Congenital diaphragmatic hernia, tracheal occlusion, thyroid transcription factor-1, and fetal pulmonary epithelial maturation

Cheryl J. Chapin,1 Robert Ertsey,1 Jyoji Yoshizawa,1,2 Akihiko Hara,1,2 Lourenco Sbragia,1,2 John J. Greer,4 and Joseph A. Kitterman1,3

1Cardiovascular Research Institute and Departments of 2Surgery and 3Pediatrics, University of California, San Francisco, California; and 4Department of Physiology, Perinatal Research Center, University of Alberta, Edmonton, Canada

Submitted 15 September 2004 ; accepted in final form 10 March 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Congenital diaphragmatic hernia (CDH) occurs in ~1:2,500 human births and has high morbidity and mortality rates, primarily due to pulmonary hypoplasia and pulmonary hypertension. Tracheal occlusion (TO), in experimental animals, distends lungs and increases lung growth and alveolar type I cell maturation but decreases surfactant components and reduces alveolar type II cell density. We examined effects of CDH and CDH+TO on lung growth and maturation in fetal rats. To induce CDH, we administered nitrofen (100 mg) to dams at 9.5 days of gestation. We compared lungs from fetuses with CDH, CDH+TO, and those exposed to nitrofen without CDH. CDH decreased lung wet weight bilaterally (P < 0.0001) and DNA content in lung ipsilateral to CDH (P < 0.05). CDH+TO significantly increased lung wet weights bilaterally; DNA content was intermediate between CDH and NC. To evaluate effects on the distal pulmonary epithelium, we examined surfactant mRNA and protein levels, type I and II cell-specific markers (RTI40 and RTII70, respectively), and transcriptional regulator thyroid transcription factor-1 (TTF-1). Decreased lung distension (due to CDH) increased SP-C mRNA and TTF-1 protein expression and reduced RTI40 (P < 0.05 for all). Increased lung distension (due to CDH+TO) reduced expression of SP mRNAs and pro-SP-C and TTF-1 proteins and enhanced expression of RTI40 (mRNA and protein; P < 0.05 for all). We conclude that CDH+TO partially reverses effects of CDH; it corrects the pulmonary hypoplasia and restores type I cell differentiation but adversely affects SP expression in type II cells. These effects may be mediated through changes in TTF-1 expression.

fetal lung development; pulmonary surfactant; alveolar type I and type II cells


FETAL LUNG GROWTH IS DEPENDENT on mechanical factors including a positive transpulmonary pressure, fetal breathing movements of normal incidence and intensity, sufficient amniotic fluid volume, and adequate intrathoracic space (22, 33, 45). Congenital diaphragmatic hernia (CDH) in human infants results in high rates of morbidity and mortality, mainly due to pulmonary hypoplasia and pulmonary hypertension (21, 29). CDH affects at least two mechanical factors that determine lung growth: abdominal viscera enter the thorax, thus limiting space available for lung growth, and the diaphragmatic defect impairs fetal breathing movements (31). In experimental animals, fetal tracheal occlusion (TO) prevents egress of fetal lung liquid, distends the lungs, and accelerates lung growth, both in the presence and absence of CDH (4, 8, 13, 25, 31, 32, 42, 46, 55). The timing and duration of TO influence its effects on the fetal lung. In fetal sheep with diaphragmatic hernia (DH), long-term TO accelerates lung growth and corrects some of the pulmonary vascular anomalies associated with DH (8, 26, 41) but adversely affects surfactant production (4, 8) and decreases the number of type II alveolar epithelial cells (5). Conversely, with short-duration TO, lung growth is not accelerated, yet postnatal lung function is improved, pulmonary vascular anomalies are corrected, and type II cell numbers are preserved (41, 46, 53). In addition, Wu and associates (54) showed an inverse relationship between the duration of TO and type II cell density in late gestation fetal rabbits with DH.

The rodent model of nitrofen-induced CDH is being increasingly used to study the effects of CDH on lung development (35). Because the diaphragmatic defect is present in early gestation, this model system more closely mimics human CDH than the surgically produced models in fetal sheep and rabbits. Kitano and associates (31) showed that TO causes increases in lung weight, DNA, and protein content and in lung volume and surface area in fetuses with nitrofen-induced CDH. Kanai and associates (26) showed that TO of CDH fetuses reversed the increases in pulmonary arterial medial and adventitial thickness, which is associated with CDH. Although these studies show that TO tends to correct the pulmonary parenchymal and vascular abnormalities associated with CDH, relatively little is known about how TO affects pulmonary epithelial maturation in rat fetuses with CDH.

Numerous factors participate in the control of lung development and maturation (reviewed in Ref. 36). Of particular interest is the homeobox-containing transcription factor, thyroid transcription factor-1 (TTF-1). Early in fetal development, TTF-1 regulates branching morphogenesis and proximal distal patterning in the lung (43). Later in gestation and postnatally, TTF-1 directs expression of the surfactant proteins (SP), the Clara cell-specific protein CC10 (52), and differentiation of type I cells (49). Thus alterations in TTF-1 during gestation affect lung maturation, both architecturally and biochemically. In the extreme case of the TTF-1 knockout mouse, lung branching morphogenesis is halted at the formation of the mainstem bronchi and a proximal phenotype, epithelium with tracheobronchiolar characteristics, is maintained (56). As noted above, increased distension of the fetal lung due to TO and decreased distension (e.g., due to CDH) have opposite effects on pulmonary epithelial development. Because TTF-1 participates in development of both type I and type II alveolar epithelial cells, we hypothesize that changes in lung distension may influence pulmonary epithelial maturation through effects on TTF-1.

In the current study, we have examined the effects of CDH and of short-duration TO in CDH in late gestation on indicators of lung growth, type I and type II cell maturation, and on TTF-1. Because nitrofen has primary effects on the lung, we have used nitrofen-exposed littermates as controls (12, 20, 27).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. All studies were approved by the Committee on Animal Research of the University of California, San Francisco, and all procedures conformed with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Timed-pregnant Sprague-Dawley (SD) rats were obtained from Charles River Laboratories (Hollister, CA) and were provided feed and water ad libitum. Dams were housed in a laminar flow facility that maintained ambient temperature at 21°C.

CDH and TO. On day 9.5 of gestation (presence of a vaginal plug indicated day 1), timed-pregnant SD rats were gavage-fed 100 mg of Nitrofen (2,4-dichloro-4'-nitrodiphenyl ether; Wako Chemicals, Osaka, Japan) dissolved in 1 ml of olive oil to produce CDH in the fetuses. On day 20 of gestation, dams and fetuses were anesthetized with an intramuscular injection of ketamine (180 mg/kg) and xylazine (2.5 mg/kg). Through a maternal midline laparotomy, TO was performed on fetuses as previously described (55). In brief, a small section of one uterine horn was exposed. Near the head of a fetus, a purse-string suture was placed in the uterus, and the fetal head and neck were exteriorized through a small hysterotomy. The neck was maintained in an extended position with wet gauze. With the use of an operating stereomicroscope (Leica Wild M691, Leica Microsystems), the trachea was exposed through a midline cervical incision. The trachea was cauterized using a handheld cautery unit equipped with a microtip (Roboz Surgical, Rockville, MD). The fetuses were then returned to the uterus, and the maternal abdomen was closed in two layers. Four to six fetuses per dam received TO by microcautery. On day 22 (term), the dam and fetuses were again anesthetized with an intramuscular injection of ketamine (180 mg/kg) and xylazine (2.5 mg/kg). The fetuses were delivered and weighed, the abdominal cavity was opened, and the presence or absence of CDH was determined by visual inspection of the diaphragm. The lungs were then removed, split into left and right lung, weighed, either snap-frozen or fixed, and stored at –80°C for future study. After all fetuses had been removed, dams were killed by intracardiac injection of pentobarbital (1 ml, 390 mg/ml solution, Euthanasia Solution; Schering-Plough Animal Health, Union, NJ) followed by production of bilateral pneumothoraces. Lungs from fetal animals were separated into three groups: CDH (animals with left-sided CDH), NC (animals exposed to nitrofen without CDH), and CDH+TO (animals with left-sided CDH and TO). Except for analysis of TTF-1 (see below), only fetuses with left-sided CDH were used for analysis. We studied a total of 266 fetuses from 54 pregnant dams.

Biochemical studies. To quantify protein and DNA content, frozen lungs were dounce homogenized in 500 µl of 50 mM NaHCO3 containing protease inhibitor cocktail (Sigma, St. Louis, MO). Protein content was measured using the bicinchoninic acid (BCA) modification of the Lowry method (Sigma), and DNA content was measured using the fluorometric method of Setaro and Morley as previously described (55).

RNA isolation and analysis. Total RNA was isolated from whole lung by the RNAzol method (Tel-Test, Friendswood, TX) as previously described (34). Multiplex RT-PCR was used for analysis of RTI40 [an apical membrane protein specific to type I cells in the lung (17)], SP-A, SP-B, or SP-C mRNA in combination with 18S rRNA levels in fetal lung utilizing Competimer technology (Ambion, Austin, TX) as previously described (55). Briefly, Total RNA was reverse transcribed using random primers. cDNA was amplified with specific oligonucleotide primers for RTI40, SP-A, SP-B, or SP-C and 18S (QuantumRNA 18S Internal Standards, Ambion). Radio-labeled PCR products were produced using direct incorporation of [{alpha}-32P]dCTP (3,000 Ci/mmol; NEN, Boston, MA) during amplification. PCR products were resolved on 6% polyacrylamide gels containing 8 M urea. After the gel was dried, PCR products were quantified using the Storm 840 phosphorimager and Imagequant software (Molecular Dynamics).

Quantification of proteins RTI40, RTII70, SP-A, SP-B, and pro-SP-C. Protein was extracted from whole fetal lung, and Western dot blots were performed as previously described (34). Briefly, serial dilutions (4, 2, 1, and 0.5 µg) of total protein diluted in 1% MEGA-8 (Calbiochem-Novabiochem, La Jolla, CA)/4.5 M urea/25 mM NaHCO3, pH 9.0 (RTI40, RTII70, SP-A, and SP-B), or 20 mM sodium acetate/20 mM 1-O-Octyl-{beta}-D-glucopyranoside, pH 4.6 (SP-C), were loaded in duplicate onto nitrocellulose membranes. Blots were blocked and incubated with monoclonal antibodies to RTI40 or RTII70 [apical membrane proteins specific to type I and type II pneumocytes, respectively (16, 18, 19)] or polyclonal antibodies to SP-A, SP-B (a kind gift of S. Hawgood), or pro-SP-C (21 kDa; Chemicon International, Temecula, CA). All of the antibodies used have been tested by Western blot and detect bands of the appropriate molecular weights for each protein. After extensive washing, blots were incubated for 30 min at room temperature in horseradish peroxidase-conjugated species and isotype-specific secondary antibodies and washed again. Blots were developed using ECL+ (Amersham Biosciences, Piscataway, NJ), visualized by autoradiography, and quantified using the Storm 840 phosphorimager equipped with a blue fluorescence/chemifluorescence detector and Imagequant software (Molecular Dynamics, Sunnyvale, CA).

Immunohistochemistry. Fetal rat lungs were immersion fixed in 4% paraformaldehyde for 24 h and processed for cryosectioning. Immunohistochemistry was performed on 3-µm sections using primary antibodies to RTI40 and RTII70, as indicated above, and were detected using indirect immunofluoresence, as previously described (34). Briefly, sections were incubated with both primary antibodies simultaneously and extensively washed before incubation with isotype-specific secondary antibodies, fluorescein-conjugated goat anti-mouse IgM (Cappel, Aurora, OH) for RTI40, and biotinylated goat anti-mouse IgG3 (Santa Cruz Biotechnology, Santa Cruz, CA) followed by Texas red-conjugated streptavidin (Zymed, South San Francisco, CA) for RTII70. Sections were mounted with Prolong antifade kit (Molecular Probes, Eugene, OR), and images were acquired using a Leica TCS SP confocal microscope (Leica Microsystems). To detect TTF-1 in the lung, immunohistochemistry was performed using primary antibody to rat TTF-1 (Clone 8G7G3/1; Dako, Carpenteria, CA). TTF-1 was detected in sections pretreated with target retrieval solution (Dako) for 10 min at 95°C followed by incubation in 1% H2O2 for 10 min to block endogenous peroxidase activity. To control for nonspecific staining, sections were also processed with omission of the primary antibody. Sections were incubated in biotinylated secondary antibody and then processed using Vectastain Elite ABC kit followed by detection with 3,3'-diaminobenzidine substrate kit for peroxidase (both from Vector, Burlingame, CA) and counterstained with Gill's No. 2 hematoxylin (Sigma). Sections were mounted with Glycergel (Dako), and images were captured using a Leitz Orthoplan 2 microscope (Leica Microsystems).

TTF-1 analysis. Because of the limited number of fetuses available, we used fetuses with either left- or right-sided CDH for the TTF-1 studies. Whole frozen lungs were dounce homogenized on ice in 1 ml of cold nuclear extract buffer B (7), and the extract was transferred to a sterile microfuge tube and ultrasonicated at 30% power for 30 s on ice. Insoluble material was pelleted by microcentrifugation at 16,000 g for 30 s, and the supernatant was assayed for protein concentration using the BCA method. Extracts (20 and 10 µg) were electrophoresed under reducing conditions through discontinuous 4% stacking/15% polyacrylamide gels. Prestained molecular weight markers (Rainbow, Amersham Biosciences) and adult lung nuclear extract were simultaneously electrophoresed with the samples. Proteins were electrophoretically transferred onto 0.45-µm nitrocellulose membranes (PROTRAN; Schleicher & Schuell, Keene, NH). Efficiency of transfer was determined by Coomassie blue staining. Membranes were washed twice with 20 mM Tris-buffered saline (TBS), pH 7.4, and placed in 5% nonfat dry milk/0.4% fish gelatin (Amersham Biosciences)/0.3% bovine serum albumin/TBS for 60 min at room temperature or overnight at 4°C. Blocked membranes were then washed once in TBS and incubated in monoclonal anti-rat TTF-1 antibody (Clone 8G7G3/1, Dako) at a dilution of 1:200 for 60 min at room temperature with rocking. Membranes were washed six times over a 60-min period with TBS containing 0.05% Tween 20 (TBS-T) and then incubated for 30 min at room temperature in biotinylated anti-mouse Ig (Pharmingen, San Diego, CA) at a dilution of 1:2,000 followed by extensive washing with TBS-T. Membranes were then incubated in streptavidin-horseradish peroxidase conjugate (Amersham Biosciences) at a dilution of 1:10,000 for 30 min at room temperature and washed before development with ECL+ (Amersham Biosciences). Blots were visualized by autoradiography and quantified using the Storm 840 phosphorimager equipped with a blue fluorescence/chemifluorescence detector and Imagequant software (Molecular Dynamics).

Statistical analyses. Values for the three experimental groups (CDH, NC, and CDH+TO) were compared by ANOVA using Fisher's protected least significant differences or Bonferroni/Dunn's post hoc test where appropriate. Because we compared results only in littermates, we used Student's paired t-test for RTI40, SP-A, SP-B, and SP-C mRNA and RTI40, RTII70, SP-A, SP-B, SP-C, and TTF-1 protein analyses. Data are presented as percents ± SD, unless otherwise stated. All statistical analyses were done using Statview (Abacus Concepts, Berkeley, CA). P < 0.05 were considered significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Incidence of CDH. CDH occurred in 31.3% of the nitrofen-exposed fetuses. Of those, left-sided CDH occurred in 57.5%, right-sided defects occurred in 30.6%, and bilateral hernias occurred in 11.9%. In nitrofen-exposed fetuses with TO, there was a 49.3% survival rate, with 25% of these animals having a diaphragmatic defect. We studied 189 NC fetuses, 62 fetuses with left-sided CDH, and 15 left-sided CDH+TO fetuses from 54 pregnant dams.

Body and lung weights, DNA, and protein content. Both CDH and CDH+TO had effects on growth of the fetal body and lungs (Table 1). Fetal body weights were slightly, but significantly, reduced in fetuses with CDH or CDH+TO compared with NC, whereas body weights from CDH and CDH+TO fetuses did not differ significantly from one another. Both ipsilateral (i.e., left) and contralateral (i.e., right) lung weights were significantly decreased by CDH and increased by CDH+TO, both in absolute terms and expressed as a percent of body weight (Table 1 and Fig. 1). CDH retarded growth of the ipsilateral lung (67% of NC) more than the contralateral lung (85% of NC). In contrast, CDH+TO increased lung weight more in the contralateral lung (156% of NC) than the ipsilateral lung (130% of NC). CDH significantly reduced DNA in the ipsilateral lung to 72% of the NC value, and CDH+TO tended to increase DNA in the ipsilateral lung (to 85% of NC; Table 2). Neither CDH nor CDH+TO affected DNA content of the contralateral lung or protein content of either lung. Protein to DNA ratios did not differ between experimental groups in both lungs (data not shown).


View this table:
[in this window]
[in a new window]
 
Table 1. Effects of CDH and subsequent TO on body weight and lung weight in fetal rats

 


View larger version (40K):
[in this window]
[in a new window]
 
Fig. 1. Effects of congenital diaphragmatic hernia (CDH) and CDH + tracheal occlusion (TO) on fetal lung size. Fetuses were delivered, the diaphragmatic defect was photographed, and the diaphragm and rib cage were removed to facilitate visualization of the lungs. A: left-sided CDH. B: lungs of fetus with left-sided CDH. C: lungs of fetus with left-sided CDH and TO at embryonic day 20. Arrow in A points to large diaphragmatic defect on the left side. The lungs are outlined for clarity. The front of the animal is shown. h in B and C indicates the position of the heart. Note that the lungs of the CDH+TO fetus are much larger than those of the fetus with CDH alone.

 

View this table:
[in this window]
[in a new window]
 
Table 2. Effects of CDH and subsequent TO on DNA and protein content in lungs of fetal rats

 
mRNA expression of type I and II cell markers. To examine more closely the effects of nitrofen-induced CDH and subsequent TO on distal epithelial cell differentiation and maturation, we measured mRNA expression of RTI40, an indicator of the type I cell phenotype, and of SP-A, SP-B, and SP-C, indicators of the type II cell phenotype, normalized them to 18S ribosomal RNA using multiplex RT-PCR, and expressed the values as percent of the same side lung of littermate fetuses. To examine the effects of CDH and CDH+TO on type I and type II cell markers in an individual lung, all genes were measured from the same RNA isolation for each lung in each animal.

Ipsilateral lung. As shown in Fig. 2, A and B, CDH did not affect expression of RTI40, SP-A, or SP-B, but significantly increased SP-C to 126% of NC (P < 0.05). CDH+TO significantly increased expression of RTI40 to 149% of NC (P < 0.05) and 168% of CDH (P < 0.01). In contrast to CDH, CDH+TO significantly reduced expression of SP-A, SP-B, and SP-C. SP-A was reduced to 58% of NC and 60% of CDH (P < 0.05 for both), SP-B was reduced to 68% of NC (P < 0.01), and SP-C was reduced to 62% of NC and 50% of CDH (P < 0.0001 and 0.01, respectively).



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 2. Effects of CDH and CDH+TO on mRNA expression of type I (RTI40) and type II cell markers [surfactant protein (SP)-A, SP-B, and SP-C]. A: representative phosphor image of RTI40, SP-A, SP-B, SP-C, and 18S PCR products, in duplicate, in the ipsilateral lung (contralateral not shown). Graphic representation of RTI40, SP-A, SP-B, and SP-C mRNA expression normalized to 18S rRNA in the ipsilateral (B) and contralateral (C) lungs. CDH significantly increased SP-C expression in the ipsilateral lung, whereas CDH+TO reversed this effect and decreased SP-A, SP-B, and SP-C expression. In addition, RTI40 was increased in these lungs, indicating accelerated differentiation of type I cells. Contralateral lungs showed a similar pattern. CDH:NC, n = 3 pairs; CDH+TO:NC, n = 4 pairs; CDH+TO:CDH, n = 3 pairs. {ddagger}P < 0.0001, {dagger}P < 0.01, *P < 0.05. NC, animals exposed to nitrofen without CDH.

 
Contralateral lung. As shown in Fig. 2C, CDH did not significantly affect the level of mRNA in any of the genes examined. Effects of CDH+TO were similar to those in the ipsilateral lung, but the magnitudes of the effects were less marked.

Protein expression of type I and II cell markers. To evaluate how the changes in mRNA expression translate into changes at the protein level in fetuses with nitrofen-induced CDH and CDH+TO, we performed quantitative Western dots blots for RTI40, SP-A, SP-B, pro-SP-C, and the type II cell-specific apical epithelial marker RTII70. To examine the effects of CDH and CDH+TO on type I and type II cell markers in an individual lung, all proteins were measured from aliquots of the same lung homogenate for each lung in each animal.

Ipsilateral lung. As shown in Fig. 3, A and B, there was a tendency for CDH to reduce RTI40 protein to 75% of NC (P = 0.06). In fetuses with CDH+TO, RTI40 was similar to NC but was increased to 266% of CDH, although the values did not reach significance (P = 0.06). The 21-kDa pro-SP-C showed a slight increase in CDH lungs (145% of NC), whereas lungs of CDH+TO fetuses had somewhat decreased pro-SP-C protein levels compared with NC (69%) and CDH (51%), although these values did not reach statistical significance. Neither CDH nor CDH+TO affected the other type II cell-related proteins, SP-A, SP-B, and RTII70.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 3. Effects of CDH and CDH+TO on protein levels of type I (RTI40) and type II cell markers (RTII70, SP-A, SP-B, and pro-SP-C). A: representative Western dot blot for RTI40 and pro-SP-C in the ipsilateral (ips) and contralateral (con) lung. The other type II cell markers were not affected (blots for ipsilateral and contralateral not shown). Graphic representation of RTI40, RTII70, SP-A, SP-B, and pro-SP-C protein levels in the ipsilateral (B) and contralateral (C) lungs. RTI40 was decreased in CDH lungs both ipsilaterally and contralaterally, whereas pro-SP-C was increased. However, CDH+TO had increased RTI40 levels over CDH alone and was similar to the NC (both ipsilaterally and contralaterally), whereas pro-SP-C was reduced compared with both NC and CDH. CDH:NC, n = 4 pairs; CDH+TO:NC, n = 3 pairs; CDH+TO:CDH, n = 3 pairs. *P < 0.05.

 
Contralateral lung. As shown in Fig. 3, A and C, the results were similar to those in the ipsilateral lung, although the decrease in RTI40 with CDH and the reduction of pro-SP-C with CDH+TO were statistically significant (P < 0.05).

To visualize the differences in RTI40 and RTII70 in situ, we performed immunohistochemistry on cryosections from the ipsilateral (left) lung of CDH, CDH+TO, and left NC lung (Fig. 4). Whereas the type II cells showed a similar pattern of staining for RTII70 (red) in all experimental groups, RTI40 staining (green) of type I cells appeared reduced in the CDH group compared with both NC and CDH+TO and defined relatively small luminal spaces. In the CDH+TO lung, RTI40- positive staining defined enlarged luminal spaces that appeared increased over CDH alone and similar to NC. These findings are consistent with data obtained by biochemical analysis for RTI40 and RTII70 (Fig. 3).



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 4. Immunohistochemical localization of RTI40 and RTII70 in the distal lung of CDH, CDH+TO, and NC fetuses. Confocal micrographs showing RTI40- positive type I cells (green) and RTII70-positive type II cells (red) in fetal lungs at 22 days (term). A: CDH lung. B: CDH+TO lung. C: NC lung. Note the similarity of the CDH+TO lung to the NC lung in staining pattern and intensity. Although the intensity of staining is similar to the other groups, the CDH lung appears quite immature with smaller lumens lined with RTI40-positive cells. L, lumen (arrows point to lumens in A); Bv, blood vessel. Bar = 100 µm.

 
TTF-1 protein in fetal lung. For measurements of TTF-1 protein, we used lungs from fetuses with both right- and left-sided CDH due to the small number of samples available.

Ipsilateral lung. As shown in Fig. 5, A and C, CDH tended to increase TTF-1 protein to 248% of NC, a value that approached statistical significance (P = 0.07). CDH+TO decreased TTF-1 to 39% of CDH (P < 0.05) and normalized TTF-1 to NC values.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 5. Effects of CDH and CDH+TO on thyroid transcription factor (TTF)-1 protein level in fetal lung. Representative Western blots showing TTF-1, 20- and 10-µg dilutions of total protein for each sample, in the ipsilateral (A) and contralateral (B) lungs of CDH, CDH+TO, and NC fetuses. C: graphic representation of relative TTF-1 protein levels in these lungs. CDH increased TTF-1 protein levels >2-fold in both ipsilateral and contralateral lungs. In contrast, TTF-1 was decreased by more than one-half in CDH+TO lungs vs. CDH lungs and was similar to NC (both ipsilaterally and contralaterally). CDH:NC, n = 6 pairs; CDH+TO:NC, n = 5 pairs; CDH+TO:CDH, n = 5 pairs. *P < 0.05.

 
Contralateral lung. As shown in Fig. 5, B and C, CDH increased TTF-1 levels to 227% of NC (P < 0.05). CDH+TO decreased TTF-1 levels to 65% of NC and 39% of CDH (P < 0.05 for both).

Qualitative assessment of TTF-1 expression in situ was done using immunohistochemistry on cryosections from the ipsilateral (left) lung of CDH and CDH+TO and left NC lung (Fig. 6). In all lungs examined, numerous TTF-1-positive cells were seen in airway epithelial and distal pulmonary epithelial cells with variation in the intensity of staining. In CDH lungs, there appeared to be more airway and distal epithelial cells that were positive for TTF-1 and more cells that stained intensely for TTF-1 than in either NC or CDH+TO lungs. TTF-1 staining pattern and intensity were similar in CDH+TO and NC lungs. There was no staining with omission of the primary antibody.



View larger version (64K):
[in this window]
[in a new window]
 
Fig. 6. Immunohistochemical localization of TTF-1 in airway and distal lung epithelium in CDH, CDH+TO, and NC fetuses. Micrographs show nuclear localization of TTF-1 in lung epithelium. A: CDH. B: CDH+TO. C: NC. D: no primary antibody. Note the appearance of numerous positive nuclei in the CDH lung, some of which show more intense staining than the other groups. In addition, note the similarity in staining pattern between CDH+TO and NC lungs. Aw, airway. Bar = 100 µm.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
It is well established that increased distension of the fetal lung accelerates lung growth, and decreased distension retards growth (1). Several studies indicate that lung distension also influences indicators of lung maturation. However, results have not been consistent, probably due to differences in species, experimental interventions, timing, and duration of the interventions and the types of control animals used in the studies. Our aim was to examine effects of decreased fetal lung distension (CDH) and of increased distension (CDH+TO) on indicators of maturation of type I and type II pneumonocytes and on TTF-1, a factor known to participate in several aspects of fetal lung development. To minimize other factors, including nitrofen, which may influence the results, experimental animals were compared only to littermates, and only fetuses with left-sided CDH were included (except for analysis of TTF-1).

Administration of nitrofen to pregnant dams at 9.5 days of gestation produced primarily left-sided DH, consistent with previous observations (11, 35). Body weights for both CDH and CDH+TO fetuses were decreased. Previous reports by others and by us had shown no differences in body weights with CDH and CDH+TO (9, 31). Those studies involved relatively small numbers of animals, whereas the current study includes much larger numbers, a factor that probably accounts for the statistical differences between the studies. It is likely that the decreased fetal weight with CDH+TO was due to the stress of surgery. However, the reasons for the decreased fetal weight with CDH are not apparent and cannot be accounted for by the decreased lung weights.

CDH lungs appeared relatively small, whereas CDH+TO produced visibly larger lungs (Fig. 1), findings reflected in the lung weights (Table 1). On the basis of lung weights and DNA content, CDH retarded lung growth more in the ipsilateral than in the contralateral lung, a finding previously noted in other species (2, 15).

Although it markedly retarded lung growth, CDH had relatively minor effects on most indicators of maturation of type I and type II cells, with the exception of SP-C mRNA and RTI40 protein. In the ipsilateral lungs, CDH increased SP-C mRNA by 26% but did not affect mRNA for other SP or RTI40, a protein specific in lung to the apical membrane of type I cells (Fig. 2). In the contralateral lung, none of these genes showed significant changes in mRNA expression. These results are similar to previous reports that showed little or no change in mRNA for SP-A, SP-B, or SP-C with nitrofen-induced CDH (12, 44, 50, 51). Although CDH tended to increase the 21-kDa pro-SP-C protein in both lungs, it did not affect the concentration of SP-A and SP-B proteins in either lung (Fig. 3), findings consistent with the mRNA data and with the report by Van Tuyl and associates (51). Furthermore, CDH had no effect in either lung on RTII70, a protein specific to the apical membrane of type II cells. In contrast, CDH decreased the type I cell protein, RTI40, a finding that we previously noted in hypoplastic lungs due to oligohydramnios (34). Therefore, based on these data, CDH has little effect on indicators of type II cell maturation, but does decrease expression of the type I cell-associated protein, RTI40.

As previously reported by Kitano and associates (31), CDH+TO increased lung weight (Table 1). In the current study, this effect was greater on the contralateral lung than on the ipsilateral (see RESULTS), a finding not previously described. The reason for this is not known but may relate to the portion of the liver that has herniated into the thorax, impeding distension of the lung ipsilateral to the CDH (37). In the ipsilateral lung, TO of CDH fetuses tended to increase DNA content, but not to the level of NC lungs (Table 2). Previously, Kitano and associates (31) reported that CDH+TO increased lung DNA to control levels. The most likely explanation for the differences between our results and theirs is that they produced TO earlier in gestation and for a longer duration, factors known to influence the effects of TO on lung growth (41, 53). Other possible factors include the method of TO (tracheal cautery in the current study vs. tracheal ligation) and the method of comparison of experimental animals to controls (paired comparisons between littermates in the current study vs. unpaired comparisons of nonlittermates). Several investigators have shown that prolonged TO in normal fetuses and in those with DH, in a variety of species, decreases production of surfactant components (4, 25, 39, 47). Furthermore, TO decreases the density of type II cells (47) and the ratio of type II to type I cells (17). This deleterious effect on type II cells can be lessened by performing TO later in gestation and for a shorter period (13, 14, 42, 46, 54, 55). In the current study, we performed TO relatively late in gestation (20 days) in fetuses with left-sided CDH. Our results were concordant with previous studies in that the mRNAs for SP-A, SP-B, and SP-C were decreased in the ipsilateral CDH+TO lung compared with CDH and/or NC lungs. There was a similar, but less marked, effect in the contralateral lung. In an apparent contrast to the mRNAs, only pro-SP-C protein tended to decrease (P = 0.1) in the ipsilateral lung and was significantly decreased in the contralateral lung (P = 0.03) of CDH+TO animals. Of note is that, despite the decreases in mRNA, CDH+TO did not affect lung concentration of SP-A and SP-B proteins. In addition, the type II cell-specific membrane protein RTII70 was also unchanged (mRNA for RTII70 was not measured as this protein has not been purified or cloned to date). In a previous report (55), we saw similar discrepancies between type II cell-specific mRNA and protein levels and speculated that they were due to entrapment of secreted SP in the fetal lung fluid in animals with TO. These results are similar to those of Kitano and associates (30), who reported that TO of CDH fetuses in the rat impaired SP mRNA expression but had little effect on SP-A and SP-B proteins. Similar findings were reported in normal sheep fetuses with short-term TO by Lines et al. (39).

CDH+TO increased mRNA of RTI40, a protein specific in the lung to type I cells (18) and essential for normal lung development and epithelial differentiation (48). In addition, the concentration of RTI40 protein was increased to >250% of the value in CDH lungs, although this change did not quite reach statistical significance (Fig. 3). We have previously shown that expression of RTI40 correlates closely with the surface area covered by type I cells (34). These biochemical results are supported by the immunohistochemical findings that CDH caused an apparent decrease in RTI40 compared with NC, and CDH+TO caused an apparent increase so that these lungs qualitatively were similar to NC lungs; the RTII70 staining pattern appeared similar in all lungs (Fig. 4).

The expression pattern of TTF-1, an important regulator of proximal-distal patterning in early embryonic development and, in late gestation, a transcriptional regulator of the SP, the Clara cell secretory protein, and type I cell differentiation have been examined in CDH lungs by several investigators with mixed results. Losada et al. (40) showed that TTF-1 is downregulated in the lungs of fetuses exposed to nitrofen independently of the presence of CDH and confirm this in vivo finding with in vitro data showing that nitrofen reduces TTF-1 expression in a time- and dose-dependent manner in cultured H441 cells. In addition, Leinwand et al. (38) showed in whole organ culture that nitrofen-exposed mouse lungs have reduced expression of TTF-1. These studies show that nitrofen has a primary effect on TTF-1 expression in lung and must be considered when making comparisons in expression levels between experimental groups.

Several investigators have examined the effect of pulmonary hypoplasia on TTF-1 in lung. CDH caused no change in total TTF-1 mRNA expression in late gestation mice with nitrofen-induced CDH (10, 12), in TTF-1 protein content in sheep with surgically produced CDH (3), or in distribution of TTF-1 protein assessed by immunohistochemistry in human infants with CDH (23). However, in hypoplastic lungs there appears to be disruption of the normal developmental proximal-distal gradient of TTF-1 expression in which there is decreasing expression in proximal airway epithelium. TTF-1 expression persists in proximal airway epithelium, whereas expression patterns in the distal epithelium were normal in CDH lungs (12, 57) and in MyoD knockout mice in which the diaphragm muscle is markedly thinned and nonfunctional, resulting in pulmonary hypoplasia due to lack of fetal breathing movements (24). These latter studies suggest that, for normal fetal lung development, the expression pattern of TTF-1 may be as important as expression levels. We hypothesized that, because TO appears to reverse abnormalities of growth and maturation of the fetal lung that occur with CDH, it may also affect TTF-1 expression. Therefore, we examined TTF-1 protein levels in the lungs of animals with CDH, CDH+TO, and NC.

In the current study, TTF-1 protein levels were increased in CDH lungs compared with NC lungs (Fig. 5). This finding is consistent with the increase in SP-C expression seen with CDH, as TTF-1 is a transcriptional regulator of the SP-C gene (28). Although TTF-1 also regulates transcription of SP-A and SP-B (6), CDH did not affect expression of these genes. Therefore, factors other than decreased lung distension and increased TTF-1 must be necessary to upregulate SP-A and SP-B. In contrast to CDH, CDH+TO decreased TTF-1 expression to the level of NC lungs (Fig. 5). The decrease in expression of the mRNAs for the three SP suggests that a reduction in TTF-1 may have a negative influence on surfactant gene transcription. By immunohistochemistry, TTF-1 was increased in CDH lungs, both in distal airway and in distal parenchymal epithelium; CDH+TO had the opposite effects. Because we did not examine proximal airways, we do not have information on the effects of CDH and CDH+TO on the proximal-distal gradient expression pattern of TTF-1 seen in normally developing fetal lungs.

Our results indicate that changes in distension affect expression of TTF-1. These changes in TTF-1 may be responsible for the observed changes in maturation of type I and type II cells. Alternatively, changes in fetal lung distension may affect indicators of type I and type II cell maturation through other mechanisms independent of TTF-1. Additional studies will be needed to resolve this point.

In the summary, our results show that decreased distension of the fetal lung due to CDH caused pulmonary hypoplasia, retarded type I cell maturation, and increased expression of SP-C and TTF-1. Increased distension of CDH lungs (i.e., CDH+TO) reversed the pulmonary hypoplasia, promoted maturation of type I cells, and reduced expression of SP and TTF-1. These studies do not allow us to determine whether the changes in the type I cell-specific protein RTI40 were due to changes in type I cell number, size, or both. In conclusion, TO of the CDH lung reverses several of the effects of CDH alone, some of which may be directed by modulations in TTF-1 expression.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This research was supported by National Heart, Lung, and Blood Institute Grant HL-24075. J. Yoshizawa and A. Hara were supported by a grant from the Department of Surgery, The Jikei University School of Medicine, Tokyo, Japan. L. Sbragia was supported by Faculdade de Ciências Medicas-Universidade Estudual de Campinas and FAPESP-The State of São Paulo Research Foundation N:99/03870-7. J. J. Greer was supported by grants from the Canadian Institutes of Health Research and March of Dimes.


    ACKNOWLEDGMENTS
 
Present address of J. Yoshizawa and A. Hara: Department of Surgery, The Jikei University School of Medicine, 3-25-8, Nishi-shinbashi, Tokyo 105-8461, Japan. Present address of L. Sbragia, State University of Campinas, School of Medicine, Division of Pediatric Surgery, Rua Alexandre Fleming 181, Campinas, São Paulo, Brazil 13087-970.


    FOOTNOTES
 

Address for reprint requests and other correspondence: C. J. Chapin, Cardiovascular Research Institute, Box 1245, Univ. of California, San Francisco, CA 94143 (E-mail: cheri{at}itsa.ucsf.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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alcorn D, Adamson TM, Lambert TF, Maloney JE, Ritchie BC, and Robinson PM. Morphological effects of chronic tracheal ligation and drainage in the fetal lamb lung. J Anat 123: 649–660, 1977.[ISI][Medline]
  2. Areechon W and Eid L. Hypoplasia of lung with congenital diaphragmatic hernia. Br Med J 5325: 230–233, 1963.[Medline]
  3. Benachi A, Chailley-Heu B, Barlier-Mur AM, Dumez Y, and Bourbon J. Expression of surfactant proteins and thyroid transcription factor 1 in an ovine model of congenital diaphragmatic hernia. J Pediatr Surg 37: 1393–1398, 2002.[CrossRef][ISI][Medline]
  4. Benachi A, Chailley-Heu B, Delezoide AL, Dommergues M, Brunelle F, Dumez Y, and Bourbon JR. Lung growth and maturation after tracheal occlusion in diaphragmatic hernia. Am J Respir Crit Care Med 157: 921–927, 1998.[Abstract/Free Full Text]
  5. Benachi A, Delezoide AL, Chailley-Heu B, Preece M, Bourbon JR, and Ryder T. Ultrastructural evaluation of lung maturation in a sheep model of diaphragmatic hernia and tracheal occlusion. Am J Respir Cell Mol Biol 20: 805–812, 1999.[Abstract/Free Full Text]
  6. Boggaram V. Regulation of lung surfactant protein gene expression. Front Biosci 8: d751–d764, 2003.[ISI][Medline]
  7. Bohinski RJ, Di Lauro R, and Whitsett JA. The lung-specific surfactant protein B gene promoter is a target for thyroid transcription factor 1 and hepatocyte nuclear factor 3, indicating common factors for organ-specific gene expression along the foregut axis. Mol Cell Biol 14: 5671–5681, 1994.[Abstract]
  8. Bratu I, Flageole H, Laberge JM, Chen MF, and Piedboeuf B. Pulmonary structural maturation and pulmonary artery remodeling after reversible fetal ovine tracheal occlusion in diaphragmatic hernia. J Pediatr Surg 36: 739–744, 2001.[CrossRef][ISI][Medline]
  9. Chapin CJ, Yoshizawa J, Sbragia L, Albanese CT, and Kitterman JA. Congenital diaphragmatic hernia (CDH) reduces and tracheal occlusion (TO) increases lung growth and type I cell marker, RTI40, without affecting type II cell function in fetal rats (Abstract). Pediatr Res 49: 31A, 2001.
  10. Chinoy MR, Chi X, and Cilley RE. Down-regulation of regulatory proteins for differentiation and proliferation in murine fetal hypoplastic lungs: altered mesenchymal-epithelial interactions. Pediatr Pulmonol 32: 129–141, 2001.[CrossRef][ISI][Medline]
  11. Cilley RE, Zgleszewski SE, Krummel TM, and Chinoy MR. Nitrofen dose-dependent gestational day-specific murine lung hypoplasia and left-sided diaphragmatic hernia. Am J Physiol Lung Cell Mol Physiol 272: L362–L371, 1997.[Abstract/Free Full Text]
  12. Coleman C, Zhao J, Gupta M, Buckley S, Tefft JD, Wuenschell CW, Minoo P, Anderson KD, and Warburton D. Inhibition of vascular and epithelial differentiation in murine nitrofen-induced diaphragmatic hernia. Am J Physiol Lung Cell Mol Physiol 274: L636–L646, 1998.[Abstract/Free Full Text]
  13. De Paepe ME, Johnson BD, Papadakis K, and Luks FI. Lung growth response after tracheal occlusion in fetal rabbits is gestational age-dependent. Am J Respir Cell Mol Biol 21: 65–76, 1999.[Abstract/Free Full Text]
  14. De Paepe ME, Papadakis K, Johnson BD, and Luks FI. Fate of the type II pneumocyte following tracheal occlusion in utero: a time-course study in fetal sheep. Virchows Arch 432: 7–16, 1998.[CrossRef][ISI][Medline]
  15. DeLorimier AA, Tierney TD, and Parker HR. Hypoplastic lungs in surgically produced congenital diaphragmatic hernia. Surgery 62: 12–17, 1967.[ISI]
  16. Dobbs LG, Pian MS, Maglio M, Dumars S, and Allen L. Maintenance of the differentiated type II cell phenotype by culture with an apical air surface. Am J Physiol Lung Cell Mol Physiol 273: L347–L354, 1997.[Abstract/Free Full Text]
  17. Flecknoe S, Harding R, Maritz G, and Hooper SB. Increased lung expansion alters the proportions of type I and type II alveolar epithelial cells in fetal sheep. Am J Physiol Lung Cell Mol Physiol 278: L1180–L1185, 2000.[Abstract/Free Full Text]
  18. Gonzalez RF and Dobbs LG. Purification and analysis of RTI40, a type I alveolar epithelial cell apical membrane protein. Biochim Biophys Acta 1429: 208–216, 1998.[ISI][Medline]
  19. Gonzalez RF and Dobbs LG. Characterization and utility of a monoclonalantibody specific to the apical surface of rat alveolar type II cells (Abstract). Mol Biol Cell 8: 1981, 1997.[ISI]
  20. Guilbert TW, Gebb SA, and Shannon JM. Lung hypoplasia in the nitrofen model of congenital diaphragmatic hernia occurs early in development. Am J Physiol Lung Cell Mol Physiol 279: L1159–L1171, 2000.[Abstract/Free Full Text]
  21. Harrison MR, Adzick NS, Estes JM, and Howell LJ. A prospective study of the outcome for fetuses with diaphragmatic hernia. JAMA 271: 382–384, 1994.[Abstract]
  22. Hooper SB and Harding R. Fetal lung liquid: a major determinant of the growth and functional development of the fetal lung. Clin Exp Pharmacol Physiol 22: 235–247, 1995.[ISI][Medline]
  23. Hosgor M, Ijzendoorn Y, Mooi WJ, Tibboel D, and De Krijger RR. Thyroid transcription factor-1 expression during normal human lung development and in patients with congenital diaphragmatic hernia. J Pediatr Surg 37: 1258–1262, 2002.[CrossRef][ISI][Medline]
  24. Inanlou MR and Kablar B. Abnormal development of the diaphragm in mdx:MyoD–/–(9th) embryos leads to pulmonary hypoplasia. Int J Dev Biol 47: 363–371, 2003.[ISI][Medline]
  25. Joe P, Wallen LD, Chapin CJ, Lee CH, Allen L, Han VK, Dobbs LG, Hawgood S, and Kitterman JA. Effects of mechanical factors on growth and maturation of the lung in fetal sheep. Am J Physiol Lung Cell Mol Physiol 272: L95–L105, 1997.[Abstract/Free Full Text]
  26. Kanai M, Kitano Y, von Allmen D, Davies P, Adzick NS, and Flake AW. Fetal tracheal occlusion in the rat model of nitrofen-induced congenital diaphragmatic hernia: tracheal occlusion reverses the arterial structural abnormality. J Pediatr Surg 36: 839–845, 2001.[CrossRef][ISI][Medline]
  27. Keijzer R, Liu J, Deimling J, Tibboel D, and Post M. Dual-hit hypothesis explains pulmonary hypoplasia in the nitrofen model of congenital diaphragmatic hernia. Am J Pathol 156: 1299–1306, 2000.[Abstract/Free Full Text]
  28. Kelly SE, Bachurski CJ, Burhans MS, and Glasser SW. Transcription of the lung-specific surfactant protein C gene is mediated by thyroid transcription factor 1. J Biol Chem 271: 6881–6888, 1996.[Abstract/Free Full Text]
  29. Kitagawa M, Hislop A, Botden EA, and Reid L. Lung hypoplasia in congenital diaphragmatic hernia. A quantitative study of airway, artery and alveolar development. Br J Surg 58: 342–346, 1971.[ISI][Medline]
  30. Kitano Y, Korimilli A, Flake A, and Guttentag S. Effects of fetal tracheal occlusion on lung growth and maturation in nitrofen-induced congenital diaphragmatic hernia (Abstract). Pediatr Res 47: 72A, 2000.
  31. Kitano Y, Davies P, von Allmen D, Adzick NS, and Flake AW. Fetal tracheal occlusion in the rat model of nitrofen-induced congenital diaphragmatic hernia. J Appl Physiol 87: 769–775, 1999.[Abstract/Free Full Text]
  32. Kitano Y, Yang EY, von Allmen D, Quinn TM, Adzick NS, and Flake AW. Tracheal occlusion in the fetal rat: a new experimental model for the study of accelerated lung growth. J Pediatr Surg 33: 1741–1744, 1998.[CrossRef][ISI][Medline]
  33. Kitterman JA. The effects of mechanical forces on fetal lung growth. Clin Perinatol 23: 727–740, 1996.[ISI][Medline]
  34. Kitterman JA, Chapin CJ, Vanderbilt JN, Porta NF, Scavo LM, Dobbs LG, Ertsey R, and Goerke J. Effects of oligohydramnios on lung growth and maturation in the fetal rat. Am J Physiol Lung Cell Mol Physiol 282: L431–L439, 2002.[Abstract/Free Full Text]
  35. Kluth D, Kangah R, Reich P, Tenbrinck R, Tibboel D, and Lambrecht W. Nitrofen-induced diaphragmatic hernias in rats: an animal model. J Pediatr Surg 25: 850–854, 1990.[CrossRef][ISI][Medline]
  36. Kumar VH and Ryan RM. Growth factors in the fetal and neonatal lung. Front Biosci 9: 464–480, 2004.[ISI][Medline]
  37. Langwieler T, Fiegel HC, Alaamian M, Mann O, Beshir I, Izbicki JR, and Kluth D. The relationship of diaphragmatic defect, liver growth, and lung hypoplasia in nitrofen-induced congenital diaphragmatic hernia in the rat. Pediatr Surg Int 20: 509–514, 2004.[ISI][Medline]
  38. Leinwand MJ, Tefft JD, Zhao J, Coleman C, Anderson KD, and Warburton D. Nitrofen inhibition of pulmonary growth and development occurs in the early embryonic mouse. J Pediatr Surg 37: 1263–1268, 2002.[CrossRef][ISI][Medline]
  39. Lines A, Nardo L, Phillips ID, Possmayer F, and Hooper SB. Alterations in lung expansion affect surfactant protein A, B, and C mRNA levels in fetal sheep. Am J Physiol Lung Cell Mol Physiol 276: L239–L245, 1999.[Abstract/Free Full Text]
  40. Losada A, Tovar JA, Xia HM, Diez-Pardo JA, and Santisteban P. Down-regulation of thyroid transcription factor-1 gene expression in fetal lung hypoplasia is restored by glucocorticoids. Endocrinology 141: 2166–2173, 2000.[Abstract/Free Full Text]
  41. Luks FI, Wild YK, Piasecki GJ, and De Paepe ME. Short-term tracheal occlusion corrects pulmonary vascular anomalies in the fetal lamb with diaphragmatic hernia. Surgery 128: 266–272, 2000.[CrossRef][ISI][Medline]
  42. Maltais F, Seaborn T, Guay S, and Piedboeuf B. In vivo tracheal occlusion in fetal mice induces rapid lung development without affecting surfactant protein C expression. Am J Physiol Lung Cell Mol Physiol 284: L622–L632, 2003.[Abstract/Free Full Text]
  43. Minoo P, Hamdan H, Bu D, Warburton D, Stepanik P, and deLemos R. TTF-1 regulates lung epithelial morphogenesis. Dev Biol 172: 694–698, 1995.[CrossRef][ISI][Medline]
  44. Mysore MR, Margraf LR, Jaramillo MA, Breed DR, Chau VL, Arevalo M, and Moya FR. Surfactant protein A is decreased in a rat model of congenital diaphragmatic hernia. Am J Respir Crit Care Med 157: 654–657, 1998.[ISI][Medline]
  45. Nobuhara KK and Wilson JM. The effect of mechanical forces on in utero lung growth in congenital diaphragmatic hernia. Clin Perinatol 23: 741–752, 1996.[ISI][Medline]
  46. Papadakis K, De Paepe ME, Tackett LD, Piasecki GJ, and Luks FI. Temporary tracheal occlusion causes catch-up lung maturation in a fetal model of diaphragmatic hernia. J Pediatr Surg 33: 1030–1037, 1998.[CrossRef][ISI][Medline]
  47. Piedboeuf B, Laberge JM, Ghitulescu G, Gamache M, Petrov P, Belanger S, Chen MF, Hashim E, and Possmayer F. Deleterious effect of tracheal obstruction on type II pneumocytes in fetal sheep. Pediatr Res 41: 473–479, 1997.[Abstract]
  48. Ramirez MI, Millien G, Hinds A, Cao Y, Seldin DC, and Williams MC. T1{alpha}, a lung type I cell differentiation gene, is required for normal lung cell proliferation and alveolus formation at birth. Dev Biol 256: 61–72, 2003.[ISI][Medline]
  49. Ramirez MI, Rishi AK, Cao YX, and Williams MC. TGT3, thyroid transcription factor I, and Sp1 elements regulate transcriptional activity of the 1.3-kilobase pair promoter of T1{alpha}, a lung alveolar type I cell gene. J Biol Chem 272: 26285–26294, 1997.[Abstract/Free Full Text]
  50. Thebaud B, Barlier-Mur AM, Chailley-Heu B, Henrion-Caude A, Tibboel D, Dinh-Xuan AT, and Bourbon JR. Restoring effects of vitamin A on surfactant synthesis in nitrofen-induced congenital diaphragmatic hernia in rats. Am J Respir Crit Care Med 164: 1083–1089, 2001.[Abstract/Free Full Text]
  51. Van Tuyl M, Blommaart PE, Keijzer R, Wert SE, Ruijter JM, Lamers WH, and Tibboel D. Pulmonary surfactant protein A, B, and C mRNA and protein expression in the nitrofen-induced congenital diaphragmatic hernia rat model. Pediatr Res 54: 641–652, 2003.[Abstract/Free Full Text]
  52. Whitsett JA and Glasser SW. Regulation of surfactant protein gene transcription. Biochim Biophys Acta 1408: 303–311, 1998.[ISI][Medline]
  53. Wild YK, Piasecki GJ, De Paepe ME, and Luks FI. Short-term tracheal occlusion in fetal lambs with diaphragmatic hernia improves lung function, even in the absence of lung growth. J Pediatr Surg 35: 775–779, 2000.[CrossRef][ISI][Medline]
  54. Wu J, Ge X, Verbeken EK, Gratacos E, Yesildaglar N, and Deprest JA. Pulmonary effects of in utero tracheal occlusion are dependent on gestational age in a rabbit model of diaphragmatic hernia. J Pediatr Surg 37: 11–17, 2002.[CrossRef][ISI][Medline]
  55. Yoshizawa J, Chapin CJ, Sbragia L, Ertsey R, Gutierrez JA, Albanese CT, and Kitterman JA. Tracheal occlusion stimulates cell cycle progression and type I cell differentiation in lungs of fetal rats. Am J Physiol Lung Cell Mol Physiol 285: L344–L353, 2003.[Abstract/Free Full Text]
  56. Yuan B, Li C, Kimura S, Engelhardt RT, Smith BR, and Minoo P. Inhibition of distal lung morphogenesis in Nkx2.1(–/–) embryos. Dev Dyn 217: 180–190, 2000.[CrossRef][ISI][Medline]
  57. Zhou H, Morotti RA, Profitt SA, Langston C, Wert SE, Whitsett JA, and Greco MA. Expression of thyroid transcription factor-1, surfactant proteins, type I cell-associated antigen, and Clara cell secretory protein in pulmonary hypoplasia. Pediatr Dev Pathol 4: 364–371, 2001.[CrossRef][ISI][Medline]




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
289/1/L44    most recent
00342.2004v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Chapin, C. J.
Articles by Kitterman, J. A.
Articles citing this Article
PubMed
PubMed Citation
Articles by Chapin, C. J.
Articles by Kitterman, J. A.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.