REPORT
Retinoic acid receptor-alpha regulates pulmonary alveolus formation in mice after, but not during, perinatal period

Gloria De Carlo Massaro1, Donald Massaro2, and Pierre Chambon3

Lung Biology Laboratory, Departments of 1 Pediatrics and 2 Medicine, Georgetown University School of Medicine, Washington, District of Columbia 20057-1481; and 3 Institute de Génétique et de Biologie Moléculaire et Cellulaire, 67404 Illkirch, Strasbourg, France


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The formation of pulmonary alveoli in mice and rats by subdivision of alveolar saccules that constitute the newborn's gas-exchange region ends by approximately postnatal day 14. However, alveoli continue to form after age 14 days until age ~40 days by means other than septation of the saccules present at birth. With the use of morphometric procedures and retinoic acid receptor (RAR)-alpha +/+ and RAR-alpha -/- mice, we now show the volume of individual alveoli (va), the number of alveoli (Na), and alveolar surface area (Sa) are the same in 14-day-old RAR-alpha +/+ and RAR-alpha -/- mice. However, at age 50 days, va is larger, and Na and Sa are smaller, in RAR-alpha -/- than in RAR-alpha +/+ mice, although total lung volume is the same in both groups. These findings, and prior data showing RAR-beta is an endogenous inhibitor of alveolus formation during, but not after, the perinatal period, indicate there are developmental period-specific regulators of alveolus formation and that total lung volume and alveolar dimensions may have different regulators.

all-trans retinoic acid; retinoic acid receptor-alpha null mice; morphometry; lung volume


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THE FORMATION OF PULMONARY ALVEOLI in mice (1) and rats (4, 5), by subdivision of the gas-exchange structures (alveolar saccules) present at birth, begins at approximately postnatal day 3-4, is mainly over by postnatal day 14 (5, 9, 10, 17), and where quantitated (rats) is responsible for approximately one-third of alveoli formed in the first two postnatal weeks (9, 10, 17). Alveolus formation by other than septation of alveolar saccules present at birth continues in mice (7) and rats (2, 3, 9) after postnatal day 14 until approximately age 5-6 wk. Alveolus formation by septation of the alveolar saccules present at birth occurs throughout the lung (5, 9, 10, 17); its cessation, but the continued formation of alveoli, suggests the molecular regulation of alveolus formation, is, at least in part, developmental period specific. A developmental period molecular specificity of the regulation of alveolus formation is supported by the demonstration that retinoic acid receptor (RAR)-beta is an endogenous inhibitor of alveolus formation during, but not after, the perinatal period (15). We now report RAR-alpha is not required for alveolus formation in mice during the first two postnatal weeks but is required for the production of alveoli of normal volume (va), number (Na), and surface area (Sa) between age 14 and 50 days.

RAR-alpha -/- mutant mice were generated in France, as previously described, on a mixed 129/Sv × C57BL/6 background (8), shipped to Georgetown University, housed in the Department of Comparative Medicine, allowed food (Rodent Chow 5001; Ralston Purina, St. Louis, MO) and water ad libitum, and maintained on a 12:12-h light-dark cycle at ~22°C. RAR-alpha -/- females were bred with RAR-alpha +/- males, and RAR-alpha +/+ females were bred with RAR-alpha +/+ males. The day of birth was considered age 1 day. Male mice were killed at ages 14 or 50 days by cutting the abdominal aorta after inducing a surgical level of anesthesia by the intraperitoneal injection of xylazine plus ketamine. Genotyping was performed, as previously described (8), on a piece of mouse tail. All procedures on animals were approved by the Georgetown University Animal Care and Use Committee and conformed to National Institutes of Health and US Department of Agriculture guidelines for the care and use of animals.

We fixed lungs by the intratracheal infusion of cold 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) at a transpulmonary pressure of 20 cmH2O (10, 11-14). We identified alveoli, estimated their volume, and calculated the number per lung as previously described (10, 12, 13, 15). Thirty alveoli were analyzed per mouse. The volume density of alveolar components and the Sa of alveoli were determined by counting points and intersections (21). The statistical significance between two groups was estimated by using an unpaired two-tailed t-test analysis (19), and P < 0.05 was considered to be statistically significant.

Fourteen-day-old RAR-alpha +/+ mice weighed 6.6 ± 0.2 g (N = 4) and were 54 ± 0.7 mm long (nose to rump); same-aged RAR-alpha -/- mice weighed 6.5 ± 0.2 g (n = 4) and were 52 ± 0.7 mm long (P > 0.05 between groups for the same parameter). At age 50 days, RAR-alpha +/+ mice weighed 24.1 ± 1.1 g and were 88 ± 0.9 mm long (n = 6); same-aged RAR-alpha -/- mice weighed 24.5 ± 1.6 g (n = 4) and were 87 ± 1.7 mm long (P > 0.05 between groups for the same parameter).

Lung volume (18) of RAR-alpha +/+ mice was 0.34 ± 0.01 cm3 (n = 4) and 0.65 ± 0.01 cm3 (n = 6) at ages 14 days and 50 days, respectively. In RAR-alpha -/- mice, lung volume was 0.33 ± 0.01 cm3 (n = 4) and 0.65 ± 0.02 cm3 (n = 4) at ages 14 and 50 days, respectively (P > 0.05 for the same parameter between groups at each age). We did not find differences in the volume density of alveolar air, alveolar duct air, or alveolar wall between RAR-alpha +/+ and RAR-alpha -/- mice or in the absolute volume of each of these parameters at either age (not shown). At age 14 days, va, Na, and Sa, respectively, did not exhibit intergroup differences (Fig. 1). However, at age 50 days, va was larger, and Na and Sa were smaller, in RAR-alpha -/- mice than in RAR-alpha +/+ mice (Fig. 1).


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Fig. 1.   Effect of deletion of retinoic acid receptor (RAR)-alpha on dimensions of the gas-exchange region. Values are means ± SE. The number of mice is given within each bar. va, volume of individual alveoli; Na, number of alveoli; Sa, alveolar surface area. At age 14 days, alveolar dimensions are the same in RAR-alpha +/+ and RAR-alpha -/- mice. At age 50 days, va is bigger, and Na and Sa are smaller, in RAR-alpha -/- mice compared with RAR-alpha +/+ mice. *P = 0.028, dagger P = 0.016, Dagger P = 0.04.

Among species that differ in body mass by several logs, lung volume is proportional to body mass. However, alveolar size (distance between alveolar walls) is inversely related to the organism's body mass-specific O2 consumption, and Sa is directly proportional to the organism's total O2 consumption (20). Thus lung volume and alveolar dimensions seem to have different regulators. This is consistent with the present study in which va, Na, and Sa, but not lung volume, were affected by the absence of RAR-alpha . Similarly, the presence or absence of RAR-beta (15) or RAR-gamma (16) does not affect lung volume, although their absence affects alveolar dimensions (15, 16).

The genetic dissection of regulatory events may be influenced by the organism's genetic background. Therefore, the universality of the present findings, and similar prior findings (15, 16), depend on, in part, obtaining the same results in mice with a different genetic background. Furthermore, in the RAR-alpha -/- mice we used, there is a high postnatal lethality (8). However, to the extent that the mice we studied at ages 14 and 50 days reflect the lungs of the dead mice, abnormal alveolar structure is not a cause of the lethality.

The genetic analysis of RAR regulation of alveolus formation in mice indicates: 1) RAR-alpha influences alveolus formation between ages 14 and 50 days but not in the perinatal period; 2) RAR-beta is an endogenous inhibitor of alveolus formation in the early postnatal period but not thereafter (15); and 3) RAR-gamma is required for normal alveolus formation at least up to age 28 days (16). Thus endogenous all-trans retinoic acid (ATRA), by virtue of its ability to bind RARs (6), could regulate alveolus formation in neonatal and in adult rodents. This possibility is supported by the evidence that exogenous ATRA induces alveolus formation in newborn as well as adult rodents (12-14). The differential regulation of alveolus formation by RARs, and in the case of RAR-alpha and RAR-gamma , possible complementary induction of alveolus formation, should be considered in attempts to use ATRA, other oxidation and isomerization products of retinol, or synthetic retinoid agonists and antagonists for therapeutic purposes in lung disease.


    ACKNOWLEDGEMENTS

We thank Dr. L. B. Clerch for reviewing the manuscript and Zofia Opalka for technical help.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-37666, HL-20366, and HL-60115.

G. D. Massaro and D. Massaro were Senior Fellows of Lovelace Respiratory Institute when this study was performed. D. Massaro is Cohen Professor at Georgetown University School of Medicine. G. D. Massaro and D. Massaro hold patents for the use of retinoids in lung diseases.

Address for reprint requests and other correspondence: G. D. Massaro, Lung Biology Laboratory, Box 571481, 3900 Reservoir Rd., NW, Washington, DC 20057-1481 (E-mail: massarog{at}georgetown.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajplung.00245.2002

Received 24 July 2002; accepted in final form 1 November 2002.


    REFERENCES
TOP
ABSTRACT
ARTICLE
REFERENCES

1.   Amy, RW, Bowes D, Burri PH, Haines J, and Thurlbeck WM. Postnatal growth of the mouse lung. J Anat 124: 131-151, 1977[ISI][Medline].

2.   Blanco, LN, Massaro D, and Massaro GD. Alveolar size, number, and surface area: developmentally dependent response to 13% O2. Am J Physiol Lung Cell Mol Physiol 261: L370-L377, 1991[Abstract/Free Full Text].

3.   Blanco, LN, Massaro GD, and Massaro D. Alveolar dimensions and number: developmental and hormonal regulation. Am J Physiol Lung Cell Mol Physiol 257: L240-L247, 1989[Abstract/Free Full Text].

4.   Burri, PH. The postnatal growth of the rat lung. Morphology. Anat Rec 180: 77-98, 1974[ISI][Medline].

5.   Burri, PH, Dbaly J, and Weibel ER. The postnatal growth of the rat lung. I. Morphometry. Anat Rec 178: 711-730, 1974[ISI][Medline].

6.   Chambon, P. A decade of molecular biology of retinoic acid receptors. FASEB J 10: 940-954, 1996[Abstract/Free Full Text].

7.   Kawakami, M, Paul JL, and Thurlbeck WM. The effect of age on lung structure in male BALB/cNNia inbred mice. Am J Anat 170: 1-21, 1984[ISI][Medline].

8.   Lufkin, T, Lohnes D, Mark M, Dierich A, Gorry P, Gaub MP, LeMeur M, and Chambon P. High postnatal lethality and testis degeneration in retinoic acid receptor alpha  mutant mice. Proc Natl Acad Sci USA 90: 7225-7229, 1993[Abstract].

9.   Massaro, D, and Massaro GD. Pulmonary alveoli: formation, the "call for oxygen," and other regulators. Am J Physiol Lung Cell Mol Physiol 282: L345-L358, 2002[Abstract/Free Full Text].

10.   Massaro, GD, and Massaro D. Formation of alveoli in rats: postnatal effect of prenatal dexamethasone. Am J Physiol Lung Cell Mol Physiol 263: L37-L41, 1992[Abstract/Free Full Text].

11.   Massaro, GD, and Massaro D. Postnatal lung growth: evidence that the gas-exchange region grows fastest at the periphery. Am J Physiol Lung Cell Mol Physiol 265: L319-L322, 1993[Abstract/Free Full Text].

12.   Massaro, GD, and Massaro D. Postnatal treatment with retinoic acid increases the number of pulmonary alveoli in rats. Am J Physiol Lung Cell Mol Physiol 270: L305-L310, 1996[Abstract/Free Full Text].

13.   Massaro, GD, and Massaro D. Retinoic acid treatment abrogates elastase-induced pulmonary emphysema in rats. Nat Med 3: 675-677, 1997[ISI][Medline].

14.   Massaro, GD, and Massaro D. Retinoic acid treatment partially rescues failed septation in rats and in mice. Am J Physiol Lung Cell Mol Physiol 278: L955-L960, 2000[Abstract/Free Full Text].

15.   Massaro, GD, Massaro D, Chan WY, Clerch LB, Ghyselinck N, Chambon P, and Chandraratna RA. Retinoic acid receptor-beta : an endogenous inhibitor of the perinatal formation of pulmonary alveoli. Physiol Genomics 4: 51-57, 2000[Abstract/Free Full Text].

16.   McGowan, S, Jackson SK, Jenkins-Moore M, Dai HH, Chambon P, and Snyder JM. Mice bearing deletions of retinoic acid receptors demonstrate reduced lung elastin and alveolar numbers. Am J Respir Cell Mol Biol 23: 162-167, 2000[Abstract/Free Full Text].

17.   Randell, SH, Mercer RR, and Young SL. Postnatal growth of pulmonary acini and alveoli in normal and oxygen-exposed rats studied by serial section reconstruction. Am J Anat 86: 55-68, 1989.

18.   Scherle, W. A simple method for volumetry of organs in quantitative stereology. Mikroscopie 26: 57-60, 1970.

19.   StatMost Statistical Analysis and Graphics. StatMost, version 3.2. Salt Lake City, UT: Dataxiom Software, 2000.

20.   Tenney, SM, and Remmers JE. Comparative quantitative morphology of the mammalian lung diffusing area. Nature 197: 54-56, 1963[ISI].

21.   Weibel, ER. Stereological Methods. New York: Academic, 1979, p. 9-196.


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