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Published ahead of print on January 31, 2003, doi:10.1165/rcmb.2002-0135OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 81-87, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0135OC

Continuous Mechanical Contraction Modulates Expression of Alveolar Epithelial Cell Phenotype

Jorge A. Gutierrez, Vincent V. Suzara and Leland G. Dobbs

Departments of Pediatrics and Medicine and the Cardiovascular Research Institute, University of California at San Francisco, San Francisco, California

Address correspondence to: Jorge A. Gutierrez, Department of Pediatrics, University of California, San Francisco, 3333 California St., Suite 150, San Francisco, CA 94118. E-mail: jgut{at}itsa.ucsf.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously reported that mechanical distention of alveolar epithelial type II cells in culture favored the expression of the type I cell phenotype and inhibited the expression of the type II cell phenotype. The objective of the present study was to investigate the effects of continuous mechanical contraction on the expression of specific markers for the type I and type II cell phenotypes in cultured alveolar type II cells. Type II cells were mechanically contracted in culture at varying amplitudes and times. Cells were analyzed for mRNA and protein content of markers of the type I (RTI40) and type II (surfactant proteins [SPs] A, B, and C) phenotypes. Continuous contraction of culture membrane surface area by 25% for a duration of 4 h resulted in an 83% increase in SP-A, a 42% increase in SP-B, and a 230% increase in SP-C, in comparison with controls. After 12 h of contraction, RTI40 mRNA content decreased to 59% of control levels. A minimal contraction of 20% of culture membrane surface area was required to modulate expression of the type II cell markers. In summary, mechanical contraction favors expression of the type II cell phenotype and inhibits expression of the type I cell phenotype in a time- and amplitude-dependent manner.

Abbreviations: phosphate-buffered saline, PBS • surfactant protein, SP • tris-buffered saline containing 0.05% Tween, TBS-T


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mechanical factors are believed to exert potent influences on lung growth and alveolar epithelial differentiation both during fetal development and in adults. The initial concept that mechanical factors are important in these processes developed from clinical observations and experiments with fetal model systems. Congenital diaphragmatic hernia, which causes underdistention of the developing lung in utero, causes pulmonary hypoplasia and surfactant deficiency (1–4). Overdistention of the lung, for example, in congenital laryngeal atresia, results in large lungs with an increased number of alveoli and a more mature architecture than expected for gestational age (5).

From studies performed on fetal lungs in utero (6, 7), there were qualitative conclusions that maintaining the fetal lung in an overdistended state by tracheal or bronchial ligation favored expression of the type I phenotype while inhibiting expression of the type II phenotype; underdistention or contraction of fetal lung in utero by chronic tracheal or bronchial drainage had the opposite effects. Although these initial studies did not report quantitative data, such as determining the numbers of type II cells or measuring biochemical or molecular markers for the type I or type II cell phenotypes, other investigators subsequently demonstrated that increased lung expansion secondary to tracheal ligation alters the proportions of type I and type II cells in the fetal lung (8, 9), led to decreased amounts of surfactant protein (SP)-A and saturated phosphatidylcholine (10), and a decrease in mRNAs for SP-A, -B, and -C mRNA (11, 12). In contrast to tracheal obstruction, underdistention of fetal lung caused by tracheal drainage of lung fluid resulted in an increase in SP-C mRNA content (12), and underdistention caused by oligohydramnios resulted in a decrease in mRNA content of RTI40, a protein specific in the lung to the apical surface of type I alveolar epithelial cells (13). In summary, data from these in vivo studies support the initial qualitative observations.

Mechanical forces are potent regulators of cell phenotype in vitro. Continuous mechanical distention in vitro of both fetal rat lung explant tissue (14) and type II cells isolated from adult rat lungs (15), favors the expression of RTI40, a marker of the type I cell phenotype, and inhibits the expression of SP-B and SP-C, markers of the type II cell phenotype. In these studies, the effects were due at least in part to changes at the transcriptional level. Type II cells cultured on detached, floating collagen gels, appear to maintain the type II cell phenotype (16). Although it is not clear whether these findings are due to cell contraction or other factors such as an air–liquid interface, contraction of the gels favored expression of the markers of the type II cell phenotype.

The experiments in this communication were designed to test the hypothesis that mechanical contraction favors the expression of the type II cell phenotype and inhibits the expression of the type I cell phenotype. The results demonstrate the plasticity of phenotypic expression of fully differentiated type II cells and its modulation by mechanical forces.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of Alveolar Type II Cells
Alveolar type II cells were isolated from the lungs of pathogen-free adult male Sprague-Dawley rats weighing 180–200 g (Charles River, Hollister, CA) by previously described methods (17). Porcine pancreatic elastase was purchased from Boehringer Mannheim, Indianapolis, IN.

Cell Culture and Mechanical Contraction
Cells were cultured on fibronectin-coated Bioflex six-well tissue culture membranes (Flexcell, McKeesport, PA) that were maintained in the distended state using the Flexcell 3,000 Strain Unit (Flexcell). Membranes were placed on a loading station and stretched continuously by applying a vacuum to the underside of the membranes. To vary the amount of subsequent contraction, cells were cultured on membranes, which were initially stretched biaxially by 25%. Once the membranes were distended, 2 x 106 cells were placed in each well and allowed to adhere to the distended membrane for 18–20 h. Control cells were placed on nonstretched membranes, as the current model system does not allow for removal of the vacuum from only a portion of the membranes on the baseplate, because all of the distended membranes would have contracted. The plating efficiency of cells was between 75 and 85%. Nonadherent cells were removed and fresh media added. The vacuum was then released, allowing the membranes to contract, and cells were harvested at 4, 12, 24, and 48 h after contraction began. To determine if there was a threshold amount of contraction, membranes were contracted 10%, 15%, 20%, and 25% for 12 h. After the experimental period was complete, tissue culture media was removed and membranes were washed twice with sterile phosphate buffered saline (PBS) at 4°C. Cells were harvested and samples processed as described below.

Cell Viability
Cell viability was determined by epiflourescent microscopy using the Live/Dead Viability/Cytotoxicity kit (Molecular Probes, Eugene, OR). Briefly, 0.23 µM ethidium homodimer-1, which enters through the damaged cell membrane of dead or damaged cells and binds to nucleic acids, and 0.12 µM calcein-AM, which is retained in living cells, was added to control and contracted cells. We evaluated cell viability at a contraction magnitude of 25% for durations of 24 and 48 h. At the end of the study period, images were captured using a Nikon Eclipse TE300 inverted microscope with a x20 objective (Scientific Instruments, Palo Alto, CA), a digital RT Slider camera (Diagnostic Instruments Inc., Sterling Heights, MI), and Spot RT software (Diagnostic Instruments Inc.). Live and dead cells were counted in three random locations in each of six control membranes and six contracted membranes. Viability was calculated as a percentage of live cells per the sum of live plus dead cells.

Preparation of RNA
Cells were harvested in RNA-STAT 60 (Tel-Test, Friendswood, TX). Total cellular RNA was extracted with chloroform, precipitated with isopropanol, and was quantitated spectrophotometrically. RNA integrity was assessed by electrophoresis and ethidium-bromide staining for rRNA.

Northern Blotting
Total RNA (10 µg/sample) was separated electrophoretically on 1% agarose gels. Total RNA was transferred to nylon membranes by downward capillary action and cross-linked with UV light (UV Stratalinker 2400; Stratagene, La Jolla, CA). Filters were probed with full-length cDNAs for rat SP-A, SP-B, SP-C, RTI40, and 18S rRNA labeled with {alpha}32P-dCTP (NEN Research Products, Boston, MA) by random-primer second strand synthesis (Random Primer Labeling Kit, GIBCO/BRL, Gaithersburg, MD). Filters were prehybridized for 10 min in QuikHyb Hybridization Solution (Stratagene) at 68°C. Filters were then hybridized in 10 ml of QuikHyb solution containing 1.25 x 106 dpm/ml for 18 h. Hybridized filters were washed under high stringency conditions and subjected to autoradiography (Hyperfilm; Amersham, Buckinghamshire, UK) before quantifying radiolabeled bands by volume integration of pixels measured by phosphorimage analysis (Imagequant Software; Molecular Dynamics, Sunnyvale, CA.). Using 18S rRNA as control ensured equal loading.

RNase Protection Assay
We utilized the RiboQuant Multi-probe RNase protection assay system (PharMingen, San Diego, CA) for the T7 polymerase-directed synthesis of [32P]-UTP labeled antisense RNA probes for SP-A, SP-B, SP-C, and RTI40. 18S rRNA probes (Ambion, Austin, Tx) were synthesized separately and 18S probe specific activity was lowered by adding cold UTP to the reaction. Probe synthesis was terminated by incubation with DNase I at 37°C for 30 min. Riboprobes were isolated using Tris-buffered phenol-chloroform-isoamyl alcohol separation and ethanol precipitation. Unprotected probes were taken up in 50 µl of hybridization buffer, and 1 µl samples were quantitated in a scintillation counter. 0.5 µg of total RNA was hybridized with 2–3 x 106 cpm of probe mix and 2–3 x 104 cpm of 18S probe at 56°C for 12–16 h in hybridization buffer. Samples then underwent RNase digestion for 45 min at 30°C, followed by Proteinase K digestion for 15 min at 37°C. Protected probes were then isolated using phenol-chloroform-isoamyl alcohol separation and ethanol precipitation. Protected probes, undigested and digested unprotected probes, and markers were then resolved on 6% denaturing polyacrylamide gels. Gels were dried under vacuum for 2 h at 80°C. Gels were then subjected to autoradiography (Kodak X-OMAT, Rochester, NY) before quantifying radiolabeled bands by volume integration of pixels measured by phosphorimage analysis (Imagequant Software; Molecular Dynamics). Equal loading was assessed by measuring 18S rRNA.

Protein Determination
Protein content was measured by the bicinchoninic acid method (Pierce, Rockford, IL).

Quantification of SP-A, SP-B, and RTI40 Protein by Dot Blotting
SP-A, SP-B, and RTI40 protein were assayed by quantitative dot blotting as previously described (13). Duplicate dots of serial dilutions of samples and standards were assayed on the same piece of nitrocellulose. Protein samples from control and contracted cells were diluted 1:100 with 50 mM NaHCO3, pH 9.0, and dot blotted onto nitrocellulose. Endogenous peroxidase activity was quenched by treatment with 15% hydrogen peroxide for 5 min, and nonspecific binding was blocked by a 1-h incubation in a solution of 1% nonfat milk, 0.4% gelatin, 0.1% bovine serum albumin, 0.9% NaCl, and 10 mM Tris-buffered saline, pH 7.2. For each marker, an appropriate primary antibody was added to blocking buffer, and blots were incubated for 20 min. We used a monoclonal antibody against RTI40 (18), and rabbit polyclonal antibodies against sheep SP-A and SP-B (a kind gift of Dr. S. Hawgood, UCSF, San Francisco, CA), which are immunoreactive to rat SP-A and rat SP-B by immunohistochemistry and Western blot analysis (13). The blots were washed 20 times with 20 mM tris-buffered saline, pH 7.4, containing 0.05% Tween (TBS-T), then incubated with a solution containing peroxidase-labeled donkey anti-rabbit secondary antibody (SP-A and SP-B; Amersham) or peroxidase-labeled goat anti-mouse antibody (RTI40; Amersham) in TBS-T (1:5,000). After a 20-min incubation, unbound secondary antibody was removed by washing the blot 10 times in TBS-T. Bound secondary antibody was detected by exposure to luminol (ECL Light Detection System; Amersham) for 1 min. Relative light units were measured immediately after exposure to luminol in a plate luminometer (Packard Instrument Co., Downers Grove, IL) before autoradiography. Quantitation was performed only within the linear range of the standard curve.

Immunocytochemistry
Control and contracted cells were fixed in place for 2 h at room temperature in 2% freshly prepared paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4).

The cells were rinsed in buffer (0.1% bovine serum albumin and 0.3% triton in PBS), blocked with 10% goat serum in PBS for 1 h at room temperature. The cells were prepared for immunofluorescence by incubation with antibodies against RTI40 (1:200) and SP-B (1:200) as described above. The cells were rinsed again before 1-h incubation with goat anti-mouse IgG1 conjugated to Alexa Fluor 594 (1:2,000) and goat anti-rabbit IgG conjugated to Alexa Fluor 488 (1:2,000; Molecular Probes, Eugene, OR). After rinsing again, the cells were reacted with goat anti-rabbit IgG (1:2,000; Molecular Probes). The cells were examined and images were captured using a Nikon Eclipse TE300 inverted microscope with a x40 objective, a digital RT Slider camera, and Spot RT software (Diagnostic Instruments Inc.).

Statistical Analysis
Results are expressed as the percent of change from noncontracted controls, mean ± SD of three experiments, each with a different preparation of cells. The effects of mechanical contraction on type II cells were compared with controls by the Student's t test. Comparison between various contraction conditions were made using ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of Mechanical Contraction on Cell Viability
Continuous mechanical contraction at an amplitude of 25% did not result in a change in cell viability in comparison with control cells. At 24 h of contraction, 95.5 ± 1.8% of cells were alive. In comparison, control cells had a viability of 96.1 ± 1.2% (n = 6, P = 0.3). At 48 h, 94.4 ± 1.4% of contracted cells and 95.3 ± 1.5% of control cells were alive (n = 6, P = 0.3).

Effect of Mechanical Contraction on the mRNA Content of the Markers for the Type I and Type II Cell Phenotypes
The data shown in Figure 1 demonstrate that mechanical contraction of membranes by 25% for 24 h resulted in an increase in expression of some markers of the type II cell phenotype and a decrease in expression of a marker of the type I cell phenotype. There was a near doubling in the content of mRNAs for both SP-B (198 ± 18%, n = 3, P < 0.0005) and SP-C (176 ± 25%, n = 3, P < 0.005), in comparison with noncontracted controls (100%). Contracted cells had less RTI40 mRNA (64%±14%, n = 3, P < 0.005). There was no difference in SP-A mRNA content (98 ± 13%, n = 3, P = 0.10) between contracted and control cells at this time point and at this level of contraction.



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  		Figure 1. Effects of mechanical contraction on expression of marker genes for the type I and type II cell phenotypes. Northern blots were performed on RNA obtained from cells from control cells (open bars) and cells contracted by 25% for 24 h (solid bars). Blots were probed with full-length cDNAs for rat SP-A, SP-B, SP-C, RTI40, and 18S rRNA. Autoradiograms were obtained before quantifying radioactivity by phosphorimage analysis. 18S RNA was used as a control to ensure equal loading. Results are expressed as the percent of change from controls, mean ± SD of three experiments, each with a different preparation of cells. *P < 0.05.

 
Effects of Contraction on Markers of the Type II Cell Phenotype Are both Amplitude- and Time-Dependent
There was no significant change in expression of SP-A, SP-B, or SP-C mRNAs with contraction of membrane surface area by 10 or 15%. As membranes were contracted beyond a threshold level of 15–20%, there were progressive effects on expression of marker genes. A decrease in membrane surface area of 20% caused an increase in mRNA content of SP-A (148 ± 10%), SP-B (165 ± 10%), and SP-C (165 ± 19%) in comparison with control cells. The differences in mRNA content of the markers of the type II cell phenotype in contracted cells in comparison with controls was greatest at 25% contraction (193% ± 19% for SP-A, 215 ± 10% for SP-B, 195 ± 7% for SP-C). The mRNA content of all three markers was significantly greater at 20% and 25% contraction in comparison with both 10% and 15% contraction. There was no statistical difference between the 20% and 25% contracted groups (Figure 2) .



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  		Figure 2. Effects of degree of contraction on markers of the type II cell phenotype. RNase Protection assays were performed on RNA obtained from control cells and cells that had undergone 10%, 15%, 20%, or 25% contraction for 12 h. Samples were analyzed for mRNA content of SP-A (black bars), SP-B (shaded bars), and SP-C (striped bars) markers of the type II cell phenotype. 18S RNA was used as a control to ensure equal loading. Results are expressed as the percent of change from controls, mean ± SD of three experiments, each with a different preparation of cells. *P < 0.05 compared with control; {dagger}P < 0.05 compared with 10% contracted; f P < 0.05 compared with 15% contracted.

 
Interestingly, the time course for effects of mechanical contraction varied among the marker genes (Figure 3) . For example, the increase in SP-A mRNA peaked by 4 h of contraction and returned to control levels by 24 h. In contrast, SP-B mRNA content was increased by similar amounts at both 4 h (142 ± 3%, n = 3, P < 0.005) and 12 h (142 ± 12%, n = 3, P < 0.005), peaked at 24 h (196 ± 18%, n = 3, P < 0.0005), and then began to decline toward control levels by 48 h. The effects on SP-C mRNA showed a still different time course, with maximal effects at 4 h (230 ± 27%, n = 3, P < 0.05), and a gradual decline to control levels over the next 44 h.



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  		Figure 3. Time course of changes in mRNA content of markers of the type II cell phenotype in response to contraction. Northern blots were performed on RNA obtained from control cells and cells from membranes that had been contracted 25% for 4, 12, 24, and 48 h. Each time point was compared with controls harvested at the same time point. 18S RNA was used as a control to ensure equal loading. Results are expressed as the percent of change from controls, mean ± SD. Black bars, SP-A; shaded bars, SP-B; striped bars, SP-C. *P < 0.05.

 
Effects of Amplitude and Duration of Contraction on RTI40, a Marker for the Type I Cell Phenotype
RTI40 mRNA content was decreased on average by 47% in comparison with control, noncontracted cells at all amplitudes at 12 h (Figure 4) . RTI40 mRNA content was decreased by 12 h, and this decrease persisted through 48 h (Figure 5) .



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  		Figure 4. Effects of degree of contraction on RTI40 mRNA content. RNA samples from control cells and cells from membranes that had undergone 10%, 15%, 20%, or 25% contraction were analyzed by RNase Protection assays for RTI40 mRNA content. 18S RNA was used as a control to ensure equal loading. Results are expressed as the percent of change from controls, mean ± SD of three experiments, each with a different preparation of cells. Open bars, control; filled bars, RTI40. *P < 0.05.

 


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  		Figure 5. Effects of contraction time on RTI40 mRNA content. RNA samples from control cells and cells from membranes that had been contracted 25% for 4, 12, 24, and 48 h were analyzed for RTI40 mRNA content by Northern blot analysis. 18S RNA was used as a control to ensure equal loading. Results are expressed as the percent of change from controls, mean ± SD. Open bars, control; filled bars, contracted. *P < 0.05.

 
Mechanical Contraction Alters RTI40, SP-A, and SP-B Protein Levels
Mechanical contraction of 25% for 12 h resulted in a 2-fold increase (310 ± 28%, n = 6, P < 0.05) in SP-A protein and a 1.5-fold increase (250 ± 15%, n = 6, P < 0.05) in SP-B protein in comparison with controls. Contracted samples had a 20% (80%±8%, n = 6, P < 0.05) decrease in RTI40 protein content in comparison to controls (Figure 6) .



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  		Figure 6. Mechanical contraction alters RTI40, SP-A, and SP-B protein levels. Quantitative dot blots were performed on protein homogenates from control and contracted cell as described in the text. Serial dilutions of protein were run on each blot to determine the linear portion of the standard curve. Results are expressed as the percent of change from controls, mean ± SD. Open bars, control; filled bars, contracted. n = 6, *P < 0.05.

 
Markers of the Type I and Type II Cell Phenotypes are Coexpressed in the Same Cells
To determine if markers of the type I and type II cell phenotypes were coexpressed in the same cells or represented different cell populations, cells were evaluated by immunocytochemistry for RTI40 and SP-B labeling. After 24 h of contraction (44 h in culture), type II cells coexpressed both RTI40 and SP-B by immunofluorescence (Figure 7) .



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  		Figure 7. Cultured type II cells coexpress RTI40 and SP-B. Type II cells were cultured on membranes for 20 h before undergoing contraction for 24 h. Cells were fixed in placed and labeled against RTI40 and SP-B. After 2 d in culture, type II cells expressed both RTI40 and SP-B. (A) Phase contrast; (B) RTI40 staining; (C) SP-B staining.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Information regarding the relationship between mechanical factors and alveolar epithelial phenotypic expression has been largely derived from studies with fetal model systems (6, 7, 11, 13, 19). Although the data in these studies suggest that both distention and contraction are important regulators of phenotypic expression in the developing lung, most previous work with mechanical forces in vitro has concentrated primarily on the effects of mechanical distention (14, 15, 20). The current studies were designed to investigate the direct effect of continuous mechanical contraction on pulmonary alveolar epithelial phenotypic expression.

In the present study, cultures of type II cells were subjected to continuous mechanical contraction. To assess alveolar epithelial phenotypic expression, we used various molecular and protein markers. As a marker for the differentiated type I cell phenotype, we used RTI40 (T1{alpha}), a gene that encodes a protein that is localized within the lung to the apical plasma membrane of type I cells (18, 21–23). SPs A, B, and C were used as markers of the differentiated type II cell phenotype. Cells were contracted after 18–20 h in culture to allow adherence to the membranes, and harvested anywhere from 28–72 h after isolation, depending on the experimental conditions. Although experiments were performed as early as possible to minimize the loss of differentiated function known to occur when type II cells are cultured on a substratum that promotes spreading, significant differentiation was evident. In these studies, analysis of freshly isolated cells demonstrated high levels of SP-A, -B, and -C expression, which fell to less than 5% of this level by Day 2, the time point when most experiments were terminated. Therefore, the approximate doubling of the type II cell markers in comparison with control cells at 24 h of contraction is an increase from {bsim} 5% of freshly isolated cell values to 10% of freshly isolated cell values.

The data in Figures 1–5 showing that contraction increases expression of markers of the type II cell phenotype and decreases expression of a marker of the type I cell phenotype demonstrate that mechanical contraction directly influences alveolar epithelial phenotypic expression in vitro. The observed changes were both time- and amplitude-dependent, and the pattern of time and amplitude dependence varied among the different marker genes. The difference in mRNA content between contracted and control cells for the marker of the type I cell phenotype became evident at 12 h and persisted through 48 h. Contraction of type II cells for 24 h resulted in a 36% decrease in the mRNA content of a marker of the type I phenotype and an approximate doubling of mRNA content of two of the markers of the type II phenotype. The time course for effects of contraction varied among the different SPs. For example, SP-A mRNA content was increased and peaked at 4 h of contraction and was back to control levels by 24 h, whereas SP-B and SP-C demonstrated different patterns of expression over time in response to contraction. The lack of coregulated expression of SPs has been observed in other model systems (24, 25, 26, 27). In a previous study evaluating the effects of distention on expression of cell-specific phenotypic markers, we demonstrated a decrease in SP-B and SP-C mRNA content after 18–20 h of stimulation, but no difference in SP-A mRNA in stretched cells in comparison with control cells (15). In retrospect, these data might be explained by differences in the time courses of the effects of mechanical factors on different genes. There was also variation in the amplitude dependence of the various markers to mechanical contraction. The decrease in mRNA content of RTI40 was stimulated with as little as 10% contraction, whereas the threshold for effects on surfactant genes was between 15 and 20%.

A recent in vivo study by Kitterman and associates (13) demonstrated that oligohydramnios, which reduces the normal distention of the fetal lung, inhibits expression of RTI40, and decreases the relative surface area of the airspaces covered by type I cells compared with type II cells. These investigators did not observe changes in mRNA content of SP-A, SP-B, or SP-C, or changes in SP-A and SP-B proteins. There are several potential reasons for the difference between this study and our findings. First, the model systems used in both studies are markedly different. Kitterman and coworkers used fetal rats, whereas the current study used primary cultures of type II cells isolated from adult rats. Also, the duration of stimuli was markedly different in each study. In experiments shown in Figure 3, the effects of chronic contraction on surfactant protein gene expression diminished by 48 h. This is also in contrast to fetal model systems of pulmonary hypoplasia, in which changes in epithelial cell phenotype are sustained over time.

Previously, the expression of markers of alveolar epithelial phenotype was felt to be influenced primarily by cell shape (16, 28), extracellular matrix (28), and hormones (29, 30). For the current studies, we used type II cells in primary culture, an accepted model for many studies of type II cell functions, such as surfactant synthesis, secretion, and reuptake (32) and ion transport (33, 34). We used type II cells cultured for up to 72 h in these studies. Type II cells in primary culture gradually (over days) cease to express markers of the type II cell phenotype (35, 36) and begin to express markers of the type I phenotype (37, 38). The cells used in this study are undergoing phenotypic change while in culture and while they are being subjected to mechanical stimuli. The effects of mechanical distention (15) on type II cells appear to accelerate the phenotypic changes that occur when cells are cultured on plastic. In contrast, the changes induced by mechanical contraction are in the opposite direction to the effects seen in cell culture. Therefore, direct mechanical contraction of type II cells in culture appears to counteract some of the changes in phenotype that occur when type II cells are allowed to flatten on a culture substratum.

The objective of the present study was to determine the effects of mechanical contraction on the phenotypic expression of markers of pulmonary alveolar cell differentiation. Prior studies of fetal lung development in vivo support the concept that mechanical contraction affects both growth and phenotypic expression of the alveolar epithelium. In these fetal model systems, the effects on phenotypic expression may be due to a number of potential factors. For example, chronic tracheal drainage or models of congenital diaphragmatic hernia, contracted lung may alter pulmonary blood flow and delivery of nutrients to the developing lung. These secondary effects may also cause changes in phenotypic expression. The experiments in this manuscript provide evidence that in mature differentiated type II cells, contraction in and of itself results in changes in expression of alveolar cell phenotype.

The relevance of these observations in vitro to mature lung is uncertain. It is possible that contraction of type II cells results in alterations in cell shape, thereby slowing the transition of type II cells toward the type I cell phenotype. The biological significance of these findings may be relevant during the process of healing after acute lung injury. During this process the type II cell serves as a progenitor cell, first undergoing cell division, followed by differentiation of some of the progeny to the type I cell phenotype (39). The results of the current studies of mechanical effects in vitro raise the possibility that mechanical forces may alter the evolution of phenotypic changes during the processes of injury and repair, and suggest topics for future investigation.


   Acknowledgments
 
The authors thank Wen Zhou for her technical assistance. This study was supported in part by RWJ 30805, NIH 1 KO1 HL04372-01, and ALA RG-046-NL.

Received in original form July 26, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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