Rowett Research Institute, Aberdeen AB21 9SB, United Kingdom
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Vitamin A is required during pregnancy for fetal lung development. These experiments monitored fetal lung morphology in normal and vitamin A-deficient rats. The expression of elastin and the growth arrest-specific gene 6 (gas6) in fetal and neonatal hearts and lungs was assessed by Northern blotting. In normal-fed rats, elastin and gas6 were expressed in the fetal lung and heart from day 19 of gestation up to day 2 postnatally. Maternal vitamin A deficiency altered fetal lung development. On day 20, the bronchial passageways were less developed and showed reduced staining for elastic fibers, and in the neonates, the relative air space and the size of the sacculi were reduced. In the fetal lung, the mRNAs for elastin and gas6 were reduced to 56 and 68% of the control values, respectively. In the fetal heart, the mRNA for elastin was reduced to 64% of the control value, whereas gas6 was increased twofold. In the neonate, there was no change in elastin expression in the lung or heart, but gas6 expression in the heart was increased twofold. These results suggest that, in the pregnant rat, vitamin A deficiency may retard fetal lung development or influence the differentiation of critical cell lines. The changes in elastin and gas6 expression may be used to identify the cell types affected.
elastin; growth arrest-specific gene 6
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
VITAMIN A IS REQUIRED during pregnancy for normal fetal development (1, 23). The vitamin is stored as retinyl esters in the liver of the mother and is mobilized during pregnancy. Animals fed a diet severely deficient in retinoids for long periods before mating remain reasonably healthy but fail to reproduce because of embryo failure on day 15 (24). The administration of retinol from day 10 allows development to continue beyond day 20 (26). In the case of less severe vitamin A deficiency, the early stages of pregnancy are supported by the release of vitamin A from retinyl esters stored in the maternal liver, but once these maternal stocks are exhausted, there is insufficient vitamin A for normal development.
We have developed an experimental rat model that causes a 50% reduction in circulating maternal retinol concentrations during late pregnancy. The lungs and hearts of fetuses and neonates carried by mothers with lower circulating retinol concentrations are relatively lighter, with higher allometric coefficients. These changes in fetal organ growth occur in the absence of changes in whole fetal and neonatal weights (Antipatis, Grant, and Ashworth, unpublished data). The hearts of fetuses from vitamin A-deficient rats have defects in the aortic arch and dysmorphic hypoplastic development of the ventricular chambers (27). In the lungs of adult experimental animals, vitamin A deficiency causes replacement of mucus-secreting epithelium, with stratified squamous keratinizing epithelium in the trachea and bronchi (28). This histological change has also been found in the airways of premature infants who are born vitamin A deficient. This may influence orderly lung repair and contribute to the development of bronchopulmonary dysplasia (4). About 50% of neonates born to vitamin A-deficient rats are born dead or die soon after birth (23), and this may be due to pathophysiological changes in the lungs.
Extensive studies with cell culture systems have shown that the active forms of vitamin A, retinol and retinoic acid, serve as signals to regulate the expression of genes during vertebrate development. The molecular mechanisms underlying the role of vitamin A in heart and lung maturation have not been fully determined. However, it is likely that the interactions of retinoic acid with its receptors ultimately produce changes in the expression of structural genes (3). Probably the most important extracellular matrix protein in organs that undergo repeated physical deformations, such as the lungs, heart, blood vessels, and skin, is elastin (18). This resilient connective tissue protein forms a key structural component of both the heart and lungs and is critical for their lifelong function. Because of its slow turnover, changes in elastogenesis during fetal development can result in near-permanent defects in the adult (22). Cell adhesion is another important process taking place during maturation of the lungs, where the formation of alveolar and capillary epithelia are important in determining the size of the gas-exchange surface (11). A gene associated with the processes of cell adhesion is the growth arrest-specific gene 6 (gas6). The protein product of gas6 is a ligand for the axl tyrosine kinase receptor (25), and treating cells that express axl with gas6 causes them to form aggregates (12). Cell adhesion also occurs during the development and maturation of cardiac myocytes, and gas6 has been shown to be expressed in the heart during fetal development (16).
There is limited information on the temporal expression of the elastin and gas6 genes during the late stages of gestation and early postnatal life. Our objectives therefore were 1) to investigate temporal changes in the expression of elastin and gas6 mRNA levels in the late fetal and neonatal hearts and lungs, 2) to examine the effects of maternal vitamin A deficiency on lung morphology, and 3) to assess the impact of maternal vitamin A deficiency on the expression of elastin and gas6.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experimental diets. The experimental diet was based on the control diet previously described (8), except that the vitamin mixture was free of vitamin A. Corn oil was bought from Strachan and Sons (Aberdeen, UK); other ingredients were from BDH Chemicals (Poole, UK) or Sigma (Poole, UK). Analysis showed that this diet (vitamin A deficient) was essentially free of vitamin A, containing <0.02 retinol equivalents vitamin A/kg. A control diet sufficient in vitamin A was prepared by adding 1,200 retinol equivalents vitamin A/kg to the vitamin A-deficient diet. The litter sizes and neonatal survival of pups from rats fed the control diet were identical to those fed the standard laboratory chow.
Experimental animals. For the time-course studies, female rats from the Rowett Hooded-Lister strain were fed normal stock diets (Cambridge rat and mouse diet, Special Diet Services, Witham, UK). Animals of ~230 g body weight were caged overnight with normal males, and mating was confirmed by the presence of a plug on the following morning (day 0). Animals were killed at different points during pregnancy, the fetuses were dissected, and the organs were frozen immediately in liquid nitrogen. Neonates were killed 6, 24, and 48 h after birth.
Sixteen female Hooded-Lister rats were divided into two groups of eight and weaned directly onto control or vitamin A-deficient diets that were available ad libitum. At 7 wk postweaning, the female rats were mated with males of the same strain. Mating was confirmed by detection of a vaginal plug, and this was denoted as day 0. The female rats continued with the same diets after mating. Four females from each group were killed on day 20 of pregnancy and the remaining four on the day of parturition. Adult rats were killed by exsanguination under terminal anesthesia with halothane (Rhone Merieux, Harlow, UK), whereas fetuses and neonates were killed within 4-6 h of birth by decapitation. Fetal and neonatal weights were recorded. All experimental procedures were approved by the appropriate ethical committee and conducted in accordance with the United Kingdom Animals (Scientific Procedures) Act, 1986.
Plasma retinol analysis. Maternal
blood was collected in a tube containing 30 units of heparin and placed
on ice for a maximum of 1 h. The blood was centrifuged (1,000 g for 10 min at 4°C), and the
plasma was stored at 70°C. Maternal plasma retinol
concentrations were determined by reverse-phase high-performance liquid
chromatography as previously described (9), with retinyl palmitate as
an internal standard. All samples were analyzed in duplicate.
Histology. Fetal and neonatal hearts
and lungs were dissected, weighed, and either rapidly frozen in liquid
nitrogen and stored at 70°C until required for RNA
extraction or immersed in 4% neutral-buffered Formalin overnight at
4°C. Fixed samples were transferred to 70% ethanol for standard
processing and wax embedding. Five-micrometer sections were dewaxed,
passed through an ethanol series, and subjected to either hematoxylin
and eosin (Cellpath, Hampstead, UK) or Miller's elastin staining. For
elastin staining, sections were treated with 0.5% potassium
permanganate (Sigma) for 5 min, bleached through 1% oxalic acid
(Sigma), and stained in Miller's elastin stain for 60 min (Raymond A. Lamb, London, UK). After treatment with 95% alcohol for 10 min to
remove excess stain, the sections were counterstained in van Gieson's
stain (Raymond A. Lamb) for 5 min. The excess stain was drained off,
and the sections were dehydrated, cleared in xylene, and mounted in Pertex.
Northern analysis. Frozen tissue
samples were transferred directly to TRI Reagent (Sigma), with ~1
ml/100 mg tissue. The sample was then disrupted by homogenization for
10 s with an Ultra-Turrax homogenizer at maximum speed. RNA was
prepared from this mixture according to the manufacturer's
instructions. Twenty micrograms of total RNA were separated on a 1.2%
agarose gel and transferred to a nylon membrane (Boehringer Mannheim,
Mannheim, Germany). Probes for elastin (a generous gift from Dr. F. W. Keeley, Children's Hospital, Toronto, ON) and
gas6 (5) were labeled with
[-32P]dCTP with a
Megaprime labeling kit (Amersham, Little Chalfont, UK). Hybridizations
were carried out according to standard protocols (20). The blots were
washed to high stringency in 0.1× saline-sodium citrate (1×
is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) plus 1% SDS at
65°C and imaged on a wire proportional counter (Packard Instant
Imager). The mRNAs were quantified by measuring the amount of
radioactivity hybridizing to the bands on the Northern blot and
corrected for loading by reprobing with a probe for 18S rRNA.
Statistical analysis. Fetal and neonatal viability was defined as the number of live fetuses or neonates expressed as a proportion of the total number of live, dead, and resorbed fetuses or neonates. Mean values for fetal and neonatal samples were calculated from all live fetuses or all live neonates, respectively, in each group with Excel 5.0 (Microsoft, Seattle, WA). These data and the comparisons of relative mRNA expression were analyzed with unpaired Student's t-test. Differences were considered as significant at the P < 0.05 level.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preliminary experiments were undertaken to determine the temporal expression of elastin and gas6 mRNAs during heart and lung development. Animals were fed normal laboratory chow and killed at different stages of pregnancy. Figure 1 shows the expression of elastin and gas6 mRNAs in the lung and heart during the later stages of gestation and early neonatal life. The elastin probe hybridized to a principal transcript of 3.2 kb in the heart and lungs (10). There were two peaks of elastin mRNA expression in the lung, the first on day 20, confirming a previous report (19), and the second on day 1 after birth, suggesting a second period of elastogenesis during postnatal development (6). Levels of elastin mRNA in the heart increased on day 20 but otherwise remained relatively constant throughout development. A single gas6 transcript of 2.7 kb (15) was detected in both the heart and lungs. There was a progressive increase in gas6 expression during development of the lungs, with levels reaching a maximum just after birth. Expression in the heart remained relatively constant, apart from a small increase just before birth. The levels of gas6 mRNA were approximately twofold higher (relative to the 18S ribosomal subunit) in the heart.
|
Maternal plasma vitamin A levels on day 20 of pregnancy were significantly lower in the vitamin A-deficient mothers (0.075 ± 0.009 µg retinol/ml plasma) compared with the control mothers (0.168 ± 0.012 µg retinol/ml plasma; P < 0.001). Table 1 illustrates that maternal vitamin A deficiency had no effect on fetal number or viability on day 20. There were, however, significant reductions in the absolute and relative weights of both the heart and lungs of fetuses recovered from mothers fed the deficient diet compared with those maintained on the control diet. By the time of birth, there was no difference in the relative weight of the hearts in neonates from vitamin A-deficient mothers, whereas the relative weight of the lungs remained significantly lower (Table 1).
|
Over half of the neonates from vitamin A-deficient mothers died within 4 h of birth (Table 1). There was evidence that mortality was due to respiratory failure, and even those animals that survived exhibited signs of respiratory distress. Histological examination (Fig. 2) revealed that lung development was retarded in both day 20 fetuses and neonates from vitamin A-deficient mothers. On day 20, the lungs from the control animals were in the canalicular stage of development, and Fig. 2A shows the characteristic formation of bronchial passageways and branching ducts. In the vitamin A-deficient group (Fig. 2B), the bronchial passageways are not as well developed and there is no centrifugal branching. In the control neonates (Fig. 2C), the lungs are in the saccular stage of development and the air spaces have increased relative to the tissue components. In live neonates from the vitamin A-deficient group (Fig. 2D), the relative air space and the size of the sacculi were reduced relative to the control group. Analysis of the fetal hearts failed to show any changes in gross morphology (data not shown).
|
Lung sections were also stained for elastic fibers with Miller's elastin stain, and a typical control section from a day 20 fetus is shown in Fig. 3A. There was extensive staining around the developing bronchial passageways and, to a lesser extent, around the sacculi in the control sections. In the vitamin A-deficient group (Fig. 3B), the staining around the walls of the sacculi was greatly reduced; however, there was still some staining around the main airways. The differences between the neonates from the control (Fig. 3C) and vitamin A-deficient (Fig. 3D) groups were less pronounced. It is likely that fetuses with more severely compromised lung and heart development failed to survive, and, consequently, Figs. 2D and 3D are typical of the less-affected animals.
|
The morphological changes were accompanied by a significant decrease in the expression of both elastin (56% of the control value; P < 0.001) and gas6 (68% of the control value; P < 0.05) in the lungs of fetuses from mothers fed the vitamin A-deficient diet (Fig. 4). In those neonates that survived, the levels of elastin and gas6 mRNAs were the same in both groups; however, the reductions in mRNA levels seen in the fetal stages may have persisted in the nonviable neonates, which were not included. In the hearts (Fig. 5), there was also a significant decrease in the expression of elastin (64% of the control value; P < 0.05) as a result of maternal vitamin A deficiency, and this too disappeared in those fetuses that survived after birth. The expression of gas6 was different in the heart where maternal vitamin A deficiency significantly increased expression twofold (P < 0.001), and this increase persisted even after birth.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The studies described in this paper show that changes in the expression of certain developmental genes in the fetal lung and heart are dependent on an appropriate maternal vitamin A status. This highlights a likely mechanism underlying the previously documented effects of maternal vitamin A deficiency on fetal lung development (26). Data presented here illustrate that a marginal vitamin A deficiency in late gestation reduces neonatal viability at birth without affecting the number of live fetuses. Our data show marked differences in the structure of fetal lungs from vitamin A-deficient mothers, and thus it is likely that respiratory problems cause a large number of neonatal deaths. The changes in morphology suggest that saccular formation has been inhibited or delayed. Vitamin A deficiency also increases the mean gestation time from 22.5 to 23.5 days (2); however, we believe that this change is related to effects on the maternal endocrine system. The effects on the fetal lungs may be due to direct effects of vitamin A on gene expression, indirect effects caused by a failure of cell development, or a delay in normal development.
In the vitamin A-deficient fetuses, the change in lung morphology is associated with a reduction in the deposition of elastin. Features such as the tidal volume of the lungs depend on the extent of elastin synthesis, the bulk of which occurs during fetal development and in the first few weeks after birth (14). Elastin synthesis is essential for the maturation of the alveoli, the number and size of which determines the total lung capacity of the neonate (11). In situ hybridization studies have shown a close association between cells expressing elastin mRNA and the elastic fibers surrounding developing terminal airways (19). It is possible that elastin synthesis is directly dependent on vitamin A because the treatment of lung fibroblast cultures with retinoic acid can increase elastin expression approximately twofold (13). However, because of the extensive changes in morphology, it is also possible that fewer elastin-producing cells are present in the fetuses from the vitamin A-deficient mothers because of either a delay or a failure in lung development. Elastin expression in the developing lung is abruptly increased on day 20 during normal pregnancy (Fig. 1), so a delay in the development of the vitamin A-deficient fetuses would produce the apparent decrease in mRNA levels. However, in the absence of additional markers, it is equally possible that a more specific failure of cell differentiation has reduced elastin deposition. The data presented in Fig. 3, A and B, also suggest that there are qualitative differences in the pattern of elastin staining. Because retinoids may not have the same effects on elastin production by bronchial smooth muscle cells as they do on the interstitial mesenchymal cells, the pattern observed in the deficient fetuses could be the result of differential regulation of gene expression in cells of different phenotype.
We have also shown that vitamin A deficiency is associated with reduced levels of gas6 mRNA in the lungs. The product of the gas6 gene has a role in the regulation of cell adhesion (12), and this may also be associated with the changed morphology of the lungs. Retinoic acid treatment of embryonal carcinoma cells in culture, which induces differentiation, produces a small increase in gas6 mRNA (5). The increase is only seen after several days of treatment, which suggests that it is an indirect consequence of altered cell contact. In this respect, gas6 can in some ways be regarded as a structural component involved in the adhesion of cells, with its expression depending on the immediate local environment of the cell. If this is the case, the changes observed in the vitamin A-deficient fetuses may be the result of a population of gas6-expressing cells failing to differentiate. We have demonstrated that the decrease in gene expression is associated with the reduction in the number of cells forming the cuboidal-columnar epithelia surrounding the bronchioles, which can be seen in Fig. 2. The product of gas6 has also been shown to be a growth factor for endothelial cells (17). Blood capillaries that are closely associated with the terminal buds are also forming during this period, and it is possible that gas6 has a role in regulating this vascular growth. Experiments are in progress to investigate the localized expression of gas6 and its receptor by in situ hybridization.
Our results also show that the changes in elastin and gas6 expression are not restricted to the lungs; there are also very significant changes in the heart. As with the lungs, the mechanical properties of the heart are also dependent on elastin deposited during fetal life. The gas6 expression pattern is reversed in the heart compared with the lung, and mRNA levels are increased in the hearts of fetuses from deficient mothers. It is possible that the reversed pattern of expression is due to changes in the timing of development. The data in Fig. 1 show an association between an increase in gas6 expression in the lungs and a decrease in the heart during late fetal and neonatal development. One may predict, therefore, that developmental immaturity would be manifest by a decrease in gas6 expression in the lung and, conversely, an increase in the heart. This may not, however, be the only explanation; it is possible that the regulation of gas6 in heart cells is similar to 3T3 fibroblasts where the gene was first identified (21). In 3T3 cells, growth arrest modifies the interactions between cells, which results in changes in gas6 expression. Recently, it has been shown that gas6 may be involved in chemotaxis in smooth muscle cells (7). Fewer cells undergoing fusion, consistent with the decrease in organ weight, could also be responsible for the change in gas6 expression.
Through the use of an animal model in which maternal stores of vitamin A were reduced but not entirely depleted, we have demonstrated changes in both fetal lung and heart. These changes in the fetal lung may explain the effects of vitamin A deficiency on perinatal lung function, which affect fetal viability. It is likely that those offspring that do not succumb to lung failure early in life may be affected by defects in the heart later. Elastin and gas6 are good markers for organ development, predicting altered function after birth, and may be useful in developing optimal vitamin A supplementation strategies.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by the Scottish Office Agriculture, Environment and Fisheries Department.
![]() |
FOOTNOTES |
---|
C. Antipatis was the recipient of a scholarship from the Greek State Foundation.
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. §1734 solely to indicate this fact.
Address for reprint requests: W. D. Rees, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, UK.
Received 17 February 1998; accepted in final form 8 September 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Antipatis, C.,
C. J. Ashworth,
and
G. Grant.
Effects of maternal vitamin A status on prenatal development in rats.
J. Reprod. Fertil. Abstr. Ser.
18:
82,
1996.
2.
Antipatis, C.,
M. G. Thompson,
R. M. Palmer,
and
C. J. Ashworth.
The effects of maternal vitamin A status on the ATP/ubiquitin-dependent proteolytic pathway in fetal and neonatal development in the rat (Abstract).
Proc. Nutr. Soc.
57:
73A,
1998.
3.
Chambon, P.
A decade of molecular biology of retinoic acid receptors.
FASEB J.
10:
940-954,
1991
4.
Chytil, F.
The lungs and vitamin A.
Am. J. Physiol.
262 (Lung Cell. Mol. Physiol. 6):
L517-L527,
1992
5.
Fleming, J. V.,
S. M. Hay,
D. N. Harries,
and
W. D. Rees.
The effects of nutrient deprivation and differentiation on the expression of growth arrest genes (gas and gadd) in F9 embryonal carcinoma cells.
Biochem. J.
330:
573-579,
1998[Medline].
6.
Foster, J. A.,
and
S. W. Curtiss.
The regulation of lung elastin synthesis.
Am. J. Physiol.
259 (Lung Cell. Mol. Physiol. 3):
L13-L23,
1990
7.
Fridell, Y.-W. C.,
J. Villa,
E. C. Attar,
and
E. T. Liu.
GAS6 induces Axl-mediated chemotaxis of vascular smooth muscle cells.
J. Biol. Chem.
273:
7123-7126,
1998
8.
Grant, G.,
P. M. Dorward,
and
A. Pusztai.
Pancreatic enlargement is evident in rats fed diets containing raw soybeans or cowpeas for 800 days but not in those fed diets based on kidney beans or lupinseed.
J. Nutr.
123:
2207-2215,
1993[Medline].
9.
Hess, D.,
H. E. Keller,
B. Oberlin,
R. Bonfanti,
and
W. Schuep.
Simultaneous determination of retinol, tocopherols, carotenes and lycopene in plasma by means of high-performance liquid chromatography on reversed phase.
Int. J. Vitam. Nutr. Res.
61:
232-238,
1991[Medline].
10.
Liu, R.,
C. S. Harvey,
and
S. E. McGowan.
Retinoic acid increases elastin in neonatal rat lung fibroblast cultures.
Am. J. Physiol.
265 (Lung Cell. Mol. Physiol. 9):
L430-L437,
1993
11.
Massaro, G. D.,
and
D. Massaro.
Formation of pulmonary alveoli and gas-exchange surface area: quantification and regulation.
Annu. Rev. Physiol.
58:
73-92,
1996[Medline].
12.
McCloskey, P.,
Y.-W. Fridell,
E. Attar,
J. Villa,
Y. Jin,
B. Varnum,
and
E. T. Liu.
GAS6 mediates adhesion of cells expressing the receptor tyrosine kinase Axl.
J. Biol. Chem.
272:
23285-23291,
1997
13.
McGowan, S. E.,
M. M. Doro,
and
S. K. Jackson.
Endogenous retinoids increase perinatal elastin gene expression in rat lung and fetal fibroblasts.
Am. J. Physiol.
273 (Lung Cell. Mol. Physiol. 17):
L410-L416,
1997
14.
McGowan, S. E.,
C. S. Harvey,
and
S. K. Jackson.
Retinoids, retinoic acid receptors, and cytoplasmic retinoid binding proteins in perinatal rat lung fibroblasts.
Am. J. Physiol.
269 (Lung Cell. Mol. Physiol. 13):
L463-L472,
1995
15.
Manfioletti, G.,
C. Brancolini,
G. Avnzi,
and
C. Schneider.
The protein encoded by a growth arrest specific gene (gas6) is a new member of the vitamin K-dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade.
Mol. Cell. Biol.
13:
4976-4985,
1993[Abstract].
16.
Nakano, T.,
K. Kawamoto,
K. Higashino,
and
H. Arita.
Prevention of growth arrest-induced cell death of vascular smooth muscle cells by a product of growth arrest-specific gene, gas6.
FEBS Lett.
387:
78-80,
1996[Medline].
17.
Nakano, T.,
K. Kawamoto,
J. Kishino,
K. Nomura,
K. Higashino,
and
H. Arita.
Requirement of gamma-carboxyglutamic acid residues for the biological activity of Gas6: contribution of endogenous Gas6 to the proliferation of vascular smooth muscle cells.
Biochem. J.
323:
387-392,
1997[Medline].
18.
Parks, W. C.,
R. A. Pierce,
and
R. P. Mecham.
Elastin.
In: Advances in Molecular and Cellular Biology, edited by H. K. Kleinman. Greenwich, CT: JAI, 1993, vol. 6, p. 133-182.
19.
Pierce, R. A.,
W. I. Mariencheck,
S. Sandefur,
E. C. Crouch,
and
W. C. Parks.
Glucocorticoids upregulate tropoelastin expression during late stages of fetal lung development.
Am. J. Physiol.
268 (Lung Cell. Mol. Physiol. 12):
L491-L500,
1995
20.
Sambrook, J.,
E. F. Fritsch,
and
T. Maniatis.
Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989.
21.
Schneider, C.,
R. M. King,
and
L. Phillipson.
Genes specifically expressed at growth arrest of mammalian cells.
Cell
54:
787-793,
1988[Medline].
22.
Shapiro, S. D.,
S. K. Endicott,
M. A. Province,
J. A. Pierce,
and
E. J. Campell.
Marked longevity of human lung parenchymalelastic fibers deduced from prevalence of D-aspartate and nuclear weapons-related carbon.
J. Clin. Invest.
87:
1828-1834,
1991[Medline].
23.
Takahashi, Y. I.,
E. S. Smith,
M. Winick,
and
D. S. Goodman.
Vitamin A deficiency and fetal growth and development in the rat.
J. Nutr.
105:
1299-1310,
1975[Medline].
24.
Thompson, J. N.,
J. M. Howell,
and
G. A. J. Pitt.
Vitamin A and reproduction in the rat.
Proc. R. Soc. Lond. B Biol. Sci.
159:
510-535,
1964.
25.
Varnum, B. C.,
C. Young,
G Elliot,
A. Garcia,
R. J. Bartley,
D. Yanagihara,
L. Benett,
M. Sylber,
L. A. Merewether,
A. Tseng,
E. Escobar,
E. T. Liu,
and
H. K. Yamane.
Axl receptor tyrosine kinase stimulated by the vitamin K-dependent protein encoded by growth-arrest-specific gene 6.
Nature
373:
623-626,
1995[Medline].
26.
Wellik, D. M.,
D. H. Norback,
and
H. F. DeLuca.
Retinol is specifically required during midgestation for neonatal survival.
Am. J. Physiol.
272 (Endocrinol. Metab. 35):
E25-E29,
1997[Abstract].
27.
Wilson, J. G.,
and
J. Warkany.
Aortic arch and cardiac anomalies in the offspring of vitamin A deficient rats.
Am. J. Anat.
85:
113-155,
1949.
28.
Zachman, R. D.
Role of vitamin A in lung development.
J. Nutr.
125:
1634S-1638S,
1995[Medline].