can enhance non-specific inflammatory responses. In vitro studies of epithelial ceils and monocytes in culture demonstrate that DEP can induce expression or secretion of IL-8, RANTES, ICAM-1 and GM-CSF.118'119 The effect of DEP on IL-8 and RANTES was linked to p38 MAK kinase activation. 119 In vitro studies of a human macrophage cell line have shown that tert-butylhydroxyxquinone (which is thought to mediate

the effect of DEPs on TH2 inflammation) mediates the activation of the RANTES gene promoter via activation of AP-1 and NF-lcB). Thus, at a cellular level, it appears that DEPs activate cytokine expression via signal transduction pathways that are activated by a number of pollutant or infective agents. These observations suggest that co-exposure with DEP and other agents may be more effective in inducing inflammation than with either alone. Numerous animal and in vitro studies have demonstrated that DEPs shift primary immune responses to neoantigens towards a TH2 phenotype, characterized by production of antigen-specific IgE, and enhance allergen induced immune responses, including increasing IgE production and enhancing cytokines involved in eosinophilic or allergic inflammation, especially IL-4, IL-5 and GM-CSF, as well as airway hyperresponsiveness. 12~ DEPs have been reported to induce B-lymphocyte immunoglobulin isotype switching to IgE. 123'124 One group of investigators has demonstrated that DEPs can promote TH2 -type inflammation in humans, including enhancement of IgE. 123 Nasal challenge studies in humans demonstrate that challenge of volunteers (4 atopic and 7 non-atopic) to DEP increased nasal IgE production 4 days after DEP challenge without any effect on IgG, IgA or IgM; 123 there were also shifts in the ratio of the 5 isoforms of IgE. These effects were very dose specific as only a dose of 0.3 mg DEP caused this resuh. 123 It has also been reported that DEP challenge of the nasal mucosa causes increased cytokine production by cells recovered in lavage fluid. 125'126 When comparing nasal lavage fluid components recovered pre- and post-challenge with 0.3 mg of DEP, pre-challenge lavage had detectable mRNA levels for 3,-interferon, IL-2 and IL-13. In contrast, lavage cells recovered post-challenge were associated with detectable levels of IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, and 3,-interferon in recovered cells. IL-4 protein was also measured in post-challenge lavage. While it is unclear which type of cells were present in lavage fluid before or after challenge in this study, a subsequent report suggests that mast cells are the primary source of these cytokines. 127'128When coupled with challenge with ragweed allergen in ragweed sensitized atopic subjects, DEP was found to yield an enhanced ragweed specific IgE and IgG response to ragweed allergen when compared to ragweed alone. This effect included increased expression of IL-4, IL-5, IL-6, IL- 10, IL- 13 and decreased expression of 3,-interferon and IL-2 and had no effect on total IgE and IgG. 125 Most recently, this group reported that DEP challenge markedly shifts primary immune responses in the nasal mucosa to keyhole limpet hemocyanin (KLH, a neoantigen rarely encountered by humans) towards a TH2 phenotype, yielding KLH-specific IgE. Without DEPs, immune response to K L H is of the TH1 type characterized by KLH-specific IgE. 129 Extracts from DEP containing the polyaromatic hydrocarbon (PAH) fraction from these particles, as well as specific PAH compounds phenanthrene and 2,3,7,8-tetracholorodibenzo-p-dioxin, are thought to mediate many of the effects

of DEP on immune response. 13~ Thus, polyaromatic hydrocarbons, by their action on B-cells, appear to be important mediators of allergic inflammation. Additionally, DEPs can promote CD80 (which is an important molecule for M H C Class II antigen presentation) expression in macrophages as well as enhanced LPS-induced IL-10 responses. 132 These effects on macrophages or other antigen presenting cells could account for their ability to present antigen in such a way as to promote TH2 responses to those antigens.

SUMMARY The neonatal period is a particularly important period for the development of allergic disease. Genetic, environmental and lifestyle factors all appear to be important factors in this process. The mechanisms by which Th2 responses in early life are augmented such that development of atopic diseases is enhanced are not completely understood. However, there is an emerging understanding of the enhancement of Th2 processes in fetal life. It seems reasonable that Th2 i m m u n e responses, perhaps via antagonism of T h l inflammatory responses, play an important role in the normal physiology of pregnancy. A better understanding of these Th2 mechanisms should enhance our understanding of why certain neonates and infants may be at higher risk towards developing an atopic phenotype. Large scale national surveys of allergen and endotoxin exposures in a variety of domestic settings should improve our understanding of the role of early life allergen exposure in development of atopy. Epiderffiological studies of both environmental pollutant exposures and genetic risk factors, coupled with complementary animal studies, will also contribute to an increased understanding of the biological and environmental risk factors involved in development of asthma. Such studies will be essential in designing strategies for the primary prevention of atopic diseases in children.

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INTRODUCTION

A substantial body of evidence indicates that exposure to current levels of ambient air pollutants is associated with a wide spectrum of acute adverse health effects. The health effects of air pollution on children's respiratory health are of clinical, public health, and regulatory concern (Table 24.1). Although short-term effects of exposure to ambient pollutants are well documented, long-term effects of chronic exposures on lung development and adverse respiratory health events have only recently been extensively investigated. The lack of understanding of which pollutants are important for acute and chronic health effects and what levels of exposure are safe inhibits rational approaches for control. Although a large number of chemical species occur in ambient air, ozone, nitrogen dioxide (NO2) , acid vapors, respirable particulate matter (PM10 and PM2.5), sulfur dioxide (SO2) and acid aerosols have been identified as presenting the greatest hazard to human populations. 1

AMBIENT

AIR

POLLUTION

Air polluted with ozone, NO 2 and respirable particles is an important public health problem in many regions of the world. These pollutants are produced by fossil fuel combustion and subsequently undergo photochemical reactions in the atmospheric aerosol. The patterns of emission and photochemistry produce an aerosol that varies in composition and particle size distribution in a complicated manner over time and space. Photochemical reactions between NO 2 *To whom correspondence should be addressed. The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

and hydrocarbons produce a diurnal ozone profile. Ozone levels rise in the late morning after heavy traffic and peak in the 10:00 a.m. to 6:00 p.m. period. 2 In the evening, scavenging of ozone (03) by NO titration in areas with heavy traffic reduces the ozone to levels below background levels at night. High ozone levels may occur in downwind communities later in the afternoon and evening, stemming from airborne transport in the setting of low NO levels in areas without heavy traffic. Ozone levels show systematic variation by day of week. High ozone areas have higher levels on weekends compared to weekdays, low ozone areas do not have higher weekend levels. Other pollutants have less pronounced diurnal variation. 2 SO 2 is produced by combustion of sulfur containing fuels, primarily coal-fired power plants and diesel engines. Because coal is a common source of fuel, SO 2 and acid aerosols are exposures of interest in many regions of the world. Acids, such as nitric, formic, and acetic acids, are also present in the gas phase of the atmosphere in some regions including southern California. Higher concentrations of pollutants in late afternoon influence exposure because children are most likely to be outdoors and physically active. The timing of high ozone levels may increase the exposure and dose for children relative to adults. Children spend more time outside during high ozone periods in the afternoon, on weekends, and during the summer months? Furthermore, children are more likely to engage in vigorous physical activity while outside, increasing the delivered dose of pollutants to the distal lung which is more sensitive to damage because it has thin or patchy respiratory extracellular lining fluid (RELF) as a protective barrier. Particulate pollution has received increased attention in recent years. PMlo levels show marked geographic variation. Annual average concentrations of PM2. 5 in southern Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

California during the 1994-1997 period ranged from 7 ~tg/m3 outside the Los Angeles air basin to 32 ].tg/m3 within the air basin. Of measured ions in PM2.5, nitrate is the most abundant, followed by ammonium, sulfate, and chloride. In addition, PM2.5 contains a number of transition metals and organic compounds that influence its toxicity. Based on associations with lung cancer and asthma outcomes, increased research attention has also been focused on bioaerosols, diesel exhaust particles and ultrafine particles (< 100 nm).

R E S P I R A T O R Y H E A L T H EFFECTS OF A M B I E N T AIR P O L L U T I O N Acute exposures to high levels of ambient pollutants have resulted in severe effects including substantial increases in morbidity and mortality as observed during pollution episodes in the Meuse Valley in 1930, Donora, Pennsylvania in 1948, and London in 1952. In the U.S. and Western Europe, regulatory efforts make such episodes a remote possibility; however, acute respiratory effects in children exposed at current ambient levels of ozone are welldocumented. 1 Acute respiratory effects from exposures to ambient levels of NO 2, SO2 and acids have been documented in studies of susceptible children and adults. Studies have established that adverse health effects occur in some groups of children exposed to ambient levels of ozone, particulates and N O 2 that occur in developed and developing nations, and have provided a limited amount of suggestive evidence for chronic health effects (Table 24.1). 1 Much of the research on the acute effects of air pollution has focused on shortterm exposures to ozone and NO 2. Studies of adults and children using controlled human exposures have documented that ozone inhalation of as little as 0.08 ppm for several hours is associated with reproducible dose-dependent (concentration, duration, and minute ventilation) effects that are enhanced by exercise. 1 NO 2 also has acute effects, but primarily at levels exceeding 2.0 to 4.0 ppm. 1 The effects

include decrements in forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC), cough and chest discomfort, lung injury and inflammation, and changes in airway responsiveness. 1 Changes in pulmonary function have also been observed in children at summer camp, military recruits, and athletes exposed to ambient air pollution during outside activities. 4 The ozone-induced acute changes in pulmonary function are reversible. The reversibility of the acute and sub-acute changes in lung function either with anesthetic or with time indicates this mechanism may not be responsible for chronic effects. 1 Airway hyperreactivity persists after ozone-induced changes in FEV 1 resolve and the degree of reactivity is not associated with the magnitude of spirometric changes, suggesting an independent mechanism for hyper-reactivity. Increased reactivity may be involved in the pathogenesis of chronic lung disease because increased airway reactivity to nonspecific stimuli such as methacholine is a characteristic of chronic respiratory diseases including asthma and chronic obstructive pulmonary disease (COPD). The inflammatory response involves increased numbers of macrophages, neutrophil infiltration, increased cellular protein permeability following the production of a broad range of inflammatory cytokines, and increased arachidonic acid metabolites. As with airway hyper-reactivity, the inflammatory response is not correlated with acute lung function decrements. In addition to the inflammatory response, some studies suggest that acute NO 2 exposure at commonly encountered ambient levels may adversely affect other aspects of immune function, including macrophage function resulting in decreased airway clearance and increased risk of infection. In the following sections the emerging evidence for chronic effects of ambient pollutants focusing on lung function development and decline and asthma incidence will be reviewed.

Chronic effects of air pollution on lung function Normal development and growth of the lungs is necessary for optimal gas exchange that is essential for aerobic life.

Alterations in lung structure during the life course can adversely affect lung function, resulting in an increased occurrence of respiratory-related morbidity and mortality. Lung function can be assessed using a broad array of tests that measure lung volume, airflow and gas diffusion. Spirometry measures how well the respiratory system functions in exhaling air. Maximal forced expiratory volume maneuvers and spirometers are used to assess FEV 1, maximum mid-expiratory flow (MMEF), and FVC. The information gathered using spirometry is useful in assessing airway obstruction and functional lung capacity. Spirometry does not measure total lung capacity or diffusion capacity. A number of insults including intrauterine growth retardation, viral infections, premature birth, inflammatory conditions, genetic mutations and environmental toxicants can disrupt lung development and growth leading to reduced lung function. Airborne environmental toxicants pose a unique threat to the development and maintenance of maximum attained lung function. Exposures to tobacco smoke and combustion-derived ambient air pollutants are common. Large volumes of air are inhaled daily, and in polluted environments substantial inhaled and deposited doses to airways and air exchange regions occur. If lung defenses are breached, normal developmental and homeostatic process can be disrupted leading to disruption of development and acute damage that can lead to chronic reduction in lung function.

Effects of environmental toxicants on lung function growth and decline

Normal growth of lung function Fig. 24.1 shows the change in lung function over the life course using FEV 1 as an example. Curve I shows the optimal growth and decline of FEV 1. Lung growth begins in the in utero period and continues until the late teens and early twenties. Lung function reaches a maximum by age 18-20 years in females and 22-25 years in males; 5 some males may show a small amount of lung function growth into the mid-20s. Among non-smokers, FEV1 and FVC show a plateau, without respiratory symptoms, that may

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last up to 10 years for males and shorter for females before beginning a slow decline. Impaired prenatal or postnatal lung growth may result from exposure to environmental toxicants including tobacco smoke and ambient air pollutants. The temporal patterns of exposures and lung function growth and development may be important in understanding the long-term adverse effects of exposures. Effects of active and passive tobacco smoke exposure have been extensively investigated, although recent studies show in utero effects of maternal smoking are important. 6'7 Reduced prenatal or postnatal growth rates result in lungs that do not reach their developmental potential, reaching a reduced level resulting in symptoms at an earlier age with normal age-related decline in function or acute respiratory conditions. The effects of toxicants on postnatal growth may be permanent; however, it is unknown whether 'catch-up' or prolonged growth occurs during late adolescence resulting in normal attained lung function levels. Normal or reduced lung function growth rates may also be followed by a shorter plateau phase and/or a period of accelerated decline that produces an early onset of chronic respiratory diseases. Superimposed on these lifetime patterns are acute episodes of reversible airflow obstruction. For a given amount of obstruction, symptoms may be more severe depending on baseline function.

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Based on studies of occupational groups and model systems, it has become recognized that a large number of toxicants have the potential to adversely affect lung function growth and decline. In order to understand the effects of environmental toxicants on lung function growth and decline, the timing of exposure must be considered. Critical windows of susceptibility occur during the fetal and childhood periods of growth and development. Exposure during these periods may have larger long-term consequences than exposure at the same level during the adulthood phase of decline. Furthermore, exposures during later life-course periods are often correlated with exposure in earlier periods. For example, active smoking, exposure to environmental tobacco smoke (ETS) and in utero exposure to maternal smoking are highly correlated. If the temporal correlation of in utero and ETS exposure during childhood are not accounted for, the effects of in utero exposure could be incorrectly ascribed to ETS exposure during childhood. 6'7 Lastly, toxicants can induce disease states that affect later exposure or recall of previous exposure history, suggesting that prospective studies may be necessary to clarify the temporal relationships between exposures and adverse respiratory outcomes.

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Fig. 24.1. Schematic representation of the life course of forced expiratory volume (not to scale). I=normal growth and decline, II=impaired prenatal or postnatal growth, Ill=normal growth but accelerated decline, IV = episodes of reversible airflow obstruction.

Tobacco smoke and lung function growth during childhood Tobacco smoke is a prototypic toxicant because its effects on lung function have been most extensively studied over the life course. 8-1~ A detailed account of the effects of

early-life tobacco smoke is given in Chapter 20. In brief, in utero exposure to maternal smoking is associated with

decreased lung function at birth, which persists into adolescence and adulthood. The effects of in utero exposure are largest in children who also develop childhood asthma. 11 Because in utero exposure also increases the risk for asthma, in utero exposure affects lung function directly during the in utero period and indirectly through increased occurrence of childhood asthma. A large number of studies have investigated the role of ETS exposure on lung function in children and ETS exposure is prospectively associated with lung function growth; however, most studies have not assessed the effects of the highly correlated exposure of maternal smoking during pregnancy. Tobacco smoke exposure affects the length of the plateau and rate of decline in lung function. Smoking and ETS exposure are associated with a shorter plateau and an accelerated decline in susceptible smokers that may lead to an early onset of disability and death from chronic lung diseases. Smoking cessation results in a rate of decline in lung function similar to that in non-smokers, even after the onset of disability.

Ambient air pollution and childhood lung function growth The fetal period appears to be a critical window for the effects of toxicants on lung function. The effects of ambient air-pollutant exposure during the in utero period on lung function at birth or during childhood have yet to be established. Recent studies showing that current levels of ambient air pollution increase the risk for low birth weight and pre-term birth suggest that lung function could be adversely affected by air pollution exposure during the fetal period. 12'13 Studies of air pollution during specific age periods and lung function levels in newborns as well as lung function growth in children will be required to address this important issue. Long-term exposure to outdoor air pollutants has been associated with reductions in the growth of lung function. ~4'15 We studied the effects of air pollution on lung function growth over a 4-year period in school children residing in 12 communities in Southern California with varying levels of air pollutants. Significant deficits in growth of lung function (FEV 1, FVC, and MMEF) were associated with exposure to particles with NO 2, PM10, PM2.5, PM10-PM2.5, and inorganic acid vapor (p < 0.05). Our results showing an association of NO 2 with lung function growth in the 12 communities are depicted in Fig. 24.2. The associations for both PM and acids were similar to those for NO2, indicating that exposures associated with mobile sources (NO x and PM) are important. The independent effects of these pollutants could not be identified due to their high degree of correlation across communities. No significant associations were observed with ozone in the cohort of fourth graders. The deficits were generally larger for children spending more time outdoors. In an analysis of a second cohort of fourth graders recruited four years later in the same 12 Southern California

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communities, reduced FEV 1 and MMEF growth was most strongly associated with vapor acids, NO2, PM2.5, and elemental carbon levels, a marker for diesel exhaust. The estimated growth rate for children in the most polluted of the communities as compared with the least polluted was predicted to result in a cumulative reduction of 3.4% in FEV 1 and 5.0% in MMEF over the 4-year study period. Across cohorts and lung function measures, we observed significant associations with both particles and gaseous pollutants. Although the correlations among pollutants were high, fine particles (PM2.5) and the elemental carbon portion of PM showed stronger associations with lung function growth than PM10 or the organic carbon portion of particulates. Two other studies, one conducted in Austria 16 and the other in Poland 17, have also reported associations between ambient air pollutants and lung function growth in children. The long-term effect of exposure to ambient air pollution on children's lung function was investigated in nine Austrian cities over a 3-year period. ~6 Repeated spirometry in a cohort of 1150 children showed significant deficits in FVC, FEV 1, and MMEF associated with ozone levels and some evidence that SO 2 and NO 2 were associated with deficits in MMEF growth. In the Polish study, lung function growth (FVC and FEV1) over 2-year period was compared for 1001 children living in two regions of Krakow with different levels of particulate air pollution. Lung function growth was significantly slower in the high pollution area. Collectively, these prospective studies strengthen earlier evidence from cross-sectional studies that long-term exposure to elevated levels of air pollution during childhood can produce deficits in lung function growth. Whether the deficits in growth result in decreased maximum attained lung function in adulthood is an active area of research. Cross-sectional studies in adults have provided some evidence that air pollution is associated with lung function level. 1 For example, a study in Switzerland reported that NO2, SO2, and PM10 were associated with deficits in FVC and FEV~, but the results for ozone were inconsistent, is

In a follow-up study of the effects of NO 2 on lung function in the same population, average home outdoor and personal exposure within a community showed a deficit in average F V C . 19 The investigators were unable to determine which, if any of the single pollutants accounted for the observed associations. There have been few truly longitudinal studies of the effects of air pollution on lung function decline and the findings have been inconsistent. 2~ In a longitudinal study of subjects living in two regions in Southern California with high and low ozone exposure, three follow-up measurements were conducted between 1977 and 1987. Lung function decline in non-smokers did not vary by chronic exposure to air pollution. This study could not separate the effects of individual pollutants. In this study, acute responses to laboratory ozone exposure were not correlated with individual long-term changes in FEV1, suggesting that acute decrements from ozone exposure may not be related to long-term effects. However, the hyper-responsiveness and acute inflammatory response to ozone is not correlated with acute decrements in FEV 1, and any chronic effects of ozone are likely to involve repeated inflammatory insults. 22 In contrast, a second study of residents of three southern Californian communities with varying levels of air pollutants, long-term exposure to polluted air was associated with decline in FEV1. 21 Again, this study could not identify the effects of individual pollutants. Associations between lung function and 20-year average concentrations of respirable particles, suspended sulfates, SO 2, ozone, and indoor particles were examined in a cohort of 1391 non-smokers. 2~ The number of days that PM10 levels exceeded 100ktg/m 3 and average ozone levels were associated with decrement in FEV 1 in males with a family history of asthma and allergy and mean SO 4 concentration was associated with FEV 1 in all males. 2~ Although effects of mixtures of ambient pollutants on lung function development and decline have been reported, these studies have not clearly identified the constituent or characteristic of the air-pollution mixture that accounts for the associations. The lack of understanding of which pollutants are important and what levels of exposure are safe inhibits rational approaches for control. Although a large number of chemical species occur in ambient air, ozone, NO 2, acid vapors, respirable particulates (PM10 and PM2.5) , SO 2, and acid aerosols have been identified as candidate pollutants for adverse effects on lung function. 1 Evidence for effects of each of these pollutants on lung function growth and decline are reviewed next.

Ozone and lung function development The acute effects of ozone on lung function, airway hyperresponsiveness, and airway inflammation in humans and animal models have led to the hypothesis that living in regions with high levels of ambient ozone is associated with chronic deficits in lung function by reducing growth and speeding lung function decline. 23 Much of the evidence derives from cross-sectional studies of attained lung function and retrospectively assessed lifetime exposure. 24-28

The effects of air pollution on lung function have been studied in a cross-sectional analysis of children and youths aged 6 to 24 years. 25 In these subjects, community ozone level was associated with decrements in FVC and FEV 1. In another study, the effects of ozone exposure on lung function level were analyzed in 130 college students using a residence-based exposure assignment for ozone; 27 a strong relationship was observed between lifetime ambient ozone exposure and mid- and end-expiratory flows. No association with FEV 1 and FVC was found, which is consistent with biologic models of chronic effects of ozone in the small airways. In a study of another group of college students using a similar design, lung function was lower in the group with high ozone exposures. 28'29 Deficits were observed for FEV 1 [-3.1%; 95% confidence interval (CI),-0.2 to-5.9%] and M M E F (-8.1%; C I , - 2 . 3 to-13.9%). However, after considering the effects of PM10 exposure, the authors concluded that living for 4 or more years in regions of the country with high levels of ozone and related co-pollutants was associated with lower lung function, but that the effects were more strongly associated with PM10 levels than ozone levels. 28'29 Cross-sectional analysis of the Children's Health Study has also found an effect of ozone on peak expiratory flow rate (PEFR, r =-0.75, p < 0.005), and an effect of PM2. 5 on M M E F (r=-0.80, p < 0.005). Ozone exposure was associated with a decreased FVC and FEV 1 in girls with asthma, and an association found between peak ozone exposures and lower FVC and FEV 1 in boys spending more time outdoors. 3~ The effects of ozone were larger for exposures earlier in life. The cross-sectional studies suggest that high lifetime ozone exposure is associated with deficits in small airway function. The effects of ozone on children's lung function growth was prospectively investigated by Austrian researchers. 16 Using repeated pulmonary function tests over a three-year period from children in nine Austrian cities, they reported associations between ozone and reduced lung function growth, although the ozone findings may be confounded by contemporaneous exposure to other pollutants. In the Children's Health Study, we did not observe significant effects of ozone on growth of FVC, FEV1, or M M E F among school aged children. However, in the second cohort of fourth graders, we found that ozone was associated with reduced growth of PEF and some evidence for reduced growth in FVC and marginally with FEV 1 (p =0.053) in the more-outdoors group of children. 15 Putting the results of longitudinal and cross-sectional studies together, the evidence is consistent with an age-dependent effect of ozone on growth of small airway function that is largest during preschool ages. N O 2 and lung function development Because NO 2 is a common indoor air pollutant arising from natural gas combustion, the effect of NO 2 on lung function has been examined free from the effects of the mixture of other ambient pollutants. 1 In a prospective study of Dutch children who were followed over a 2-year period with serial

lung function measurements, NO 2 exposure showed a weak, negative association with MMEF, but there was no consistent relationship between growth of lung function and a single measurement of indoor NO2 .31 In early analyses of data from the Six Cities studies, lower levels of FEV 1 and FVC were observed in children living in homes with gas stoves, 32'33 but in subsequent analysis there was no evidence that lung function growth was correlated with gas stove exposure. 34 In a subsample of children from the Six Cities study for whom indoor NO 2 was measured in homes, there was no effect of NO 2 on lung function level. 35 Other studies of the effect of indoor sources of NO 2 on lung function in children have also been inconsistent. 1 The data from these studies, the Children's Health Study and the Swiss Study on Air Pollution and Lung Disease in Adults suggest that NO 2 at ambient levels may not have an independent effect on lung function level or growth; however, ambient NO 2 level may be associated with lung function growth in the context of the other pollutants that occur with ambient NO 2. In this regard, it is uncertain whether NO 2 itself is the active pollutant that interacts with other pollutants, such as ozone, or whether it serves as a surrogate for high levels of fresh emissions from combustion sources such as motor vehicles.

Particulates, acids and lung function development The acute effects of nitric acid vapor on lung function have not been extensively studied. In a clinical study, exposure to 50ppb resulted in modest acute reductions in FEV 1 among children with asthma. 36 In an epidemiologic study of Dutch children, reduced flow rates were associated with same-day exposure to ambient nitrous acid which is in equilibrium with nitric acid. 37 Although cross-sectional associations of PMlo and PM2. 5 mass concentration with lung function have been inconsistent, total suspended particulate level has been associated with decreased lung function growth in children in Poland and PMlo and PM2. 5 levels with lung function in children in Southern California. 1'14'15'17'34'38 The effect of PM may be due, in part, to particle acidity, as particle strong acidity, characterized by sulfur dioxide-derived acidic sulfate particles, has been associated cross-sectionally with lung function level. 26 In the Children's Health Study, we observed a strong association with vapor phase acids and deficits in lung function growth; however, this association was not due to particle acidity as Southern California had low concentrations of SO 2 and acidic sulfate particles during the study period. High ambient concentrations of NO 2 were the primary source of nitric acid vapor in southern California. These findings suggest that the effects of gaseous nitric acid and acid sulfate aerosols on lung function level and growth may be mediated, in part, by the H § produced in the lung; however, the findings in the Children's Health Study suggest that vapor acids may be a surrogate marker for other species that occur in a polluted atmosphere in which vehicle emissions have undergone significant photooxidation.

Other ambient pollutants and lung function development Although motor vehicle exhaust is the primary source of many of the ambient air pollutants associated with adverse effects on lung function, the role of high levels of exposure to freshly emitted motor vehicle exhaust in lung function growth or level is an important unanswered question. There is some evidence supporting an effect of fresh exhaust on lung function based on experimental studies, studies of children who live near highways with heavy traffic volumes, and studies of exposures in tunnels. In a cross-sectional study of 1191 Dutch children living near busy roadways, deficits in lung flow rates were observed in children living within 300 m of a busy roadway. 39 The deficits were larger for traffic counts of trucks, which were powered primarily by diesel, than for automobiles, which were powered primarily by gasoline, and stronger for girls than for boys. 39 In a study of 4320 fourth grade children in Munich, traffic density diminished forced expiratory flow. 4~ In preschool children in Leipzig, exposure to heavy traffic was cross-sectionally associated with lower FVC and FEV1.41 In contrast, a study using repeated cross-sectional surveys of 200 non-smoking women living in each of 3 areas in Tokyo within 20 m of major roads, 20-150 m from the same roads, and in a separate suburban low traffic neighborhood, traffic exposure was not associated with lung function. 42'43 The effects of living near heavily traveled roadways may be related to NO 2 exposure; however, a number of other pollutants that are emitted in exhaust are of interest, including diesel exhaust and ultrafine particles. Diesel exhaust is a traffic-related pollutant that contains high levels of NO 2, fine particles, and organic compounds. Diesel exhaust appears to have acute and chronic effects on lung function. 44 In the Children's Health Study, we have reported that elemental carbon levels, a marker for diesel exhaust, was associated with reduced lung function growth in children. 15 Further work is needed to investigate the components or characteristics of diesel exhaust that affect lung function development and decline. Urban particulate matter consists of three size modes: ultrafine particles of <0.1 ktm diameter, accumulation mode particles (which together form the fine particle mode, <2.5 ~tm diameter) and coarse mode particles between 2.5 and 10 ktm diameter. Ultrafine particles contribute very little to the overall mass of fine particles, but are very high in number especially within 100m of roadways. These particles are of interest because they have high deposition in the distal lung, have large surface areas coated in organic compounds and transition metals, and have the ability to induce oxidative stress and inflammation in the lung. 45 No investigations of the chronic effects of ultrafine particles on lung function growth or decline have been reported. Although ultrafine particles may be important, determining the particle size distribution of fine particles in general may be required. For example, the number of accumulation mode but not ultrafine particles was consistently inversely associated with PEFR and no associations were observed

with large particles or particle m a s s . 46'47 Given the biological effects of ultrafine particles on the lung, studies of the effects on lung function are a high priority.

Susceptibility to ambient pollutants and lung function development Because airway defenses to inhaled oxidants are interacting systems, a number of host and genetic factors may contribute to fetal and children's lung function responses to air pollutants. Asthma and other respiratory conditions appear to be important determinants of lung function growth and decline following exposure to elevated levels of air pollutants. Time-activity patterns and sex may also modify the effects of air pollution on lung function growth and development. Dietary factors may affect lung growth as well as responses to air pollutants. 48-51A growing number of susceptibility genes have been identified as participants in the pathogenesis of persistent lung damage. 52'53Genotypes that result in a higher intensity oxidative stress, inflammatory responses or altered tissue response to damage appear to be associated with increased susceptibility to respiratory effects from acute and chronic exposure to air pollutants. 53-56

AIR P O L L U T I O N A N D A S T H M A OCCURRENCE Asthma is a large and growing threat to children's health and well-being. 57 In some communities, the prevalence of asthma in school-age children exceeds 25% and the prevalence has been rapidly rising in many regions of the developed world. 3~ Although asthma is the subject of intense research efforts, its etiology and the explanation for the increase in prevalence have yet to be firmly established. 57'62-65 The role of air pollutants as etiologic agents and their roles in the asthma epidemic are controversial; however, emerging evidence suggests that the effects of ambient air pollutants on asthma occurrence warrant renewed research attention. Ambient air pollutants are clearly related to wheezing and other asthma-related symptoms across the life course. A wide spectrum of pollutants including ozone, PM10 and PM2. 5, NO 2, and SO 2 has been associated with exacerbation of asthma in children and adults. 1 Although it is accepted that combustion-related air pollution exacerbates asthma, little evidence is available to support a role for ambient air pollutants on prevalence or incidence of new onset asthma and it is generally thought that air pollution does not cause new cases of asthma. The consensus that combustion related air pollution does not cause asthma is supported by a large number of crosssectional studies. 66 For example, a comparison of asthma prevalence between East and West Germany showed a lower rate in East Germany, where pollution from coalburning was much higher. 67 In analyses of asthma prevalence at the beginning of the Children's Health Study, we also found that exposure to air pollution was associated

with exacerbation of chronic symptoms of asthma, 68 but that there was no association between asthma prevalence and any ambient air pollutant measured including ozone, PMlo and PM2.5 o r NO2 .69 The effect of air pollution on exacerbation of asthma, but lack of association with asthma prevalence or incidence is somewhat paradoxical based on our understanding of asthma pathobiology. This apparent paradox may be resolved by consideration of issues of interpretation of findings made from cross-sectional studies about the relationships of air pollution and asthma. A case in point is the relationship between ozone and asthma. In clinical toxicology studies using ambient levels of exposure, acute effects of ozone have been observed on lung function, airway responsiveness and measures of inflammation. 1 Notably, the effects were largely observed in conditions involving moderate exercise. The lung function and inflammatory changes observed in experimental studies are thought to explain the associations of ozone exposure with exacerbations including increased symptoms, respiratory hospitalization and medication use in individuals with asthma. However, the acute effects of ozone at ambient levels are present in children with and without asthma, and the lack of association between ozone and prevalence seen in epidemiologic studies might be explained, in part, by the fact that levels of physical activity were not considered in these studies. The dose of outdoor air pollutants deposited in the lung depends not only on local pollutant concentrations, but also on time-activity patterns including the usual frequency, duration and intensity of physical activity. Timing and location of exercise is also important, as children often exercise outdoors in the afternoon when pollutants such as ozone are at their highest levels. It follows that children who engage in high levels of physical activity and experience the highest doses of air pollutants to the lung are at greatest risk for the adverse effects of ozone. Because the onset of asthma may result in lower levels of physical activity and subsequently lower doses of ozone given a fixed ambient level of ozone, crosssectional studies of asthma prevalence may not provide valid estimates of the relationship between air pollution and asthma occurrence. Prospective studies are needed to investigate the effects of air pollution on asthma occurrence and account for the effects of exercise in high and low pollution environments. Very few prospective studies of the relationship between air pollution and incident asthma have been reported. We investigated the relationship between air pollution, physical activity levels and newly diagnosed asthma among school-aged children residing in 12 Southern California communities. We used participation in team sports to classify usual levels of vigorous physical activity. We examined the association of team sport participation with the subsequent development of asthma during five years of follow-up of 3535 fourth, seventh, and tenth grade children who were asthma-free and participated in the Children's Health Study. Study communities were selected based on high and low ambient ozone exposure, in combination with

varied levels of other pollutants. 69 In the Children's Health Study, the risk of new onset asthma was associated with the highest doses of ozone (Table 24.2). Within communities with high ozone (mean 59.6 ppb), the relative risk (RR) for newly diagnosed asthma among children playing 3 or more sports was 3.3 (95% CI, 1.9-5.8), compared with children playing no sports. There was no effect of physical activity in low ozone communities (mean 40.0ppb) (RR, 0.8; 95% CI, 0.4-1.6). Children who spent more time outside also had a higher incidence of asthma in high ozone communities (RR, 1.4; 95% CI, 1.0-2.1) but not in low ozone communities. Exposure to pollutants other than ozone did not modify the relationship between team sports, ozone, and new onset asthma. The Children's Health Study demonstrates that high levels of physical activity in a high ozone environment are associated with an increased risk for subsequent new onset asthma. The fact that the effect of high energy expenditure sports was larger than that of low expenditure sports, and that the risk in children who spent more time outdoors was higher in high ozone communities, strengthens the inference that ambient levels of exposure to ozone increases the likelihood of development of asthma in children with the largest lung doses. Exercise-induced asthma by itself was unlikely to account for these results, because asthma onset was associated with exercise only in polluted communities. In a second, large prospective study of asthma and air pollution, an increased risk for new onset of asthma was observed among non-smoking adult Seventh Day Adventists in communities with high ozone concentrations. 70 For males, but not females, the risk of new doctor-diagnosed asthma was associated with 20-year mean 8 h average ozone levels ( R R - 2 . 0 9 for a 27 ppb increase in ozone concentration, 95% CI-1.03-4.16). Taken together with the clinical data showing that ozone exposure causes inflammation and airway hyper-responsiveness, the findings from these two large prospective studies indicate that ozone exposure is associated with new onset asthma in groups with higher exposure from being outdoors and engaging in recreational and occupational physical activity.

While ozone appears to be related to new onset asthma in school-aged children and adults, clinical and experimental studies suggest that asthma could be caused by exposure to elevated levels of other pollutants in addition to ozone. High levels of NO 2 and PMlo are candidates based on the enhanced response of asthmatics to bronchial allergen challenge with dust mite allergen after exposure. 71 We found limited evidence for an effect of physical activity on asthma in communities with high levels of NO 2 and PMso; however, it should be noted that the statistical power of the study to identify an independent association of NO 2 and PMxo with the development of newly diagnosed asthma, or to identify such a multi-pollutant interaction between sports, ozone and other pollutants w a s l o w . 72 The current research questions of greatest interest concern the role of diesel exhaust and ultrafine particles on the incidence and prevalence of asthma. In cross-sectional studies, residential or school proximity to heavy traffic has been identified as a risk factor for asthma and for wheezing and other respiratory symptoms in children. 39'73-79 In one of the few experimental studies examining fresh vehicular traffic exhaust exposure, asthmatic response to allergens was reported to be increased in the presence of short-term exposure to air pollution in a road tunnel. Studies with better exposure assessment have been conducted in Holland, where environmental traffic maps have been validated against experimental and field measurements of NO 2 in a model which is calibrated yearly. In one study, children living along busy streets were found to have a higher prevalence of chronic respiratory symptoms and to take more medication for respiratory conditions than children living along quieter streets, 73 and in another study children living within 100 m of freeways were found to have increased rates of self-reported doctor diagnosed asthma. 76 In a British study, children admitted to the hospital for asthma were more likely to live in an area with high traffic flow than children admitted for non-respiratory illness. 75 However, other cross-sectional and case-control studies have concluded that traffic activity in the school locality or near homes is not a major determinant of wheeze or asthma in children.

There is increasing toxicologic and limited epidemiologic evidence supporting a role for ultrafine particles less than 0.1 ktm in diameter in the acute effects of particulates on asthma outcomes. There are few epidemiologic studies that have examined the association specifically between ultrafine particles and asthma outcomes, and the results are not consistent. One such study demonstrated an increase in cough and a decrease in peak expiratory flow among a panel of asthmatic patients as the number of ultrafine particles increased. 8~ The association was stronger than that observed for PM10 or for mass of particles of 0.1-0.5 ktm diameter. However, a study in Finland found that ultrafine particle number was not more strongly associated with variations in PEF than PM10 .81 There are no studies that specifically examine the chronic effect of ultrafine particles on children's health. Such research is needed to evaluate the implications of recent animal toxicological studies for potentially sensitive populations like children and subjects with asthma.

CONCLUSIONS Exposures to environmental toxicants are associated with a substantial burden of adverse respiratory health effects including abnormal lung function development, reduced lung function growth, and more rapid lung function decline over the life course. In the coming decades, as urban development continues and the number of motor vehicle burning fossil fuels increases, populations exposed to elevated levels of a wide variety of ambient air pollutants will grow and the burden of adverse effects on lung function growth and obstructive lung diseases will continue to increase. Because the cost of controlling environmental hazards such as ambient air pollution is often high, defining the full spectrum of adverse effects from ambient pollution and identification of the components or characteristics of air pollution that produce adverse effects is a high priority.

ACKNOWLEDGEMENT This work was supported in part by the National Institute of Environmental Health Science (Grants #SPO1ES11627, 1POLES11627 and #5P30ES07048), the Environmental Protection Agency (Contract #CR82670801), the National Heart, Lung and Blood Institute (Grant 1RO1HL61768) and the Hastings Foundation.

REFERENCES 1. Anonymous. Health effects of outdoor air pollution. Committee of the Environmental and Occupational Health Assembly of the American Thoracic Society. Am. J. Respir. Crit. Care Med. 1996; 153:3-50. 2. Peters J. Epidemiologic investigation to identify chronic health effects of ambient air pollutants in Southern California: Phase II final report. Report prepared for the California

Air Resources Board, Sacramento, CA by the University of Southern California School of Medicine, Department of Preventive Medicine, Los Angeles, CA. 1997. 3. Wiley J. Study of Children's Activity Patterns. Survey Research Center, University of California, Berkley, CA. 1991. 4. Lippmann M. Health effects of ozone: a critical review. J. Air Pollut. Control Assoc. 1989; 39:672-95. 5. Tager IB, Segal MR, Speizer F E e t al. The natural history of forced expiratory volumes. Effect of cigarette smoking and respiratory symptoms.Am. Rev. Respir. Dis. 1988; 138:837-49. 6. Gilliland FD, Berhane K, McConnell R et al. Maternal smoking during pregnancy, environmental tobacco smoke exposure and childhood lung function. Thorax 2000; 55:271-6. 7. Tager IB, Ngo L, Hanrahan JP. Maternal smoking during pregnancy. Effects on lung function during the first 18 months of life.Am. J. Respir. Crit. Care Med. 1995; 152:977-83. 8. U.S. Department of Health and Human Services. The Health Consequences of Involuntary Smoking. Report of the Surgeon General. Public Health Service, Washington, DC. 1986. 9. US Environmental Protection Agency. Respiratory Health Effects of Passive Smoking: Lung Cancer and Other Disorders. Washington, DC. 1992. 10. Samet JM, Lange P. Longitudinal studies of active and passive smoking.Am. J. Respir. Crit. Care Med. 1996; 154:$257-65. 11. Li YF, Gilliland FD, Berhane K et al. Effects of in utero and environmental tobacco smoke exposure on lung function in boys and girls with and without asthma. Am. J. Respir. Crit. Care Med. 2000; 162:2097-104. 12. Ritz B, Yu F. The effect of ambient carbon monoxide on low birth weight among children born in southern California between 1989 and 1993. Environ. Health Perspect. 1999; 107:17-25. 13. Ritz B, Yu F, Chapa G et al. Effect of air pollution on preterm birth among children born in Southern California between 1989 and 1993. Epidemiology 2000; 11:502-11. 14. Gauderman WJ, McConnell R, Gilliland F et al. Association between air pollution and lung function growth in southern California children. Am. J. Respir. Crit. Care Med. 2000; 162:1383-90. 15. Gauderman WJ, Gilliland GF, Vora H etal. Association between air pollution and lung function growth in southern California children: results from a second cohort. Am. J. Respir. Crit. Care Med. 2002; 166:76-84. 16. Frischer T, Studnicka M, Gartner C etal. Lung function growth and ambient ozone: a three-year population study in school children. Am. J. Respir. Crit. Care Med. 1999; 160: 390-96. 17. Jedrychowski W, Flak E, Mroz E. The adverse effect of low levels of ambient air pollutants on lung function growth in preadolescent children. Environ. Health Perspect. 1999; 107:669-74. 18. Ackermann-Liebrich U, Leuenberger P, Schwartz J et al. Lung function and long term exposure to air pollutants in Switzerland. Study on Air Pollution and Lung Diseases in Adults (SAPALDIA) Team. Am. J. Respir. Crit. Care Med. 1997; 155:122-9. 19. Schindler C, Ackermann-Liebrich U, Leuenberger P etal. Associations between lung function and estimated average exposure to NO 2 in eight areas of Switzerland. The SAPALDIA Team. Swiss Study of Air Pollution and Lung Diseases in Adults. Epidemiology 1998; 9:405-11. 20. Abbey DE, Burchette RJ, Knutsen SF etal. Long-term particulate and other air pollutants and lung function in nonsmokers.Am.J. Respir. Crit. Care Med. 1998; 158:289-98. 21. Tashkin D, Detels R, Simmons Met al. The UCLA population studies of chronic obstructive respiratory disease: XI. Impact of air pollution and smoking on annual change in forced expiratory volume in one second. Am. J. Respir. Crit. Care Med. 1994; 149:1209-17.

22. Gong H Jr, Simmons MS, Linn WS eta1. Relationship between acute ozone responsiveness and chronic loss of lung function in residents of a high-ozone community. Arch. Environ. Health 1998; 53:313-19. 23. Lippmann M. Health effects of ozone. A critical review. Japca. 1989; 39:672-95. 24. Ackermann-Liebrich U, Leuenberger P, Schwartz J e t al. Lung function and long term exposure to air pollutants in Switzerland. Study on Air Pollution and Lung Diseases in Adults (SAPALDIA) Team. Am. J. Respir. Crit. Care Med. 1997; 155:122-9. 25. Schwartz J. Lung function and chronic exposure to air pollution: a cross-sectional analysis of NHANES II. Environ. Res. 1989; 50:309-21. 26. Raizenne M, Neas LM, Damokosh AI et al. Health effects of acid aerosols on North American children: pulmonary function. Environ. Health Perspect. 1996; 104:506-14. 27. Kunzli N, Lurmann F, Segal M. Association between lifetime ambient ozone exposure and pulmonary function in college freshmen - results of a pilot study. Environ. Res. 1997; 72:8-23. 28. Galizia A, Kinney PL. Long-term residence in areas of high ozone: associations with respiratory health in a nationwide sample of nonsmoking young adults (see comments). Environ. Health Perspect. 1999; 107:675-9. 29. Kinney PL, Chae E. Diminished lung function in young adults is associated with long-term PM10 exposures. Proc. 14th Con. Int. Soc. Environ. Epidem. 2002:43. 30. Peters JM, Avol E, Gauderman WJ etal. A study of twelve southern California communities with differing levels and types of air pollution. II. Effects on pulmonary function. Am.J. Respir. Crit. Care Med. 1999; 159:768-75. 31. Dijkstra L, Houthuijs D, Brunekreef B etal. Respiratory health effects of the indoor environment in a population of Dutch children. Am. Rev. Respir. Dis. 1990; 142:1172-78. 32. Speizer FE, Ferris B Jr, Bishop YM et al. Respiratory disease rates and pulmonary function in children associated with NO z exposure.Am. Rev. Respir. Dis. 1980; 121:3-10. 33. Ware JH, Dockery DW, Spiro A III et al. Passive smoking, gas cooking, and respiratory health of children living in six cities. Am. Rev. Respir. DIS. 1984; 129:366-74. 34. Berkey CS, Ware JH, Dockery DW et al. Indoor air pollution and pulmonary function growth in preadolescent children. Am.J. Epidemiol. 1986; 123:250-60. 35. Neas LM, Dockery DW, Ware JH et al. Association of indoor nitrogen dioxide with respiratory symptoms and pulmonary function in children.Am. J. Epidemiol. 1991; 134:204-19. 36. Koenig JQ, Covert DS, Pierson WE. Effects of inhalation of acidic compounds on pulmonary function in allergic adolescent subjects. Environ. Health Perspect. 1989; 79:173-8. 37. Hoek G, Brunekreef B, Hofschreuder P et al. Effects of air pollution episodes on pulmonary function and respiratory symptoms. Toxicol. Ind. Health 1990; 6:189-97. 38. Pope CA III. Particulate pollution and health: a review of the Utah valley experience. J. Expo. Anal. Environ. Epidemiol. 1996; 6:23-34. 39. Brunekreef B, Janssen NA, de Hartog J etal. Air pollution from truck traffic and lung function in children living near motorways. Epidemiology 1997; 8:298-303. 40. Wjst M, Reitmeir P, Dold S etal. Road traffic and adverse effects on respiratory health in children. BMJ. 1993; 307:596-600. 41. Fritz GJ, Herbarth O. Pulmonary function and urban air pollution in preschool children. Int. J. Hyg. Environ. Health 2001; 203:235-44. 42. Maeda K, Nitta H, Nakai S. Exposure to nitrogen oxides and other air pollutants from automobiles. Public Health Rev. 1991; 19:61-72.

43. Nakai S, Nitta H, Maeda K. Respiratory health associated with exposure to automobile exhaust. III. Results of a cross-sectional study in 1987, and repeated pulmonary function tests from 1987 to 1990. Arch. Environ. Health 1999; 54:26-33. 44. Sydbom A, Blomberg A, Parnia S et al. Health effects of diesel exhaust emissions. Eur. Respir. J. 2001; 17:733-46. 45. Oberdorster G. Pulmonary effects of inhaled ultrafine particles. Int. Arch. Occup. Environ. Health 2001; 74:1-8. 46. Penttinen P, Timonen KL, Tiittanen P e t al. Number concentration and size of particles in urban air: effects on spirometric lung function in adult asthmatic subjects. Environ. Health Perspect. 2001; 109:319-23. 47. Penttinen P, Timonen KL, Tiittanen P etal. Ultrafine particles in urban air and respiratory health among adult asthmatics. Euro. Respir. J. 2001; 17:428-35. 48. Schunemann HJ, Grant BJ, Freudenheim JL etal. The relation of serum levels of antioxidant vitamins C and E, retinol and carotenoids with pulmonary function in the general population. Am. J. Respir. Crit. Care Med. 2001; 163:1246-55. 49. Schunemann HJ, McCann S, Grant BJ et al. Lung function in relation to intake of carotenoids and other antioxidant vitamins in a population-based study. Am. J. Epidemiol. 2002; 155:463-71. 50. Romieu I, Trenga C. Diet and obstructive lung diseases. Epidemiol. Rev. 2001; 23:268-87. 51. Romieu I, Sienra-Monge JJ, Ramirez-Aguilar M e t a l . Antioxidant Supplementation and Lung Functions among Children with Asthma Exposed to High Levels of Air Pollutants.Am. J. Respir. Crit. Care Med. 2002; 166:703-9. 52. Sandford AJ, Joos L, Pare PD. Genetic risk factors for chronic obstructive pulmonary disease. Curr. Opin. Pulmon. Med. 2002; 8:87-94. 53. He JQ, Ruan J, Connett JE etal. Antioxidant gene polymorphisms and susceptibility to a rapid decline in lung function in smokers. Am. J. Respir. Crit. Care Med. 2002; 166:323-8. 54. Gilliland FD, McConnell R, Peters J e t al. A theoretical basis for investigating ambient air pollution and children's respiratory health. Environ. Health Perspect. 1999; 107 (Suppl. 3):403-7. 55. Arbour NC, Lorenz E, Schutte BC et al. TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nat. Genet. 2000; 25:187-91. 56. Bergamaschi E, De Palma G, Mozzoni P e t al. Polymorphism of quinone-metabolizing enzymes and susceptibility to ozone-induced acute effects. Am. J. Respir. Crit. Care Med. 2001; 163:1426-31. 57. Redd SC. Asthma in the United States: burden and current theories. Environ. Health Perspect. 2002;ll0(Suppl. 4): 557-60. 58. Asher MI, Barry D, Clayton T et al. The burden of symptoms of asthma, allergic rhinoconjunctivities and atopic eczema in children and adolescents in six New Zealand centres: ISAAC Phase One. N. Z. Med. J. 2001; 114:114-20. 59. ISAAC Steering Committee. Worldwide variations in the prevalence of asthma symptoms: the International Study of Asthma and Allergies in Childhood (ISAAC) [see comments]. Eur. Respir. J. 1998; 12:315-35. 60. Mannino DM, Homa DM, Redd SC. Involuntary smoking and asthma severity in children: data from the third national health and nutrition examination survey. Chest 2002; 122:409-15. 61. Pekkanen J, Xu B, Jarvelin MR. Gestational age and occurrence of atopy at age 31 - a prospective birth cohort study in Finland. Clin. Exp. Allergy 2001; 31:95-102. 62. Pearce N, Pekkanen J, Beasley R. How much asthma is really attributable to atopy? Thorax 1999; 54:268-72.

63. Pearce N, Douwes J, Beasley R. The rise and rise of asthma: a new paradigm for the new millennium? J. Epidemiol. Biostat. 2000; 5:5-16. 64. Committee on the Assessment of Asthma and Indoor Air. Clearing the Air: Asthma and Indoor Exposures. Washington, DC: National Academy of Sciences, 2000. 65. Carter S, Platts-Mills T. Searching for the cause of the increase in asthma. Curt. Opin. Pediatr. 1998; 10:594-9. 66. Clark NM, Brown RW, Parker E etal. Childhood Asthma. Environ. Health Perspect. 1999; 107(Suppl. 3):421-9. 67. Wichmann HE, Heinrich J. Health effects of high level exposure to traditional pollutants in East G e r m a n y - review and ongoing research. Environ. Health Perspect. 1995; 103 (Suppl. 2):29-35. 68. McConnell R, Berhane K, Gilliland F et al. Air pollution and bronchitic symptoms in southern California children with asthma. Environ. Health Perspect. 1999; 107:757-60. 69. Peters JM, Avol E, Navidi W e t al. A study of twelve Southern California communities with differing levels and types of air pollution. I. Prevalence of respiratory morbidity. Am. J. Respir. Crit. Care Med. 1999; 159:760-7. 70. McDonnell WF, Abbey DE, Nishino N etal. Long-term ambient ozone concentration and the incidence of asthma in nonsmoking adults: the AHSMOG Study. Environ. Res. 1999; 80:110-21. 71. Jenkins HS, Devalia JL, Mister RL et al. The effect of exposure to ozone and nitrogen dioxide on the airway response of atopic asthmatics to inhaled allergen: dose- and time-dependent effects.Am. J. Respir. Crit. Care Med. 1999; 160:33-9. 72. McConnell R, Berhane K, Gilliland F et al. Asthma in exercising children exposed to ozone: a cohort study. Lancet 2002; 359:386-91.

73. Ciccone G, Forastiere F, Agabiti N etal. Road traffic and adverse respiratory effects in children. Occup. Environ. Med. 1998; 55:771-8. 74. Duhme H, Weiland S, Keil U et al. The association between self-reported symptoms of asthma and allergic rhinitis and self-reported traffic density on street residence in adolescents. Epidemiology 1996; 7:578-82. 75. Edwards J, Waiters S, Griffiths RK. Hospital admissions for asthma in preschool children: relationship to major roads in Birmingham, United Kingdom. Arch. Environ. Health 1994; 49:223-7. 76. van Vliet P, Knape M, de Hartog J et al. Motor vehicle exhaust and chronic respiratory symptoms in children living near freeways. Environ. Res. 1997; 74:122-32. 77. Venn A, Lewis S, Cooper M etal. Local road traffic activity and the prevalence, severity, and persistence of wheeze in school children: combined cross sectional and longitudinal study. Occup. Environ. Med. 2000; 57:152-8. 78. Waldron G, Pottle B, Dod J. Asthma and the motorways - one District's experience.J. Public Health Med. 1995; 17:85-9. 79. Weiland S, Mundt K, Ruckmann A et al. Self-reported wheezing and allergic rhinitis in children and traffic density on street residence.Ann. Epidemiol. 1994; 4:243-7. 80. Peters JM. Epidemiologic investigation to identify chronic health effects of ambient air pollutants in Southern California: Phase II final report. California Air Resources Board Contract #A033-186. University of Southern California, Los Angeles. 1997. 81. Pekkanen J, Timonen KL, Ruuskanen J etal. Effects of ultrafine and fine particles in urban air on peak expiratory flow among children with asthmatic symptoms. Environ. Res. 1997; 74:24-33.

EXPOSURE

TO

TOXICANTS

The respiratory system is a target for a wide range of toxic environmental contaminants. While the acute and chronic effects of a large number of these substances have been well characterized for the respiratory system of adult mammals, there is significantly less known about the impact of these lung-targeted compounds on the developing respiratory system. This chapter summarizes what is currently known about the majority of these compounds, addressing first environmental tobacco smoke, a well-recognized lung toxicant mixture; bioactivated environmental contaminants; oxidant gases; therapeutic glucocorticosteroids; and other miscellaneous compounds which have been shown to alter lung development.

ENVIRONMENTAL

TOBACCO

SMOKE

The health consequences of exposure to environmental tobacco smoke (ETS) among children have been the subject of much public concern (see Chapter 20). Animal studies are very valuable because the indirect effects of in utero ETS exposure can be separated from the direct effects of postnatal ETS exposure. Exclusive in utero exposure to ETS has been shown to accelerate the developmental pattern of Clara cell secretory protein expression in the rat, 1 suggesting a potential acceleration of airway epithelial differentiation in the lung. Whether this accelerated development is maintained after birth is unknown. In utero exposure does not increase cytochrome P450 gene expression unless it is combined with an early postnatal exposure. 2 Exclusive postnatal ETS exposure in rats does not alter Clara cell secretory protein expression. 3 It does, however, *To whom correspondence should be addressed. The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

decrease cell kinetic activity in rat distal airways and increase cytochrome P450 1A1 protein distribution throughout the airways; 3 these changes were maintained for up to 100 days (with ongoing ETS exposure). Acute postnatal exposure to ETS in juvenile ferrets increased the ability of the lungs to metabolize (-)-trans-benzo[a]pyrene-7,8-dihydrodiol. 4 When slightly older rats (weanling age) were exposed to tobacco smoke, emphysematous changes were reported in the lungs. 5 Postnatal exposure to ETS has also been reported to affect the neurophysiologic responses of the lung. Chronic ETS exposure during the period of postnatal lung development in guinea pigs has been shown to increase lung C-fiber sensitivity. 6 In addition, ETS can increase the sensitivity of C-fiber activated neurons in the nucleus tractus solatarius (NTS). 7'8 A combination of in utero and postnatal exposure to ETS appears to have the greatest effect on developing lungs. Rats exposed to both in utero and postnatal ETS have decreased lung compliance, increased reactivity to methacholine and an increase in the number of neuroendocrine ceils per cm of basal lamina. 9 These changes were not seen in rats exposed to ETS in utero only or postnatal only. The increased airway hyper-responsiveness that is set up during postnatal exposure does not resolve even after an extensive period of no exposure to ETS. 1~ In non-human primates, in utero plus postnatal exposure to ETS increases pulmonary adenyl cyclase activity. 11 Whether alterations induced in utero are the result of direct or indirect effects of ETS is unknown.

BIOACTIvATED

COMPOUNDS

The lungs of mammals are selectively injured by many chemically diverse compounds including aromatic Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

hydrocarbons, furans, halogenated ethylenes and indoles. 12 Many of these target airway epithelium, especially Clara cells. In all cases, the metabolic activation of the chemically inert parent compound has been demonstrated to be an important factor in selective lung injury. It is generally assumed that the Clara cell is susceptible due to its high expression of cytochrome P450 monooxygenases. Despite the extensive documentation of the susceptibility of Clara cells to P450-mediated cytotoxicants in the lungs of adults, 13-22 little is known of the susceptibility to these compounds of undifferentiated and developing cells in the neonate. The few studies that are available regarding neonates suggest that lower pulmonary P450 activity is associated with greater susceptibility to P450-activated toxicants. 23-26 In utero exposure to bioactivated compounds produces embryotoxic or teratogenic effects, including chromosomal aberrations. 27-32 The latter appears to be the case for a number of pro-carcinogens which, when given to pregnant mothers, produce Clara cell tumors in adult offspring.31,33,34 The herbicide dichlobenil specifically injures olfactory nasal mucosa in fetal and neonatal mice just as it does in adult mice. 35 The toxicity of dichlobenil increases in neonatal mice with the development of Bowman's glands. In fetal mice, there was more irreversible binding of 14C-dichlobenil in the nasal cavity if the dichlobenil was given to the mother rather than injected directly into the fetus. This suggests that maternal metabolism may be important in the fetus. While the toxicity of dichlobenil increases with development of the target organ, this is not always true for other bioactivated compounds in the developing lung. Neonatal rabbits are much more sensitive than adults to the P450-bioactivated furan 4-ipomeanol; 23 in neonates, distal airway epithelium was injured at doses that did not affect adult airway epithelium at all. This seems to be a contradiction because development of the P450 system is a postnatal event, and in neonatal rabbits, P450 activity is very low. This phenomenon is not restricted to the rabbit. Studies in our laboratory have shown that neonatal mice are also more susceptible than adult mice to the Clara cell cytotoxicant naphthalene. 25 In vitro metabolism studies of neonatal and adult airways show that P450 activity is lower in neonatal mouse lung than it is in adult mouse lung. 26 Gender may also play a role in heightened postnatal sensitivity to pulmonary toxicants. Weanling male and female mice are reported to be more susceptible to 1,1-dichloroethylene-induced pulmonary injury than adult male mice, but not more susceptible than adult females. 36 The exact mechanisms of these increases in sensitivity of postnatal animals have yet to be defined. In some cases, levels of injury positively correlate with specific P450 monooxygenase activity, 36 while in other cases Phase II enzyme activity may be key to increased susceptibility. 37 The mechanisms may also involve as yet undefined factors specific to differentiating cells. In addition to postnatal exposure to bioactivated compounds, in utero exposure may also affect lung development.

In mice, high levels of trichloroethylene exposure on gestational day 17 can cause a decrease in fetal lung weight and total lung phospholipid content, while not changing total DNA content. 3s Benzo[a]pyrene causes lung tumors in offspring of mice treated at days 18 and 19 gestatiom 39 Both males and females have increased incidence of tumors and increased numbers of tumors per animal. When the offspring were followed over 5 generations of inbreeding, the females of the F2 generation had a higher incidence of lung tumors and both males and females of the F2 generation had an increase in the total number of tumors per animal. T h e tumor incidence was not statistically different from controls in the F3 through F5 generations, but the number of tumors per animal remained high.

O X I D A N T GASES In contrast to our understanding of P450-activated lung toxicants, the susceptibility of the lungs of postnatal animals to oxidant gases is much better understood. For the best studied oxidant gas environments (hyperbaric oxygen, ozone and NO2) two fundamental characteristics have been defined. First, in general, postnatal animals prior to weaning are less susceptible to pulmonary injury than are adults. Second, exposure to oxidant gases retards postnatal maturation of the lung. The tolerance of postnatal animals to hyperoxia appears to be species-specific 4~ and is based on differences in (1) the ability of neonatal animals to elevate pulmonary antioxidant defense systems in response to hyperoxic stress; 43-45 (2) the composition of lung polyunsaturated fatty acids; 46 or (3) the presence of antioxidant compounds, including iron chelators. 47 A common factor appears to be the ability to increase the intracellular glutathione pool and to upregulate the enzymes whose antioxidant functions depend on it, including superoxide dismutase, catalase, glutathione peroxidase and glucose 6-phosphate dehydrogenase. 41'42'4s-52 Undernutrition and premature weaning have also been shown to alter susceptibility. 47 ' 53'54 Pharmacologic intervention by administration of steroids (dexamethasone) or endotoxin reduces neonatal susceptibility to hyperoxia but has a mixed effect on antioxidant enzyme activity (endotoxin elevates them, dexamethasone does not). 55'56 Hyperoxia has been shown to delay lung morphogenesis, including alveolarization and vascularization, 4s'57-6~and differentiation of Clara cells in postnatal rats. 6~ Treatment with retinoic acid does not prevent hyperoxia-induced alterations in alveolarization, however, it does result in later improvement in alveolarization. 62'63 Despite the alterations in lung development, neonatal rats have been reported to survive hyperoxia longer than adult rats. This may partially be due to the fact that, in neonatal rats, there is a delay in pulmonary neutrophil influx. 64 Compared to adult rats, neonates have fewer overall lung tissue neutrophils, even though they have higher levels of neutrophils in bronchoalveolar lavage. This suggests that neonatal rats retain fewer neutrophils than adults. 64

When neonatal rats exposed to hyperoxia were treated with antibodies to cytokine-induced neutrophil chemoattractant-1 (CINC-1) to block neutrophil influx, they had increased lung compliance and no change in alveolar volume or surface density compared to control antibody-treated neonates. 65 In addition, blocking neutrophil influx reduces DNA damage in the neonatal lung. 66 The retardation of alveolar development in neonatal lung may also be related to the timing of the hyperoxia and subsequent exposure to leukotrienes 67 or a reported increase in the number of apoptotic cells in the lungs of hyperoxia exposed neonates. 68 For some parameters, neonates appear to be less susceptible to ozone or NO 2 exposure. They have fewer alterations in pulmonary enzymes and markedly reduced cellular injury in the central acinus compared to adults. 69-73 Weaning appears to be the critical time point for changes in responsiveness. Preweaning animals are much less sensitive than postweaning animals. 7~ As in hyperoxia, ozone exposure reduces the postnatal morphogenesis of the gas exchange area, TM impairs bronchiolar formation, 75 and retards the differentiation of the mucociliary apparatus of proximal airways. 76 A potential mechanism for the age-related differences in ozone-induced injury may be the way in which neonates control their ventilation during exposure. Neonatal rats have higher baseline minute ventilation than adults, and during ozone exposure, adult rats reduce their minute ventilation while neonates do not. 77 This indicates that neonatal rats receive a higher delivered dose of ozone than adults and may explain increased indices of acute injury such as increased bronchoalveolar lavage protein and prostaglandin E 2 levels. What can be concluded from these studies with oxidant gases is that the two aspects of postnatal lung development which involve Clara cells, the rate of differentiation of bronchiolar epithelium, and the organization and differentiation of the centriacinus, are impeded by oxidant gas injury. Whether this is true for other classes of pulmonary toxicants such as organic chemicals metabolized by the cytochrome P450 system has not been investigated.

CORTICOSTEROIDS Corticosteroids are commonly used to accelerate pulmonary maturation when preterm labor is imminent. 78 This prevents the respiratory distress syndrome in prematurely born infants 79 and more recently, has been recommended as a treatment for asthma in children under 5 years of age. s~ Although corticosteroids are beneficial in the short term, very little is known about their long-term effects on lung development and growth. The systemic side effects of corticosteroid treatment has recently been reviewed. 81 Most of the information concerning corticosteroids and lung growth are from studies of prenatal exposure. Administration of betamethasone to pregnant animals has been shown to have mixed effects. It results in no changes in lung compliance or lung volume in postnatal term-born lambs, 82 however, in preterm lambs it increases lung function by 50% over

controls 83 and also increases pulmonary antioxidant levels. 84'85 Maternal corticosteroids also decrease the overall number of alveoli, thereby increasing the average alveolar volume and resulting in an emphysematous lung. 83'86 The effects of maternal treatment with glucocorticoids in rats are similar to those found in sheep. Adult rats born to dams treated with dexamethasone during late pregnancy have fewer, larger alveoli. 87-89 Studies in fetal rhesus monkeys suggest that effects on the lung from prenatal corticosteroid treatment are time-dependent and possibly steroid-specific. Prenatal exposure prior to 133 days gestational age (term is 168 days) to betamethasone has been reported to increase the number of alveoli in the lung, but to impair overall lung growth in rhesus monkeys. 9~ Studies in our laboratory have shown that exposure to betamethasone at gestational age 121-127 days (mid-canalicular stage) does not accelerate the maturation of alveolar type II ceils, nor does it alter the morphogenesis of the gas exchange region. 91 However, another corticosteroid, triamacinolone, did induce structural alterations in the fetal lungs of rhesus monkeys when given during the pseudoglandular (63-65 days gestational age) or mid-canalicular (110-112 days gestational age) phase of lung development. 92 Postnatal treatment of mice with dexamethasone has been reported to increase pulmonary gene expression of vascular endothelial growth factor (VEGF), hypoxia-induciblelike factor (HLF) and murine homologue fetal liver kinase (Flk-1) without altering cell specific protein expression. 93 As in the non-human primate, steroid-specific differences have been noted: hydrocortisone and dexamethasone both alter alveolar development in the rat, but the extent of alteration with hydrocortisone is not as great. 94 In addition, postnatal treatment of rats with dexamethasone increases the susceptibility of those rats as adults to experimentally induced pulmonary hypertension. 95

MISCELLANEOUS C O M P O U N D S The developmental effect of nonbioactivated compounds has not been widely studied in postnatal lung. One study has examined the effects of bromodeoxyuridine (BrDU), a thymidine analog used to evaluate DNA synthesis, on alveolar development. 96 Rats were exposed to BrDU at 6 days after birth and then to an excess of thymidine to remove BrDU from the rats. The BrDU incorporates most heavily into alveolar cells. Two weeks after treatment, the lungs appeared normal. Eight weeks after treatment the rats had enlarged airspaces and decreased numbers of alveoli. In another study, 97 male and female mice were exposed to BrDU on the 1st, 3rd and 7th days after birth. There was no increase in tumor development with BrDU exposure alone, but additional exposure to urethane caused significant increases in lung adenoma incidence and an increase in the number of tumors per mouse. Congenital diaphragmatic hernia can be experimentally induced by exposing fetuses to the herbicide nitrofen

(2,4-dicholro-4'-nitrodiphenyl ether). In utero exposure to nitrofen causes functional impairment of the lungs of male rats. 73 At 3 weeks of age, no differences between treated and control rats were detected, but at 6 weeks of age, decreases in tidal volume, vital capacity, total capacity and compliance were reported; in addition, non-homogeneous alveolar ventilation was observed. These effects continued to become more apparent as the rats matured. Although nitrofen is metabolized to mutagenic intermediates in adult rodents, metabolic activation does not appear to play a role in teratogenicity. 98 Distribution studies performed in pregnant rats with labeled nitrofen indicate that while maternal tissues contain nitrofen metabolites, fetal tissue contains only the parent compound. 99 The fetal tissue is actually a sink for nitrofen. Nitrofen may cause congenital diaphragmatic hernia by interfering with thyroid hormone levels or with thyroid hormone receptors. Glucocorticoids can ameliorate some of the abnormalities induced by nitrofen. Dexamethasone has been shown to reverse the nitrofen-induced reduction of thyroid transcription factor gene expression (a marker of lung morphogenesis) and surfactant protein-B (a marker of lung maturity). 1~176176 Nitrofen also induces an increase in the number of pulmonary neuroendocrine cells (PNECs) in the lung, and this number is augmented by treatment with dexamethasone.102 Exposure of mice at 13-16 days gestation to ethylnitrosourea caused an increase in lung tumor incidence. 33'1~176 T u m o r nodules were noticed as early as 7 days after birth and the number of tumors continued to increase until a threshold was reached at 90 days postnatal. The percentage of tumors derived from Clara cells increased up to 60 days after birth, while the diameter of the tumors continued to increase throughout the time of the study (1 year). Other compounds that have been studied for their effect on the lung include ethanol 1~176 and cocaine. 1~ Maternal exposure to ethanol throughout pregnancy decreased the amount of insulin-like growth factor II compared to control-fed mothers. 1~176 The difference was not as great in pair-fed dams. This suggests that a nutritional deficit is also involved. Cocaine increases lung catecholamine and glucocorticoid levels and also fetal hypoxemia. 1~

CONCLUSIONS The impact of lung-targeted toxicants on the developing respiratory system of pre- and postnatal animals is not welldefined. The pattern of lung development itself may play a significant role in modulating the toxic response. Significant portions of lung morphogenesis and cytodifferentiation occur during the postnatal period. The enzyme systems responsible for bioactivation and detoxification differentiate during the perinatal period, with the majority of differentiation activity occurring for an extended period of time after birth. In addition, each enzyme system has a different pattern of differentiation during pre- and postnatal lung development. The risk of injury from an environmental contaminant

which is known to target the lung in adults must be evaluated with two considerations: (1) the toxicant may have its effect by altering morphogenesis and cytodifferentiation, resulting in differential expression or organization of the lung in the adult; (2) the stage of morphogenesis and differentiation of various subcompartments of the lungs are at the time of exposure to a toxicant may significantly affect the severity of the response. It is therefore necessary to separate the effects produced by maternal exposure, either prior to parturition or during the suckling phase of postnatal growth, from the effects of direct exposure on the postnatal animal. Alteration of the differentiated expression of a particular enzyme system or other functional protein by exposure during lung development which does not lead to direct changes in lung function may not indicate that the impact of a particular toxicant is harmful to the respiratory system. While there is extensive literature on the toxic potential of a wide range of environmental contaminants when the adult is exposed, there is a dearth of information on the response of the respiratory system to exposure during development. The majority of studies suggest that: (1) the respiratory system in developing pre- and postnatal animals is more susceptible to injury from lung-directed toxicants than in adults of the same species; (2) the differences in toxic response to respiratory-targeted compounds among species are amplified when responses are evaluated during lung development. Current data in humans suggest that those most at risk of being affected by respiratory-targeted environmental contaminants are fetuses and neonates.

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LUNG DEVELOPMENT AND THE ENVIRONMENT

Lung development is both a pre and a postnatal process. The lung is largely formed during gestation; however, following birth there is a significant period of cellular differentiation and proliferation as the lung continues to grow until adolescence. Before birth, the fetal lungs are largely, but not entirely, protected and buffered from exposure to environmental toxicants (any toxicant circulating in the maternal systemic system has the potential to cross the placenta). After birth the respiratory system is exposed to all the toxic particles, oxidant gases, smoke, dusts and allergens that make up our normal environment in the twenty-first century. It is becoming apparent from epidemiologic and animal studies that the developing lung is especially susceptible to toxic pollutants present in the environment and that, in many cases, it has limited repair potential compared to the adult lung. Lung repair in the growing individual is complicated by the fact that reparative and developmental phases overlap following injury; this may result in alterations in the normal course of lung maturation. Repair after exposure to environmental toxicants has usually been defined only in the adult. This review focuses on what little is known about repair of injury in the normal, full term postnatal lung and compares it to repair in the adult lung.

WHAT

IS REPAIR?

'Repair' is defined as all the processes that are involved in resolving an injury, and includes both cellular and acellular *To whom correspondence should be addressed. The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

components. The temporal sequence of events for the cellular components generally occurs in the following order: injury, loss of injured cells, shape changes in the surviving cells, recruitment and activation of leukocytes, proliferation and migration of uninjured cells, re-differentiation and restoration of the normal cellular population. Within the lung, the temporospatial distribution of these events is important because all parts of the lung do not respond similarly (nor are they equally susceptible to injury). The resolution from injury may result in return to the steady state, so-called 'normal repair', or it may result in a disease state with a defined pathology (i.e. 'abnormal repair'). For the most part, in experimental studies, the definition of repair is based on histologic assessment and is the return to steady state and resolution of injury. Parameters that are often examined to establish the phases of repair include: cell injury, cell loss, cell proliferation and apoptosis, cell recruitment, distribution and abundance of repopulating cells, return of steady state function and differentiation markers.

FACTORS THAT INFLUENCE AND DEVELOPMENT

REPAIR

Both repair and development can be thought of as a complex set of events that are dictated by the epithelial mesenchymal trophic unit. 1-3 Using the conducting airway as an example, the trophic unit is composed of epithelial and mesenchymal cells, extracellular matrix, nerves and inflammatory cells that interact both directly and indirectly (through peptide mediators) to regulate the local repair and developmental responses. The challenge of studying repair in the developing lung is to understand how these multiple factors interact normally so that any abnormalities during repair can be identified. Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

Epidermal growth factor (EGF) and transforming growth factor-beta (TGF-I3) are well-studied examples of the sort of peptide mediators that interact with the epithelial mesenchymal trophic unit during normal repair and development. The EGF receptor (EGF-R) is present at both prenatal and postnatal periods of human lung development and has been found in bronchial epithelium, bronchiolar epithelium, alveoli, and vascular smooth muscle. 4 Ligands of EGF-R include EGF as well as transforming growth factor-alpha (TGF-~). The EGF-R is associated with cellular differentiation during lung development. I n utero exposure of rhesus monkeys to EGF accelerated the biochemical, physiologic, and cellular maturation of the lungs compared to age-matched controls. 5 Addition of EGF to fetal lung explant cultures increased SP-A production. 6 EGF-R is found in airway epithelium following lung damage and during repair in the adult. 7'8 Aerosolized EGF given to sheep after tracheal damage shortens the time course of epithelial repair. 9 Activation of the EGF-R through ligand binding stimulates cell proliferation and cytoskeletal changes related to movement. 8 Both proliferation and cell movement, in the form of migration, are key components of repair as well as lung development. EGF-R and its ligands are associated with lung diseases of infants and children; it is, for example, upregulated and found throughout the epithelium of asthmatics. 2 Furthermore, human fetuses (26 to 34 weeks) and infants (6 weeks to 7 months) with advanced bronchopulmonary dysplasia (BPD) were found to have elevated levels of EGF in bronchiolar epithelium. 1~In contrast, EGF, TGF-a, and EGF-R protein have been found in bronchiolar and bronchial epithelium of normal and BPD lung. 4 However, premature infants with BPD had significantly more alveolar macrophages, and these macrophages co-localized EGF, TGF-a and EGF-R. Abnormalities with EGF-R activation and subsequent signal transduction have also been linked with diseases of the developing lung. In asthma, EGF-R is upregulated but epithelial proliferation does not correlate with the level of upregulation. 2 Benzo[a]pyrene, a constituent of cigarette smoke, was found to significantly decrease EGF binding to cells taken from early gestational period human placenta. 11 This may be a contributing factor to the association between in utero exposure to cigarette smoke and fetal growth retardation and decreases in postnatal lung function. Increased activation of the EGF-R has been implicated in the development of pulmonary fibrosis and alterations of lung morphogenesis. A transgenic mouse model overexpressing a TGF-c~ construct had pulmonary fibrosis and enlarged alveoli within the first month of life. 12 TGF-I3 is another important growth factor that interacts with the epithelial mesenchymal trophic unit in lung development and repair. TGF-13 is generally associated with growth arrest and cellular differentiation. 6'13 During lung development, TGF-I3 type I and II receptors are temporospatially expressed. 14'15 TGF-13 regulates the production

of extracellular matrix proteins such as collagen, 16'17 and is believed to be instrumental in causing fibrosis; TM it is also found in the lavage fluid of infants with BPD. 13Prior to the development of fibrotic alveolar septa, in infants suffering BPD, the lavage fluid contains high numbers ofalveolar macrophages with TGF-I3 protein, compared to age-matched controls. When fibrotic alveolar septa become recognizable, TGF-13 levels have decreased to the level of controls, 19 perhaps indicating a role for TGF-13 in the formation of fibrotic alveolar septa. TGF-I3 is elevated in neonatal rats subjected to hyperoxia and normal alveolarization is stunted. 13 TGF-13 also regulates fibronectin, 2~ an extracellular matrix protein instrumental in morphogenesis, cellular migration, wound healing, and cellular differentiation. 21 Lavage levels of fibronectin are elevated in infants with BPD. 22 Pathology studies have found that TGF-13 protein and mRNA levels mirror the development of fibrosis in this disease. 23 The interactions of mediators such as EGF and TGF-~ with the epithelial mesenchymal trophic unit are not the only important factors to affect the repair response; many other factors influence repair. The type of injurious substance and the developmental stage of the epithelial mesenchymal trophic unit are important. Most toxicants target particular cell types. For example, naphthalene, a component of cigarette smoke and diesel exhaust, preferentially injures the Clara (nonciliated bronchiolar) cell. 24 This is likely due to the Clara cell having high levels of cytochrome P450 monooxygenase enzymes which metabolize naphthalene to its toxic form. 25'26 Epithelial maturation in the lung is temporospatially controlled; the many cell phenotypes differentiate at different rates and maturation occurs along the complex branching airway tree in a proximal to distal direction. Thus, as is shown in the examples below, the extent of repair during development can be related to the cell type that is injured, the location of the cell type, and its stage of maturation. Confounding this already complex picture is the effect of immune cells, which are key for proper resolution of most types of cellular injury. Inflammatory cells themselves undergo major changes as the developing lung is exposed to the environment. Immune system maturation at the time of injury is likely a key component of long-term responses and even airway remodeling, yet very little is known about even the normal maturational processes of immune cells as well as other lung cells. Lastly, the gender and genetic background of the exposed individual, as well as the history of exposure (acute, repeated, chronic or episodic) to the injurious agent, also influence repair. As can be seen, several factors may influence both susceptibility and the capability of the lung to properly repair injury. The alterations seen in repair of the developing lung following exposure to environmental toxicants may be due to a temporary or permanent disruption of the epithelial mesenchymal trophic unit resulting in either an inability to heal the initial wound or disruption of critical morphogenic events.

T O X I C I T Y / R E P A I R STUDIES IN THE MATURE A N D D E V E L O P I N G L U N G The following is a discussion of repair in the mature and developing lung after injury by environmental toxicants. More studies have been done with the mature lung because defining repair is straightforward in the absence of changes due to development. Studies on toxicity and repair fall into two general categories: acute and chronic. Acute exposure is rarer in real life, but repair is easy to study because the lesion usually resolves. Chronic exposure is truer to life but more complicated to study due to the overlapping events of injury and repair associated with a continued exposure.

P450-MEDIATED TOXICITY Cytochrome P450 monooxygenases metabolize a number of environmental chemicals such as aromatic hydrocarbons, 27'28 chlorinated ethylenes 29 and furans. 3~ In the lung, cytochrome P450 is found predominantly in the Clara cell, a nonciliated epithelial cell that lines the conducting airways. 32 Clara cells have the highest levels of P450 of any lung cell 33 and therefore are often the primary injury target of metabolically activated cytotoxicants. 33 There are several studies in the adult that examine airway morphology after acute exposures to P450-activated Clara cell toxicants such as 1,1-dichloroethylene,34'35trichloroethylene, 36 4-ipomeanol,37 bromobenzene 38 and naphthalene. 39-41Of these toxicants, the two best studied in both adults and neonates are naphthalene and 1,1-dichloroethylene; for simplicity, naphthalene is described here. In both the mature and the developing lung, a single acute exposure to Clara cell toxicants results in a characteristic lesion, recovery from which is shown in Fig. 26.1. Injury is characterized by vacuolization and exfoliation of nonciliated bronchiolar epithelial (Clara) cells. Cell loss is focal, affecting terminal bronchioles at the lowest doses and larger airways with increasing dose. At very high doses, the entire epithelial population of an airway generation may exfoliate, and the basement membrane may be exposed. In the mature lung, as injured Clara cells are lost, the ciliated cells squamate to cover the basement membrane, 41'42 and they lose their cilia in this process (Fig. 26.1). 38,42 The repair process does not include pronounced recruitment of inflammatory ceils, although this has not been well characterized. In the mature lung, squamation of surviving cells is followed by proliferation in several sites: the injured region, 41 in more proximal, non-injured regions of airway epithelium, 41'43'44 and in the interstitium immediately adjacent to the site of injury. 41 The proliferating cell phenotypes include surviving Clara cells in proximal airways, 43 undefined cells in the injury zone which may be ciliated cells 38'41'42 and cells at bifurcations which have been described variously as 'pollutant resistant' Clara ceils and pulmonary neuroendocrine cells. 44--46The proliferative phase is followed by a period of re-differentiation of both Clara cells

and ciliated cells as the markers and morphology of differentiated Clara and ciliated cells appear. 41'43 Proliferation and re-differentiation overlap a period of cell migration when cells from uniniured proximal airways may push down into the iniured airways to assist with repopulation of the epithelium. 41'42 Support for this concept comes from the widespread cell proliferation that occurs in adiacent, non-iniured, airway generations and the fact that iniured regions regenerate an undifferentiated epithelium very quickly. 41 Repair is usually complete in two to three weeks. 7'41 The repair pattern in the developing lung is different compared to that in the mature lung (Fig. 26.2). After iniury, surviving cells squamate and cover the basement membrane. However, one or more of the subsequent steps of proliferation, migration, and redifferentiation, which in the adult resolve iniury is altered or fails to occur. For example, when six-day-old mice were given a single dose of naphthalene, 35 days post-treatment the distal bronchioles were found to have an abnormal population of squamous cells and areas of hyperplasia. 47 At 22 weeks post-treatment, the bronchiolar epithelium of the adult mice was normal. However, nodules with disorganized nuclei were present at alveolar-duct iunctions. 48 The extent of lung repair in rabbits was found to depend upon the stage of Clara cell differentiation at which the iniury took place, even when iniury was determined to be equivalent at all stages. 49 Rabbit kits treated with 4-ipomeanol at early stages of Clara cell differentiation (3- to 5-days-old) or at intermediate stages (7- to 9-days-old) had abnormal populations of simple squamous and irregularly shaped cuboidal cells in the bronchioles at four weeks of age. In contrast, animals iniured during late stages of Clara cell differentiation (21-days-old) had bronchiolar epithelium similar to age-matched controls at four weeks of age. At 3 months post-treatment, animals treated with 4-ipomeanol at early stages of Clara cell differentiation still had squamous cells populating the terminal bronchioles. One mechanism for age-related differences in repair following Clara cell iniury may be inhibition of proliferation. Proliferating nuclear cell antigen (PCNA) was used to evaluate cell proliferation in rabbits during the first week of repair. Animals treated at intermediate stages of Clara cell differentiation (day 7) had significantly decreased cell labeling in distal bronchioles for the first four days of repair compared to animals treated at late stages of Clara cell differentiation (day 21 ).50 During lung development, Clara cells serve as progenitor cells for themselves and ciliated cells 51 and differentiate postnatally in a number of species, including primates. 52-55 The acute studies mentioned here were conducted when the Clara cell was differentiating postnatally. The epithelial abnormalities found in the mouse and rabbit following treatment with cytochrome P450-mediated cytotoxicants during the neonatal period may be attributed to progenitor Clara cells being reduced in number by iniury and compromising the normal development of the epithelial population.

Fig. 26.1. Scanning electron micrographs of the phases of epithelial repair after exposure to naphthalene to cause Clara cell injury. Terminal bronchiolar epithelium of normal mice (A) and mice 1 day (B and C), 2 days (D), 7 days (E) and 14 days (F) after exposure. (A) At steady state, the epithelium has a regular arrangement of Clara and ciliated cells. (B) Some injured and exfoliating Clara cells are still present at 1 day after naphthalene treatment. (C) The remaining ciliated cells squamate. (D) As cells enter the active proliferation phase (2 days after naphthalene treatment) they lose markers of cell surface differentiation, and ciliated cells engulf their cilia (arrowhead). (E) Bronchiolar epithelium is still in an active re-differentiation phase with both differentiated and undifferentiated cells present 7 days after injury. (F) By 14 days, the epithelium is approaching steady state levels of Clara and ciliated cell abundance. CC, Clara cell; Ci, ciliated cell. Bar in F = 20 lam.

Many factors regulate the repair process following injury by P450-mediated toxicants, including various soluble cytokines and growth factors, 7 interactions with integrins and matrix elements 56'57 as well as cell-cell interactions. 58 The above mentioned studies demonstrate that when the developing lung does not repair, abnormalities may persist into adulthood.

Ozone Ozone is one of the most abundant and widespread environmental pollutants with 92.5 million Americans living in areas that exceed the national ozone standard of 0.12 ppm in 1998. 59 There are many studies of acute and chronic ozone exposures in the adult because of the large number of people exposed and the deleterious effects of ozone exposure on respiratory health, especially in sensitive groups.

Studies of the effects of acute ozone exposure primarily involve an ozone exposure that lasts one day or less, and the lesion involves trachea, bronchi, distal airways and the gas exchange region. The cell types targeted are primarily the ciliated and alveolar type I cell, although basal cells are also injured in larger airways. 6~ Acute ozone toxicity generates a reversible lesion without a defined long-term pathology but involving a substantial inflammatory cell response. Repair following acute injury is characterized by both proliferation and ciliary regeneration detectable in the surviving cells 24 h after exposure. 6~ The repair response has a different temporal pattern and sequence of events that varies by airway level. 6~ Recruitment of neutrophils was found to be key to the resolution of injury. When neutrophil migration was blocked using a CD18 monoclonal antibody, there was an increase in necrotic epithelial cells and exposed basement

STEADYSTATE DIFFERENTIATION~

~

~ ( CLARA INJURCYELL

.! CILIATEDCELL SQUAMATION PROLIFERATION Fig. 26.2. Altered repair cycle in the developing lung following P450-mediated injury of the bronchiolar epithelium. Failure to return to the steady state may result when a repair stage(s) is blocked (represented by X) after injury. Abnormal bronchiolar epithelium results and persists into adulthood.

membrane in ozone exposed animals. 61 Blocking neutrophil recruitment also inhibited cell proliferation 8 h after cessation of ozone exposure. 62 Proper recruitment of inflammatory cells appears to be key to resolution of acute ozone injury, particularly the removal of injured cells and early onset of cell proliferation. Cell proliferation following ozone injury was found to peak 24-48 h after cessation of a 6-h ozone exposure (0.8 ppm), 63 and the proliferation was found in bronchiolar nonciliated and alveolar type 2 cells. Re-differentiation of epithelium after ozone injury using cell differentiation markers has been little studied. Most chronic studies of ozone exposure in adults have examined endpoints obtained after long-term exposure that typically ranges from 3 to 90 days or longer. The literature is especially complicated by the fact that ozone effects have been studied in many species as well as with a variety of experimental designs that vary in timing, dose and endpoints. The overall response to chronic ozone exposure is characterized by a large influx of inflammatory cells into the lung. This influx is predominately neutrophils in the early phase while elevated macrophages persist throughout exposure. Depending upon the length of the study, changes include: the formation and resolution of intraluminal exudate containing injured cells and serum proteins, epithelial proliferation, steady increases in interstitial collagen and fibroblasts, hyperplasia of bronchiolar epithelium, elevated macrophages in lavage, and increased numbers of mast and smooth muscle cells. 64'65 Species with respiratory bronchioles, such as primates, are especially affected by long-term ozone exposure and show remodeling of the respiratory bronchiole including a thicker airway wall, narrower airway lumen and increased epithelial cell mass. 66 Mucous cell metaplasia is found in the nasal epithelium. 67'68 Thus, while the ozone exposure continues, repair back to the steady state does not occur. Repeated chronic ozone exposure results in the lung becoming tolerant to acute ozone injury. One study of a 60-day exposure in rats to 0.96ppm ozone quantitatively

evaluated repair in the trachea at 0, 7 and 42 days after exposure and found little change at 0 and 7 days with the exception of increased abundance of cells with shortened cilia. 69 By 42 days after exposure, tracheal epithelium was indistinguishable from controls. Compared to the adult, little is known about repair in children following exposure to ozone. Both short-term 7~ and long-term 72 epidemiologic studies show a relationship between ozone exposure and decrease in pulmonary function in children such as lower FEV 1 and FVC. Data suggest that ozone exposure changes the development of the respiratory system, particularly epithelial development. Children living where ozone concentrations are chronically elevated have been found to have abnormal nasal epithelium 73 with DNA damage 74 and increased p53 staining. 75 Infant rhesus monkeys exposed episodically to ozone plus allergen have an altered mucous cell population; mucous cell composition as well as mucous cell location in distal bronchioles was changed compared to controls. 76 Lambs exposed to ozone for 5 days during the first week of life following birth have an altered development of the mucociliary apparatus. 77 Total epithelial cell density is decreased, and the epithelial population has an increased density of mucous cells. In addition, ozone exposure alters the carbohydrate composition of the mucus produced. 78 In order to understand how ozone affects repair and development of the lungs in children, more research is needed to define the sequence of events during acute and chronic exposure to ozone, especially focusing on inflammatory cells. Cigarette smoke The injury and repair response in adults and children in response to tobacco smoke exposure is largely undefined. Exposures to tobacco smoke are either to the mainstream smoke that is taken in from the cigarette by the smoker or to sidestream smoke, which is composed of the smoke that

is emitted from a lit cigarette between puffs and that which is exhaled by the smoker. Very young children are primarily exposed to sidestream smoke, which is the principal component of environmental tobacco smoke (ETS). A recent analysis of tobacco smoke products in the urine of elementary school students found levels of cotinine > 5 ng/ml in 35% of the samples, indicating significant exposures even in this very young age group. 79 In adults, ETS exposure is associated with lung cancer, s~ wheezing, and cough, s2 Children exposed to tobacco smoke have more coughing, wheezing, airway reactivity, respiratory illness, and decreases in lung function compared to children not exposed to tobacco smoke, s3-s7 Data suggest that decreases in lung function are especially prevalent and long lasting in children exposed in utero or in early childhood, sS'ss's9 but the mechanism by which tobacco smoke exposure impairs repair and leads to these disease states remains undefined. Animal studies have been undertaken to pinpoint when and what changes occur following exposure to environmental tobacco smoke that compromise lung health (see Chapter 20). In the adult, ETS has little effect on the lungs of rodents, although it is a known carcinogen for humans. Tumors and defined pathology in adult animals have been very difficult to produce with the exception of some strains of mice, which are susceptible to tumors. 9~ However, ETS has been shown to impair bronchiolar epithelial repair in adult mice challenged with naphthalene. 9~ In this model, a week of prior exposure to ETS impairs bronchiolar repair; some cells in the terminal bronchiole remain in an undifferentiated state (lacking cilia and Clara cell characteristics including secretory granules) for 3 weeks. It is not known if these squamated cells will resolve over time, thereby returning the epithelium to steady state. This in vivo finding in mice is supported by recent in vitro work that shows both cigarette smoke condensate and the volatile components of tobacco smoke (acrolein and acetaldehyde) impair cellular functions important for repair such as chemotaxis in human epithelial cells in culture. 92 If squamated cells similarly persist in the lungs of humans exposed to ETS and then exposed to other air pollutants, this could lead to decreased clearance and increased cough. Rat studies have shown that the timing of exposure to sidestream smoke is key to the development of airway hyperreactivity. When rats were exposed chronically to sidestream smoke either just before or after birth, pulmonary function and airway reactivity were not altered. 93'94However, when rats were exposed both pre- and postnatally, lungs were less compliant, and the airways were more reactive compared to controls. 94 Moreover, if the rats were given a 5-week respite from exposure to sidestream smoke, airway hyper-responsiveness was retained. The responsiveness persisted into adulthood. 95 In the rat, a combination of pre and postnatal exposure overrides the lung's ability to repair changes that occur from exposure to sidestream smoke. Chronic exposure to sidestream smoke during lung development also alters normal cellular maturation. When rats were exposed postnatally to indoor levels of sidestream

smoke for the first 100 days of life, the maturation of bronchiolar epithelium was accelerated. 96 Epithelial differentiation normally occurs in a proximal to distal direction along the airway tree and indications of a mature epithelium include decreased cell proliferation as well as adult levels of epithelial enzymes such as cytochrome P450 monooxygenase. Exposed rats had significantly decreased cell labeling in terminal bronchioles compared to control animals during the first two weeks of life. Furthermore, expression of the cytochrome P450 isozyme 1A1 was significantly increased from 7 to 100 days of postnatal life in bronchioles and bronchi. The decline in cell labeling and the increased expression of 1A1 indicate an earlier maturation of the epithelium than is normal. This same trend occurs if rats are exposed prenatally to sidestream smoke. Beginning on gestational day 5, pregnant rats were exposed to sidestream smoke for 6h each day; pups were then killed on gestational days 14, 18 and 21. Clara cell secretory protein and mRNA in bronchiolar epithelium were significantly increased above the control group by gestational day 21 ;97 this protein regulates pulmonary inflammation and its expression increases with Clara cell maturity. The pulmonary morbidity experienced by children exposed to tobacco smoke is probably a result of changes, such as those mentioned above, that occur when repair and maturation of the pulmonary system occur simultaneously.

UNANSWERED QUESTIONS REGARDING

REPAIR F R O M

ENVIRONMENTAL LUNG INJURY IN C H I L D R E N There are many other environmental toxicants that adversely affect the adult lung (and potentially the developing lung) besides those discussed above; they include particulates, nitrogen dioxide, sulfur dioxide, endotoxin, and viruses. However, little is known about lung repair in the infant or child following exposure to these toxicants. There is now a real need to understand the repair process in children because often children do not repair like adults, and perturbations in normal lung development may persist into adulthood. For some exposures, the developing lung is more susceptible to injury than the adult lung. Even doses that produce equivalent injury in both adults and infants can result in failure of repair in the infant, particularly when the exposure occurs at certain times. One hypothesis regarding both injury and repair due to environmental exposures is that the period of postnatal lung morphogenesis contains one or more 'windows of susceptibility' during which the lung is more susceptible to either injury or failure of repair or both. For example, when a single dose of monocrotaline was given to 3 day (neonatal), 8 day (infant) and 8 week old (adult) rats a 'window of susceptibility' was found. Two weeks post-treatment, all groups suffered equivalent pulmonary vascular changes. However, the neonatal group had less than one-third the number of alveoli of

controls and did not survive to three weeks of age. There was no significant decrease in alveolar number or deaths in the infant and adult groups. In the rat, alveolar multiplication occurs between postnatal days 3 and 8. Neonatal deaths were attributed to monocrotaline inhibiting the development of the alveoli (both alveolar number and blood supply were reduced). In contrast, older animals, beyond the alveolar multiplication stage, were able to compensate. 9s 'Windows of developmental susceptibility' are still largely undefined. Key questions to answer are: (1) What is the spatial and temporal distribution of these "windows of susceptibility" during postnatal lung growth and maturation? and (2) What are the intervention strategies that can minimize susceptibility at these time points (or enhance repair)? While most studies of lung repair are based on histopathology or related molecular measurements, many epidemiologic studies that establish potential links between environmental exposures in children and lung disease are based on altered lung function in the adult. There is a need for studies that bridge the gap between the relatively shortterm pathologic changes observed in experimental animals and the possible downstream effects of lung injury observed in older children and adults with lung disease, especially altered lung function. Long-term studies that integrate standard histopathology and lung function testing with temporal sampling are especially needed to answer the question: Which effects on repair are transient and reversible and which are not reversible and have long-term detrimental consequences? While epidemiologic studies have clearly linked exposure to both indoor and outdoor environmental pollution with lung disease, the nature of the exposure needed to cause these effects is still largely unknown especially in children. Dose, duration and timing of exposure are important issues as well as the effects of co-exposures to mixtures of pollutants on repair processes. The question that needs to be answered experimentally is: Which environmental exposures in children are most likely to lead to, or exacerbate, adult lung diseases such as asthma? Since many of these lung diseases have a genetic component, a related question is: Which genes influence repair and make children more susceptible to environmental pollution as well as lung diseases? The multiplicity of events involved in lung growth and differentiation provide numerous targets for environmental exposures to disrupt the normal maturational process; this can present as abnormal repair. While the lack of repair may be attributable to the presence of some factor that is present only at critical points during growth and differentiation, it is just as likely that failure of repair following environmental exposures in children is linked to disruption of growth processes. This failure can only clearly be attributed to the factors involved in postnatal maturation when the same repair process has been studied and has been shown to occur successfully in the adult. Studies are currently needed to examine the repair process in both adults and children, a key question being: How do similar exposures in children and adults result in disparate effects?

CONCLUSIONS The lung is composed of a series of microenvironments both within the dichotomous branching structures of the conducting airways and in the gas exchange regions, the parenchyma and the pleura. These microenvironments are populated by different cell populations that have separate and distinct potentials to repair environmental injury. The injury target site and cell type within the lung, as well as the extent of the injury are important determinants of the temporospatial pattern of repair. Certain cell types are easier to replace than others. A large injury that encompasses much of the lung is more likely to have a prolonged repair response. A concern when doing any sort of study that examines repair in the lung is to clearly define at the outset the pattern of toxicity that leads to that repair response as studies cannot be adequately interpreted and compared unless these factors are known. Important questions that still need to be addressed relate to whether certain cell types and lung regions are more susceptible to failure of repair following lung injury. Furthermore, what are the key timepoints in postnatal development that these cells and regions are susceptible, and how important is the extent of injury in relation to repair potential? Clearly, there are many unanswered questions regarding repair of environmental lung injury in children. To answer key questions about repair will require a more detailed knowledge of the effects of postnatal growth and differentiation on the lung's repair responses. While there have been a number of insightful and exciting studies, they are dwarfed by the large quantity of work that remains to be done to further elucidate the origins of lung disease.

ACKNOWLEDGEMENTS Supported in part by NIH grants ES00628, ES06700, ES05707 and ES04311 as well as the University of California's Tobacco-Related Diseases Research Program (grants 11RT-0258, 6KT-0306 and 121T-0191).

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cells capable of epithelial regeneration. Am. J. Pathol. 2000; 156:269-78. 45. Peake JL, Reynolds SD, Stripp BR etal. Alteration of pulmonary neuroendocrine cells during epithelial repair of naphthalene-induced airway injury. Am. J. Pathol. 2000; 156:279-86. 46. Hong KU, Reynolds SD, Giangreco A et al. Clara cell secretory protein-expressing cells of the airway neuroepithelial body microenvironment include a label-retaining subset and are critical for epithelial renewal after progenitor cell depletion. Am. J. Respir. Cell Mol. Biol. 2001; 24:671-81. 47. Voit M, Buckpitt A, Plopper C. Naphthalene injury to Clara cells during early postnatal lung development alters the maturation of conducting airway epithelial cells. Amer. J. Respir. Crit. Care Med. 1996; 153:A555. 48. Fanucchi MV, Murphy ME, Plopper CG. Long-term effects of acute naphthalene injury during postnatal development. Am.J. Respir. Crit. Care Med. 1997; 155:A45. 49. Smiley-Jewell SM, Liu FJ, Weir AJ et al. Acute injury to differentiating Clara cells in neonatal rabbits results in age-related failure of bronchiolar epithelial repair. Toxicol. Pathol. 2000; 28:267-76. 50. Smiley-Jewell SM, Liu FJ, Plopper CG. Age-related differences in cell proliferation and repair following neonatal lung injury in rabbits. Am. J. Respir. Crit. Care Med. 1998; 159:A553. 51. Plopper CG, Nishio SJ, Alley JL et al. The role of the nonciliated bronchiolar epithelial (Clara) cell as the progenitor cell during bronchiolar epithelial differentiation in the perinatal rabbit lung.Am. J. Respir. Cell Mol. Biol. 1992; 7:606-13. 52. Massaro GD, Davis L, Massaro D. Postnatal development of the bronchiolar Clara cell in rats. Am. J. Physiol. 1984; 247:C197-203. 53. Plopper CG, Alley JL, Serabjitsingh CJ et al. Cytodifferentiation of the nonciliated bronchiolar epithelial (Clara) cell during rabbit lung maturation: an ultrastructural and morphometric study.Am. J. Anat. 1983; 167:329-57. 54. Ten Have-Opbroek, AA, De Vries EC. Clara cell differentiation in the mouse: ultrastructural morphology and cytochemistry for surfactant protein A and Clara cell 10 kD protein. Microsc. Res. Tech. 1993; 26:400-11. 55. Tyler NK, Hyde DM, Hendrickx AG et al. Cytodifferentiation of two epithelial populations of the respiratory bronchiole during fetal lung development in the rhesus monkey. Anat. Rec. 1989; 225:297-309. 56. Lawson G, Parks WC, Van Winkle L et al. Distribution of matrilysin in acute bronchiolar injury/repair. Am. Rev. Respir. Crit. Care Med. 1998; 157:A267. 57. Lawson G, Sheppard D, Van Winkle L etal. Bronchiolar epithelial repair in beta 6 knockout mice following Clara cell specific injury by naphthalene. Am. Rev. Respir. Crit. Care Med. 1998; 157:A553. 58. Helton C, Murphy ME, Van Winkle L e t al. Measurement of gap juntion intracellular communication (GHC) in situ by dye coupling in tracheal epithelium. Am. J. Respir. Crit. Care Med. 1997; 155:A957. 59. EPA Air Quality and Emission Trends Report, 1998. United States Environmental Protection Agency, 2000, pp. 67. 60. Hyde DM, Hubbard WC, Wong V. Ozone-induced acute tracheobronchial epithelial injury: relationship to granulocyte emigration in the lung. Am. J. Respir. Cell Mol. Biol. 1992; 6:481-97. 61. Hyde DM, Miller LA, McDonald RJ etal. Neutrophils enhance clearance of necrotic epithelial cells in ozoneinduced lung injury in rhesus monkeys. Am. J. Physiol. 1999; 277:L1190-8. 62. Vesely KR, Schelegle ES, StovaU MY et al. Breathing pattern response and epithelial labeling in ozone-induced airway

injury in neutrophil-depleted rats. Am. J. Respir, Cell Mol. Biol. 1999; 20:699-709. 63. Vincent R, Adamson IY. Cellular kinetics in the lungs of aging Fischer 344 rats after acute exposure to ozone. Am. J. Pathol. 1995; 146:1008-16. 64. Dungworth D. Noncarcinogenic responses of the respiratory tract to inhaled toxicants. In: McClellan R, Henderson R (eds) Noncarcinogenic Responses of the Respiratory Tract to Inhaled Toxicants. Hemisphere Publishing, New York, 1989, pp. 273-98. 65. Paige R, Plopper C. Acute and chronic effects of ozone in animal models. In: Hogate S, Samet J, Koren H, Maynard R (eds) Acute and Chronic Effects of Ozone in Animal Models. Academic Press, San Diego, 1999, pp. 531-58. 66. Moffatt RK, Hyde DM, Plopper CG etal. Ozone-induced adaptive and reactive cellular changes in respiratory bronchioles of bonnet monkeys. Exp. Lung Res. 1987; 12:57-74. 67. Harkema JR, Plopper CG, Hyde DM etal. Effects of an ambient level of ozone on primate nasal epithelial mucosubstances. Quantitative histochemistry. Am. J. Pathol. 1987; 127:90-6. 68. Harkema JR, Barr EB, Hotchkiss JA. Responses of rat nasal epithelium to short- and long-term exposures of ozone: image analysis of epithelial injury, adaptation and repair. Microsc. Res. Tech. 1997; 36:276-86. 69. Nikula KJ, Wilson DW, Girl SN et al. The response of the rat tracheal epithelium to ozone exposure. Injury, adaptation, and repair.Am. J. Pathol. 1988; 131:373-84. 70. Linn WS, Shamoo DA, Anderson KR etal. Short-term air pollution exposures and responses in Los Angeles area schoolchildren. J. Expo. Anal. Environ. Epidemiol. 1996; 6:449-72. 71. Kopp MV, Bohnet W, Frischer T etal. Effects of ambient ozone on lung function in children over a two-summer period. Eur. Respir. J. 2000; 16:893-900. 72. Peters JM, Avol E, Gauderman WJ etal. A study of twelve Southern California communities with differing levels and types of air pollution. II. Effects on pulmonary function. Am. J. Respir. Crit. Care Med. 1999; 159:768-75. 73. Calderon-Garciduenas L, Valencia-Salazar G, RodriguezAlcaraz A et al. Ultrastructural nasal pathology in children chronically and sequentially exposed to air pollutants. Am. J. Respir. Cell Mol. Biol. 2001; 24:132-8. 74. Calderon-Garciduenas L, Osnaya N, Rodriguez-Alcaraz A etal. DNA damage in nasal respiratory epithelium from children exposed to urban pollution. Environ. Mol. Mutagen 1997; 30:11-20. 75. Calderon-Garciduenas L, Rodriguez-Alcaraz A, Valencia-Salazar G et al. Nasal biopsies of children exposed to air pollutants. Toxicol. Pathol. 2001; 29:558-64. 76. Fanucchi M, Tan L, Gershwin LJ et al. Postnatal remodeling of the mucous cell population of the epithelial-mesenchymal trophic unit in airways of infant rhesus monkeys exposed to ozone and allergen. Am. J. Respir. Crit. Care Med. 2001; 163:A93. 77. Mariassy AT, Abraham WM, Phipps RJ et al. Effect of ozone on the postnatal development of lamb mucociliary apparatus. J. Appl. Physiol. 1990; 68:2504-10. 78. Mariassy AT, Sielczak MW, McCray MN et al. Effects of ozone on lamb tracheal mucosa. Quantitative glycoconjugate histochemistry.Am. J. Pathol. 1989; 135:871-9. 79. Hecht SS, Ye M, Carmella SG et al. Metabolites of a tobaccospecific lung carcinogen in the urine of elementary schoolaged children. Cancer Epidemiol. Biomarkers Prev. 2001; 10:1109-16. 80. Tredaniel J, Boffetta P, Saracci R et al. Exposure to environmental tobacco smoke and risk of lung cancer: the epidemiological evidence. Eur. Respir. J. 1994; 7:1877-88.

81. Pershagen G. Passive smoking and lung cancer. In: Samet JM (ed.) Passive Smoking and Lung Cancer. Dekker, New York, 1994, pp. 109-30. 82. Leuenberger P, Schwartz J, Ackermann-Liebrich U etal. Passive smoking exposure in adults and chronic respiratory symptoms (SAPALDIA Study). Swiss Study on Air Pollution and Lung Diseases in Adults, SAPALDIA Team. Am. J. Respir. Crit. Care Med. 1994; 150:1222-8. 83. Forastiere F, Corbo GM, Michelozzi P etal. Effects of environment and passive smoking on the respiratory health of children. Int. J. Epidemiol. 1992; 21:66-73. 84. Dijkstra L, Houthuijs D, Brunekreef B etal. Respiratory health effects of the indoor environment in a population of Dutch children. Am. Rev. Respir. Dis. 1990; 142:1172-8. 85. Frischer T, Kuehr J, Meinert R etal. Maternal smoking in early childhood: a risk factor for bronchial responsiveness to exercise in primary-school children. J. Pediatr. 1992; 121:17-22. 86. Wang X, Wypij D, Gold DR et al. A longitudinal study of the effects of parental smoking on pulmonary function in children 6-18 years. Am. J. Respir. Crit. Care Med. 1994; 149:1420-5. 87. Tager IB, Weiss ST, Munoz A et al. Longitudinal study of the effects of maternal smoking on pulmonary function in children. N. Engl. J. Med. 1983; 309:699-703. 88. Martinez FD, Antognoni G, Macri F et al. Parental smoking enhances bronchial responsiveness in nine-year-old children. Am. Rev. Respir. Dis. 1988; 138:518-23. 89. Cunningham J, Dockery DW, Speizer FE. Maternal smoking during pregnancy as a predictor of lung function in children.Am. J. Epidemiol. 1994; 139:1139-52.

90. Witschi H, Pinkerton K. Pulmonary carcinogenicity of cigarette sidestream smoke in A/J mice. Fundam. Appl. Toxicol. 1996; 30:203 (Abstract). 91. Van Winkle LS, Evans MJ, Brown CD et al. Prior exposure to aged and diluted sidestream cigarette smoke impairs bronchiolar injury and repair. Toxicol. Sci. 2001; 60:152-64. 92. Wang H, Liu X, Umino T etal. Cigarette smoke inhibits human bronchial epithelial cell repair processes. Am. J. Respir. Cell Mol. Biol. 2001; 25:772-9. 93. Joad JP, Pinkerton KE, Bric JM. Effects of sidestream smoke exposure and age on pulmonary function and airway reactivity in developing rats. Pediatr. Pulmonol. 1993; 16:281-8. 94. Joad JP, Ji C, Kott KS et al. In utero and postnatal effects of sidestream cigarette smoke exposure on lung function, hyperresponsiveness, and neuroendocrine cells in rats. Toxicol. Appl. Pharmacol. 1995; 132:63-71. 95. Joad JP, Bric JM, Peake JL et al. Perinatal exposure to aged and diluted sidestream cigarette smoke produces airway hyperresponsiveness in older rats. Toxicol. Appl. Pharmacol. 1999; 155:253-60. 96. Ji, CM, Plopper CG, Witschi HP et al. Exposure to sidestream cigarette smoke alters bronchiolar epithelial cell differentiation in the postnatal rat lung.Am. J. Respir. Cell Mol. Biol. 1994; 11:312-20. 97. Ji CM, Royce FH, Truong U etal. Maternal exposure to environmental tobacco smoke alters Clara cell secretory protein expression in fetal rat lung. Am. J. Physiol. 1998; 275 :L870-6. 98. Todd L, Mullen M, Olley PM et al. Pulmonary toxicity of monocrotaline differs at critical periods of lung development. Pediatr. Res. 1985; 19:731-7.

THE

SURFACTANT

SYSTEM

IN ADULTS

We have already described the composition, function and regulation of the normal surfactant system, and how this system can be altered by the environment and disease in the developing fetus and neonate (Chapter 10). In this chapter we concentrate on the adult pulmonary surfactant system and describe how it can be affected by the aging process, respiratory diseases and environmental factors. We have also synthesized current knowledge of the interaction between the pulmonary surfactant system of an aged or diseased lung and some specific environmental factors. The natural aging process has profound effects on both lung function and lung architecture (see Chapter 28). These effects include enlargement of air spaces, decrease in exchange surface area and loss of supporting tissue for peripheral airways ('senile emphysema'), resulting in decreased static elastic recoil, increased residual volume and functional residual capacity and decreased expiratory flow rates. 1 However, the effects of the natural aging process on the pulmonary surfactant system are relatively poorly known; nor is it known whether age-related changes to the surfactant system contribute to or ameliorate structural and functional changes in the aged lung.

NATURAL AGING EFFECTS ON THE PULMONARY SURFACTANT SYSTEM

Surfactant in the alveolar space exists in two major forms: functional large aggregates (LA), which demonstrate signi*To whom correspondence should be addressed. The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

ficant surface activity, and non-functional small aggregates (SA), which are poorly surface active. Developmental aspects of these two forms have been investigated in newborn, young, middle-aged and aged rats. 2 Alveolar lavages from aged rats contained a higher proportion of LA than did those from young or newborn rats. There was also an age-related decrease in the rate of conversion from LA to SA in vitro. The amount of saturated phosphatidylcholine (PC) in the lavage decreased with age, but the surfactant protein A (SP-A) content was similar in young and aged rats. The decreased conversion between forms may contribute to the maintenance of functional surfactant pool sizes in the lungs of aged rats. 2 The proportion of total PC in surfactant increased with age in the bronchoalveolar lavage (BAL) from beagles aged between 3 months and 12 years. 3 The proportions of phosphatidylserine and sphingomyelin were significantly lower in old beagles compared to young dogs. Although an age-related decrease in saturated PC was found, 2 this is not necessarily in conflict, as only the saturated form of PC was measured. In addition to compositional changes, the minimal surface tensions of BAL fluids from old beagles was lower than that of young dogs. 3 Overall, however, the surface tensions obtained were very high, possibly due to the small amount of sample examined. Nevertheless, it was speculated that the shift in surfactant lipid composition could be one of the mechanisms that permits efficient lung function in old dogs, despite changes in morphology and mechanics in the aging lung. In another study, age-related changes in lung elastic recoil and changes in lavage surface activity were compared in hamsters ranging in age from 11 to 60 weeks. 4 Despite an increase in the mean linear intercept (a measure of the size of the air space) and a decrease in the extensibility of the alveolar wall, lung elastic recoil did not change. Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

Therefore, the changes in surface tension may counteract the changes in lung architecture, thereby abolishing the expected age-related changes in lung elastic recoil. It is also possible that pulmonary surfactant production may be impaired with aging. For example, in macaques, both the number of lamellar bodies per type II alveolar epithelial cell (AEC) and the volume density of lamellar bodies/cytoplasmic volume decreased with age between 1 month and 31 years (lifespan= 35 years). 5 In older animals, there was also an increase in the volume density of a vacuolelike dilatation of the endoplasmic reticulum in type II AECs. In contrast, a study on humans aged from 13 months to 80 years did not find any differences in the amount of saturated PC in lung tissue obtained after autopsy, relative to either body weight, lung protein or lung DNA. 6 Lavage material obtained from 10 human lungs at autopsy demonstrated that the amounts of saturated PC and SP-A from alveoli were similar and unusually low, compared with other species. It is possible that the relatively small surfactant pools in the human may make the lung more susceptible to injuries that interfere with surfactant function. While very few studies have investigated the effect of aging on the pulmonary surfactant system, some evidence indicates that changes in surfactant may ameliorate the negative aging effects on lung architecture and function. Furthermore, aging has other effects, which could contribute to the effects of aging on surfactant composition, function and regulation. In particular, the autonomic nervous system (e.g. cholinergic and adrenergic receptor number and function), 7-1~ the immunological status of the lung (e.g. numbers of alveolar macrophages, leukocytes and neutrophils, extent of phagocytosis, concentration of inflammatory mediators, etc.) 11-13 and alveolo-capillary clearance 14'15 all change with age and could impact on the composition and function of the surfactant system. Finally, it is important to note that studies on aging between different mammalian species may not be readily comparable because of differences in maximum ages; e.g. rats are senescent at 24 months, dogs at 12 years, and humans at 70 years. Recently, however, animal models have been developed to specifically explore physiological aging effects as well as the underlying molecular mechanisms. One such model is the senescence accelerated mouse (SAM) which includes both 'senescence-prone strains' (SAM-P) and 'senescence-resistant strains' (SAM-R). 16'17 This model has also recently been used to determine aging effects on the lungs and the interactive effects of aging and smoking. TM

S U R F A C T A N T RELATED DISEASES IN THE A D U L T L U N G The most common, serious surfactant-related disease is the acute respiratory distress syndrome (ARDS), in which pulmonary surfactant function is severely impaired, contributing to the high mortality rate of this disease. In addition there are a number of subacute pulmonary diseases

that are associated with alterations in surfactant composition, structure and/or function. 19'2~ The extent to which surfactant dysfunction contributes to the genesis of these diseases, is unknown. Surfactant dysfunction may be a secondary complication, but if it can be treated, the severity of the disease may be reduced and the outcome for the patient improved.

Acute lung injury/ARDS Acute respiratory distress syndrome is a generalized severe respiratory failure of rapid onset. It represents the most severe subset of the syndrome known as acute lung injury (ALI), which is characterized by an increase in alveolocapillary permeability, pulmonary inflammation, pulmonary oedema, surfactant abnormalities and dysfunction, a decrease in lung compliance and severe hypoxemia. The degree of disturbance of oxygenation is the basis for the classification of severity. 21 Fig. 27.1 illustrates the alterations in an injured alveolus in the acute phase of ARDS. The causes of ARDS are diverse and include pulmonary infection (e.g. bacterial or viral pneumonia), toxic chemical insult of the airways, burns, severe trauma and sepsis. Due to the wide variety of causes, the progression, severity and specific pathogenesis of ALI and/or ARDS varies from patient to patient. For example, it is still unclear whether surfactant dysfunction is a primary or secondary cause of the lung injury. The level and timing of the surfactant dysfunction is also likely to depend on whether the initial insult occurred via the airways or via the circulation. 22 Nevertheless, whatever the aetiology of the lung injury, surfactant abnormalities occur in ARDS, 23-3~ and there is some evidence that exogenous surfactant therapy is of potential benefit in ARDS. 31-34

Effects on pulmonary surfactant Since the early discoveries of surfactant abnormalities in humans with ALI/ARDS, a large number of animal models of this disease have been developed, covering some of the aetiologies of the disease documented in humans (e.g. infection, toxic inhalation, sepsis). These models have demonstrated that differences in the pathophysiology of the disease depend on the type of initial insult; however, some conclusions about the effects on surfactant composition and function can be drawn.

Phospholipids Perturbations in phospholipid composition are common and include decreased PC and phosphatidylglycerol (PG) concentrations associated with concomitant increases in sphingomyelin and phosphatidylinositol. 23'26'3~Such changes may occur in response to the oxidative, chemical or inflammatory damage to the alveolar lining, which alter type II AEC number and surfactant homeostasis. 22 In addition, the influx of macrophages and neutrophils may lead to an increase in lipid clearance and degradation, thereby leading to alterations in surfactant pools. 35 In particular, the ratio of highly surface-active large aggregates to the less surface-active

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Fig. 27.1. The normal alveolus (left side) and the injured alveolus in the acute phase (right side) of acute lung injury and acute respiratory distress (ARDS). In the acute phase of the syndrome, there is a sloughing of both the bronchial and alveolar epithelial cells, with the formation of proteinrich hyaline membranes and fibrin on the denuded basement membrane. Neutrophils are shown adhering to the injured capillary endothelium and migrating through the interstitium into the air space, which is filled with protein-rich edema fluid. In the airspace, an alveolar macrophage is secreting cytokines and other inflammatory mediators, which act locally to activate neutrophils to produce oxidants, proteases and other pro-inflammatory mediators, as well as activate fibroblasts to produce extracellular matrix material. The injured capillary endothelium leads to interstitial and alveolar edema. The influx of protein-rich edema fluid into the alveolus leads to the inactivation of pulmonary surfactant. (Figure modified from Ware LB, Matthay MA. The acute respiratory distress syndrome. N. Engl. J. Med. 2000; 342:1339.)

small aggregates decreases markedly. 27'29'3~ Furthermore, the surface activity of the large aggregate surfactant is also severely compromised. 37 Marked increases in lyso-phosphatidylcholine (LPC) occur in the BAL fluid of ARDS patients in the early stages of the disease, because dipalmitoylphosphatidylcholine (DPPC) is hydrolysed, z7'38 The hydrolysis is catalysed by increased levels of phospholipase A2, which is secreted by type II AECs in response to the pulmonary inflammation. 38 The increased levels of LPC are highly detrimental to lung function as LPC directly interferes with the surface activity of the surfactant film, preventing it from reaching the necessary low surface tensions. 39 Furthermore, LPC is highly injurious to the membranes of type I AECs 4~ as well as capillary endothelial cells,41 resulting in a manifold increase in alveolo-capillary permeability. Hence, this specific phospholipid imbalance may represent the most severe and damaging aspect of the surfactant lipid abnormalities in ARDS. Proteins Major imbalances in total protein as well as surfactantspecific proteins occur during the progression of ARDS. Total protein is always markedly increased, due to the influx of plasma proteins. 25'28'3~ This influx exacerbates

the poor surfactant function, as plasma proteins (in particular albumin and fibrinogen) directly interfere with the surface tension lowering ability of the surfactant film. 42-44 On the other hand, the surfactant proteins (SP) generally appear to decrease in the BAL fluid of ARDS patients and in various animal models with ARDS, 25'27'29'30'36'37'45'46although the findings are not always consistent. Importantly however, SP gene expression was markedly decreased in diseased lungs, whereas genes that are associated with oxidative stress, antiprotease function, cell proliferation and extracellular matrix repair demonstrated enhanced expression. 47'48 In addition to the reduced SP gene expression, the surfactant proteins also appear to be damaged by proteolysis within the alveolar compartment, caused by the release of elastase from activated neutrophils. 49 These surfactant alterations promote atelectasis (partial alveolar and small airway collapse) and create hydrostatic forces that favour edema formation, 24 resulting in further surfactant alterations. Fig. 27.2 summarizes the role of altered endogenous surfactant in the pathogenesis of ARDS and indicates the mechanisms contributing to altered surfactant function, as well as the relationship between surfactant abnormalities and lung function. 31 Although it is clear that the surfactant abnormalities outlined are present in patients with established ARDS, significant surfactant

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Fig. 27.2. Flow diagram summarizing the role of altered endogenous surfactant in the pathogenesis of ARDS. Also indicated are mechanisms contributing to altered surfactant function which include damage to the alveolar type II cells, direct damage to alveolar surfactant which influences surfactant activity, as well as the influx of protein-rich edema fluid, which inactivates surfactant. The influence of surfactant dysfunction on lung function is also indicated and includes atelectasis, which leads to reduced functional residual capacity (FRC), ventilationperfusion imbalance, which leads to intrapulmonary shunting with hypoxemia and reduced lung compliance, which leads to increased work of breathing. (Figure reproduced with permission from Lewis JF, Jobo AH. Surfactant and the adult respiratory distress syndrome. Am. Rev. Respir. Dis. 1993; 147:221.)

abnormalities also occur in mild-to-moderate cases of ALI, 37 as well as in patients considered to be at risk of ARDS. 45 Hence, surfactant is abnormal in patients with varying degrees of inflammatory lung injury, associated with different aetiologies. Furthermore, the presence of abnormal surfactant may play a role in the development of the lung injury, and not simply be a consequence of cellular dysfunction. However, the extent to which changes in surfactant contribute to the development of ALI requires further investigation.

Subacute pulmonary diseases Only in the past 10 years has it become known that pulmonary surfactant dysfunction is associated with a number of subacute pulmonary diseases. Diseases associated with surfactant abnormalities include obstructive lung diseases (e.g. asthma, bronchiolitis, chronic obstructive pulmonary disease, and following lung transplantation), infectious lung diseases (cystic fibrosis, pneumonia, and human immunodeficiency virus), interstitial lung diseases (sarcoidosis, idiopathic pulmonary fibrosis, and hypersensitivity pneumonitis), pulmonary alveolar proteinosis, following cardiopulmonary bypass, and in smokers. Moreover, we have only recently become aware of the potential importance of airway surfactant dysfunction (as a secondary consequence of airway inflammation) in the function of small airways in obstructive lung diseases (e.g. asthma and chronic obstructive pulmonary disease). Fig. 27.3 summarizes the possible role of pulmonary surfactant in the pathogenesis of airway obstruction. 5~ The specific surfactant abnormalities of some of the most common or most understood diseases are described below. For a more complete description the reader is referred to several recent reviews. 19'2~

,

Impaired I m m u n o m o d u l a t o r y ~ iucos%Edema Properties ( ~ LuminalF : l u i d a n d ~ ~( Pr~ Accumulati~ ""X,

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Inflammation Increased Permeability

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Fig. 27.3. Flow diagram summarizing the possible role of pulmonary surfactant dysfunction in the pathogenesis of airway obstruction. It is proposed that the dysfunction specifically of airway (as opposed to alveolar) surfactant (as a secondary consequence of airway inflammation) can have a severe impact on small airway function. This would lead to increased airway resistance, thereby further exacerbating obstructive lung diseases, such as asthma and chronic obstructive pulmonary disease. (Figure reproduced with permission from Hohlfeld Jet al. The role of pulmonary surfactant in obstructive airways disease. Eur. Respir. J. 1997; 10:486.)

Asthma As asthma is predominantly a disease of the conducting airways, the possibility that pulmonary surfactant may play a significant role in its pathogenesis has been overlooked until recently. 5~As a very comprehensive review on the role of pulmonary surfactant in asthma exists, 51 we will summarize the surfactant abnormalities reported. Impaired surfactant function, attributable to increased plasma protein leakage, has been demonstrated in animal models of asthma 52 and in BAL fluid of asthmatic patients. 53 The ratio of small to large aggregate surfactant increases. This structural change is further supported in the murine chronic asthma model, which demonstrates decreased surfactant pool sizes, a decreased ratio of saturated PC to total protein in BAL fluids, decreased surface activity and accelerated surfactant subtype conversion in vitro. These changes were similar to the pattern of acute lung injury in humans, suggesting that alveolar inflammation might be involved in the pathogenesis of chronic asthma. 54 Treatment, either prophylactically, or after onset of airway damage, with exogenous surfactant, in animal models 55'56 and in adult asthmatic patients after an asthma attack 57 has had some success. If these data are supported by larger clinical trials, exogenous surfactant treatment may become a therapeutic tool in patients with chronic asthma.

Cystic fibrosis Cystic fibrosis leads to chronic inflammation of the airways, with significant airflow limitations; patients are also extremely susceptible to infection. BAL fluid of adults suffering from cystic fibrosis demonstrates a reduced phospholipid, PC,

PG and SP-A content, as well as impaired surface activity. 58 The reduction in SP-A appears to be as a result of proteolytic cleavage. 59 Children with cystic fibrosis also demonstrate greatly reduced levels of both SP-A and SP-D, with relatively normal PL molecular composition. 6~As SP-A and SP-D have important host defence functions, an inactivation of these proteins, may place these patients under further risk of lung infections.

Air pollution is an increasingly serious health issue facing every major industrialized city. The lung is the only internal organ exposed to the atmosphere, and with its huge surface area, is a prime target for the toxic effects of air pollutants. Not unexpectedly, there is a correlation between the levels of air pollution and mortality due to aggravation of preexisting respiratory conditions, or hospital admissions with respiratory or respiratory-related complaints. 65-67 Within the lung, the pulmonary surfactant system is a crucial, susceptible target for air pollutants.

The extent to which a pollutant can infiltrate the lung is influenced by the size, solubility, and reactivity of the compounds, as well as the ventilatory pattern. 69 Every time the air changes direction within the bronchial tree, inertia keeps particles continuing on the same trajectory, causing them to impact into the respiratory walls where they can be quickly cleared by the mucociliary escalator. 7~ Larger and heavier particles tend to impact more at junctions while smaller particles follow the path of the air around bends. As the air velocity decreases deeper in the bronchial tree, gravity induces particles to sediment. Smaller particles will remain suspended in the inspired air for longer and hence travel further. 7~ Pre-existing conditions (such as asthma), that cause obstruction of the respiratory airways, can greatly enhance the deposition of particles. 71'72 This can lead to excessive particle burdens in individuals with respiratory diseases, even when they are exposed to conditions that are acceptable for healthy individuals. The physicochemical properties of a pollutant, such as solubility and reactivity, are of particular importance for gaseous compounds. Gases with a high solubility are readily removed from the air and absorbed by the first tissue with which they come in contact. Therefore, most uptake of these gases occurs in the upper airways. 73'74The toxic effects are minimized as the fluid lining is relatively thick in these regions with high levels of antioxidants and the pollutant does not reach the epithelium. Insoluble gases are more uniformly distributed along the respiratory system, as their uptake is mainly dependent on reactivity. 73'74 Generally pollutants with low solubility, low reactivity and small diameter will travel furthest into the respiratory system. The toxic effect of a pollutant is also dependent on concentration, duration of exposure and penetration and retention within the respiratory system. Furthermore, the reactivity of a pollutant will influence its ability to cause biochemical damage to the lipids and proteins with which it comes into contact. Most non-reactive particulate matter deposited in the upper airways is rapidly removed (<24 h) by the mucociliary escalator. 7~ Smaller particles that reach the gas exchange regions can be phagocytosed by macrophages and transported through the epithelium and deposited into lymphatic vessels, but this process takes significantly longer.

Common pollutants Some atmospheric agents occur naturally from biological degradation of organic matter (ammonia), volcanism (ash, SO2) and wind erosion (dust, sea salt). However, in urban areas, increased anthropogenic activities such as industry, power plants, vehicles and domestic households release substantially greater amounts of pollutants into the atmosphere. The six most common pollutants found in urban regions are oxides of nitrogen (NOx), sulphur dioxide (SO2), carbon monoxide (CO), respirable particulate matter (PMlo), ozone (03) and lead (Pb) while other hazardous pollutants include benzenes, xylenes, chlorine, asbestos and heavy metals. 68

Pollution effects on pulmonary surfactant There have been numerous laboratory investigations into the effects of individual pollutants, particularly gaseous pollutants such as NO 2, SO 2 and 03, on lung function. These studies expose animals to a single pollutant over a relatively short period of time at concentrations many times higher than those found in urban air. As there is a comprehensive, recent review on this topic, 75 we will provide only a brief account of the most recent literature on the effects of common gaseous pollutants, particulate pollutants and cigarette smoke on the pulmonary surfactant system. Finally, we provide information on interactive effects of pollutants with aging and disease.

Pulmonary alveolarproteinosis This is a rare idiopathic disease, characterized by massive accumulation of surfactant in the alveoli, which impedes gas exchange and rapidly leads to respiratory failure. 61 Granulocyte-macrophage colony stimulating factor (GM-CSF) knockout mice develop alveolar proteinosis. Mutations in the genes for GM-CSF or its receptor have been identified as the cause for pulmonary alveolar proteinosis. 62 Apart from the large excess of alveolar surfactant, which is thought to be caused by reduced clearance, 2~ the biochemical abnormalities include an increase in SP-A and SP-B relative to disaturated phospholipid, and a 7-fold increase in the cholesterol to disaturated phospholipid ratio. 63 Furthermore, the phospholipid composition is abnormal, with a significant decrease in PG and a concomitant increase in phosphatidylinositol (PI), resulting in a decreased PG to PI ratio. 64 However, whether these surfactant abnormalities are secondary effects, or whether they are involved in the pathogenesis of the disease is unknown.

E N V I R O N M E N T A L INFLUENCES O N THE M A T U R E S U R F A C T A N T SYSTEM

Common gaseous pollutants Nitrogen dioxide The effects of nitrogen dioxide (NO2) inhalation on the lung epithelium, lung antioxidant status and the biochemical composition and function of the surfactant system are perhaps the most thoroughly documented of all pollutants. This is possibly because, apart from specialized factory work places, the highest NO 2 concentrations are found in cigarette smoke, which can reach up to 250 ppm. 76 It appears that the initial reaction to short-term NO 2 exposure is an increase in the pool size of surfactant phospholipid in the alveoli, which appears to be dose and time-dependent (reviewed by Ref.75). However, after chronic exposure, the alveolar phospholipid content is either normal or reduced. 77 These changes also correlate with changes observed in the enzymes responsible for phospholipid synthesis. After acute exposure, enzyme activity is elevated, 78 whereas during the recovery time after chronic exposure, lipid synthesis is reduced. 79 Despite the overall increase in phospholipid content, the concentration of PC, and also that of its saturated form (DPPC) are substantially reduced. 77'8~ These changes appear to correlate with a decrease in the surface activity of the surfactant after both short term (< 30 h) and chronic (9 months) exposure. 77'8~ However, an increase in the level of unsaturated fatty acids also o c c u r s . 77'81 Short-term NO 2 exposure (24-72 h) increases the size of the SP-A pool in the alveoli, although the in vitro activity of this protein is diminished. 82 NO 2, like ozone (03) also affects the alveolar epithelium. These effects occur in two phases. The first is a degenerative phase during which type I AECs are eliminated, 83'84 and the lamellar bodies of the type II AECs increase in size and number. 85'86 This may be caused by solute influx through membrane leakage. 75 The second, regenerative phase, is characterized by greatly enhanced rates of type II cell proliferation 87-9~and differentiation into type I AECs. 87'91 Ozone (0 3) Low concentrations (0.4-0.5 ppm) of ozone for either medium term (2 weeks) or long term (9-22 months) have surprisingly little effect on either lipid peroxides, levels of antioxidants or the surfactant lipids, although there are some species differences. 92-94 Acute exposure (2 and 12h) to 0.8ppm 0 3 did not change the content, composition or surface activity of pulmonary surfactant, but did cause an alteration in the structure of surfactant s u b t y p e s . 95,96

Relatively low concentrations of 0 3 (0.1-0.5ppm) in chronic exposure (up to 18 months) did not change the phospholipid content in lavage. 97 However, the incorporation of fatty acids into lung tissue phospholipids decreased. 97 Furthermore, in isolated type II cells acutely exposed to 0 3 (2 ppm, 4 h) the activity of one of the PC synthetic enzymes decreased; however, this reverted to normal very rapidly after continued cell culture. 98 A decrease in the incorporation of precursors into PC in isolated type II cells, with a corresponding decrease in enzyme activity was also described. 99'1~176 The level of unsaturated fatty acids in PC also changed, but the findings are not consistent. 75

The combined effect of the two oxidants ( 0 3 and NO2) , each at similar concentrations (0.4-0.5 ppm), produced increased levels of lipid peroxides and altered alveolar surfactant composition. Alveolar PC content increased, while phosphatidylethanolamine (PE) decreased. Furthermore, there was an increase in the level of saturated fatty acids and a decrease in unsaturated fatty acids. 92 However, significant species differences were observed. The changes in phospholipids and fatty acids may represent a possible adaptive mechanism to avoid further lipid peroxidation. On the other hand, that unsaturated fatty acids increase after either NO 2 or ozone exposure appears counterintuitive, as these oxidants produce free radicals that should attack double bonds, forming ozonides and peroxides, thereby reducing the levels of unsaturated fatty acids. 75 However, an intriguing recent finding about the influence of surfactant monolayers on NO 2 absorption and NO 2 flux may shed light on some of these apparent conflicts. 1~ Phospholipid monolayers are able to restrict NO 2 absorption in vitro, and this monolayer-induced resistance to NO 2 flux is directly related to the physicochemical properties of the surfactant film. Hence, the lower the surface tension of the compressed surfactant monolayer, the lower is the NO 2 absorption by the aqueous subphase. 1~ Pulmonary surfactant may therefore influence the intrapulmonary gas phase distribution of inhaled N O 2 and if uptake of N O 2 w e r e sufficiently restricted, cell injury would not necessarily correlate with the local gas phase concentration of the oxidant. Furthermore, as in vitro exposure models do not include a surfactant film, the dose response data cannot easily be extrapolated to the in vivo situation. A further complication from extrapolation is the presence in vivo of antioxidants and other chemicals which protect against oxidation (including SP-A and SP-DI~ 75 Although it is not known whether the resistance to absorption also applies to ozone, it is intriguing to speculate that the ozone-induced increase in surface activity (i.e. decrease in surface tension) observed in the BAL of rats 1~ may represent an adaptive protective mechanism to low level ozone exposure. On the other hand, acute exposure of ozone (2ppm up to 8 h) demonstrated decreased ability of the surfactant to maintain small airway patency, which was attributed to the increased plasma 1 protein contamination. 104,05 Ozone exposure also has significant damaging effects on surfactant proteins. In vitro exposure of SP-A to ozone caused oxidation damage to the methionine and tryptophan residues, and diminished the aggregation and binding properties of S P - A . 106 These in vitro results may explain the structural changes in secreted surfactant forms observed after in vivo exposure. 107 Furthermore, in vitro exposure to ozone also significantly compromised the immune function of S P - A , 108'109 which may explain the increased risk of infections in areas of high air pollution. 110 However, in vivo, SP-A enhanced alveolar macrophage activity. Ozone exposure therefore induced either structural changes in the protein, or altered its ability to interact with other

surfactant components. The level of SP-A mRNA remained unaffected by either acute or repeated ozone exposure. 111

Sulfur-related compounds Long-term exposure (400 days) to either sulfate or sulfite did not alter the amount, extracellular metabolism or surface active properties of pulmonary surfactant. 1~2 Similarly, short-term exposure of rat lungs to sulfuric acid aerosol (4 h) did not produce any biochemical or biophysical effects on surfactant, although half the dose in guinea pigs caused acute lung injury with secondary effects on surface activity, due to protein leakage. 113 Furthermore, beagle dogs exposed for 13 months to both neutral sulfite and acidic sulfate aerosol did not demonstrate any significant changes to alveolo-capillary permeability, oxidant burden of the BAL fluid or lactate dehydrogenase in BAL, nor did surfactant function change. However, there was some evidence for increased type II cell proliferation. 114

Particulate pollutants Particulate air pollution, which results primarily from the combustion of fossil fuels, is classified as either PM10 or PM2.5 particulate matter (i.e. < 10 or <2.5 ~tm in aerodynamic diameter, respectively). 115 The fine particulate matter (PM2.5) has been associated with increased cardiopulmonary and lung cancer mortality. 67 Due to the greater size of these particles, relative to gaseous pollutants, damage to the alveolar epithelium, characterized by hyperplasia and hypertrophy of the type II cells, 116 occurs predominantly in the most proximal alveolar duct bifurcations. 117 The two most common diseases resulting from deposition of mineral particles are silicosis and asbestosis. Whereas these mineral dusts are characterized by only one component, dusts such as cigarette smoke or diesel exhaust comprise many different components, which makes it difficult to assign the toxic effects to one particular component. 75

Mineral dustpoUutants In general, the mineral-induced diseases such as silicosis and asbestosis are characterized by an excessive accumulation of alveolar pulmonary surfactant, without significant changes to the lipid or protein composition. 75 The particles that enter the alveolar space are coated with surfactant material, which is thought to either limit the toxicity of the material, or aid in the removal of the particles. 11s The increase in phospholipid content is linked to increased phospholipid synthesis, 11s-121 which in turn is linked to an increase in phospholipid synthetic enzyme activity in a subset of type II A E C s . 122'123 In terms of the protein content, the amount of SP-A is increased both in BAL fluid and in type II AECs, 75 as is the SP-A mRNA. 124 The elevation of SP-A in acute silicosis may serve as a normal host response to prevent lung cell injury after exposure to silica, as SP-A protects macrophages from the damaging effect of silica toxicity. 125

Cigarette smoke and diesel exhaust Diesel exhaust exposure is associated with an increased level of lavage phospholipids,

which exhibit an elevated PC content accompanied by an increase in palmitate content of PC. 126 A comparison of several types of particles 127 demonstrated that diesel exhaust particles caused only minimal changes in surfactant amount and composition, by inducing a transient elevation in protein, and causing a very small and transient increase in lung permeability, lung inflammation and cellular damage. 127 In comparison, the slightly larger carbon black particles did not cause any detectable changes, whereas the much smaller, but highly reactive silicon dioxide particles caused very significant increases in lung permeability and inflammation, increases in the amount of pulmonary surfactant in the alveoli and damage to the alveolar and bronchiolar epithelium. Hence, surface chemistry appears a more potent stimulus for lung damage than particle size. 127 On the other hand, the inhalation of cigarette smoke is associated with a reduced yield of surfactant phospholipid, although lipid composition remains unaltered. Following cessation of smoking, phospholipid levels return to normal within 2 weeks. 12s Although phospholipid synthesis in dogs 129 was not altered after exposure to cigarette smoke, secretion of PC from isolated type II cells was reduced after the cell cultures were exposed to cigarette smoke. 13~Despite unaltered lipid composition, the surface activity of lavage fluid from chronic smokers is significantly reduced. TM This is likely to be related to the increase in albumin contamination found in BAL fluid of smoke-exposed rats, as well as the reduction in SP-B levels. 132 A reduced level of SP-A occurs in rat lavage after exposure to tobacco smoke and in BAL from chronic smokers. Surfactant from smokers also demonstrated a decrease in SP-D. 133 Although the reason for this reduction is not known, the fact that neither SP-A, nor SP-B mRNA levels were affected in rat lung tissue after exposure suggests that the mechanism involves either an inhibitory effect on the secretory process, or a localized destruction of surfactant proteins in the alveolar space. 132 Whether the in vivo function of surfactant is affected by tobacco smoke is unknown. However, a recent study evaluating the efficacy of different treatments for acute respiratory failure following severe smoke inhalation in rats, discovered that the mortality rate after 24 h markedly declined in the group which was lavaged, treated with exogenous surfactant and ventilated mechanically. The improved survival was accompanied by improved surface activity of BAL, static lung compliance and oxygenation. TM These findings, therefore, indicate a localized destruction of surfactant components, which when removed and replaced, restore lung function.

Summary of effects of poUutants The majority of studies have examined the effect of a single pollutant on the pulmonary surfactant system. While this is important in enabling us to assign toxicological effects to particular components of pollutant mixtures, this approach is nevertheless an oversimplification. For example, while NO 2 tends to cause an increase in alveolar surfactant phospholipids and proteins, cigarette smoke, which is the

major contributor of NO2, causes a reduction in alveolar surfactant phospholipids and proteins. Hence, there is likely to be a very important interaction between different gaseous and particulate pollutants, yet few studies have addressed the issue of additive or cumulative effects. An exception is a study showing that exposure to a combined mixture of nitrogen dioxide and ozone produced a more significant response than exposure to either gas individually. 92-94 Only one study, has addressed the question of natural levels of pollution and their effect on the surfactant system. This study demonstrated differences in the morphological characteristics of the type II cells between a city-dwelling and a country-dwelling population of pigeons. 135 Other major issues facing this area of research are the choice of species, dose and time of exposure.

Interactive effects of pollutants on the aging or diseased lung How and whether pollutants can affect the aging or diseased (immuno-compromised) lung is poorly understood. The lungs of aging (24 month old) Fisher 344 rats respond differently to ozone-induced (0.4-0.8 ppm ozone for 2-6 h/day for 1-4 days) lung injury than either juvenile or adult rats. This effect was manifest as a greater inter-individual variation in alveolar protein transudation, increased levels of inflammatory mediators and lysosomal enzymes, as well as decreased levels of the antioxidant, ascorbic acid. 136 Furthermore, when adult and senescent animals were compared during the recovery period after ozone exposure (0.8 ppm for 6 h), the cellular events during repair are essentially similar in both age groups, but the senescent rats have a significantly higher level of initial injury (including type I AEC necrosis) from inhalation of ozone. 137 The long-term (9-month) effects of cigarette smoke inhalation in young (starting from 2 months old) and old (starting from 8 to 10 months old) mice revealed an interaction between smoke inhalation and aging. Several characteristics prominent only in the smoke-exposed old animals include a reduction of alveolar space with a concomitant increase in lung cellularity and thickened alveolar septa, an accumulation of intra-alveolar surfactant and decreased pulmonary function. These abnormalities were restrictive in nature and conform most closely to pulmonary fibrosis. 138 Furthermore, the lungs of the senescence-prone mouse were more susceptible to damage by tobacco smoke than the senescence-resistant mouse. Adverse effects include lung permeability and inflammation, antioxidant level and epithelial structure (e.g. hyaline membrane formation). 16 An epidemiological study of a well-defined, multiethnic population in Northern California observed a correlation between smoking and the incidence of ARDS, 139 but not between alcohol consumption and ARDS. Furthermore, there appeared to be a dose-dependent effect, as the correlation was greater for people who smoked more than 20 cigarettes per day. Assuming a causal relationship, approximately 50% of ARDS cases were attributable to cigarette smoking. 139

Effects of hyperoxia on pulmonary surfactant In a clinical setting, elevated oxygen tension can also be viewed as a gaseous pollutant; in the treatment of respiratory distress the use of high oxygen concentrations can result in free radical formation and consequently surfactant impairment. Exposure to hyperoxia (80% for 24 h or 100% for 64 h) results in reduced amounts of BAL phospholipid, PC and the PC saturated fraction. 14~ Similar findings and a decrease in PG were reported after 12, 24 and 48h of normobaric hyperoxia. 142 These findings correlate with the observation that activity of one of the PC synthetic enzymes is decreased in type II AECs following hyperoxia. 143 Furthermore, hyperoxia directly affects type II AECs to decrease choline incorporation into PC. TM On the other hand, adult hamsters exposed to 100% 0 2 for up to 8 days, showed 3-fold increases in the amount of BAL surfactant and in the percentage of disaturated PC, but the surface tension lowering ability was not altered. 145 Over the course of the 8-day exposure to 100% 02145 in adult hamsters, SP-A levels increased transiently, before decreasing below control levels, while SP-B and SP-C decreased at different rates. However, the lavage content of three of the surfactant proteins, SP-A, -B and -C increased in oxygen-exposed rats, depending on the duration of hyperoxia exposure. 146-148 In each of these studies, SP mRNA content of the lung also increased. 146-14s Increased SP-B expression occurs in bronchiolar epithelium of different mouse strains that differ in their sensitivity to hyperoxic injury. 149 Hence, it appears that both the surfactant phospholipids and proteins are regulated independently during hyperoxia, 14s and that the species, dose and cell type affect the hyperoxic response of the surfactant proteins. The degree of induction of SP-A, -B and -D was greater in bronchiolar epithelial cells (Clara cell), compared with alveolar epithelial cells. 15~ Furthermore, the time course of hyperoxic exposure also appears important. Variable periods of exposure (95% for 12, 36 or 60h) demonstrated that all SP mRNAs were diminished at 12h and rose to or exceeded control by 60h. TM In the case of SP-B, this also translated to the secreted protein level, such that there was a 50% decline of SP-B in lavage at 12 h. SP-A and SP-C protein levels in type II AECs also declined at 12 h. Such sharp declines in SP expression after 12h of 95% 0 2 may affect local alveolar stability. TM Moreover, if hyperoxia is applied to a lung with underlying lung injury, the surfactant effects on lung stability may be exacerbated. The cause of the biphasic response to hyperoxia may be related to different threshold levels for different genes. 151 Effects of hypoxia or altitude on pulmonary surfactant Acute hypoxia changes the surfactant lipids, is2 including a decrease in phosphatidylcholine 153'1s4 and an increase in lysophosphatidylcholine 154 and lysocompounds 153 with a concomitant increase in phospholipase activity. 153 Surface activity can decrease, 15e'154'155and edema of the interalveolar

septa can occur. TM While PC levels decrease in lavage surfactant, the incorporation of 32p into lung PC and PE increased, ~56 suggesting that the reduced alveolar content may be due to changes in secretion and/or local inactivation, and not synthesis. Furthermore, during the recovery period, the intracellular surfactant fraction returned to normal levels before the extracellular fraction. 155 One exciting area of research is the response of the surfactant system to chronic hypoxia caused by altitude. Rats raised at an altitude of 3500 m demonstrated a reduction in the number of macrophages and in the amount of lavage and lung tissue phospholipid, especially PC. 157 Llamas on the other hand, born at an altitude of 4720 m demonstrated numerous prominent Clara cells with large 'apical caps', many of which had been extruded into the terminal bronchioles. 15s Although the extruded material was not analysed, it is possible that the hyperactivity of the Clara cells was an adaptive response to chronic hypoxia. 15s These novel findings are likely to lead to further research.

CONCLUSIONS The central role played by the pulmonary surfactant system in the healthy functioning of the lung is evidenced by the diverse range of genetic, environmental and disease factors that can affect the system throughout fetal, neonatal and adult life. Abnormalities of the surfactant system are implicated in almost every known pulmonary disease or condition. These abnormalities involve both the lipid and the protein components of surfactant, which result in perturbations in surface activity and hence lung function. These perturbations include physical factors such as reduced lung compliance, resulting from reduced surface activity, increased alveolar permeability, which leads to pulmonary edema, and altered immune function, which may lead to increased risk for secondary lung infections. In many cases the abnormalities of the surfactant system are secondary to an inflammatory assault on the lung, and are not the primary cause of the pathology. However, surfactant dysfunction often contributes substantially to the progression of the disease/condition and is frequently the ultimate cause of death through respiratory failure. Hence, a thorough understanding of the surfactant system and its complex interactions with the neurohormonal, immunological and cardiovascular systems, and with its developmental and/or environmental conditions, is essential for the development of effective therapeutic strategies in pulmonary conditions.

ACKNOWLEDGEMENT This work was funded by an Australian Research Council (ARC) fellowship to SO, and an ARC grant to CBD. The authors thank Joshua Griffiths for assistance with the pollution section of this review.

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INTRODUCTION

The purpose of this chapter is to review the ways in which environmental factors interact with the aging lung. In Chapter 15, we showed that the lung ages in a predictable way in mammals, and that aging involves both anatomic and physiologic correlates. Distinguishing between physiologic aging and the additional effects of environmental factors is difficult. As our knowledge of normal aging is based upon data from populations with different genetic susceptibilities to lung disease when exposed to air pollutants and respiratory tract pathogens, it has proven difficult to distinguish aging p e r se from the accumulated effects of environmental insults and genetic susceptibility. 1 Table 28.1 summarizes the major age-related changes in the mammalian respiratory system, the environmental factors that influence them and associated diseases. In general, environmental factors enhance aging effects which, in turn, lead to clinical disease in susceptible subjects. Not all changes progress to disease with age. Adaptive responses have also been described. For example, changes in the composition of alveolar surfactant have been shown to partially offset age-related declines in lung mechanical properties in rats 2'3 and dogs. 4 Thus, adaptive changes may occur with age, and these may mitigate the aging process. Lung diseases can be separated into age-related diseases and age-dependent diseases: 5 the first are opportunistic (for example, tuberculosis) and take advantage of age-related changes in *To whom correspondence should be addressed. The Lung: Development, Aging and the Environment ISBN 0 12 324751 9

the host; the second group comprises diseases that appear to be exaggerations of the normal aging process, for example, declines in lung function associated with cigarette smoking. Obtaining accurate data on disease prevalence and severity can be difficult in the elderly. Epidemiologic studies have shown under-diagnosis of diseases, such as asthma, in the elderly. 6 Furthermore, with advancing age, the 'survivor' population becomes progressively less representative of the population as a whole. An age-related change that has proven difficult to separate from environmentally induced disease is 'senile' emphysema. 7,s The human lung loses approximately 30-50% of its tissue mass over an average lifetime. This is associated with loss of elastic fibres and capillaries in the alveolar walls. 9 Morphometric studies show an increase in alveolar duct volume, mean alveolar linear intercept, and size and number of interalveolar pores. 1~ These changes meet some of the criteria for the diagnosis of emphysema, defined as a permanent abnormal enlargement of airspaces distal to the terminal bronchiole, accompanied by destruction of their walls. The functional correlates are identical. Lung aging is associated with increased closing volumes, 12 reductions in FVC and FEV 1 and loss of elastic recoil pressure. 13 Senile emphysema does however differ from 'environmental' emphysema in several respects. Senile emphysema, unlike environmentally induced emphysema, involves the lung diffusely and is not readily apparent to naked eye inspection at autopsy. In contrast, environmentally induced emphysema is readily apparent on visual inspection of the lung as it is patchy and occupies distinct anatomic compartments. Senile emphysema Copyright 9 2004 Elsevier All rights of reproduction in any form reserved.

apparently results from changes in collagen composition characterized by a progressive shift towards insoluble collagen with fewer crosslinks 1~ and changes in elastin. TM Furthermore, changes similar to those described in humans with senile emphysema have been described in rats, mice, Syrian hamsters, dogs and non-human primates (see also Chapter 15). 15 The morphologic and physiologic changes in lungs of aging beagle dogs 16 are virtually identical to those reported for aging human lungs. As environmental exposures in laboratory animals are usually controlled (with the possible exception of subclinical infections), it seems likely that the changes described in these animals reflect the aging process rather than a specific environmental effect. It would appear that the slow decline of FEV 1 with age and its anatomic and biochemical correlates are true age-related phenomena. These changes form the platform on which environmental factors operate.

FACTORS T H A T I N F L U E N C E SUSCEPTIBILITY OF THE A G I N G L U N G TO DISEASE Aging is accompanied by changes that affect the whole body, not just the lung. In order to understand the relationship between aging of the lung and the environment, it is necessary to briefly review some of the general factors involved in aging.

Cellular homeostasis Many hypotheses have been proposed to explain the basis for aging; such hypotheses must take into account that different

species have markedly different longevities. Evolutionary theory predicts that longevity is determined by age of reproductive maturation. Programmed senescence is thus a driving force behind aging. The cumulative effects of oxidative stress underlie many theories of the mechanism of aging. 17 Mitochondria are a major source of intracellular free radicals. TM A gradual impairment of recognition and repair of oxidized proteins, DNA and lipids and the cumulative effects of lipid peroxides are important in the aging process. Mice deficient in DNA repair and transcription mechanisms age prematurely. 19 Oxidative damage to DNA is extensive even under normal physiologic conditions. Estimates of damage range from one base modification in 130,000 bases in nuclear DNA to 1 per 8000 bases in mitochondrial DNA. 2~ It is important to bear in mind that oxygen and other free radicals also play important physiologic roles in defence against microbial pathogens and as second messengers in cell signaling. 21 Carcinogenesis is also closely linked to the aging process and epidemiologic data indicate that aging contributes more to risk of cancer than environmental factors. 22 Replicative senescence occurs in cells where chromosomal telomeres are shortened. 23 Accumulation of post-mitotic cells reduces the ability of an organ to repair damaged cellular structures and contributes to the abnormalities associated with aging including neoplasia. Although degradation of DNA appears to be central to aging, post-translational modification of proteins with age may also be important. 24 It is likely that aging involves an interplay between various factors, including endogenous species-specific factors and exogenous agents (Fig. 28.1).25 Studies of individuals with exceptional longevity are also beginning to identify longevity-enabling genes. 26

Exogenous agents Radiation, chemical carcinogens

Endogenous agents Ros, alkylating agents

Altered chromatin Single-stranded DNA

Damaged DNA maintenance

Compromised genome maintenance

/ Persistent DNA lesion, single-stranded DNA -~" Altered chromatin

l

r

Transcriptional interference (pleiotrophic phenotype?)

\ Response

Mutation . accumulation "

Increased cancer incidence

?

Apoptosis Cellular senescence

Age-related functional decline (normal organismal senescence?)

Fig. 28.1. DNA damage in aging. Endogenous and exogenous agents cause continuing damage to DNA over the lifespan of the individual. Defects in genome maintenance may result in defective protein transcription, decreased gene activity and elicit cellular responses such as apoptosis and senescence. Accumulated errors during DNA repair replication or recombination of damaged DNA may lead to the accumulation of mutations and an increased risk for cancer and other aging-related phenomena. (Reprinted with permission from Hasty P, Vijg J. Genomic priorities in aging. Science 2002; 296:1250-1 .)

Lung antioxidant defenses The lung has a remarkable array of responses and defenses to oxidative stress. 27 Low level stress may invoke adaptive responses including growth arrest and preferential expression of genes responsible for damage repair. 28 Oxidative stress results in the transcription of over 40 mammalian genes. 2~ Deletion of P66shc, a signaling protein that maintains antioxidant levels, extends the lifespan of mice. 3~ Antioxidant enzymes synthesized in response to lung oxidant injury include superoxide dismutase (SOD), glutathione peroxidases, and catalases. Aqueous phase lung antioxidants include vitamin C, uric acid, ferritin, and ceruloplasmin. Lipid-associated antioxidants include vitamin E, surfactant, beta-carotene and ubiquinone. Age-related changes in antioxidant defenses include decreased lung tissue ascorbic acid 31'32 and reduction of glutathione. 33-35 However, not all antioxidant defenses decrease with age;

for example, the vitamin E content of rat lungs increases with age. 36 The lung is continuously exposed to oxidative stress, and a progressive reduction in antioxidant defenses might contribute to the decline of FEV 1 and increased risk for emphysema with increasing age. Some particulates, such as cigarette smoke, 37'38 coal dust and silica, 39 have intrinsic free radical activity. Others, such as diesel emissions, contain compounds that catalyze the generation of reactive oxygen species (ROS) 4~ and most exogenous toxins activate lung resident cells to produce ROS. 41 Many of these agents have been associated with are accelerated decline in lung function. 42,43

Innate and acquired immunity Immune mechanisms underlie many chronic degenerative diseases of the lung. Both innate and acquired immunity

tend to decline with age. 44 Innate responses, mediated via pathogen-specific recognition receptors, are not well studied as a function of age. The elements of innate immunity include the functions of antigen presenting cells (such as dendritic cells, B-cells, and macrophages), phagocytic cells (including macrophages and neutrophils), inflammatory mediators released from mast cells, polymorphonuclear leukocytes and macrophages, natural killer lymphocytes, antimicrobial molecules (such as nitric oxide), surfactant associated proteins, defensins, lactoferrin, and complement. 44 The impact of age on adaptive or specific immune responses is summarized in Table 28.2. It is likely that some of these changes account for the increased risk for infectious disease in the elderly; furthermore, the elderly produce a less vigorous response to vaccination. 44 Changes in broncho-alveolar lavage fluid have also been noted in association with aging (Table 28.3). Some of these parameters

Table

28.2.

Involution of the thymus Na'fve T-cell output Altered thymocyte differentiation Peripheral blood memory T-cells Hyporesponsive memory cells Proliferative responses to mitogens or antigens T-cell receptor repertoire diversity Shift of Thl to Th2 cytokine profile HLA-DR+ Fas-mediated apoptosis

Humoral Immunity Helper T-cell function B-cell number Germinal center formation Altered B cell repertoire expression Antibody responses to specific antigens Altered generation of primary B cells Impaired generation of memory B cells Ability to generate high-affinity protective antibody IgG and IgA F'~,-~.~ n _ ~ , - , , - - ; ~ c | , -.

~'~,f,-...~n ~ - ; k ~ , - I ; , - , c

Matrix remodeling A general feature of aging is decreased turnover of protein 46 combined with increased catabolism. 47 Elastin is very stable in the human lung with relatively little turnover. 48 The number and size of elastic fibers in the alveolar walls decrease with age whereas type III collagen increases. 49 With increasing age, serum elastin peptides, a marker for turnover of mature elastin, decrease 5~ indicating that impaired repair mechanisms may play a role in the pathogenesis of senile emphysema.

Nutrition, body mass and physical activity

Altered immunity in the elderly.

Cell-mediated immunity

reflect innate as well as acquired immune responses. On balance, the data indicate that the aging lung may be suffering a low grade inflammation compared with that of younger individuals. 45 Inflammation is central to the pathogenesis of obstructive lung diseases, including asthma, chronic bronchitis, and emphysema.

Changewith age

Body mass increases with age and increased body mass is associated with reduced longevity. 51 Body mass is positively associated with airway hyperresponsiveness, 52 and with progressive reductions in FVC, FEV 1 and FEV1/FVC ratio. 53 Cross-sectional 54'55 and longitudinal 56 studies have shown a relationship between asthma and obesity, particularly in women. 57'58 Static lung volumes significantly increased following weight loss in middle-aged and older obese men (46-80 years); 59 in contrast, aerobic exercise had no effect on pulmonary function but did increase maximal oxygen uptake (VO 2 max). The contribution of body composition, physical activity and smoking to lung function in older humans has been investigated; 6~ fat free mass and physical activity both exerted significant independent effects on FEV 1. These results, in contrast to the former study, 59 indicate that heavy intense physical activity may be more important in contributing to forced expiratory function than previously recognized. Table 28.3. Changes in bronchoalveolar lavage fluid in healthy aged individuals.

Parameter Lymphocytes CD4 +/CD8 + T-cell ratio H LA-D R+ T-cells B-cells IgM, IgA and IgG

Change ..... 1" 1" 1" ,I, 1"

~:~: i

Animal studies have established that caloric restriction is associated with increased longevity. Metabolic studies in mice indicate that caloric restriction enhances and maintains protein turnover into old age.61 Calorie restriction may thus slow the well-documented decline in protein renewal which occurs in the elderly and provides a scientific basis for understanding the contribution of calorie restriction to longevity. By contrast, severe calorie-protein restriction in rodents produces an emphysema-like lesion after several weeks of restricted diet. 62 This animal model of nutritional emphysema is characterized by destruction of collagen and elastin. Severe malnutrition may be a factor in the acceleration of disease in patients with advanced emphysema. 63,64

Early lung injury Several developmental abnormalities may affect the rate of onset of age-related lung diseases. Among these are diaphragmatic hernia, renal agenesis, oligohydramnios, cystic fibrosis, immotile cilia syndrome and Down's syndrome. In addition, low birth weight 65 and early environmental influences, such as maternal smoking and oxidative injury resulting in bronchopulmonary dysplasia (BPD), may increase susceptibility to environmentally related diseases such as asthma. 66 Acute lung injuries produced by common pathogenic bacteria, for example Staphylococcus aureus, are usually associated with complete recovery and subsequent normal lung function. Viral infections, however, may produce more long lasting effects; notable among these are respiratory syncytial virus (RSV), adenovirus, and chlamydia. These agents increase risk for childhood asthma 67 and may be involved in the pathogenesis of COPD later in life. 68

Regulation of breathing The elderly breathe with smaller tidal volumes and greater frequency than younger subjects, with the result that minute ventilation is unchanged. 69 The ventilatory response to hypoxia and, to a lesser extent, hypercapnea is blunted in the elderly 7~ as is the cardiac response to hypoxia. 71 Paradoxically, during exercise, elderly subjects appear more responsive to hypercapnia than younger subjects. 72 The prevalence of sleep disordered breathing increases sharply with age in adults. In the middle-aged, the prevalence of sleep apnea is --4% in females and ---9% in males. 73 In elderly subjects, the prevalence may be as high as 44%. 74 The increased prevalence of sleep disordered breathing in the elderly may, in part, result from changes in circadian sleep rhythms and altered cognitive function. 75 Diminished respiratory effort in response to upper airway occlusion and impaired perception of bronchoconstriction has been observed in older subjects. 76

Gas exchange Regional heterogeneity in the ventilation-perfusion ratio increases with age, possibly due to small airway closure in dependent parts of the lung. 77 Mean arterial PO 2 declines

with age78 as does the pulmonary transfer factor for carbon monoxide (TLCO). 76 These changes in gas exchange could result from a variety of factors including increased heterogeneity of ventilation-perfusion ratio, reduction of alveolar surface area and/or decreased density of lung capillaries. 79 A longitudinal study of middle-aged men showed that the single breath TLCO did not decline in male non-smokers over a 22-year period; however, significant reductions occurred in smokers. 8~

Hormonal status and diurnal rhythm Aging is associated with changes in hormonal status and blunting of diurnal rhythms. 75 Age-related changes have been demonstrated for growth hormone, sl glucocorticolds, 82 insulin, testosterone, s3 and pulmonary venous epinephrine, s4 The age-related decrease in growth hormone is paralleled by changes in body composition (i.e. decreased lean body mass, bone mineral density and increased visceral fat). 81 Increasing levels of cortisol with age, particularly in men, contribute to these effects. Glucocorticoid hypersecretion plays a role in accelerated aging in several animal models. 85 Declines in growth hormone, testosterone and insulin with age contribute to a generalized catabolic state. Endogenous cytokines such as interleukin-1 and TNF-~ associated with chronic inflammation in the lung in COPD may also contribute to generalized protein catabolism. s6 Protein degradation in catabolic states is mediated primarily through the ubiquitin-proteosome proteolytic pathway. 47 Induction of proteosome expression by glucocorticoids may result from down-regulation of nuclear factor kappa B. 47 These hormonal changes may in part explain the loss of lung tissue mass with age. Impaired respiratory and peripheral muscle function and reduced capacity for exercise are seen with age, particularly in patients with COPD. 83 Changes in the hypothalamic pituitary-adrenal axis occur with age and diurnal variability is attenuated, s7 Blood cortisol levels have been studied in 86 healthy men free of chronic illness who denied chronic use of medications; a single blood cortisol determination was made with the subjects in the supine position at 8:00 a.m. s2 Longitudinal analysis of the relationship between basal plasma cortisol concentration and FEV1 over an average of 4.7 years revealed a significant (p=0.008) relationship between the plasma cortisol concentration and the rate of decline of FEV1 after adjustment for age, height, smoking status, and initial FEV 1. Perhaps most surprisingly, the authors' multivariate model predicted that subjects with cortisol concentrations one standard deviation (23.3ng/ml) below the mean would experience FEV 1 declines of 71.6m1/year greater than subjects with cortisol concentrations one standard deviation above the mean. The difference was comparable to the estimated 69.5 ml/year difference between current smokers and never-smokers. These data indicate that physiological concentrations of cortisol may modulate the process responsible for the deterioration of ventilatory function with aging.

A separate study of 631 male participants in the normative aging study (age range 44-85 years) showed that 2 h urinary excretion of serotonin, but not 5-hydroxy indole acetic acid (5-HIAA), decreased with age. Current smokers secreted significantly more serotonin than never-smokers. Former smokers did not differ significantly from never-smokers in these respects, ss

Deposition, retention and clearance of particulates In children, upper airway (mouth, larynx or pharynx) deposition of fine therapeutic aerosols is greatly increased compared to adults, and deep lung deposition is correspondingly decreased, s9'9~This may explain why plasma concentrations of budesonide administered by pressurized metered dose inhaler were similar in children (2-6 years) and adults (20-41 years) when administered the same nominal dose. 91 Adult aging per se does not appear to alter the regional deposition fraction 92 but changes in inspiratory peak flow may impair deposition of drugs in the elderly. 7s Pre-existing disease, more common in the elderly, will also affect deposition patterns. Asthma and chronic bronchitis are associated with enhanced central deposition due to increased turbulence and high local velocities both during inspiration and expiration. 9~ In a model of experimental emphysema decreased retention of diesel soot in the lung has been observed. 93 Some studies suggest a slowing of clearance with increasing age, 94'95 but other studies indicate that clearance velocity is retained in old age in the absence of disease. 9~ Aging is associated with accumulation of particles and metals in the mammalian lung. A non-occupationally exposed urban dweller in North America accumulates, on average, more than 5 • 10 6 mineral dust particles per dried gram of lung tissue. 96 The concentrations of carcinogenic metals, including oxides of chromium, nickel and cadmium, increase with age. 97'98 Exogenous carbonaceous particles also appear to accumulate progressively with age, but accurate quantification has not been achieved.

SUSCEPTIBILITY OF THE A G I N G LUNG TO E N V I R O N M E N T A L INJURY Ozone Numerous studies in both humans and animals have shown an effect of age on the biological response to ozone: these include changes in pulmonary eicosanoid metabolism in rabbits and rats, 99 altered ventilatory responses to CO 2 in rats 1~176 and altered hydroxylation of salicylate in lungs of Fischer 344 rats. 1~ Overall, the cellular and biochemical effects of ozone exposure appear greater in senescent Fisher 344 rats (24 months old) compared to juvenile or adult rats. 32'1~ In humans, pulmonary toxicity to ozone appears to be dependent on the effective dose which, in turn, depends upon ventilatory rates. 1~176 Thus, in acute exposure studies younger individuals appear to show greater adverse effects than older individuals. 1~ The long-term effects of ozone on the human lung appear to enhance aging. 1~

Cigarette smoke Cigarette smoking is associated with premature aging and increased death from age-associated cardiovascular disease, cancer and COPD. It is proposed that the aging effect is mediated by the reaction of components of cigarette smoke with plasma and extracellular matrix proteins to form covalent adducts resulting in advanced glycation end products (AGE). 1~ AGEs have been implicated in a variety of degenerative diseases associated with aging. Smoking has other effects on the lung that enhance lung aging, including accelerated maturation of the fetal lung, impairment of lung growth, shortening of the plateau phase of FEV 1 and acceleration of age-related declines in FVC and FEVx .1~ These effects are also shown for cigar and pipe smoking. 1~ Smoking, by impairing host defense mechanisms, increases the risk of bacterial pneumonias, 11~a factor that contributes to the incremental declines in FEV 1 associated with COPD. Animal models provide insights into the pathogenesis of these clinical observations. The senescent mouse is more sensitive to cigarette smoke. 111 Enhanced susceptibility is also described in a genetically engineered senescence-prone mouse. 112'113 In older rats, the ability to resist oxidative damage by cigarette smoke is seriously impaired, whereas the activation of PAHs to their carcinogenic forms remains intact. 114 Thus, the balance is shifted in favor of carcinogenesis in the aged rat. Whether this imbalance exists in the human is not known. Older humans who smoke have been shown to be at greater risk for developing adult respiratory distress syndrome (ARDS) than younger smokers. 115

Particulates The relationship between inhaled particulate matter (PM10 and PM2.5) in animals and humans has been studied as a function of age. There is a general consensus that the very young and the very old are more susceptible to the effects of particulate pollution. 15 Mortality and hospital admissions for cardiorespiratory diseases are associated with increasing age. 116-12~To what extent this increased risk results from aging per se, or to pre-existing illnesses (for example, emphysema and cardiovascular disease) or cumulative increases in dust burden in the l u n g 97'98 is not clear. It is likely that many factors play a role, including the systemic effects of aging discussed earlier. The effects of particles on age-associated changes and loss of lung surface area have been studied in normal rats and rats with elastase-induced emphysema. The rats were exposed to whole diesel exhaust for up to two years at a particle concentration of 3500 Bg/m 3 beginning at 18 weeks of age. In this model, it was shown that the presence of emphysema protected the rats from particle-associated effects (measured by functional and biochemical parameters) and that there was a reduction in retained lung dust in the emphysematous animals. 93 In another study, the pulmonary responses of young (4 months) and aged (20 months) male Fisher 344 rats were compared following 3-day exposures, 5 hours/day to concentrated Boston ambient particulate

matter at an average concentration o f 100].tg/m3.TM The inflammatory responses, as measured by the concentration of cells in broncho-alveolar lavage fluid, were greater in both control and particulate-exposed young rats than in older rats. Because these were acute exposure studies, longterm effects on lung injury and remodeling were not studied. Therefore, it is not possible to determine whether the more brisk response in the younger rats would lead to a protective or damaging response in the long term. Bronchoalveolar lavage parameters and oxygen released from lavaged cells have been studied in 8 and 20+ month old male Fisher 344 rats exposed for 6 h/day to ultrafine carbon at 100~tg/m 3, ozone at 1 ppm or a combination of carbon and o z o n e . 122 The pathologic effects of the individual agents or combinations were less in younger animals compared with the older group. These short-term exposure studies indicate decreased cellular responses in older rats but do not provide information on the relationship between these findings and development of chronic disease nor on differences between young and old rats during long-term exposures.

Infectious agents Most forms of respiratory infection are more common in the elderly. Sub-optimal nutrition, deteriorating lung mechanics and depressed immunity all appear to play a role. Group housing also plays a role in the elderly, contributing to the spread of diseases associated with influenza A and LegioneUa.44 Patients aged 65 or more account for 30-80% of hospital admissions for community acquired pneumonia. Pneumococcal pneumonia is the most common cause of pneumonia in the very young and the elderly. However, the intracellular pathogen mycoplasma pneumonia is rare in the elderly, 123 possibly reflecting the changes in innate and acquired immunity associated with aging (see Tables 28.2 and 28.3). Tuberculosis causes 3• deaths per year worldwide. During the twentieth century, the incidence of tuberculosis fell from over 200/100,000 to less than 10/100,000 in developed nations primarily as a result of improvements in public health, nutrition and, to a lesser extent, by screening programs and chemotherapy. The proportion of tuberculosis in the elderly has risen in recent decades TM where it is frequently overlooked, being commonly diagnosed post mortem. 125 Age is an adverse risk factor for outcome in miliary tuberculosis which is also more common in the elderly.

N O N - N E O P L A S T I C DISEASES OF THE L U N G ASSOCIATED W I T H A G I N G Chronic obstructive pulmonary disease (COPD) Definition of COPD COPD is a term used to describe a variety of chronic lung disorders characterized by chronic airflow obstruction, the main symptoms of which are progressive shortness of breath, cough, sputum and wheeze. In North America, there

are approximately 3-17 million people with a diagnosis of COPD. It is responsible for 2.2 • 10 6 disability adjusted life years and 0.5 • 106 potential years of life lost in the United States. 126 Its impact on the economy for work loss alone is estimated at $9.9 billion 127 and total costs may reach $18 billion annually. 126 COPD is the fourth leading cause of death. In the United Kingdom, --10% of medical hospital admissions are for COPD, mostly the elderly. TM Although COPD is considered a disease of the elderly, COPD was present in 8.6% of a sample of participants in the 3rd National Health and Nutrition Examination Survey, a population that included males and females aged 18-65 (mean age 37.9 years). 128 Increasing age, male sex, white race and smoking exposure were all significant risk factors for self-reported COPD, as was employment in particular industries and occupations. 128 The COPD has three components: reversible airway obstruction, mucous hypersecretion (simple chronic bronchitis), and emphysema. There is considerable clinical overlap between asthma, chronic bronchitis and emphysema, and many subjects with a diagnosis of COPD show features of more than one of these, leading to diagnostic confusion. 129 Furthermore, the predominant manifestation may change over time. 13~ Reversible airflow obstruction, primarily associated with asthma, is found in many patients with chronic bronchitis and emphysema. Asthma also has an irreversible component. TM The natural history of COPD has been inferred from cross-sectional, longitudinal, and autopsy studies. Pathology studies have been particularly useful in identifying the underlying anatomic features of COPD (Table 28.4). An anatomical feature that is common to the component diseases of COPD is altered small airways. Studies of expiratory flow volume curves indicate that small airway obstruction is also a cardinal feature of the aging lung in non-smokers. 132 Over the years attempts have been made to unify the family of diseases that comprise COPD. These have focused on airway obstruction or airway hyperresponsiveness; however, these theories have tended to lack biological plausibility. Airway obstruction can result from such diverse processes as mucous plugs, airway thickening by edema or fibrosis, constriction of airway smooth muscle, or loss of elastic recoil due to destruction of lung parenchyma. Similarly, airway hyperresponsiveness can result from diverse causes ranging from geometric changes in airway wall dimensions (due to muscle hypertrophy, mucosal edema, and inflammation) to changes in sensitivity of airway smooth muscle to agonists. The concept that airway hyperresponsiveness and atopy underly accelerated declines in FEV 1 is the basis for the Dutch hypothesis. 13~ This approach has proven useful in epidemiologic studies TM and has highlighted endogenous factors in the pathogenesis of these diseases; however, the hypothesis does not adequately account for the important role of exogenous factors, such as cigarette smoking, or for the strong evidence that the pathogenetic mechanisms are complex and better pursued

by separating the component features. 135 One line of evidence that would point to a unifying theory for COPD is latent viral infections. 136 Double-stranded DNA viruses can persist in airway epithelial cells long after the acute infection has cleared, and viral genes may be expressed at the protein level without replication of a complete virus. Expression of the adenoviral trans-activating protein may act synergistically with exogenous agents like cigarette smoke to produce a heightened inflammatory response leading, in experimental models, to emphysema. 137 In support of this theory is the demonstration that adenoviral DNA is increased in the lungs of patients with COPD. 136

for non-smokers than smokers, after which smokers show accelerated declines in FEV 1 compared with non-smokers. The value of FEV 1 at a given point in adulthood is determined by three factors: (1) lung function attained during early adulthood, (2) duration of the plateau phase, and (3) rate of decline of lung function thereafter. 1~ Because these factors are largely unknown for a given individual, prediction equations for estimating lung function suffer from considerable error. Age, height and sex account for 40-50% of forced vital capacity (FVC), 144 the remainder is unexplained. Equations based on cross-sectional data tend to underestimate reductions in FEV 1 compared with equations based

Effect of aging on airflow limitation The decline of FEV 1 with age is due in part to loss of elastic recoil secondary to emphysema and in part to changes in airway smooth muscle tone. 78'1~ The evolution of changes in lung volumes with age is shown in Fig. 28.2, and the timeframe of these changes, as they affect airways and parenchyma, is shown in Table 28.5. The relationship between FEV 1 and age is shown in Fig. 28.3. This figure shows the different possibilities for failure to attain normal FEV 1 at age 20 and for accelerated declines thereafter. Exposure to cigarette smoke in adolescents, especially girls, 139'14~appears to affect the growth phase of the lungs as does the presence of persistent asthma. 141 In healthy nonsmokers, there appears to be a plateau phase of lung function from 25-35 years. 142'143 Most studies of aging on airflow limitation have focused on FEV 1 largely because of ease of measurement and good reproducibility. 108 Peak lung function occurs between ages 18 and 25 (Fig. 28.3), followed by a plateau phase, after which FEV 1 declines with age. The peak appears to be later

Fig. 28.2. Changes of lung volumes with age. TLC, total lung capacity; VC, vital capacity; IRV, inspiratory reserve volume; ERV, expiratory reserve volume; FRC, functional residual capacity; RV, residual volume. (Adapted with permission from Crapo RO, Crapo JD, Morris AH. Lung tissue and Capillary block volumes by rebreathing and morphometric techniques. Respir. Physiol. 1982; 49(2):175-86.)

Fig. 28.3. FEV1 plotted as a percentage of peak value at age 20 against age. Line a = healthy normal subjects, Line b=submaximal growth but normal decline, Line c= premature or early decline, Line d=accelerated decline in lung function compared with normal subjects (Line a). The horizontal line indicates levels below which clinical impairment might be anticipated. The figure illustrates the important point that agerelated declines in FEV1 have more than one cause and that more than one may be operating in a given individual. (Reproduced with permission from Weiss ST, Ware JH. Overview of issues in the longitudinal analysis of respiratory data. Am. J. Respir. Crit. Care Med. 1996; 154:$208-11 .)

on longitudinal study data. 1~ There are many prediction equations for FEV1; some assume a linear relationship with age, others do not. 146 In non-smokers there is a steady decline in FEV 1 with age 147-15~ (Table 28.6). Smoking accelerates this age-

dependent decline in FEV1.150-152 Age-related reductions in FEV 1 were studied in 1397 men and women aged 51-95 years to determine the influence of age and smoking cessation on FEV1.153 The decreases in FEV1 per year reported by Frette et al. 153 among never-smoking men (34ml/y) and

never-smoking women (28ml/y) are similar to values reported for subjects of the same age in other cross-sectional or longitudinal studies, with reductions of 28-34 ml/y for men 154-156 and 21-32 ml/y in women. 154'155 The results from some longitudinal studies of FEV 1 with age in smokers and non-smokers are shown in Table 28.6. From these data, it appears that, in moderate to heavily smoking men, FEV 1 declines by --15 ml/y more than in non-smokers. Comparable declines, based on normalized data, are seen for women. Only a small proportion of smokers appear to develop clinically significant airway obstruction. Some studies of annual declines in FEV 1 as a function of age have noted a slowing of the declines in FEV1 in older men and women, both smokers and non-smokers. 153'157'15s Other studies have reported an accelerated decline of FEV 1 with age. 159 An apparent positive relation of FEV 1 in the oldest (81 years and older) current smokers has been noted; 153'154 however, the numbers of subjects in the older categories are small and thus the population probably shows a survivor selection bias. Another problem with studies in the elderly is the large proportion of men and women unable to perform ventilatory function tests that meet ATS standards. Spirometry test failure is an index of poor health status and has been shown to be associated with a lower FEV1, a faster rate of annual loss of ventilatory function, increased respiratory symptoms and excess mortality. 7s

Genetic and familial factors in decline in lung function Epidemiologic studies have shown that lung function (FVC, FEV1) is predictive of longevity. 1~176 The relationship between lung function and mortality is to all causes, not just to lung specific diseases, x'162 Simple lung function tests appear to provide important information on the general health of elderly subjects and offer a good overall marker of aging. These relationships remain after known risk factors, such as smoking and occupational exposures, are taken into account. 1 The heritability of lung function has been studied in heterozygotic and monozygotic twin pairs (aged 18-34 years) living in Australia. The heritability in females was ---0.8 and slightly lower in males, ---0.6. The difference between males and females was attributable to environmental influences. 163 This study showed that ---9% of the variance in women was associated with Pi polymorphism. No effect of the Pi locus was found in males. A similar twin study from Sweden gave heritability estimates of 0.48 and 0.67 for VC and FEV1, respectively, but no gender differences were noted. TM Genetic effects appeared equally important in predicting FEV 1 in older and younger populations. Other studies have confirmed the heritability of pulmonary function. 164-166 Pulmonary function has even been related to longevity of parents. 167 Putative genes identified to date include alpha-l-antitrypsin (tzl-AT), tumour necrosis factor tz (TNF-~) and surfactant protein B (SP-B) genes. 168

Effect of smoking cessation on age-related decline in lung function Smoking cessation has been shown to reverse the accelerated decline in lung function associated with cigarette

smoking. Intermittent smoking is associated with rates of decline between those of active current smokers and nevers m o k e r s . 146'156'169 Age of quitting has a dramatic effect on subsequent declines in lung function. 153 Smokers (men and women) who quit before 40 years of age had age and height adjusted FEV 1 that did not differ from never-smokers; those who quit between the ages of 40 and 60 had greater FEV 1 values than current smokers but lower than those of never-smokers. However, in smokers who quit after age 60, FEV 1 was similar to that of current smokers.

Risk factors for accelerated declines in lung function Of the attributable risks for COPD, cigarette smoking accounts for approximately 80-90%. 142 By contrast, heavy habitual marijuana smoking does not appear to accelerate FEV 1 decline with age. 17~A longitudinal study of 1171 randomly chosen U.S. steel workers demonstrated that age, weight gain, smoking, trauma, pneumonia and a history of allergy, asthma or hay fever were independently related to the risk of accelerated declines in FEV1.171 Occupational dust exposure is another well-recognized cause of accelerated declines in FEV 1. In a review of 13 studies between 1966 and 1991 encompassing four different cohorts of miners, a relationship was observed between lung function decline and dust exposure when corrected for age and other risk factors; 172 other reviews also have arrived at the same conclusion. 42'43 Air pollution has also been linked to accelerated rates of decline of FEV1.173 Asthma 174 and airway hyperresponsiveness have been shown to be independent risk factors for accelerated declines in FEV 1 in a number of large cohort studies. 142'175-178 On average, having airway hyperresponsiveness adds ---10ml to the annual decline in FEV1; 142 the effect is largest in elderly subjects. 176 No interaction between airway hyperresponsiveness and smoking has been demonstrated. 134'x42 Chronic mucous hypersecretion is also an independent risk factor for accelerated declines in FEV1.179 Gender does not appear to affect rates of decline. 156 Studies of Innuit indicate that adoption of a modern lifestyle, particularly the use of snowmobiles, increases the rate of decline of FVC and FEV1; this decline was less in those who exercised regularly. 18~

Markers of accelerated decline in lung function Urinary excretion of the amino acid, desmosine (DES), a specific marker for degradation of mature cross-linked elastin, is elevated in healthy smokers compared with healthy non-smokers. TM A nested case control study of elastin and collagen degradation in current smokers with and without rapid decline of lung function showed that rapid decline in pulmonary function was associated with increased urinary excretion of desmosine. 182 Antioxidant gene polymorphisms may also serve as markers for rapid decline in lung function. 183 Blood elastin peptide has not proven useful as a predictor of rate of decline of FEV1, TM but may have some value in evaluating remodeling in emphysema and fibrosis. 5~

Emphysema

Pathogenesis In humans, cigarette smoking is by far the most common cause of emphysema. Dusts and fumes associated with specific occupations are also major causes of emphysema in selected populations. 43'185 Emphysema due to environmental agents, for example smoking and coal mine dust exposure, is usually centriacinar in type. 186 There are several genetically determined forms of emphysema, the most common of which is oil-AT deficiency. ctl-AT exists as more than 70 biochemical variants (the PI system) which are inherited as autosomal codominant alleles. 187 Homozygosity for t~I-AT deficiency occurs in about 6% of the population. Affected individuals often develop emphysema at an earlier age (<40 years) than is seen in the general population in which clinically significant emphysema rarely develops before 50 years of age. o~I-AT deficiency is associated with panacinar emphysema involving all lobes of the lung. In contrast, environmental emphysema caused by cigarette smoke or occupational dusts is usually more pronounced in the upper zones and starts in a centriacinar location, o0-AT deficiency impairs the antiproteolytic defense mechanisms of the lung, predisposing to protease damage of the connective tissue framework. The development of emphysema in t~I-AT is associated with age and exposure to environmental agents, including cigarette smoke and workplace dusts and fumes. Subjects who are heterozygous (PiMZ) for o~I-AT deficiency are found in - 1 0 % of the population. These individuals are also at risk of developing pulmonary emphysema, lss A ten-year study of 28 PiMZ subjects compared to 28 normal PiMM subjects showed significant declines in FEV 1, TLCO and static transpulmonary pressures. Total lung capacity and residual volume were increased in the heterozygous group and trypsin inhibitory capacity was decreased (0.65 _ 0.17 mg/ml) compared to 1.52 _ 0.3 mg/ml in the PiMM group. Changes in the PiMZ group exceeded the values expected as the result of physiologic aging.

Asthma

Mortality, morbidity and severity Asthma in the elderly is often severe and refractory to therapy 6 and is frequently underdiagnosed and undertreated. 189 Mortality rates appear increased in elderly asthmatics. 6 A Japanese study showed that the median age for asthma-related deaths was 60 years; males predominated up to 60 years, and female deaths were greater thereafter. 19~ Although more deaths from asthma occur in older populations, children and young adults have shown the greatest increase in death rates in recent decades. TM Data on the prevalence of asthma among the elderly are scant. Asthma was reported by 4.1% of all adults in Michigan, 192 and by 11.1% of men and 9.1% of women aged 65-95 years in Arizona. 189,193 Bronchial hyperresponsiveness (BHR) has been described in infants and very young children, and it has

been postulated that BHR is present in all children but that genetic and environmental factors determine which children will lose their BHR. TM In a longitudinal study of children, BHR was related to slower growth of FEV1 TM and increased risk for developing asthma. 195 BHR is related to blood eosinophilia in the young and the elderly. 177'196-198 A pathologic study designed to determine the effect age and duration of disease on airway wall remodeling in fatal asthma compared a group of young individuals who died of asthma (age range 20-23 years) with a group of older individuals who died of asthma (age range 40-49 years). 199 The study showed an increase in airway wall area, and smooth muscle thickness in the older asthmatics compared to the younger group. These data support the concept that mesenchymal remodeling is a progressive process and accounts for the irreversible airflow obstruction demonstrated in longitudinal studies of asthma. 131'2~176176 To what extent these changes are a function of age or duration of disease cannot be determined from these studies. A morphologic change in asthma that does not appear to be influenced by age or duration of asthma is subepithelial collagen thickness. 199'2~176

Atopy Age-related differences in the manifestations of asthma are well recognized. Asthma is influenced by geometric factors associated with the developing lung, hormonal influences in adolescence and age-related changes in IgE and specific immune and non-specific inflammatory responses. Allergen priming begins in utero. House dust mite-specific T-cells have been identified in cord blood, z~ A longitudinal study of T-helper (Th) lymphocyte cytokine responses was performed prospectively in blood mononuclear cell responses from birth to two years in atopic and non-atopic infants. 2~ At birth, both groups showed Th 2 skewed allergenspecific responses to house dust mite antigen, which were greater in the non-atopic group. In the first year of life, there was a rapid down-regulation of the Th 2 response in the non-atopic group, whereas the atopic group showed consolidation of their Th 2 responses. This study supports the hypothesis that patterns of immune deviation in the first year of life determine atopic status in later years. Sensitization to allergens is also age dependent. Atopy, as expressed by serum immunoglobulin E (IgE), is a strong predictor of asthma, 2~176 even in the elderly. 6 Serum IgE declines with age 2~176 and is influenced by race, 2~176 smoking history, 21~ occupation, 211'212 and exposure to aeroallergens. 211 Reductions in IgE with age are associated with decreased eosinophilia. 13~ The relationship between serum IgE and age is different for men and women. Studies have repeatedly shown that in women, IgE levels fall with age, whereas in men, IgE levels remain relatively stable. These changes are seen even when adjusted for the effects of smoking and environmental factors. TM The relationship between serum IgE and the rate of decline of pulmonary function in the general population has produced conflicting

results. Several studies have shown an inverse relationship between serum IgE and rate of decline in FEV1; 176,213-215 however, other studies failed to show such a relationship 177'216 or revealed that the effect was confined to individuals with a diagnosis of asthma. 217 Specific IgE is also related to age, 218'219 smoking 22~ and occupational exposures. 211 Specific IgE levels decline with age in both men and women. 211 Skin test reactivity to common aero-allergens has been shown to be a significant predictor of annual rates of decline of FEV 1 and FEV1/FVC ratio. 176'221 In a study of rates of decline of lung function in 1025 men using regression models, the excess rate of decline in FEV 1 associated with skin test reactivity to house dust, mixed grasses, mixed trees and/ or ragweed was 9.45 ml/y. 222 Surprisingly, no relationship was found between serum IgE level and annual FEV 1 decline in a longitudinal analysis of 790 men from the same cohort. 177 There is evidence that environmental agents, particularly diesel exhaust particulates and cigarette smoke, are capable of augmenting IgE immune responses 21~ and pro-inflammatory cytokine production. 223 A longitudinal study of 778 elderly men aged 41-86 years showed that current smokers with at least one positive skin test reaction to common aero-allergens displayed significantly greater methacholine dose-response slope than smokers with negative skin test results. A relationship between skin test positivity and methacholine responsiveness was also seen in non-smokers, but the effect was not statistically significant. The data indicate that smoking and atopy may act synergistically to increase airway hyperresponsiveness in an older male population. A study of 250 men and women aged 65-91 years showed that, after allowance for age and gender, serum IgE and smoking interacted synergistically as risk factors for airflow obstruction. 214 This synergism was also observed in subjects who showed no evidence of airway hyperresponsiveness to inhaled methacholine. These findings indicate that the role of IgE in the pathogenesis of airflow obstruction is not confined to asthmatics.

Triggers Two classes of environmental factors can trigger an asthma attack: sensitizing agents (e.g. house dust mite, cat dander) and irritants (e.g. fine particles, gases and fumes). The dominant triggers vary with age; asthmatics aged 2-20 years were more likely to have asthma triggered at night and by respiratory infections, whereas older asthmatics more commonly reported exposure to allergens and exercise as triggers. 224 Other triggers, such as cigarette smoke exposure, air pollution, stress or changes in weather showed no relationship to age. 224 A cross-sectional study of asthmatics in Holland showed that the number of specific agents that asthmatics reported triggering their asthma and the degree of reaction to those agents increased until 25 years of age and then started to decline. Furthermore, during childhood positive tests for

indoor allergens predominated, but after age 15 sensitization to outdoor allergens (such as grass pollen) were more common. 225

C O N C L U S I O N S A N D FUTURE DIRECTIONS Aging appears simple, but in reality the situation is highly complex. It is not defined by one variable, time, but by a multitude of processes, which appear to interact. The latter include genes, environmental stressors and pre-existing disease. This chapter has provided an overview of these factors and their interactions. Much, however, remains to be discovered. There is a remarkable shortage of information on the effects of aging on the adult human lung. 168'226 Many longitudinal epidemiologic studies designed to detect effects of environmental pollutants on lung disease have noted or controlled for age-related effects. Few of these have tested for interactions between the environmental factor of interest and age. Where this has been done (for example, in studies of decline of FEV 1 associated with cigarette smoking or smoking cessation), non-linear effects have been noted. Useful additional information on the interactions between environmental factors and aging might be obtained by re-analyzing existing databases. There is a need for more longitudinal studies of hormonal status and age-related decline in lung function; for example, the findings of Sparrow et al. 82 need replicating. Their observation that a plasma cortisol concentration one standard deviation below the mean was associated with an annual decline in FEV 1 equivalent to current smoking is quite remarkable. Furthermore, endocrine deficiencies are potentially treatable. Although the annual decline of FEV 1 is an accurate marker of lung aging and has some predictive value for identifying individuals at risk for pulmonary impairment, there is a need to develop biochemical tests for identifying high risk individuals. In this regard, breath analysis for indices of oxidative stress, for example, carbon monoxide (produced from the stress protein, heme-oxygenase) and ethane (an end product of lipid peroxidation) look promising. 227 There is a need to determine the underlying structural basis for the declines in FEV 1. Emphysema and changes in airways are only poorly correlated with FEV1228 suggesting that other, as yet, undefined factors may be important. There is a need to determine the cause of the variability in rates of decline of FEV 1 with age in both smokers and nonsmokers. 16s Several genetic and environmental risk factors for rapid decline of FEV 1 have been identified; however, the majority of the risk cannot be attributed to known factors. 168 There is a need to correlate new insights from COPD research into studies of the normal aging process, specifically the roles of proteases, oxidant injury, viral infection and apoptosis. 16s Finally, there is a need for studies of lung function and anatomy in individuals with exceptional longevity.

ACKNOWLEDGEMENT Special thanks to Marnie Cudmore and T a m e r E1 Mays.

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Note: The italicized page numbers refers to tables and figures.

Acid vapors, 338-9 Acute lung injury (ALI), 364-6 Acute respiratory distress syndrome (ARDS), 242, 364-6, 382 Adenosine, 209 Adhesion receptors, 82, 306 Adrenergic agonists, 156 Adrenocorticotropic hormone (ACTH), 158 Adventitial fibroblasts, 84 Aging: as natural process, 231 body mass, 380 complexity of, 389 deposition, retention, clearance of particulates, 382 dog, 228-31 effect of pollutants on, 370 effect on airflow limitation, 384-5 effects on surfactant system, 363-4 factors influencing susceptibility to lung disease, 377-82 gas exchange, 381 hormonal status/diurnal rhythm, 381 influence on genetic susceptibility, 285 life span characteristics, 214-15 mammalian, 213-14, 230 mouse, 213-19 non-neoplastic diseases, 383-9 nutrition, 380, 383 physical activity, 380 rat, 219-28 regulation of breathing, 381 repair differences, 355 susceptibility to environmental injury, 382-3 Agranular endoplasmic reticulum (AER), 23-5 Air pollution: ambient, 333-4, 336-7 and asthma occurrence, 339-41 chronic effects, 334-5 common, 367 effect of hypoxia/altitude on pulmonary surfactant, 370-1 effect on aging/diseased lung, 370 effects on normal lung function growth, 335 effects on pulmonary surfactant, 367-70 environmental toxicants, 335 NO z, 337-8 ozone, 337 particulates and acids, 338-9

respiratory effects of, 334-9 susceptibility to, 339 tobacco smoke, 335-6 vehicle exhausts, 338 Airway epithelium: branching morphogenesis, 13 composition of the wall, 13 development, 13-28 differences in phenotypic expression, 13-15 enlargement, 13 epithelial differentiation, 17-25 regulation of differentiation, 26-8 Airway innervation, 33 anatomy, morphology, distribution, 33-6 canalicular stage, 39 density, 43 efferent nerves, 43-4 function during fetal life, 41 mapping, 36-41 muscarinic receptors, 46-8 origin (fetal mouse lung), 346 postnatal lung, 41-4 pseudoglandular stage, 37, 39 pulmonary neuroendocrine cells, 44 and reflex control of ASM, 45-6 saccular stage, 39, 41 Airway smooth muscle (ASM), 33-5, 37, 41 activation of, 47-8 functional consequences, 45 muscarinic receptors, 46-8 ontogeny, 45 orientation of bundles, 44 postnatal, 45 prenatal, 45 reflex control in newborn lung, 45-6 Airways: preterm birth, 241 resident immune/inflammatory cells, 171-2 Allergens, 296 dizygotic (DZ) twins, 322 food, 323 genetic influence on response to exposure, 314 hygiene hypothesis, 315 immune modulation by helper T-cell subsets, 311 influence of in utero exposure on atopic phenotype, 312-14

Allergens - contd. interaction with environmental factors during development, 316-17 juvenile period exposure, 315-16 maternal influences on immune status of fetus, 312-14 monozygotic (MZ) twins, 322 neonatal exposure, 314-15 postnatal exposure and development of atopy, 323-4 principles of sensitization, 311, 317-18 role of dietary factors, 314 role of infectious diseases during neonatal period, 314 sensitization to, 388 in utero, 388 Allergic rhinitis, 326 Altitude: acclimatisation/adaptation to, 267, 268 adolescent/adult, 269-71 effect on pulmonary surfactant, 370-1 pregnancy, the fetus, postnatal growth, 268-9 reduced inspired PO2, 267 ultra-structural/cellular considerations, 271-3 Alveolar-capillary recruitment, 191-2 Alveolar duct, 55, 377 Alveolar epithelial cells (AEC), 56-60, 66, 136, 149, 201, 202, 364, 370 Alveolar formation, 258 Alveolar hypoxia, 194 Alveolar interstitium, 61-3 Alveolar macrophages, 171, 173, 228 Alveolar myofibroblasts (AMF), 64-5 Alveolar oxygen tension, 93 growth of vascular units in low, 96 mural cells/change in, 96, 98-9 Alveolar sacs, 55 Alveolar septum, 65-6 Alveolar type I cell, 3, 9, 59, 304 Alveolar type II cell, 3, 9, 59, 240-1,243, 303, 304, 364 Alveolarization, 17, 68, 240-1 chronology of morphologic changes, 57-8 developmental defects, 66 effect of nicotine exposure on, 302, 304-305, 306 growth, 58 interspecies comparison, 58 regulation of, 9, 66-7 remodeling of alveolar wall, 58-9 secondary septation, 57 Angioblasts, 105-106 Angiogenesis (expansion), 84 Angiogenesis (intussusceptive microvascular growth/splitting), 82, 84 Angiogenesis (sprouting), 82 Antigen presenting cells (APCs), 173-4, 324 Antioxidant enzyme system, 177, 182-3, 379 activity in aging mouse, 219 development, 178-9 Apoptosis, 65-6, 305-307 Asthma, 277, 284, 322, 323, 366, 381,383 and air pollution, 339-41 allergic, 311,314, 317-18 diesel exposure, 326-8 effect of exposure to ETS, 294-6 and ETS, 325 mortality, morbidity, severity, 388 ozone exposure, 326 triggers, 389 Atopy, 388-9 diesel exhaust, 326-8 gene mapping, 322 genetic factors, 321-2

influence of cytokines, 321 innate immunity, 322 lifestyle, 324-8 ozone exposure, 325-6 postnatal allergen exposure, 323-4 prenatal immunological factors, 322-3 tobacco smoke, 325 Autonomic agonists, 154-5 Autonomic neurotransmitters, 156 Basement membrane zone (BMZ) development, 77-8 functions, 76-7 structure/composition, 75-6 BCG vaccination, 324 ..... Bioactivated compounds, 345-6 dichlobenil, 346 and neonates, 346 postnatal, 346 and selective lung injury, 346 in utero exposure, 346 Bone morphogenetic protein (BMP), 8, 113 Branching morphogenesis, 5, 56, 177 early, 15-16 epithelial-mesenchymal interactions, 5 FGF signaling as driving mechanism, 5-8 left-right asymmetry, 8 self-similarity characterization, 187 signaling molecules, 7-8 Bromodeoxyuridine (BrDU), 347 Bronchial hyperresponsiveness (BHR), 388 Bronchiole-alveolar duct junction (BADJ), 216 Bronchoalveolar lavage (BAL) fluid, 363, 365, 369 Bronchopulmonary dysplasia (BPD), 45, 66, 159-60, 172, 244-5, 246, 262, 354, 380, 381 Bronchus-associated lymphoid tissue (BALT), 171-2 Calmodulin (CAM), 47 Canalicular stage, 56, 239-40 cholinergic neurons, 39 neurotransmitters, 39 spatial separation of ganglia, 39 vasoactive intestinal peptide/substance P positive fibres, 39 Cancer, 277, 278-9, 283, 382 Carbon monoxide (CO), 111,209, 367 Carcinogenesis, 378 Catalase (Cat), 179, 219 Cell differentiation, 177 alveolar type I cell, 59 alveolar type II cell, 59 surfactant phospholipids, 59-60 surfactant proteins, 60-1 Cell phenotypes, 14 Cellular homeostasis, 378 Ceruloplasmin, 179 Chemokines, 172 Children's Health Study, 337, 338, 339-40 Cholesterol, 59-60, 149, 150 Cholinergic agonists, 156 Chronic beryllium disease (CBD), 283 Chronic bronchitis, 301,381,384 Chronic lung disease (CLD), 159-60, 244-6, 262 Chronic obstructive pulmonary disease (COPD), 272, 277, 334, 381,382, 383-4, 387 Cigarette smoking see environmental tobacco smoke; maternal nicotine exposure; nicotine Clara cells, 3, 182 adaptive response to chronic hypoxia, 371 differentiation, 27

environment toxicants, 346, 347, 348 exposure to ETS, 293 mouse, 216 NEBs, 44 repair of lung injury, 354 secretory protein, 23, 24-5 Collagen, 76, 77, 78, 226-7, 260, 380 Conducting airways, 55 Congenital diaphragmatic hernia (CDH), 93, 134, 188, 347-8 Continuous positive airway pressure (CPAP), 242 Corticosteroids, 195, 243, 347 beneficial effects, 143 effects on fetal lung, 142-3 Cystic fibrosis, 366-7 Cystolic Ca2+, 47-8 Cytochrome P450 (CYP) monooxygenases, 23-5, 179-80 Cytokines, 172, 325, 381 allergen reactions, 312, 316 development of atopy, 321 Cytoplasmic glycogen, 20, 23 Cytoplasmic plaque proteins, 82 Dendritic cells, 173-4 Dexamethasone, 246 Diesel exhaust (DE), 316-17, 326-8, 338, 369, 389 Diesel exhaust particles (DEP), 327-8 Dipalmitoylphosphatidylcholine (DPPC), 59, 150, 365 Disaturated phospholipids (DSP), 150 Distal respiratory system (trachea, bronchi, lungs), 177 DNA, 135, 137, 378 Dog aging: body mass, 214-15 general characteristics of lungs, 228 human lung similarity, 231 tracheobronchial tree, 230 Drosophila, 5, 6, 7, 8

Ductus arteriosus (DA), 112 closure at birth, 207-208 -Elastic laminae, 84-6 Elastin, 62-3, 245, 259-60, 380 Emphysema, 68, 301, 381,387-8 End-expiratory lung volume (FRC), 209 Endothelial cells, 107, 228 Endothelial channel formation, 87 Endothelial nitric oxide synthase (eNOS), 108-11, 113, 245 Endothelin-1 (ET-1), 111, 114, 273 Endothelium-derived hyperpolarizing factor (EDHF), 111 Energy metabolism, 302-304 Environment: effect on allergens, 315-17 effect on disease, 277 effects of air pollution, 333-41 effects of altitude, 267-73 effects on lung aging, 377-8, 382-3 effects on pulmonary surfactant development, 158-60 Energy metabolism, 182 influences on mature surfactant system, 367-71 interaction with genes, 278-9, 280-3 toxicant repair, 356-9 Environmental tobacco smoke (ETS), 254, 291,345, 369 active/passive exposure, 335 development of atopy, 325 effect of cessation on age-related decline, 387 effect on aging, 382 effect on lung function growth during childhood, 335-6 effects on asthma, 294-6, 325 effects on development of allergy, 296 effects on sudden infant death/obstructive apnea, 296-7

inhibition of fibroblasts, 304 injury/repair response, 357-8 lung function changes in children, 294 postnatal respiratory symptoms, 293 in utero respiratory effects, 292 see also maternal nicotine exposure; nicotine Enzyme systems, 15, 177-82 Epidermal growth factor (EGF), 26, 27-8, 195, 196, 354 Epithelial cells: dog, 230 mouse, 216 rat, 220-6 Epithelial differentiation: embryonic period, 19 loss of, 26-7 mesenchymal interactions, 26 midcanalicular stage, 20 mucus glycoprotein biosynthesis and secretion, 21-2 overview, 17-18 pattern in bronchioles, 23-5 pattern in trachea and bronchi, 18-23 postnatal period, 21 and proliferation, 22 pseudoglandular stage, 19-20 regulation, 26-8 and repopulation of epithelial cells, 22-3 ultrastructural features, 19 and vitamin A, 27 Epoxide hydrolase (EH), 181-2 Ethanol, 348 Ethylnitrosourea, 348 Extracellular matrix (ECM) proteins, 107, 137-8, 259, 306 Extracellular signal-regulated kinase (ERK), 141 Extremely low birth weight, 246 Fetal breathing movement (FBM), 131,136, 188, 206 detection of, 209 during active labor, 209 during parturition, 209 and fetal growth restriction, 257-8 Fetal growth restriction (FGR) airway development, 260 alveolar formation, 258 during childhood, 255 during infancy, 255 effects in the adult, 255-6 effects of hypoxia, 261 effects on surfactant system, 260 expansion of fetal lung, 257 experimental evidence, 257-61 extracellular matrix, 259-60 fetal breathing, 257-8 fetal causes, 254 fetal lung growth, 258 lung defense, 261 maternal causes, 253-4 micronutrients, 261-2 nutrition after birth, 256-7 and organ development, 254-5 placental causes, 254 pulmonary blood-air barrier, 258-9 respiratory effects following preterm birth, 256 Fetal hypoxemia, 261 Fetal hypoxia, 158 Fetal lung development, 131-2 clinical evidence, 134 clinical treatments for inappropriate, 143-4 DNA content, 135, 137 experimental evidence, 135-6

Fetal lung development- contd. mechano-transduction mechanisms, 137-8 regulation of basal degree of expansion, 132-4 response to changes in expansion, 135 role of breathing movements in growth, 136-7 role of endocrine/other circulating factors, 142-3 role of expansion in growth, 134-6 role of fluid, 132-4 role of growth factors, 138-42 role of physical factors in regulating growth, 132 stretch/distension, 157 and tracheal obstruction, 135-6 Fetal lung maturation, 201-202 Fibroblast growth factor (FGF), 5-8, 27-8, 77, 86-7, 106, 141 Fibroblasts, 84, 86, 259, 304 Fibrosis, 283 Flavin-containing monooxygenases, 182 Fluid transport see solute transport Following environmental lung injury in children, 358-9 toxicity studies in mature/developing lung, 355-8 Forced expiratory volume in one second (FEV1), 335, 336-7, 338, 379, 380, 381,384-7, 388 Forced vital capacity (FVC), 335, 336, 337 Ganglia, 35-6 long preganglionic fibres, 43-4 separation, 39, 41 short postganglionic fibres, 43-4 Gas-exchange area, 38, 55 Gene-environment interaction, 278-9, 280-3 Genetic factors, 277-9, 285-6 acute lung injury, 280-3 contribution of age to airborne pollutants, 285 development of atopy, 321-2 effect on decline in lung function, 385, 387 identification of candidate disease susceptibility genes, 279-80 inter-individual differences, 280-3 meiotic mapping, 279-80 quantitative trait locus (QTL), 279 response to allergens, 313 susceptibility to environmental stimuli, 280-3 susceptibility to occupational lung disease, 283-5 Glandular mucus cells, 17 Glial-derived neurotrophic factor (GDNF), 36 Glucocorticoid receptor (GR), 202 Glucocorticoids, 66-7, 155-6, 201-202 Glucose, 302-303 Glucose transporter (GLUT), 303 Glucuronyl transferase, 182 Glutathione (GSH) system, 179, 219 Glutathione peroxidase (GPx), 178 Glutathione S-transferase (GST), 180-1,279, 285 Glycoproteins, 82 Granular endoplasmic reticulum (GER), 19, 20, 23, 25 Growth factors, 82, 195 role in fetal lung development, 138-42 Growth hormone (GH), 143 GTPases, 47 Heparin-binding epidermal growth factor, 107 Hepatocyte growth factor, 194 High altitude pulmonary edema (HAPE/mountain sickness), 272-3 Homeodomain transcriptional factor, 26 House dust mite (HDM), 315-16, 388 Hox proteins, 8 Hyaline membrane disease (HMD), 159 Hygiene hypothesis, 315, 323-4 Hyperoxia, 94-5, 98, 346, 347, 370, 371 Hyperventilation, 155

Hypoxemia, 158, 209 Hypoxia, 113, 155, 158, 259, 261,268, 271,272, 370-1 Hypoxia-inducible-like factor (HLF), 347 IgE antibodies, 311,312, 313, 315, 317, 388-9 Immunological factors, 322-3 Infectious agents, 383 Innate/acquired immunity, 379-80 Interleukins (ILs), 26, 87, 174, 283, 312, 314, 321,322, 324, 381 Interstitial fibroblasts (IF), 61, 84, 96, 98 elastin, 62-3 migration, 62 proliferation, 61-2 reduction in, 65 structural proteins in alveolar wall, 62 Interstitial mesenchyme, 61 Intracellular protein, 23-5 Intrauterine growth retardation (IUGR), 158 Kinase activity, 303, 306 Kyphoscoliosis, 188 Lamina densa, 76-8 Lamina lucida, 75, 76 Lamina reticularis, 75-8 Lectin reactivity, 16, 22 Leukocytes, 173, 242 Ligands, 86-7, 354 Lipid interstitial cell (LIC), 63-4 Lipids, 149-51 Lung antioxidant defenses, 379 Lung development: alveolarization, 240-1 branching morphogenesis, 56, 239 canalicular stage, 56, 239-40 fetal stages, 55--6 formation of trachea, 4-5 impaired nutrition, 257 initial, 3-4 lobular organization, 35 lung bud formation, 55-6 mechanical forces, 187-9 mesenchymal-derived structures, 3-4 molecular regulation of alveolarization, 9 molecular regulation of lung bud initiation, 4 multi-event process, 177 nicotine exposure, 302-307 onset, 3-5 preterm birth, 239-46 primary buds, 3, 4 pseudoglandular stage, 57, 239 regulators, 6 relationship with body mass, 214-15 retinoids, 4, 5 saccular stage, 56, 240 signaling molecules, 7 thyroid transcription factor 1 (Ttfl), 4 Lung diffusing capacity (DL) measurement, 191 source of physiologic compensation, 191-3 Lung function: effect of genetic/familial factors, 387 effect of smoking cessation on age-related decline, 387 effects of ambient air pollution, 336-7, 338-9 effects of environmental toxicants, 335 effects of NO2, 337-8 effects of ozone, 337 effects of particulates and acids, 338 effects of tobacco smoke during childhood, 335-6

impairment during prenatal/postnatal growth, 335 markers of accelerated decline, 387 normal/reduced growth rates, 335 risk factors for accelerated declines, 387 Lung inflammation/injury: acute, 282-3 aging/environmental susceptibility, 382-3 dichlobenil, 346 early, 380-1 and ETS, 304 0 3 induced, 281,282 particle-induced, 281-2 repair, 353-9 Lung liquid, 242 alterations in fetal, 158-9 clearance before birth, 202-203, 204 clearance mechanisms during labor and birth, 203 impacts on, 134 integral role in development, 131 re-absorption at birth, 203 role of chest wall in maintaining volumes, 133-4 role of trans-pulmonary pressure gradient in regulating, 132-3 Lung vascular growth/development, 105-107 angiogenesis, 105-106, 107 vasculogenesis, 105 Lymphocytes, 311 after birth, 171 childhood into adulthood, 169-70, 171 neonatal, 170, 172 trafficking, 172-3 Lysophosphatidylcholine (LPC), 150, 370 Maternal disease: alcohol (ethanol) intake, 254 cigarette smoking, 254 diabetes, 253-4 hypoxemia, 254 infectious, 254 renal insufficiency, 254 thrombophilia, 254 vascular, 253 Maternal nicotine exposure, 292 absorption, distribution, metabolism during pregnancy, 301-302 and development of asthma, 325 effect on energy metabolism, 302-304 effect on lung structure, 304 effects during gestation/lactation on placenta/fetal growth, 302 effects on lung development, 302-305 nicotine/cell signaling: apoptosis/lung development, 305-307 see also environmental tobacco smoke; nicotine Matrix remodeling, 380 Maximum mid-expiratory flow (MMEF), 335, 336, 337 Mechanical forces: after pneumonectomy, 189-90 airway growth, remodeling, function after pneumonectomy, 194-5 effect of nicotine exposure on, 307 effect of stretch of alveolar septa in vitro, 188 importance of lung strain as signal for septal growth, 193-5 lung development, 187-9 lung-thorax interaction, 188 post-pneumonectomy compensatory response, 190-3 postnatal lung stretch in vivo, 188-9 regulatory patterns during developmental/compensatory growth, 196 Mechanical ventilation, 242-3, 246 Mechano-transduction mechanisms: potential cytoplasmic second messenger systems, 137-8 potential role of growth factors, 138 role of ECM/"outside-in" cell signaling, 137

P450-Mediated toxicity, 355-8 Meiotic mapping: association, 280 linkage, 279, 280 Mesenchymal cells, 6-7, 107, 305 Micronutrients, 261-2 Mineral dust pollutants, 369 Mitogen-activated protein kinase (MAPK), 141-2, 306 Morphogenesis, 3-9 soluble factors, 3 transcription factors, 3, 4, 8 Mouse: allergen exposure, 316 alveolarization, 58 body mass, 214 life span characteristics, 215 senescence-accelerated mouse (SAM), 215, 218, 364 tight-skin/pallid, 66 Mouse aging: antioxidant enzyme activity, 219 changes in lung volume, 215-16 lung parenchymal structure, 216-18 macrophages, 219 tracheobronchial airways, 216 Mucosal innervation, 41 Mural cells: and change in oxygen tension, 96, 98-9 development, 87-8 origin/role, 84-6 Muscarinic receptors: classification and roles of, 46 control of ASM excitability via Ca+2 sensitivity, 47 development/pharmacological antagonists, 46-7 importance in asthma, 46 monomeric GTPases, 47 PAK and/or ROK as cause of Ca+2 sensitization, 47-8 prejunctional, 46 Musculoskeletal disorders, 93, 134 NADH CYP, 179-80 Naphthalene, 355 National Maternal and Infant Health Survey (USA), 296 Nerves: in developing airways, 43-4 Neural crest-derived cells (NCCs), 34-6 Neural tissue, 33-4, 35-6 Neuroendocrine bodies (NEBs), 44 Neuroendocrine cells (NECs), 44 Neutrophils, 173, 242 Nicotine: daily intake, 301 maternal exposure, 301-307 uptake of, 301 see also environmental tobacco smoke; maternal nicotine exposure Nitric oxide (NO), 108-11, 112-13, 114, 195 NO-cGMP cascade, 112, 114 Nitrofen, 348 Nitrogen dioxide (NO2), 333, 334, 337-8, 367, 368 Nutrition, 245, 253 and aging, 380, 382 causes of restricted fetal growth, 253-5 effects on developing lungs, 257-61 fetal hypoxemia, 261 prenatal growth, 255-6 restricted growth after birth, 256-7 restriction after maturity, 262-3 role of micronutrients, 261-2 Nutritional emphysema, 262, 381

Obstructive apnea, 296-7 Occupational lung disease, 283-5 Oligohydramnios, 134 Oxidant gases, 346-7 Oxygen toxicity, 245 Ozone (03), 281,316, 325-6, 333, 334, 337, 356-7, 368-9, 382 P21-activated kinase (PAK), 47-8 Parathyroid hormone-related protein (PTHrP), 141 Particulate matter (PM) pollution, 333-4, 338-9, 367, 369, 382 urban, 338-9 Peribronchial innervation, 40-1 Pericytes, 84 Persistent pulmonary hypertension of the newborn (PPHN), 105, 112, 113-14 Phosphatidylcholine (PC), 150, 156, 363-4 Phosphatidylglycerol (PG), 150, 364 Phosphatidylinositol (PI), 150-1 Phosphatidylserine (PS), 150-1 Phospholipids (PL), 149-50, 201-202, 364-5 Plasma cells, 171 Platelet-derived growth factor (PDGF), 9, 67, 86-8, 106, 107, 138, 195 Pneumoconiosis, 284 Pneumonectomy (PNX), 189-90 airway growth, remodeling, function post-PNX, 195-6 compensatory response post-PNX, 190-3 effect of lung strain on post-PNX, 193-5 EGF post~ 196 re-initiation of septal growth, 192-3 recruitment of physiologic reserves, 191 septal remodeling, 191-2 surfactant protein expression post-PNX, 196 Pneumonia, 382-3 Pollutants s e e air pollution; toxicants Polyunsaturated fatty acids (PUFA), 160 Positive end-expiratory pressure (PEEP), 209 Post-pneumonectomy syndrome, 190 Preterm birth, 160 as environmental influence on lung development, 239-46 causes, 237-9 definition, 237 effects of FGR on, 256 factors leading to, 237-8 incidence, 237 morbidities/adverse outcomes, 238-9 and perinatal mortality, 238-9 Prostacyclin (PGI2), 272 Protein, 151-2, 365-6, 380 deficiency, 259-60 detection, 23-5 matrix, 82 Proteinases, 245-6 Proteoglycans, 76, 77, 78, 260 Proximal-distal differentiation, 8-9 Pseudoglandular stage, 239, 57 171 airway innervation, 37, 39 airway Schwann cells, 39 epithelial differentiation, 19-20 ganglion formation, 36 nerve trunk development, 37, 39 Pseudostratified cuboidal epithelium, 16, 17 Pulmonary alveolar proteinosis, 367 Pulmonary circulation, 105, 206 control of the ductus arteriosus, 111 fetal/adult comparison, 206 growth/development, 105-107 mechanisms of pulmonary vasodilation at birth, 112-13 mechanisms that cause failure at birth, 113-14 physiology of the fetal, 107-11

Pulmonary edema, 153-4, 242 Pulmonary hypertension, 271 Pulmonary hypoplasia, 134, 144 Pulmonary immune system: defense mechanisms during infancy, 173-4 developmental expression of cytokines/chemokines, 172 maturation of postnatal, 169-72 Pulmonary surfactant: autonomic agonists, 154-5 at birth, 157 composition, 149-52 development of system, 155-7 effect of nicotine exposure on, 303 effects of hypoxia/altitude, 370-1 environmental influences on development, 158-60 environmental influences on mature system, 367-71 formation/release of, 149 functions, 152-4 genetic causes of deficiencies during development, 157-8 immune functions, 154 lipids, 149-51 lung stability, 152 natural aging effects, 363-4 nutritional effects, 260, 263 physical properties, 152 prevention of pulmonary edema/maintenance of airway patency, 153-4 proteins, 151-2 regulation of development, 155-7 regulation of secretion, 154-5 related diseases in adult lung, 364-7 SP-B deficiency, 157 static lung compliance, 152 surface tension, 152 variations of protein B deficiency, 157-8 ventilation, 155 Pulmonary transition at birth: change from liquid-filled to air-filled lung, 204-206 changes in blood flow, 205-207 closure of ductus arteriosus, 207-208 fetal lung maturation/glucocorticoids, 201-202 lung liquid clearance, 202-204 onset of continuous breathing, 209 Pulmonary vascular bed, 82 alveolar oxygen tension/vascular density in adult lung, 93 bronchial artery/pulmonary artery connections, 91 cell-cell interactions/signaling, 86-91 cellular basis of vessel morphogenesis, 82 conventional/supernumerary arteries/veins, 89-90, 91, 92 development of bronchial arteries/veins, 91 development of vascular mural cells, 84-6 distribution of intra-acinar vessels, 90, 91, 92 early vessel development, 88-9 embryonic/fetal, 88-91 failure to develop normal quota of units, 92 formation/growth of endothelial channels, 82, 84 growth of vascular units in low alveolar oxygen tension, 96 growth/reorganization in the adult, 91-2 loss of vascular units by hyperoxia, 94-6 mural cells/change in oxygen tension, 96, 98-9 musculoskeletal disorders, 93 normal lung, 88-91 postnatal development/growth, 91 repair of congenital diaphragmatic hernia, 93 single lung in agenesis, 92-3 vessel wall reorganization in aging, 92 vessel wall structure, 90, 91, 92 Pulmonary vascular resistance (PVR), 105, 107-11,205-207

Quantitative trait locus (QTL), 279, 280-1,282-3 Rat: alveolarization, 58 body mass, 214 collagen, 226-7 endothelial cells, 228 epithelial cells, 220--6 exposure to smoke, 358 interstitial changes, 225-7 life span characteristics, 219 Rat aging, 219 alveolar macrophages, 228 alveolar tissue compartments, 222-8 lung parenchymal structure, 221 tracheobronchial tree and epithelium, 220 Receptor tyrosine kinase (RTK), 86, 141-2 Reflex responses, 45-6 Regulation of pulmonary immune/inflammatory cell trafficking, 172-3 resident immune/inflammatory cells of developing airways, 171 Repair: definition, 353 factors influencing, 353-4 Respiratory bronchioles, 14, 16-17, 55 Respiratory distress syndrome (RDS), 159, 160, 161,241-4 Respiratory syncytial virus (RSV), 160, 314-15, 381 Respiratory toxicants, 14 Retinoic acid (RA), 8, 67, 261 Retinoids, 8, 9, 143 Rho-associated kinase (ROK), 47-8 Saccular stage, 56, 171,240, 241 glial ensheathment, 39, 41 increase in bronchial mucosal circulation, 41 Selenium, 262 Senile emphysema, 377-8, 380 Sensory reflexes, 41 Septal thinning, 65-6 Serous cells, 17 Signaling: effect of nicotine, 305-307 focal adhesion sites, 138 molecules, 7-9 reduction, 36-7 retinoids, 4 Simple cuboidal epithelium, 16 Six Cities studies, 338 Sleep disordered breathing, 381 Smooth endoplasmic reticulum (SER), 180 Smooth muscle cells (SMC), 82-92, 96, 97, 98 Solute transport, 119-22 adult airway epithelium, 123 adult type II cells, 123 fetal airways, 122 fetal lung explants, 121 intact adult lung, 122-3 intact fetal lungs, 120-1 mechanisms of, 119-20 perinatal switch, 125-6 primary cultures of fetal lung epithelium, 121-2 switch from liquid secretion to absorption, 123-5 Sonic hedgehog (Shh), 7 Spherical mitochondria, 19 Sphingomyelin (S), 150 Starvation, 262-3 Stem cells, 58

Subacute pulmonary disease, 366-7 Submucosal glands, 14, 17 carbohydrate content, 15 morphogenesis of, 18 Substance P (SP), 39 Sudden infant death syndrome, 296-7 Sulfotransferases, 182 Sulfur-related compounds, 369 Sulphur dioxide (SO2), 333, 367 Superoxide dismutase (SOD), 178, 219 Surfactant phospholipids, 59-60 Surfactant proteins (SP), 60-1, 196, 241 Surfactant replacement therapy, 244 Surfactant see pulmonary surfactant Terminal bronchioles, 14 T-helper cells (T-cells), 321,322-3, 324, 388 type 1,311,312, 313, 316 type 2, 314 Thyroid hormones, 156, 243-4 T-lymphocytes, 323 Tobacco smoke, exposure to see environmental tobacco smoke; maternal nicotine exposure; nicotine Toxicants: bioactivated compounds, 345-6 corticosteroids, 347 ETS, 345 exposure to, 14, 345 miscellaneous compounds, 347-8 oxidant gases, 346-7 see also air pollution Trachea, 4-5, 55, 177 dorsal trachea, 34 Tracheo-esophageal fistula, 5 Tracheobronchial epithelial cell differentiation, 26-7 Tracheobronchial tree: dog, 228-30 mouse, 216-18 rat, 220 Transforming growth factor-beta (TGF-[3), 8, 9, 107, 113, 141,354 Tropoelastin, 62-3, 245 Tuberculosis, 383 Tumor necrosis factor alpha (TNF), 283-4 Tumors, 82, 347-8 Unsaturated phospholipids (USP), 150 Urate levels, 179 Vagus nerves, 209 Vascular endothelial growth factor (VEGF), 67, 86-7, 106-107, 109, 138, 141,245, 272, 347 Vasculogenesis, 82 Vaso-mediators, 82 Vasoactive intestinal peptide (VIP), 39 Vasoconstrictors, 111 Ventilator-induced lung injury, 242 Vitamin A (retinol), 27, 245, 261 Vitamin C (ascorbate), 179 Vitamin D, 261 Vitamin E (tocopherol), 245, 261-2 Winged-helix family transcription factor, 26 Xenobiotic metabolizing enzyme systems, 14, 15, 177 development, 179-82 Zinc oxide (ZnO), 282