Tracheal occlusion stimulates cell cycle progression and type I cell differentiation in lungs of fetal rats

Jyoji Yoshizawa,1,3 Cheryl J. Chapin,1 Lourenço Sbragia,1,3 Robert Ertsey,1 Jorge A. Gutierrez,1,2 Craig T. Albanese,1,3 and Joseph A. Kitterman1,2

1Cardiovascular Research Institute, and Departments of 2Pediatrics and 3Surgery, University of California, San Francisco, California 94118

Submitted 15 August 2002 ; accepted in final form 31 March 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fetal tracheal occlusion (TO) has been reported to stimulate lung growth but decreases number and maturation of type II cells, effects that vary with gestational age and duration of TO. We examined effects of a novel method of TO (unipolar microcautery to seal the trachea) produced at 19.5–20 days (d) of gestation in fetal rats; fetuses were delivered at term, 22 d. Controls were sham operated and unoperated littermates. TO increased wet lung weight but not dry lung weight or lung DNA and protein. To evaluate further the effects of TO, we examined the cell cycle regulators, cyclins D1 and A, in fetal lungs. Cyclin D1 increased with TO (P < 0.005). TO also increased expression of the type I epithelial cell marker RTI40 (mRNA and protein). TO decreased mRNA for surfactant proteins (SP)-A and -C but did not affect protein levels of SP-A and -B and of RTII70, a type II epithelial cell marker. We conclude that TO by microcautery, even of short duration, has diverse pulmonary effects including stimulating increased levels of cyclin D1 with probable cell cycle progression, type I cell differentiation, and possibly inhibiting type II cell function.

cyclins; fetal lung development; lung growth; pulmonary epithelial differentiation; pulmonary surfactant


LATE IN GESTATION, the fetal lung rapidly changes in appearance from an almost glandular tissue into a lacy, thin-walled organ capable of efficient gas exchange and surfactant production. During this period lung growth, septal thinning, maturation of the surfactant system, and differentiation of alveolar type I and type II cells occur. Normal fetal lung development is crucial for survival in extrauterine life, but factors that control these developmental processes and their interdependence have been incompletely defined.

Lung growth and maturation of the surfactant system are the most widely studied aspects of late fetal lung development. Fetal lung growth is dependent on several mechanical factors, including adequate intrathoracic space, sufficient amniotic fluid volume, normal fetal breathing movements, and a positive transpulmonary distending pressure (21, 27). In contrast, maturation of the surfactant system is highly dependent on endocrine factors (4, 50). There is relatively little information about factors that are responsible for differentiation of alveolar epithelial type I and type II cells in vivo.

Several investigators have shown that occlusion of the trachea in experimental animals increases lung growth but has adverse affects on type II cell number and function (2, 10, 11, 15, 40). These changes occur when tracheal occlusion (TO) is produced relatively early in gestation and maintained for a prolonged period. In contrast, when TO is produced later in gestation for a relatively short period, lung growth is stimulated, and adverse affects on the type II cell population are diminished. In 1996, Keramidaris and associates (24) showed a clear relationship between the TO-induced growth response and gestational age in fetal sheep lung, with older fetuses showing a greater response. Other studies have confirmed this observation in fetal sheep (29), rabbits (10), and rats (25). There is also evidence, from studies in fetal rabbits (10) and sheep (11), that the type II cell population may be spared some of the adverse effects of TO when it is performed late in gestation for a short duration.

TO also influences development of type I cells. Benachi and associates (6) reported that, in fetal sheep with diaphragmatic hernia, relatively long-term TO adversely affected type II cells and increased the percentage of type I cells compared with controls and fetuses with diaphragmatic hernia alone. These authors observed numerous indeterminate cells that displayed some characteristics of both type I and type II cells in lungs of fetuses with TO. More recently, Flecknoe and associates (15) showed a time-dependent increase in type I cell percentage with TO in fetal sheep over a 10-day period; they also observed intermediate cells that exhibited traits of both type I and type II cells. The proportion of this intermediate cell type increased fourfold within 2 days of TO. Presumably, these cells contributed to the increase in the type I cell population through terminal differentiation.

The studies cited above indicate that the effects of increased distension of the fetal lung, caused by TO, are dependent on the maturity of the lung and the duration of the occlusion. We hypothesized that TO of short duration would stimulate lung growth and maturation without adverse affects on type II cell function or population. Because we had technical difficulties with the tracheal ligation method (26), we used a novel method (microcautery) to produce TO. In the current study, we report the effects of short-term TO, produced by microcautery, on the fetal rat lung late in gestation.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
All studies were approved by the Committee on Animal Research of the University of California, San Francisco, and all procedures conformed with the NIH Guide for the Care and Use of Laboratory Animals. Timed-pregnant Sprague-Dawley rats were obtained from Charles River Laboratories (Hollister, CA) on day 18 of gestation and were provided feed and water ad libitum. Dams were housed in a laminar flow facility, which maintained ambient temperature at 21°C.

Surgical procedure. We studied a total of 281 fetuses from 54 pregnant dams. At 19.5–20 days of gestation (presence of a vaginal plug indicated day 1), dams and fetuses were anesthetized with an intramuscular injection of ketamine (90 mg/kg; Parke-Davis, Morris Plains, NJ) and xylazine (1.25 mg/kg; Butler, Columbus, OH) administered to the dam. Through a maternal midline laparotomy, a small section of one uterine horn was exposed. Near the head of a fetus, a purse string was placed in the uterus using 6-0 polypropylene suture, and the fetal head and neck were exteriorized through a small hysterotomy. The neck was maintained in an extended position with wet gauze. Using an operating stereomicroscope (Leica Wild M691, Leica Microsystems), we exposed the trachea through a midline cervical incision. We cauterized the trachea using a hand-held cautery unit equipped with a microtip (Roboz Surgical, Rockville, MD). The cervical incision was not closed, the fetal head and neck were returned to the amniotic sac, and the purse string suture was closed. Four fetuses per dam received TO by microcautery (TO group), whereas two fetuses from the same dam received sham operations in which the trachea was not cauterized (Sham group). Controls were unoperated littermates. The maternal abdominal incision was closed in two layers with silk sutures, and the dam was allowed to recover. At 22 days of gestation (term), the dam and fetuses were again anesthetized with an intramuscular injection of ketamine (180 mg/kg) and xylazine (2.5 mg/kg). One uterine horn was exposed at a time, and the fetuses were removed individually by hysterotomy, pithed, and weighed. The fetal thorax was opened, the lungs were examined, and signs of distension, indentations on the pleural surface of the lung from the rib cage, and/or fluid accumulation were noted. The fetal lungs were dissected away from heart and trachea, weighed, and snap-frozen in liquid nitrogen. Some litters were managed for use in immunohistochemical studies described below in Immunohistochemistry. After all fetuses had been removed, dams were killed by intracardiac injection of pentobarbital (1 ml, 390 mg/ml Euthanasia Solution; Schering-Plough Animal Health, Union, NJ) followed by production of bilateral pneumothoraces.

Biochemical studies. To estimate dry lung weight, we dried previously weighed fetal lungs to constant weight in a vacuum oven at 86°C. To quantify protein and DNA content, we dounce homogenized frozen lungs. Protein content was measured using the bicinchoninic acid (BCA) modification of the Lowry method (Sigma, St. Louis, MO) (46). DNA content was measured using the fluorometric method of Setaro and Morley (44).

Nuclear extracts. Whole frozen lungs were dounce homogenized in 1 ml of nuclear extract buffer B (8), 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 by the BCA method.

Analysis of cyclins D1 and A. Nuclear extracts from whole lung (100 and 50 µg) were electrophoresed under reducing conditions through 4% stacking-15% polyacrylamide gels. Prestained molecular weight markers (Rainbow; Amersham Pharmacia, Piscataway, NJ), adult lung nuclear extract (negative control), and MOLT-4 cell lysate (BD PharMingen, San Diego, CA) (positive control) 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, pH 7.4 (TBS), and placed in 5% nonfat dry milk-0.4% fish gelatin (Amersham Pharmacia)-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 either mouse anti-cyclin D1 antibody (1:200) or rabbit anti-cyclin A antibody (1:500) (both from Santa Cruz Biotechnology, Santa Cruz, CA) 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) then incubated for 30 min at room temperature in species-appropriate horseradish peroxidase-linked secondary antibody (1: 3,000, Amersham Pharmacia) followed by extensive washing with TBS-T. Blots were then developed with ECL(+) (Amersham Pharmacia), visualized by autoradiography, and quantified with the Storm 840 phosphorimager equipped with a blue fluorescence-chemifluorescence detector and Imagequant software (both from Molecular Dynamics, Sunnyvale, CA). Specificity of both primary antibodies was determined by blots containing recombinant cyclin D1 and cyclin A (Santa Cruz Biotechnology).

To evaluate effects of TO and sham operation on the distal pulmonary epithelium, we used methods described below to measure RTI40 and RTII70, proteins specific in the lung to the apical membranes of rat type I and type II pneumonocytes, respectively (13, 14, 16), and mRNA for RTI40 (48), as well as surfactant proteins (SP)-A and -B, and mRNAs for SP-A, -B, and -C.

RNA isolation and analysis. Total RNA was isolated from whole lung by the RNAzol method (Tel-Test, Friendswood, TX) as previously described (28). Multiplex RT-PCR utilizing Competimer technology (Ambion, Austin, TX) was used for analysis of RTI40, SP-A, SP-B, or SP-C mRNA in combination with 18S rRNA levels in fetal lung. Total RNA was reverse transcribed with random primers. cDNA was amplified with specific oligonucleotide primers for RTI40, SP-A, SP-B, or SP-C, as previously described (18), and 18S (QuantumRNA 18S Internal Standards, Ambion). For each gene, we determined the linear range and the optimal 18S Primer-Competimer ratio (RTI40 = 1:9, SP-A = 3:17, SP-B = 3:17, and SP-C = 2:8). Radiolabeled PCR products were produced by direct incorporation of [{alpha}-32P]dCTP (3,000 Ci/mmol; NEN, Boston, MA) during amplification (15 cycles for all genes). PCR products were resolved on 6% polyacrylamide gels containing 8 M urea. After drying the gel, we quantified PCR products with a Storm 840 phosphorimager and Imagequant software (Molecular Dynamics).

Quantification of proteins RTI40, RTII70, SP-A, and SP-B. Protein was extracted from whole fetal lung as previously described (28). In brief, 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, were loaded in duplicate onto nitrocellulose membranes. Blots were blocked and incubated with monoclonal antibodies to RTI40 (14, 16) or RTII70 (13) or polyclonal antibodies to SP-A or SP-B (23, 28). 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 Pharmacia), visualized by autoradiography, and quantified using the Storm 840 phosphorimager equipped with a blue fluorescence/chemifluorescence detector and Imagequant software (Molecular Dynamics).

Immunohistochemistry. Fetal rat lungs were fixed in 4% paraformaldehyde either by immersion for 24 h or in situ at a constant intratracheal pressure of 10 cmH2O for 4 h and processed as previously described (28). In brief, immunohistochemistry was performed on 3-µm sections of fixed, cryo-protected lungs using primary antibodies to cyclin D1 (Santa Cruz Biotechnology) or to RTI40 and RTII70 as indicated above. Cyclin D1 was detected in sections pretreated with target retrieval solution (Dako, Carpinteria, CA) 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, we also processed sections with omission of either the primary antibody or the primary and secondary antibodies. Sections were then processed with the Vectastain Elite ABC kit followed by detection with the DAB substrate kit for peroxidase (both from Vector, Burlingame, CA) and counter-stained with Gill's no. 2 hematoxylin (Sigma). Sections were mounted with Glycergel (Dako) and photographed under a Leitz Orthoplan 2 microscope (Leica Microsystems). RTI40 and RTII70 proteins were detected by indirect immunofluorescence (28). Sections were incubated 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,) 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 under a Leica TCS SP confocal microscope (Leica Microsystems).

Statistical analyses. Values for the three experimental groups (TO, Sham, and control) were compared by analysis of variance using Fisher's protected least significant difference or Bonferroni/Dunn post hoc test where appropriate. Student's t-test for paired data was used when TO or Sham values were compared with control littermate values. Data are presented as ratios ± SE, except where stated. All statistical analyses were done with Statview (Abacus Concepts, Berkeley, CA). P values < 0.05 were considered significant.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To examine the effects of increased lung distension, we occluded the trachea of fetal rats at 19.5 or 20 days of gestation by monopolar microcautery. Data were analyzed from three experimental groups: TO, Sham, and unoperated (control) littermates. Fetuses were delivered by hysterotomy at term (22 days of gestation). The appearance of large, distended lungs in an animal that had undergone a TO operation (Fig. 1) indicated the procedure was successful. The survival rate of TO fetuses was 48%, with a success rate in the surviving fetuses of 70%. Sham fetuses had a 94% survival rate. We studied 79 TO fetuses, 43 Sham, and 159 control fetuses from 54 pregnant dams.



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Fig. 1. Gross appearance of fetal rat lung at 22 days (d) of gestation. Fetal rats were delivered, and the rib cage was cut away to visualize the lung. A: control lung; B: lung exposed to tracheal occlusion (TO) from 20- to 22-d gestation. The left lungs are outlined for clarity. Note that the lungs of the TO fetus are larger than those of the control fetus.

 

Fetal body weights were similar among the three experimental groups. Compared with both Sham and control fetuses, TO significantly increased wet lung weight, both in absolute terms (g) and as a percentage of body weight (Table 1). Lung weights of Sham fetuses (expressed as a percentage of body weight) were significantly less than controls. Dry lung weight (expressed as a percentage of body weight) of TO fetuses was greater than Sham; dry lung weight of controls did not differ significantly from TO or Sham (Table 1). Lung DNA and protein contents did not differ among the three experimental groups (Table 2).


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Table 1. Effects of tracheal occlusion and sham operations on body weight and lung weight in fetal rats

 

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Table 2. Effects of tracheal occlusion and sham operations on DNA and protein in lungs of fetal rats

 

To evaluate further the effects of TO, we measured the lung contents of cyclin D1, an early G1-phase cell cycle regulatory protein, and cyclin A, an S-phase regulatory protein. Cyclin D1 was significantly increased in TO lungs compared with control (TO/control = 133 ± 8%, n = 10 pairs, P < 0.005). Conversely, cyclin D1 was reduced in lungs of Sham fetuses compared with controls (Sham/control = 77 ± 5%, n = 6 pairs, P < 0.005). In the same fetuses, TO also caused increases in cyclin A, but the changes did not reach statistical significance (TO/control = 121 ± 12%, n = 10 pairs, P = 0.13). Cyclin A levels were similar in control and Sham fetuses. These results are illustrated in Fig. 2.



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Fig. 2. Effects of TO and sham operations (Sham) on cell cycle progression in lungs of fetal rats. Representative Western blots of fetal lung nuclear extracts (100 µg total lung protein) for cyclin D1, a G1 phase protein (A), and cyclin A, an S phase protein (B). C: graphic representation of the data. Values are shown as percent ± SE. *P < 0.005.

 

To identify which cells were being affected by TO or Sham, we localized cyclin D1 in the lungs of these fetuses by immunohistochemistry (Fig. 3). No immunostaining was evident with omission of the primary antibody (Fig. 3D) or the primary and secondary antibodies (results not shown). Although all compartments of the lung showed some degree of positive nuclear staining, the airway and distal epithelia showed strong staining. Airway epithelial staining was similar among experimental groups, whereas both distal epithelial and interstitial cell staining were dramatically increased both in number and in intensity in TO fetuses, represented in Fig. 3B by numerous arrows, compared with control and Sham fetuses (Fig. 3, A and C, respectively). Because lungs for these studies were fixed by immersion, no conclusions can be made regarding the morphological effects of TO and Sham.



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Fig. 3. Immunohistochemical localization of cyclin D1 in fetal rat lung. Sections of lung from littermate rat fetuses exposed to control (A), TO (B), or Sham (C). No immunostaining was evident with omission of the primary antibody (D). Airway epithelial (AW) staining was similar among experimental groups, whereas both distal epithelial and interstitial cell staining were dramatically increased both in number and in intensity with TO (numerous arrows in B). Bar = 100 µm.

 

We evaluated the effects of increased lung distension by TO on type II cell function by measuring mRNA expression of SP-A, -B, and -C in fetal lungs (Fig. 4). mRNA expressions in TO and in Sham fetuses were expressed as ratios to littermate control values. TO decreased expression of SP-A (TO/control = 72 ± 5%, n = 8 pairs, P < 0.01) and SP-C (TO/control = 72 ± 8%, n = 8 pairs, P < 0.01) and tended to decrease expression of SP-B (TO/control = 87 ± 8%, n = 8 pairs, P = 0.15). Expression of surfactant protein mRNAs were similar in Sham and control fetuses (n = 7 pairs). In contrast to mRNA levels, SP-A and SP-B protein levels were similar in TO, Sham, and control fetuses, although SP-A tended to decrease in TO lungs compared with control (Table 3).



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Fig. 4. Effects of TO or Sham on surfactant protein (SP) mRNA expression in lungs of fetal rats. A: RT-PCR products, in duplicate, for SP-A, SP-B, and SP-C. B: graphic representation of the data. Values shown are percent ± SE of TO or Sham to littermate control fetuses (C) fetuses. {ddagger}P < 0.001; *P < 0.01.

 

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Table 3. Effects of tracheal occlusion and sham operations on surfactant proteins A and B in lungs of fetal rats

 

To examine in more detail the effects of increased distension of the fetal lung on distal pulmonary epithelial differentiation, we evaluated the expression of RTI40 mRNA, as well as RTI40 and RTII70 proteins (Fig. 5). These proteins are specific markers in the lung for type I and type II alveolar epithelial cells, respectively, and both are integral membrane proteins located on the apical surface of the cells. RTI40 mRNA expression levels tended to increase with TO compared with control (TO/control = 130 ± 13%, n = 8 pairs, P = 0.05); there were no differences for TO vs. Sham and Sham vs. control. RTI40 protein levels in whole lung homogenates were significantly increased in TO fetuses compared with control (TO/control = 142 ± 8%, n = 13 pairs, P < 0.001) and with Sham fetuses (TO/Sham = 156 ± 17%, n = 6 pairs, P < 0.05). RTII70 levels were unaffected by TO compared with control and Sham, whereas Sham-operated animals had significantly less RTII70 compared with control (Sham/control = 90 ± 4%, n = 8 pairs, P < 0.05).



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Fig. 5. Effects of TO or Sham on type I and type II cell development. A: RT-PCR product, in duplicate for RTI40 mRNA. B: representative Western dot blot for RTI40 and RTII70 proteins. C: graphic representation of the data. Values shown are percent ± SE. *P < 0.05; {ddagger}P < 0.001.

 

To visualize differences in RTI40 and RTII70 proteins in situ, we performed immunohistochemistry on sections of lungs exposed to TO, Sham, and control conditions, utilizing the same antibodies used to measure relative protein levels in the lungs (Fig. 6). Lungs from Sham and control fetuses were fixed at an inflation pressure of 10 cmH2O. Because of the technical difficulties in cannulating the occluded trachea, lungs from TO fetuses were immersion fixed. Images were obtained by confocal microscopy. In TO lungs, the RTI40 staining was more intense than in Sham and control lungs, whereas the intensity and distribution of RTII70 staining appeared to be similar among the three experimental groups. Because TO lungs were fixed by immersion and Sham and control lungs by inflation at 10 cmH2O, no conclusions were made regarding possible morphological differences between the experimental groups.



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Fig. 6. Immunohistochemical localization of RTI40 and RTII70 in lungs from 22-d rat fetuses: control (A), TO (B), and Sham (C) fetuses. Monoclonal antibodies against RTI40 and RTII70, apical membrane proteins specific to alveolar type I and type II cells, respectively, were used to visualize type I and type II cells using confocal microscopy. Control and Sham lungs were fixed at an inflation pressure equal to 10 cmH2O. TO lungs were immersion fixed. Note the intense green labeling of type I cells in the TO lung compared with the control or Sham, whereas the red type II cell (arrows) pattern appears similar among all groups. Yellow and orange staining represents autofluorescence of red blood cells (arrowhead in A). Bar = 50 µm.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study we have described a novel method of using microcautery to produce TO in the fetal rat. The method is technically practical and effective with acceptable rates of success of the operative procedure (70%) and of fetal survival (48%). In our experience, it is technically less difficult than tracheal ligation as described by Kitano and associates (26).

TO was judged to be complete if the lungs appeared larger than normal and there were indentations from the ribs on the pleural surfaces. We did not directly test the completeness of TO at postmortem examination, but we believe that microcautery produced complete TO on the basis of the presence of large distended lungs, increased wet lung weight, and increased lung water (calculated from lung dry weight), findings similar to those of Kitano and associates (26), who used tracheal ligation to perform TO. In addition, previous studies have shown that even a tiny leak in the occluded trachea results in normal, and not increased, lung size and wet weights (23, 26).

Previous studies in a variety of experimental animals have shown that TO is a potent stimulus for growth of the fetal lung (e.g., 1, 2, 7, 10, 26) but has little effect on fetal body growth. In the present study, body weights among the three experimental groups did not differ significantly (Table 1); the small decrease in body weights among TO and Sham fetuses may have been due to the stress of the operative procedure. Wet lung weights of TO fetuses were significantly larger than those of Sham and controls (Table 1). However, TO did not significantly affect either dry lung weights or lung contents of DNA or protein, and the increased wet lung weight with TO was due mainly to an increase in total lung water, a previously reported effect of TO (23, 26). Therefore, in contrast to previous reports, TO did not stimulate increased lung growth in this study despite the apparent complete occlusion of the trachea. The increase in wet lung weight was due to distension of the lung with fetal lung liquid.

The results for dry lung weights and lung contents of DNA and protein in the current study differ from those published by Kitano and associates (26), who used tracheal ligation with fine suture to produce TO in fetal rats at 19 days of gestation and harvested the fetuses at 21.5 days, a 2.5-day period of TO. They reported significant increases in lung dry weight and in DNA and protein in TO fetuses. In the present study, TO was produced later in gestation, between 19.5 and 20 days, and lungs were harvested just before birth at term (22 days), a 2–2.5-day period of TO. Although the reasons for the discrepancies between the results of the present study and those of Kitano and associates (26) are not known, possible causes include the type of operative procedure used to produce TO and the gestational ages at operation and at harvest. Both of the operative procedures, tracheal ligation (26) and occlusion by microcautery, resulted in similar significant increases in wet lung weight and in the appearance of the lungs, which were large and distended after TO (Fig. 1). As for the differences in gestational age between the two studies, there are data indicating that the ability of the fetal rodent lung to respond to TO is dependent on gestational age. Kitano and associates (25) recently reported that TO in fetal rats at 20 days of gestation for a 1.5-day period resulted in greater increases in wet and dry lung weights than TO at 18.5 days for the same duration. In that study, they did not report data for lung DNA and protein. De Paepe and associates (10) used labeling with bromodeoxyuridine (BrdU) to assess the effects of TO on cellular proliferation in lungs of fetal rabbits, and they reported that early TO (77% of term) resulted in an initial decrease in BrdU labeling for 3 days followed by a dramatic increase for the following 3 days. In contrast, TO later in gestation (87% of term) resulted in a lower rate of BrdU labeling, which was only slightly higher than in control animals. Thus it is clear that gestational age does influence the response of the lung to TO, and this may be a reason why there was no effect of TO on total lung content of DNA in the present study. This gestational effect may be related to the ability of the lung to respond to mitogenic signals produced by mechanical distension, which include growth factor and hormonal stimulation (9, 20, 23, 31, 38).

The possible contribution of pulmonary blood flow to the effects of TO has not been defined. Acute changes in postnatal lung gas volume markedly affect pulmonary vascular resistance. As lung volume expands from residual volume, pulmonary vascular resistance first decreases and then increases at higher lung volumes (41). In acute studies in exteriorized fetal sheep, Walker and associates (49) found that pulmonary vascular conductance is inversely proportional to lung liquid volume. Because TO increases lung liquid volume, it is possible that pulmonary blood flow was decreased in the TO fetuses in our study. However, it is not known whether the effects of increased lung liquid volume in chronically prepared fetuses in utero are similar to those in the acute studies described above.

To examine in more detail the effects of TO on the fetal lung, we looked at components of the early phase of cell proliferation by examining lung content of the cell cycle regulators cyclin D1 (G1 phase) and cyclin A (S/G2 phases) (for reviews, see Refs. 45, 47, 51). Compared with control and Sham fetuses, TO caused a significant increase in cyclin D1 protein levels and a tendency for cyclin A to increase (Fig. 2). This finding is supported by the immunohistochemical localization of cyclin D1 (see discussion below). The increase in cyclin D1 and the apparent rise in cyclin A levels are consistent with the notion that stretch of lung cells (produced in vivo by lung distension) activates mechanotransductive signaling pathways, which, in this case, involve entry into and progression of the cell cycle and an apparent commitment to growth. Despite the changes in cyclin content in the lungs, as noted above, TO did not increase lung dry weight or contents of DNA and protein. The likely explanation for this is that progression of the cell cycle stimulated by TO had not advanced far enough into S phase for us to detect an increase in lung dry weight, DNA, and protein.

In the present study, wet weights of lungs, expressed as a percentage of body weight, from Sham fetuses were significantly less than those from control littermates (Table 1). Most likely, this was due to loss of amniotic fluid during the surgical procedure, which acutely decreases the volume of fluid in the potential airways and air spaces (43) and, if prolonged, leads to pulmonary hypoplasia (12, 28, 34, 35). Because there were no significant differences in dry lung weight and in total lung protein and DNA between Sham and control fetuses, the Sham lungs were not truly hypo-plastic. However, the sham operation did have an inhibitory effect on stimuli to lung growth, based on the significant decrease in cyclin D1 (Fig. 2).

Using immunohistochemistry, we examined the spatial distribution of cyclin D1 in the lung. TO markedly increased expression of cyclin D1, both in the intensity of the signal and in the number of positive nuclei in lung parenchyma, particularly in the periphery. Conversely, compared with control fetuses, lung parenchyma from Sham fetuses showed decreased expression of cyclin D1 (Fig. 3). All three experimental groups had a similar staining pattern in airway epithelial cells, indicating that the effect of distension on fetal lung growth was primarily on the distal parenchyma. These observations are concordant with previous studies that showed that growth occurs mainly in the lung periphery, not only in fetuses with TO (11), but also in normal fetal rats (36) as well as postnatally (32).

To evaluate the effects of increased lung distension produced by TO on alveolar epithelial type II cells, we measured SP-A, -B, and the type II cell-specific protein RTII70 (13). Neither TO nor sham operation affected lung levels of the proteins SP-A and -B (Table 2) and RTII70 (Fig. 5). However, with TO, mRNAs for SP-A and -C decreased significantly (28% for both), whereas SP-B mRNA tended to decrease (by 13%) (Fig. 4). A possible explanation for these apparent discrepancies between message and protein levels is the effect of TO on the efflux of fetal lung fluid. In the normal fetus, the lung produces fluid that flows out of the trachea and carries with it surfactant secreted by type II cells (33). Obstruction of the egress of lung fluid by TO not only increases lung distension but also traps surfactant that has been released from type II cells into the luminal spaces of the lung. Our measurements of SP-A and -B were done on homogenates of whole lung and thus included surfactant that had been secreted into the potential airways and air spaces and trapped there by TO, as well as that contained in type II cells. Therefore, although TO caused decreases in message levels for surfactant proteins, the levels of the proteins themselves were similar to control and Sham fetuses, in which lung fluid was allowed to flow out of the trachea and carry the secreted surfactant with it. These results are consistent with those of Lines and associates (30), who studied the effect of TO on surfactant proteins in fetal sheep. They report that, compared with control animals, SP-A, -B, and -C mRNA levels were dramatically reduced after 2, 4, and 10 days of TO, whereas SP-A protein levels were equal to controls at 2 days, somewhat decreased at 4 days, and not significantly decreased until 10 days after occlusion. Consequently, for a short period of TO, whole lung levels of type II cell protein products are maintained despite decreased type II cell function at a molecular level.

To evaluate effects of tracheal occlusion on type I cells, we examined expression of RTI40, a type I cell-specific apical membrane protein (16). In contrast to the lack of change in proteins produced by type II cells, TO had a major effect on type I cells with a significant increase in RTI40 protein level compared with both control and Sham fetuses (increases of 42 and 50%, respectively) (Fig. 5). In addition, RTI40 mRNA levels were increased above control by 30%, a value that almost reached statistical significance (P = 0.05) (Fig. 5).

In studies in fetal sheep (2) and in fetal rats with diaphragmatic hernia (25), TO resulted in increased relative size and volume of potential air spaces. In contrast, Kitano and associates (26), who performed TO by tracheal ligation in normal fetal rats, reported no qualitative morphological differences when lungs from control and TO fetuses were fixed in inflation. Because of technical difficulties with cannulating the trachea after TO by microcautery in the present study, those lungs were fixed by immersion. Lungs from control and Sham fetuses were fixed at a distending pressure of 10 cmH2O to obtain uniform inflation. There are no data for tracheal pressure in fetal rats after TO, but other investigators have reported that tracheal pressure increases by ~4 cmH2O in fetal sheep with TO (38) and after bronchial ligation in whole organ culture of mouse lungs (7). Thus it is likely that, in the present study, the control and Sham lungs were fixed at a higher distending pressure than the TO lungs. Consequently, we are unable to assess possible morphological effects of TO in our study due to the differences in methods of lung fixation.

Immunofluorescent studies revealed that the potential air spaces in the TO lungs were lined with intensely stained RTI40-positive type I cells, whereas Sham and control lungs had relatively less intensely stained type I cells (Fig. 6). The staining of individual RTII70-positive type II cells appeared similar among the different experimental groups. Overall, the qualitative appearance of RTI40 and RTII70 in TO lungs is consistent with the biochemical and molecular data.

There is mounting evidence that mechanical factors play key roles in differentiation of both type I and type II alveolar epithelial cells. Gutierrez and associates found that tonic mechanical stretch (which mimics the effects of increased lung distension in vivo) increased RTI40 mRNA and decreased SP-B and -C mRNAs in cultures of adult rat type II cells (18) and fetal rat lung explants (17), results consistent with the effects of TO in the current study. Gutierrez and associates (19) also reported that tonic mechanical contraction of cultured adult rat type II cells inhibits expression of RTI40 mRNA and stimulates increases in SP-A, -B, and -C mRNAs. Recently we reported that decreased distension of the lung in fetal rats (produced by oligohydramnios) reduces the expression of RTI40 (mRNA and protein), as well as the relative surface area covered by type I cells; in contrast to the results of in vitro studies quoted above, there was no effect on type II cells on the basis of measurements of RTII70 and pulmonary surfactant phospholipid, protein, and mRNA levels (28). Other investigators have reported that changes in distension of the fetal lung in vivo influence pulmonary epithelial differentiation. Fetal TO in sheep and rabbits decreases the number of type II cells (2, 10, 11, 15, 40) and reduces various indicators of pulmonary surfactant, whereas decreased fetal lung distension tends to have opposite effects (23, 30). These parallel findings in vitro and in vivo emphasize the importance of lung distension, not only for lung growth but also for other aspects of fetal lung maturation. It is important to note that in the in vitro studies quoted above, the stimulus was either tonic stretch or tonic contraction. Cyclic stretch of lung cells has different effects and can stimulate increased expression of surfactant proteins (37, 42).

In summary, we have found that TO in the fetal rat in late gestation increases cyclin D1 levels in lung cells. Most likely, this increase, along with the tendency for cyclin A to increase, represents entry into and progression of the cell cycle, although we cannot exclude the possibility that these increases represent increases in cyclins without concurrent entry into the cell cycle. However, in view of other studies in which TO increases DNA synthesis (10, 20, 24, 38), we speculate that TO in the fetal rat stimulates cell cycle progression in distal lung cells. In addition, TO decreases expression of SP-A and -C mRNAs and tends to inhibit SP-B mRNA production. In the current study we have not determined the effect of TO on surfactant function, which may or may not be affected by decreased expression of surfactant proteins at the RNA level. TO also promotes differentiation of alveolar type I cells, although our study does not distinguish whether TO increases the number or size of type I cells but, in combination with previous reports, suggests that TO increases the surface area of potential air spaces covered by type I cells.

The mechanisms that mediate the effects of changes in distension of the fetal lung have not been defined. Indeed, it is likely that the cellular effects of mechanical stimuli are complex (3, 31) and represent responses to force-dependent changes in components of the cytoskeleton (5), a system based on tensegrity architecture (22).


    ACKNOWLEDGMENTS
 
Present addresses: J. Yoshizawa, Jikei Univ. School of Medicine, Dept. of Surgery, 3-25-8 Nishi-Shinbashi, Minato-ku, Tokyo 105-8471, Japan; C. T. Albanese, Div. of Pediatric Surgery, Lucile Packard Children's Hospital, Stanford Univ. Medical Center, 725 Welch Rd., Palo Alto, CA 94304; L. Sbragia, State Univ. of Campinas School of Medicine, Dept. of Surgery, Div. of Pediatric Surgery, Rua Alexandre Fleming 181, Cidade Univeristaria-Barao Geraldo, Campinas-São Paulo, Brazil CEP: 13087-970.

DISCLOSURES

This research was supported by National Heart, Lung, and Blood Institute (NHLBI) Program Project Grant HL-24075. L. Sbragia was supported by Faculdade de Ciencias Médicas-Universidade Estudual de Campinas and Fundação de Amparo a Pesquisa do Estado de São Paolo-The State of São Paulo Research Foundation Grant 99/03870-7. Jorge Gutierrez received support from NHLBI Grant KO1 HL-04372 and American Lung Association Grant RG 046-NL.


    FOOTNOTES
 

Address for reprint requests and other correspondence: C. J. Chapin, Univ. of California, San Francisco, Cardiovascular Research Institute, Box 1245, 3333 California St., Ste. 150, San Francisco, CA 94118 (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.


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