1 Department of Physiology, Monash University, Clayton, Victoria 3168, Australia; and 2 Department of Obstetrics and Gynaecology, University of Western Ontario, London, Ontario, Canada N6A 5A5
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ABSTRACT |
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Obstruction of the fetal trachea is a potent stimulus for fetal lung growth, and it has been suggested that this procedure may be used therapeutically to reverse lung growth deficits in human fetuses with lung hypoplasia. However, little is known about the effects of increased lung expansion on other aspects of lung development. Our aim was to determine the effect of increased and decreased lung expansion on the mRNA levels encoding surfactant protein (SP) A, SP-B, and SP-C in ovine fetal lungs. Lung tissue samples were collected from fetuses exposed to 2, 4, or 10 days of increased lung expansion caused by tracheal obstruction. The mRNA levels for SP-A, SP-B, and SP-C were determined by Northern blot analysis with specific ovine cDNA probes; SP-A protein levels were determined by Western blot analysis. Compared with age-matched (128-day gestational age) control fetuses, SP-A, SP-B, and SP-C mRNA levels in fetal lung tissue were significantly reduced at 2 days of tracheal obstruction and remained reduced at 4 and 10 days. However, SP-A protein levels were not reduced at 2 days of tracheal obstruction, tended to be reduced at 4 days, and were almost undetectable at 10 days. In contrast to tracheal obstruction, 7 days of lung liquid drainage significantly increased SP-C, but not SP-A, mRNA levels in fetal lung tissue compared with age-matched control fetuses. Our results demonstrate that increases in fetal lung expansion, induced by obstruction of the fetal trachea, cause large simultaneous reductions in SP-A, SP-B, and SP-C mRNA levels in the fetal lung as well as a decrease in SP-A protein levels. These data suggest that expression of the genes encoding SPs in the fetal lung are specifically responsive to the degree of lung expansion.
messenger ribonucleic acid; fetus; fetal lung liquid; fetal lung liquid volume
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INTRODUCTION |
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THE FETAL LUNG IS FILLED with a liquid that is secreted across the pulmonary epithelium into the lung lumen (22) and leaves the lungs via the trachea (5). Retention of this liquid within the future airways maintains the lungs in an expanded state that is critical for normal lung growth and development (1, 6, 12). Indeed, deflation of the lung caused by drainage of lung liquid abolishes lung growth and eventually results in a structurally immature and markedly hypoplastic lung (1, 11, 18, 19). Conversely, increases in fetal lung expansion induced by obstruction of the fetal trachea is a potent stimulus for lung growth, although the mechanisms involved are largely unknown (1, 11, 18, 19). It has been suggested that the increase in fetal lung expansion induced by tracheal obstruction could be used to reverse lung growth deficits in human fetuses with severe lung hypoplasia (2, 9, 11, 19, 28) because this procedure is a potent stimulus for fetal lung growth. However, little is known about the effects of increased lung expansion on other aspects of pulmonary physiology and, in particular, whether growth-stimulated lungs would be functional in terms of gas exchange after birth.
After birth, the respiratory function of the lung is critically dependent on the surface-active properties of surfactant, which acts to lower surface tension forces at the air-liquid interface (8). This greatly reduces the likelihood of alveolar collapse during expiration and facilitates lung inflation during inspiration. Surfactant protein (SP)-A, SP-B, and SP-C play essential roles in the function of surfactant, including extracellular processing of phospholipids and recycling of surfactant constituents as well as adsorption of phospholipids into the surface monolayer (8). Recent studies have shown that prolonged periods of tracheal ligation reduce SP-A protein (13) and SP-C mRNA levels (23) in fetal lung tissue. Both studies suggested that the reduction resulted from a reduction in the number of differentiated type II alveolar epithelial cells (AECs).
The increase in lung growth induced by tracheal obstruction follows a specific time course and is completed within 7 days (11, 21). In addition, although prolonged periods of tracheal obstruction reduce the proportion of differentiated type II AECs (1, 23), we have been unable to detect a reduction before day 10 (20). Thus, after tracheal obstruction, it is possible that the period of accelerated lung growth (0-7 days) precedes the period (>10 days) when the proportion of type II AECs decreases, possibly due to differentiation into type I cells (23). Our aim was to determine whether tracheal obstruction affects SP-B mRNA levels in fetal lung tissue as well as to determine the changes in SP-A, SP-B, and SP-C mRNA levels at specific time points during tracheal obstruction: 1) at 2 days when lung growth is at a maximum, 2) at 4 days when the growth rate is reduced but still elevated, and 3) at 10 days when the rate of lung growth has returned to the control value (21). We hypothesized that the mRNA encoding SP-A, SP-B, and SP-C in fetal lung tissue would not be reduced until after the lung growth response to tracheal obstruction was complete (i.e., after 7 days). We have also compared the time course for the changes in fetal lung SP-A, SP-B, and SP-C mRNA levels in response to tracheal obstruction because, although expression of SP-C is specific for type II AECs, SP-A and SP-B are not, and these proteins are known to be regulated by different factors (8). We have also determined the changes in fetal lung SP-A levels after tracheal obstruction and related these to the changes in mRNA levels encoding this protein. This comparison provides an indication of the time delay before a decrease in SP-A gene expression results in a decrease in mature protein levels in vivo. Finally, we have also determined the effect of lung deflation on fetal lung SP-A and SP-C mRNA levels and have shown that at least SP-C gene expression appears to be intimately linked to the degree of lung expansion.
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METHODS |
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Experimental preparation. Fetal lung tissues were obtained from experiments performed previously on chronically catheterized fetal sheep in our laboratory (11, 14, 21). Briefly, aseptic surgery was performed on pregnant ewes at 113-120 days of gestation. Under general anesthesia (1.5% halothane in O2-N2O, 50:50 vol/vol), two large-diameter saline-filled silicone rubber catheters were inserted into the midcervical trachea of each fetus; one catheter was directed toward the lungs, and the other was directed toward but did not enter the larynx. These catheters were externalized and joined together to form an exteriorized tracheal loop that maintained the normal flow of tracheal fluid (10). The ewe and fetus were allowed to recover from surgery for at least 5 days before the start of experiments.
The fetal trachea was obstructed by occluding the exteriorized tracheal
loop for either 2 (n = 5), 4 (n = 5), or 10 (n = 5) days, with all experimental
periods finishing on day 128 of
gestation; the number of animals used in each analysis depended on RNA
quality. Each group of trachea-obstructed fetuses had a separate age-
and treatment-matched control group of fetuses
(n = 5/group) in which the trachea
remained unobstructed and the normal flow of lung liquid was
maintained. In an additional group of fetuses, lung liquid was drained
into a sterile bag for 7 days with the use of gravity, again with the
experimental period ending on day 128 of gestation (11); this group of fetuses also had an age- and treatment-matched group of control fetuses
(n = 5/group). At the end of the
experimental period, all ewes and fetuses were painlessly killed with
an intravenous injection of pentobarbital sodium administered to the
ewe. At postmortem, lung tissue was collected, frozen in liquid
nitrogen, and stored at 70°C.
Northern blot analysis. Fetal lung
SP-A, SP-B, and SP-C mRNA levels were quantified by Northern blot
analysis as previously described (11). Total RNA was extracted from
fetal lung tissue with a modified guanidine thiocyanate-cesium chloride
method (3). Total RNA (20 µg/lane) was denatured and electrophoresed
in a 1% agarose gel containing 2.2 M formaldehyde before the RNA was transferred to a nylon membrane (Duralon, Stratagene, La Jolla, CA) by
capillary action; the RNA was cross-linked to the membrane with
ultraviolet light (Hoeffer UVC 500, AMRAD). The membrane was incubated
in hybridization buffer [50% (vol/vol) deionized formamide, 7%
(wt/vol) SDS, 5× saline-sodium phosphate-EDTA, and 0.1 mg/ml of denatured and fragmented salmon sperm DNA] for
3-4 h at 42°C and then hybridized in the same buffer
containing a 32P-labeled SP-A,
SP-B, or SP-C cDNA probe (2 × 106
counts · min1 · ml
1)
for 24-48 h at 42°C. The ovine SP-A cDNA probe was a 322-bp Sac
I-Kpn I fragment encoding the globular
portion of ovine SP-A. The SP-B cDNA probe was a 330-bp
Pst
I-BamH I fragment encoding a portion
of the preprotein flanking the mature protein. The SP-C cDNA probe was
an 802-bp EcoR I fragment encoding the
entire mature protein. The cDNA probes were labeled with
[
-32P]dCTP by a
random-priming technique with a labeling kit (Oligolabeling Kit,
Pharmacia) and then purified with Sephadex G-50 DNA grade columns (NICK
columns, Pharmacia). Not all cDNA probes were labeled with
[32P]dCTP from the
same batch.
After hybridization with the labeled probe, the membranes were washed once in 1× standard saline-sodium citrate (SSC)-0.1% SDS for 10 min at room temperature, once in 1× SSC-0.1% SDS for 30 min at 42°C, and once in 0.1× SSC-0.1% SDS for 30 min at 42°C. The membranes were then air-dried, sealed in airtight bags, and exposed to a storage phosphor screen for 24-48 h at room temperature. To standardize the amount of total RNA loaded onto each lane, the blot was stripped by washing in 0.01× SSC-0.5% SDS at 90°C for 30 min to 2 h and was reprobed with a 32P-labeled cDNA probe for 18S rRNA. The relative levels of SP-A, SP-B, and SP-C mRNA were quantified with ImageQuant (Molecular Dynamics, Sunnyvale, CA) and are expressed as a ratio of the level of 18S rRNA. The molecular sizes of the transcripts were determined with a 0.24- to 9.5-kb RNA ladder (GIBCO BRL).
Slot blot analysis. The levels of SP-A and SP-C mRNA in lung tissue collected from fetuses exposed to 7 days of lung liquid drainage and age-matched control fetuses were determined by slot blot analysis. For each tissue sample, 5 and 10 µg of total RNA were separately made up in 100 µl of sterile diethyl pyrocarbonate-treated water and then to a final volume of 400 µl with a 1:1 mixture of 37% formaldehyde and 20× SSC. The 400-µl samples were loaded into individual wells of the slot blot apparatus (Hoefer) and transferred onto a nylon membrane (Duralon, Stratagene) by vacuum. Each well was washed with 400 µl of 10× SSC before the membrane was removed and washed for 10 min in 10× SSC. The membrane was air-dried, and the RNA was cross-linked to the membrane as described in Northern blot analysis. The membrane was prehybridized and then hybridized with 32P-labeled SP-A and SP-C cDNA probes as described in Northern blot analysis.
Western blot analysis. Approximately 200-400 mg of lung tissue were homogenized (Ultra Tarrax T25, Janke and Kunkel) in 0.9% NaCl (1 ml/100 mg tissue) with phenylmethylsulfonyl fluoride (1 mM) and a protease inhibitor cocktail (5 µl/ml saline; Sigma). The homogenate was sonicated for 30 s and cooled on ice for 1 min. This process was repeated three times before the samples were centrifuged at 3,000 rpm for 20 min. The supernatant was removed, and the pellet was resuspended in 500 µl of saline. The total protein content of each pellet was determined with a standard protein assay (Bio-Rad). According to the protein concentration determined for each sample, 15 µg of total protein were dissolved in the sample buffer (35 mM Tris · HCl, 30% glycerol, 10% SDS, 0.6 mM dithiothreitol, and 0.012% bromphenol blue) used for SDS-PAGE, then denatured by boiling for 5 min and placing on ice for 5 min. The protein within each sample was separated by SDS-PAGE with a mini-electrophoresis apparatus (Hoefer Mighty Small II).
After separation by electrophoresis, the proteins were transferred to a nitrocellulose membrane (Protran BA-83 nitrocellulose membrane, Schleicher & Schuell) by electrotransfer (2 h at 90 V), which was confirmed by staining the blot with Ponceau S (0.5% in 0.02% glacial acetic acid). The membrane was then incubated overnight at 4°C in blocking buffer (1× PBS, 0.05% Triton X-100, 0.02% sodium azide, and 3% skim milk powder) to reduce nonspecific binding. The blot was incubated with a rabbit anti-sheep SP-A primary antibody (kindly donated by Dr. S. Hawgood, Cardiovascular Research Institute, San Francisco, CA) diluted 1:400 in blocking buffer for 1 h at room temperature. The blot was then washed four times (5 min each), with fresh blocking buffer each time. The blot was incubated with a secondary antibody (goat anti-rabbit, diluted 1:10,000 in blocking buffer; Boehringer Mannheim) conjugated to horseradish peroxidase for 45 min at room temperature. The blot was rinsed four times (once for 5 min, once for 15 min, and twice for 5 min each) in wash buffer (1× PBS and 0.05% Triton X-100). Enhanced chemiluminescence reagents (1:1 volume; Amersham) were added to the blot for 1 min. The blot was then exposed to film for between 1 min and 2 h depending on the strength of the signal. The density of the SP-A protein bands were analyzed with an image-analysis system.
Statistical analysis. All data are presented as means ± SE. In all of the Northern and Western blots presented, each lane contained samples from different fetuses. The total integrated density of each SP-A, SP-B (the density of the two SP-B transcripts were summed), or SP-C transcript was divided by the total integrated density of the 18S rRNA band for each lane. This gave a corrected density for each band that accounted for minor RNA loading differences between lanes. Comparisons were only made between samples from control and trachea-obstructed fetuses run in the same Northern or Western blot and, therefore, were subjected to the same hybridization conditions and exposure times. Statistical analyses were performed with Student's unpaired t-test with a computerized statistics package. The accepted level of significance for all statistical analysis was P < 0.05. SP-A, SP-B, and SP-C mRNA levels are presented as a ratio of the optical density of the 18S rRNA band and, therefore, have no units.
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RESULTS |
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Transcript size. Single mRNA transcripts were detected for both SP-C and SP-A, whereas two transcripts were identified for SP-B. The sizes of the transcripts were ~0.84 and 2.0 kb for SP-C and SP-A, respectively. The densities of both SP-B transcripts were found to vary between fetuses in an identical manner, and, therefore, the densities of both transcripts were summed for each fetus.
SP-A mRNA levels. At 2 days of
tracheal obstruction, the SP-A mRNA level in lung tissue was reduced
from a mean value of 30.2 ± 15.9 in control fetuses to 5.5 ± 1.2 (Fig. 1). Similarly, the levels of mRNA
encoding SP-A in lung tissue were reduced from mean values of 26.1 ± 5.6 and 71.8 ± 11.9 in control fetuses to 2.7 ± 1.0 and
24.2 ± 5.9 at 4 and 10 days, respectively, of tracheal obstruction
(Fig. 1). Expressed as a percentage of the control values, SP-A mRNA
levels were reduced to 18.0 ± 9.0, 10.3 ± 5.2, and
33.7 ± 16.8% at 2, 4, and 10 days, respectively, of tracheal obstruction (Fig. 2).
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Seven days of lung liquid drainage did not significantly alter the SP-A
mRNA levels in fetal lung tissue; SP-A mRNA levels were 0.24 ± 0.07 in control fetuses and 0.23 ± 0.06 in fetuses subjected to 7 days
of lung liquid drainage (Fig.
3).
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SP-B mRNA levels. At 2 days of
tracheal obstruction, the fetal lung SP-B mRNA level was reduced from a
mean control value of 117.2 ± 22.7 to 27.7 ± 1.5 (Fig.
4). The SP-B mRNA level in fetal lung
tissue was reduced from a mean control value of 15.8 ± 1.2 to 2.0 ± 0.4 at 10 days of tracheal obstruction (Fig. 4). Expressed as a
percentage of the control values, the SP-B mRNA levels in fetal lung
tissue were reduced to 23.6 ± 1.3 and 12.6 ± 2.3% at
2 and 10 days, respectively, of tracheal obstruction (Fig. 2).
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SP-C mRNA levels. At 2 days of
tracheal obstruction, the fetal lung SP-C mRNA level was reduced from a
mean control value of 10.2 ± 2.1 to 2.7 ± 1.9 (Fig.
5). Similarly, SP-C mRNA levels in fetal
lung tissue were reduced from mean control values of 153.5 ± 39.8 and 3.1 ± 0.4 to 11.7 ± 7.1 and 0.4 ± 0.2 after 4 and 10 days, respectively, of tracheal obstruction (Fig. 5). Expressed as a
percentage of the control values, the SP-C mRNA levels in fetal lung
tissue were reduced to 26.1 ± 13, 7.6 ± 3.4, and 14.4 ± 7.2% at 2, 4, and 10 days, respectively, of tracheal obstruction (Fig. 2).
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In contrast to tracheal obstruction, 7 days of lung liquid drainage significantly increased the SP-C mRNA level in fetal lung tissue from a mean control value of 0.39 ± 0.10 to 0.89 ± 0.13 (Fig. 3).
SP-A protein levels. Using Western
blot analysis, we detected four bands of immunoactivity, the densities
of which were summed (Fig. 6).
The protein level for SP-A in control fetuses [34.8 ± 7.8 density units (DU)] was similar to that in fetuses exposed to 2 days of tracheal obstruction (35.3 ± 0.8 DU). At 4 days of tracheal
obstruction, the SP-A level (23.8 ± 4.4 DU) tended to be reduced
compared with that in age-matched control fetuses (39.6 ± 7.6 DU),
although this reduction just failed to reach significance. In contrast,
at 10 days of tracheal obstruction, the protein level for SP-A in the
fetal lung was reduced from 41.2 ± 13.3 DU to almost undetectable
levels (6.6 ± 1.5 DU; Fig. 6). Thus SP-A levels were reduced to
51.1 ± 11.4 and 15.4 ± 4.1% of control values at 4 and 10 days, respectively, of tracheal obstruction (Fig. 2).
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DISCUSSION |
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The results of this study demonstrate that tracheal obstruction causes a rapid (within 2 days) and large reduction (to ~20% of control values) in the mRNA levels encoding SP-A, SP-B, and SP-C in fetal lung tissue. Furthermore, the reduction in SP-A, SP-B, and SP-C mRNA levels induced by tracheal obstruction was similar in magnitude and appeared to follow a similar time course (see Fig. 2), indicating that transcription of the genes encoding these proteins may be controlled by similar mechanisms in response to increases in fetal lung expansion. Despite the rapidity of the gene response to increases in lung expansion, SP-A protein levels were not reduced at 2 days, tended to be reduced at 4 days, and were reduced by 85% at 10 days of tracheal obstruction. On the other hand, 7 days of lung deflation induced by lung liquid drainage caused a large increase in SP-C mRNA levels but had no effect on SP-A mRNA levels. Thus it appears that gene expression, at least for SP-C, is closely and inversely related to the degree of fetal lung expansion. Furthermore, our results indicate that there is an ~4-day time lag between a reduction in SP-A gene expression and a detectable reduction in mature SP-A protein levels after tracheal obstruction.
Our results confirm and extend the previous finding that tracheal occlusion reduces fetal lung SP-C mRNA levels (23). In that previous study, it was suggested that tracheal obstruction had a "deleterious" effect on pulmonary epithelial type II cells and that the reduction in fetal lung SP-C mRNA levels resulted from a reduction in type II cell number (23). The findings of our study demonstrate that the reduction in fetal lung SP-A, SP-B, and SP-C mRNA levels induced by tracheal obstruction occurs within the first 2 days, which is well before we have been able to detect a change in the proportion of type II AECs by electron microscopy (identified by the presence of lamellar bodies; Nardo and Hooper, unpublished observations). Thus the effect of increased lung expansion on SP mRNA levels may initially be due to a direct effect of stretch on type II AECs leading to a decrease in gene transcription or, perhaps, to a decrease in mRNA stability. After prolonged periods of tracheal obstruction, however, the reduction in SP-C mRNA levels may eventually result from a reduction in the number of type II AECs as first described by Alcorn et al. (1). Whatever the mechanism, it is likely that SP-C is not a good cellular marker for type II AECs during the rapid lung growth phase induced by tracheal obstruction.
In the present study, we found that SP-A, SP-B, and SP-C mRNA levels were reduced at 2 days of tracheal obstruction and remained reduced at 4 and 10 days. Hooper et al. (11) and Nardo et al. (21) previously demonstrated that, after tracheal obstruction, fetal lung DNA synthesis rates increase to a maximum at 2 days, are reduced but still elevated at 4 days, and return to control levels by day 10. Consequently, the reduction in SP-A, SP-B, and SP-C expression in fetal lung tissue appears to be unrelated to the growth response (in terms of DNA and protein) of the fetal lung to an increase in lung expansion induced by tracheal obstruction. That is, mRNA levels for all three proteins in fetal lung tissue were low on day 2 when DNA synthesis rates were greatly elevated and remained at low levels during and after the time that DNA synthesis rates had decreased to control values (21).
We detected three bands of immunoactivity for SP-A in the ovine fetal lung, which is similar to that previously observed in the developing rat lung (25). Fetal lung SP-A protein levels were not reduced at 2 days of tracheal obstruction but tended to be reduced at 4 days of tracheal obstruction. Thus the turnover of SP-A in fetal lung tissue is relatively slow at this stage of gestation because it took up to 4 days before a reduction in gene expression was translated into a detectable reduction in mature protein content. After 10 days, however, the levels of the mature SP-A protein were reduced by 85% and were almost undetectable. This is consistent with the previous finding of Joe et al. (13), who showed, using immunohistochemistry, that prolonged periods (~30 days) of tracheal obstruction cause a reduction in SP-A-positive cells. It is interesting that these authors also observed that SP-A protein levels were increased after transection of the fetal spinal cord. This procedure was designed to inhibit fetal breathing movements, but it also causes a reduction in the volume of fetal lung liquid (7). Thus the increase in fetal lung SP-A levels induced by spinal cord transection (13) may have been due to a decrease in fetal lung expansion. In the present study, we found that SP-C mRNA levels increased in response to lung deflation, whereas SP-A mRNA levels were not altered. However, the period of lung deflation was only 7 days in our study, whereas in the study of Joe et al. (13), the period of lung deflation associated with fetal spinal cord transection was >22 days. Thus it is possible that if the period of fetal lung deflation in the present study had persisted longer, an increase in SP-A mRNA levels may have been observed. A possible explanation for the finding that 7 days of lung liquid drainage caused an increase in SP-C, but not in SP-A, mRNA levels is that SP-C is type II AEC specific, whereas SP-A is not. That is, the type II AECs may be more sensitive to reductions in lung expansion than the other cell types within the lung that produce SP-A. Furthermore, it is possible that the amount of SP-C mRNA per type II AEC varies widely due to variability in the degree of stretch experienced by individual cells. Indeed, the degree of stretch, or lack thereof, experienced by individual cells is likely to differ markedly depending on their location within the structural framework of the lung.
The mechanisms by which alterations in fetal lung expansion affect
SP-A, SP-B, and SP-C mRNA levels are unknown. It is possible that
increases in fetal lung expansion directly affect gene expression in
type II AECs due to activation of extracellular matrix receptors resulting from distortion of the extracellular matrix. Indeed, increases in fetal lung expansion increase insulin-like growth factor
II (IGF-II) mRNA levels in the fetal lung. In contrast, reductions in
fetal lung liquid volumes, whether the reduction results from lung
liquid drainage (11) or spinal cord transection (7), reduce fetal lung
IGF-II mRNA levels. These studies indicate that IGF-II expression in
fetal lung is directly regulated by the degree of lung expansion.
Similarly, stretch of fetal lung cells in culture has been shown to
activate platelet-derived growth factor expression via activation of
protein kinase C, which is considered to mediate the strain-induced
increase in DNA synthesis in vitro (15, 16). It is also possible that
the effect of alterations in fetal lung expansion on SP-A and SP-C mRNA
levels is mediated via an alteration in the synthesis and/or
release of growth factors that act locally on type II AECs.
Transforming growth factor-1 has been shown to reduce mRNA levels
for SP-A (26, 27) and SP-C (17, 29) in pulmonary epithelial cells in
culture. Conversely, epidermal growth factor has been shown to
increase SP mRNA levels (4, 24). Whatever the mechanism, an important
observation from this study is that all three SPs are inhibited to the
same degree and over the same time frame after tracheal obstruction.
This most probably indicates that the inhibitory mechanism is common to
all three SPs. It should also be noted that Clara cells of the
bronchiolar epithelium also express SP-A and SP-B and, therefore,
contribute to total lung mRNA levels for these proteins (8). Thus it is
possible that alterations in lung expansion affect SP expression in
these cells in an identical manner as that observed in type II AECs.
Tracheal obstruction is a potent and rapid stimulus for fetal lung growth (11, 21), and, therefore, it has been suggested that this procedure may be used therapeutically to reverse lung growth deficits in human fetuses with lung hypoplasia. Indeed, only 6 days of tracheal obstruction are required to reverse a severe lung growth deficit in fetal sheep (19). However, the finding that tracheal obstruction decreases SP-A, SP-B, and SP-C mRNA levels indicates that this procedure may have some detrimental effects on the ability of the lung to function postnatally. Indeed, although hypoplastic fetal lungs are considered to have a greater proportion of type II AECs than control lungs (1), if increases in fetal lung expansion switches off gene expression for SP-A, SP-B, and SP-C directly, even a brief exposure of these lungs to increases in lung expansion could reduce SP levels. However, the timing may be the critical factor because the significant time lag between the decrease in SP gene expression and the decrease in protein levels may provide a window of opportunity to stimulate lung growth without greatly reducing SP content. Consequently, more research is required to assess the relative impact of increases in lung expansion on the pulmonary surfactant system. For instance, total phospholipid, phosphatidylcholine, and disaturated phosphatidylcholine contents of lung tissue are not affected by prolonged alterations in fetal lung expansion (18). This may indicate that alterations in fetal lung expansion affect the synthesis of SPs without altering the synthesis of the phospholipids. In any event, our data indicate that increases in fetal lung expansion could reduce the biological activity of surfactant because SPs are essential for the biological function of surfactant.
Our study demonstrates that increases in fetal lung expansion cause large reductions in fetal lung SP-A, SP-B, and SP-C mRNA levels within 2 days of obstructing the fetal trachea and that this reduction persists for up to 10 days. On the other hand, 7 days of lung deflation caused by draining the lungs of liquid induces a large increase in fetal lung SP-C mRNA levels. This indicates that at least SP-C mRNA levels are inversely related to the degree of fetal lung expansion. Furthermore, our data also indicate that if this procedure is to be used therapeutically in humans, it may be important to determine the time required for SPs to be reexpressed after a period of increased fetal lung expansion.
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ACKNOWLEDGEMENTS |
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We are indebted to Alison Thiel for expert technical assistance and to Dr. S. Hawgood (Cardiovascular Research Institute, San Francisco, CA) for the kind donation of the surfactant protein A primary antibody.
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FOOTNOTES |
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This work was supported by the National Health and Medical Research Council of Australia and the Medical Research Council of Canada (F. Possmayer).
Address reprint requests to A. Lines.
Received 1 October 1997; accepted in final form 15 October 1998.
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