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Annu. Rev. Physiol. 2005. 67:623–61 doi: 10.1146/annurev.physiol.67.040403.102229 Copyright c 2005 by Annual Reviews. All rights reserved First published online as a Review in Advance on October 26, 2004 LUNG V ASCULAR DEVELOPMENT: Implications for the Pathogenesis of Bronchopulmonary Dysplasia Kurt R. Stenmark and Steven H. Abman Developmental Lung Biology Laboratory 1 , Pediatric Heart Lung Center 2 , Sections of Critical Care 1 and Pulmonary Medicine 2 , Department of Pediatrics, University of Colorado Health Sciences Center and The Children’s Hospital, Denver, Colorado 80262; email: [email protected]; [email protected] Key Words angiogenesis, pulmonary circulation, pulmonary hypertension Abstract Past studies have primarily focused on how altered lung vascular growth and development contribute to pulmonary hypertension. Recently, basic studies of vas- cular growth have led to novel insights into mechanisms underlying development of the normal pulmonary circulation and the essential relationship of vascular growth to lung alveolar development. These observations have led to new concepts underlying the pathobiology of developmental lung disease, especially the inhibition of lung growth that characterizes bronchopulmonary dysplasia (BPD). We speculate that understand- ing basic mechanisms that regulate and determine vascular growth will lead to new clinical strategies to improve the long-term outcome of premature babies with BPD. INTRODUCTION The lung is a complex organ system whose basic physiologic function is to perform gas exchange across a thin blood-gas interface. The lung has the largest epithelial surface area of any mammalian organ and is capable of supporting a systemic oxygen consumption of between 250 ml/min at rest to 5500 ml/min during exer- cise (1). To create such a large, diffusible interface with the circulation, the lung epithelium must not only undergo a series of proliferative, branching, and mor- phogenetic steps, but also must interact continuously and in a well-coordinated manner with mesenchymal tissue to ensure the development of functioning vas- cular and lymphatic systems. The lung originates as a pair of invaginations from foregut endoderm. These endodermal buds branch and differentiate within the sur- rounding mesoderm, ultimately giving rise to the airways, alveoli, blood vessels, and lymphatics of the mature lung. Lung branching morphogenesis and epithe- lial development and differentiation have been the subject of intense investigation over the past 50 years, and the processes involved have been the subject of many comprehensive recent reviews (2–9). However, studies of the mechanisms that 0066-4278/05/0315-0623$14.00 623 Annu. Rev. Physiol. 2005.67:623-661. Downloaded from arjournals.annualreviews.org by UNIVERSITY OF MANITOBA on 02/19/06. For personal use only.

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Annu. Rev. Physiol. 2005. 67:623–61doi: 10.1146/annurev.physiol.67.040403.102229

Copyright c© 2005 by Annual Reviews. All rights reservedFirst published online as a Review in Advance on October 26, 2004

LUNG VASCULAR DEVELOPMENT: Implicationsfor the Pathogenesis of BronchopulmonaryDysplasia

Kurt R. Stenmark and Steven H. AbmanDevelopmental Lung Biology Laboratory1, Pediatric Heart Lung Center2, Sections ofCritical Care1 and Pulmonary Medicine2, Department of Pediatrics, University ofColorado Health Sciences Center and The Children’s Hospital, Denver, Colorado 80262;email: [email protected]; [email protected]

Key Words angiogenesis, pulmonary circulation, pulmonary hypertension

■ Abstract Past studies have primarily focused on how altered lung vascular growthand development contribute to pulmonary hypertension. Recently, basic studies of vas-cular growth have led to novel insights into mechanisms underlying development of thenormal pulmonary circulation and the essential relationship of vascular growth to lungalveolar development. These observations have led to new concepts underlying thepathobiology of developmental lung disease, especially the inhibition of lung growththat characterizes bronchopulmonary dysplasia (BPD). We speculate that understand-ing basic mechanisms that regulate and determine vascular growth will lead to newclinical strategies to improve the long-term outcome of premature babies with BPD.

INTRODUCTION

The lung is a complex organ system whose basic physiologic function is to performgas exchange across a thin blood-gas interface. The lung has the largest epithelialsurface area of any mammalian organ and is capable of supporting a systemicoxygen consumption of between 250 ml/min at rest to 5500 ml/min during exer-cise (1). To create such a large, diffusible interface with the circulation, the lungepithelium must not only undergo a series of proliferative, branching, and mor-phogenetic steps, but also must interact continuously and in a well-coordinatedmanner with mesenchymal tissue to ensure the development of functioning vas-cular and lymphatic systems. The lung originates as a pair of invaginations fromforegut endoderm. These endodermal buds branch and differentiate within the sur-rounding mesoderm, ultimately giving rise to the airways, alveoli, blood vessels,and lymphatics of the mature lung. Lung branching morphogenesis and epithe-lial development and differentiation have been the subject of intense investigationover the past 50 years, and the processes involved have been the subject of manycomprehensive recent reviews (2–9). However, studies of the mechanisms that

0066-4278/05/0315-0623$14.00 623

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regulate lung vascular and lymphatic development and that link capillary growthwith alveolarization are relatively recent and limited in scope.

Until recently, most of the information regarding pulmonary vascular devel-opment has been largely descriptive in nature and often based on model systemsand methodologic techniques that may preclude accurate assessment of the di-verse processes governing pulmonary vascular development. This is especiallytrue with regard to the origin, differentiation, and maturation of the various cellstypes within the vascular wall of lung blood vessels. This fact is important becauseit has become increasingly clear that airway and vascular development are closelyinteractive processes and that disruption of one system may have catastrophic con-sequences on the development of the other and, ultimately, of lung structure andfunction.

Developmental abnormalities of the pulmonary circulation contribute to thepathogenesis of several neonatal cardiopulmonary disorders, including diseasesassociated with persistent pulmonary hypertension of the newborn (10–12). Morerecently, however, there has been growing recognition that the importance of un-derstanding basic mechanisms of lung vascular growth in the context of human dis-ease may be best highlighted in the setting of bronchopulmonary dysplasia (BPD)(13, 14). Premature birth with injury to the immature lung disrupts normal lunggrowth and causes severe chronic lung disease, or BPD, which remains a ma-jor cause of late morbidity and mortality of premature newborns. Histologically,BPD is characterized by arrested lung growth, with decreased alveolarization anda dysmorphic vasculature. Recent studies suggest that disruption of normal lungvascular growth may play a central role in the pathogenesis of BPD; however, littleis known about basic mechanisms of pulmonary vascular injury in the immaturelung, the impact of this injury on subsequent growth and development of eitherthe pulmonary or bronchial circulations, and the contribution of abnormalities ofpulmonary and/or bronchial vascular growth to either the pathogenesis of BPD orfunctional abnormalities that characterize the disease. The purpose of this review isto examine lung vascular development in the context of understanding how growthof the pulmonary circulation and lung structure is impaired in BPD, with an eyetoward restoring the injured lung to normal.

LUNG BLOOD VESSELS: NORMAL STRUCTURE ANDFUNCTION OF THE PULMONARY AND BRONCHIALCIRCULATIONS

The vascular system of the lung is divided into the pulmonary and bronchial sys-tems. The pulmonary arteries supply the intrapulmonary structures and ultimatelyregulate gas exchange; vessels branch with the airways but branch into an exten-sive capillary network only at the level of respiratory bronchioles and alveoli. Thebronchial system is the nutrient supply to the lung and perfuses the capillary bedwithin the bronchial wall and the structures of the perihilar region, including lymph

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LUNG VASCULAR DEVELOPMENT 625

nodes and the adventitia of elastic and large muscular pulmonary vessels. All in-trapulmonary structures drain to the pulmonary veins, whereas the hilar structuresdrain to the so-called true bronchial veins and then to the azygos system (15, 18).

The pulmonary artery accompanies the airways but gives off many more bran-ches than the airway. In fact, two main types of pulmonary arterial vessels have beendescribed: (a) conventional vessels, which are the long pulmonary artery branchesthat run within an airway, dividing as the airway divides and finally distributing tothe capillary bed beyond the level of the terminal bronchioles; (b) supernumeraryvessels, which are additional branches that arise from the pulmonary artery be-tween the conventional branches, run a short course and supply the capillary bedof alveoli immediately around the pulmonary artery (9, 16, 17). Supernumeraryarteries are thought to be a prominent component of the mature lung with theratio of conventional to supernumerary vessels in the order of 1:2 in the prelob-ular region and 1:13 in the pre- and intra-acinar region (16). There appear tobe distinct functional differences between supernumerary and conventional arter-ies. For instance, serotonin-induced vasoconstriction in supernumerary vessels is30 times more potent than in conventional vessels (18). Others have suggested thatplexiform lesions selectively develop in supernumerary vessels (19).

The pattern of veins in the lung resembles the arteries in that there are many morevenous tributaries than airway branches (9, 15, 16). Similarly, at least two types ofveins can be identified. Conventional veins arise from the points of division of anairway, pass to the periphery of a given lung unit, and combine to form increasinglylarge venous tributaries. Tributaries to the pulmonary veins also arise from thepleura and connective tissue septa. Within the lung, the anatomic distribution ofthe arteries and veins is characteristic. The broncho-arterial bundle includes theairway with the bronchial artery capillary bed and the adjacent pulmonary arteryand lymphatic vessels, all within a single adventitial sheath. The veins always runat the periphery of any unit, whether it is the acinus, lobule, or segment. At thehilum, the veins join to form superior/inferior veins that drain to left atrium, givingthem a distribution within the mediastinum different from that of the pulmonaryartery.

A systemic blood supply to the lung was suggested at least 500 years ago byGalen. Its presence was confirmed by Ruysch in 1732, who designated the lung’ssystemic vessel as the bronchial artery (15). It is now known that the bronchialarteries may arise from the descending aorta, intercostals, subclavian, or internalmammary arteries. The bronchial arteries may be classified as extrapulmonaryor intrapulmonary. The extrapulmonary artery gives off small branches to theesophagus, to mediastinal tissues, to hilar lymph nodes, and the lobar bronchus. Theintrapulmonary bronchial artery distributes to the supporting tissue and structuresof the intralobal bronchi (mucous membrane, muscle, perichondrium, secretoryglands), to pulmonary pleura, to lymph nodes, to walls of the pulmonary artery,and to veins and nerves. Bronchial arteries on the walls of the pulmonary artery andvein are functionally vasa vasorum. They are not as dense as on the bronchial wallsand cannot be easily observed on the walls of small vessels. The extrapulmonary

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bronchial artery blood drains into extrapulmonary bronchial veins and connect tothe azygous or hemiazygous veins, which ultimately drain to the right atrium. Theintrapulmonary bronchial artery drains into intrapulmonary bronchial veins and/orpulmonary veins, which then connect to the left atrium.

There are close and important relationships between the pulmonary and bron-chial vascular systems, which become most evident in the diseased lung or in thesetting of aberrant cardiovascular development (9, 15). For example, enlargementof the bronchial circulation is especially striking in association with congenitalheart diseases, such as pulmonary atresia or transposition of the great vessels. In-terestingly, ligation of the pulmonary arteries produces enlargement and dilationof the bronchial artery resulting in the increase of precapillary anastomosis andthe development of various abnormal flow routes (20, 21). Similarly, severe pul-monary thromboembolic disease can result in proliferation of bronchial vessels inand around the obstructed pulmonary artery (15, 22). In bronchiectasis, a markedproliferation of bronchial vessels, which anastomosis with the pulmonary vascularsystem via precapillary, capillary, or postcapillary networks, is observed (23, 24).In emphysema, the normal structure of the alveolar capillary networks often dis-appears and enlarged branches of bronchial vessels are observed to occupy thoseareas, a situation obviously unfavorable to gas exchange (15). A similar prolifer-ation of bronchial vessels is noted in the setting of chronic atelectasis (24). Thusmany observations would support the idea that conditions leading to the narrowingor stenosis of blood vessels of the pulmonary vascular system are generally relatedto a proliferation and dilation of vessels of the bronchial vascular system. Thisrelationship will be examined in the setting of BPD (see below).

Because the primary function of the lung is gas exchange, it is not surprisingthat keeping the airspaces and lung interstitium free of excess fluid is a criticalcomponent of normal lung function. The lymphatic system plays a critical rolein fluid clearance, as well as in host defense, and in clearing solid particles andcells from the lung (26–28). It is also involved in the genesis of diseases such astuberculosis and the metastasis of lung cancer. The lymph vessels form a closedcirculatory system lined by endothelium. According to size, they are called lymphcapillaries, lymph vessels, and collecting lymph vessels (15, 26). Lymph vesselsare clearly evident at the level of the alveolar ducts (none is observed in directrelation to alveoli) and extend proximally (15, 26). Interestingly, the caliber oflymph vessels does not necessarily increase as they move toward the central lung,with lymph capillaries sometimes being larger than collecting lymph vessels. Thecritical nature of the lung lymphatic system is illustrated in human cases in whichthere is a primary developmental defect of the lung lymphatics, such as pulmonarylymphangiectasia. In the majority of neonatal cases, effective respiration is neverestablished at birth, and the infants are stillborn or die in the first weeks of lifedespite aggressive support (29). In patients with bilateral pulmonary lymphang-iectasia who survive, many develop chronic respiratory disease with pulmonaryhypertension and cor pulmonale. Thus an understanding of the development of thelung and lung vasculature must take into account lymphatic development.

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MORPHOGENESIS OF THE PULMONARY ANDBRONCHIAL CIRCULATIONS

The most proximal part of the pulmonary circulation, the pulmonary trunk, isderived from the truncus arteriosus, which becomes divided into the aorta andpulmonary trunk by 8 weeks of gestation in humans by growth of the spiralaorticopulmonary septum (30). The pulmonary trunk connects to the pulmonaryarch arteries, which are derived from the sixth branchial arch arteries, the mostcaudal of the brachial arteries, by 7-weeks gestation in humans (31). There is noconsensus as to the mode of development of the sixth branchial arch artery, but re-cent reappraisal suggests that these pulmonary arch arteries originate from a strandof endothelial precursors that connect the ventral wall of the dorsal aortae to thepulmonary trunk (32, 33). These strands become lumenized, initially at the sites ofconnection with pulmonary trunk and dorsal aortae, then in between these sites ofattachment. The origin of the intrapulmonary arteries has been variously describedby early investigators as endothelial sprouts from either the aortic sac (31) or fromthe dorsal aortae (34), or as originating from a network of capillaries around theforegut (35, 36). The latter observation comes closest to the results of more recentstudies suggesting that the pulmonary artery and vein develop in situ within thesplanchnic mesoderm surrounding the foregut via at least two processes that likelyoccur concurrently: (a) vasculogenesis, in which new blood vessels form in situfrom angioblasts and (b) angiogenesis, which involves sprouting of new vesselsfrom existing ones.

The idea that some blood vessels arise de novo in the mesoderm surround-ing the protruding endoderm may actually not be so new because it was initiallyraised some 70 years ago by Chang (37). Strong support for this idea has comefrom recent studies using molecular markers of endothelial progenitor cells, aswell as endothelial differentiation markers (38–42). Cells expressing primitive en-dothelial markers appear in the mesoderm at early stages of lung development(embryonic and early pseudoglandular), which proceeds any documented connec-tion with the established circulatory system. Lung vascular development continuesthroughout lung development, and requires epithelial-mesenchymal cross talk ateach stage of development (8). Studies combining light and transmission electronmicroscopy with scanning electron microscopy of mercox vascular casts furthersuggest that large pulmonary arteries originate via the process of angiogenesis ofcentral vessels, whereas distal vascular development requires vasculogenic mech-anisms within the lung mesenchyme (43, 44). In addition to angiogenesis andvasculogenesis, a third process, fusion, is necessary to ultimately connect the an-giogenic and vasculogenic vessels and expand the vascular network (43, 45).

At least two studies suggest similar mechanisms apply to human lung vasculardevelopment. DeMello et al. studied human embryos from the Carnegie Collectionof Human Embryos and presented evidence to support the idea that both vascu-logenesis and arteriogenesis operate cooperatively to form the pulmonary vessels(45). Using different techniques, Hall et al. also proposed that vasculogenesis is a

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628 STENMARK � ABMAN

primary mechanism for intrapulmonary vascular development (46). Both investi-gations suggested that the venous circulation develops in a fashion similar to thearterial circulation and, importantly, both showed that the venous circulation is thefirst to be established.

On the other hand, this concept has recently been challenged by the suggestionthat the lung vasculature is formed by distal angiogenesis, a process in whichthe formation of new capillaries from pre-existing ones occurs at the peripheryof the lung (47). In this model epithelial/endothelial interactions are decisive ininducing angiogenesis and ensure the coordinate expansion of a vascular networkas branching proceeds. Newly formed vessels remodel dynamically to form theafferent (arterial) and efferent (venous) vascular systems. Therefore, questionsremain regarding the process that may be related to methodologic approachesand/or semantics. Future studies using new and improved techniques will likelyresolve these issues.

At the end of 16 weeks of gestation in humans, all preacinar bronchi are in placeand are accompanied by the pulmonary and bronchial arteries (48, 49). However,although the conducting airways and arteries are formed by the end of this so-called pseudoglandular stage (5–17 weeks) of lung development, the developmentof the gas-exchanging surface of the lung has just begun. The canalicular stage(17–26 weeks) encompasses the early development of the pulmonary parenchymaduring which there is a great increase in the number of lung capillaries (9, 50). Thecapillaries begin to arrange themselves around the air spaces and come into closeapposition with the overlying cuboidal epithelium. At sites of apposition, thinningof the epithelium occurs, to form what will be the first air-blood barrier. Duringthe saccular stage (24 weeks to birth), the airways end in clusters of thin-walledsaccules and by term have formed the last generations of airways, the alveolarducts. Capillaries form a bilayer within the relatively broad and cellular intersac-cular septa at this stage (9, 50). The period of alveolarization is largely a postnatalphenomenon, with more than 90% of all alveoli being formed after birth in humans(6, 9). During the period of alveolarization the initially thick interalveolar septaare attenuated and the double capillary layer fuses to form a single layer adultform. Secondary septae initially form as low ridges that protrude into primitiveairspaces to increase their surface area. During this time, the lung is undergoingmarked microvascular growth and development as well (51). Multiple stimuli con-tribute to alveolar and vascular growth and development, and in part, involve crosstalk of paracrine signals between epithelium and mesenchyme (52). For example,vascular endothelial growth factor (VEGF) is expressed in developing lung ep-ithelium, whereas VEGF receptor-2 (VEGFR-2) is localized to angioblasts withinthe embryonic mesenchyme (53–57). Diffusion of VEGF to precursors of vascularendothelial cells within the mesenchyme leads to angiogenesis by stimulating en-dothelial proliferation, a key step in vessel development. In addition, the processof septation involves alternate upfolding and growth of capillary layers withinprimary septa (54). This mechanism of alveolarization suggests that the failureof capillary network formation and growth, or disruption of the infolding of the

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LUNG VASCULAR DEVELOPMENT 629

double capillary network, could potentially cause failed alveolarization. Less isknown of the mechanisms contributing to development of the bronchial circula-tion. However, molecular marker studies suggest that intrapulmonary bronchialarteries also form in situ from the mesenchyme surrounding the epithelial buds(32, 33). Then, early in development (12 weeks in humans) connections to thesystemic circulation are made. Presently, it is unclear as to what directs theproximal and distal connections between the systemic versus pulmonary vascu-lar beds, especially when in early lung development the intrapulmonary vascularplexus are virtually indistinguishable. The question is of obvious importance be-cause the behavior of these two circulations is vastly different. Current studies pro-vide little insight into how the marked differences in endothelial cell and smoothmuscle phenotype and function arise within the lung and its two circulations. Fur-ther, they do not adequately address how heterogeneity in phenotype and functionof endothelial and smooth muscle cells arise even along the longitudinal axis ofthe respective circulations.

ROLE OF GROWTH AND TRANSCRIPTION FACTORSIGNALING IN CONTROL OF LUNG VASCULAR GROWTH

Tremendous advances in our understanding of blood vessel formation within theembryo and various organs and tissues have taken place over the past 10 years.Many of the molecules involved seem to be highly conserved both across speciesand among various organs (Figure 1, Table 1). However, there may be subtledifferences, and a complete understanding of the signaling pathways that regulateand coordinate vessel development and differentiation in a cell-specific fashion inthe lung will be critical to understanding the unique functions and responses that thepulmonary and bronchial circulations exhibit in response to many pathophysiologicstimuli. Molecular programs important for the development of the lung vasculaturehave only recently begun to be elucidated. Among the different regulators of vesselformation, those that are specifically expressed in the endothelial lineage are bestcharacterized during vascular development. This is the case for the ligand-receptorpairs VEGF/VEGFR, angiopoietin/TIE, ephrins/eph receptors, and notch/jagged.

Several studies have documented the appearance of VEGFR-2 (Flk-1) at veryearly time points of lung development in the mesenchyme (38, 58–60). VEGF alsoappears early in the developing epithelium and mesenchyme (58–63). It is knownthat VEGF-A is absolutely required for embryonic vascular development. Loss ofeven a single VEGF allele results in early embryonic death, before embryonic day9.5. VEGF-A expression and function is tightly regulated during lung development.At least in the mouse, VEGF-A exists as three predominant isoforms, VEGF-A120, 164, and 188. Each isoform has differing properties, including affinity forthe heparin sulfate component of the extracellular matrix, as well as for VEGFR-1(Flt-1) and VEGFR-2 (Flk-1). VEGF-120 is highly diffusible because of lack ofbinding to heparin sulfate and probably serves a key early role in vascular formation

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Figure 1 A proposed model for the development of a mature vessel wall. (A) Inthis model, early expansion of mesoderm, perhaps driven by bFGF, is followed byan emergence of angioblasts or endothelial precursor cells within this mesenchyme asidentified by Flt-1 and Flk-1. VEGFA is critical in the expansion of this cell population.(B) In the lung, epithelial-mesenchymal interactions are critical for the developmentof the vasculature, which involves diverse molecules [adapted from Ng et al. (88)]. Re-cruitment of mural cells follows endothelial tube formation. Angiopoietin, producedby undifferentiated mesenchymal cells, binds to and activates the TIE2 receptor onendothelial cells (C), resulting in release of a recruitment/chemotactic signal (e.g.,PDGF, HB-EGF) for mesenchymal cells. Migration of mesenchymal cells to the de-veloping endothelial tube and subsequent contact with the endothelium may activatesignals (D) (e.g., TGF-β) that are necessary for the commitment of mesenchymal cellsto SMC-specific lineages. The developing vessel then undergoes structural assembly(E) that includes inhibition of endothelial cell proliferation, rapid SMC proliferation,and extracellular matrix deposition. Throughout this period, differentiation to a moremature cell occurs as SMC gradually accumulate contractile and cytoskeletal proteins.Importantly, the vessel remains surrounded by mesenchymal cells or fibroblasts, whichmay serve as a continued source for mural cells. It is possible that progenitor cells residein even the mature adventitia. Heterogeneity in both SMC and fibroblast populations isestablished and, as the mature vessel morphology is achieved (F), SMC become quies-cent, respond poorly to mitogenic stimulation, and produce minimal matrix proteins.The adventitia may provide a reservoir of cells for vessel repair after injury in postnatallife.

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LUNG VASCULAR DEVELOPMENT 631

TABLE 1 Factors likely to be involved at different stages of blood vessel formationa

Molecules Localization

Mesoderm formation (Stage A)VEGF-A EndodermBFGF EndodermVEGFR-2, VEGFR-1 Mesodermal cellsFGFR(s) Mesodermal cellsFibronectin Extracellular matrix

Aggregation of angioblasts (Stage A)VEGFR-2 AngioblastsVEGFR-1 AngioblastsVE-cadherin Angioblaststie-2/tek Angioblastsets-1 AngioblastsPECAM-1 AngioblastsFibronectin Extracellular matrixVEGF Endoderm

Endothelial differentiation and formation of primary capillary plexus(Stage B)

VEGFR-2 Endothelial cellsVEGFR-1 endothelial cells Endothelial cellsVE-cadherin Endothelial cellstie-2/tek Endothelial cellstie-1 Endothelial cellsE-selectin Endothelial cellsets-1 Endothelial cellsPECAM-1 Endothelial cellsIntegrins (e.g., αVβ3) Endothelial cellsNotch Endothelial cellsdelta/jagged Endothelial cellsEphrins Endothelial cellsEphs Endothelial cellsFibronectin Extracellular matrixLaminin Extracellular matrixCollagen IV Extracellular matrixVEGF-A Endoderm

Smooth muscle cell recruitment and differentiation (Stages C, D)tie-2 Endothelial cellsPDGF-BB Endothelial cellsPDGF-AA Endothelial cellsHB-EGF Endothelial cellsBFGF Endothelial cellsThromboxane A2 Endothelial cellsAngiotensin II Endothelial cellsEndothelin Endothelial cells

(Continued)

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TABLE 1 (Continued)

Molecules Localization

Leukotrienes Endothelial cellsSubstance P Endothelial cellsSerotonin Endothelial cellsAngiopoietin Endothelial cellsTGF-β Endothelial cellsIFG-II MesenchymeTissue factor MesenchymeWNT MesenchymeLaminin Basement membraneType IV collagen Basement membranePerlecan Basement membraneHeparan sulfate Basement membrane

aStages correspond to those illustrated in Figure 1.

through driving endothelial commitment and expansion (61). The importance ofVEGF-164 and 188 isoforms was demonstrated in mice engineered to express onlythe VEGF-120 isoform. VEGF-120 only animals had fewer air-blood barriers anddecreased airspace to parenchyma ratios compared with that of wild-type litter-mates. Thus as development proceeds, the pattern of VEGF isoform expressionbecomes more restrictive, and the VEGF isoforms serve different roles (64).

VEGF has other family members including VEGF-C and -D. These VEGFshave different affinities for specific VEGF receptors with both VEGF-C and -Ddemonstrating an ability to bind to VEGFR-3 (65–67). It is now recognized thatVEGF-C and -D(?)/VEGFR-3 interactions are probably crucial for development ofthe lymphatic vascular system (65, 66). Recent studies have evaluated temporal andspatial pattern of VEGF-D expression during mouse lung development (62). Thepattern of expression is distinct from that of VEGF-A, suggesting a unique func-tion for each VEGF during lung development. In addition, the finding of VEGF-Dexpression in the mesenchyme by cells distinctly different from endothelial cellsand smooth muscle cells SMC), i.e., fibroblasts, suggests that mesenchymal cellscan influence endothelial phenotype during lung development through expressionof either VEGF-A or VEGF-D (and possibly VEGF-C). Thus it is likely thatsubsets of cells within the mesenchyme must have specific roles pertaining to in-ductive events between mesenchymal and endothelial compartments and thereforemay also exert influence on the generation of endothelial heterogeneity describedabove.

The angiopoietins (Ang) and their major receptor Tie-2 (tyrosine kinase withimmunoglobulin and EGF-like domains) are critical for normal vascular develop-ment because Ang 1−/− or Tie 2−/− mice are embryonic lethal owing to the failureof vascular integrity (68, 69). Ang 1 is produced by lung mesenchyme and smoothmuscle, whereas Tie 2, its receptor, is restricted to endothelial expression (69–71).

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Ang 1 binding to Tie 2 causes receptor tyrosine phosphorylation and down-stream signals for endothelial cell survival through phosphatidylinositol 3-kinasePI3K/Akt signaling (72, 73). Angiogenic actions of Ang 1 require endothelium-derived NO (74). Ang 1 promotes interactions between endothelial cells, extra-cellular matrix, and pericytes that are required for vessel maturation (68). Ang1-VEGF interactions are critical for normal vascular maturation, but their inter-active effects are complex and dependent upon multiple factors. In some settings,Ang1 treatment attenuates VEGF-induced angiogenesis by increasing intercellu-lar endothelial cell junctions, but Ang 1 also makes vessels resistant to VEGFwithdrawal (70, 75, 76). Ang 2 also binds Tie 2, but blocks the function of Ang1 (72). Ang 2 is expressed only at sites of vessel remodeling where it destabi-lizes vessels, perhaps via Ang 1 inhibition, which may increase endothelial cellresponses to VEGF. The combination of low Ang 2 and low VEGF may enhanceendothelial cell death (72). Low Ang 1 to Ang 2 ratios favor decreased Tie 2 phos-phorylation, which leads to weaker endothelial connections and perhaps increasedresponsiveness in VEGF. Little is known about Ang expression during develop-ment, especially in models of lung hypoplasia, but it is increased in nitrofen modelof congenital diaphragmatic hernia (77). In addition, the role of Ang 1 in pul-monary hypertension has been extremely controversial (78). Overexpression ofAng 1 causes severe pulmonary hypertension and is increased in lungs from hu-man patients with pulmonary hypertension (79). However, cell-based gene transferstudies with Ang 1 protected against experimental pulmonary hypertension causedby monocrotaline (80). These differences may be related to the mixed effects ofAng 1 on isolated cells: Ang 1 stimulates smooth muscle cell proliferation butinhibits endothelial cell apoptosis.

Less is known about the other ligand-receptor pairs with regard to lung vasculardevelopment. The notched/jagged pathways seem to lie downstream from VEGFsignaling and be important during embryonic vascular development (81). Taichmanet al. performed a systematic evaluation of notch-1/jagged-1 gene and proteinexpression in the developing mouse lung and found that the mRNA transcripts fornotch-1 and jagged-1 increased progressively from early to later lung development,accompanied by simultaneous rise in endothelial cell-specific gene expression, apattern somewhat unique to the lung (82). Notch-1 and jagged-1 appeared initiallyon well-formed larger vessels within the embryonic lung and were progressivelyexpressed on smaller developing vascular networks (82). Notch signaling maymediate its effects, at least in part, through the forkhead box (Fox) f-1 transcriptionfactors (81). That this transcription factor family plays a critical role in developmentof the pulmonary vasculature was recently shown in studies demonstrating thatFox-f1 haploinsufficiency is capable of disrupting pulmonary expression of genesin the notch-2 signaling pathway resulting in abnormal development of the lungmicrovasculature (83).

Another autocrine/paracrine pathway shown to be important in lung vasculardevelopment is Wnt. Wnt proteins are homologs of the Drosophila wingless geneand have been shown to play important roles in regulating cell differentiation,

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proliferation, and polarity (84, 85). Several Wnt genes have been shown to beexpressed in the developing lung including Wnt-2, Wnt-2b, Wnt-7b, Wnt-5a, andWnt-11. Of those only Wnt-7b is expressed at high levels exclusively in the devel-oping airway epithelium during early lung development (86). Morrisey et al. haverecently reported the generation and characterization of a Wnt-7b (lacz) knock-inmouse (87). These mice exhibit severe pulmonary hypoplasia and lung-specificvascular defects. The vascular defects include dilation of large blood vessels inthe lung with subsequent cell death and degradation of the vascular SMC layerleading to pulmonary hemorrhage. Defects in the lungs of Wnt-7b (lacz−/−) em-bryos, including early defects in mesodermal proliferation, suggest that Wnt-7bis required for lung mesodermal development. It has been demonstrated that thetranscription factor Fox-f2, which is expressed in the developing lung mesoderm,is down-regulated in Wnt-7b (lacz−/−) lung tissue. Because Fox-f2 is related toFox-f1, which, as mentioned above, has been shown through loss-of-functionexperiments to regulate lung vascular development (83), this suggests that Wntsignaling through forkhead box transcription factors is critical for developmentof vascular SMC from the lung mesoderm and for mesodermal thickening. Thesestudies are among the first to specifically evaluate the factors involved in SMCrecruitment/differentiation during lung vascular development.

Other factors have been shown to be involved in regulating the recruitment ofmural cells (pericytes or SMC) to the vessel wall. However, few of these studieshave been specifically performed in the developing lung. It is known that proliferat-ing endothelial cells secrete platelet-derived growth factor-b (PDGF-b), which actsas a chemoattractant and mitogen for mural cell precursors derived from the mes-enchyme surrounding the endothelial tubes (88). Other molecules thought to be in-volved in mural cell recruitment include angiopoietic tissue factor, notch/jagged 1,ephrin/eph, and COUP-TFII (89, 89a). Because the molecules involved in muralcell recruitment across organs appear similar, it has been assumed by many thatorgan-specific mesodermal cells contribute to the mural or smooth muscle layers ofdeveloping vessels, which likely results in tissue-specific functional and regulatoryproperties of vascular SMC and pericytes. The anatomical position of developingvessels may also contribute to mural cell heterogeneity. SMC in the pulmonarytrunk appear to be largely derived from neural crest tissues, whereas mural cellsin the parenchymal lung blood vessel appear to have a distinct mesenchymal ori-gin. However, because bronchial and pulmonary vessels are apparently derivedfrom similar (identical ?) mesenchyme and have similar anatomic locations withinthe lung, the cell- and tissue-specific factors that lead to the emergence of SMCsaround the pulmonary and bronchial arteries with distinct functional phenotypesare unclear. Even more intriguing is how the differences in functional phenotypebetween SMC of conventional versus supranumery arteries arise.

Upon recruitment to the endothelial tube, newly recruited mesenchymal pro-genitor cells are induced toward a smooth muscle fate. This process seems to bemediated, at least in part, by the activation of transforming growth factor-β (TGF-β). Although the exact mechanisms of how TGF-β is activated and signals to cause

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mural cell differentiation are unknown, it is clear that this signaling system playsa critical role in vascular development (90, 91). TGF-β response elements havebeen identified in the promoter regions of smooth muscle (SM) genes, such asSM-α-actin, SM-22, and calponin (92). TGF-β can also induce differentiation viathe up-regulation of the transcription factor serum response factor (SRF) (93). SRFbinds the serum response elements in the promoter regions of mural cell–specificgenes, including SM-α-actin, SM-γ -actin, SM-22α, and calponin and inducestheir coordinated expression (94). The acquisition of differentiated contractile SMor SM-like cells is critical not only to stabilize and maintain the integrity of the de-veloping endothelial tubes but also to control blood flow through these tubes. Thismay be particularly important in the pulmonary circulation where early intensevasoconstriction capabilities must be acquired to limit blood flow to the develop-ing lung. The mechanisms involved in this mural cell/endothelial cell interaction,which results in the acquisition of SMC with properties differing from systemicvascular smooth muscle and critical for lung function, remain to be elucidated.

Knockout studies of gap junction proteins, including CX43 and CX45, suggestthat gap junction communication is required for endothelial-induced mural celldifferentiation and function (95, 96). To our knowledge, developmentally regulatedexpression of these genes in the lung circulation has not been studied. Otherrequirements include cell-cell adhesion and appropriate cell-matrix interactionsmediated via integrin interactions. Unfortunately, very little is known about theregulation of integrin expression, either by endothelial cells or mural cells, duringpulmonary vascular development.

Several studies have suggested that homeobox genes may be involved in earlyvessel development and mural cell recruitment. Homeobox genes, including Hex,are important in early vasculogenic or angiogenic processes (97, 98). Other home-obox genes, such as the paired-related homeobox gene, PRX-1, may be involved incell differentiation and stabilization of the vessel wall (99, 100). In the developingsystemic vasculature, Prx-1 appears early and is highly evident in prospective con-nective tissues, including the endocardio-cushions, the epicardium, and the wallsof great arteries and veins (99). In the chick embryo, Prx-1 mRNA expression isfirst evident within the primary vessel wall of coronary and pulmonary arteries.As the vessels mature and thicken, the pattern of expression becomes restrictedto nonmuscle cells in the adventitial and outer medial regions (100). These datasuggest that Prx-1 may control the expression of genes involved not only in earlydifferentiation of endothelial cells but also in the assembly and segregation ofdifferent cell types within the differentiating blood vessel wall.

In addition to proangiogenic stimuli, normal vascular development may alsorequire the expression of angiostatic molecules. Schwartz et al. have describedthe presence in the lung of a protein, termed endothelial monocyte-activatingpeptide-2 (EMAP-2), with potent angiostatic properties both in vitro and in vivo(101, 102). It appears to act by specifically causing endothelial cell apotosis.Studies suggest that its presence may balance and therefore help shape the pul-monary vasculature in the developing mouse lung. Overexpression of EMAP-2

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markedly inhibits neovascularization and also inhibits alveolar type II cell devel-opment. Other angiostatic proteins, including Ang-2, and monokine-induced byinterferon-γ (MIG) and interferon-γ inducible protein (IP-10), have been identifiedand shown to block the angiogenic effects of angiogenic factors including VEGF.The role of these chemokines in normal lung vascular development remains to bedetermined.

ROLE OF OXYGEN TENSION IN THE REGULATIONOF LUNG VASCULAR MORPHOGENESIS

Oxygen tension appears to be an important physiologic mediator of embryonic andfetal development, and hypoxia is known to be an important regulator of both vas-culogensis and angiogenesis. As such, it is not surprising that studies of mammaliandevelopment in vitro demonstrate that proper embryonic development is depen-dent on low oxygen tension (3–5%) and that even short exposures to normoxicenvironments (20%) can be detrimental to embryonic development. The criticalrole of oxygen tension in lung development is strongly supported by observationsregarding the development of the tracheal system of Drosophila (103). Initiationof tubulogenesis in Drosophila depends on the expression of two basic helix-loop-helix PAS transcription factors, trachealess and single-minded (trh and sim),which dimerize with the tango (tgo) gene product to direct expression of genes thatgovern tracheal invagination (103). These Drosophila genes share high sequenceand functional homology with members of the mammalian HIF-1α/ARNT familywhere both trh and sim act as transcriptional initiators homologues to HIF-1α,which depends on an ARNT homolog, (tgo), for nuclear transport and the forma-tion of the transcriptional complex (104). Experimental knockout of the complexresults in complete failure of tracheal system development in Drosophila. Nullmutation of HIF-1α in mice has no effect until embryonic day 9, a period thatcoincides with the initiation of lung development and vasculogenesis. These micedisplay apparently enlarged vascular structures, fail to initiate lung morphogenesisfully, and die by E 10.5 (105). In addition, there is a regulated loss of mesenchymaltissue suggesting a role for HIF-1α in regulating the survival and differentiationof mesenchymal progenitor cells into vascular structures (105, 106).

Recent work utilizing mammalian fetal lung explants also provides evidencethat the process of lung budding and airway bifurcation is oxygen dependent (106–108). In these studies, budding and bifurcation is accelerated at PO2s that mimicthose in the fetal environment. Interestingly, the increases in branching morpho-genesis appear to be confined to the periphery in explant culture, suggesting thatthe locus of hypoxia dependence is within the region of active airway bifurca-tion and vascular cell proliferation (106, 108). This effect is reversible, a findingthat provides insight into the changes in gene expression and lung structure thatoccur at the time of birth when the lung becomes immediately exposed to alve-olar (very high compared with the fetal lung) oxygen concentrations. Embryonic

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lung explants maintained at alveolar PO2s show traits of alveolarization (i.e., mes-enchymal thinning, epithelial flattening) and saccularization, and also express thesurfactant protein C gene. Removal of these explants to fetal PO2 for as little as 24 hresults in the rapid loss of surfactant protein C expression and regrowth of airwaysinto the saccular spaces and an increase in airway surface complexity (108). Thisis consistent with observations showing that neonatal mice or rats exposed tomoderate hypoxia exhibit arrested postnatal lung maturation and alveologenesis,as well as surfactant protein expression and the perinatal increase in epithelialsodium transport (109–111). Thus O2 tension may modulate both physiologic andstructural characteristics of lung vascular and airway development.

Several oxygen-regulated genes appear to be involved in this process. Fibroblastgrowth factor (FGF)-9 expression is increased at fetal PO2, whereas FGF-10,which is also known to be involved in branching morphogenesis, demonstrates nosuch O2 dependency (108). The FGF-9 promoter has a putative binding site forC/EBP-β. Importantly, studies demonstrate that low oxygen tension results in thenuclear translocation and DNA binding of C/EBP-β. Thus O2 regulation of FGF-9via C/EBP-β may be a critical mediator of lung and lung vascular development.Other factors, including members of the hepatocyte nuclear factor (HNF) family,a homolog of Drosophila forkhead gene, may play roles in determining epithelialcell lineage fates by acting as dimerization partners for crucial developmentaltranscription factors such as NKX-2.1 (113, 114). The role of HNFs in hypoxicresponses is demonstrated by their involvement in erythropoietin gene expression,suggesting that interactions, at least between HNF-4 and HIF-1α are necessary forregulation of erythropoietin gene expression. Similarly, Sp1 transcription factors,known to be activated by hypoxia, are dimerization partners for HNF-3. Thus HNFtranscription factors may integrate the association between oxygen availability andlung development (106).

TGF family genes (TGFβ-1, -2, -3) and their receptors (TGF-βR1, 2, 3) promoteproliferation of mesenchyme tissue and thereby inhibit the rate of differentiationof the lung into branched airway structures (116). TGFβ-3 is a HIF-1α−regulatedgene that mediates mesenchymal proliferation under many conditions, both devel-opmental and pathophysiologic. In embryonic lung explants, HIF-1α and TGFβ-3appear to be up-regulated, thus supporting a role for both low oxygen concentra-tions and TGFβ-3 in lung development. Neutralizing antibody against TGFβ-3 re-sults in mesenchymal thinning and arrest of airway bifurcation. On the other hand,and very importantly, exposure of neonatal rats to reduced oxygen concentrations(9.5%) results in a dramatic increase in TGFβ signaling and TGFβ-R1 receptorexpression that collectively act to diminish alveolarization (117). Other groupshave reported that TGFβ-1 is an effective inhibitor of stress-invoked induciblenitric oxide synthase (iNOS) activity (118). Nitric oxide generated endogenouslyand exogenously can increase branching morphogenesis by as much as twofold(119). All three NOS isoforms are expressed in fetal pulmonary tissues with NOSexpression increasing dramatically within the final trimester (120). Thus it is pos-sible that endogenous NO production is required for normal airway growth and

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development both pre- and postnatally. Although hypoxia is known to evoke andsustain NO synthesis and release, it is possible that the simultaneous augmenta-tion of the TGFβ pathway results in inhibition of airway growth and epithelialmaturation through direct repression of iNOS activity and expression.

Recent studies have begun to elucidate the signaling pathways through whichhypoxia might act to modulate vascular cell proliferation and angiogenesis. Withregard to hypoxia-induced angiogenesis, it is clear that PI3K activity is essentialand that downstream targets, independent of Akt, are important (121). Activationof mTOR appears essential for hypoxia-mediated amplification of cell prolifera-tion and angiogenesis and activation may occur independently of Akt activationunder hypoxic conditions (122). This observation demonstrates a unique signalingaspect of hypoxia compared with that of other mitogenic stimuli. mTOR can actas an important upstream regulator of both HIF-1α and C/EBPβ and thus mayrepresent a key point of convergence in the sensing pathways that regulate cellcycle progression under hypoxic conditions (123).

In addition, recent studies also suggest that purinergic signaling is critical inhypoxia-induced fibroblast proliferation and differentiation as well as in SMCmigration and endothelial proliferation (124). This purinergic signaling loop maybe autocrine in nature because hypoxia induces the release of ATP from vascularwall cells. In addition, sympathetic or sensory nerves may be an additional sourceof purine nucleotides and thus may exert significant trophic effects on both thenascent and the mature vasculature. Recent studies have clearly demonstrated therole of the nervous system in directing vascular development (125). However,whether this is true in the lung or just for proximal lung vessels as opposed todistal lung vessels is currently unclear.

Thus it appears unequivocal that hypoxia activates unique cellular signalingpathways critical for growth and differentiation of cells within the pulmonaryvasculature. Studies in the future need to be directed at utilizing the effects ofoxygen to study lung vascular development. Furthermore, we need to utilize thisinformation to better understand the response of the premature lung, which isremoved from this hypoxic environment to a hyperoxic environment. That this tran-sition can inhibit lung development as well as vascularization often leading to sig-nificant cardiopulmonary dysfunction is clear. How the effects are mediated is not.

IMPAIRED VASCULAR GROWTH INBRONCHOPULMONARY DYSPLASIA

Perhaps the most relevant example of how disruption of lung vascular developmentcontributes to human disease lies in the clinical problem of BPD. Abnormalities ofthe pulmonary circulation, especially the development of pulmonary hypertension,have long been recognized as playing an important role in the pathophysiologyand clinical outcomes of premature infants with BPD (126, 134). More recent datafrom animal and clinical studies suggest that impaired vascular growth may also

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contribute to abnormalities of lung architecture, especially decreased alveolariza-tion, and may play a critical role in the pathogenesis of BPD. In the followingsection, we provide a brief overview of problems related to abnormalities of thepulmonary circulation in BPD and review recent data from animal models andclinical studies of BPD that may provide insight into how normal developmentalprocesses are interrupted by premature delivery.

BPD–the Clinical Problem

BPD is the chronic lung disease of infancy that follows mechanical ventilation andoxygen therapy for acute respiratory distress after birth in premature newborns (14,135, 136). As first characterized by Northway and colleagues in 1967, BPD hastraditionally been defined by the presence of persistent respiratory signs and symp-toms, the need for supplemental oxygen to treat hypoxemia, and an abnormal chestradiograph at 36-weeks corrected age (135) (Figure 2). Despite improvements inperinatal care, chronic lung disease after premature birth remains a major clin-ical problem. With increasing survival of extremely premature newborns, BPDremains as one of the most significant sequelae of neonatal intensive care, withan estimated 10,000 affected infants in the United States each year. The medicaland socio-economic impact of BPD is substantial, with many infants requiring fre-quent physician visits and hospitalizations due to recurrent respiratory infections,reactive airways disease, upper airway obstruction, cor pulmonale, and exerciseintolerance. In many cases, signs and symptoms of chronic lung disease continueinto adolescence and adulthood.

With the introduction of surfactant therapy, maternal steroids, new ventilatorstrategies, aggressive management of the patent ductus arteriosus, improved nutri-tion, and other treatments, the clinical course and outcomes of premature newbornswith RDS have dramatically changed over the past 30 years. During the “presur-factant era,” BPD was directly related to the severity of acute respiratory distresssyndrome (RDS) and was often present in premature infants who were born atrelatively large birth weights and gestational ages. For example, in the originalreport of Northway and coworkers, the premature infants with BPD were born at34-weeks gestation, weighed 2200 g, and mortality was 67% (135). Since that time,mortality of preterm infants has markedly decreased, with survival increasing fromless than 10% to presently over 50% in even the most extremely preterm newborn(24–26-weeks gestation). The risk of BPD rises with decreasing birth weight, withan incidence up to 85% for newborns between 500–699 g (135, 136). In extremelyimmature infants, even minimal exposure to oxygen and mechanical ventilationmay be sufficient to contribute to BPD (137–139). A recent report demonstratedthat about two thirds of infants who develop BPD have only mild respiratory dis-tress at birth (137). These findings suggest that developmental timing of lung injuryis a critical factor in the etiology of BPD.

In parallel with this changing epidemiologic and clinical pattern, key featuresof lung histology in BPD have also changed. Original studies of BPD described a

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continuous process through distinct stages of disease, progressing from acute lunginjury, or an exudative phase with diffuse pulmonary edema, proteinacous debris,and inflammation, to a proliferative phase with structural features of chronic lungdisease. Older reports described the gross cobblestone appearance of the lungs,representing alternating areas of atelectasis, marked scarring, and regional hy-perinflation (or emphysema). Typical histologic features of BPD included markedairway changes, such as squamous metaplasia of large and small airways, increasedperibronchial smooth muscle and fibrosis, chronic inflammation and airway edema,and hyperplasia of submucosal glands. Distal parenchymal lung disease was char-acterized by heterogenous changes, including regions of volume loss from atelec-tasis and septal fibrosis alternating with areas of overdistension or emphysematousregions. Mesenchymal thickening with increased cellularity and destruction of sep-tae with alveolar hypoplasia were also noted in early autopsy studies, along withhypertensive structural remodeling of small pulmonary arteries, including smoothmuscle hyperplasia and distal extension of smooth muscle growth into vessels thatare normally nonmuscular.

There is now growing recognition that infants with persistent lung disease afterpremature birth have a different clinical course and pathology than was tradition-ally observed in infants dying with BPD during this presurfactant era (140, 141).The classic progressive stages that first characterized BPD are often absent ow-ing to changes in clinical management, and BPD has clearly changed from beingpredominantly defined by the severity of acute lung injury to its current characteri-zation, which is primarily defined by a disruption of distal lung growth. In contrastto classic BPD, the new BPD develops in preterm newborns who generally requireminimal ventilator support and relatively low FiO2 (fraction of inspired oxygen)during the early postnatal days. Pathologic signs of severe lung injury with strik-ing fibroproliferative changes have become rare. At autopsy, lung histology nowdisplays more uniform and milder regions of injury, and signs of impaired alve-olar and vascular growth are more prominent. These features include a pattern ofalveolar simplification with enlarged distal airspaces and reduced growth of thecapillary bed, with vessels that are often described as dysmorphic because of theircentralized location in the thickened mesenchyme (Figure 2).

Thus, the so-called new BPD of the postsurfactant period represents inhibitionof lung development with altered lung structure, growth, and function of the dis-tal airspaces and vasculature (see below). Physiologically, these findings suggesta marked reduction in alveolo-capillary surface area, potentially contributing toimpaired gas exchange with increased risk for exercise intolerance, pulmonaryhypertension, and poor tolerance of acute respiratory infections (133, 142–145,see below).

Pathogenesis of BPD

BPD represents the response of the lung to injury during a critical period of lunggrowth, that is, during the canalicular period (17 to 26 weeks in the human), a

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Figure 3 Abnormal pulmonary circulation in chronic lung disease of in-fancy (BPD) Schematic illustrating pathogenetic abnormalities of the pul-monary circulation in BPD.

time during which airspace septation and vascular development increase dramat-ically (9). Several factors increase the susceptibility of the premature newborn tothe development of BPD, including surfactant deficiency, decreased antioxidantdefenses, impaired epithelial ion and water transport function, and lung struc-tural immaturity. Lung injury after premature birth and the subsequent arrest oflung growth results from complex interactions between multiple adverse stimuli,including inflammation, hyperoxia, mechanical ventilation, and infection, of thepoorly defended developing lung (Figure 3). Most recently, prenatal exposure toproinflammatory cytokines, such as TNF-α, IL-6, IL-8, and others, due to mater-nal chorioamnionitis, have been shown to enhance lung maturation in utero, butincrease the risk for BPD (146–148).

Hyperoxia and oxidant stress are critical factors in the development of BPD(149, 150). The transition of the premature newborn from the low-oxygen ten-sion environment of the normal fetus to the relative hyperoxia of extrauterine lifeincreases the risk for BPD with decreased alveolarization and a dysmorphic vas-culature. This premature change in the oxygen environment is likely to impedenormal epithelial-mesenchymal interactions leading to alterations in endothelialcell survival, differentiation, and organization in the microvasculature, the mech-anisms of which are partly described above. The premature infant is especiallysusceptible to reactive oxidant species (ROS)-induced damage owing to the lackof adequate antioxidants after premature birth. Antioxidant enzymes [e.g., super-oxide dismutase (SOD), catalase, and glutathione peroxidase] markedly increaseduring late gestation (151). In addition, the ability to increase synthesis of antioxi-dant enzymes in response to hyperoxia is decreased in preterm animals, suggestingthat premature birth may precede the normal up-regulation of antioxidants, which

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persists during early postnatal life. Experimental studies have shown that hyper-oxia, even in the absence of ventilation, can induce lung injury that mimics RDSand late sequelae that are similar to BPD (152, 153; see below). Endothelial andalveolar type II cells are both extremely susceptible to hyperoxia and ROS-inducedinjury, leading to increased edema, cellular dysfunction, and impaired cell survivaland growth (152–154). The critical role of host antioxidant defenses in protectingthe developing lung from hyperoxia-induced injury is further shown by studies oftransgenic SOD mice and treatment with exogenous SOD (155–157).

Even in the absence of overt signs of baro- or volutrauma, treatment of prematureneonates with mechanical ventilation initiates and promotes lung injury with in-flammation and permeability edema, and contributes to BPD. Ventilator-associatedlung injury (VALI) results from stretching distal airway epithelium and capillaryendothelium, which increases permeability edema, inhibits surfactant function,and provokes a complex inflammatory cascade (158). Experimental studies haveclearly shown that overdistension, and not pressure per se, is responsible for lunginjury in the surfactant-deficient lung (159, 160). Even brief periods of positive-pressure ventilation, such as during resuscitation in the delivery room, can causebronchiolar epithelial and endothelial damage in the lung, setting the stage forprogressive lung inflammation and injury (161, 162).

As described above, lung inflammation, whether induced prior to birth (fromchorioamnionitis) or during the early postnatal period (due to hyperoxia or VALI)plays a prominent role in the development of BPD (163–165). Numerous clinicaland experimental studies have shown that the risk for BPD is associated with sus-tained increases in tracheal fluid neutrophil counts, activated macrophages, highconcentrations of lipid products, oxidant-inactivated α-1-antitrypsin activity, andproinflammatory cytokines, including IL-6 and IL-8, and decreased IL-10 levels(166–168). Release of early response cytokines, such as TNF-α, IL-1β, IL-8, andTGF-β, by macrophages and the presence of soluble adhesion molecules (i.e., se-lectins) may impact other cells to release chemoattractants that recruit neutrophilsand amplify the inflammatory response (169, 170). Elevated concentrations ofproinflammatory cytokines in conjunction with reduced anti-inflammatory prod-ucts (i.e., IL-10) usually appear in tracheal aspirates within a few hours of life ininfants subsequently developing BPD. Increased elastase and collagenase releasefrom activated neutrophils may directly destroy the elastin and collagen frameworkof the lung, and markers of collagen and elastin degradation have been recoveredin the urine of infants with BPD (171). Infection from relatively low virulence or-ganisms, such as airway colonization with Ureaplasma urealyticum, may augmentthe inflammatory response, further increasing to the risk for BPD (172). Finally,other factors, such as nutritional deficits and genetic factors, such as vitamin A andE deficiency or single nucleotide polymorphism variants of the surfactant proteins,respectively, are likely to increase risk for BPD in some premature newborns (173,174). Thus multiple stimuli act on the susceptible lung at a critical stage of devel-opment after premature birth, leading to disruption of normal vascular growth andimpaired alveolarization.

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Pulmonary Circulation in Human BPD

In addition to adverse effects on the airway and distal airspace, acute lung injuryalso impairs growth, structure, and function of the developing pulmonary circu-lation after premature birth (13, 133, 153). Endothelial cells have been shown tobe particularly susceptible to oxidant injury through hyperoxia or inflammation(152, 153, 157). The media of small pulmonary arteries may also undergo strikingchanges, including smooth muscle cell proliferation, precocious maturation of im-mature mesenchymal cells into mature smooth muscle cells, and incorporation offibroblasts/myofibroblasts into the vessel wall (176, 177). Structural changes in thelung vasculature contribute to high pulmonary vascular resistance (PVR) throughnarrowing of the vessel diameter and decreased vascular compliance. In additionto these structural changes, the pulmonary circulation is further characterized byabnormal vasoreactivity, which also increases PVR (127, 130). Finally, decreasedangiogenesis may limit vascular surface area, causing further elevations of PVR,especially in response to high cardiac output with exercise or stress.

Overall, early injury to the lung circulation leads to the rapid developmentof pulmonary hypertension, which contributes significantly to the morbidity andmortality of severe BPD. Even from the earliest reports of BPD, pulmonary hyper-tension and cor pulmonale were recognized as being associated with high mortality(128, 131). Walther et al. showed that elevated pulmonary artery pressure in pre-mature newborns with acute RDS (determined from serial echocardiograms) wasassociated with severe disease and high mortality (131). Past studies have alsoshown that persistent echocardiographic evidence of pulmonary hypertension be-yond the first few months of life is associated with up to 40% mortality in infantswith BPD (128). High mortality rates have also been reported in infants with BPDand pulmonary hypertension who require prolonged ventilator support (132). Al-though pulmonary hypertension is a marker of more advanced BPD, elevated PVRalso causes poor right ventricular function, impaired cardiac output, limited oxygendelivery, increased pulmonary edema and, perhaps, a higher risk for sudden death.

Physiologic abnormalities of the pulmonary circulation in BPD include elevatedPVR and abnormal vasoreactivity, as evidenced by the marked vasoconstrictor re-sponse to acute hypoxia (127, 130, 132, 178). Cardiac catheterization studies haveshown that even mild hypoxia causes marked elevations in pulmonary artery pres-sure in infants with modest basal levels of pulmonary hypertension. Treatmentlevels of oxygen saturations above 92–94% effectively lower pulmonary arterypressure (130). Strategies to lower pulmonary artery pressure or limit injury to thepulmonary vasculature may limit the subsequent development of pulmonary hyper-tension in BPD. Recent data suggest that high pulmonary vascular tone continuesto elevate PVR in older patients with BPD, as demonstrated by responsiveness toaltered oxygen tension and inhaled nitric oxide (178).

In addition to pulmonary hypertension, clinical studies have also shown thatmetabolic function of the pulmonary circulation is impaired, as reflected by theimpaired clearance of circulating norepinephrine (NE) across the lung (179).

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Normally, 20–40% of circulating NE is cleared during a single passage throughthe lung, but infants with severe BPD have a net production of NE across thepulmonary circulation. It is unknown whether impaired metabolic function of thelung contributes to the pathophysiology of BPD by increasing circulating cate-cholamine levels, or if it is simply a marker of severe pulmonary vascular disease.It has been speculated that high catecholamine levels may lead to left ventricularhypertrophy or systemic hypertension, which are known complications of BPD(179–181).

Prominent bronchial or other systemic-to-pulmonary collateral vessels werenoted in early morphometric studies of infants with BPD and can be readily iden-tified in many infants during cardiac catheterization (182, 183). Although thesecollateral vessels are generally small, large collaterals may contribute to significantshunting of blood flow to the lung, causing edema and the need for higher FiO2.Interestingly, this enlargement of the bronchial circulation is similar to that de-scribed in adults with emphysema, chronic atelectasis, and/or high PVR, againsupporting the notion that obstruction to blood flow in the pulmonary circulationis a significant stimulus for growth of the bronchial circulation (see above). Collat-eral vessels have been associated with high mortality in some patients with severeBPD who also had severe pulmonary hypertension. Some infants have improvedafter embolization of large collateral vessels, as reflected by a reduced need forsupplemental oxygen, ventilator support, or diuretics. However, neither the actualcontribution of systemic collateral vessels to the pathophysiology of BPD nor thecellular mechanisms driving their enlargement is known.

Finally, pulmonary hypertension and right heart function remain major clinicalconcerns in infants with BPD. However, it is now clear that pulmonary vasculardisease in BPD also includes reduced pulmonary artery density owing to impairedgrowth, which contributes to physiologic abnormalities of impaired gas exchange,as well as to the actual pathogenesis of BPD (184, 185). As discussed below,experimental data support the hypothesis that impaired angiogenesis can impedealveolarization (186, 187) and that strategies preserving and enhancing endothelialcell survival, growth, and function may provide new therapeutic approaches forthe prevention of BPD.

Altered Signaling of Angiogenic Factors in Human BPD

As described above, multiple growth factors and signaling systems have beenshown to play important roles in normal lung vascular growth (7, 13) (Table 1).Several studies have examined how premature delivery and changes in oxygentension, inflammatory cytokines, and other signals alter normal growth factor ex-pression and signaling and thus lung/lung vascular development. The majority ofstudies have focused on VEGF. Impaired VEGF signaling has been associated withthe pathogenesis of BPD in the clinical setting (184, 185). Bhatt and coworkersfirst demonstrated decreased lung expression VEGF and VEGFR-1 in the lungs ofpremature infants who died with BPD (184). In another study, VEGF was found

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to be lower in tracheal fluid samples from premature neonates who subsequentlydevelop BPD than those who do not develop chronic lung disease (185). Exper-imentally, hyperoxia down-regulates lung VEGF expression, and pharmacologicinhibition of VEGF signaling in newborn rats impairs lung vascular growth andinhibits alveolarization (188–191; see below). Thus the biologic basis for impairedVEGF signaling leading to decreased vascular growth and impaired alveolariza-tion is well established. Additionally, lung VEGF expression is impaired in primateand ovine models of BPD induced by mechanical ventilation after premature birth,further supporting the hypothesis that impaired VEGF signaling contributes to thepathogenesis of BPD.

In addition, a role for TGF-β in the pathogenesis of BPD has been raised. Forexample, high levels of TGF-β inhibit lung morphogenesis, and increased levelsof TGF-β-1 are found in tracheal fluid samples early in the course of infants devel-oping BPD, which may predict high risk for BPD (192). Thus while TGF-β maybe necessary for certain stages in normal lung development, excessive expression,particularly at the wrong time, may induce an inhibition of lung morphogenesisand cause progressive pulmonary fibrosis (193). In fact, overexpression of TGF-β-1 inhibits alveolar growth and remodeling during the neonatal period (194).These observations make TGF-β a particularly intriguing target when consideringstrategies to ameliorate the structural abnormalities that characterize BPD.

Ongoing studies of the regulation and activities of diverse growth factors arelikely to lead to greater understanding of the pathobiology and treatment of BPD.

Relationship of Vascular Growth and Alveolarization

As described above, close coordination of growth between airways and vessels isessential for normal lung development. Thus we and others have hypothesized thatfailure of pulmonary vascular growth during a critical period of lung growth (sac-cular or alveolar stages of development) could decrease septation and ultimatelycontribute to the lung hypoplasia that characterizes BPD.

To determine whether angiogenesis is necessary for alveolarization, we studiedthe effects of the antiangiogenesis agents, thalidomide and fumagillin, on lunggrowth in the newborn and infant rat (187). In comparison with vehicle-treatedcontrols, postnatal treatment with these inhibitors of angiogenesis reduced lungvascular density, alveolarization, and lung weight. These findings suggest that an-giogenesis is necessary for alveolarization during lung development and that mech-anisms that injure and inhibit lung vascular growth may impede alveolar growthafter premature birth. Because VEGF has potent angiogenic effects during lungdevelopment and may synchronize vascular growth with neighboring epithelium,we also studied the effects of SU5416, a selective VEGF receptor inhibitor, onalveolarization during early postnatal life (191). Treatment of neonatal rats witha single injection of SU5416 caused pulmonary hypertension, reduced lung vas-cular density, and reduced alveolarization in infant rats (191) (Figure 4). Thusinhibition of lung vascular growth during a critical period of postnatal lung growth

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Figure 4 Inhaled NO increases lung vascular and alveolar growth in rats treated withthe VEGF receptor inhibitor, SU5416. The upper panels illustrate improved vascu-lar growth after inhaled NO therapy of SU5416-treated rats. Inhaled NO improvedpulmonary artery wall thickness, vascular growth, and alveolarization (radial alveolarcounts, RAC) (88).

impairs alveolarization, suggesting that endothelial-epithelial cross talk, especiallyvia VEGF signaling, is critical for normal lung growth following birth.

The mechanisms through which impaired VEGF signaling inhibits vasculargrowth and alveolarization are uncertain, but, in part, may be mediated by alteredNO production. Past in vitro and in vivo studies have shown that VEGF stimulatesendothelial NO synthase (eNOS) expression in isolated endothelial cells from thesystemic circulation (195–201), but whether VEGF is an important regulator oflung eNOS expression, especially during development, and what role NO playson lung growth are incompletely understood. In addition to its effects on vasculartone, NO can alter angiogenesis, but data are conflicting on its effects. AlthoughNO inhibits endothelial cell proliferation in some models, most studies have shownthat NO mediates the angiogenic effects of VEGF via activation of VEGFR-2 andstimulation of the Akt-PI3K pathway (196, 198–201). Proliferating bovine aorticendothelial cells express greater eNOS mRNA and protein than confluent cells,but NOS inhibition does not apparently affect their rate of proliferation. Studieswith the eNOS−/− fetal mouse model suggest that NO plays a critical role in

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vascular and alveolar growth in utero and that eNOS−/− newborns are more sus-ceptible to hypoxia-induced inhibition of alveolarization (202–205). Interestingly,lung eNOS expression is down-regulated in primate and ovine models of BPD(206, 207). More recently, treatment of newborn rats with SU5416, the VEGFreceptor antagonist, was shown to decrease eNOS expression and NO productionduring infancy, and that prolonged treatment with inhaled NO prevented the de-velopment of pulmonary hypertension, improved vascular growth, and enhancedalveolarization (208) (Figure 4). Previous studies have shown that inhaled NOattenuates hyperoxia-induced acute lung injury (209–211), which may enhancesubsequent vascular and lung growth. These studies suggest that decreased VEGFsignaling down-regulates lung eNOS expression, and that impaired NO produc-tion may contribute to abnormal lung growth during development. Importantly,a recent randomized single-center study has shown that inhaled NO treatmentreduced the combined endpoint of BPD and death in human premature newbornswith moderate RDS (212). Ongoing multicenter clinical trials are evaluating thepotential efficacy of inhaled NO in reducing BPD in larger population studies.

MODELS OF BPD

For more than 20 years, exposure of newborn rats to hyperoxia has been usedas a model to study mechanisms of BPD due to the effects of enriched oxygenon alveolarization and vascular growth. During the alveolar phase of lung devel-opment, the first 2 weeks of postnatal life in the rat, the lung also undergoes astriking proliferation of vascular growth. Exposure of newborn rats to either 100%or 60% FiO2 markedly decreases capillary density when compared with that ofair-breathing controls (153, 175). During the room air recovery period after rel-atively short exposures to hyperoxia, the number of capillaries and alveoli tendto increase toward normal values in infant rats. However, if the period of oxygenexposure is more prolonged, the decrease in pulmonary vascular density persistsdespite room air recovery.

Although multiple mechanisms are likely involved in the arrest of lung growthafter hyperoxia, several studies have demonstrated marked and sustained down-regulation of lung VEGF and VEGFR-2 expression, suggesting that impairedVEGF signaling contributes to these long-lived abnormalities in lung structure.Thus hyperoxic exposure provides a useful model for the study of basic mecha-nisms that disrupt normal pulmonary vascular growth and development.

Recently, the gene expression profile induced by prolonged oxidative stress hasbeen studied. Using DNA-microarray analysis, which was then largely confirmedby real-time RT-PCR, Wagenaar et al. demonstrated that prolonged oxidativestress induces changes in a complex orchestra of genes involved in inflammation,coagulation, fibrinolysis, extracellular matrix turnover, cell cycle, signal trans-duction, and alveolar development (213). The changes in gene expression, bothup-regulated as well as down-regulated, appear consistent with the pathologic

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changes in immature lungs developing a BPD-like picture. One of the majoreffects of hyperoxia on postnatal lung development in the rat is the reductionof secondary septation and the enlargement of alveoli. This phenomenon wasaccompanied by a down-regulation of FGFR-4 from day 3 onward and of theFlk-1 receptor later in the course (day 10). These observations are consistentwith previous findings demonstrating that the lungs of FGFR-3−/−/FGFR-4−/−

mice are normal at birth but then develop a complete block in alveologenesisand do not form secondary septa (214). Down-regulation of VEGFR-2 (or Flk-1) confirmed in this array analysis on day 10 coincided with the presence ofenlarging alveoli and is consistent with the role of VEGF/VEGFR-2 signalingin the maintenance of alveolar structures (215). These findings provide furthersupport for a critical role of VEGF/VEGFR-2 signaling in the pathogenesis ofBPD.

Another set of genes up-regulated by hyperoxia is linked to the inflammatoryresponse and is consistent with observations in the human as described above. Theinflux of leukocytes appears to be mediated by chemokines such as IL-8, CINC-1,and MIP-2 via the activation of the CXCR2 receptor. The Wagenaar study con-firms the up-regulation of these molecules observed in other hyperoxic modelsand implicates them in the disease process. The importance of these chemokinesin hyperoxia-induced lung injury is demonstrated by the fact that antichemokinetreatment, which reduces neutrophil influx into the lung, preserved alveolar devel-opment in newborn rats exposed to hyperoxia (216, 217).

Several studies have demonstrated that extravascular fibrin deposits are fre-quently observed in the septa and alveoli of infants with BPD. Again, in thehyperoxic model described, gene array analysis demonstrates the up-regulation ofthe procoagulant tissue factor (TF), down-regulation of the anticoagulant throm-bomodulin (TM), and up-regulation of the fibrinolytic inhibitor, PAI-1. Thesechanges would lead to a procoagulant and antifibrolytic environment resulting infibrin deposition. The significance of PAI-1 in hyperoxia-induced fibrin depositionhas been demonstrated in PAI-1-deficient mice, which fail to develop intra-alveolarfibrin deposits and show a less severe phenotype in response to hyperoxia-inducedinjury (218). The accumulation of fibrin can contribute to injury in several ways.It can function as a ligand for receptors on circulating leukocytes, including theintegrin Mac-1, and can promote both their migration and activation (219). Fibrincan also activate and induce proliferation and migration of fibroblasts, probablyvia activation of NF-κB and AP-1 (220). Therefore, unwanted activation of leuko-cytes and fibroblasts can contribute to the abnormalities of lung vascular growththat characterize BPD.

The combination of rat models and gene array analysis thus provides importantnew insights into the mechanisms through which interrupted fetal lung develop-ment can lead to significant abnormalities in postnatal pulmonary vascular andlung development.

Extensive work from the Bland laboratory has shown that prolonged mechani-cal ventilation of premature lambs after cesarean-section delivery at 120–130 days

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gestation (term 147 days) for 2–3 weeks causes a chronic lung disease (CLD) thatshares many features of human BPD (221). In addition to airway abnormalities,pulmonary vascular growth, structure, and function are impaired after chronic ven-tilation. In comparison with controls, CLD lambs have reduced pulmonary arterydensity with reduced alveolarization and hypertensive pulmonary artery remodel-ing. Although the mechanisms of altered lung growth are unclear, lung eNOS andsoluble guanylate cyclase expression are reduced in CLD lambs, suggesting thatimpaired NO-cGMP signaling may contribute to pulmonary vascular disease inthis model.

Primate Model of BPD

An extremely important model of BPD in primates has been developed underthe guidance of Coalson (141). In this model, premature baboons are deliveredby cesarean section at 140 days (or 0.75 term) and treated with mechanicalventilation with high FiO2 (222). These animals develop severe chronic lung dis-ease, with histologic lesions that mimic human BPD (222). A newer and perhapsmore relevant model for the new BPD is one in which extremely premature ba-boons are delivered at 125 days (or 0.67 term; which is roughly equivalent to humangestation of 26–27 weeks) and are treated with exogenous surfactant and mechan-ical ventilation, but are managed with lower (as needed) levels of supplementaloxygen. These animals develop structural lesions characterized by severe alveolarhypoplasia with decreased and dysmorphic vascular growth. As in the prematurelamb model of BPD, lung eNOS expression is impaired (206). In addition, lungVEGF mRNA content is markedly decreased, and the localized epithelial patternof VEGF expression is absent (223). Expression of mRNA for VEGFR-1 alsodecreases by 30–40% in treated animals but the VEGFR-2 mRNA expression re-mained unchanged. Whether interventions such as inhaled NO or VEGF treatmentcan reduce the abnormalities of lung vascular growth and impaired alveolarizationin this model is uncertain.

CONCLUSIONS

Past studies have primarily focused on how altered lung vascular growth anddevelopment contributes to pulmonary hypertension. Recently, basic studies ofvascular growth have led to novel insights into mechanisms underlying develop-ment of the normal pulmonary circulation and the essential relationship of vas-cular growth to lung alveolar development. These observations have led to newconcepts underlying the pathobiology of developmental lung disease, especiallythe inhibition of lung growth that characterizes BPD. We speculate that under-standing basic mechanisms that regulate and determine vascular growth will leadto new clinical strategies to improve the long-term outcome of premature babieswith BPD.

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The Annual Review of Physiology is online athttp://physiol.annualreviews.org

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LUNG VASCULAR DEVELOPMENT C-1

Figure 2 Radiographic and histologic features of bronchopulmonary dysplasia (BPD).This chest radiograph demonstrates late features of BPD, including marked hyperinfla-tion (upper left panel). Pulmonary angiography illustrates striking pruning of the pul-monary circulation from this patient, who also had severe pulmonary hypertension(lower left panel). Marked fibroproliferative changes with interstitial thickening anddecreased septation are shown (right panels). Abnormal vascular growth, as reflectedby factor VIII staining of endothelium, is shown in the lower right panel.

HI-RES-PH67-24-Stenmark.qxd 1/25/05 2:15 PM Page 1

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January 20, 2005 12:12 Annual Reviews AR237-FM

Annual Review of PhysiologyVolume 67, 2005

CONTENTS

Frontispiece—Michael J. Berridge xiv

PERSPECTIVES, Joseph F. Hoffman, Editor

Unlocking the Secrets of Cell Signaling, Michael J. Berridge 1

Peter Hochachka: Adventures in Biochemical Adaptation,George N. Somero and Raul K. Suarez 25

CARDIOVASCULAR PHYSIOLOGY, Jeffrey Robbins, Section Editor

Calcium, Thin Filaments, and Integrative Biology of Cardiac Contractility,Tomoyoshi Kobayashi and R. John Solaro 39

Intracellular Calcium Release and Cardiac Disease, Xander H.T. Wehrens,Stephan E. Lehnart and Andrew R. Marks 69

CELL PHYSIOLOGY, David L. Garbers, Section Editor

Chemical Physiology of Blood Flow Regulation by Red Blood Cells:The Role of Nitric Oxide and S-Nitrosohemoglobin, David J. Singeland Jonathan S. Stamler 99

RNAi as an Experimental and Therapeutic Tool to Study and RegulatePhysiological and Disease Processes, Christopher P. Dillon,Peter Sandy, Alessio Nencioni, Stephan Kissler, Douglas A. Rubinson,and Luk Van Parijs 147

ECOLOGICAL, EVOLUTIONARY, AND COMPARATIVE PHYSIOLOGY,Martin E. Feder, Section Editor

Introduction, Martin E. Feder 175

Biophysics, Physiological Ecology, and Climate Change: Does MechanismMatter? Brian Helmuth, Joel G. Kingsolver, and Emily Carrington 177

Comparative Developmental Physiology: An InterdisciplinaryConvergence, Warren Burggren and Stephen Warburton 203

Molecular and Evolutionary Basis of the Cellular Stress Response,Dietmar Kultz 225

ENDOCRINOLOGY, Bert O’Malley, Section Editor

Endocrinology of the Stress Response, Evangelia Charmandari,Constantine Tsigos, and George Chrousos 259

vii

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viii CONTENTS

Lessons in Estrogen Biology from Knockout and Transgenic Animals,Sylvia C. Hewitt, Joshua C. Harrell, and Kenneth S. Korach 285

Ligand Control of Coregulator Recruitment to Nuclear Receptors,Kendall W. Nettles and Geoffrey L. Greene 309

Regulation of Signal Transduction Pathways by Estrogenand Progesterone, Dean P. Edwards 335

GASTROINTESTINAL PHYSIOLOGY, John Williams, Section Editor

Mechanisms of Bicarbonate Secretion in the Pancreatic Duct,Martin C. Steward, Hiroshi Ishiguro, and R. Maynard Case 377

Molecular Physiology of Intestinal Na+/H+ Exchange,Nicholas C. Zachos, Ming Tse, and Mark Donowitz 411

Regulation of Fluid and Electrolyte Secretion in Salivary GlandAcinar Cells, James E. Melvin, David Yule, Trevor Shuttleworth,and Ted Begenisich 445

Secretion and Absorption by Colonic Crypts, John P. Geibel 471

NEUROPHYSIOLOGY, Richard Aldrich, Section Editor

Retinal Processing Near Absolute Threshold: From Behaviorto Mechanism, Greg D. Field, Alapakkam P. Sampath, and Fred Rieke 491

RENAL AND ELECTROLYTE PHYSIOLOGY, Gerhard H. Giebisch, Section Editor

A Physiological View of the Primary Cilium, Helle A. Praetoriusand Kenneth R. Spring 515

Cell Survival in the Hostile Environment of the Renal Medulla,Wolfgang Neuhofer and Franz-X. Beck 531

Novel Renal Amino Acid Transporters, Francois Verrey, Zorica Ristic,Elisa Romeo, Tamara Ramadam, Victoria Makrides, Mital H. Dave,Carsten A. Wagner, and Simone M.R. Camargo 557

Renal Tubule Albumin Transport, Michael Gekle 573

RESPIRATORY PHYSIOLOGY, Carole R. Mendelson, Section Editor

Exocytosis of Lung Surfactant: From the Secretory Vesicle to theAir-Liquid Interface, Paul Dietl and Thomas Haller 595

Lung Vascular Development: Implications for the Pathogenesis ofBronchopulmonary Dysplasia, Kurt R. Stenmark and Steven H. Abman 623

Surfactant Protein C Biosynthesis and Its Emerging Role inConformational Lung Disease, Michael F. Beers and Surafel Mulugeta 663

SPECIAL TOPIC, CHLORIDE CHANNELS, Michael Pusch, Special Topic Editor

Cl− Channels: A Journey for Ca2+ Sensors to ATPases and SecondaryActive Ion Transporters, Michael Pusch 697

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CONTENTS ix

Assembly of Functional CFTR Chloride Channels, John R. Riordan 701

Calcium-Activated Chloride Channels, Criss Hartzell, Ilva Putzier,and Jorge Arreola 719

Function of Chloride Channels in the Kidney, Shinichi Uchidaand Sei Sasaki 759

Physiological Functions of CLC Cl− Channels Gleaned from HumanGenetic Disease and Mouse Models, Thomas J. Jentsch,Mallorie Poet, Jens C. Fuhrmann, and Anselm A. Zdebik 779

Structure and Function of CLC Channels, Tsung-Yu Chen 809

INDEXES

Subject Index 841Cumulative Index of Contributing Authors, Volumes 63–67 881Cumulative Index of Chapter Titles, Volumes 63–67 884

ERRATA

An online log of corrections to Annual Review of Physiology chaptersmay be found at http://physiol.annualreviews.org/errata.shtml

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