Retinoic acid receptor-ß: an endogenous inhibitor of the perinatal formation of pulmonary alveoli
GLORIA DE CARLO MASSARO1,
DONALD MASSARO2,
WAI-YEE CHAN1,
LINDA B. CLERCH1,
NORBERT GHYSELINCK3,
PIERRE CHAMBON3 and
ROSHANTHA A. S. CHANDRARATNA4
1 Lung Biology Laboratory, Department of Pediatrics, Georgetown University School of Medicine, Washington District of Columbia 20007-2197
2 Lung Biology Laboratory, Department of Medicine, Georgetown University School of Medicine, Washington District of Columbia 20007-2197
3 Institut de Génétique et de Biologie Moléculaire et Cellulaire, 67404 Illkirch, Strasbourg, France
4 Retinoid Research, Allergan, Inc., Irvine, California 92623-9534
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ABSTRACT
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Pulmonary alveoli are formed, in part, by subdivision (septation) of the gas-exchange saccules of the immature lung. Septation is developmentally regulated, and failure to septate at the appropriate time is not followed by delayed spontaneous septation. We report retinoic acid receptor (RAR) ß knockout mice exhibit premature septation; in addition, they form alveoli twice as fast as wild-type mice during the period of septation but at the same rate as wild-type mice thereafter. Consistent with the perinatal effect of RARß knockout, RARß agonist treatment of newborn rats impairs septation. These results 1) identify RARß as the first recognized endogenous signaling that inhibits septation, 2) demonstrate premature onset of septation may be induced, and 3) show the molecular signaling regulating alveolus formation differs during and after the period of septation. Suppressing perinatal RARß signaling by RARß antagonists may offer a novel, nonsurgical, means of preventing, or remediating, failed septation in prematurely born children.
bronchopulmonary dysplasia; mice; knockout; very low birth weight infant; pulmonary emphysema; morphogenesis
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INTRODUCTION
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PULMONARY ALVEOLI are formed, in part, by subdivision (septation) of the saccules that constitute the gas-exchange region of the architecturally immature lung (7). Septation is developmentally regulated, occurring in rats and mice from postnatal day 4 through 14 (2, 7) and in humans during the last month of gestation and the first few postnatal years (22, 41). Septation helps establish the link, highly conserved among mammals, between alveolar dimensions and the organisms oxygen requirements (3, 38). However, when septation ends, alveoli continue to form (5, 32), suggesting, as proposed earlier (27), the formation of alveoli is regulated differently during and after the period of septation (for review, see Ref. 28).
The endogenous molecular signals that initiate or end septation are poorly understood, but retinoid involvement is suggested because: 1) treatment of newborn rats with all-trans retinoic acid (RA) results in increased septation and prevents the glucocorticosteroid-induced inhibition of septation (30); 2) RA administered to adult rats, in which the prior intratracheal administration of elastase had caused emphysema, induces septation (31) and abrogates key features of emphysema (39); 3) addition of RA to explants of fetal mouse lung induces septation (8); and 4) retinol (vitamin A) accelerates the formation of alveoli in prematurely delivered lambs (1).
Here, we test the hypothesis that signaling via RA receptor (RAR) ß has a regulatory role in septation. We chose RARß because, among the RARs, its expression is linked to diminished cell replication (23), an effect associated with impaired septation (27). Our findings indicate signaling via RARß prevents the premature onset of septation and slows septation but does not regulate the postseptation formation of alveoli. These observations 1) identify the first endogenous signaling that inhibits septation, 2) demonstrate the presence of different molecular signaling for the formation of alveoli during and after septation, and 3) suggest the use of RARß antagonists might result in septation in prematurely born infants.
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METHODS AND MATERIALS
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Animals.
We purchased timed-pregnant, specific pathogen-free, Sprague-Dawley rats (Taconic Farms, Germantown, NY). RARß-/- mutant mice were generated as previously described on a mixed 129/Sv x C57BL/6 background (16). The RARß-/- mice are slightly growth deficient but have a normal life span (16). The RARß mutant is fertile and homozygous (RARß-/-). Thus backcrossing will not generate a wild type or heterozygote. We therefore used wild-type (RARß+/+) mice from which the RARß-/- were generated. In rats and mice, the day of birth was considered postnatal day 1.
Treatment.
We injected rats intraperitoneally daily with AGN-193174 (1.66 µmol/kg; Allergan, Irvine, CA), an RARß-selective agonist (21), or an equal volume (1 µl/g body wt) of the vehicle for AGN-193174 (cottonseed oil), from postnatal day 3 through 13. The molar concentration of AGN-193174 was the same as that of all-trans RA that induces alveolus formation in rats (29). Mice were untreated. All animals were killed by cutting the abdominal aorta after inducing a surgical level (toe-pinch) of anesthesia by the intraperitoneal injection of xylazine (
10 mg/kg) plus ketamine (
75 mg/kg). We killed rats at age 14 days and mice at age 4, 21, or 68 days.
Fixation and sampling tissue.
Lungs were fixed at a transpulmonary pressure of 20 cmH2O by the intratracheal infusion of cold 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Further processing and sampling of tissue, as well as the distinction of alveolar saccules from alveolar saccule ducts and of alveoli from alveolar ducts, were as previously described in detail (29). We designated gas-exchange structures bounded by septa as alveolar saccules in 4-day-old mice and as alveoli in 21-day-old and 68-day-old mice (7). Saccules and alveoli were identified as structures with a mouth through which they communicate with a common air space. We considered small structures occasionally seen opening into a saccule or alveolus to be part of these structures, not independent structures. We distinguished alveolar saccule and alveolar air space from, respectively, alveolar saccule duct air space and alveolar duct air space by analysis of serial sections of the lung.
Stereology.
We used the selector method (10) to choose alveoli for analysis. For volume determination, we selected saccules or alveoli that appeared on a reference plane (print) within a defined counting frame that did not appear on a plane (print) a convenient distance from the reference plane. This procedure allows the selection of structures unbiased by size, shape, or orientation (10). Thirty saccules or alveoli were analyzed per animal.
The volume of individual saccules or alveoli was estimated by the point-sample intercept method (17). To do this, a system of parallel lines with test points was superimposed on each of the selected prints that contained the relevant alveolar images. The position and orientation of each print under the test system was determined following two sets of random numbers. Each transection hit by a test point was classified with an arithmetic ruler; the corresponding segment was cubed, and their average was used to establish the individual alveolar volume according to the expression vest =
/3
o3, where vest is the individual estimated volume and
o3 is the average cubed segment. We calculated the number of saccules or alveoli per lung (Na) using the identity Na = VL x Vva/
a, where VL is lung volume determined by fluid displacement (34), Vva is the volume density of saccule airspace or alveolar airspace, and
a is the mean volume of a saccule or of an alveolus. Vva was determined by point counting on three prints using a square lattice test system that had points 0.4 cm apart. More than 900 points were counted per animal, thereby attaining a relative error of between 5% and 10%. Saccule surface area and alveolar surface area were determined by counting points and intersections and the distance between alveolar walls (Lm), and the surface-to-volume ratio (S/V) of gas-exchange units was calculated (40).
Statistical methods.
For each parameter measured or calculated from measurements, values for individual animals were averaged per experimental group, and the SE was calculated. The statistical significance of intergroup differences was determined by nonparametric analysis using the Mann-Whitney test (37).
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RESULTS
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RARß agonist.
At age 14 days rats treated with an RARß agonist (AGN-193174, Allergan) (21) from postnatal day 3 through 13 exhibited substantially diminished septation of the gas-exchange saccules. This could be appreciated by the presence of larger and fewer alveoli in RARß agonist-treated rats than in rats treated with cottonseed oil (the vehicle for the agonist) (Fig. 1). These differences, apparent on histological sections, were so large we could quantitate them by measuring the distance between alveolar walls (Lm) and the surface-to-volume ratio (S/V) of gas-exchange structures, even though these are relatively insensitive probes of alveolar size. The Lm in cottonseed oil-treated rats was 80 ± 2.8 µm [mean ± SE; number of rats, (n) = 4] and 110 ± 7.3 µm in RARß agonist-treated rats (n = 3, P = 0.03). The S/V was 504 ± 18 cm-1 in cottonseed oil-treated rats and 365 ± 23 cm-1 in RARß agonist-treated rats (P = 0.03). Lung volume (Table 1) was the same in both groups, but surface area was larger (634 ± 19 cm2) in cottonseed oil-treated rats than in RARß agonist-treated rats (492 ± 28 cm2, P = 0.03).

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Fig. 1. Histological section of lungs of 14-day old rats. Rats were injected intraperitoneally daily from age 3 through 13 days with cottonseed oil (A) or with an equal volume (1.0 µl/g body wt) of a selective retinoic acid receptor (RAR) ß-agonist (1.66 µmol/kg body wt) (B) and killed at age 14 days. Lungs were fixed at a transpulmonary pressure of 20 cmH2O. Scale bars = 50 µm.
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RARß knockout mice.
The present generation of retinoid analogs is selective, not specific. However, we have provided specificity by examining the lungs of RARß knockout and wild-type mice (16) at age 4, 21, and 68 days. Lung volume did not differ between wild-type and knockout mice at any age (Table 1). Examination of histological sections clearly demonstrated alveolar saccules were substantially larger at age 4 days in wild-type than in RARß knockout mice (Fig. 2). Because the intergroup differences were less clear at age 21 (Fig. 2) and 68 days (not shown), at all ages we made the more sensitive and specific measurement of the volume of individual alveoli, rather than the less sensitive measurement of Lm or S/V. At each age, alveolar volume (
a) was smaller in knockout than in wild-type mice (Fig. 3). The frequency distribution of alveolar dimensions demonstrated substantially fewer large saccules in 4-day-old knockout and 21-day-old knockout mice than in wild-type mice of the same respective ages (Fig. 4). The number of alveoli was higher at each age in RARß knockout mice than in wild-type mice (Fig. 5). There was not an intergroup difference (P > 0.05) in alveolar surface area at any age: surface area for wild-type at ages 4, 21, and 68 days, and the number of mice (n) studied were, respectively, 62 ± 2.7 (n = 3), 223 ± 11.1 (n = 4), and 274 ± 8.5 cm2 (n = 3); for RARß knockout mice at the same ages, surface area was, respectively, 65 ± 2.9 (n = 3), 242 ± 9.6 (n = 4), and 293 ± 19.4 cm2 (n = 3). The rate of alveolus formation was two times faster in knockout than in wild-type mice between age 4 and 21 days, but the rates were the same in wild-type and RARß mice between age 21 and 68 days (Fig. 6). Thus the absence of signaling through RARß regulated the formation of alveoli only during the perinatal period.

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Fig. 2. Histological sections of lungs of 4-day-old and 21-day-old mice. Lungs from 4-day-old wild-type (A) and RARß knockout (B) mice and 21-day-old wild-type (C) and RARß knockout (D) mice were fixed at a transpulmonary pressure of 20 cmH2O. Scale bars = 50 µm.
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Fig. 3. Volume of individual alveoli ( a). Wild-type and RARß knockout mice were killed at age 4, 21, or 68 days. Their lungs were fixed at a transpulmonary pressure of 20 cmH2O. Values are means ± SE; n = 3 for each group at age 4 days, 4 for each group at age 21 days, and 3 for each group at age 68 days. *P < 0.05, **P = 0.02, and ***P < 0.05 between wild-type and RARß knockout (RARß k.o.) mice at age 4, 21, and 68 days, respectively.
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Fig. 5. Number of individual alveoli (Na). The number of alveoli per rat was calculated as described in METHODS AND MATERIALS. Values are means ± SE; the number of animals are as in the legend to Fig. 3. *P < 0.05, **P = 0.02, and ***P < 0.05 between wild-type and knockout mice at age 4, 21, and 68 days, respectively.
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Fig. 6. The rate of formation of alveoli. The rates of alveolus formation were calculated from the mean values in Fig. 5 by dividing the differences in alveolar number by the number of days.
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DISCUSSION
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Endogenous regulation of the formation of alveoli.
The gas-exchange region of the architecturally immature lung is composed of structures termed alveolar saccules and alveolar saccule ducts (7). Septation of alveolar saccules gives rise to alveoli; the architecturally mature, but not necessarily full-sized gas-exchange region, is then, by definition, composed of alveoli and alveolar ducts (7). Analysis of serial lung sections and use of newer stereological procedures (10, 17) have allowed unbiased estimates of the volume of individual alveolar saccules and alveoli from which the number of saccules or alveoli may be calculated (5, 29, 30, 31, 33). Enumeration of alveoli gave clear evidence (5), supporting earlier data (27), that alveoli continue to be formed after septation of alveolar saccules present at birth has ended. This confirmatory information has raised the previously posed question (27) of whether the formation of alveoli, during and after the period of septation, is regulated by the same signaling pathways.
Our studies with mutant mice provide key insights into this regulation by demonstrating a role for endogenous retinoid signaling in the formation of alveoli. The presence at age 4 days, the normal start of septation in mice (2), of smaller, more numerous saccules in RARß knockout mice than in wild-type mice indicates the premature onset of septation occurs in the absence of RARß receptors. This shows endogenous signaling through RARß exerts an inhibitory effect on septation prior to its normal onset in wild-type mice. The more rapid formation of alveoli between ages 4 days and 21 days in RARß knockout mice than in wild-type mice establishes that signaling via RARß is also a modulator of alveolus formation during septation. By contrast, the identical rate of alveolus formation in mutant and wild-type mice between age 21 days and 68 days, in view of the different rates of alveolus formation in these two groups during the period of septation, demonstrates RARß does not influence the formation of alveoli after the period of septation. This shows different signaling paths regulate alveolus formation during and after septation.
Our studies using a selective RARß agonist in rats complement and strengthen those with mutant mice by demonstrating excess ligand impairs septation, an effect opposite the enhanced septation in the absence of receptor in RARß knockout mice. In addition, the studies with the RARß agonist extend to a second species a role for RARß in the regulation of the perinatal formation of alveoli, thereby increasing the universality of our findings.
Exogenous regulation of the formation of alveoli.
All-trans RA induces the formation of alveoli in newborn rats (30), prevents the inhibition by dexamethasone of alveolus formation in newborn rats (30), induces septation in mouse fetal lung in culture (8), and induces septation in adult rats in which elastase-induced emphysema had been previously induced (31). Treatment of prematurely delivered lambs with retinol (vitamin A) increases the formation of alveoli (1). Furthermore, treatment of adult rats with 9-cis RA, in which emphysema had been previously induced by treatment with elastase, induces the formation of alveoli (4).
It is clear from the studies just cited that treatment with pharmacological doses of retinoids (all-trans RA, 9-cis RA, and retinol) induces the formation of alveoli (4, 8, 15, 30, 31). By contrast, we now demonstrate RARß is a physiological endogenous inhibitor of septation. These observations are not contradictory, considering the complex nature of retinoid signaling and of the action of retinoids on cellular functions. For example, it is possible that RA-induced septation results from a balance between inhibition through RARß and promotion via other RARs. Treatment with pharmacological doses of RA might displace this equilibrium in favor of promotion. This notion is supported by reports showing the temporal and cellular pattern of expression of RARs differ (12, 13), suggesting the subtypes regulate different functions. Each RAR gene has two promoters, which give rise to two distinct transcripts that generate receptor isoforms with amino termini that are different and hence may have different functions (for review, see Ref. 25). To add to the complexity of retinoid signaling, promoter architecture, cofactors, and orphan receptors can determine the response to all-trans RA (15), there can be antagonism among the RARs (19), one isoform may signal via another isoform (35), one RAR isoform may control the expression of another RAR isoform (14), the induction of RARs may be cell specific (11, 15) and thereby functionally specific, and RA may induce the degradation of and thereby diminish the concentration of its receptors (41). Finally, because all-trans RA and retinol may be converted in vivo to 9-cis RA, the RA-induced formation of alveoli may be an RXR-mediated event.
Individual alveoli, alveolar number, and the regulation of alveolar surface area.
RARß-/- mice have more but smaller alveoli than RARß+/+ mice, but do not have greater alveolar surface area. We believe there are at least two explanations for these findings. If alveolar shape is the same in both groups, as appears to be the case, then surface area is related to volume to the power
and a change in volume would be accompanied by a change in surface area only about 66% as large. Therefore, a change of alveolar volume would be detected before a change of surface area. Viewed biologically, the absence of intergroup differences of alveolar surface area, together with differences of alveolar size and the number of alveoli, raise interesting questions about the regulation of the eruption of septa, their eventual length, and hence the size of alveoli. An explanation for the lack of a difference of alveolar surface area between RARß+/+ and RARß-/- mice is that RARß regulates the rate of eruption of septa but not their ultimate length and therefore not alveolar surface area. This notion of a dichotomy between eruption of a septum and the length of a septum is consistent with the demonstration that alveoli enlarge after the period of septation ends (5). We propose another regulator, perhaps oxygen consumption, which is tightly linked to alveolar size (37), determines septal length and thereby determines alveolar surface area. If this is the case, then there must be feedback inhibition of septal elongation to prevent excess surface area, in the face of more septa, when additional alveolar surface is not needed.
Potential clinical relevance.
Relatively recent advances in the therapy of premature infants have changed the characteristics of bronchopulmonary dysplasia from a syndrome in which damage to conducting airways was predominant to one in which the arrest of alveolus formation plays an increasing role in mortality (18, 20). The basis for the failure to form alveoli is uncertain, but prematurely born infants are commonly treated with corticosteroid hormones (31) and oxygen (31), which in rats impair septation (6, 32). However, even in the absence of corticosteroid treatment, prematurely born human infants (26, 35) and baboon infants (9) may fail to form alveoli. Thus, it is increasingly apparent that the prevention or remediation of the arrested formation of alveoli will be a target of therapy. The present work, by demonstrating signaling via RARß is an endogenous inhibitor of alveolus formation in the perinatal period, allows the hypothesis that the use of RARß antagonists (24) would induce the premature onset of alveolus formation in babies.
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ACKNOWLEDGMENTS
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This research was supported in part by National Heart, Lung, and Blood Institute Grants HL-37666, HL-20366, HL-59432, HL-60115, and HL-47413 and by an American Lung Association Career Investigator Award to L. B. Clerch. G. D. Massaro and D. Massaro are Senior Fellows of the Lovelace Respiratory Institute, Albuquerque, NM. D. Massaro is Cohen Professor of Pulmonary Research, Georgetown University.
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FOOTNOTES
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Address for reprint requests and other correspondence: G. Massaro, Lung Biology Laboratory, Georgetown Univ., School of Medicine, 3900 Reservoir Rd., NW, Washington, DC 20007-2197 (E-mail: massarog{at}georgetown.edu)
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