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
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ABSTRACT |
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fetal lung development; pulmonary surfactant; alveolar type I and type II cells
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).
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MATERIALS AND METHODS |
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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 [-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--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.
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RESULTS |
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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).
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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).
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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.
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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).
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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.
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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.
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DISCUSSION |
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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.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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