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1Department of Biomedical Engineering, Northwestern University, Evanston, Illinois; and Departments of 3Physiology and 2Medicine, The University of Tennessee Health Science Center, Memphis, Tennessee
Submitted 10 February 2005 ; accepted in final form 13 June 2005
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ABSTRACT |
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mechanotransduction; ventilator-induced lung injury
Increased ROS production in response to mechanical stress has been described in a variety of cell types. Endothelial cell production of ROS has been shown to increase in response to shear stress (4, 6, 7, 10, 19, 30, 45, 50) and to cyclic mechanical stretch (CMS; see Refs. 2, 29, 47, 48). CMS also stimulates ROS production in vascular smooth muscle cells (13, 22, 31) and cardiac myocytes (1). Although superoxide (O2–) appears to be the initial species generated in these cell types, there is disagreement as to the source of ROS production. The NADPH oxidase system (7, 13, 29, 30), mitochondrial production (2, 21), and the xanthine oxidase system (30) have all been implicated as potential sources for increased O2– production in response to mechanical stress. However, whether these potential sources are activated directly or indirectly by mechanical stress is unclear. Little data are available demonstrating increased ROS production by stretched alveolar or airway epithelial cells. A 25% increase in ROS production, as measured by 2,7-dichlorodihydrofluorescein (DCFH) fluorescence caused by interaction with H2O2, was measured in response to 30% biaxial stretch of A549 cells, a human alveolar type II-like cell line (42). In another study, A549 cells subjected to cyclic stretch produced significantly increased levels of isoprostane (a marker of oxidant injury) after 0.5 h, and levels of reduced glutathione (GSH, an endogenous antioxidant) were significantly decreased after 1 h of CMS (23). Pretreatment with antioxidants prevented the change in isoprostane levels in this study. However, the source(s) of increased ROS production and species generated in pulmonary epithelial cells exposed to mechanical stretch have not been determined.
In the current study, we examined whether cyclic mechanical strain induced production of O2– in the main epithelial constituents of the lungs, airway and alveolar epithelial cells. O2– production was significantly enhanced in an SV40-transformed human airway epithelial cell line (16HBE), A549 cells, and isolated primary rat alveolar type II (rat AT II) epithelial cells undergoing CMS. Increased ROS production was dependent on the time and magnitude of stretch. The source of the O2– was a combination of the NADPH oxidase system and the mitochondria (complex I). Mitochondrial involvement may be initiated by a direct distention of the mitochondria resulting from mechanical stretch.
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MATERIALS AND METHODS |
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Cell culture. 16HBE cells were generously provided by Dr. Deiter Gruenert and were cultured in DMEM (GIBCO, Carlsbad, CA) with 4 mM glutamine supplemented with 10% FBS (Cellgro, Herndon, VA or GIBCO), 20 mM HEPES (Sigma), and 2.5 µM Plasmocin (Invivogen, San Diego, CA). These cells were first treated for 2 wk with 25 µM Plasmocin. A549 cells were cultured in DMEM with 4 mM glutamine supplemented with 10% FBS (Cellgro) and 1% penicillin/streptomycin solution (GIBCO). Both cell types were cultured until 85–100% confluence before being seeded on the BioFlex plates coated with collagen type I (Flexcell International, Hillsborough, NC) for studies on confluent monolayers. The medium was changed every other day for all cultures.
Isolation and culture of rat AT II cells. Primary rat AT II cells were isolated according to established procedures (8, 26, 32). Briefly, male Sprague-Dawley rats were anesthetized with phenobarbital, killed by exsanguination, and their lungs were excised. The trachea was catheterized, and the pulmonary vasculature was perfused via the pulmonary artery with solution II (in mM: 140 NaCl, 5 KCl, 2.5 Na2HPO4, 10 HEPES, 1.3 MgSO4, and 2.0 CaCl2; pH 7.4) to remove the blood. The air spaces were then lavaged with solution I (in mM: 140 NaCl, 5 KCl, 2.5 Na2HPO4, 6 glucose, 0.2 EGTA, and 10 HEPES; pH 7.4) to remove free, nonepithelial cells. Elastase (4.3 U/ml in solution II; Worthington Biochemical, Lakewood, NJ) was instilled in the air space and incubated at 37°C for 10 min. This was repeated, and the large airways and heart were removed. The remaining lung tissue was then minced in 5 ml FBS and 250 µl of 250 µg/ml DNase (Sigma) per 4 lungs. The minced lungs were filtered through gauze followed by a nitrocellulose membrane, and the cell suspension was collected. The suspension was centrifuged and resuspended in AT II culture medium [DMEM with 10% heat-inactivated FBS (HyClone, Logan, UT), 4 mM glutamine, 1% penicillin/streptomycin, and 0.25 µM amphotericin B (Sigma)] and plated on untreated petri dishes coated with IgG. The plates were incubated for 1 h to allow nonepithelial cells such as macrophages to bind to the IgG. The plates were "panned" to loosen nonspecifically bound cells, pooled, and counted. BioFlex plates coated with collagen type I by the manufacturer were then coated with 32.3 µg/ml human fibronectin (Roche Life Sciences, Indianapolis, IN), and cells were seeded to confluence at 3.0 x 106/well in AT II culture medium. Experiments were performed on day 2 after isolation. AT II cells were identified using Nile Red (Sigma) staining of lamellar bodies, and >90% of the cells were Nile Red positive on day 2.
Application of mechanical strain. Strain was applied using a Flexercell strain unit 4000 (Flexcell International), a vacuum-driven device that applies biaxial strain to cells cultured on Silastic-bottomed well plates. The device allows control of the frequency of strain and magnitude up to 30% linear strain. 16HBE and A549 cells were strained at 20% elongation and 30 cycles/min (cpm) for the initial studies. This strain profile was previously found to maximally inhibit wound closure in airway epithelial cells (38). Rat AT II cells were strained at 15% elongation and 15 cpm because higher levels of strain and frequency resulted in increased cell detachment from the culture plate.
Measurement of O2– production. Intracellular O2– production was measured by the fluorescent intensity of oxoethidium after dihydroethidium (DHE; Molecular Probes, Eugene, OR) was oxidized by O2–. Oxoethidium binds to DNA, becoming highly fluorescent. After treatment, cells were rinsed two times with warm serum- and phenol red-free (PR-Free) DMEM and loaded with 5 µM DHE in PR-Free DMEM at 37°C in the incubator for 30 min. The cells were then rinsed one time with room temperature PR-Free DMEM, fresh PR-Free DMEM at room temperature was added, and the cells were imaged immediately. Images were taken on an inverted fluorescent microscope (TE300; Nikon Instruments, Lewisville, TX) outfitted with a x20 PlanFluor objective (Nikon Instruments), rhodamine filter set for excitation and emission (Chrorma Technology, Rockingham, VT), and digital CCD camera (Roper Scientific, Tucson, AZ). Images were acquired using MetaMorph software (Universal Imaging, Downingtown, PA) for 10 s with a x4 neutral density filter engaged. Four separate images were taken per well, avoiding areas of low confluence and areas near the edge of the well. The average pixel intensity per image was calculated, and an average intensity per well was determined. To assess the involvement of the mitochondria, treatment with the pharmacological inhibitor of the mitochondrial complex I, rotenone (25 nM), was initiated at the beginning of the stretch protocol.
Measurement of reduced and oxidized glutathione levels. A protocol from Kamencic et al. (25) adapted for a fluorescence plate reader was used to determine levels of reduced and oxidized GSH (GSSG). 16HBE cells were stretched or held static for various periods of time and then placed on ice. The cells were rinsed two times with ice-cold Ca2+/Mg2+-free Dulbecco's phosphate-buffered saline (DPBS). Ice-cold cell lysate buffer (0.5 ml, 0.2 mM EDTA in Ca2+/Mg2+-free DPBS) was added to each well. The cells were scraped and transferred to centrifuge tubes at 4°C. Samples were sonicated on ice for 20 s. The samples were centrifuged for 10 min at 10,000 g and 4°C. Two 100-µl aliquots of each sample were diluted with 200 µl deionized H2O/aliquot. One diluted aliquot from each sample was completely reduced by the addition of 15 µl of 4 M triethanolamine (TEA) to determine the total amount of GSH. A standard curve of GSH with and without TEA was created. Wells of a black 96-well plate were loaded with 50 µl of diluted sample and 150 µl assay solution (100 µM monochlorobimane, 1 U/ml glutathione-S-transferase, and 0.2 mM EDTA in Ca2+/Mg2+-free DPBS), and the plate was incubated in the dark for 30 min at room temperature. The plate was then read in a fluorescence plate reader (Molecular Devices, Sunnyvale, CA) with excitation at 380 nm and emission at 470 nm. To calculate GSSG, the amount of reduced GSH was subtracted from the TEA-treated sample and divided by two to account for the dimerization. The ratio of reduced GSH to GSSG was then calculated to monitor the redox state of the cells.
NADPH oxidase assay. The assay used to determine NADPH oxidase activity was adapted from previous studies (7, 51). Cells were stretched or held static, placed on ice, and rinsed three times in ice-cold DPBS. Cells were placed in 0.5 ml homogenization buffer (1 mM EGTA, 10 µg/ml aprotinin, 0.5 µg/ml leupeptin, and 0.5 mM phenylmethanesulfonyl fluoride in Ca2+/Mg2+-free DPBS), scraped, homogenized manually, and placed in tubes. Cell homogenate (50 µl) was added to 3 ml warm chemiluminescence buffer (10 µM Lucigenin, 1 mM EGTA, and 150 mM sucrose in Ca2+/Mg2+-free DPBS) containing NADPH (50 µM) in a scintillation vial. Chemiluminescence was measured (in duplicate) for 3 min in a Packard scintillation counter. Diphenylene iodonium (DPI, 10 µM) was included in the chemiluminescence buffer for selected static and stretched samples.
Staining and imaging of mitochondria of stretched cells.
16HBE cells were cultured on collagen I-coated Silastic membranes for the StageFlexer (Flexcell International), which is a single membrane stretching device adapted for use on a microscope stage. Live 16HBE cells were stained with 500 nM MitoTracker Orange (Molecular Probes) for 30 min and rinsed two times with warm DPBS. Membranes were placed in the StageFlexer, and images were acquired with a x60 objective (Nikon) before and after the application of 
17% biaxial strain. Because images included contributions of structures from multiple planes, we selected structures that were likely to be in thin or confined regions of the cells such as beneath the nucleus and near the edges of the cells. The total length of each mitochondrion was measured by tracing a line down the center line of each structure to determine the overall length before and during strain.
Statistical analysis.
All values are presented as means ± SE. All statistical analyses were performed with the SigmaStat statistical package (version 2.03; Jandel Scientific, San Rafael, CA). One-way ANOVA was performed for comparisons of multiple treatments. If this showed significant difference between the treatments, a Student's t-test with the Bonferroni correction was performed to determine significant differences between the individual conditions. All of the ANOVA tests were run with multiple comparisons against the static control except for the data collected using the mitochondrial inhibitor rotenone. These experiments employed multiple comparisons between all of the different treatments. Significant differences are based on a threshold of P 
0.05.
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RESULTS |
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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CMS of A549 cells has been shown previously to increase generation of ROS. Upadhyay et al. (42) demonstrated that A549 cells stretched for 1 h at 30% biaxial elongation and 30 cpm increased generation of H2O2 by 25% compared with static controls (42). In that study, the cells were loaded with DCFH, which is oxidized to the fluorescent compound dichlorofluorescein by H2O2. H2O2 is one of several downstream metabolites of O2– generation. We measured a greater increase in ROS production by A549 cells using DHE after 2 h of CMS (Fig. 1B). The lower increase in ROS production detected by Upadhyay et al. (42) may be because of the metabolism of O2– to other pathways not detected by DCFH. In addition, DCFH has been reported to leak out of cells (37) and in preliminary experiments we too observed leakage of DCFH from cells. Also, stretching cells loaded with this dye may cause further leakage through transient, nonlethal strain-induced membrane breaks (44). Oxidized DHE binds to DNA and fluoresces, and thus the fluorescent dye remains inside the cells. We loaded the dye after the stretch concluded, reducing potential leakage of the DHE dye. Differences between our study and previous studies could also be because of the duration of the stretch. We measured changes in ROS production over time in 16HBE cells (Fig. 2) and found that CMS of 0.5 h did not elicit an increase in O2– production, but 2 h caused a significant increase. Although we did not measure ROS production after 1 h of CMS in A549 cells, the response may be similar to that reported by Upadhyay et al. (42).
Other investigators have used DHE to monitor changes in O2– production in human lung endothelial cells exposed to hyperoxia (35), nutrient-deprived human aortic endothelial cells (28), and ANG II treatment of bovine aortic endothelial cells (9). Lopez et al. (28) used images of DHE staining to quantify changes in the generation of O2– with nutrient deprivation. We used a similar method to quantify changes in the current study. Parinandi et al. (35) used cell lysate fluorescence from DHE-loaded cells to measure changes in O2– production. O2– production has also been measured by an HPLC method in cells and intact tissues (9) based on the fluorescence of oxoethidium. This study also demonstrated the specificity of DHE for O2– by showing that fluorescence was not increased after treatment with H2O2, peroxynitrite, or hypochlorous acid.
ROS generation caused by CMS resulted in a significant reduction of the ratio of GSH to GSSG in 16HBE cells, indicating an oxidant stress that occurred as early as 0.5 h, and the ratio remained significantly reduced through 24 h (see Fig. 3). An increase in ROS production as a result of CMS was not observed in 16HBE cells until 2 h of strain, as measured by DHE fluorescence (see Fig. 4), but the GSH-to-GSSG ratio indicated an oxidant stress after only 0.5 h of stretch. This implies that the cellular antioxidant defenses were able to absorb the excess ROS before being overwhelmed by 2 h, resulting in an increase in DHE fluorescence. However, after 24 h, DHE measurements returned to basal levels, whereas the GSH-to-GSSG ratio remained significantly reduced. Jafari et al. (23) measured GSH levels in A549 cells and found that levels were significantly reduced after 1 h of CMS at 15% strain and 20 cpm, returned to basal levels after 2 and 3 h, but showed a significant increase after 4 h of CMS. Our results corroborate their findings of an early oxidant stress caused by CMS. However, in our study, although the ratio of GSH to GSSG decreased, reduced GSH levels were maintained at a constant level in 16HBE cells subjected to CMS with no significant changes over 24 h, whereas total GSH levels increased significantly after 24 h, resulting in the continued reduction in the ratio (data not shown). This may not fully explain the reduction in O2– after 24 h, but we did not investigate activity or protein levels of antioxidants more specific to O2– such as superoxide dismutase. Increased levels of superoxide dismutase after 24 h might account for the decrease in O2– production with the sustained decrease in the GSH-to-GSSG ratio. Differences between antioxidant systems in 16HBE and A549 cells may also contribute to the observed differences. However, measurement of both GSH and GSSG levels is important to assess the redox state of the cells.
We explored the effects of varying the magnitude of strain on ROS production of pulmonary epithelial cells and found that application of strain levels <15% elongation resulted in O2– production that was not significantly different from control. Application of strain levels 
15% caused significant increases in ROS production (Fig. 4). To our knowledge, this is the first time ROS production has been studied as a function of the magnitude of strain in pulmonary epithelial cells. These results suggest that a threshold level of strain exists above which elevated ROS production occurs. The strain levels experienced by the lungs in vivo during either spontaneous breathing or mechanical ventilation are difficult to determine. Tschumperlin and Margulies (41) measured the changes in epithelial basement membrane surface area (EBMSA) of lungs fixed at different inflations and found the expected nonlinear response. Inflation from 24% to total lung capacity (TLC) caused a 40% increase in EBMSA, whereas inflation from 42% of TLC to TLC resulted in a 34% increase. This change in surface area corresponds to a linear strain of 18%. In injured lungs, some regions may be collapsed or fluid-filled, functionally reducing the volume of the lungs. Therefore, during mechanical ventilation, the remaining volume of the lungs may be significantly overinflated, causing even greater distention and injury (20). However, the actual levels of strain in the airways and alveoli in injured lungs have not been well characterized. Some authors have suggested that linear strain of 10% occurs during normal breathing and that higher levels occur during high tidal volume mechanical ventilation, but there are no direct measurements of this. Our results showed that ROS production was significantly increased at strains of 15% or greater. During mechanical ventilation at high tidal volumes or even during lower tidal volumes, if the lung volume is effectively reduced by injury, the average distention of the tissue may be above this threshold and cause generation of ROS from the epithelium.
In this study, we were unable to stretch the primary rat AT II cells at 20% elongation and 30 cpm because significant detachment of the cells from the membrane occurred. Therefore, the values reported in Fig. 1B for rat AT II cells are from cells stretched at 15% strain and 15 cpm on day 2 of culture, and the cells remained attached under these conditions. Oswari et al. (33) showed a substantial loss of viability (49%) in rat AT II cells cultured for 1 day on fibronectin-coated Silastic and exposed to 1 h of CMS of 25% change in surface area (12% linear strain) and 15 cpm. However, treatment with keratinocyte growth factor or culture of the cells on a matrix deposited by AT II cells for 5 days resulted in levels of viability similar to static controls. We are currently adapting our culture methods to better promote attachment and survival at increased strain and frequency. Despite the lower strain and frequency of stretch, we observed a significant stimulation of ROS production, albeit at a lower level than that seen in the cell lines tested.
The source of mechanical strain- or shear stress-induced ROS is currently debated, and potential sources in different cell types include NADPH oxidase (7, 13, 29, 30), xanthine oxidase (30), nitric oxide synthase, and the mitochondria (2, 21). Previous studies with endothelial cells concluded that the source of ROS was primarily NADPH oxidase, since treatment with the flavoprotein inhibitor DPI abolished the response. However, DPI is a nonspecific inhibitor of NADPH oxidase that also inhibits other potential sources of ROS generation, including the enzymes of the electron transport chain in mitochondria. Recent studies have demonstrated that mitochondria are involved in strain-induced production of ROS in endothelial cells. Ichimura et al. (21) and Ali et al. (2) have shown that the complex I inhibitor rotenone eliminated strain-induced ROS production. Ali et al. also used ethidium-treated human umbilical vein endothelial cell cultures to eliminate mitochondrial DNA and the electron transport chain proteins and found that the CMS-induced increase in ROS production was eliminated. A more specific pharmacological inhibitor of NADPH oxidase, apocynin, was employed and was found to have no effect on CMS-induced ROS generation. We found that CMS of 16HBE cells stimulated NADPH oxidase activity and that ROS production was partially sensitive to inhibition of mitochondrial complex I. The activity of NADPH oxidase, measured by Lucigenin chemiluminescence, increased significantly after 2 h of CMS (Fig. 6). Treatment of the cell lysates with DPI (10 µM) inhibited the increase in activity and reduced the activity in static cell lysates to similar levels. The specificity of the response was tested by comparing results with and without an excess of NADPH (50 µM) in the reaction mixture. Chemiluminescent readings were similar to background when NADPH was not included in the reaction cocktail (data not shown). Rotenone partially inhibited CMS-induced increases in DHE fluorescence (Fig. 5). Taken together, our results suggest that CMS-induced increases in ROS production are the result of both NADPH oxidase and mitochondrial generation in pulmonary epithelial cells.
If mitochondrial production of O2– is stimulated by mechanical stretch, then what is the initiating event? We hypothesized that global strain applied to the cells may lead to direct distention of the mitochondria, since mitochondria are attached to both the actin (39) and microtubule (16, 17) components of the cytoskeleton. We observed substantial deformation of mitochondrial structures when cells were stretched (Fig. 7). We speculate that mechanical distention of mitochondrial structures may activate pathways that lead to generation of ROS. For example, mitochondrial ATP-sensitive K+ (
) activation has been linked to generation of ROS (11, 15), and activation of the mitochondrial channel has been linked to mitochondrial swelling (18). However, the mechanism of ROS production through activation of the mitochondrial channel has not been determined, and no studies have connected the distention caused by mitochondrial swelling to ROS production. One of the major limitations of our measurements of mitochondrial deformation is that the images that were analyzed (Fig. 6) were from a single plane of focus. Because we did not collect images at different z-planes, we cannot exclude the possibility that the change in length that was observed was due to the appearance of mitochondrial structures from out-of-focus planes in the images of stretched cells. However, these images were not taken with a confocal microscope, and thus contributions from multiple planes are more likely to appear in the image. In addition, 16HBE cells are quite thin, and we specifically identified mitochondrial structures that were in more confined areas near the cell boarder or beneath the nucleus, making the possibility of out-of-plane regions appearing in the focal plane less likely. These considerations, however, still do not rule out the possibility that apparent deformation was the result of the appearance of out-of-focus structures.
One proposed mechanism for VILI is that increased mechanical deformation of lung tissue stimulates an acute inflammatory response characterized by increased levels of proinflammatory cytokines and inflammatory cell infiltration. In previous studies, high tidal volume mechanical ventilation without underlying lung injury led to both increased cytokine production (46) and enhanced neutrophil infiltration (5). CMS has been shown to stimulate production of proinflammatory cytokines in both airway and alveolar cells. CMS of BEAS-2B, an airway epithelial cell line, resulted in an increased production of interleukin (IL)-8 (34). A549 cells when subjected to CMS increased production of proinflammatory cytokines IL-8 (23, 27, 43, 49), IL-6 (23), and transforming growth factor-
(49). Primary cultures of rat AT II also exhibited CMS-induced production of IL-8 (36). Exposure to H2O2 or exogenous O2– production by xanthine/xanthine oxidase and exposure of rat AT II cells to hyperoxia also caused increased production of the proinflammatory cytokines IL-1, IL-6, and tumor necrosis factor-
(14). Ali et al. (2) demonstrated that stretch-induced expression of vascular cell adhesion molecule-1 and activation of NF-
B could be abrogated by blocking mitochondrial generation of ROS in endothelial cells. Thus inflammatory responses may be initiated by stretch-induced production of ROS. We demonstrated in this study that stretch stimulated increased ROS production in both airway and alveolar epithelial cells, that the response was both time varying and magnitude dependent, and that ROS are derived from both NADPH oxidase and mitochondria.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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