Department of Pediatrics, Women & Infants' Hospital of Rhode Island and Brown Medical School, Providence, Rhode Island 02905
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ABSTRACT |
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Development of the pulmonary air sacs is crucial for extrauterine survival. Late fetal lung development is characterized by a thinning of the mesenchyme, which brings pneumocytes and endothelial cells into apposition. We hypothesized that mechanical stretch, simulating fetal breathing movements, plays an important role in this remodeling process. Using a Flexercell Strain Unit, we analyzed the effects of intermittent stretch on cell proliferation and apoptosis activation in fibroblasts isolated from fetal rat lungs during late development. On day 19, intermittent stretch increased cells in G0/G1 by 22% (P = 0.001) and decreased in S phase by 50% (P = 0.003) compared with unstretched controls. Cell proliferation analyzed by 5-bromo-2'-deoxyuridine incorporation showed a similar magnitude of cell cycle arrest (P = 0.04). At this same gestational age, stretch induced apoptosis by two- to threefold over controls, assayed by DNA flow cytometry, terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick-end labeling, and caspase-3 activation. These results indicate that mechanical stretch of fibroblasts isolated during the canalicular stage inhibits cell cycle progression and activates apoptosis. These findings are cotemporal with the mesenchymal thinning that normally occurs in situ.
flow cytometry; 5-bromo-2'-deoxyuridine incorporation; programmed cell death; terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick-end labeling assay; caspase-3
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INTRODUCTION |
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DURING FETAL LUNG DEVELOPMENT, the distal lung undergoes dramatic anatomic and functional changes required for optimal postnatal gas exchange. Developmentally timed, specific temporal-spatial interactions between mesenchymal and epithelial cells permit gradual establishment of an effective air-blood barrier. This process, initiated in utero during the transition from pseudoglandular to canalicular/saccular stages of lung development, is characterized by epithelial and endothelial cell proliferation and differentiation and by thinning of the mesenchyme. These events remodel the distal air sacs and bring pneumocytes, mesenchymal cells, and capillary endothelial cells into apposition (1). This remodeling process is completed after birth during the alveolar stage.
The importance of mechanical forces in fetal lung development is well established. During intrauterine life, the fetus makes episodic breathing movements starting in the first trimester and increasing in frequency to 30% of the time by birth (20). The fetal lung also actively secretes fluid into the tissue lumen, creating a constant transpulmonary pressure in the potential airway and air spaces (41). Studies in several species have demonstrated that both forces are necessary for normal pulmonary growth and maturation (25, 29, 36). Recently, Schittny et al. (44) elegantly demonstrated that fetal airway smooth muscle contraction exerted throughout gestation was associated with forward displacement of the lung liquid and maintenance of a positive intraluminal pressure, suggesting a role for mechanical distortion of the surrounding fetal pulmonary epithelium and mesenchyme on lung growth.
Tissue remodeling requires coordinate regulation of cell proliferation and cell death or apoptosis. Glucksmann (16), in 1965, first emphasized the concept of apoptosis or programmed cell death in embryology. Since then, numerous studies have demonstrated the central role of apoptosis in normal development of the kidney, heart, immune, and nervous systems, etc. (9, 22, 27, 28).
Apoptosis as a physiological event during lung development was initially described by Scavo et al. (42) and Kresch et al. (26) in 1998. They demonstrated the presence of interstitial cell apoptosis in fetal lungs during transition from the pseudoglandular to saccular stages of lung development. Mesenchymal apoptosis is also significantly increased around birth (26) and during the third postnatal week in conjunction with alveolar septal formation (6, 43). In contrast, apoptosis occurs at very low levels during earlier stages of lung morphogenesis (23).
The role of stretch-induced apoptosis in lung development has been suggested by De Paepe et al. (14), who showed that the depletion of type II cells after tracheal ligation in fetal rabbits was due to an increase in apoptotic activity. However, information about the effect of physiological cyclic stretch on fetal lung interstitial cells is lacking.
This study was undertaken to assess the role of intermittent stretch in the mesenchymal remodeling process during late fetal lung development. Using an in vitro model system, we demonstrate that mechanical stretch, simulating fetal breathing movements, inhibits proliferation and induces apoptosis in isolated fetal rat lung fibroblasts. The developmentally timed responsiveness of fibroblasts to mechanical forces corresponds to the transition from the canalicular to saccular stages of lung development.
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MATERIALS AND METHODS |
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Fibroblast isolation and mechanical distention. Fetal lungs were obtained from timed-pregnant Sprague-Dawley rats (Charles River, Wilmington, MA) at different gestational ages (days 18-20; term = 22 days). The time of copulation was designated as day 0. After hysterotomy, fetal lungs were dissected under sterile conditions and cleared of major airways. The tissues were finely minced and digested with collagenase type I and type IA (each 0.5 mg/ml, Sigma, St. Louis, MO) with vigorous pipetting for 15 min at 37°C as previously described (2). The suspension was centrifuged at 400 × g for 5 min, and the pellet was resuspended in Dulbecco's modified Eagle's medium (DMEM) with 10% (vol/vol) fetal bovine serum (FBS) and sequentially filtered through 100-, 30-, and 20-µm nylon meshes. The filtrate was plated into 75-cm2 flasks and incubated at 37°C in an atmosphere of 95% air-5% CO2 for 30-60 min to allow fibroblasts to adhere. The attached fibroblasts were maintained overnight in DMEM with 1% ITS+ (Collaborative Biomedical Products, Bedford, MA).
After overnight culture, fibroblasts were harvested with 0.25% (wt/vol) trypsin in 0.4 mM EDTA and plated at a density of 5 × 105 cells/well (~60% confluence) on Bioflex multiwell plates precoated with collagen I (Flexcell, Hillsborough, NC). Cultures were maintained for 24 h in serumless defined medium containing DMEM with 1% ITS+ (2 ml/well) and then were mounted in a Flexercell FX-3000 Strain Unit (Flexcell) atop flat-head Delrin cylinders. Application of vacuum stretches each membrane over the central cylinder post, creating a uniform radial and circumferential strain across the membrane surface. Radial elongation of 5% was applied at intervals of 60 cycles per min (cpm), 15 min/h for fixed intervals up to 24 h. This regimen was chosen based on mean hourly rate of human fetal breathing movements observed by Patrick et al. (37) and ultrasound studies by Harding and Liggins (19) as has been described by Liu and colleagues (31). Cells grown in stationary nonstrained Bioflex collagen-coated plates were otherwise treated in an identical manner and served as controls. Cultures were examined by phase-contrast microscopy and trypan blue exclusion to verify cell attachment and viability after mechanical distention. Purity (>95%) was assessed by light microscopy and by immunochemical staining for the cytoskeletal marker vimentin.Cell cycle analysis. The percentage of cells in each phase of the cell cycle was analyzed by DNA flow cytometry. Cultured fibroblasts from stretched and control conditions on each gestational day (days 18-20) were harvested and centrifuged at 400 × g for 5 min. The resulting pellets were then washed with cold PBS and resuspended in 0.5 ml of propidium iodide (PI) solution [7.5 µM PI, 0.1% (vol/vol) Nonidet P-40, 0.1% (wt/vol) sodium citrate] and incubated for 30 min at room temperature in the dark. Samples were placed on ice and immediately analyzed on a FACS Sort flow cytometer (Becton-Dickinson Immunocytometry Systems, San Jose, CA) equipped with an argon laser set at 488 nm and Modifit LT software (Verity Software House, Topsham, ME) to separate G0/G1, S, G2/M, and hypodiploid (apoptotic) nuclei.
5-bromo-2'-deoxyuridine incorporation.
Measurement of cell proliferation was analyzed by DNA incorporation of
the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) as described by the
manufacturer (Boehringer Mannheim, Mannheim, Germany). Briefly,
cultures (~60% confluence) were maintained in the mechanically
active conditions or not, and immediately before each experiment, fresh
medium containing 10 µM of BrdU labeling reagent was added to each
well. At the end of each experiment, monolayers were washed with PBS
and then fixed in 70% ethanol for 20 min at 20°C. Cells were then
washed and incubated with mouse anti-BrdU antibody (negative controls
were incubated with PBS) followed by incubation with
fluorescein-conjugated secondary antibody and mounting with Vectashield
mounting medium with 4,6-diamidino-2-phenylindole (DAPI, Vector
Laboratories). Slides were examined, photographed, and counted
under an Olympus bright-field fluorescence microscope.
Terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick-end labeling assay. Detection and quantification of apoptotic cells was performed by use of terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick-end labeling (TUNEL), by a fluorescein label apoptosis detection system (Promega, Madison, WI). On the day of isolation, fibroblasts from different gestational ages were cultured on Bioflex plates and subjected to mechanical stretch or parallel-unstretched controls for 24 h. Cells were then washed with PBS and fixed in freshly prepared 4% paraformaldehyde in PBS for 25 min at 4°C. After a second wash, cells were permeabilized by immersion in 0.2% Triton X-100 in PBS. Positive controls consisted of cells treated with DNase I (0.5 µg/ml) in DNase buffer [40 mM Tris · HCl (pH 7.9), 10 mM NaCl, 6 mM MgCl2, and 10 mM CaCl2] to induce DNA fragmentation. Monolayers were incubated at 37°C for 60 min in equilibration buffer, 2-deoxynucleotide 5'-triphosphates, and terminal deoxynucleotidyltransferase (TdT) enzyme as per manufacturer's protocol. A negative control was prepared omitting the TdT enzyme. Samples were washed in PBS, mounted with Vectashield mounting medium with PI (Vector Laboratories), and immediately analyzed by fluorescence microscopy. For quantification of apoptotic cells, a minimum of 25 high-power fields per sample was analyzed (~100 cells/field). Areas from each membrane quadrant were randomly chosen and photographed. Two persons masked to the experimental conditions examined each sample. Cells containing green fluorescence and either nuclear condensation or chromatin fragmentation (without nuclear morphological changes) were identified as apoptotic cells. Results were expressed as apoptotic index (number of apoptotic cells per number of total cells).
Detection of cleaved caspase-3 activity by Western blot.
Fibroblasts obtained from different gestational days (days
18-20) were processed as described in the TUNEL assay method
and subjected to experimental conditions for different lengths of time.
At the end of each experiment, monolayers were washed with PBS and
lysed by adding extraction buffer (1% Triton X-100, 10 mM Tris-base pH
7.6, 5 mM EDTA, and 50 mM NaCl) containing protease inhibitors (10 µg/ml leupeptin, 10 µg/ml aprotinin, and 143.5 µM aminoethyl
benzenesulfonyl fluoride). Lysates were centrifuged, and the
supernatants were stored at 80°C until processing. Total protein
contents were determined by the BCA protein assay (Pierce Chemical,
Rockford, IL). Protein samples (30 µg/lane) were separated by 16.5%
SDS polyacrylamide gel electrophoresis (PAGE) and transferred to
polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA). Membranes were incubated for 1 h at room temperature in blocking buffer (Tris-buffered saline + Tween 20 with 5% nonfat dry milk) to reduce nonspecific binding. To detect the active isoform of caspase-3, blots were then incubated with a cleaved caspase-3 primary
antibody (Cell Signaling, Beverley, MA) diluted 1:1,000 in blocking
buffer for 2 h at room temperature. After washing, secondary
antibody (donkey anti-rabbit-horseradish peroxidase, diluted 1:2,000 in
blocking buffer) was added for 1 h at room temperature.
Immunoreactive caspase-3 was detected by enhanced chemiluminescence
(Amersham, Piscataway, NJ). The densities of the cleaved caspase-3
protein bands were analyzed by densitometry.
Ribonuclease protection assay.
Total cellular RNA was isolated from lung fibroblasts using the
single-step method as previously described (40).
Ribonuclease protection assay (RPA) was performed using the RPA II
procedure (Ambion) with minor modifications.
[-32P]UTP-labeled antisense cRNA probes were
synthesized from a rat apoptosis multi-probe template set
(rAPO-1; PharMingen, San Diego, CA) using an in vitro transcription kit
(Promega), RNA polymerase, and [
-32P]UTP (Amersham).
Unincorporated nucleotides were separated from the RNA probes by
affinity chromatography on an Elutip column (Schleicher & Schuell). For
each experimental tube, 10-20 µg of total RNA were mixed with
106 cpm of probe, EtOH precipitated, resuspended in 20 µl
of hybridization buffer [80% deionized formamide, 100 mM sodium
citrate (pH 6.4), 300 mM NaAc (pH 6.4), and 1 mM EDTA], denatured, and
hybridized overnight at 45°C. Unhybridized RNA was digested to free
ribonucleotide triphosphates and short oligonucleotides by the addition
a 1:100 dilution of RNase A+ T1 mix and incubated for 30 min at 37°C. The RNases were inactivated in the same tubes
and the RNA EtOH was precipitated. Protected fragments (RNA:RNA
probe:target duplexes) were resolved using a 5% polyacrylamide/8 M
urea gel. For each experiment, control lanes contained probes
hybridized to sheared yeast tRNA. The gel was mounted on filter paper
and exposed to X-ray film with intensifying screens. Differences in
band signal intensity were adjusted to intensities of the
constitutively expressed L32 and glyceraldehyde-3-phosphate
dehydrogenase RPA bands.
Statistical analysis. Results are expressed as means ± SD of three to five experiments, using different litters each time. Stretched samples were compared with controls by unpaired Student's t-test. For multiple-group comparisons, one-way analysis of variance (ANOVA) followed by the post hoc Fisher's test were used. P < 0.05 was considered statistically significant.
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RESULTS |
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Effects of mechanical stretch on fibroblast cell cycle regulation.
To assess the effect of mechanical stretch on fibroblast cell
proliferation, we analyzed the distribution of timed-gestation isolated
pulmonary fibroblasts in each phase of the cell cycle by flow
cytometry. Five samples obtained from different litters were cultured
in serumless medium ± the mechanically active environment on
gestational days 18, 19, and 20, which
correspond to late intrauterine periacinar remodeling in the rat. On
day 18 of gestation (corresponding to pseudoglandular stage
of fetal lung development), stretched fibroblasts showed no changes in
G0/G1, S, or G2/M phases of the cell cycle or in the hypodiploid (apoptotic) population compared with unstretched matched controls (Fig.
1A). In contrast, on day 19 (early canalicular stage), stretched fibroblasts showed a
statistically significant 22% increase in cell accumulation in
G0/G1 (P = 0.001) and a 54%
decrease in S phase (P = 0.003) compared with
unstretched controls. Mechanical stretch also increased the
apoptotic fraction from 0.1 to 3% (P = 0.03) (Fig.
1B). Day 20 fibroblast cultures (saccular stage),
like the day 18 cultures, did not show significant stretch-inducible changes in cell cycle kinetics or apoptosis, assessed by these methods (data not shown).
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Effects of intermittent stretch on lung fibroblast cell
proliferation: developmental timing.
To investigate further this inhibitory effect of mechanical distension
on G1 to S phase transition, we directly assayed cell proliferation. Fetal lung fibroblasts isolated on days
18-20 were analyzed by fluorescence immunocytochemistry to
assess DNA incorporation of the thymidine analog BrdU. As seen in Fig.
2, on day 18, stretch had no
effect on BrdU incorporation (n = 5). In contrast, on
day 19, stretch decreased BrdU incorporation by 42%
compared with unstretched controls (n = 5, from 5 different litters, P = 0.04). On day 20,
BrdU incorporation also was not affected by cyclic mechanical stretch
(n = 4). Figure 3 depicts
representative fluorescence immunocytochemistry fields from day
19 fibroblast cultures. Figure 3A (unstretched
fibroblasts) shows that approximately half of these cells have entered
S phase, indicated by nuclear labeling with a Texas red-conjugated
anti-BrdU antibody. Nuclei were counterstained with DAPI (blue). In
contrast, stretched fibroblast cultures (Fig. 3B)
incorporated less BrdU, shown as fewer and less intense red-staining cells.
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Developmental timing of mechanical stretch-inducible lung
fibroblast apoptosis activation.
Because apoptosis plays an important role in fetal lung
morphogenesis, we studied the role of mechanical stretch in inducing apoptosis. In these experiments, 3-4 different litters
were analyzed at each gestational age. On days 18 (pseudoglandular stage) and 20 (saccular stage), mechanical
stretch did not alter the number of TUNEL-positive cells, compared with
controls. In contrast, on day 19 (early canalicular stage),
intermittent stretch increased the number of TUNEL-positive cells by
twofold, compared with controls (P = 0.01) (Fig.
4). A representative day
19 TUNEL fluorescence photomicrograph is shown in Fig.
5. Apoptotic cells, which
show nuclear fragmentation and DNA condensation, are stained green, indicating incorporation of FITC-conjugated dUTP. Nuclear and mitochondrial DNA and ribosomal RNA are labeled red by PI. Figure 5A shows a representative unstretched sample. In Fig.
5B, stages of apoptosis are apparent by intensity of
the FITC label and morphology of the cells. Figure 5, C
and D, shows a positive and negative control,
respectively. These fluorescence immunocytochemistry findings were
confirmed by examining cell ultrastructure. Figure 6 illustrates a representative electron
microphotograph of stretched fibroblasts on day 19. We
observed fragmentation and condensation of chromatin characteristic of
apoptosis. This apoptotic cell has been engulfed by a
neighboring fibroblast.
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Effect of intermittent stretch on caspase-3 activation.
To confirm further the gestational age-dependent activation of
apoptosis by mechanical stretch, we analyzed fibroblasts
isolated from different gestational ages (days 18-20)
by Western blot to detect the cleaved, active caspase-3 isoform. As
observed in Fig. 7, on day 18,
mechanical stretch did not affect caspase-3 activation, compared with
unstretched control samples. On day 19, however, after
16 h of intermittent stretch, activated caspase-3 was increased compared with matched unstretched samples. On day 20, we
observed an increase of basal apoptotic activity and activated
caspase-3 in unstretched samples compared with earlier gestational
days. However, mechanical stretch did not further induce caspase-3
activation.
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Effect of mechanical stretch on Fas/Fas ligand mRNA expression.
To assess if activation of the Fas/Fas ligand (FasL) system was
involved in stretch-induced apoptosis in fetal lung
fibroblasts, we analyzed fibroblast RNA isolated on different
gestational ages (days 18-20) and subjected to several
durations of stretch (1-24 h). Using multiplex RPA, we detected no
significant differences in Fas mRNA expression between unstretched and
stretched samples. FasL mRNA was not detectable in any of our samples.
These data suggest that Fas/FasL is not involved in stretch-induced
apoptosis in isolated fetal lung fibroblasts. A representative
RPA blot from fibroblasts isolated on day 19 of gestation is
depicted in Fig. 8.
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DISCUSSION |
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The transition from late pseudoglandular to saccular stages of lung development is characterized by a specific spatial-temporal pattern of apoptotic activity (13, 14, 26). During this period, interstitial cell apoptotic activity increases as the periacinar mesenchyme thins and capillaries and epithelium approximate. From the canalicular stage onward, fetal type II cell apoptosis also contributes to late gestational and early postnatal lung remodeling.
In this study, we hypothesized that mechanical forces are important in inducing thinning of the periacinar mesenchyme during late fetal lung development. To test this hypothesis we isolated fetal rat lung fibroblasts on different gestational ages and determined the effects of cyclic stretch using distention and frequencies similar to fetal breathing patterns. Our first approach was to assess the effect of stretch on cell cycle distribution by flow cytometry. To minimize phenotypic changes in cultures, cells were maintained in serum-free defined medium before and during the experimental conditions. We observed that cyclic distention of fibroblasts isolated during the canalicular stage (day 19) inhibited cell cycle progression; this was manifested by an increase in the percentage of cells in G0/G1 and a decrease in S phase. The effect of intermittent stretch on inhibition of cell proliferation was confirmed by BrdU incorporation. Our data suggest that late-gestation fetal lung fibroblasts respond to stretch with a developmentally time dependent decrease in proliferation.
An important observation derived from our experiments was the significant increase in the percentage of BrdU-positive cells on unstretched day 19 samples compared with days 18 and 20, suggesting that mechanical stretch may interfere with a normal developmental increase in proliferation and contribute to mesenchymal thinning in vivo by interfering with fibroblast G0/G1 to S phases transition. These findings differ from those of Bishop et al. (5), who found that 10% elongation increased cell proliferation in IMR-90 human fetal lung fibroblasts. They observed that medium from mechanically deformed cells was mitogenic for IMR-90 cells, suggesting that stretched fibroblasts release autocrine growth factors. Differences between the two studies might be attributable to the greater magnitude of distension used by these investigators and/or the use of a transformed cell line. Liu et al. (32), using organotypic cultures of fetal rat lung cells isolated on day 19 of gestation, showed mechanical stretch stimulated epithelial cell and fibroblast proliferation. This apparent discrepancy with our results might be explained by their use of organotypic cultures (which show automaturation in culture) instead of fibroblast monolayers and the different experimental conditions. However, the same group of investigators (46) also demonstrated that the effect of mechanical stretch on fibroblast proliferation was gestational-age dependent, peaking at the early canalicular stage and decreasing thereafter.
Our study demonstrates the importance of timing on mechanical stretch-induced lung development. Caniggia et al. (7) investigated whether the regression of the mesenchyme during late development was controlled by epithelial-mesenchymal interactions. They observed that conditioned medium from rat lung epithelial cells isolated during the canalicular stage of lung development (but not from the pseudoglandular or saccular stages) inhibited fetal lung fibroblast proliferation, suggesting that fetal lung epithelial cells elaborate factor(s) that inhibit fetal lung fibroblast proliferation. Our data indicate that mechanical distension of fetal lung fibroblasts also has direct effects on cell proliferation in the absence of epithelial cells or serum factors. Our findings demonstrate that the responsiveness of lung mesenchymal cells to mechanical stretch is developmentally regulated. Similar inhibitory effects of physiological cyclic stretch on cell proliferation have recently been reported in cultured vascular smooth muscle cells (10, 21) and in endothelial cells subjected to laminar shear stress (30).
The activation of apoptosis by mechanical forces has been shown in several cell types (8, 12). Stretch-induced apoptosis in lung development was demonstrated by De Paepe et al. (14). They showed that tracheal ligation and consequent fetal lung distension in rabbit pups induced a progressive increase of interstitial and epithelial apoptotic activity with advancing gestation. Edwards et al. (15) observed that cyclic stretch with 22% radial elongation in cultured rat type II cells induced apoptosis. However, in both studies, tissue and cell distention were greater than those considered "physiological" during gestation.
In the current study, we used cyclic intermittent stretch to simulate in vivo conditions. Apoptosis was distinguished by DNA flow cytometry, TUNEL assay, and caspase-3 activation. Our data show that culture of pseudoglandular and saccular stage lung fibroblasts in a mechanically active environment did not increase apoptotic index. In contrast, in canalicular stage fibroblasts, mechanical stretch increased apoptosis, assayed by several different endpoints.
The initiation and execution phases of apoptosis involve activation of a family of cytoplasmic aspartate-specific cysteine proteases known as caspases (17). Caspases cause cell death by degrading critical structural elements and activating proteolytic enzymes. Caspase-3 is seen as one of the key executioners of apoptosis, being responsible for the cleavage of crucial substrates in the final degradation phase. Activation of caspase-3 requires proteolytic cleavage of its inactive zymogen into active p17 and p12 subunits. To confirm our flow cytometry and TUNEL assay findings, we used Western blot detection of the active (cleaved) caspase-3 isoform. On days 18 and 20, mechanical stretch did not affect caspase-3 activation. On day 19, intermittent stretch consistently increased activated caspase-3 accumulation. The discrepancy between gene and protein expression on caspase-3 activation may be explained by posttranscriptional activation. As shown by Raff and colleagues (38), several cell types can express the cell death machinery constitutively at all times, and upon removal of survival signals, intrinsic death program is activated without de novo gene expression.
An intriguing question is why fibroblasts isolated from early canalicular stage of lung development specifically respond to mechanical stretch with cell cycle arrest and activation of apoptosis. Scavo et al. (42) did not observe any differences in the incidence of apoptosis of fetal lung cells in vivo from days 16 through 22 of gestation. The low apoptotic index (between 0 and 3%) and efficient clearance mechanisms might explain this lack of in vivo detection. Early canalicular lung undergoes critical architectural changes. We and others (32, 39, 46) have demonstrated that mechanical forces exert maximal effects on epithelial cell proliferation and pulmonary surfactant production during this period of lung development. Although caution is warranted in extrapolating from these in vitro experiments, our results suggest that fibroblasts become responsive to mechanical deformation at a particular point during lung development so that interstitial cell proliferation slows, apoptosis is activated, and tissue remodeling takes place.
The signaling pathways by which mechanical stretch induces apoptosis remain to be elucidated. Activation of the Fas/FasL system results in type II cell apoptotic remodeling in the developing pulmonary acinus (13) and after injury in adult lung (4, 33). During early pulmonary development, FasL production is localized in primitive bronchial epithelium. With increasing architectural and cellular maturation, FasL is detected in bronchial and bronchiolar Clara cells and in a majority of type II cells (13). We recently reported that Fas and FasL are not detectable in fetal lung interstitial cells by immunohistochemical techniques (13). Using more sensitive assays, in the present studies we detected Fas but not FasL mRNA in isolated fetal lung fibroblasts. Consequently, although the Fas/FasL system appears to play a pivotal role in late-gestation fetal type II cell apoptosis, mechanical forces induce fetal lung fibroblast apoptosis via signaling pathways independent of Fas ligation.
The cell-adhesion integrin receptors influence cell-cycle progression, cell survival, and gene expression (45). The survival of many anchorage-dependent cell types requires integrin-mediated adhesion to the extracellular matrix (ECM) (35), and fibronectin peptides may differentially regulate lung epithelial and fibroblast apoptosis via interaction with integrin-mediated survival signaling (3, 18). White et al. (47) showed that disruption of actin filament integrity induced apoptosis in airway epithelial cells. The loss of anchorage to the ECM (anchorage-dependent cell-cycle progression) (34), changes in cell shape (11), or direct changes in the nucleus through the cytoskeleton induced by mechanical forces may be important factors to trigger apoptosis.
In summary we demonstrate that intermittent mechanical stretch of fetal fibroblasts inhibits cell proliferation and induces apoptosis. These events take place during early canalicular lung development, a period when dramatic thinning of mesenchyme occurs in situ. The mechanism(s) by which mechanical forces induces apoptosis in fetal lung development remain to be determined.
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ACKNOWLEDGEMENTS |
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The authors thank Virginia Hovanesian for technical assistance with image analysis and Regina Allen for manuscript preparation.
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FOOTNOTES |
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This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-55268.
Address for reprint requests and other correspondence: J. Sanchez-Esteban, Dept. of Pediatrics, Women & Infants' Hospital, 101 Dudley St., Providence, RI 02905 (E-mail: jsesteba{at}wihri.org).
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.
10.1152/ajplung.00399.2000
Received 13 November 2000; accepted in final form 25 April 2001.
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