INTRODUCTION

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THE LUNG IS AN ORGAN in which there are large, ever changing states of mechanical tension (30). With each inspiration, the lung expands and increases tension on the load-bearing components of the lung. The large vessels and airways remain open because of radial traction, longitudinal strain is exerted on the scaffolding of the alveolar walls, and surface tension is generated as the lung maximally inflates. Besides controlling the physical act of breathing, mechanical tension is thought to provide regulatory signals that govern the composition of the pulmonary vasculature and airways (5). This type of stimulus is critical in the developing lung as the fetus initiates independent breathing. The mechanical act of breathing stimulates new cell proliferation, lung growth, and surfactant production (13). Furthermore, the ability to restore new lung tissue has been reported in young adults and animals after pneumonectomy (14, 28). The driving force for this response most likely stems from the mechanical signal generated by overinflation or stretch of the remaining lung (21, 28). However, an imbalance in the tensional state of the lung is found in many pathological conditions (5, 23, 35). Furthermore, mechanical ventilation commonly used in many hospital settings can result in unnatural overinflation of regions of the lung, thereby applying tension to the network of capillaries and large vessels (34). In an extreme case, a large increase in mechanical tension may ultimately lead to stress failure of the capillary wall and result in pulmonary edema and hemorrhage (36).

Pulmonary remodeling in response to hypoxic pulmonary vasoconstriction is well known to result in a thickening of the large and medium pulmonary artery walls (23). This is a result of the proliferation of smooth muscle cells and fibroblasts accompanied by increased gene expression of extracellular matrix (ECM) proteins, ₁(I) procollagen and tropoelastin (22, 27). Furthermore, the direct application of circumferential tension to pulmonary arteries in vitro has also revealed increases in type I collagen and elastin (2, 15, 33) and proliferation predominantly in the adventitial fibroblast population (15). Increased mechanical strain exerted on lung parenchymal regions is also thought to result in tissue remodeling. For example, the study by Berg et al. (3) indicates that high lung inflation in vivo increases mRNA levels for ECM components and transforming growth factor-ß₁ in the outer parenchymal region of rabbit lungs. Furthermore, in an isolated perfused lung model, it has been demonstrated that increased airway pressures or lung overinflation results in increased mRNA levels for the ECM components ₁(I) and ₂(IV) procollagen and laminin B chain (25). More recently, Zhang et al. (38) increased mechanical strain in unanesthetized ferret lungs by ventilation with continuous positive airway pressure over an extended period of 2 wk. Ventilation at modestly high lung volumes resulted in a 40% increase in total lung capacity associated with increased lung weight, total protein, and total cellular DNA content. These biochemical changes further support the idea that pulmonary remodeling is an adaptive response to increased mechanical strain.

In the lung, the fibroblast represents a dynamic cell type. Fibroblasts, located in the interstitial space of the alveolar septal edges throughout the lung parenchyma and within the large vessels and airways, are a high-collagen-producing cell. It is hypothesized that the pulmonary fibroblast can sense changes in mechanical tension directly and respond by altering the level of gene expression of ₁(I) procollagen. Furthermore, transmission of the mechanical signal requires specific cell interactions with ECM proteins. Evidence that the pulmonary fibroblast has the potential to respond to mechanical strain has been reported in in vitro model systems. Bishop et al. (6) were the first to report that IMR-90 pulmonary fibroblasts are stimulated to proliferate in response to cyclic mechanical strain. Preliminary studies also suggest that IMR-90 cells increase their level of procollagen synthesis after 1 or 24 h of exposure to increased mechanical tension (4). Furthermore, collagen type I gene expression is augmented in mechanically strained cardiac fibroblasts, and the mechanism of this response requires the presence of serum growth factors (7).

In the present study, the response of pulmonary fibroblasts in vitro to cyclic mechanical strain was further investigated. The gene expression of ₁(I) procollagen in mechanically strained IMR-90 cells was evaluated at the level of mRNA and total procollagen synthesis. In addition, the influence of the ECM environment to modulate the pulmonary fibroblast response was elucidated. These findings were correlated to the proliferative state of the cell.

	METHODS

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Fibroblast cell cultures. The human fetal lung cell line IMR-90 (American Type Culture Collection, Rockville, MD) was routinely cultured in MEM (GIBCO BRL, Grand Island, NY) containing 10% fetal bovine serum (FBS), penicillin (50 U/ml), and streptomycin (50 U/ml). Cells were propagated in a 5% CO₂-95% air humidified incubator at 37^°C. For passage, cells were release with 0.25% trypsin solution. Experiments were performed by using cells from passages 10-20.

Application of mechanical strain. The Flexercell strain apparatus (Flexcell International, McKeesport, PA) was used to expose cell cultures to increased cyclic mechanical strain (1). In this system, cells are grown on a flexible-bottom elastomer membrane coated with ECM protein Flex I culture plates. These culture plates were prepared by adsorption of a 1.5 M solution of fibronectin, laminin, or elastin peptides (Flexcell International). The fibronectin and elastin plates are prepared with synthesized peptides, and the elastin protein is isolated from bovine ligament by Flexcell International. For each experiment, fibroblasts were counted with a hemocytometer and seeded at an initial density of 3 × 10⁴ cells/cm². Cells were allowed to attach for 48 h. After the first 24 h, the media were exchanged for MEM containing 1% FBS. At the initiation of cyclic mechanical strain, cells were replenished with MEM-10% FBS and placed in the Flexercell baseplate. Strained cells were exposed to a maximum 20% elongation or 13-kPa pressure at a cycle of 1 stretch/s over a period of 48 h, unless otherwise stated. The Flexwell is exposed to a gradient of force that is minimal in the center of the well and maximal at the periphery. Control cells were cultured under identical conditions but remained stationary.

Morphology. Fluorescein-labeled phalloidin (Molecular Probes, Eugene, OR) was used according to the manufacturer's directions to reveal F-actin-containing filaments. Cells were washed with PBS, pH 7.4, and fixed in 3.7% formaldehyde solution in PBS for 10 min at room temperature. Cells were again rinsed in PBS followed by permeabilization with an acetone solution at 20^°C for 5 min. After a PBS rinse, cells were stained with FITC-phalloidin for 20 min at room temperature. The cells were rinsed with PBS, and the entire Flexwell elastomer was removed from the well and mounted on a slide cell side down in a 1:1 solution of PBS and glycerol. Slides were viewed immediately by fluorescent microscopy by using a ×25 objective and ×10 magnification on the camera attachment and were photographed (Kodak Extrachrome 400HC film, Eastman Kodak, New Haven, CT).

Northern blot analysis. Total cellular RNA was isolated by the method of Chomczynski and Sacchi (8). Two six-well Flex I culture plates were used for RNA isolation for each sample. RNA preparations were quantitated by absorbance at 260 nm, and intactness was assessed by ethidium bromide staining following separation in a 6.6% formaldehyde-1% agarose gel. Fractionated RNA was transferred by Northern blot to Zeta probe membrane (Bio-Rad, Hercules, CA). RNA was cross-linked to the membrane by ultraviolet irradiation and stored at 4^°C. The blots were then probed with oligolabeled [-³²P]dCTP cDNA probes, which have a specific activity of at least 1 × 10⁹ disintegrations · min¹ · µg¹ DNA (Prime-It II Kit, Stratagene, La Jolla, CA). The mRNA level for ₁(I) procollagen was detected by using the cDNA recombinant plasmid, p1R1, from Dr. David Rowe (10). Prehybridization and hybridizations were performed in 50% formamide, 5× SSC (20× SSC = 0.3 M sodium chloride, 0.3 M sodium citrate), 10× Denhardt's solution (100× Denhardt's solution = 2% Ficoll, 2% polyvinyl pyrrolidone, 2% BSA), 50 mM sodium phosphate, pH 6.5, 1% SDS, and 200 µg/ml salmon sperm DNA at 42^°C. Blots were washed under high-stringency conditions of 0.1× SSC and 0.1% SDS at 65^°C. The signal was obtained by exposure to XAR-5 X-ray film (Eastman Kodak, New Haven, CT) by using a Cronex Lightning Plus screen at 80^°C. Autoradiographs were quantitated by densitometry within the linear range of signals and normalized to 28S or 18S ribosomal RNA levels.

Assay for cell proliferation. Three wells in each of two six-well Flexwell plates were pulsed with 5 µCi/ml of [³H]thymidine, specific activity 6.7 Ci/mmol (248 GBq/mmol) (NEN DuPont) 1 h before the end of the period of applied cyclic mechanical strain. The Flexercell apparatus was then stopped, and each well was washed three times with assay medium (140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM Na₂HPO₄, 25 mM glucose, 25 mM HEPES/NaOH, 0.5 mg/ml BSA). Ice-cold 15% TCA (500 µl) was added to each Flexwell, and the dishes were stored at 4^°C for 30 min. Wells were rinsed with water, and individual Flexwell membranes were removed from the plates and placed in scintillation vials. Scintillation fluid (Ecoscint, National Diagnostics, Atlanta, GA) was added to each vial, shaken, left in the dark overnight, and counted the next day. In the other three wells of the same plate, the total micrograms of DNA were determined by fluorometry by using Hoescht 33258 and a calf thymus DNA standard (17).

Procollagen and noncollagen protein synthesis. Procollagen and noncollagen synthesis were estimated by using the bacterial collagenase digestion assay, as previously described (24). IMR-90 fibroblasts were supplemented with 1 × 10⁵ M ascorbic acid at the initiation of mechanical strain. One hour before the cells were harvested, the growth medium was removed, and the cells were rinsed twice with serum-free medium at 37^°C. To each 25 mM well, serum-free medium containing 0.1 mM ß-aminopropionitrile (Sigma Chemical, St. Louis, MO) and 1 × 10⁵ M ascorbic acid were added, and the cells were incubated for 15 min in a 37^°C CO₂ incubator. The cells were then labeled by adding 30 µCi/ml of L-[2,3,4,5-³H]proline, specific activity 20-50 Ci/mmol (925 GBq/mmol to 1.85 TBq/mmol) (DuPont NEN) and continued to be exposed to cyclic mechanical strain for 1 h. This amount of [³H]proline was within the linear range of incorporation for this assay. At the end of the labeling period, the cell layer was collected. Aliquots were digested with bacterial collagenase form III (Advanced Biofactures, Lynbrook, NY) or incubated with buffer at 37^°C, as described by Newman and Cutroneo (24). Total DNA levels were determined by fluorometry by using Hoescht 33258 (17).

Statistical analysis. Statistical significance was evaluated by using the Student's t-test. P values <0.05 were considered significant (18).

	RESULTS

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Morphology. Our study confirms previous reports that pulmonary fibroblasts subjected to cyclic mechanical strain undergo a strain-dependent change in morphology (6). Strained fibroblasts have a more extended shape compared with control cells and align perpendicular to the force vector in the outer periphery of the circular plate. It is in this region of the well that the force or percent elongation of the surface is greatest. Parallel to the cells, the cytoskeletal actin filaments also align perpendicular to the force vector, and this is revealed by staining the F-actin-containing microfilaments with FITC-conjugated phalloidin (Fig. 1).

View larger version (97K): [in this window] [in a new window]   Fig. 1.   Alignment of cytoskeletal actin filaments perpendicular to force vector in mechanically strained pulmonary fibroblasts. Pulmonary fibroblasts were exposed to cyclic mechanical strain resulting in 20% elongation at 60 counts/min for 48 h on laminin-coated Flexwells. Orientation of F-actin-containing filaments in control (A) and strained (B) fibroblasts was observed by staining with FITC-conjugated phalloidin (magnification ×250). Arrow shows direction of force vector.

Effect of matrix environment on ₁(I) procollagen mRNA levels. Cells were cultured on fibronectin, laminin, or elastin and exposed to cyclic mechanical strain. On a fibronectin substratum, there was a strain-dependent decrease in the level of ₁(I) procollagen mRNA (Fig. 2). In contrast, when cells were plated on a laminin matrix, there was an increase in ₁(I) procollagen mRNA level in response to mechanical strain (Fig. 2). A 2.7-fold increase in mRNA levels for ₁(I) procollagen was observed in mechanically strained cultures compared with unstrained, control fibroblasts. A similar response was observed when pulmonary fibroblasts were cultured on elastin. Fibroblasts cultured on an elastin matrix revealed an increase in the ₁(I) procollagen mRNA level, which was greatest at an initial seeding density of 3 × 10⁴ cells/cm² (2.3-fold). The observed difference between mechanically strained cells and control cells decreased with increasing cell densities from 4-6 × 10⁴ cell/cm² (data not shown). Thus the potential for the pulmonary fibroblasts to increase the level of mRNA for the ECM protein ₁(I) procollagen is dependent on signals from the ECM environment.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Role of extracellular matrix in response of pulmonary fibroblasts to increased mechanical strain. Pulmonary fibroblasts were seeded on a 2-dimensional matrix of fibronectin (FN), laminin (LN), or elastin (ELN) and subjected to an increased cyclic mechanical strain. A: 1(I) procollagen mRNA levels were determined by Northern analyses for control, unstretched fibroblasts (C) compared with mechanically strained fibroblasts (S). B: mRNA levels were quantitated by densitometry and normalized to 28S rRNA. Values are expressed as average percent of control ± SE; n = 5. * Statistical differences, P < 0.05.

Time course of increased ₁(I) procollagen mRNAs. A further examination of the time course of this response was studied in pulmonary fibroblasts, which were cultured on an elastin matrix. Cells were seeded at an initial density of 3 × 10⁴ cells/cm², and, at various times after the initiation of mechanical strain, the cells were harvested for RNA isolation (Fig. 3). The mRNAs for ₁(I) procollagen increased in mechanically strained fibroblast compared with control cells as early as 6 h after the initiation of mechanical tension. The mRNA levels for ₁(I) procollagen remained increased for 48 h and returned to control levels after 72 h of mechanical stimulation. There was only a 30% increased in ₁(I) procollagen mRNA levels in mechanically strained fibroblasts compared with control cells at 72 h, whereas there was a 2.3-fold increase at 12 h.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Time course of 1(I) procollagen mRNA levels in response to increased cyclic mechanical strain. Pulmonary fibroblasts seeded on an elastin matrix were exposed to increased cyclic mechanical strain (S) for 6, 12, 24, or 72 h and compared with fibroblasts cultured for same time periods in absence of strain (C). 1(I) procollagen mRNA levels were measured by Northern analysis (A) and quantitated by densitometry and normalized to 18S rRNA levels (B).

Proliferative response. The next experiment examines the dependence of increased collagen production on ongoing cell division. Pulmonary fibroblasts were again cultured at densities of 3 × 10⁴ cells/cm² on fibronectin-, laminin-, or elastin-coated Flexwell plates (Fig. 4). At the end of the 48-h period of increased mechanical strain, the rate of DNA synthesis was assayed by [³H]thymidine incorporation. On each ECM protein, there was no significant change in proliferation in mechanical strain cells under the conditions that led to an increase in procollagen gene expression.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Proliferative response of fibroblasts exposed to increased cyclic mechanical strain. Pulmonary fibroblasts were seeded at an initial density of 3 × 104 cells/cm2 on FN-, LN-, or ELN-coated Flexwell culture dishes. One hour before the end of mechanical stimulation, cells were pulsed with 5 µCi/ml of [3H]thymidine. Incorporation of TCA precipitable counts (dpm) per microgram of total cellular DNA was determined in 6 wells. Values are means ± SE; n = 6.

[³H]proline incorporation into newly synthesized procollagen. The increase in ₁(I) procollagen gene expression at the mRNA level was also correlated with increased total procollagen synthesis (Fig. 5). Pulmonary fibroblasts were cultured on either an elastin, laminin, or fibronectin matrix. The cells were then mechanically stimulated at the same force, producing a 20% elongation for 48 h. At the end of the straining period, the cells were labeled with [³H]proline, and the incorporation into newly synthesized procollagen was measured by using the bacterial collagenase digestion assay. When fibroblasts were cultured on elastin, a strain-dependent 114% increase in the incorporation of [³H]proline into the procollagen protein fraction was observed. A similar 190% increase in [³H]proline incorporation into procollagen was also detected in strained fibroblasts cultured on a laminin matrix. In contrast, cells mechanically strained on a fibronectin matrix did not reveal a significant difference in levels of [³H]proline-labeled procollagen (Fig. 5). Increased proline incorporation into noncollagen protein was observed in cells cultured on fibronectin, laminin, and elastin matrices. These studies suggest a similar pattern of procollagen gene expression at the levels of both mRNA and protein synthesis in pulmonary fibroblasts exposed to increased mechanical tension.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Procollagen synthesis in mechanically strained fibroblasts. Pulmonary fibroblasts were seeded on FN-, LN-, or ELN-coated Flexwell culture dishes. One hour before the end of mechanical stimulation period, cells were pulsed with 30 µCi/ml of [3H]proline in presence of ascorbic acid. Incorporation of [3H]proline into collagen (A) and noncollagen protein (B) was determined by collagenase digestion. Values represent average counts per minute per milligram of DNA ± SE; n = 5-6. Significant differences in strained cell values (S) from control (C) were observed at * P < 0.05, # P < 0.06, and + P < 0.10.

	DISCUSSION

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Modulation of cellular collagen production by the ECM. Cell types in other organs, such as heart, bone, and kidney, have displayed the capability of responding to increased mechanical strain by altered proliferative properties and collagen production (12, 31, 37); however, very little information is known about the cellular units of the lung. In the systemic circulation, there have been reports that both cardiac fibroblasts and aortic smooth muscle cells increase the rate of collagen synthesis in in vitro models of increased mechanical tension (7, 11, 19). Furthermore, mesangial cells have demonstrated the ability to increase the expression of several ECM proteins at the transcriptional and translational levels in response to increased cyclic mechanical strain (31, 37).

Strain-induced type I procollagen expression is independent of cell proliferation. Cells in culture undergo a transition from a proliferative phase in which the subset of growth-related genes, including the immediate early genes (c-fos and c-myc) and histones, is maximally expressed to a quiescent phase in which proteins characteristic of a more differentiated cell type are produced. Collagen synthesis is highest during the transitions from a sparse, actively proliferating to a confluent, quiescent cell culture (26). It is at this cellular state or log phase of growth that a mechanical strain-induced increase in procollagen gene expression was observed. The observed increase in procollagen gene expression did not correlate with an increase in the rate of cellular proliferation on any of the ECM matrices on which the fibroblasts were cultured. Interestingly, a decrease in mechanical strain-induced collagen production with increasing cell density has been reported in mesangial cells by Riser et al. (31). The ability of metabolically active fibroblasts to further increase collagen synthesis in response to mechanical strain is also supported by the requirement of serum growth factors in the mechanical strain-induced production of ₁(I) procollagen observed in cardiac fibroblasts (7).

Role of the ECM in transmitting a mechanical signal. The ECM environment provides important signals for many cellular functions, including morphogenesis, differentiation, angiogenesis, and remodeling (32). The ECM modifies the ability of the cell to adhere to a surface and influences cell shape. Specific focal adhesions at the cellular surface allow mechanical tension generated in the system to be transduced to the cytoskeletal network. Thus the cell is an integrated system in terms of mechanical force transduction. A change in the cytoskeletal architecture is transmitted to the nuclear matrix, ultimately allowing the expression of a subset of gene products (20). In our study, the ability of a mechanical signal to alter ₁(I) procollagen gene expression was modulated by contact with various ECM proteins. Whether this results in the interaction with a specific subset of adhesion molecules or a change in the number or distribution of interactions remains to be determined. In the lung, the fibroblast may be exposed to matrix environments of various compositions. For instance, in wound healing or injury, a provisional fibronectin matrix is first laid down to allow the attachment and migration of cells (9). Laminin, a major component of the basement membrane, provides an important signal for capillary morphogenesis (16). Elastin is located in the interstitial space in the alveolar septal region and in the large vessel. These are some of the main support structures in the lung that would represent load-bearing elements resistant to changes in transmural pressure or longitudinal tension of the lung parenchyma. Future studies to elucidate the differences in the mechanotransduction pathways initiated by ECM proteins at the cell surface may give further insight into pulmonary conditions in which the balance of mechanical forces is disturbed.