H2O2 inhibits alveolar epithelial wound repair in vitro by induction of apoptosis
Thomas Geiser,1,2,3
Masanobu Ishigaki,1,2
Coretta van Leer,3
Michael A. Matthay,2 and
V. Courtney Broaddus1,2
1Lung Biology Center and 2Cardiovascular Research Institute, University of California, San Francisco, California 94143; and 3Division of Pulmonary Medicine, University of Bern, CH-3010 Bern, Switzerland
Submitted 24 April 2003
; accepted in final form 24 April 2004
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ABSTRACT
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Reactive oxygen species (ROS) are released into the alveolar space and contribute to alveolar epithelial damage in patients with acute lung injury. However, the role of ROS in alveolar repair is not known. We studied the effect of ROS in our in vitro wound healing model using either human A549 alveolar epithelial cells or primary distal lung epithelial cells. We found that H2O2 inhibited alveolar epithelial repair in a concentration-dependent manner. At similar concentrations, H2O2 also induced apoptosis, an effect seen particularly at the edge of the wound, leading us to hypothesize that apoptosis contributes to H2O2-induced inhibition of wound repair. To learn the role of apoptosis, we blocked caspases with the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (zVAD). In the presence of H2O2, zVAD inhibited apoptosis, particularly at the wound edge and, most importantly, maintained alveolar epithelial wound repair. In H2O2-exposed cells, zVAD also maintained cell viability as judged by improved cell spreading and/or migration at the wound edge and by a more normal mitochondrial potential difference compared with cells not treated with zVAD. In conclusion, H2O2 inhibits alveolar epithelial wound repair in large part by induction of apoptosis. Inhibition of apoptosis can maintain wound repair and cell viability in the face of ROS. Inhibiting apoptosis may be a promising new approach to improve repair of the alveolar epithelium in patients with acute lung injury.
reactive oxygen species; acute lung injury; hydrogen peroxide; caspase inhibition; neutrophil
ACUTE LUNG INJURY (ALI) and the acute respiratory distress syndrome (ARDS) are characterized by extensive alveolar epithelial damage (4). The loss of epithelial integrity with increased permeability of the alveolar-capillary barrier leads to the influx of protein-rich edema fluid and accumulation of neutrophils in the alveolar space (27).
Neutrophils are the predominant inflammatory cells in the alveolar space of patients with ALI/ARDS (23) and contribute to alveolar tissue injury by releasing proteases and reactive oxygen species (ROS). Several experimental and clinical studies have provided evidence for oxidant-mediated injury to the lung, characterized by increases in endothelial and epithelial permeability (9, 19). Loss of the alveolar epithelial barrier has been well documented in morphologic studies of patients dying with ALI/ARDS (4). Release of ROS may be one important mechanism of alveolar epithelial cell death in ALI.
There is increasing evidence that alveolar epithelial cell apoptosis is a major mechanism of cell death in the acute phase of ALI/ARDS. Moreover, there are some reports suggesting a role for apoptosis in the repair phase after ALI for both the clearance of excess alveolar epithelial cells (5) and the removal of excess mesenchymal cells (24). However, the role of apoptosis in the repair of the injured alveolar epithelium has not been well studied.
Efficient repair of the alveolar epithelium is crucial for the restoration of the alveolar epithelial barrier and recovery from ALI/ARDS (18). Moreover, disorganized or insufficient epithelial repair may lead to fibrosis (7). The alveolar epithelial type II cell is the progenitor for reepithelialization of the denuded alveolar epithelium (1, 4). Alveolar type II epithelial cells restore the integrity of the alveolar epithelium by proliferation, spreading, and migration, and finally by differentiation to alveolar type I cells, restoring the normal morphology and functional properties of the alveolar epithelium.
To study the effect of ROS on alveolar epithelial repair in vitro, we used our model of alveolar epithelial repair (12, 14, 16). In our initial studies we found that H2O2 inhibits in vitro alveolar epithelial wound repair. We found that alveolar epithelial cell apoptosis is an important mechanism of H2O2-induced inhibition of alveolar epithelial repair in vitro. Moreover, inhibition of the apoptosis restored repair to a large degree, suggesting a potential future role for manipulation of apoptotic mechanisms in therapy of lung injury.
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METHODS
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In vitro alveolar epithelial wound repair assay.
Epithelial repair was determined using our in vitro epithelial wound repair assay as described before (3, 12, 14, 16). Briefly, A549 alveolar epithelial-like cells (American Type Culture Collection, Rockville, MD) were cultured to confluence in 24-well plates in DMEM containing 10% fetal bovine serum and then mechanically wounded with a pipette tip. In addition, primary human distal lung epithelial cells (Cambrex) were cultured in complete growth media (SAGM, Cambrex) according to the manufacturer's protocol before wounding. Both A549 epithelial cells and primary lung distal epithelial cells were plated on untreated culture plates. Four hours after wounding, H2O2 (Fisher Scientific, Pittsburgh, PA) was added in a range of concentrations to the wounded alveolar epithelial monolayers, and the area of the denuded surface was measured immediately after addition of H2O2 and again after 24 h. The plates were placed on an inverted microscope (Axiovert 35; Zeiss, Thornwood), and the cell monolayer was photographed with a digital camera (C 2400; NEC, Hawthorne, CA) connected to the microscope. The image was later captured by an image-analyzing frame-grabber card (LG-3 Scientific Frame Grabber; Scion, Frederick, MD) and analyzed with image analysis software (NIH Image 1.55). Repair was expressed as the percentage of the original wound area covered by cells. All experiments were performed in triplicate.
Detection of alveolar epithelial cell apoptosis/late necrosis.
Apoptotic cells as a percentage of the total were quantified by flow cytometry after double staining of the alveolar epithelial cells with annexin V-green fluorescent protein (GFP) and propidium iodide (PI), as described earlier (8). Briefly, A549 epithelial cells were exposed to H2O2 for 18 h and then detached with trypsin (0.25%) and EDTA (0.5 mM). The detached cells were combined with the floating cells of each well and resuspended in HEPES buffer containing 2 mM CaCl2. After incubation with a GFP-annexin V fusion protein [final concentration 3 µg/ml, prepared as described (10)] and PI (final concentration 15 µg/ml, Sigma Chemicals), the cells were analyzed by flow cytometry. Annexin V-positive cells were considered to be apoptotic if PI negative (normal permeability) or as either late apoptotic or necrotic if PI positive (increased permeability) (8). The presence of apoptosis was confirmed by visualization of condensed nuclei with acridine orange staining (8).
Apoptosis was inhibited by addition of the tripeptide N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk; R&D Systems, Minneapolis, MN) a broad-spectrum caspase inhibitor, at the concentrations indicated (40120 µM) to the cells 1 h before addition of H2O2.
Detection of apoptotic cells in wounded monolayers.
A549 epithelial cells were plated in standard 24-well, Corning Costar* brand tissue culture plates (Fisher Scientific). The cells were cultured, wounded, and exposed to H2O2 for 18 h as described above with the exception that phenol red-free medium was used to improve fluorescence detection. After 600 µl of medium were gently removed from the top of each well, annexin V-Cy3 (2 µl, Apoptosis Detection Kit; MBL, Nagoya, Japan) was added to the remaining 400 µl and incubated at room temperature for 5 min in the dark. Fluorescent and phase images were captured [Nikon Eclipse TE300 with charge-coupled device (CCD), Hamamatsu], recorded on a Macintosh G4 with capture software (Openlab), and analyzed under blinded conditions as images (Adobe Photoshop, Adobe Systems). The recordable CCD area of 1,022 x 1,280 pixels was adjusted to the area of 800 µm (parallel to the wound edge) x 1,000 µm (perpendicular to the wound edge). For counting, the area was divided into strips of 250-µm-width from the edge of the wound (0250, 250500, and 500750 µm from edge). In each of these strips of 800 x 250 µm, total cells were counted with phase contrast, and fluorescent cells were counted and expressed as a percentage of the total.
Cell spreading and migration.
Cell spreading and migration were determined as described (3, 14, 16). Briefly, wounded A549 alveolar epithelial cells were stained with Diff-Quik (Dade Behring, Duedingen, Switzerland) after the experiment, and images were obtained with the computerized imaging system described above. Thirty different internuclear distances in three randomly chosen high-power fields were measured in each condition at the edge of the wound and in the monolayer 250 µm away from the wound of the same well.
Mitochondrial potential difference.
A549 alveolar epithelial cell monolayers were wounded and incubated with different concentrations of H2O2 in presence and absence of zVAD-fmk, as described. After 18 h, adherent and floating cells were harvested and stained for flow cytometry with the ApoAlert Mitochondrial Membrane Sensor kit (Clontech) following the manufacturer's protocol. In this assay, a cationic dye is aggregated in normal mitochondria where it fluoresces red; when mitochondria lose their normal potential difference, the dye remains unaggregated in the cytoplasm where it fluoresces green. We measured the percentage of cells in the FL1 green channel as a measure of abnormal mitochondrial function.
Statistics.
Data are presented as means ± SE. Statistical analysis was done by unpaired Student's t-test or ANOVA where appropriate. The results were considered significant if P < 0.05.
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RESULTS
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H2O2 inhibits alveolar epithelial repair in vitro.
H2O2 decreased in vitro alveolar epithelial wound repair in a concentration-dependent manner in A549 alveolar epithelial cells (Fig. 1). In the presence of 200 µM H2O2, alveolar epithelial wound closure over 24 h was reduced from 95 ± 4% of the total in control monolayers to 27 ± 5% (P < 0.01).

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Fig. 1. H2O2 inhibits in vitro alveolar epithelial repair. Alveolar epithelial repair was determined by an in vitro alveolar epithelial wound healing assay with human A549 alveolar epithelial cells. H2O2 inhibited alveolar epithelial wound repair in vitro in a concentration-dependent manner. All results are reported as means ± SE. **P < 0.01; ***P < 0.001 compared with medium control (n = 5).
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H2O2 induces apoptosis/late necrosis in alveolar epithelial cells.
H2O2 induced cell death in a concentration-dependent manner in A549 alveolar epithelial cells (Fig. 2). Both early apoptosis (PI-negative cells) and late apoptosis or necrosis (PI-positive cells) contributed to cell death in the presence of H2O2. Early apoptosis was the predominant mechanism of cell death at these concentrations (200400 µM H2O2), whereas late apoptosis or necrosis was the predominant mechanism of cell death at higher H2O2 concentrations (500600 µM) (data not shown).

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Fig. 2. H2O2 induces alveolar epithelial cell apoptosis. Wounded alveolar epithelial cell monolayers were exposed to the H2O2 concentrations indicated and early apoptosis [annexin V positive, propidium iodide (PI) negative], and late apoptosis/necrosis (annexin V positive, PI positive) was determined after 18 h by annexin V/PI staining and flow cytometry. H2O2 increased apoptosis in a concentration-dependent manner. Results are shown as means ± SE. *P < 0.05; **P < 0.01 compared with medium control (n = 3).
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zVAD, a general caspase inhibitor, reduces H2O2-induced apoptosis and inhibition of wound repair.
To test the hypothesis that apoptosis is the major mechanism of H2O2-induced inhibition of alveolar epithelial wound repair, we tested the effect of a general caspase inhibitor, zVAD, on alveolar epithelial cell apoptosis and in vitro alveolar epithelial wound repair. In the presence of zVAD (60 µM), apoptosis of A549 cells from exposure to H2O2 (200 µM and 300 µM) was significantly reduced (Fig. 3, P < 0.05).

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Fig. 3. H2O2-induced apoptosis can be inhibited by a general caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp (zVAD). Wounded alveolar epithelial cell monolayers were preincubated for 1 h with zVAD and exposed to H2O2 for 18 h. zVAD inhibited H2O2-induced apoptosis. Results are shown as means ± SE. *P < 0.05 compared with H2O2-exposed without zVAD (n = 3).
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Of importance, this decrease of apoptosis was associated with an increase in wound repair (Fig. 4). At a concentration of H2O2 of 200 µM, in vitro alveolar epithelial wound repair was improved by zVAD in a concentration-dependent manner (Fig. 4). At this concentration of H2O2 (200 µM), the highest dose of zVAD (120 µM) restored alveolar epithelial repair from 27 ± 5 to 60 ± 6% of total (P < 0.01). zVAD alone (up to 120 µM) had no effect on in vitro alveolar epithelial repair (Fig. 4).

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Fig. 4. Inhibition of apoptosis partly maintains alveolar epithelial repair in vitro. Despite continued exposure to H2O2, alveolar epithelial repair was partly maintained by zVAD in a concentration-dependent manner. zVAD alone had no effect. Results for 3 independent experiments are reported as means ± SE. *P < 0.05; **P < 0.01 compared with H2O2 without zVAD.
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Our results were confirmed in primary human distal lung epithelial cells (Fig. 5). Lung epithelial wound repair was decreased in the presence of H2O2 in a concentration-dependent manner. In the presence of zVAD (40 µM), lung epithelial wound repair was restored from 25.7 ± 5 to 65.5 ± 7% of total (P < 0.01). Higher concentrations of zVAD (80 µM) did not further increase epithelial repair (data not shown).

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Fig. 5. H2O2 induces a concentration-dependent decrease of epithelial repair in primary distal lung epithelial cells (DLEC) that can be partly prevented by zVAD. DLEC repair was determined by an in vitro epithelial wound healing assay. Results are shown as means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001 compared with medium control, #P < 0.05 compared with 200 µM H2O2 without zVAD (n = 3).
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These results indicate that H2O2 inhibits in vitro alveolar epithelial repair in part by inducing alveolar epithelial cell apoptosis and that this H2O2-induced apoptosis is partially preventable.
Apoptotic alveolar epithelial cells are located mainly at the edge of the wound.
We suspected that cells at the wound edge were more susceptible to H2O2-induced cell death. To detect apoptotic cells, we incubated wounded alveolar epithelial cell monolayers with medium alone or 200 or 300 µM H2O2 and, after 18 h, stained cells with annexin V, photographed them, and counted them for each area (Fig. 6A). In wounded monolayers exposed to H2O2, the percentage of apoptotic cells was significantly increased at the edge of the wound (0250 µm from edge) compared with the intermediate zone (at 250500 µm from the edge) and the intact area of the monolayer (>500 µm from the edge) (Figs. 6 and 7). In the presence of zVAD, the percentage of apoptotic cells at the wound edge was significantly reduced so that there was no longer a difference between the edge, the intermediate zone, and the intact monolayer (Figs. 6B and 7).

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Fig. 6. H2O2-induced alveolar epithelial cell apoptosis is higher at the edge of the wound. A: phase-contrast images of the wounded monolayers are presented on the left and the corresponding fluorescent images are shown on the right. Annexin V-positive cells are more numerous at the edge of the wound (0250 µm). B: fluorescent images of the wounded monolayers. In the presence of zVAD (80 µM), H2O2-induced apoptosis is reduced, especially at the wound edge. Bar represents the 0- to 250-µm region considered the edge of the wound.
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Fig. 7. H2O2 induces alveolar epithelial cell apoptosis at the edge of the wound that can be reduced by zVAD. Wounded alveolar epithelial monolayers were incubated with H2O2 ± zVAD, and the percentage of apoptotic cells was determined 18 h later by fluorescent microscopy of annexin V-positive cells at the edge of the wound (0250 µm from edge), in an intermediate zone (250500 µm from the edge), and within the monolayer (>500 µm from the edge). Alveolar epithelial cell apoptosis was highest at the edge of the wound and was reduced in presence of zVAD. All results are reported as means ± SE. *P < 0.05; **P < 0.01, n = 3. n.s., Not significant.
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H2O2-induced inhibition of epithelial cell spreading and migration at the edge of the wound is reduced by zVAD.
We showed in previous studies that wound closure in our in vitro model is due to cell spreading and cell migration (14, 16). We therefore investigated the effect of H2O2 and H2O2-induced apoptosis on epithelial cell spreading and migration at the edge of the wound. Compared with the medium control, H2O2 (200 µM) significantly reduced internuclear distance at the edge of the wound (Fig. 8). In the presence of zVAD, internuclear distance at the edge was partially maintained, indicating that the effect of H2O2 in reducing cell spreading was due to apoptosis. In contrast, in the monolayer away from the wound edge, H2O2 did not alter internuclear distances.

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Fig. 8. H2O2-induced reduction in cell spreading and/or migration at the edge of the wound is partially blocked by zVAD. Internuclear distance of cells at the edge of the wound (0250 µm) and within the monolayer away from the wound edge (250500 µm) was measured. In cells exposed to H2O2, cell spreading and/or migration was reduced; with blockade of caspases with zVAD, cell spreading and/or migration was partially maintained despite continued exposure to H2O2. Results for 3 independent experiments are reported as means ± SE. *P < 0.05 compared with no H2O2 (medium control); #P < 0.05 compared with H2O2 without zVAD.
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H2O2-induced loss of the mitochondrial membrane potential in alveolar epithelial cells is reduced by zVAD.
Because inhibition of caspases may not protect cell viability if caspases are activated distal to mitochondrial involvement (21), we measured mitochondrial membrane potential as a marker of cell viability. In the presence of H2O2 (200 µM), a loss of the mitochondrial membrane potential was detected (30.1 ± 5.5% of total cells shifted from red to green fluorescence compared with 4.2 ± 0.9% of cells in medium control, P < 0.01). This effect was decreased in presence of zVAD in a concentration-dependent manner (Fig. 9).

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Fig. 9. H2O2-induced loss of the mitochondrial membrane potential in alveolar epithelial cells is reduced by zVAD. The H2O2-induced loss of the mitochondrial membrane potential was partially blocked in presence of the pan-caspase inhibitor zVAD in the concentrations indicated. zVAD alone had no effect on the mitochondrial membrane potential compared with medium control (data not shown). *P < 0.05, **P <0.01 compared with 200 µM H2O2; n = 4.
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DISCUSSION
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In this study, we have found that H2O2 inhibits in vitro alveolar epithelial repair in a dose-dependent manner. The effect of H2O2 was mediated in large part by apoptosis, especially by apoptosis of alveolar epithelial cells at the edge of the wound. Inhibition of alveolar epithelial cell apoptosis by the general caspase inhibitor zVAD improved repair of the wound in the setting of H2O2, indicating that inhibition of epithelial apoptosis in vivo may offer a valid therapeutic strategy for improving alveolar epithelial repair in patients with lung injury.
There is evidence that oxidative stress may contribute to clinical ALI/ARDS (19); the accumulating neutrophils and phagocytes release ROS in the alveolar space and may contribute to the damage of the alveolar epithelial and endothelial barriers, leading to alveolar edema. However, the role of ROS in alveolar epithelial repair and resolution of ALI/ARDS is not known. In some studies of epithelial cells, H2O2 has been shown to impair epithelial cell growth, proliferation, and repair of human tracheal epithelial cells (29) and to inhibit wound repair in gastric epithelial cells (28), suggesting that ROS may modulate alveolar epithelial repair as well. Using an in vitro alveolar epithelial wound repair model, we found that H2O2 decreases the rate of wound closure in a concentration-dependent manner both in A549 alveolar epithelial cells and in primary distal lung epithelial cells, indicating that ROS may delay efficient alveolar epithelial repair in the intact lung.
Recent studies indicate that apoptosis contributes to the damage of the alveolar epithelium in animal models of acute lung injury and in patients with ALI/ARDS. Bronchoalveolar lavage fluid (BALF) from patients with ALI/ARDS induced apoptosis of distal lung epithelial cells, whereas BALF from patients at risk for ALI/ARDS did not (20). In lung tissue obtained at autopsy from patients with ALI/ARDS, apoptotic alveolar epithelial cells were more prevalent in patients who died with ALI/ARDS compared with patients who died without pulmonary disease (2). Although there is some evidence for apoptosis in the pathophysiology of ALI/ARDS, the role of apoptosis in alveolar epithelial repair is not known. We demonstrate that induction of alveolar epithelial cell apoptosis by H2O2 inhibits in vitro alveolar epithelial repair. Inhibition of H2O2-induced apoptosis by zVAD, a general caspase inhibitor, partially restores lung epithelial repair in vitro as shown with A549 alveolar epithelial cells and then confirmed with primary distal lung epithelial cells. The partial restoration is compatible with the partial inhibition of apoptosis, perhaps due to incomplete inhibition of caspases by zVAD in the setting of H2O2 or to caspase-independent modes of cell death such as necrosis. By measuring internuclear distances, we were able to show a decrease in cell spreading and migration due to H2O2 that was also in part preventable by inhibition of caspases, showing that some of the decrease in cell migration or spreading was also due to apoptosis. Thus apoptosis may affect epithelial cell monolayer repair in different ways, by reducing cell numbers or by interfering with cell migration or cell spreading.
Inhibition of caspases may block the morphologic appearance of apoptosis without necessarily blocking cell death (21). Such cell death without caspase activity may result if mitochondrial involvement leads to loss of cytochrome c and metabolic activity at a step proximal to caspase activation. In these experiments, we believe that inhibition of caspases did protect cell viability in the face of ongoing ROS activity. Evidence for cell viability included the sustained cell migration and spreading at the wound edge and the maintenance of a more normal mitochondrial potential difference. However, this was possible only in some of the cells; perhaps the inhibition of caspases was incomplete if zVAD was not fully active in the face of H2O2, or perhaps the inhibition of caspases could not prevent all H2O2-induced damage. It was of interest that the cells most sensitive to H2O2 and most responsive to protection by zVAD were at the edge of the wound. These cells presumably play the greatest role in repair, so their response to therapeutic intervention is encouraging.
Inhibition of epithelial wound repair by ROS has been described in other in vitro models. For example, H2O2 was shown to inhibit wound repair in gastric epithelial cells (28), and, in parallel with our results, H2O2-induced apoptosis of gastric epithelial cells was mainly detected at the edge of the wound. Similarly, NO2 was shown to cause apoptosis selectively in epithelial cells migrating into a wound (11), indicating that apoptosis may interfere with wound healing. With these studies and our own, we would suggest that the response to ROS is likely to differ between an epithelium undergoing repair and an intact epithelium. It is reasonable to suppose that the cells migrating into a wound may be deprived of important survival signals usually provided by normal cell-cell and cell-extracellular matrix contacts and thus be more prone to apoptosis than the cells in the intact epithelium. Protecting those susceptible cells from apoptosis may allow repair to proceed even in abnormal alveolar environments.
In fact, inhibition of apoptosis has been attempted with some success in both acute and subacute lung injury animal models. For example, in an LPS-induced ALI model in mice, apoptosis of alveolar epithelial cells was reduced and survival was increased in presence of the caspase inhibitor zVAD (15). In bleomycin-induced lung injury, zVAD reduced the number of apoptotic epithelial cells and the subsequent accumulation of collagens in the lung, attenuating lung fibrosis (17, 26). In a recently published study, apoptosis-dependent ALI was induced in rats by intratracheal administration of norepinephrine (25). In the presence of zVAD, alveolar epithelial cell apoptosis was reduced and the formation of alveolar edema was attenuated. Interestingly, an increasing number of apoptotic cells were detected within the alveoli compared with the alveolar wall over time, suggesting that apoptotic alveolar epithelial cells were shed from the alveolar wall surface to the alveolar spaces. These data suggest that blocking apoptosis may increase the regeneration of the alveolar barrier by promoting alveolar epithelial repair, although the authors did not show the distribution of apoptotic cells in presence of zVAD (25). Because the efficient repair of the alveolar epithelium is crucial to the recovery of patients with ALI/ARDS, inhibition of epithelial apoptosis may be a logical strategy for epithelial cell protection (6).
Some limitations of this study should be discussed. First, A549 cells were used instead of primary type II alveolar epithelial cells, in part because the primary type II cells isolated from rats had a high background level of apoptosis. With respect to epithelial wound healing, we have found in our previous studies using our in vitro epithelial wound repair model that human A549 cells behave similarly to primary alveolar type II cells (13, 14). In addition, we performed the critical experiments using human primary distal lung epithelial cells, thereby confirming our results in a different cell model. These cells were previously shown to have characteristic features of alveolar type II cells (lamellar-like bodies) and undergo apoptosis after incubation with apoptotic stimuli (20). Similar to A549 alveolar epithelial cells, primary distal lung epithelial cells showed reduced epithelial wound repair in the presence of H2O2 that was partly restored in the presence of the pan-caspase inhibitor zVAD. Second, our model is simplified by the absence of other cell types. Nonetheless, this model has permitted the discovery of several basic mechanisms that are critical for alveolar epithelial repair (3, 1214, 16, 22). Finally, H2O2 was used as only one of several possible toxic materials of neutrophils or macrophages. Nonetheless, it is meant to represent a more clinically relevant situation, without reproducing the full complexity of the clinical environment.
In summary, we have found that H2O2 inhibits in vitro alveolar epithelial repair, in part by induction of apoptosis. At the edge of the wound, H2O2 led to an increase in apoptotic cells and a decrease in internuclear distance. Despite the continued exposure to H2O2, inhibition of caspases partly blocked these effects and partly restored in vitro alveolar epithelial repair. These data indicate that, in the presence of ROS, in vitro alveolar epithelial repair is determined in part by apoptosis. Inhibition of lung epithelial apoptosis may therefore be a promising strategy for future treatment in patients with ALI/ARDS.
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GRANTS
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This work was supported by Swiss National Science Foundation Grant 3200-100497 (T. Geiser) and National Institutes of Health Grants HL-51854 and HL-51856 (M. A. Matthay) and RO1 ES-08985 (V. C. Broaddus).
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FOOTNOTES
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Address for reprint requests and other correspondence: V. C. Broaddus, Box 0854 UCSF, Univ. of California, San Francisco, San Francisco, CA 94143-0854 (E-mail: sfcourt{at}itsa.ucsf.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.
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