Retinoids in Lung Development and Regeneration

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Retinoids in Lung Development and Regeneration Malcolm Maden MRC Centre for Developmental Neurobiology King’s College London London SE1 1UL, United Kingdom

I. Stages of Lung Development A. Embryonic Period (22 d to 6 Weeks) B. Pseudoglandular Stage (6–16 Weeks) C. Canalicular Phase (16–26 Weeks) D. Saccular Phase (26–36 Weeks) E. Alveolar Phase (36 Weeks to 2 Years Postnatal) II. What is Retinoic Acid (RA) III. RA is Required for Budding of the Foregut Tube IV. Branching Morphogeneis is Inhibited by RA V. RA is Required during the End of the Foetal Period (Pseudoglandular, Canalicular, and Terminal Saccule Stages) VI. RA is Required for the Postnatal Septation (Alveologenesis) VII. RA Induces Alveolar Regeneration VIII. Speculations of the Mode of Action of RA IX. Clinical Implications and Future Possibilities Acknowledgment References

This review considers the role that retinoids, the family of molecules derived from vitamin A, play in lung development and regeneration.The stages of lung development are described using rodents and where the information is available, humans, as model systems. Because vitamin A is a dietary component it has long been known that early retinoid deprivation of pregnant animals results in abmormalities such as lung agenesis and later deprivation results in defective alveologenesis. The presence of retinoids, the presence of the transducers of the retinoid signal, and the experiments that have been performed both in vivo and in vitro to investigate the role of retinoids are described for each of the stages of lung development from the initial lung bud stages through to alveologenesis. Recent data on the induction of alveolar regeneration by retinoic acid is also described and its possible modes of action via diVering cell types, namely type II pneumocytes or haematopoietic stem cells is discussed. The potential roles of retinoic acid in alleviating human conditons, notably the failure of alveologenesis in premature infants and as Current Topics in Developmental Biology, Vol. 61 Copyright 2004, Elsevier Inc. All rights reserved. 0070-2153/04 $35.00

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a therapy for alveolar regeneration to treat diseases involving the loss of alveoli such as emphysema is considered. The adult lung is a highly eYcient gas-exchanging organ whose surface area in humans is approximately 70 m2. This surface, across which oxygen diVuses to enter the pulmonary circulation and carbon dioxide leaves to enter the expired air, consists of about 300 million alveoli. The diVusion barrier of the alveolar walls whose thickness varies from 0.1 mm to several microns consists of three tissue layers: capillary endothelium, alveolar epithelium, and interstitial tissue. Lung development is a long and complicated process that takes up 90% of the period of gestation and then continues well into childhood in humans. Once formed, however, the adult lung is not a static, unchanging organ but a dynamic one that rapidly responds to pathogens and can change remarkably rapidly in terms of alveolar number according to environmental influences such as calorific intake (Massaro et al., 2004) or altitude (Massaro and Massaro, 2002). There is a slow and irrevocable decline in alveolar number with age. If the lung is not given the chance to develop properly, such as in low-birth-weight premature infants, then the reduced number of alveoli present often result in lung disorders such as bronchopulmonary dysplasia. In adults, there is a severe and dramatic decline in alveolar number in diseases such as emphysema, which is irreversible and currently incurable. This review deals with the role that retinoids, the family of molecules derived from vitamin A, play in lung development and regeneration. Because vitamin A is a dietary component, the role of retinoids in lung development is particularly relevant to the respiratory problems of premature infants. Most excitingly, recent data from animal studies suggest that retinoids can induce regeneration of alveoli in young adults, which, if relevant to humans, might be the first potential treatment for diseases such as emphysema or bronchopulmonary dysplasia. Therefore, it is particularly timely to review this subject. The review first describes the events and stages of lung development; then discusses what retinoids are, how they are synthesized, and how they act; then describes the role of retinoids in each stage of lung development; and finally discusses its role in lung regeneration and the clinical implications. C 2004, Elsevier Inc.

I. Stages of Lung Development Lung development occurs over an incredibly extended period of time, from day 22 of gestation to perhaps as far as 8 years after birth in humans. At birth, after more than 8 months of development the lung is functional, albeit not at full eYciency, and during the extensive postnatal period the alveoli mature and increase in number. In humans, lung development is classically divided into several phases, as detailed in Table I.

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7. Retinoids in Lung Development and Regeneration Table I Summary of the Stages and Events of Human Lung Development Stage of development

Period

Embryonic

22 d–6 weeks

Pseudoglandular

6–16 weeks

Canalicular

16–26 weeks

Saccular

28–36 weeks

Alveolar

36 weeks to term and beyond

Events Lung bud arises as a ventral outgrowth of the foregut endoderm, undergoes three rounds of branching, producing primordia of two lungs, lung lobes, and bronchopulmonary segments. Respiratory trees undergo 16 more rounds of branching resulting in formation of terminal bronchioles. Each terminal bronchiole divides into two or more respiratory bronchioles to form acini. Type I and type II cells diVerentiate. Vasculature develops. Respiratory bronchioles subdivide into terminal sacs. Alveolar ducts and alveolar sacs form. Secondary septa with a double capillary network divide the sacs into alveoli. Alveolar walls thin as a result of apoptosis and develop a single capillary structure.

From Larsen, W. F. (1993). ‘‘Human Embryology.’’ Churchill Livingstone, New York, with permission.

A. Embryonic Period (22 d to 6 weeks) The lung first makes its appearance on day 22 of human development as an outgrowth that buds oV the foregut endodermal tube (Fig. 1A) to invade the surrounding splanchnic mesoderm (Burri, 1997; Larsen, 1993; Thurlbeck, 1975). This process takes place in the mouse embryo on embryonic day 9 (E9) and in rats on E12. Separation of the two tubes is achieved by two lateral inpushings: the laryngotracheal grooves that appear and move in a caudocranial direction. Like all the other branches from the gut tube, the epithelium of lungs is of endodermal origin. On days 26–29 in the human embryo, the lung bud undergoes its first bifurcation into left and right bronchial buds, which are the rudiments of the two lungs (Fig. 1A). By 4.5 weeks, the lung anlage forms five saccules, the secondary bronchial buds, two on the left and three on the right, thus performing the future lobar bronchii and the corresponding lung lobes (Fig. 1B). By continuing dichotomous divisions up to the end of the sixth week, the tertiary bronchial buds form (Fig. 1C) and thereby generate the bronchopulmonary segments of the mature lung. The structure of these buds consists of a columnar endoderm (epithelium) surrounded by splanchnic mesoderm (Fig. 1E), and the mesoderm plays a

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Figure 1 Drawings of the stages of human lung development, with comments on their requirement for RA added. (A) The initial budding from the foregut tube (arrow) takes place on day 22 in humans and day 9 in mice. Left drawing shows a lateral view of the gut tube from the pharynx (ph) to the yolk sac (ys), showing the location of the lung bud. The next drawing shown

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crucial role in inducing this branching activity. [For a review, see Shannon and Deterding (1997).] Removal of the mesenchyme results in death of the epithelium and foreign mesenchyme (somatic, mesonephric) keeps the epithelium alive, but normal branching does not occur. The mesenchyme at the branching tip is inductively unique because if it is replaced by a more proximal mesenchyme such as that surrounding the trachea, then further branching is inhibited. Conversely, the mesenchyme from the tip of a bud transplanted to the side of the future trachea induces outgrowth of a new bud. The type of branching is also mesenchymally determined because if a chicken lung mesoderm is transplanted to a mouse lung epithelium, the latter branches in an avian fashion. We now know that the mesenchyme at the budding tip expresses important inducing molecules such as FGF10 (Bellusci et al., 1997b; Park et al., 1998; Weaver et al., 2000), but obviously other factors are involved because chicken FGF10 is the same as mouse FGF10 yet the branching patterns are diVerent. B. Pseudoglandular Stage (6–16 Weeks) By 7 weeks of gestation, the human lung has the appearance of a primitive gland (Fig. 1D): airways lined by columnar epithelium and separated by thick undiVerentiated mesenchyme (Fig. 1E). Branching of the airways continues during this phase and formation of terminal bronchioles and primitive acinar structures is complete by the end of this period. About 65–75% of bronchial branching occurs between the 10th and 14th weeks. The epithelial cells become loaded with glycogen and ciliated cells and goblet cells appear in the central airways and spread to the more peripheral tubules. The surfactant proteins A, B, and C within epithelial cells are first detected a ventral view of the same bud. The next drawing shows the bifurcation of the bud into a left and right bud as it invades the splanchnic mesoderm (red). The next drawing shows the two buds continuing to elongate. (B) 4.5 weeks of human development showing that the left bud has branched into two secondary bronchial buds and the right bud has branched into three secondary bronchial buds as the mesoderm (red) continues to surround the branches. (C) Formation of tertiary bronchial buds by the end of the sixth week. (D) Pseudoglandular stage at 7 weeks. Further branching into the mesoderm (red) has continued. (E) Histological appearance of lung buds at the pseudoglandular stage is of a columnar epithelium in an acinar arrangement surrounded by mesoderm (red), which is richly invaded with capillaries (yellow). (F) Histological appearance of the canalicular phase when the epithelium of the acini has thinned out and the cells have diVerentiated into flat type I pneumocytes. (G) Alveolization. Acini have thinned and expanded into alveolar ducts (ad) (two shown here). Secondary septa (dotted lines) grow out from the walls of the alveolar ducts to cut oV smaller-diameter regions, the alveoli (a). Further proximal the respiratory epithelium is more columnar with a thin coating of mesoderm. The mesoderm between the acini [as in (F)] has now been reduced to the extracellular matrix of the alveolar walls. Capillaries are present in the alveolar walls (not shown).

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during this phase and the mesenchyme starts to diVerentiate cartilage and smooth muscle. C. Canalicular Phase (16–26 Weeks) This phase is characterized by the appearance of acini consisting of an airway stem and a spray of short tubules arranged in a cluster surrounded by a covering of loose and thinning mesenchyme (Fig. 1F). The mesenchyme becomes riddled with capillaries and is said to be ‘‘canalized.’’ The epithelial cells lining the tubules in the distal regions of the lung begin to flatten out and diVerentiate into type I and type II pneumocytes. Type II cells start to accumulate lamellar bodies, which represent the intracellular storage form of the components of surfactant. Proximal bronchiolar epithelial cells begin to synthesize the Clara cell secretory protein. At the end of this phase, gas exchange in the 26- to 28-week human infant can be supported, especially when surfactant is provided exogenously. Surfactant synthesis and mesenchymal thinning are enhanced by glucocorticoids administered to mothers to prevent respiratory distress syndrome after premature birth. D. Saccular Phase (24–36 Weeks) The distal airways form terminal clusters of widened airspaces called saccules, and there is a massive expansion of airspace volume. The terminal sacs give rise, on average, to three generations of prospective alveolar ducts and one generation of alveolar sacs. As a result of the expansion, the interstitial tissue between the airspaces is compressed and its volume proportion markedly decreases. The structure of the pulmonary capillary bed is changed as a result: the capillaries get closer together, and the walls between the distal airspaces contain a double capillary network. E. Alveolar Phase (36 Weeks to 2 Years Postnatal) In some species with little locomotive ability at birth, notably the mouse and rat, true alveoli are not present at birth (Fig. 2A). Instead, saccules with smooth walls whose dimensions are much larger than those of alveoli are present (Amy et al., 1977). Thus, the entire alveolar stage takes place postnatally, from day 4 to 14. In guinea pigs and other range mammals that have great locomotive ability at birth, alveolization takes place in utero. In humans, it seems that a considerable number of alveoli (approximately 20 million) are present at birth, as alveolization starts around 26 weeks of gestation and continues postnatally, probably terminating by 18 or 24

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Figure 2 (A)–(C) micrographs of postnatal alveolization in mice, all at the same magnification. (A) A P4 lung showing large alveolar ducts or saccules with thick walls. (B) By P9 the alveolar size as decreased as seconday septa (arrowheads) grow out from the walls of the saccules. (C) A P15 lung showing that many thin-walled, small-diameter alveoli have been generated, along with some alveolar ducts still present. Note the vastly increased number of alveoli compared with that in (A). (D) Formation of secondary septa. Ridges (arrows) are formed by outpushings of a capillary (yellow). These septa grow out with a double layer of capillaries and also depositions of elastin (red dots), which are closely related to the leading edge of the septa. From Burri, P. H. (1997). In ‘‘Lung Growth and Development’’ (J. A. McDonald, Ed.), pp. 1–35. Marcel Dekker, New York, with permission.

months (Burri, 1997) as by 18 months the alveolar walls have assumed an adult structure (Zeltner and Burri, 1987). There are about 300 million alveoli in the adult human. It is possible that alveoli are continually added into adulthood as, at least in rats, new alveoli are added at the periphery of the lung (Massaro and Massaro, 1993). Alveolization starts with the appearance of low ridges along both sides of the saccular walls (Fig. 2B, arrowheads), which grow and divide the saccules into smaller units, the alveoli. These new walls are called secondary septa and at their leading edge is a deposition of elastin (Fig. 2D). Both the saccular walls and secondary septa contain a double capillary layer and

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the bulging out of a capillary is thought to be the instigating factor in the outgrowth of the secondary septa. The alveolar surface area therefore increases substantially as the diameter of the airspaces decreases (Fig. 2D). The final remodeling of the alveolar walls to assume the adult morphology represents the last stage in lung development. This involves changes in the capillary structure, the extracellular matrix, and cell numbers. The double capillary layer is replaced with a single capillary layer, thereby decreasing the diVusion distance between the alveolar gas and the pulmonary capillaries. The extracellular matrix undergoes a reduction in mass (Vaccaro and Brody, 1978) as do the cells responsible for its production, the interstitial fibroblasts. Two populations of fibroblasts are present in the alveolar walls with two diVerent proliferation rates (Awonusonu et al., 1999) and with no evidence of interchange between the two (Brody and Kaplan, 1983). In one there are abundant lipid droplets, the lipid-laden interstitial fibroblasts (LIFs), which store retinoids, and the other population is the non-lipid interstitial fibroblasts (NLIFs). Together, these cells are the major contributors to the synthesis of the extracellular matrix, which provides tensile strength and elasticity to the gas-exchanging surface (McGowan and Torday, 1997). LIFs are often found at the base of the outgrowing septa during alveologenesis (Vaccaro and Brody, 1978) and increase in number during early alveologenesis and then decrease (Maksvytis et al., 1981) because of apoptosis (Awonusonu et al., 1999). The other cells in the alveolar walls are the type I and type II pneumocytes. Type II cells produce surfactant and are regarded as a lung progenitor cell population, or stem cells for the type I cell (Adamson and Bowden, 1975; KauVman et al., 1974). Ultrastructural analysis has demonstrated that LIFs are often in close contact with type II cells (Sannes, 1991) and that the type II cell may be involved in the eruption of secondary septa. The number of type I and II cells also decrease as alveologenesis is completed because there is an eight-fold increase in apoptosis at the end of the third week in rat lungs. The highest levels of apoptosis are found in fibroblasts and type II cells (Schittny et al., 1998), the latter additionally declining in number because of diVerentiation into type I cells.

II. What is Retinoic Acid (RA) Retinoic acid (RA) is an endogenous molecule in the embryonic and adult vertebrate that is derived from vitamin A. It is of low molecular weight (300 Da) and is lipophilic. In the adult, vitamin A is obtained from the diet in the form of retinyl esters present in animal meat or -carotene present in plants. After absorption through the gut, retinyl esters are transported in chylomicrons to the liver for storage. There are several extrahepatic sites of

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retinyl ester storage, including the lung where LIFs present in the alveolar walls serve this function (Okabe et al., 1984). Cells of the embryo or adult that require RA for their function obtain retinol from the blood system, where it circulates bound to the retinol-binding protein, having been metabolized from retinyl esters in the liver hepatocytes. Inside cells that require RA, the sequestered retinol is bound to cellular retinol binding protein (CRBP) and then enzymatically converted first to retinal by retinol or alcohol dehydrogenases (ADHs) and then to RA by retinal dehydrogenases (RALDHs; Duester, 2000). RA is further metabolized by cytochrome P450 enzymes called Cyp26s to inactive products such as 4-oxo-RA, 4-OH-RA, 18-OH-RA, and 5,18-epoxy-RA (Abu-Abed et al., 1998; Fujii et al., 1997; White et al., 1996) and finally excreted. Two isomers of RA, all-trans-RA and 9-cis-RA, act via diVerent receptors (see later), but whether they have separate enzymatic pathways from all-trans-retinol and 9-cis-retinol or isomerization takes place as a last step is not known. Once RA has been synthesized in the cell, it enters the nucleus and establishes or changes the pattern of gene activity by binding to ligand-activated nuclear transcription factors. There are two classes of these transcription factors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). In humans, rats, and mice, there are three RARs, , , and , each having multiple isoforms (Kastner et al., 1990) and three RXRs,  , and , (Kliewer et al., 1994), again each having several isoforms. RARs and RXRs act as heterodimers (e.g., RAR/RXR ) and recognize consensus sequences known as retinoic acid response elements (RAREs) in the upstream promoter sequences of RA-responsive genes. A summary of the retinoid pathway is shown in Fig. 3.

III. RA is Required for Budding of the Foregut Tube In the early part of the twentieth century, studies were conducted on the role that vitamins and minerals played in the diet. To this end, the components were individually removed from the diet of a variety of farm animals and rats and the eVect both on the adult body and the subsequent development of embryos from pregnant animals was observed. When vitamin A was removed from the diet of rats, the classical observation of widespread epithelial keratinization was made (Wolbach and Howe, 1925). This included the respiratory tract, where the normal mucociliary lining of the trachea and bronchi was replaced with stratified keratinizing epithelium. It is now well established that RA is required for epithelial diVerentiation (e.g., Rosenthal et al., 1994). Subsequent studies on the embryonic defects caused by the absence of vitamin A in the maternal diet revealed underdeveloped lungs, and, occasionally, agenesis of one lung (the left) and tracheoesophageal

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Figure 3 Diagram to show the how retinoids are stored in the liver, get to the cells of the body and are then metabolised in the cytoplasm of cells to generate retinoic acid when then enters the nucleus to activate gene transcription and is finally catabolised. RBP ¼ retinol binding protein, which is the carrier of retinol in the blood. CRBP ¼ cellular retinol binding protein. CRABP ¼ cellular retinoic acid binding protein. ADH ¼ alcohol dehydrogenases which are the enzyme that metabolise retinol to retinal. RALDH ¼ retinaldehyde dehydrogenases, which are the enzymes that metabolise retinal to RA. RA ¼ retinoic acid. RAR ¼ retinoic acid receptor. RXR ¼ retinoid  receptor. Cyp26 ¼ the family of cytochrome P450 enzymes that catabolise RA. Small dots ¼ catabolic products leaving the cell.

fistula (Kalter and Warkany, 1959; Warkany et al., 1948; Wilson et al., 1953). Most recently, these observations on the eVects of vitamin A deficiency on rat embryos have been repeated and extended. Under conditions of acute retinoid deficiency from E14, embryos displayed agenesis of the lung buds and persistent laryngeal-tracheal groove (Dickman et al., 1997). This confirmed the importance of RA in stimulating the initial budding of the lungs (Fig. 1A). If RA is required for the initial induction of lung buds, then it should be present at the right time and place. The presence of RA has been assayed by using a transgenic mouse that expresses a transgene comprising the RARE from the RAR gene, the heat shock promoter, and the lacZ gene. Wherever there are areas of the embryo that generate RA, the transgene will be activated and can be detected after histochemical staining for the lacZ gene product -galactosidase. Using this mouse, at E9.5 prominent lacZ staining was detected throughout all the layers of the foregut where the trachea and lung primordia were forming (Malpel et al., 2000). Expression of the RA synthesizing gene, Raldh2, was also determined at this stage and shown to be

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present in the mesenchyme of the prospective trachea and lung primordia (Malpel et al., 2000). Put together, these two pieces of data suggest that RA is made in the mesenchyme and diVuses out to activate target genes in all layers, including the epithelium. RARs and other transducers of the RA signal are also present at the right time and place in the mouse embryo. RAR and CRBP are expressed in the early and midsomite stage foregut endoderm; RAR is expressed in the splanchnopleur mesoderm of the upper trunk whereas RAR is ubiquitous at these stages (Ruberte et al., 1991). By E12.5, RAR and CRBP are strongly expressed in the tracheal epithelium and mesoderm (Dolle et al., 1990) whereas RAR is expressed only in the mesoderm surrounding the tracheal and bronchial epithelium, the cells from which the tracheal and bronchial cartilages diVerentiate (Ruberte et al., 1990). Of the RXRs, both RXR and RXR seem to be ubiquitously expressed and therefore are presumably involved in these early phases of lung development (Dolle et al., 1994). The crucial role that these receptors play in transducing the RA signal was shown in experiments in which they were knocked out either singly or doubly in mice made null mutant. Various combinations of these genotypes mimicked the eVects of vitamin A deficiency. RAR/RAR double mutants (Ghyselinck et al., 1997; Luo et al., 1996; Mendelsohn et al., 1994) displayed agenesis of the left lung with hypoplasia of the right lung, which remarkably is the same as the eVects caused by the absence of vitamin A (see previously). The esophageal septum was also absent, resulting in the complete lack of separation between the esophagus and trachea and the replacement of the normally stratified squamous epithelium of the esophagus with ciliated epithelium of the trachea. Cartilages of the trachea and bronchi are also disrupted in some of these mutants. In the RAR single mutant, the tracheal rings were fused in the ventral plane (Lohnes et al., 1993), in RAR /RAR double mutants the rings were severely malformed, in RAR/RAR double mutants they were completely disorganized, and they disappeared altogether in RAR/RAR double mutants (Mendelsohn et al., 1994). Double null mutants involving RARs and RXRs also result in these early lung abnormalities. RXR/RAR double mutants had hypoplastic lungs and lacked the esophagotracheal septum (Kastner et al., 1997). Tracheal cartilages were disorganized in RXR/RAR and RXR/RAR double mutants. These analyses showed that RXR is the heterodimeric partner predominantly involved in the action of the RARs during lung development. A third experimental regime that has been used to show the requirement for RA during the early stages of lung budding is use of inhibitors of RA signaling. Such compounds act as antagonists of the RARs (RAR, , and

) and prevent RA from inducing gene activity. Mollard et al. (2000) used one such compound, BMS493, on E8 mouse embryos. When control embryos

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were cultured for 48 h, the left and right lung buds branched from the foregut during this period of time. But embryos cultured in 106 M BMS493 showed no evidence of lung buds or esophagotracheal fold formation. These defects were partially prevented by the simultaneous addition of 107 MRA and the BMS compound to the culture medium. It is clear therefore that RA and the signaling pathway components are required for the appearance of the esophagotracheal septum and the initial budding of the lungs from the foregut (Fig. 1A and B).

IV. Branching Morphogenesis is Inhibited by RA As the developing lung begins the phase of branching morphogenesis, RARs and RXRs continue to be expressed, but in the mouse embryo there is a subtle redistribution such that RAR (both the 1/3 and the 2/4 isoforms) is found in only the epithelium and mesenchyme of the proximal bronchi and not the distal bronchi (Chazaud et al., 2003; Dolle et al., 1990). RAR thus seems related to the diVerentiation status of the developing lung. RAR (specifically the 1 and 2 isoforms) is expressed homogeneously, as is RAR (the 1 isoform) initially, but the latter by E12 becomes strongly associated with the epithelium of both the proximal and distal tubules (Malpel et al., 2000). CRBP transcripts are restricted to the mesenchyme. In the human embryo (13–16 weeks of gestation) a similar picture emerged when antibodies were used to study the distribution of the RAR and RXR proteins (Kimura et al., 2002). RAR, RAR , RXR, RXR , and RXR were present throughout the epithelium and mesenchyme at both proximal and distal sites. In a distribution somewhat similar to that of the mouse, RAR is present in proximal cells and the distal mesenchyme but not in the distal epithelium. Again, RA is present during these stages as determined by the RARE– lacZ transgenic reporter mouse, but with an interesting diVerential distribution (Chazaud et al., 2003; Malpel et al., 2000). Using this mouse, that activity could be detected in the lung epithelium up to E11.5, and then during the pseudoglandular stage (up to E14.5) was seen in the pleural mesothelium and in the epithelium of the proximal lobules and not in the mesenchyme of the lobules. The loss of lacZ staining preceded the appearance of secondary airway branching. Raldh2 expression was also downregulated in regions of the lung that were undergoing budding and remained high in proximal regions of the lobes. Raldh1 is also expressed by the epithelium of the proximal bronchi from E12.5 to 14.5 (Chazaud et al., 2003). By day 16.5, the only region of the lung expressing Raldh2 was the pleural mesothelium, by which time lacZ staining had also disappeared (Malpel et al., 2000). These changes in RA synthesis correlate with the

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changes in RAR expression, which, as detailed previously, becomes associated with proximal diVerentiating tubules, but the rest of the receptors, RAR, RAR , RXR, and RXR , do not change their expression patterns in the absence of endogenous RA. Lack of RA activity in branching distal regions of the lung at this stage may well be because of the presence of an enzyme that catabolizes RA, namely, Cyp26A1. This enzyme is expressed in the epithelium of the lungs coincident with the appearance of secondary buds and the extinction of epithelial RARE–lacZ transgene expression (Malpel et al., 2000). From E12 to 14.5, a proximodistal gradient of Cyp26A1 is established, with highest levels in distal buds, by which time expression has expanded to include the mesenchyme between the buds. However, as revealed above distal buds still express some RARs and RXRs which, although present, are not activated. This might be because of the absence of ligand caused by its rapid catabolism in the presence of Cyp26A1, but in addition an inhibitory transcription factor, COUP-TFII, is also present in the lung at these stages, with highest levels in the distal mesenchyme (Malpel et al., 2000), which would prevent the activity of any RA that might be present in distal regions. The reason for the inhibition of RA activity in distal branching regions of the lung at these stages, either through its catabolism or the presence of negative regulators, is that RA is inhibitory to distal branching morphogenesis. Therefore, for the lung to continue to develop correctly RA must now be switched oV. Having been present initially to induce outgrowth of lung buds from the foregut, RA now acts as a diVerentiating agent for proximal buds. This conclusion was reached on the basis of several experiments that showed this phenomenon in explant culture. Thus, Mollard et al. (2000) cultured lung explants from E11.75 and E12.5 mouse embryos for 4 d. At concentrations of 107 M and 106 M, RA decreased the average terminal bud number in a dose-dependent manner. The same inhibitory results were obtained by Volpe et al. (2000) and Packer et al. (2000). Conversely, the addition of the RAR antagonist BMS493 increased the number of terminal buds in a dose-dependent manner and this eVect was prevented by the simultaneous addition of RA and BMS493, suggesting the specificity of the eVect of BMS493 on RA signaling (Mollard et al., 2000). The phenomenon was time dependent, because early treatment with RA (E11–12 lungs) aVected branching but late treatment (3 d later) did not. Similarly, Cardoso et al. (1995, 1996) had shown that E13.5 rat embryo lungs cultured in a high concentration of RA (105 M) for 3–7 d showed considerably less diVerentiation than controls in that they still looked like E13.5 lungs and the development of distal tubules had been inhibited in favor of proximal-like tubules. Proliferation still continued in the presence of RA, there was no necrosis, and the eVects were reversible when the RA was removed from the medium. Expression of each surfactant protein (A, B,

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and C) was inhibited in a dose-dependent fashion. As described previously, this eVect of RA is stage dependent, because when E13.5 lungs were first cultured for 5 d in control medium and then the RA added, there was no inhibitory eVect (Cardoso et al., 1995). Oshika et al. (1998) also demonstrated that E14 rat embryo lungs cultured in 105 M RA for 4 d had their distal branch development inhibited and the eVect decreased as the concentration of RA decreased to 107 M. However, a converse conclusion was reached by Schuger et al. (1993), who concluded that at 106 M RA there was an increased branching activity of E12 mouse lung explants cultured for 48 h. However, the branches generated in these experiments were of a proximal nature and resembled those described in previous studies; no distal buds appeared in these cultures. Therefore, it does seem that RA is inhibitory to distal branching during these early stages of lung development. The inhibitory eVect of RA seems to be mediated by RAR . When RA is added to E11.75 lung explants, after 24 h RAR is induced throughout the pulmonary tree of the explant, including the distal buds, whereas BMS493 decreases RAR expression in proximal buds (Mollard et al., 2000). When lung explants from RAR / null mutant mice were used in the same experimental design, RA failed to decrease the average number of terminal buds, confirming the involvement of the RAR receptor. We might expect, conversely, that the BM493 eVect would also disappear in RAR / mutant lungs, although this experiment was not reported. RAR / lungs, however, were investigated for the BMS eVect because RAR 1 is expressed preferentially in the distal bud epithelium (see previously). They were found to respond to BMS493 by increasing the average terminal bud number just as wild-type lungs do (Mollard et al., 2000). Another way of demonstrating that RA signaling is inhibitory to distal branching is to use overexpression constructs of RAR and RAR linked to the surfactant protein-C promoter to ensure their expression in the distal branches of the lung (Wongtrakool et al., 2003). Overexpression of RAR resulted at birth in the inhibition of saccule formation; inhibition of type I cells; inhibition of SP-A and SP-B expression; increased epithelial apoptosis as well as increased proliferation; upregulation of the genes Tft1, Fgf10, and GATA6; and the formation of tubule-like structures. This phenotype resembled immature lungs at the pseudoglandular stage. RAR -overexpressing lungs showed the presence of type I and type II cells, increased epithelial apoptosis and proliferation, and a thick mesenchymal layer inappropriate for gas exchange. These authors conclude that RAR is an important receptor that required downregulation for the formation of distal budding. This lung explant system has also been very valuable for examining the relationship between RA and other genes known to be involved in

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branching morphogenesis and that might be potential downstream gene targets of RA. Raldh1 is downregulated by RA and upregulated by BMS493 (Chazaud et al., 2003). The expression of Tgf 3 overlaps with RAR , and, like RA, TGF 3 is an inhibitor of lung branching (Bragg et al., 2001). Tgf 3 is downregulated after BMS493 treatment and upregulated after RA treatment, suggesting that RA positively regulates Tgf 3 to inhibit branching (Chazaud et al., 2003). On the other hand, Cftr, another gene expressed in the lung epithelium, was upregulated after BMS493 treatment and downregulated after RA treatment, suggesting a negative regulation. Bmp4 is expressed in the epithelium of the distal lung buds, where it maintains the distal character (Weaver et al., 1999). RA downregulated Bmp4 expression (made the buds more proximal) (Malpel et al., 2000) and BMS493 upregulated Bmp4 expression specifically in the distal tips (Chazaud et al., 2003). Fgf10 is also expressed in the distal buds, but in the mesenchyme and is though to stimulate bud formation (Bellusci et al., 1997b; Park et al., 1998). RA downregulated Fgf10 expression, in line with its role in reducing budding (Bellusci et al., 1997b; Malpel et al., 2000) and BMS493 upregulated its expression (Chazaud et al., 2003). Shh is expressed in the epithelium, with its highest levels distally, and it seems to be involved in mesenchymal proliferation rather than branching morphogenesis because overexpressing Shh in the distal epithelium results in smaller lungs at birth, with an overproliferation of the mesenchyme (Bellusci et al., 1997a). There was no eVect on Bmp4 levels, but Fgf10 (branching inducer) was downregulated (Bellusci et al., 1997b). RA increased the expression of Shh (Bellusci et al., 1997b; Cardoso et al., 1996) and BMS493 decreased its expression (Chazaud et al., 2003), consistent with an inhibition of branching. Hox genes are also downstream targets of RA, because several of them contain RAREs and the newborn mouse lung expresses 15 Hox genes (Bogue et al., 1994). During branching morphogenesis, 8 Hox genes have been identified by Bogue et al. (1996)—Hoxa1, a3, a5, b3, b4, b6, b7, and b8—and others have identified a2, a4, and b5, making 11 in all. Their relative levels vary during the stages of lung development and some of them show both temporal colinearity (Hoxa1 is transcribed before a4, which is transcribed before a7) and spatial restrictions (Hoxa1 and b7 display proximodistal gradients in expression levels). In the developing chick lung, Hoxb genes show the classical nested expression patterns typical of these genes (Sakiyama et al., 2000). Hoxb5 and b6 are expressed in the trachea, bronchial tree, and air sacs, and b6 to b9 expression corresponds to the morphological subdivisions of the air sacs along the proximodistal axis. Hox genes in the lung are upregulated by RA as in all the other systems of the body, but are any of them involved in branching morphogenesis? Cardoso et al. (1996) showed that Hoxa2, which is normally expressed in the mesenchyme and at lower levels in the distal mesenchyme than the proximal, is upregulated by

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RA so that its expression becomes homogeneously distributed. Presumably this gene is inhibitory to the development of distal tubules. Hoxb6 was also upregulated, although its normal expression pattern suggests that it is involved in distal branching and thus one would expect it to be downregulated by RA during inhibition. Hoxa4, a5, and b5 were also upregulated by RA during the inhibition of branching morphogenesis (Bogue et al., 1994; Kim and Nielsen, 2000; Packer et al., 2000; Volpe et al., 2000). Hoxa5 and b5 are restricted to the proximal mesenchyme and RA spreads its expression to the distal regions (Packer et al., 2000). Interestingly, Hoxb5 antisense oligonucleotides inhibit proximal tubule development, an eVect opposite to that of RA, suggesting that these Hox genes indeed play a role in branching morphogenesis, mostly in the diVerentiation of proximal tubules and their ectopic expression in distal regions inhibits distal development by promoting proximal development. Conversely, the Hoxa5 null mutant displays highly abnormal lung development (Aubin et al., 1997). There are reduced levels of surfactant proteins and less extensive and reduced size of the branches at the pseudoglandular stage with excessively thick mesenchyme and a complete lack of saccule development. Hoxa5 thus seems to be required for distal diVerentiation. The roles of these and other transcription factors in the lung have been recently reviewed (Costa et al., 2001). Other genes involved in the RA eVects on the lung include a novel transcription factor, Rcd1. This gene is a required for RA-induced diVerentiation of F9 cells, and antisense oligonucleotides to Rcd1 block diVerentiation. Rcd1 forms a complex with the RARs. When RA inhibits branching morphogenesis in culture (E11.5), Rcd1 antisense inhibits the inhibitory eVect of RA (Hiroi et al., 2002). It is also possible that RA acts through EGF and the EGF receptors because RA upregulates EGFR in mixed primary cultures of lung cells (Schuger et al., 1993) and EGF itself might play a role in lung development (Warburton et al., 1992). Finally, an RA-responsive extracellular molecule known as midkine (MK), which is a heparin-binding growth factor and known to be involved in several other developing systems involving epithelial/mesenchymal interactions, is upregulated by RA in cultures of E21 rat foetal lung cells (Kaplan et al., 2003). Interestingly, the lungs of the CRBP/ null mutant responded abnormally to these experimental interventions in culture, even though they seem to develop normally in situ (Ghyselinck et al., 1999). The pseudoglandular stage of the lung is a major site of CRBPI expression (Dolle et al., 1990), and when wild-type lungs are cultured at this stage in the presence of RA or BMS493 CRBPI expression is increased and decreased, respectively. Explants of CRBPI/ lungs respond excessively to BMS493 treatment; at a concentration that has no eVect on wild-type lungs (107 M), BMS493 increases the average terminal bud number.

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V. RA is Required during the End of the Foetal Period (Pseudoglandular, Canalicular, and Terminal Saccule Stages) It was shown by HPLC that in rat lungs retinyl palmitate and total retinyl ester levels peak in the middle of the last third of gestation followed by a decline that continues postnatally (Masuyama et al., 1995; Shenai and Chytil, 1990; Zachman et al., 1984). These data from whole lungs were replicated by studying the retinyl ester content of LIFs alone, and this declines after birth as well, suggesting that these are the important retinoid-containing cells in the lung (McGowan et al., 1995). Retinol levels in the lung peak on embryonic E19 in rats (Geevarghese and Chytil, 1994) and after dexamethasone treatment on E18, rat embryo lungs show a more rapid postnatal decline in retinyl palmitate and the prenatal peak of retinol is lower (Geevarghese and Chytil, 1994), suggesting that the inhibitory eVect of dexamethasone on the lung could be mediated by lack of retinoids. Conversely, a single large dose of retinyl palmitate given on E16 of rat development results in a two- to seven-fold higher concentration of retinyl esters in the lungs, which persists throughout the 14-d postnatal period (Shenai and Chytil, 1990). Clearly, retinoids are important in this late period of development, but is RA playing the role? When pregnant rats are fed on a vitamin A-deficient diet supplemented with RA and then given one dose of retinol on day 10, the pups continue through development and are born, but die immediately of respiratory failure. The lungs fail to develop distal branched terminal structures and remain immature (Wellik et al., 1997). There seem to be no signs of squamous metaplasia of the trachea, which is a classical sign of RA deficiency, even though lung development fails. This suggests that another metabolite of retinol allows the lungs to continue development from Day 10, because apparently RA is suYcient. Other studies of vitamin A-deficient embryos have confirmed that the E20 foetal lung does not complete distal branching and the neonatal lung resembles a much younger one with thick walls and fewer saccules (Antipatis et al., 1998). There are also fewer elastin fibres in histological sections of the deficient lungs and a downregulation of tropoelastin mRNA occurs along with another gene, gas6, which has a role in the regulation of cell adhesion. During this period, RARs and RXRs continue to be expressed in the lungs (Masuyama et al., 1995). The transcript levels of most of these receptors peak after birth, during the period of alveologenesis (see later), but one species of RAR peaks on E17 in the rat (Grummer et al., 1994). In rats made retinol deficient, levels of RAR in the lungs decline (Masuyama et al., 1995). RAR seems to be the most responsive receptor to declining levels

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of retinoids, because the same result is obtained with adult rats made deficient—levels of RAR decline 65–70% whereas there is no change in the levels of RAR or RAR in the lungs (Haq et al., 1991). The level of RAR rises again 1 h after refeeding retinol. These late stages of gestation are the periods of normal development when the levels of surfactant proteins rise in preparation for birth. Coincidentally, the peak of retinyl palmitate that occurs in late gestation just precedes the appearance of the first intracellular forms of surfactant and this simultaneous increase suggests a possible causal relationship between the two. However, RA inhibits surfactant production (or at least two of the three surfactants) but increases phospholipid synthesis. For example, in cultured pieces of midtrimester human lung, RA decreased SP-A, increased SP-B, and decreased SP-C production (Metzler and Snyder, 1993). A decrease of SP-C was also seen in E14 and E15 rat embryo lungs cultured with 105 M RA (Oshika et al., 1998). Other work on explant cultures of E17 rat embryo lungs has suggested that RA stimulates mRNA levels of each of the surfactant genes and each with diVerent dose-dependent characteristics (Bogue et al., 1996). These results were obtained after only 4 h of RA treatment, whereas other studies used considerably longer culture periods in the presence of RA; therefore, it is possible that a rapid stimulation is followed during subsequent days by a decrease. In lung explants (Masuyama et al., 1995) and isolated type II cells, RA stimulated the incorporation of choline into phosphatidylcholine (Fraslon and Bourbon, 1992). Chronic administration of retinyl palmitate to rat embryos from E16 to E20 resulted in a decrease in SP-A protein content of the lungs, but synthesis of the phospholipid moiety was increased (Fraslon and Bourbon, 1994). Conversely, administration of citral, an inhibitor of RA synthesis, decreased phospholipid synthesis, and total phospolipids were decreased by 21% in rat foetuses which developed under reduced vitamin A conditions (Chailley-Heu et al., 1999). These rat foetuses also showed decreased levels of SP-A and SP-B proteins, which does not accord with RA administration itself decreasing surfactant protein (Fraslon and Bourbon, 1994; Metzler and Snyder, 1993), but others have not found any eVects on SP-A or SP-B mRNA levels during vitamin A deficiency (Zachman and Grummer, 1998). The positive eVects of RA on SP-B were confirmed and explained in studies on two cell lines, a human pulmonary adenocarcinoma line and a mouse lung epithelial cell line (George et al., 1998; Yan et al., 1998). The SP-B gene contains a RARE, which is activated by cotransfection with a RAR and RXR . RAR, RAR , and RXR were detected by immunocytochemistry in adenocarcinoma cells. Therefore, it seems a consistent finding that phosholipid synthesis is stimulated by RA, but there is considerable contradictory data on what the eVects of RA are on surfactant production.

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Other eVects of RA on type II cells include the inhibition of proliferation, at least in E19.5 cultured rat lung cells (Fraslon and Bourbon, 1992), despite there being an increase in the expression of the CCAAT/enhancer binding protein (C/EBP), which is a transcription factor involved in the proliferation and diVerentiation of type II lung cells (Barlier-Mur et al., 2003). Conversely, expression of this transcription factor was decreased in vitamin Adeficient foetuses (Barlier-Mur et al., 2003). This inhibitory eVect of RA on type II cell proliferation is also contradictory to its eVect on late foetal lung fibroblasts, in which RA stimulated proliferation (Liebeskind et al., 2000). Because RA also upregulated the platelet-derived growth factor (PDGF) ligand and receptor and a neutralizing antibody to PDGF reduced the eVect of RA, it was suggested that RA had its eVect on these fibroblasts through a PDGF-based autocrine loop. Indeed, in the PDGF-A null mutant mouse, postnatal alveolar formation does not occur (Lindahl et al., 1997). Even though it is not clear precisely what the cellular function of RA is at these later stages of lung development, these animal studies on the changes in retinoid levels at the end of gestation have had a considerable impact on concepts and treatments of premature human infants. Preterm infants frequently develop lung problems in the form of bronchopulmonary dysplasia (BPD). The changes in the epithelium of the tracheobronchial tree of such infants consist of necrolizing tracheobronchitis followed by squamous metaplasia in advanced stages of the disease (Shenai, 1999; Shenai et al., 1985). These histopathological changes are remarkably similar to those in vitamin A deficiency, the classical symptom being squamous metaplasia of the conducting airways (Wolbach and Howe, 1925 and see previously). Squamous metaplasia is followed by narrowing of the airway lumen, with a resultant increase in airway resistance and loss of mucociliary transport, which results in a predisposition to airway infection. It is possible therefore that preterm infants could be born before the full reserves of retinoids have been obtained from the maternal circulation and built up within the foetal liver and lungs, with the result that abnormally low levels of retinoids would be available to the lung. Indeed, studies showed that preterm neonates (less than 32 weeks of gestation) who developed BPD had suboptimal plasma retinol and retinol-binding protein concentrations for extended periods of time postnatally (Brandt et al., 1978; Hustead et al., 1984; Shah and Rajalakshmi, 1984; Shenai et al., 1981, 1985). The same is true for the liver stores of retinoids in very low birth weight neonates: they are well below normal, rendering the body susceptible to vitamin A deficiency (Montreewasuwat and Olson, 1979; Olson et al., 1984; Zachman, 1989). If this is the case, then supplementing the preterm neonate with retinoids should improve BPD. A clinical trial of such supplementation in

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26- to 30-week gestational age neonates showed that the need for supplemental oxygen, mechanical ventilation and intensive care, airway infection, and retinopathy of prematurity were less frequent in the supplemented group (Shenai et al., 1987). However, this has not been a consistent result. Another study showed that the same dose of retinyl palmitate did not reduce the incidence of BPD (Pearson et al., 1992), but in this study all the infants also received surfactant and half of them received dexamethasone in the postnatal period, which confounds the interpretation with regard to vitamin A intake. Indeed, the supplemented group and the control group both had plasma vitamin A levels suggesting suYciency. Nevertheless, a large multicenter trial using retinyl palmitate dosing three times a week for 4 weeks lowered the incidence of death or chronic lung disease (Tyson et al., 1999); therefore, it seems that these preterm infants suVer from retinoid deficiency and supplementation is beneficial. [For a review see Shenai (1994).]

VI. RA is Required for Postnatal Septation (Alveologenesis) RARs and RXRs continue to be expressed postnatally and in the adult (Grummer et al., 1994). The level of each RAR peaks postnatally and at slightly diVerent days (Hind et al., 2002b; McGowan et al., 1995). RAR1 (the only  isoform present) and RAR 2 (the only isoform present) peak at postnatal Day 4 and the two RAR isoforms, 2 and 4, peak slightly later (Hind et al., 2002b), perhaps reflecting a diVerent function for each RAR (see later). Dexamethasone treatment (which inhibits septation) lowered the levels of RAR , whereas O2 treatment (which also inhibits septation) increased RAR levels (Grummer and Zachman, 1995). Studies on the expression of the receptors in adult human have shown that RAR, RAR , RXR, and RXR are expressed in all adult cells. RXR is weak in the adult, and RAR is expressed in the tracheal and bronchial epithelium but not in the mesenchyme or alveoli (Kimura et al., 2002). Levels of CRBPI and CRABPI also peak postnatally (Hind et al., 2002b; Ong and Chytil, 1976; Whitney et al., 1999). Strong evidence for a role for RA in septation comes from experiments in which either RA is administered to septating newborns or RA synthesis is inhibited. In the former case, giving RA to rat pups from day 3 to day 13 resulted in a 50% increase in alveolar number and the alveoli were 47% smaller when examined on day 14 (Massaro and Massaro, 1996). In the latter case, there was a 26% increase in mean alveolar diameter (Lm) after disulfiram, an inhibitor of RA synthesis, was administered to mice for 10 days between postnatal days 2 and 14 and the lungs examined on P23 (Maden and Hind, 2004). RA also prevented the deleterious eVects of dexamethasone on the septating lung. Instead of the appearance of fewer,

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larger alveoli caused by dexamethasone, RA administered at the same time returned the alveolar number and size to almost normal (Massaro and Massaro, 1996). This correction by RA involves the downregulation of 35 genes and the upregulation of 11 genes, and it is suggested that an endothelial gene Flk-1 is causal in the inhibiting eVect of dexamethasone on alveologenesis and in the rescue by RA (Clerch et al., 2004). This is a very interesting finding because the induction of capillary outgrowth has long been considered the earliest event in secondary septa formation (Fig. 2D). As mentioned previously, dexamethasone is well known to inhibit the process of septation (Blanco et al., 1989) when applied during the critical 2-week postnatal period in rats (Massaro et al., 1985), but does this have anything to do with the role of retinoids? Some evidence suggests that the glucorticoid pathway and the retinoid pathway interact. For example, dexamethasone administration to newborn rat pups resulted in a 60% decrease in retinyl palmitate in the lungs, decreased the RAR levels (McMenamy and Zachman, 1993), and decreased the levels of CRBPI and CRABPI (Whitney et al., 1999). Elastin is the most important extracellular matrix component of the lung, and its deposition is maximal early in postnatal life during the period of rapid alveolar septal growth. Elastin is produced by NILFs and RA controls its transcription, emphasizing the importance of retinoids during alveologenesis. Application of inhibitors of retinoid metabolism to postnatal day 8 rat lung fibroblast cultures inhibited tropoelastin mRNA but not 1 procollagen mRNA (McGowan et al., 1997). Conversely, incubation of these fibroblasts with RA increased the rate of elastin transcription and the production of soluble and insoluble elastin in the medium (Liu et al., 1993). The other fibroblasts, the LIFs, contain the retinyl esters whose concentration declines postnatally from a prenatal peak, retinol whose concentration peaks between days 2 and 8 postnatally, and all-trans-RA and 9-cis-RA whose concentration peaks on day 2 (McGowan et al., 1995). LIFs can convert retinol into RA and release it in the form of all-trans-RA and 4-oxo-RA (Dirami et al., 2004). Dexamethasone treatment of LIFs halves the amount of RA released into the medium. They express RAR, RAR , and RXR and the temporal changes in the expression of these receptors mimic those in the whole lung; they express CRBPI whose concentration peaks on postnatal day 10; and they express CRABP and RALDH1 (Hind et al., 2000a, 2002b; McGowan et al., 1995; Ong and Chytil, 1976). Because the cells that contain the retinoids and LIFs and the cells that produce the elastin are the NLIFs, this suggests that RA acts in a paracrine fashion to induce septation (Fig. 4A). However, another cell type, the pulmonary microvasculature endothelial cell (PMVC), has been shown to respond to the RA released by LIFs. The response mounted in PMVCs was upregulation of CRBPI. The response was inhibited by the addition of an RAR antagonist (Dirami et al., 2004). Thus,

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Figure 4 Two hypotheses of the origin of new cells and structures during alveolar regeneration. (A) The type II origin. RA enters the LIFs (blue cells) as these are known to store retinoids and contain all the components of the RA signaling pathway. The LIF signals through RA the type II pneumocyte (orange cell) to proliferate and diVerentiate into type I pneumocytes (red cell). (B) Stem cell origin. Hemaotopoetic stem cells enter the alveoli through the capillaries and diVerentiate into type II pneumocytes, which then proliferate and generate type I pneuomcytes (upper pathway) or stem cells diVerentiate straight into type I cells (lower pathway).

perhaps any cell in the vicinity of an LIF might be able to respond to the released RA, not just an elastin producing NLIF. Type II cells, the putative stem cells, for example, might be maintained by the presence of RA. Indeed, RA stimulates proliferation of a neonatal type II cell line with a maximum eVect at 106 M and it may act by antagonizing the inhibitory eVect of TGF 1 (Nabeyrat et al., 1998). The function of the individual receptors during alveologenesis has been deduced from the phenotypes of the receptor knockouts. In RAR / mice, LIFs isolated from postnatal day 12 lungs showed a twofold reduction in tropoelastin expression. The lungs themselves contained less elastin, and there was a decrease in alveolar wall volume density, alveolar surface area, and number of alveoli, with a corresponding increase in Lm (McGowan et al., 2000). RAR/ mice, by contrast, had normal alveolar volumes, number of alveoli, and alveolar surface areas at postnatal day 14, showing that RAR has no role in postnatal alveologenesis. But by day 50, alveolar volumes were higher, there were fewer alveoli, and the alveolar surface area was lower in the knockouts than in normals. This demonstrated that RAR plays a role in the continuing process of alveologenesis, which progresses slowly after postnatal day 14 until the age of 5–6 weeks in rodents (Massaro

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et al., 2003b). However, expression of a dominant-negative RAR during septation (from P1 to P21), using an inducible construct, resulted in increased air spaces, decreased alveolar surface area, and larger and fewer alveoli (Yang et al., 2003). These lungs showed characteristic pulmonary emphysema, revealing a function of RAR in septation rather than postseptation events. The role of RAR was even more surprising in that the alveolar volumes of RAR / mice were smaller at days 4, 21, and 64; there were more alveoli at each age; and the alveolar surface area was larger (Massaro et al., 2000). This suggests that RAR is an endogenous inhibitor of alveolar septation. Indeed, administration of an RAR agonist to normal postnatal rats from days 3 to 13 produced the opposite phenotype—there were larger and fewer alveoli. These three results reveal the roles of the RARs—the balance between RAR (þve) and RAR (ve) regulates postnatal (days 4 to 14) alveologenesis and then RAR regulates the subsequent slower period of septation, which continues into adulthood. Interestingly, it is very likely that the adult lung requires vitamin A (perhaps in the form of RA) for its maintenance in the adult state, perhaps involving continued low-level repair and turnover of alveoli. When rats were maintained on a vitamin A-deficient diet, several alterations were seen in the lungs (Baybutt et al., 2000). Some areas showed marked thinning of the septa and disappearance of the parenchyma so that emphysema became apparent. Phosphatidylcholine synthesis in type II cells was significantly lower. This same phenomenon of emphysematous appearance coupled with an increase in Lm and a downregulation of tropoelastin transcription has also been seen in other studies on rats (Maden and Hind, 2004). During this period of retinoid deficiency, RAR declines to the greatest extent in the lungs (80%), followed by RAR and RAR (Verma et al., 1992). Administration of RA to these deficient rats resulted in lung RAR increasing after 1 h and reaching a maximum that was 16-fold higher than normal after 4 h (Haq et al., 1991). Maintenance of the adult lung is regulated by the balance between proteases and antiproteases, which play a crucial role in tissue destruction and remodeling. When this balance is disturbed, for example, with an excess of metalloproteases, lung destruction in the form of emphysema can result. It is very relevant to these cases of destruction induced by lack of vitamin A described previously that RA selectively downregulates MMP9 and upregulates TIMP1 in human bronchoalveolar lavage cells (Frankenberger et al., 2001).

VII. RA Induces Alveolar Regeneration If RA is an essential component of the postnatal lung and it switches on developmental gene pathways during the period of alveologenesis, it is conceivable that these pathways could be reawakened by the administration

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of RA to a damaged lung in order to ‘‘redevelop’’ the alveoli. This remarkable possibility was first observed by Massaro and Massaro (1997). Alveoli were destroyed by the intratracheal instillation of elastase, which resulted in an emphysematous lung with increased Lm and loss of alveolar surface area. This emphysema became progressively worse over subsequent months and was not spontaneously reversible. When these rats were treated with 500 mg/kg RA for 12 d and examined the next day, the lung volume, Lm, and gas-exchanging surface area had returned to normal (Massaro and Massaro, 1997). There have been two repeats of this study. In one (Belloni et al., 2000), all-trans-RA induced a 50% reversal of the elastase damage and using 9-cis-RA there was a 70% reversal. In a second repeat, only a mild improvement in lung volume was obtained, without any eVect on compliance or forced flows (Tepper et al., 2000). Elastase treatment has also been used in mice to destroy alveoli, and tRA (500 mg/kg) given daily for 12 d was suYcient when the lungs were examined immediately to give a 44% reduction in Lm (Ishizawa et al., 2004). Elastase has also been used to damage cultured cells, notably primary cultures of human tracheal cells, a human airway epithelial cell line, and a human alveolar epithelial cell line. The damage that elastase causes to cultured cells is to decrease viability and to induce apoptosis. When RA was added to the cultures, it prevented the decrease in viability, inhibited apoptosis, and inhibited caspase 3 induction (Nakajoh et al., 2003). It was also demonstrated that RA acts by inhibiting the elastase activity itself rather than through transcription of the elastase gene. Other methods of reducing the alveolar surface area have also produced positive results after RA administration. Dexamethasone treatment of postnatal rat pups from P4 to P13, when alveologenesis occurs, results in fewer, larger (three times larger) alveoli with larger individual volumes than normal. When these rats were treated from P24 to P36 with 500 mg/kg all-trans-RA and examined the next day, these parameters were partially recovered (alveolar volumes were 1.6 times lower with RA; Massaro and Massaro, 2000). Dexamethasone treatment of mouse pups from P3 to P14 has the same eVect in massively decreasing the gas-exchanging surface area. When these animals were treated with 2 mg/kg tRA daily from Day 30 to Day 42 and examined 4 weeks later, complete regeneration of the lung structure in terms of surface area per gram body weight was observed (Hind and Maden, 2004). An equally dramatic eVect was seen in the tight skin mouse. This is an autosomal dominant mutation characterized by multiple connective tissue defects, such as increased growth of cartilage and bone and hyperplasia of tendon sheaths (Rossi et al., 1984; Szapiel et al., 1981), and caused by a tandem duplication within the fibrillin-1 gene (Siracusa et al., 1996). The homozygous mutant is lethal but the heterozygotes show enlarged air spaces with thinned or broken alveolar walls, and this mouse represents a genetic model of emphysema. These mice received 500 mg/kg tRA daily from days

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40 to 51 and were examined the next day. Alveoli were 2.7-fold smaller and 3.5-fold more numerous after RA treatment, showing that RA could also induce septation in these adult mice (Massaro and Massaro, 2000). Another positive eVect of RA was observed in rats that had received a pneumonectomy. Partial pneumonectomy or lung-volume-reduction surgery is one of the few treatments for human emphysema, and this involves removing some of the emphysematous lung tissue. This procedure was thought to enhance the elastic recoil of the remaining lung, although following a clinical trial it now seems that this procedure does more harm than good (Drazen, 2001). In these rat experiments (Kaza et al., 2001), the left lung was removed completely and the right lungs responded by expanding in volume, weight, volume of respiratory airspace, and induced proliferation to compensate for the loss. When tRA (approximately 200 mg/d) was administered as well, both after 10 d and 21 d, these parameters were significantly higher than normal. These authors considered that RA could be acting through EGF, because they measured the levels of the EGF receptor and showed that this too was higher than normal in RA-treated rats. Developmental arrest and hence a loss of gas-exchanging surface area also occurs under conditions of high O2 during the critical period of postnatal development. For example, exposing rats to 90% O2 from postnatal Days 3 to 13 results in a 50% increase in lung volume, a more than twofold increase in mean air space size, and greatly increased alveolar sizes in histological sections 4 weeks later (Veness-Meehan et al., 2002). If tRA was administered (500 mg/kg) at the same time as O2, that is, from postnatal Days 3 to 13, and the lungs examined on day 14, no diVerence was observed between O2 and (O2 þ RA) treatment groups in terms of lung volume or air space size, although there was an increase in type I collagen staining in the alveolar walls induced by RA (Veness-Meehan et al., 2000). However, by day 42, the RA-treated lungs had completely recovered in terms of these volume parameters and histological structure (Veness-Meehan et al., 2002). This work reinforces the conclusion that the lung needs time to complete the process of alveolar wall regeneration after the end of the administration of RA, and this might be very significant in some of the data reported later, in which negative results have been reported when regeneration is assessed at the end of RA dosing. Therefore, it seems that RA can repair the damage caused by elastase, dexamethasone, the genetic model of emphysema, pneumonectomy, and high O2. However, these remarkable findings have not always been repeatable in other studies on rats and mice. For example, Srinivasan et al. (2002) admininstered dexamethasone and tRA (500 mg/kg) simultaneously from days 3 to 13 to postnatal rats. After 1 month, they found no diVerences between the dexamethasone-treated group and the (dexamethasone þ RA) group with regard to various measures of resting breathing parameters such

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as respiratory rate and minute ventilation. Similarly, lung volumes, lung compliance, and alveolar dimensions were increased by dexamethasone over untreated controls, but not altered by RA treatment. No histological data were reported in this work, so it is not known whether there was any alveolar regeneration, but if there was, it would suggest that the regenerated surface area does not function adequately. Similarly, mice were treated with elastase to generate an emphysematous lung. They were then given tRA at two doses (0.5 and 2 mg/kg) for 12 d. One day after the last RA injection, the animals were killed and the lungs examined. There was no diVerence between the elastase-treated and either of the two RA-treated lungs in terms of Lm or lung volumes or elastin or collagen expression (Lucey et al., 2003). A similar failure of regeneration using 2 mg/kg tRA on elastase-treated mice was recently reported by Fujita et al. (2004). They used precisely the same protocols as those used by Ishizawa et al. (2004), and therefore the complete contrast between these two studies is diYcult to comprehend. Rabbits too do not seem to respond to tRA. They were made emphysematous with elastin and treated with RA (0.5 or 1.5 mg/kg/day) for 14 d. Lung function, lung volume, surface density, and total surface area were not improved by RA over a 35-d period (Nishi et al., 2003). In two other diVerent damage model systems, no eVect of RA was seen. The damage induced by bleomycin administration in rats is a model for idiopathic pulmonary fibrosis. Rat pups were given tRA (0.5 or 2 mg/kg) from days 0 to 14, at the same time as bleomycin. At day 14, they were killed immediately and various inflammatory and fibrotic parameters were examined, although these were not particular markers of regeneration. Bronchiolavages, inflammation, and fibrosis and collagen levels were examined, and there was no diVerence between bleomycin-damaged and RAtreated animals (Segel et al., 2001). The transgenic mouse overexpressing the tumor necrosis factor- (NF-) under the SP-C promoter develops air space enlargement, loss of elastic recoil, and increased lung volumes. tRA (2 mg/kg) administered for 12 d had no eVect on this emphysema model when examined immediately after the end of RA administration; in fact, it seemed to induce a further deterioration in lung function (Fujita et al., 2004). In sheep too, there was no eVect of RA detected. A single high dose of tRA (20 mg) was given on day 115 or day 121 of gestation and the foetuses were delivered at either 125 d or 146 d (i.e., observing the lungs either 10 or 25 d later). There was no change in any measured lung parameter: compliance, lung gas volume, phosphatidylcholine accumulation, and volume of parenchyma of alveolar structure (Willet et al., 2000). Presumably, a single dose of RA that would be metabolized rapidly is not enough to have a long-lasting eVect and repeated daily doses are required. In a smoking guinea pig model, tRA did not reverse emphysematous changes

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in lung volume, airspace volume, or tissue volume (Meshi et al., 2002). Emphysematous changes similar to those seen in mild human emphysema were seen after cigarette smoke was administered for 13–16 weeks. tRA (500 mg/kg) was administered at the same time as the smoke and had no beneficial eVect; on the contrary, there was an opposite eVect because it seemed to be responsible for a significant number of deaths in these experiments.

VIII. Speculations of the Mode of Action of RA It is interesting to consider on which cell type RA is likely to act to induce regeneration of alveoli and why new septa appear where they do. A reasonable assumption would be that it is the type II pneuomcytes, because these are generally considered to be a stem cell population responsible for generating the type I cell during normal development and for maintaining a relatively embryonic-like phenotype in the fully diVerentiated lung. Perhaps LIFs are the stimulating cells because they contain retinoids, are known to be capable of signaling via RA (Dirami et al., 2004), and contain all the RA signaling components (see previously). The type II cell would be the responding cell (Fig. 4A). Thus, the location of the new alveolar septum would be determined by the location of these cells, the stem site model of alveolization (Pierce and Michael, 2000). This is a perfectly testable hypothesis with transgenic technology using a type II heritable cell marker. An alternative hypothesis is that the new cells are derived from circulating hematopoetic stem cells, which could invade the damaged lung and transform directly into type I pneumocytes (Fig. 4B). Indeed, bone marrow cells transplanted to irradiated hosts can be found in the lung, albeit rarely (Krause et al., 2001), and when given to mice with bleomycin-induced lung damage such cells could be detected in the lung as type I cells with no evidence for the presence of type II cells (Kotton et al., 2001). However, stem cells have been shown to be capable of fusion with other cells types, casting doubt on whether these transformations of cell type are real (Terada et al., 2002; Vassilopoulos et al., 2003; Wang et al., 2003; Ying et al., 2002). Nevertheless, mouse embryonic stem cells can be induced to diVerentiate into the occasional type II pneumocyte after 30 d in culture (Ali et al., 2002). A recent in vivo test of this stem cell origin of pneumocytes has, surprisingly, proved positive. Ishizawa et al. (2004) whole-body irradiated recipient mice and then reconstituted their bone marrow using foetal liver cells from a green fluorescent protein (GFP) expressing transgenic mouse. The lungs of these hybrid mice were then treated with elastase to induce emphysema. After 3 weeks, groups of mice were treated daily for 12 d with tRA (500 mg/kg), granulocyte colony-stimulating factor (G-CSF), or both, and

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examined immediately afterwards. G-CSF is a factor known to mobilize hematopoetic and mesenchymal lineage cells from the bone marrow. Each treatment induced histological regeneration, with G-CSF reducing the Lm by 44% and the combination of G-CSF and RA reducing Lm by 73%. Each treatment resulted in GFPþve cells appearing in the alveolar walls, which were double labeled with a cytokeratin antibody. This work not only shows that another factor in addition to RA, namely G-CSF, can induce alveolar regeneration, but also suggests that the source of regenerated tissue is, at least in part, circulating hematopoetic stem cells.

IX. Clinical Implications and Future Possibilities Emphysema is an untreatable disease characterized by airway destruction distal to the terminal bronchioles, gradual loss of lung recoil, decreased surface area, and impaired gas exchange (Thurlbeck, 1975). Emphysema and chronic bronchitis together comprise the clinical syndrome of chronic obstructive pulmonary disease (COPD), predicted to become the third commonest cause of death worldwide by 2020 (Lopez and Murray, 1998). Currently, there are no eVective treatments for COPD except for supplemental oxygen and lung transplantation and the outlook for aVected patients is bleak. Other respiratory problems involving loss of alveoli include the irreversible and gradual age related loss of alveoli and BPD. BPD is a chronic problem associated with lung damage in low-birth-weight preterm infants (see previously) and is characterized by progressive respiratory insuYciency, hypoxemia, and hypercapnea. There would be a dramatic impact on the health of both infants and the elderly, therefore, if alveoli could be induced to regenerate in humans as shown in the various animal studies described previously. An initial clinical trial that involved a cross-over regime only showed that RA is well tolerated in patients with emphysema, setting the scene for trials evaluating higher doses or longer treatment times (Mao et al., 2002). So one could imagine RA or one of the RAR selective agonists being used as a treatment for emphysema or a RAR agonist (Massaro et al., 2000) for BPD. The pathogenesis of emphysema is complex, but it most often results from tobacco smoking. Other causes include long-term exposure to occupational dust and a genetic deficiency of 1-antitrypsin. The alveolar destruction is generally considered to be caused by the release of elastase from neutrophils which appear in response to tobacco smoke-induced inflammation. This elastase upsets the balance of protease–antiprotease activity in the lung and breaks down the extracellular elastin in the alveolar walls, resulting in their gradual destruction. However, it is interesting to note that there is also a relationship between tobacco smoke and retinoid levels. For example,

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studies have found that cigarette smoke lowered the levels of retinol or RA in the lungs of ferrets (Liu et al., 2003) and rats (Li et al., 2003) and that this was due to the induction of the RA catabolic enzymes, the CYPs (Liu et al., 2003). In the rat study, there was a significant inverse relationship between vitamin A levels in the lungs and the severity of emphysema (Li et al., 2003). The feeding of benzopyrene, a carcingogenic combustion product found in cigarette smoke, to adult rats resulted in a decline in liver and lung vitamin A levels (Edes et al., 1992). In human patients with moderate and severe COPD serum retinol concentrations were lower than normal, and when a group of patients with mild COPD were given vitamin A for 30 d their lung functions improved by 23% (Paiva et al., 1996). This latter result already suggests that there might be some human relevance to the animal studies of retinoids and alveolar regeneration described previously. We can only hope that this might be the case.

Acknowledgment I thank Dr. Asa Apelqvist for her critical reading of the manuscript and very helpful comments.

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