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Am J Physiol Lung Cell Mol Physiol 293: L769-L778, 2007. First published June 29, 2007; doi:10.1152/ajplung.00127.2007
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High tidal volume mechanical ventilation with hyperoxia alters alveolar type II cell adhesion

Leena P. Desai,1 Scott E. Sinclair,1,2 Kenneth E. Chapman,1 Aviv Hassid,1,3 and Christopher M. Waters1,2

1Department of Physiology, 2Department of Medicine, 3Vascular Biology Center, University of Tennessee Health Science Center, Memphis, Tennessee

Submitted 30 March 2007 ; accepted in final form 28 June 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Patients with acute respiratory distress syndrome undergoing mechanical ventilation may be exposed to both high levels of stretch and high levels of oxygen. We hypothesized that the combination of high stretch and hyperoxia promotes loss of epithelial adhesion and impairs epithelial repair mechanisms necessary for restoration of barrier function. We utilized a model of high tidal volume mechanical ventilation (25 ml/kg) with hyperoxia (50% O2) in rats to investigate alveolar type II (AT2) cell adhesion and focal adhesion signaling. AT2 cells isolated from rats exposed to hyperoxia and high tidal volume mechanical ventilation (MVHO) exhibited significantly decreased cell adhesion and reduction in phosphotyrosyl levels of focal adhesion kinase (FAK) and paxillin compared with control rats, rats exposed to hyperoxia without ventilation (HO), or rats ventilated with normoxia (MV). MV alone increased phosphorylation of p130Cas. RhoA activation was increased by MV, HO, and the combination of MV and HO. Treatment of MVHO cells with keratinocyte growth factor (KGF) for 1 h upon isolation reduced RhoA activity and restored attachment to control levels. Attachment and migration of control AT2 cells was significantly decreased by constitutively active RhoA or a kinase inactive form of FAK (FRNK), whereas expression of dominant negative RhoA in cells from MVHO-treated rats restored cell adhesion. Mechanical ventilation with hyperoxia promotes changes in focal adhesion proteins and RhoA in AT2 cells that may be deleterious for cell adhesion and migration.

keratinocyte growth factor; RhoA; focal adhesion kinase

MECHANICAL VENTILATION with high levels of supplemental oxygen is an important supportive therapy to patients suffering from the acute respiratory distress syndrome (ARDS). However, both mechanical ventilation (19) and hyperoxia (13, 34) can independently damage normal lung tissue. Mechanical ventilation with large tidal volumes to recruit areas of the lung with low compliance leads to overdistension (high stretch) of areas of the lung with normal compliance (1, 3) and, in combination with hyperoxia, could exacerbate lung injury (5, 42, 52). Although patients receiving mechanical ventilation typically receive supplemental oxygen, very few studies have examined the combined effects of mechanical stretch and hyperoxia in the generation of ventilator-induced lung injury. In addition, recovery from acute lung injury requires repair of the alveolar-capillary membrane and restoration of barrier integrity, but the individual effects of either mechanical stretch or hyperoxia on epithelial repair have not been extensively investigated. In the present study we hypothesized that the combination of high tidal volume mechanical ventilation and hyperoxia promotes loss of cell adhesion and inhibition of repair mechanisms of alveolar epithelial cells following injury.

Maintenance and repair of the pulmonary alveolar epithelium is critical for preserving normal alveolar structure and function. After denudation of injured alveolar epithelial cells, the type 2 cells (AT2) initiate repair mechanisms involving cell spreading, migration, and proliferation (16, 22, 38, 65). These processes include interactions with the extracellular matrix via integrin receptors and growth factor-mediated activation of signaling pathways relevant to cell adhesion and migration (15, 16, 38, 39). Prominent among these signaling pathways are focal adhesion proteins such as focal adhesion kinase (FAK), paxillin, and p130Cas that coordinate cell adhesion and migration through phosphorylation/dephosphorylation events. Rho-GTPases such as RhoA and Rac1 also contribute to these processes by regulating cytoskeletal remodeling (6, 17, 25, 26). Although mechanical forces have induced changes in these signaling pathways in studies using many different types of cultured cells, few studies have examined these pathways in alveolar epithelial cells in response to stimulation in vivo. In this study we examined signaling mechanisms important in AT2 cell repair mechanisms following in vivo exposure to hyperoxia and high tidal volume ventilation.

Keratinocyte growth factor (KGF) has been shown to provide protection against many different types of lung injury, especially injury involving reactive oxygen species (23, 40, 68, 71, 72). Previous studies typically utilized treatment before injury, but some studies suggest that KGF is also active in the repair mechanisms. The potential mechanisms underlying the protective effects of KGF are multiple and include a mitogenic effect on AT2 cells (58), enhanced epithelial gap junctions (27), increased epithelial cell production of surfactant and other proteins (70), enhanced strain tolerance (37), and decreased apoptosis (11). Although the effects of KGF on maintenance of the AT2 cell phenotype (10) and enhanced epithelial cell migration (21, 66) are well known, the role of KGF in modulating signaling pathways related to cell adhesion and migration has not been investigated.

In the current study we determined the role of tyrosine phosphorylation of focal adhesions proteins FAK, paxillin, and p130Cas and activation of RhoA on adhesion of AT2 cells isolated from rats exposed to hyperoxia and high tidal volume mechanical ventilation. Our results indicate that changes in the phosphorylation of FAK, paxillin, and p130Cas together with activation of RhoA are important signaling events associated with decreased adhesion of AT2 cells following exposure to hyperoxia and high tidal volume ventilation.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation of AT2 cells from mechanically ventilated rats. Sprague-Dawley rats were anesthetized with phenobarbital. All rats in the ventilated groups were ventilated via tracheostomy with high tidal volume of 25 ml/kg (rodent ventilator model 683; Harvard Apparatus, Holliston, CA) and positive end-expiratory pressure of 3 cmH2O. The respiratory rate was adjusted to maintain end-tidal PCO2 of 40–45 mmHg (Novametrix Medical Systems, Wallingford, CT) under conditions of normoxia (20% O2; MV) or hyperoxia (50% O2; MVHO) for 2 h. Body temperature was maintained between 37 and 38.2°C (Thermalert TH-5; Physitemp, Clifton, NJ) using a rectal probe (Ret 2; Braintree Scientific, Clifton, CA). Spontaneously breathing rats were exposed to either normoxia (control) or hyperoxia (50% O2; HO) using a sealed Lucite chamber. Following 2 h of exposure to a given experimental condition, rats were given an overdose of ketamine/xylazine (87/13 mg/kg ip) and exsanguinated. Primary rat AT2 cells were then isolated according to established procedures (14, 18). Viability was assessed by trypan blue staining, and there were no significant differences in viability among the treatment groups. All experiments were performed in triplicate using AT2 cells pooled from two rats per experimental condition from at least three different isolations.

Expression of adenoviral mutants. Replication deficient (E-1 deleted) recombinant type 5 adenoviruses expressing wild-type or mutant proteins were prepared as described previously for enhanced green fluorescent protein (EGFP) (17), FAK-related non-kinase (FRNK) (54), constitutively active RhoA (G14V-RhoA, or CA-RhoA) and dominant negative RhoA (T19N-RhoA, or DN-RhoA) (17), wild-type p130Cas (WT-Cas) and substrate-deleted p130Cas (SD-Cas) (31), and EGFP-FRNK (54), and adenoviruses were infected in freshly isolated AT2 cells. Adenoviruses were amplified using HEK-293 cells and purified using an adenovirus purification kit (Clontech, Palo Alto, CA). Adenoviral titers were determined via a standard procedure by measurement of their cytopathic effect in HEK-293 cells and expressed in AT2 cells immediately after isolation.

Experimental cells were infected immediately following isolation with recombinant adenovirus in 5% serum-containing medium, at a multiplicity of infection (MOI) index of 6-8. This MOI had a transduction efficiency of at least 80% and typically ranged from 80 to 90% as determined by the EGFP fluorescence observed after 48 h of expression. In experiments comparing cells isolated from control rats and rats treated with MVHO, all measurements were made in time-matched cells 24 h after infection. To assure confluent monolayers, measurements of cell migration were performed using control cells infected for 48 h. For vectors not expressing fluorescent tags, expression was maximal between 24 and 48 h as determined by Western blotting for the hemagglutinin (HA) tag (CA-RhoA and DN-RhoA) or by protein expression (WT-Cas and SD-Cas).

Cell adhesion assay. In preliminary studies we compared adhesion of AT2 cells to a fibronectin matrix with adhesion to a matrix deposited by a rat lung fibroblast (RLF) cell line. Since the RLF cells provide a more complete matrix for attachment, and since there was a 26.8% increase in adherence of cells to RLF matrix compared with fibronectin (data not shown), RLF matrix was used for all subsequent studies.

To examine adhesion of freshly isolated AT2 cells, we used a spectrophotometric measurement of cell adhesion according to a previously published protocol (4). For cell adhesion after 24 h and for migration experiments 48 h after isolation, 96- or 12-well tissue culture-treated plates (Costar, Corning, NY) coated with RLF matrix were used. Plates were seeded with either 1.75 x 106 (12-well plates) or 1 x 104 cells/well (96-well plates) of isolated cells and infected with the different adenoviral vectors described in Expression of adenoviral mutants.

For preparation of RLF matrix, 96-well plates (Costar) were seeded with 1 x 104 cells/well of rat lung fibroblast cells (RLF-6; ATCC, Manassas, VA). After 4 days, removal of the confluent RLF-6 cells was performed by incubation with ammonium hydroxide (100 mM) for 10 min at 37°C. Wells were washed five times with Dulbecco's phosphate-buffered saline (DPBS) and seeded with 8.75 x 103 cells/well of AT2 cells in medium containing 5% serum. In some experiments AT2 cells were infected with the adenoviral vectors or treated with 50 ng/ml KGF. After 24-h incubation at 37°C, nonadherent cells were removed by washing twice with DPBS. Adherent cells were fixed with 3.7% formaldehyde for 10 min and stained with 1% methylene blue (Fisher Scientific, Fair Lawn, NJ) for 30 min. Washing with DPBS was followed by addition of 200 µl of a 1:1 solution of 0.1 M HCl and 95% ethanol to each well and measurement of the absorbance at 630 nm. Preliminary measurements confirmed a linear relationship between the number of cells seeded in a well, as determined by counting with a hemacytometer and measurement of the methylene blue absorbance at 630 nm after 24 h of attachment. All experiments were done in triplicate and repeated using cells from three different isolations.

Measurement of cell migration: epithelial wound healing. Adenoviral infections were performed on freshly isolated cells on RLF matrix in medium containing 5% serum for a period of 48 h, at which time the AT2 cell monolayers were confluent. Wounds of 730 ± 8 µm were produced by scraping the cell monolayers with a pipette tip across the diameter of the well.

Cells were washed with (DPBS), and medium containing 5% serum was added and maintained for the duration of the experiment. Images were obtained at the initial time of wounding and then at 6, 12, 18, and 24 h postwounding.

Images were collected with a Cool Snap cooled charge-coupled device camera (Roper Scientific, Trenton, NJ) mounted on an Eclipse TE300 inverted microscope with a x10 phase-contrast objective (Nikon, Melville, NY) using MetaMorph imaging software (version 4.6; Universal Imaging, West Chester, PA). Image dimensions were converted from pixels to micrometers by using a calibration image as described previously (17). All results reported are from three independent wells from three separate experiments.

Immunoprecipitation and immunoblotting. All experiments using freshly isolated AT2 cells were performed immediately after removal of AT2 cells from IgG-coated plates by panning and cell counting. Briefly, 1 x 107 cells were treated with Dulbecco's modified Eagle's medium (DMEM) containing 5% serum in the presence or absence of 50 ng/ml KGF at 37°C for 1 h and lysed with RIPA buffer (150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, and 50 mM Tris; pH 7.2) containing 1 mM sodium vanadate, 1 mM PMSF, 5 µg/ml aprotinin, and 5 µg/ml leupeptin (31). Protein concentration in lysates was determined using the bicinchoninic acid method. Immunoprecipitation was done by incubation of 1 mg of protein with 2.5 µg of mouse monoclonal antibody directed against p130Cas (Transduction Laboratories, San Jose, CA) or mouse IgG (control; Sigma, St. Louis, MO) for 2.5 h at 4°C, followed by the addition of protein G-Sepharose beads (50% slurry; Amersham Biosciences, Piscataway, NJ) and further incubation for 2 h at 4°C. Sepharose beads were then washed three times with 50 mM Tris buffer (pH 7.2) containing 150 mM NaCl, 1 mM sodium vanadate, and 1 mM PMSF, followed by boiling of beads in 2x Laemmli sample buffer and loading of supernatant onto gels for SDS-PAGE. Proteins were transferred electrophoretically onto nitrocellulose (NC) membranes (Invitrogen, Grand Island, NY). After the transfer, blots were probed with primary antibody against phosphotyrosine (1:2,000; MP Biomedicals, Aurora, OH) followed by horseradish peroxidase (HRP)-coupled secondary anti-mouse antibody (1:1,000; Jackson ImmunoResearch, West Grove, PA). The blots were then stripped and reprobed with anti-p130Cas (1:2,000) to confirm equal protein loading.

For immunoblotting experiments to detect phosphorylated FAK and paxillin, 50 µg of the RIPA cell lysates were loaded on gels for SDS-PAGE, transferred onto NC membranes, and probed with anti-p-FAK (Tyr397) (1:500; Biosource, Camarillo, CA) and anti-p-paxillin (Tyr118) antibodies (1:1,000; Cell Signaling Technology, Beverly, MA). For detection of expression of CA-RhoA or DN-RhoA and EGFP-FRNK, blots were probed with HA (1:1,000; Covance, Berkeley, CA) and GFP antibodies (1:500; Santa Cruz Biotechnology, Santa Cruz, CA). Secondary antibodies used were either anti-rabbit HRP (1:1,000; Amersham) or anti-mouse HRP, respectively. The blots were then stripped and reprobed with anti-FAK (1:500; Transduction Laboratories) and anti-paxillin antibodies (1:1,000; Transduction Laboratories) to confirm equivalent loading of proteins. Blots were developed using the SuperSignal West Dura extended duration substrate (Pierce, Rockford, IL) and exposed using a MultiImager chemiluminescence imaging system (Bio-Rad Fluor-S; Bio-Rad, Hercules, CA). Quantitation of Western blots was performed using the Bio-Rad Quantity One software.

RhoA activity assay. Briefly, for detection of activated RhoA, 1 x 107 cells were incubated in DMEM containing 5% FBS in the presence or absence of 50 ng/ml KGF at 37°C for 1 h and lysed. Similarly, for detection of activated RhoA in AT2 cells expressing FRNK, 1 x 107 cells were lysed after 24 h in culture. Cell lysates were incubated with Rhotekin Rho binding domain bound to glutathione-agarose beads at 4°C for 60 min. GTP-bound RhoA was solubilized in Laemmli sample buffer and detected by Western blot analysis, using anti-RhoA (1:200; Santa Cruz Biotechnology). Blots were then incubated with anti-mouse HRP for 1 h at room temperature.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Hyperoxia and high tidal volume ventilation decreased adhesion of AT2 cells. To determine whether mechanical ventilation altered adhesive properties of AT2 cells, we measured the adhesion of freshly isolated AT2 cells to matrix deposited by RLF following ventilation with room air (MV) or 50% O2 (MVHO) compared with adhesion from control rats and spontaneously breathing rats exposed to 50% O2 (HO). AT2 cell adhesion after 24 h was significantly decreased in MVHO cells compared with control cells (Fig. 1). Treatment with KGF following isolation abrogated the decreased cell adhesion induced by MVHO and significantly increased adhesion of both MV and MVHO cells compared with untreated cells under the same conditions at 24 h (Fig. 1). KGF did not affect adherence of control and HO cells. AT2 cell viability determined by trypan blue staining was >90% in all groups upon isolation, indicating that cell death did not lead to differential adhesion of MVHO cells.


Figure 1
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  		Fig. 1. Mechanical ventilation with hyperoxia significantly decreased adhesion of freshly isolated alveolar type II (AT2) cells; keratinocyte growth factor (KGF) restored adhesion. AT2 cells isolated from control rats or rats exposed to hyperoxia (HO; 50% O2), high tidal volume ventilation with normoxia (MV), or high tidal volume mechanical ventilation with hyperoxia (MVHO) were plated (1 x 104 cells/well) on 96-well plates coated with rat lung fibroblast (RLF) matrix. Cell adhesion was measured after 24 h in the presence or absence of KGF (50 ng/ml). Data are means ± SE measured in 16 independent wells from 3 separate isolations. *P < 0.05 vs. control. #P < 0.05 vs. untreated corresponding condition.

 
Reduction in FAK and paxillin phosphotyrosyl levels. Since focal adhesion proteins are critically important for adhesion and migration of cells in response to injury, we determined the phosphorylation of FAK, paxillin, and p130Cas under conditions of normoxia or hyperoxia and mechanical ventilation. In addition, to test whether KGF treatment altered phosphorylation levels of FAK, paxillin, and p130Cas, we quantified p-FAK (Tyr397), p-paxillin (Tyr118), and pTyr-p130Cas levels in the presence and absence of KGF. Phosphorylation of FAK (Tyr397; Fig. 2A) and paxillin (Tyr118; Fig. 2C) was significantly decreased in cells from the MVHO group compared with cells from control, MV, and HO experimental conditions. We measured an overall decrease in phosphorylation of FAK (68.1%) and paxillin (57.5%) levels in MVHO cells compared with control cells. KGF did not affect phosphorylation of either FAK or paxillin. As shown in Fig. 2D, we measured an increase in phosphorylation of p130Cas in MV cells that did not occur in HO or MVHO cells. KGF did not alter pTyr-p130Cas levels of any of the conditions and did not prevent the increase in MV cells.


Figure 2
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  		Fig. 2. MVHO causes dephosphorylation of focal adhesion kinase (FAK) and paxillin, and MV increases phosphorylation of p130Cas in AT2 cells. Freshly isolated AT2 cells for each condition were treated with or without KGF (50 ng/ml) at 37°C for 1 h. Cells were then lysed, and phosphorylation of FAK on Tyr397 (pFAK; A) or paxillin on Tyr118 (pPaxillin; C) was assessed by Western blotting using phospho-specific antibodies. Equal protein was confirmed by reprobing blots for total FAK or total paxillin. *P < 0.05 vs. control. ¶P < 0.05 vs. KGF-treated control. B: phosphorylation of FAK on Tyr397 determined using phospho-specific antibody followed by reprobing blots for total FAK. *P < 0.05 vs. control at the corresponding time point. D: tyrosine phosphorylation of p130Cas was determined by immunoprecipitation (IP) via an antibody directed against p130Cas followed by probing of the blot (IB) with antibody directed against phosphotyrosine (pTyr; top blot) and reprobing with antibody directed against p130Cas (bottom blot). *P < 0.05 vs. control. ¶P < 0.05 vs. KGF-treated control. Blots in each panel are representative of 3 separate isolations (4 for p130Cas), and bar graphs summarize densitometric data as means ± SE, n = 3 or 4 independent experiments. All lanes shown in the individual panels (A, C, and D) are from the same blot, but unrelated lanes were removed for clarity.

 
To determine whether the decrease in FAK phosphorylation was persistent, we measured p-FAK (Tyr397) levels in control and MVHO cells after 24 h of attachment. Decreased levels of p-FAK were observed in both freshly isolated cells and cells 24 h after isolation (Fig. 2B). p-FAK levels were unchanged in control cells.

KGF treatment blocked increase of RhoA activity. To examine the role of RhoA in adhesion of AT2 cells exposed to normoxia and hyperoxia with high tidal volume ventilation, we determined the levels of activated RhoA (GTP-RhoA) immediately following isolation. Each condition (MV, HO, and MVHO) stimulated an increase in RhoA activation (Fig. 3). Treatment with KGF for 1 h prevented the increase in RhoA activity in all three conditions.


Figure 3
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  		Fig. 3. KGF blocks MV- and HO-induced increase in RhoA activation. Freshly isolated cells were treated without or with KGF (50 ng/ml) at 37°C for 1 h and then lysed. RhoA activity was determined via a pull-down assay, and a representative blot is shown at top (all lanes are from the same blot, but unrelated lanes were removed for clarity). Bar graph summarizes the data as means ± SE from 4 independent experiments, the values of which are expressed as the ratio of active GTP-bound RhoA to total RhoA. *P < 0.05 vs. control. #P < 0.05 vs. untreated corresponding condition.

 
DN-RhoA enhanced adhesion of MVHO cells. To determine whether cell adhesion was altered by RhoA activation, we infected cells with a recombinant adenovirus expressing either CA-RhoA or DN-RhoA. Both mutant forms were labeled with HA, and expression of these mutants was detected by probing for HA (Fig. 4). Cell viability following infection was >85% as determined by trypan blue staining. Cell adhesion was reduced in MVHO cells relative to control, as shown above, and this effect was also observed in cells infected with a control vector (EGFP). Expression of CA-RhoA in control cells resulted in a 39.9% reduction in adhesion compared with uninfected cells. In cells isolated from MVHO rats, expression of CA-RhoA also resulted in decreased adhesion relative to controls. On the other hand, expression of DN-RhoA significantly increased adhesion of MVHO cells to levels similar to untreated cells. Thus downregulation of RhoA activity caused enhanced attachment of MVHO cells.


Figure 4
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  		Fig. 4. Expression of dominant negative RhoA (DN-RhoA) enhanced adhesion, whereas constitutively active RhoA (CA-RhoA) inhibited adhesion of control and MVHO cells. Freshly isolated control and MVHO cells were plated on 96-well plates, infected with adenovirus expressing control enhanced green fluorescent protein (EGFP) virus, CA-RhoA, or DN-RhoA mutants, and cell adhesion was determined 24 h after seeding. Cells in 96-well plates were fixed and stained, and absorbance was measured. Western blot shows expression of CA-RhoA or DN-RhoA in control and MVHO cells using HA-tagged antibody. The blot was reprobed with antibody directed against GAPDH to confirm loading. Data are means ± SE from triplicate measurements from 3 separate experiments. *P < 0.050 vs. untreated control. #P < 0.05 vs. untreated MVHO.

 
Expression of FRNK inhibits adhesion of MVHO cells. To determine whether decreased FAK phosphorylation was sufficient to cause decreased cell adhesion, we infected cells with adenovirus expressing an inactive form of FAK (FRNK) labeled with EGFP for verification of expression. When either control or MVHO cells were transfected with adenoviral EGFP-FRNK, there was a decrease in phosphorylation of FAK coincident with a large increase in FRNK expression (Fig. 5A). Expression of FRNK led to significantly decreased attachment of control (71.8% inhibition) and MVHO (46.7% inhibition) cells (Fig. 5A).


Figure 5
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  		Fig. 5. Overexpression of FAK-related non-kinase (FRNK) reduced cell adhesion without affecting RhoA activation. A: control and MVHO cells infected for 24 h with adenovirus expressing either control EGFP virus or EGFP-FRNK mutant were fixed and stained, and adhesion was determined by measuring absorbance. Representative Western blot depicting FAK Tyr397 levels was probed for expression of EGFP-FRNK using either phospho-specific or GFP antibody, followed by reprobing of blots for total FAK. *P < 0.05 vs. untreated control. #P < 0.05 vs. untreated MVHO. B: control cells infected for 24 h with control EGFP virus or EGFP-FRNK mutant were lysed, and RhoA activity was determined via a pull-down assay. Representative blots show levels of GTP-bound RhoA and total RhoA using antibody directed against RhoA. Bar graph indicates the activated RhoA normalized to total RhoA. No significant differences were determined. C: FAK Tyr397 levels in control cells expressing EGFP virus or CA-RhoA for 24 h. No significant differences were determined. Representative blots and bar graphs summarize data as means ± SE; n = 3.

 
FAK activation has been demonstrated to be dependent upon activation of the RhoA/Rho-associated coiled coil-containing protein kinase (ROCK) signaling pathway, and it has been reported that FRNK expression upregulates RhoA activity (41). Since we demonstrated that both RhoA activation and decreased FAK phosphorylation occur in MVHO cells and that either condition is sufficient to cause decreased cell adhesion, we investigated whether decreased FAK phosphorylation caused RhoA activation or whether increased RhoA activation caused decreased FAK phosphorylation. As shown in Fig. 5B, expression of FRNK in control cells did not significantly increase RhoA activation. Similarly expression of CA-RhoA in control cells had no effect on p-FAK (Tyr397) levels (Fig. 5C). These results suggest that downregulation of p-FAK and upregulation of RhoA activity in MVHO cells may be independent events.

Expression of p130Cas restored cell adhesion. p130Cas is an adaptor protein associated with focal adhesion proteins. Since p130Cas phosphorylation was increased by mechanical ventilation with normoxia but not with hyperoxia, we investigated whether expression of a mutant form of p130Cas with deleted phosphorylation sites (SD-Cas) would decrease cell adhesion. As shown in Fig. 6, we observed inhibition of adhesion of cells expressing SD-Cas compared with control cells. Furthermore, overexpression of WT-Cas in MVHO cells resulted in a significant increase in cell adhesion comparable to that of control cells. Together with the results in Fig. 2D, these results suggest that MV cells may retain adherence by increasing phosphorylation of p130Cas, which does not occur in MVHO cells.


Figure 6
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  		Fig. 6. Overexpression of wild-type p130Cas (WT-Cas) increases cell adhesion, whereas expression of substrate-deleted p130Cas (SD-Cas) decreased cell adhesion. Control and MVHO cells infected for 24 h with control EGFP virus, WT-Cas, or SD-Cas mutants were fixed and stained, and adhesion was determined by measuring absorbance. Representative Western blot was probed for expression of WT-Cas and SD-Cas protein levels using an antibody directed against p130Cas and reprobed for GAPDH. Bar graph summarizes data as means ± SE of 3 independent experiments. *P < 0.05 vs. untreated control. #P < 0.05 vs. untreated MVHO.

 
Effects on cell migration. We hypothesized that KGF provides protection against hyperoxia- and high tidal volume ventilation-induced lung injury by enhancing AT2 cell adhesion and by promoting epithelial repair mechanisms such as migration and proliferation. Therefore, we examined whether KGF treatment would enhance migration of AT2 cells in a scratch wound assay. As shown in Fig. 7, wounds in control cells closed to 28.4% of the original wound width by 24 h, and cell migration was unaffected by infection with an EGFP-expressing adenovirus. By comparison, treatment with KGF (50 ng/ml) increased the speed of wound closure (14.1% of original wound width at 24 h). Cell migration was also enhanced by expression of DN-RhoA or overexpression of p130Cas. Cell migration was significantly inhibited in cells expressing CA-RhoA, FRNK, or SD-Cas. Thus, under these experimental conditions, FRNK, CA-RhoA, and SD-Cas inhibited cell migration, whereas DN-RhoA and KGF accelerated wound closure, of AT2 cells. These results support the hypothesis that downregulation of FAK together with upregulation of RhoA activity can impair adhesion and repair mechanisms of AT2 cells.


Figure 7
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  		Fig. 7. AT2 cell migration was stimulated by KGF treatment and expression of DN-RhoA or WT-Cas, whereas migration was inhibited by expression of FRNK, CA-RhoA, or SD-Cas. Confluent monolayers of AT2 cells cultured on RLF matrix and infected for 48 h with control EGFP, CA-RhoA, DN-RhoA, WT-Cas, SD-Cas, or FRNK were wounded with a pipette tip, and wound closure was measured at 6, 12, 18, and 24 h. Data are means ± SE of 3 independent experiments. *P < 0.05 vs. control. Wound closure in EGFP-infected cells was not significantly different from that in uninfected control cells. Wound widths were significantly different from control cells in all treatment groups at 12, 18, and 24 h; wound widths for FRNK- and CA-RhoA-infected cells were significantly different after 6 h.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Because of the reduction in mortality in ARDS patients ventilated with a lower tidal volume identified by the ARDSnet trial (1), there has been substantial interest in determining the mechanisms by which mechanical stretch induced by higher tidal volumes contributes to lung injury (reviewed in Refs. 20, 24, 32, 62). Since damage to the alveolar epithelium is a predominant feature of ARDS and acute lung injury, much of the focus on stretch-induced mechanisms has centered on the response of alveolar epithelial cells. Although ARDS patients can be ventilated with varying levels of supplemental oxygen, few studies have examined the possibility that the combination of high stretch and hyperoxia exacerbates the underlying injury (5, 42, 52). We hypothesized that this combination not only contributes to the alveolar injury but that repair mechanisms may also be diminished. To test this hypothesis, we utilized a novel approach in which AT2 cells were isolated following in vivo mechanical ventilation, and signaling molecules relevant to cell adhesion and migration were investigated.

When AT2 cells were isolated from rats following high tidal volume mechanical ventilation with hyperoxia (MVHO), cell adhesion after 24 h was significantly decreased relative to control rats, rats exposed to hyperoxia alone, or rats that were mechanically ventilated with normoxia (Fig. 1). Consistent with this finding, levels of phosphorylated FAK and paxillin were significantly decreased in MVHO cells but not in any of the other treatment groups (Fig. 2). FAK becomes activated when it localizes into focal adhesions and autophosphorylates at Tyr397 (12, 44), and FAK facilitates the phosphorylation of other focal adhesion proteins such as paxillin and p130Cas (48, 49, 63). The decrease in FAK phosphorylation in our study indicates a loss of focal adhesions in AT2 cells following MVHO treatment. This is a novel finding given that both mechanical forces and oxidant stress have independently been shown to activate FAK in other types of cultured cells. Stimulation of FAK phosphorylation has been demonstrated in response to mechanical stretch in cardiac myocytes (50, 55, 56) and fibroblasts (64) and in response to shear stress (30) or stretch (2) in endothelial cells. FAK phosphorylation is also increased in endothelial cells in response to oxidative stress (7, 60, 61). Neither high stretch (MV) nor hyperoxia (HO) alone affected FAK phosphorylation in our studies, suggesting tight regulation of FAK signaling under these conditions in vivo. However, this regulation appears to be lost in the presence of both high tidal volume and hyperoxia. None of the previous studies examined the combined effect of mechanical stimulation and hyperoxia on FAK, and it is possible that the combination results in activation of phosphatases. In one of the few studies of focal adhesion signaling in cultured alveolar epithelial cells, it was shown that mechanical dissociation of AT2 cells from in vitro matrix resulted in decreased FAK phosphorylation that was likely the result of activation of phosphatases (36). We considered the possibility in our studies that the enzymatic digestion used to dissociate AT2 cells from the lungs following treatments resulted in the activation of phosphatases, but if this were the case, then all treatment groups would have been affected in the same way. FAK phosphorylation was decreased only in the MVHO group, and this decrease persisted for 24 h (Fig. 2B). By 48 h, FAK phosphorylation in the MVHO group increased to the level of control cells, and there was no difference from control cells in cell adhesion at this time (data not shown). In contrast, freshly isolated cells from control rats had consistent levels of FAK phosphorylation after 24 and 48 h, suggesting that the observed differences were not caused by the isolation procedure. To support our findings that decreased FAK phosphorylation results in decreased adhesion of AT2 cells, we decreased FAK phosphorylation in control cells by expressing FRNK and measured decreased adhesion (Fig. 5A). Expression of FRNK also significantly decreased AT2 cell migration in our wound assay (Fig. 7), suggesting that loss of FAK phosphorylation in MVHO cells results in impaired cell migration and wound healing in AT2 cells as well as decreased adhesion.

Our studies provide some of the first results demonstrating the consequences of in vivo exposure of alveolar epithelial cells to high tidal volume and hyperoxia on focal adhesions. In contrast with our findings in AT2 cells, Bhattacharya et al. (8) measured increased FAK phosphorylation in freshly isolated lung microvascular endothelial cells following mechanical ventilation of an isolated perfused rat lung. High tidal volume mechanical ventilation also induced P-selectin expression in endothelial cells in this study, suggesting a proinflammatory response to high stretch. That study raises the possibility that our measurements may be the result of paracrine signaling from other pulmonary cells or from immunologic responses, but the explanation for diverse responses in endothelial and AT2 cells is unclear. However, the combined effects of hyperoxia and high tidal volume were not determined in that study. Quinn et al. (42) demonstrated in a rat model that the combination of hyperoxia and high stretch tidal volume resulted in increased levels of macrophage inflammatory protein-2 (MIP-2) and corresponding increases in neutrophil migration into alveoli, and Bailey et al. (5) showed that exposure of mice to hyperoxia for 48 h before high tidal volume mechanical ventilation resulted in decreased lung compliance and increased levels of IL-6 and TNF-{alpha} in bronchoalveolar lavage. Using a similar approach in rats, Lesur et al. (29) showed that high tidal volume mechanical ventilation for 2 h increased lung injury and bronchoalveolar lavage levels of protein and Clara cell protein (CC-16) but that preexposure to hyperoxia had little effect. In another study, rabbits exposed to high tidal volume mechanical ventilation and hyperoxia experienced accelerated lung injury and early loss of alveolar-capillary barrier function that was not associated with an increase in proinflammatory mediators (52). In that study, rabbits were exposed to 50% oxygen, which is generally considered a safe level by clinicians. Although AT2 cell focal adhesions were not examined in these studies, the combination of high stretch and hyperoxia induced significant changes in several end points associated with injury.

Although both paxillin and p130Cas are potential substrates for FAK, the phosphorylation pattern of each of these focal adhesion-associated proteins was different following in vivo treatments. Whereas paxillin phosphorylation was similar to that of FAK (Fig. 2C), with decreased phosphorylation following MVHO treatment, the phosphorylation of p130Cas was increased in the MV group but was not significantly affected by HO or MVHO treatment (Fig. 2D). Since previous studies have demonstrated that FAK-mediated activation of paxillin and p130Cas involves association with FAK (28, 48, 49, 63), these results suggest that paxillin may be associated with FAK under these conditions but that p130Cas is not. It is interesting to note that overexpression of wild-type p130Cas was sufficient to restore the adhesion of MVHO cells to that of control levels, whereas the expression of a substrate-deleted mutant of p130Cas decreased adhesion of control cells to that of MVHO cells (Fig. 6). The increase in phosphorylation of p130Cas in response to mechanical ventilation may be a compensatory response to promote cell adhesion, but this mechanism was ineffective in MVHO cells. Furthermore, overexpression of WT-Cas accelerated wound repair, whereas expression of SD-Cas significantly decreased wound repair (Fig. 7). Together, these results suggest an important but not previously recognized role of p130Cas in AT2 cell adhesion and repair mechanisms.

Several groups have proposed that mechanical force-induced changes in focal adhesions are mediated through RhoA-dependent mechanisms (33, 45, 51, 56). We found that RhoA activity was elevated in AT2 cells following in vivo exposure to high tidal volume, hyperoxia, or a combination of the two (Fig. 3). Mechanical stretch previously has been shown to stimulate RhoA activation in cardiac myocytes (56), vascular and airway smooth muscle cells (35, 53), and endothelial cells (9), but to our knowledge there are no prior studies demonstrating increased RhoA activation in AT2 cells in response to in vivo mechanical stretch or hyperoxia. It is interesting to note that although RhoA was activated in all three treatment groups, cell adhesion was decreased only in the MVHO group (Fig. 1). Expression of CA-RhoA was sufficient to decrease both cell adhesion and cell migration in control cells, whereas expression of DN-RhoA restored the adhesion of MVHO cells to control levels and increased cell migration (Figs. 4 and 7). Since FAK phosphorylation was decreased only in the MVHO group, and since focal adhesion assembly and turnover has been demonstrated to depend on regulatory signaling between RhoA and FAK (6, 25, 43, 57), we investigated whether activation of RhoA or suppression of FAK were independent in AT2 cells. Expression of CA-RhoA had no effect on the phosphorylation of FAK (Fig. 5C), whereas expression of FRNK had no effect on the activation of RhoA (Fig. 5B). These results, coupled with the finding that RhoA was activated in MV and HO cells without a corresponding decrease in FAK phosphorylation, suggest that the observed decrease in FAK phosphorylation in the MVHO group was independent of RhoA activation. In addition, since adhesion was decreased only in the MVHO group, there are likely compensatory mechanisms that offset the effects of increased RhoA activity to either mechanical stretch (MV) or hyperoxia alone (HO) in this model. Phosphorylation of p130Cas is one possible explanation for this finding, since this was elevated in the MV group but not in the MVHO group (Fig. 2D). This is supported by the finding that overexpression of wild-type p130Cas restored the adhesion of MVHO cells to control levels (Fig. 6). Our data suggest that the combination of increased RhoA activation and decreased FAK and paxillin phosphorylation without a corresponding increase in p130Cas phosphorylation results in decreased cell adhesion. However, we did not investigate all potential pathways, and it is possible that other signaling molecules are also altered by the combination of high stretch and hyperoxia.

Our approach in this study was to determine phenotypic changes in AT2 cells isolated immediately following experimental treatments in vivo, but there are limitations to this approach. For example, epithelial cell attachment and migration in vivo would likely occur on a substrate undergoing mechanical deformation during the respiratory cycle, and this would likely affect these processes. We previously demonstrated that mechanical stretch inhibits wound healing of airway epithelial cells (46, 47, 67), and we are currently investigating these processes using AT2 cells. Thus our experimental approach may have minimized the functional effect of the biochemical and biological changes that we observed in freshly isolated AT2 cells. Another limitation is that the treatment with KGF occurred in a mechanically static and normoxic environment, and it is possible that the activity and functional effects of KGF may differ under conditions of mechanical deformation and/or hyperoxia. Hyperoxia may also directly affect cell adhesion and cell migration, but we did not perform these measurements under hyperoxic conditions. However, it is important to note that the in vivo treatments used in our studies were sufficient to induce changes in signaling pathways and functional consequences in the isolated cells that were persistent for as long as 24 h. Finally, it is important to note that the isolation procedure itself may expose AT2 cells to transient levels of high shear stress and mechanical deformation, but as noted above, cells from all treatment groups were isolated using the same process. Therefore, it is unlikely that the differences we observed were caused by the isolation procedure, but it is possible that other differences may have been obscured.

KGF has been shown to reduce lung injury in experimental models of acute lung injury, including exposure to hyperoxia (40) and mechanical ventilation (29, 69), primarily through mechanisms involving AT2 cells (11, 23, 40, 59, 69, 71, 72). We found that KGF enhanced the adhesion of AT2 cells isolated from rats following mechanical ventilation and increased the adhesion of MVHO cells beyond that of control cells (Fig. 1). In addition, KGF stimulated the migration of AT2 cells in our wound assay (Fig. 7). KGF had no effect on the phosphorylation of FAK, paxillin, or p130Cas in any of the treatment groups (Fig. 2) but reduced RhoA activity in both MV and MVHO cells (Fig. 3). To our knowledge, this is the first demonstration that KGF affects RhoA activity and may provide insight into how KGF promotes cell migration and enhances repair processes. Since KGF did not affect the phosphorylation of the focal adhesion proteins that we examined, this raises the question of what pathways either upstream or downstream from RhoA may be important. One likely candidate is integrin pathways, but we did not explore these pathways in the current study. Another possibility is that there were differences in other cytoskeletal signaling molecules such as Rho kinase and myosin light chain kinase that resulted in diminished adhesion. It is also important to note that the enhancement of cell adhesion occurred in cells treated with KGF after in vivo exposure to mechanical ventilation and hyperoxia, suggesting that postinjury KGF treatment may be beneficial.

Repair mechanisms involving AT2 cells are essential for the restoration of epithelial barrier integrity following acute lung injury. Our results are significant because they suggest that the combination of high stretch mechanical ventilation and hyperoxia that can occur in patients with ARDS promotes an AT2 cell phenotype that is less adherent and less motile. KGF restored cell adhesion and enhanced cell migration through mechanisms involving RhoA.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL064981 (to C. M. Waters), HL004479 (to S. E. Sinclair), and HL63886 and HL72902 (to A. Hassid).


   ACKNOWLEDGMENTS
 
We thank Alice-Corina Ceacareanu, Bogdan Ceacareanu, and Daming Zhuang for suggestions and Charlean Luellen for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. M. Waters, Dept. of Physiology, Univ. of Tennessee Health Science Center, 894 Union Ave., Nash 426, Memphis, TN 38163 (e-mail: cwaters2{at}utmem.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.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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293/3/L769    most recent
00127.2007v1
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