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Mitochondrial cytochrome c release is a key event in hyperoxia-induced lung injury: protection by cyclosporin A.

PAGANO AURRAND-LIONS, Alessandra, et al.

Abstract

Hyperoxia is known to induce extensive alveolar cell death by still poorly defined mechanisms.

In this study, the mitochondria-dependent cell death pathway was explored during hyperoxia-induced lung injury in mice. We observed a progressive release of cytochrome c from the mitochondria into the cytosol of alveolar cells. This release was accompanied by the translocation of the proapoptotic protein Bax from cytosol to mitochondria without detectable activation of caspase-3. As cytochrome c release can be induced by mitochondrial membrane alteration and permeability transition (MPT), mice were treated with cyclosporin A, which specifically inhibits MPT. Cyclosporin A treatment prevented mitochondrial release of cytochrome c during hyperoxia and concomitantly preserved mitochondria from extensive swelling and crista disorganization, as assessed by electron microscopy analysis of alveolar epithelial cells. These morphological and biochemical observations correlated with decreased lung tissue damage, as evaluated by morphological score and lung weight. In conclusion, mitochondrial damage and cytochrome c release are important [...]

PAGANO AURRAND-LIONS, Alessandra, et al . Mitochondrial cytochrome c release is a key event in hyperoxia-induced lung injury: protection by cyclosporin A. American Journal of Physiology. Lung Cellular and Molecular Physiology , 2004, vol. 286, no. 2, p. L275-83

DOI : 10.1152/ajplung.00181.2003 PMID : 14527930

Available at:

http://archive-ouverte.unige.ch/unige:55494

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Mitochondrial cytochrome c release is a key event in hyperoxia-induced lung injury: protection by cyclosporin A

Alessandra Pagano, Yves Donati, Isabelle Me´trailler, and Constance Barazzone Argiroffo Departments of Pathology and Pediatrics, University of Geneva Medical School, 1211 Geneva 4, Switzerland Submitted 6 June 2003; accepted in final form 21 September 2003

Pagano, Alessandra, Yves Donati, Isabelle Me´trailler, and Con- stance Barazzone Argiroffo. Mitochondrial cytochrome c release is a key event in hyperoxia-induced lung injury: protection by cyclosporin A. Am J Physiol Lung Cell Mol Physiol 286: L275–L283, 2004. First published October 3, 2003; 10.1152/ajplung.00181.2003.—Hyperoxia is known to induce extensive alveolar cell death by still poorly defined mechanisms. In this study, the mitochondria-dependent cell death path- way was explored during hyperoxia-induced lung injury in mice. We observed a progressive release of cytochrome c from the mitochondria into the cytosol of alveolar cells. This release was accompanied by the translocation of the proapoptotic protein Bax from cytosol to mitochon- dria without detectable activation of caspase-3. As cytochrome c release can be induced by mitochondrial membrane alteration and permeability transition (MPT), mice were treated with cyclosporin A, which specifi- cally inhibits MPT. Cyclosporin A treatment prevented mitochondrial release of cytochrome c during hyperoxia and concomitantly preserved mitochondria from extensive swelling and crista disorganization, as assessed by electron microscopy analysis of alveolar epithelial cells.

These morphological and biochemical observations correlated with de- creased lung tissue damage, as evaluated by morphological score and lung weight. In conclusion, mitochondrial damage and cytochrome c release are important linked events in hyperoxia-induced lung injury and can be efficiently blocked by cyclosporin A.

mice; apoptosis; mitochondria; type II epithelial cells; Bax

EXPOSURE TO HIGH OXYGEN TENSIONhas been used as a valuable model of acute respiratory distress syndrome in rodents. It is characterized by extensive alveolar cell death, leading to the disruption of the alveolo-capillary barrier and pulmonary edema. Alveolar leak occurs when epithelial cells are damaged, suggesting that these cells are crucial in maintaining the barrier integrity (6, 7). It is generally assumed that lung damage results from the direct action of increased intracellular reactive oxygen species (ROS), which can be amplified by a secondary inflam- matory response (17, 20). We previously showed that, in mice, hyperoxia (100% oxygen) induces extensive alveolar cell death characterized by apoptosis and necrosis (7). The mechanisms by which hyperoxia mediates cell death are not completely defined, although they have been widely investigated both in vitro and in vivo (2).

During oxygen exposure mitochondria actively participate in increasing the production of intracellular ROS (19). The mitochondrion plays a pivotal role in the regulation of cell death responses, since it maintains the cellular levels of ATP and is able to releases death-promoting factors, such as cyto- chrome c, when cells are exposed to proapoptotic signals.

Although several death stimuli induce the mitochondrial re- lease of cytochrome c, the molecular mechanisms of this process are not completely understood (16, 30). It has been shown that the activation and translocation from the cytosol to the mitochondrial membrane of the proapoptotic members of the Bcl-2 family, such as Bax, lead to the formation of channels through which cytochrome c is released without alteration of the morphology of mitochondria (3, 30). Alternatively, strong cell death signals or even Bax itself can affect directly the permeability of the mitochondrial membrane and induce the opening of the high-conductance channel permeability transi- tion pore (PTP) (30, 47). Irreversible PTP opening induces large amplitude mitochondrial swelling, outer membrane break, and subsequent release of cytochrome c (24, 37). Once into the cytosol, cytochrome c is able to trigger the apoptotic signaling cascade by activating the caspase-9 and downstream effector caspases, in particular caspase-3, responsible for the nuclear fragmentation and cell death (23).

Morphological studies have described that mitochondria from hyperoxia-injured alveolar cells undergo structural changes such as swelling and crista disorganization (27, 32).

Such alterations have been correlated with perturbation of the mitochondrial membrane permeability in vitro (1). However, a direct relationship between mitochondrial morphological changes, cytochrome c release, and apoptotic/necrotic signal- ing during hyperoxia in vivo has not been established yet.

In the present study, we explored the role of mitochondria- dependent cell death signaling in the pathogenesis of hyperox- ia-induced lung injury. Exposure of mice to 100% oxygen induced the release of cytochrome c in high amounts from the mitochondria into the cytosol. This was accompanied by Bax translocation to the mitochondrial membrane. Besides the ini- tiation of the mitochondrial apoptotic machinery, the active form of caspase-3 was not detectable. To investigate whether cytochrome c release was dependent on mitochondrial perme- ability transition, we injected mice with cyclosporin A (CsA), a specific blocker of PTP opening, before and during oxygen exposure (38, 48). CsA treatment significantly prevented cy- tochrome c release from mitochondria into the cytoplasm of alveolar cells. Electron microscopy (EM) analysis of alveolar type II cells, where mitochondria are abundant and easily recognizable, showed that mitochondria from CsA-treated mice were more electron dense and less swollen and had less disorganization of cristae compared with vehicle-treated mice.

Concomitantly, CsA treatment ameliorated hyperoxia-induced lung damage, as shown by macroscopical lung injury score and lung weight. These results suggest that preventing from mito-

Address for reprint requests and other correspondence: C. Barazzone Argi- roffo, Dept, of Pathology, Centre me´dical universitaire, 1211 Geneva, 4, Switzerland (E-mail: constance.barazzone@hcuge.ch).

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.

First published October 3, 2003; 10.1152/ajplung.00181.2003.

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chondrial structure alteration cytochrome c release in alveolar epithelial cells can protect from hyperoxia-induced lung injury.

MATERIALS AND METHODS

Mice. C57BL/6 female mice were purchased from Iffa Credo (Labresle, France) and bred in our animal facility. Experiments were performed on 8–10-wk-old mice weighing 20–25 g.

Hyperoxia exposure and in vivo treatment. Mice were placed in a sealed Plexiglas chamber and exposed to 100% O2 for 72 h, as described (5). Control mice were exposed to room air under the same conditions. CsA was injected intraperitoneally at the dose of 25 mg

kg1

day1 (Sandimmune, Novartis), diluted in vehicle, com- posed of 650 mg/ml cremaphor (Sigma-Aldrich, St. Louis, MO), 250 mg/ml ETOH, and 0.9% NaCl. The untreated group was injected with the vehicle only. This dosage regimen was shown to be effective in previous studies (33). The treatment with CsA or vehicle was started 3 days before oxygen exposure and continued during the time of air or 100% O2 exposure. After death, lung damage was macroscopically evaluated, according to the extension of hemorrhages, as described by Christofidou-Solomidou et al. (11), and a score ranging from 0 (no lesions) to 5 (complete hemorrhagic lung) was given by three expe- rienced independent examiners. We determined pulmonary edema by measuring right wet lung weight, as described previously (5). The right lung was immediately frozen in liquid nitrogen and stored at

80°C until further analysis. The left lung was fixed and stored for histological analysis. Bronchoalveolar lavage (BAL) was performed as described (6). BAL fluid was recovered immediately on ice under negative hydrostatic pressure. After centrifugation, cells were counted, and protein concentration was determined.

All study protocols were approved by the local ethical committee on animal experiments (Office Ve´te´rinaire Cantonal of Geneva).

Light and electron microscopy. Left lungs were fixed intratrache- ally with 4% paraformaldehyde in phosphate buffer, with an hydro- static pressure of 20 cmH2O, and embedded in paraffin. Sections (4

m) were stained with hematoxylin and eosin for histological evalu- ation or processed for immunohistochemistry (IHC). For EM studies, lungs were fixed by the intratracheal instillation of 2% glutaraldehyde (in 0.1 M cacodylate buffer) and processed as described (6). Sections were embedded in Epon and then examined with a Philips CM10 (Philips, Zurich, Switzerland) 400 electron microscope at 70 kV.

IHC. Paraffin-embedded lung tissue sections (5 m) were mounted on slides pretreated with 3-aminopropylxylane (Merck, Darmstadt, Germany), baked overnight at 55°C, deparaffinized, and rehydrated. Sections were cooked 35 min in microwave to facilitate the access of the antibody. After cooling, samples were blocked with 5% bovine serum albumin in Tris-buffered solution and incubated overnight with an anti-cytochrome c monoclonal antibody (1:250–1:500, clone 7H8.2C12; BD Pharmingen, Frank- lin Lakes, NJ) or with an anti-cleaved-caspase-3 rabbit polyclonal antibody (1:100, 9661-S; Cell Signaling Technology, Beverly, MA). As secondary antibody, a biotinylated anti-mouse IgG, Fc-specific antibody (Jackson Laboratories, San Diego, CA) or a biotinylated anti-rabbit total Ig antibody (Santa Cruz Biotechnol- ogy, Santa Cruz, CA) was used and then labeled with streptavidin- biotin-alkaline phosphatase complex (AB kit; Vector Laborato- ries, Burlingame, CA). The phosphatase reaction was revealed with the fast red substrate system (DaKo, Carpinteria, CA), and slides were counterstained with Hemalun. Normal mouse or rabbit IgG was used instead of the primary antibody as negative control for nonspecific binding. To quantify the mitochondrial content of cytochrome c, a score was established as follows: 1) the number of lung parenchymal positive cells was counted at100 magnifica- tion in 25 fields for each lung section (2–3 different sections per animal, 7–8 mice in each group); 2) for each positive cell a range of positivity was given between 1 and 4, 1 corresponding to25%

of the cell surface covered by the granular pattern, 2 between 25 and 50%, 3 between 50 and 75%, and 475%. The total score was expressed as the number of positive cellsthe range of positivity.

This value was divided by the number of positive cells to calculate a mean score per positive cell (mean SD). This evaluation was established independently by three different investigators.

Cytosolic and mitochondrial subcellular fractionations. Subcellu- lar fractions were prepared from total lungs extracts, as described by Bossy-Wetzel and Green (9) with some modifications (22). Briefly, frozen lungs were minced into small pieces with a scalpel and suspended in 15 volumes of cold buffer (250 mM sucrose, 20 mM HEPES-KOH, pH 7.4, 10 mM KCl, 1.5 mM Na-EGTA, 1.5 mM Na-EDTA, 1.0 mM MgCl2, 1.0 mM DTT, and protease inhibitors).

Tissue samples were then homogenized with a glass Dounce homog- enizer and a tight Teflon pestle. Homogenates were first centrifuged (800 g for 10 min at 4°C) to eliminate nuclei and debris. The postnuclear supernatant was filtered through gauze and centrifuged at 9,500 g for 15 min at 4°C. Resulting pellets were designated as mitochondria-enriched heavy membrane fractions (HMF), according to Dubrez et al. (14), while supernatants were further ultracentrifuged at 100,000 g for 1 h, at 4°C, to obtain cytosolic fractions. To verify the integrity of mitochondrial membranes and exclude cytosol contami- nation by mitochondrial proteins, we blotted each fraction with a mouse monoclonal anti-cytochrome oxidase subunit IV (COX, A-6431; Molecular Probes, Eugene, OR) as a mitochondrial marker.

A rabbit polyclonal antibody anti-actin (AL-20, a kind gift from the laboratory of G. Gabbiani) was used as a control for protein loading.

Western blot analysis. Samples were analyzed for protein concen- tration by Bio-Rad DC protein assay kit (ref. 500–0111; Bio-Rad Laboratories, Hercules, CA). To analyze the localization of cyto- chrome c, we electrophoresed 15 g of total proteins from the cytosolic and mitochondrial fractions on a 12% SDS-polyacrylamide gel. We used 70g of protein for the analysis of Bax. All gels were blotted to nitrocellulose membranes (Amersham International, Amer- sham, UK), except for detection of COX, where samples were transferred to a polyvinylidene difluoride membrane (porablot; Ma- cherey-Nagel, Du¨ren, Germany). Membranes were blocked overnight in TBST buffer (0.2 M Tris, pH 7.6, 1.5 M NaCl, and 0.1% Tween 20) and 5% milk and incubated with the following antibodies: rabbit polyclonal anti-cytochrome c antibody (1:200 dilution, sc-7159; Santa Cruz Biotechnology), mouse monoclonal anti-COX antibody (1:500 dilution), rabbit polyclonal anti-actin antibody (1:2,000, AL-20; gift from G. Gabbiani’s laboratory), rabbit polyclonal anti-Bax antibody (1:200 dilution, sc-493; Santa Cruz Biotechnology), and rabbit poly- clonal anti-cleaved caspase-3 (1:1,000, 9661-S; Cell Signaling Tech- nology). Horseradish peroxidase-conjugated anti-mouse and anti-rab- bit (1:3,000 dilution, Bio-Rad Laboratories) were used as secondary antibodies. Bands were visualized with a chemiluminescent substrate (Amersham International). Scanning and quantification of signal in- tensity were performed on subsaturated films by with the Imagequant software.

Statistical analysis. For all parameters measured, the values for all animals in different groups were averaged, and the SD of the mean was calculated. The significance of differences between the values of the groups was determined with unpaired Student’s t-test. Where appropriate, two-way ANOVA with multiple comparisons followed by unpaired t-test was used. Significance levels were set at P0.05.

RESULTS

Hyperoxia induces the release of cytochrome c from alveo- lar cell mitochondria. We analyzed the intracellular distribu- tion of cytochrome c in air- and hyperoxia-exposed lungs by IHC and Western blot (WB) analysis. Cytochrome c was easily detected in lung sections of air-breathing mice and in particular in large alveolar cells located at septal junctions. These cells

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displayed a strong intracellular, granular (mitochondria-like) staining pattern (Fig. 1, C and E). During hyperoxia most of the cells exhibited a reduced granular distribution, associated with a diffuse and weak intracellular labeling, suggesting a redistri- bution of the protein into the cytosol (Fig. 1, D and F).

Quantification of the number of positive cells and evaluation of the granular staining pattern confirmed that the number of positive cells (not shown) and the cellular score were signifi- cantly lower in hyperoxia-exposed mice compared with air- breathing mice (mean score/cell: 1.6 ⫾ 0.19 in hyperoxia- exposed mice compared with 2.3⫾0.16 in air-breathing mice, P0.001, see Fig. 5E).

As a complementary approach, we also analyzed the cellular distribution of cytochrome c by WB. In total lung extracts, the content of cytochrome c was similar in air- breathing and oxygen-exposed mice (Fig. 2A). However, its intracellular distribution was significantly affected by hy- peroxia. In air-breathing mice, cytochrome c was almost undetectable in lung cytosolic fractions, whereas it was present in mitochondria-enriched HMF (Fig. 2B). During oxygen exposure, cytochrome c appeared in the cytosol at 48 h, and the signal was even higher at 72 h (Fig. 2B).

Quantification of four different experiments (n ⫽ 11 ani- mals) showed that the cytosolic level of cytochrome c was significantly higher at 72 h of exposure compared with

control lungs (P0.001; Fig. 2C, right). In some experi- ments, the release of cytochrome c into the cytosol was accompanied with a decrease, although not significant, of the signal in HMF fraction (Fig. 2C, left). These results demonstrate that hyperoxia induces the release of cyto- chrome c from mitochondria into cytosol of alveolar cells.

Hyperoxia-induced release of cytochrome c is associated with Bax translocation to the mitochondria without caspase-3 activation. It has been described that during apoptosis Bax protein translocates from the cytosol into the mitochondria, inducing the release of cytochrome c (3). Therefore, we ana- lyzed whether cytochrome c release was accompanied by Bax translocation. As shown by WB, Bax protein content increased significantly in HMF fractions of hyperoxia-exposed compared with control lungs (Fig. 3, A and B, left, P⬍0.05), suggesting a translocation of the proapoptotic protein to mitochondria.

However, the cytosolic content of Bax was not significantly reduced by oxygen exposure (Fig. 3, A and B, right).

To further investigate whether cytochrome c release was able to trigger caspase activation, we analyzed the expres- sion of the cleaved (active) form of caspase-3, which is one of the major downstream effector caspases. As shown in Fig. 4, no positive signal was detected in the cytosol from air- and hyperoxia-exposed lung extracts, even when 150␮g of proteins were loaded. Similarly, no signal for cleaved

Fig. 1. Intracellular distribution of cyto- chrome c (Cyt c) in mouse lungs. Paraffin- embedded tissue sections from air-breathing (A) and hyperoxia (Hox)-exposed mice (B) were incubated with normal mouse IgG as negative control for nonspecific binding.

When stained with rabbit anti mouse Cyt c, alveolar cells from air-breathing mice pre- sented a strong and punctuate staining (C and E), whereas cells from mice exposed to 100% oxygen for 72 h (D and F) showed weak and diffuse staining (D and F). Micro- graphs of representative fields are shown.

Magnifications: A–D,40; E and F,100.

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caspase-3 was detected on hyperoxic lung sections by IHC (not shown).

CsA treatment prevents hyperoxia-induced cytochrome c release and mitochondrial damage. Morphological studies have shown marked structural changes in mitochondria from hyperoxia-exposed alveolar cells (1, 27). Therefore, we hy- pothesized that hyperoxia could induce the opening of the mitochondrial PTP, leading to mitochondria swelling, mem- brane disruption, and secondary cytochrome c release. To verify this hypothesis, we explored whether CsA treatment of mice exposed to hyperoxia could prevent mitochondrial dam- age and cytochrome c release.

Fig. 3. Analysis of Bax subcellular localization. A: mitochondrial and cyto- solic distribution of Bax during Hox. Western blot analysis was performed with HMF and cytosolic fractions of lungs from air-breathing and oxygen- exposed mice for 72 h (Hox 72 h). For each lane, 70g of protein were loaded.

B: quantification of 3 different experiments (n 7 animals/group) was performed by densitometry. Left: values represent the meansSD of Bax/

COX ratio in HMF (*P0.05, air vs. 72-h Hox). Right: cytosolic Bax content is normalized to actin.

Fig. 2. Western blot analysis of Cyt c in total lung extracts, heavy membrane fractions (HMF), and cytosolic fractions. A:

Cyt c content in total lung extracts from air-breathing and Hox-exposed mice (48 and 72 h). Thirty micrograms of total proteins were loaded in each lane. B: mitochondrial and cyto- solic distribution of Cyt c during Hox. HMF and cytosol were obtained from lung extracts of mice exposed to air or Hox for 24, 48, and 72 h and analyzed for Cyt c content. Fifteen micrograms of proteins were loaded in each lane. Actin was used as a protein loading control, and cytochrome oxidase subunit IV (COX) was used as mitochondrial marker. C: results of densitometric analysis of 4 independent experiments (n11 animals/group). Left: values are expressed as ratio of Cyt c normalized to COX content (meanSD) in HMF fraction.

Right: Cyt c is normalized to actin content (meanSD) in cytosolic fraction; ***P0.001, air vs. 72-h Hox.

Fig. 4. Western blot for cleaved caspase-3. Cytosolic extracts (100g) from lungs of air-breathing or oxygen-exposed mice (Hox 72 h) were analyzed with anti-cleaved caspase-3 (active form, 17 kDa) specific antibody. Each lane corresponds to a different animal. No signal was detected in lung extracts from air-breathing or Hox-exposed mice. Conversely, a strong signal was seen in thymus extracts (15g of protein) from a mouse treated with 100g of dexamethasone (Dex) and killed 6 h later. C, thymus extract from an untreated animal.

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As analyzed by IHC, CsA treatment significantly prevented the loss of mitochondrial cytochrome c staining observed in vehicle-treated mice exposed to hyperoxia (compare Fig. 5, B and D vs. A and C). Indeed, the number of positive cells in 25 fields (not shown) and the mean of positive granules per cell were significantly higher in CsA-treated animals compared with vehicle-treated animals exposed to hyperoxia (mean score/cell: 2.0⫾0.4 in CsA-treated vs. 1.6⫾0.19 in vehicle- treated group exposed to hyperoxia, P0.01, Fig. 5E). To note, in air-breathing mice, treatment with vehicle alone or CsA did not affect the strong intracellular staining pattern (not shown) that was observed in untreated animals (see Figs. 1, C and E, and 5E).

We then analyzed the mitochondrial morphology in lungs of vehicle- and CsA-treated animals by EM. In air-breathing mice, a high number of small, electron-dense mitochondria were easily recognized in alveolar type II cells (Fig. 6, A and D). Major mitochondrial alterations were observed during hyperoxia in these cells, according to previous morphological studies in hyperoxia-exposed rats (27). After 72 h of oxygen

exposure, almost all mitochondria exhibited marked swelling, diminished matrix density, and disorganized cristae (Fig. 6, B and E). Mitochondria of alveolar cells from CsA-treated mice were in large part compact and more electron dense compared with those from vehicle-treated mice (Fig. 6, C and F, arrows).

However, mitochondria with a large, swollen aspect and dis- rupted cristae were also found within the same cell (Fig. 6, C and F, arrowhead). Together, these observations suggest that CsA partially prevented, at least in a subset population of alveolar cells, hyperoxia-induced mitochondrial damage and consequent cytochrome c release.

CsA treatment reduces hyperoxia-induced lung damage.

Finally, we analyzed whether the effects on mitochondria observed with CsA treatment were associated with lung dam- age protection. Macroscopical examination of the lungs showed significantly lower extent of hemorrhages in CsA- treated compared with vehicle-treated mice (Fig. 7, compare C with B, and Table 1). Accordingly, the right lung weight and BAL protein content were significantly reduced in the CsA- treated group compared with the vehicle-treated group (Table

Fig. 5. Immunohistochemistry of Cyt c in lung sections from vehicle- and cyclosporin A (CsA)-treated mice exposed to Hox for 72 h. CsA treatment (B and D) partially avoided the loss of the punctuate mitochon- drial staining during Hox (A and C; see also Fig. 1, D and F). Compare also the staining with the air-breathing lung sections in Fig. 1, C and E, for the typical Cyt c distribution.

Vehicle and CsA treatment did not affect the labeling in air-breathing lungs (not shown).

Magnification: A and B, 40; C and D,

100 (enlargements from boxed areas in A and B). E: quantification of the staining for Cyt c. Positive cells were counted in 25 areas at100 magnification (2–3 sections/animal, 7–8 animals/group). Values represent the mean score per positive cell ( SD) (see

MATERIALS AND METHODS for details). To note, in the vehicle-treated air-breathing (n7,F) and oxygen-exposed group (n 8,) were included untreated (3–4 animals) and vehicle-treated animals (4 animals).Œ, CsA-treated mice (n8) exposed to Hox.

Score/cell: 2.30.16 in air-breathing mice (F), 1.60.19 in vehicle-treated/untreated group exposed to Hox (), 2.0 0.4 in CsA-treated animals exposed to Hox (Œ);

***P0.001, vehicle-treated, air vs. Hox,

**P0.01, vehicle- compared with CsA- treated animals exposed to Hox.

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1). However, the number of cells recovered by BAL was similar in both groups (Table 1). Lung histology of hyperoxia- exposed CsA-treated mice showed some extent of alveolar septa thickening but less edema and hyaline membrane forma- tion compared with vehicle-treated mice (Fig. 7, compare C vs.

B). CsA treatment in itself affected neither the macroscopical and microscopical aspect (not shown) nor the number of cells recovered by BAL from air-exposed mice (Table 1).

DISCUSSION

We previously reported that lungs of mice exposed to 100%

oxygen are characterized by extensive epithelial and endothe- lial cell death, which is not dependent on cell death receptor activation [TNF, Fas, CD40 (4, 8)].

Mitochondria play a central role in regulating cell death, as they control calcium homeostasis and intracellular oxidants levels (18, 47), produce ATP, and release apoptogenic factors (30). In the present study we analyzed the mitochondria- dependent cell death pathway and in particular the intracellular distribution of cytochrome c in lungs of mice exposed to air or to hyperoxia. The granular staining seen by IHC in control lungs was highly suggestive of mitochondrial protein distribu- tion, in agreement with previous descriptions (22). The staining was mainly evident in large alveolar cells located at septal junctions. Their localization and morphology were compatible with epithelial type II cells, which are known to have high mitochondria content. After 72 h of hyperoxia, the cytochrome

c-specific staining of these cells lost its granular aspect, sug- gesting that cytochrome c spilled out of the mitochondria into the cytosol. By biochemical analysis, we confirmed that mito- chondrial cytochrome c was released into the cytosol in a small amount after 48 h of oxygen exposure and in a significantly higher amount after 72 h. The presence of cytochrome c in cytosolic extracts during hyperoxia was not always correlated with a detectable decrease in its mitochondrial quantity. How- ever, since cytochrome c is normally absent in the cytoplasm, it is far easier to detect its release in this compartment than its evasion from mitochondria where cytochrome c is very abun- dant. Indeed, equivalent amounts of proteins for cytosolic and mitochondrial extracts (15 ␮g) were loaded onto the gels, whereas in most reports higher amounts of cytosol were used compared with mitochondria [i.e., 30␮g of cytosol vs. 4␮g of mitochondrial extracts in (22)].

To decipher the mechanism leading to cytochrome c release, we analyzed the modulation of Bax protein in our experimental model. In vivo oxygen exposure has been associated with a significant increase in Bax lung mRNA, without any modifi- cation of the protein expression (7, 36). The cellular fraction- ation approach allowed us to determine that Bax was signifi- cantly increased in mitochondrial fractions after 72 h of hy- peroxia, indicating the activation and translocation of this protein from the cytosol to the mitochondria. These results suggest a proapoptotic role for Bax in hyperoxia and are in agreement with a previous report showing that Bak/bax(⫺/⫺)

Fig. 6. Electron microscopic illustration of mitochondrial damage in alveolar type II cells during Hox. In air-breathing mice, mitochondria (m) were electron dense and compact and presented organized cristae (A and D). After oxygen exposure, mitochondria were swollen and round-shaped, and cristae appeared very disrupted (B and E). In CsA-treated mice, a mixed population composed of still electron-dense (arrows) and swollen mitochondria (arrowhead) was present (C and F). Magnifications:

A–C,10,000; D–F,36,000 (enlargements from boxed areas in A–C). lb, surfactant-producing lamellar bodies.

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embryonic fibroblasts were more resistant to cell death upon oxygen exposure (10). Surprisingly, the translocation of Bax was not evident at 48 h of oxygen exposure, as predicted by the assumption that Bax would induce cytochrome c release (30).

Two different hypotheses could explain the asynchrony be- tween Bax translocation and cytochrome c release: 1) the anti-Bax specific antibody used was not sensitive enough, precluding the detection of a small increase in mitochondria- associated Bax at 48 h; 2) Bax translocation may be restricted to some mitochondria, whereas others could have their mem-

branes damaged directly by the high concentration of free radicals with subsequent release of cytochrome c indepen- dently of Bax translocation. Unfortunately, we could not reli- ably determine the subcellular localization of Bax by IHC and hence could not define whether it was translocated in the same cells as those in which cytochrome c was released.

A recent report demonstrated that the activation of the caspase-8/Bid signaling pathway was involved in the apoptotic response during hyperoxia, since gene disruption of Bid pro- tected against cell death, both in vivo and in vitro (44). It has

Fig. 7. Lung injury during Hox in vehicle- and CsA-treated mice. Mice were injected intraperitoneally with the vehicle or 25 mgkg⫺1day⫺1of CsA 3 days before and during oxygen exposure until death. Left: representative macroscopical view of air-breathing and vehicle-treated lungs (A), oxygen-exposed (72 h) and vehicle-treated lungs (B), oxygen-exposed and CsA- treated lungs (C). CsA treatment significantly decreased the extent of hemorrhage (C) compared with vehicle injection (B).

Right: lung sections of the same mice showing less extent of alveolar wall thickness, edema, and hyaline membrane forma- tion in CsA-treated (C) compared with vehicle-treated animals (B). Arrows indicate hyaline membranes.

Table 1. Lung injury score in mice treated with vehicle or CsA

Air Hyperoxia

Vehicle CsA Vehicle CsA

Macroscopical score 00 00 3.610.94 1.830.96*

Right lung weight, g 0.0830.006 0.070.005 0.200.04 0.150.04‡

Proteins, mg/ml 0.060.05 0.060.06 2.770.74 1.581.0†

Cell count103/ml 31.21.7 32.514.1 61.229.1 54.55.3

Results represent the mean valueSD for each group (n10 animals/group). Mice treated with vehicle or cyclosporin A (CsA) were exposed to air or hyperoxia for 72 h. Macroscopical aspect was evaluated and a score from 0 (absence of hemorrage) to 5 (completely hemorrhagic lung) was assigned. The right lung was weighed. Bronchoalveolar lavage was performed by instilling 2 ml of PBS intratracheally and recovered by hydrostatic pressure. Protein content was measured in the supernatant, and cells were counted. *P0.001, †P0.01, ‡P0.05 CsA-treated compared with vehicle-treated group exposed to hyperoxia.

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been proposed that, once activated, Bid can assist the confor- mational change of Bax into its active form leading to cyto- chrome c release (13, 44). However, Bax insertion to the mitochondrial membrane has been observed in Bid-null fibro- blasts, suggesting an alternative Bid-independent pathway in Bax activation (39). It cannot be excluded that other BH3-only proteins, such as Bim or Bad, or other upstream molecules, might induce Bax translocation.

Cytochrome c release into the cytosol has been associated with caspase signaling activation and apoptotic cell death in several human diseases, such as heart failure, neurodegenera- tive diseases, and traumatic brain injury (35, 40, 41). We were unable to detect any active caspase-3 during hyperoxia, sup- porting our previous results, where no significant increase in caspase activity was measured in total lung extracts. In addi- tion, the administration of specific caspase inhibitors (ZVAD) did not prevent mouse lung injury (7). These data are in contrast with a recent report from Zhang et al. (49) in which activated caspase-3 was detected by IHC in lung epithelial and bronchial cells of mice exposed to hyperoxia. This discordance could account for the different sensitivity of the antibodies and the different technical approaches used in either study. Never- theless, these results leave open the question whether, besides the possible activation of caspase-3 in alveolar cells, hyper- oxia-induced lung injury is dependent on the classical caspase- 3-mediated apoptotic cascade. Indeed, a clear-cut answer might be provided by studying the survival of caspase-3(⫺/⫺) mice in hyperoxia (29). Moreover, several in vitro results indicate that the signaling pathways used to initiate cell death are not predicting the final outcome of cell necrosis or apoptosis. For instance, activation of caspase-9 upon cytochrome c release and phenotypic features of apoptosis have been shown in cultured Rat-1 cells and endothelial cells after 40–72 h of oxygen exposure (10, 25). Conversely, A549 cells exposed to 100% oxygen, although presenting caspase-8 and caspase-9 activation, did not undergo apoptotic cell death but presented morphological features of necrosis (26, 44). Our previous results based on EM analysis suggest that most of the apoptotic features occurred in endothelial cells, although some alveolar cells presented overlapping features of apoptosis and necrosis (7). This indicates that hyperoxia-induced epithelial cell death might involve the initiation of a caspase-mediated, mitochon- dria-dependent apoptotic pathway, despite a final outcome of cellular necrosis (44).

CsA treatment was previously described to be effective in protecting lungs from hyperoxia-induced injury (33). However, no mechanism was proposed for its beneficial effect. Accord- ingly, our study shows that CsA attenuated lung damage, as evaluated by different injury markers such as macroscopical injury score, lung weight, and BAL protein content. Impor- tantly, such a protective effect of CsA correlated with a preserved morphological aspect of mitochondria in type II cells and with a decrease of cytochrome c release, as demonstrated by IHC. These results are in agreement with previous reports where CsA prevented irreversible damage to mitochondria (21) and cytochrome c release in several pathological conditions (34, 46). It is interesting to note that alveolar type II cells from CsA-treated mice presented a mixed population of still elec- tron-dense and swollen mitochondria, suggesting that the num- ber of functional mitochondria and the level of ATP are crucial for cell survival and lung damage.

Morphological mitochondria alterations under hyperoxia have been mainly studied in type II and type II-like epithelial cells (1, 12, 27). These cells have been shown to play a critical role in the maintenance of the alveolar space, since they are more resistant to oxidative stress compared with other alveolar cells and are the precursors of type I cells (31). Adaptation to hyperoxia and survival after lung injury may depend on the capacity of alveolar progenitor cells to proliferate and reestab- lish the integrity of the alveolar epithelium. For this reason, type II cells have been targeted for the overexpression of protecting molecules, to prevent hyperoxia-induced lung injury (43). For instance, overexpression of mitochondrial antioxidant enzyme Mn-superoxide dismutase in surfactant protein (SP)- C-expressing cells conferred protection from oxygen-induced lung damage in mice and correlated with prevention of mito- chondrial injury and preservation of ATP content in those cells (45). Accordingly, our results emphasize the importance of epithelial type II cells in maintaining critical epithelial func- tion.

However, it cannot be excluded that CsA would also prevent cytochrome c release and mitochondrial damage in endothelial and epithelial type I cells. Alternatively, type II cells could indirectly influence the sensitivity to oxygen of endothelial and epithelial type I cells. For example, a deficiency of SP-B, a protein specifically synthesized by type II cells, sensitized mice to hyperoxia. Increased lung permeability and protein content in BAL were observed in these mice, suggesting that epithelial type II cells affect the physiological function of endothelial and epithelial type I cells (42). Additional evidence of cross talk between type II cells and endothelial cells has been provided by in vitro studies, where A549 and primary type II cells were able to modulate transendothelial migration of leukocytes un- der specific stimuli (15, 28). It is likely that these cells may also contribute to changes of endothelial function in vivo and that preserving the integrity of type II cells might exert a protective effect on alveolo-capillary barrier.

In conclusion, this report indicates that CsA, by preventing mitochondrial alterations and cytochrome c release in type II cells, confers protection against hyperoxia-mediated lung in- jury.

ACKNOWLEDGMENTS

We thank Michel Aurrand-Lions, Marc Chanson, Didier Trono, and Pierre- Franc¸ois Piguet for scientific advice; Patrick Muzzin for scientific help; and Philippe Henchoz, Jean-Claude Rumbeli, and Coralie Reverdin for technical assistance.

GRANTS

This work was supported by Fonds National de la Recherche Scientifique Grant 3200-067865.02 and by the Wo¨lfermann-Na¨gele, Novartis, and Lancar- dis Foundations.

REFERENCES

1. Ahmad S, White CW, Chang LY, Schneider BK, and Allen CB.

Glutamine protects mitochondrial structure and function in oxygen toxic- ity. Am J Physiol Lung Cell Mol Physiol 280: L779–L791, 2001.

2. Albertine KH and Plopper CG. DNA oxidation or apoptosis: will the real culprit of DNA damage in hyperoxic lung injury please stand up?

Am J Respir Cell Mol Biol 26: 381–383, 2002.

3. Antonsson B, Montessuit S, Lauper S, Eskes R, and Martinou JC. Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria. Biochem J 345:

271–278, 2000.

L282 MITOCHONDRIA-DEPENDENT CELL DEATH IN HYPEROXIA

AJP-Lung Cell Mol PhysiolVOL 286 • FEBRUARY 2004 •www.ajplung.org

(10)

4. Barazzone Argiroffo C, Donati YR, Boccard J, Rochat AF, Vesin C, Kan CD, and Piguet PF. CD40-CD40 ligand disruption does not prevent hyperoxia-induced injury. Am J Pathol 160: 67–71, 2002.

5. Barazzone C, Belin D, Piguet PF, Vassalli JD, and Sappino AP.

Plasminogen activator inhibitor-1 in acute hyperoxic mouse lung injury.

J Clin Invest 98: 2666–2673, 1996.

6. Barazzone C, Donati YR, Rochat AF, Vesin C, Kan CD, Pache JC, and Piguet PF. Keratinocyte growth factor protects alveolar epithelium and endothelium from oxygen-induced injury in mice. Am J Pathol 154:

1479–1487, 1999.

7. Barazzone C, Horowitz S, Donati YR, Rodriguez I, and Piguet PF.

Oxygen toxicity in mouse lung: pathways to cell death. Am J Respir Cell Mol Biol 19: 573–581, 1998.

8. Barazzone C and White CW. Mechanisms of cell injury and death in hyperoxia: role of cytokines and Bcl-2 family proteins. Am J Respir Cell Mol Biol 22: 517–519, 2000.

9. Bossy-Wetzel E and Green DR. Assays for cytochrome c release from mitochondria during apoptosis. Methods Enzymol 322: 235–242, 2000.

10. Budinger GR, Tso M, McClintock DS, Dean DA, Sznajder JI, and Chandel NS. Hyperoxia-induced apoptosis does not require mitochondrial reactive oxygen species and is regulated by Bcl-2 proteins. J Biol Chem 277: 15654–15660, 2002.

11. Christofidou-Solomidou M, Kennel S, Scherpereel A, Wiewrodt R, Solomides CC, Pietra GG, Murciano JC, Shah SA, Ischiropoulos H, Albelda SM, and Muzykantov VR. Vascular immunotargeting of glu- cose oxidase to the endothelial antigens induces distinct forms of oxidant acute lung injury: targeting to thrombomodulin, but not to PECAM-1, causes pulmonary thrombosis and neutrophil transmigration. Am J Pathol 160: 1155–1169, 2002.

12. Crapo JD, Barry BE, Foscue HA, and Shelburne J. Structural and biochemical changes in rat lungs occurring during exposures to lethal and adaptive doses of oxygen. Am Rev Respir Dis 122: 123–143, 1980.

13. Degli Esposti M and Dive C. Mitochondrial membrane permeabilisation by Bax/Bak. Biochem Biophys Res Commun 304: 455–461, 2003.

14. Dubrez L, Coll JL, Hurbin A, Solary E, and Favrot MC. Caffeine sensitizes human H358 cell line to p53-mediated apoptosis by inducing mitochondrial translocation and conformational change of BAX protein.

J Biol Chem 276: 38980–38987, 2001.

15. Eghtesad M, Jackson HE, and Cunningham AC. Primary human alveolar epithelial cells can elicit the transendothelial migration of CD14 monocytes and CD3lymphocytes. Immunology 102: 157–164, 2001.

16. Ferri KF and Kroemer G. Organelle-specific initiation of cell death pathways. Nat Cell Biol 3: E255–E263, 2001.

17. Fox RB, Hiodal JR, Brown DM, and Repine JE. Pulmonary inflamma- tion due to oxygen toxicity: involvement of chemotactic factors and polymorphonuclear cells. Am Rev Respir Dis 123: 521–523, 1981.

18. Freeman BA and Crapo JD. Biology of disease: free radicals and tissue injury. Lab Invest 47: 412–426, 1982.

19. Freeman BA and Crapo JD. Hyperoxia increases oxygen radical production in rat lungs and lung mitochondria. J Biol Chem 256: 10986–10992, 1981.

20. Freeman BA, Topolosky MK, and Crapo JD. Hyperoxia increases oxygen radical production in rat lung homogenates. Arch Biochem Bio- phys 216: 477–484, 1982.

21. Friberg H, Ferrand-Drake M, Bengtsson F, Halestrap AP, and Wie- loch T. Cyclosporin A, but not FK 506, protects mitochondria and neurons against hypoglycemic damage and implicates the mitochondrial perme- ability transition in cell death. J Neurosci 18: 5151–5159, 1998.

22. Guegan C, Vila M, Rosoklija G, Hays AP, and Przedborski S. Re- cruitment of the mitochondrial-dependent apoptotic pathway in amyotro- phic lateral sclerosis. J Neurosci 21: 6569–6576, 2001.

23. Hengartner MO. The biochemistry of apoptosis. Nature 407: 770–776, 2000.

24. Hirsch T, Marzo I, and Kroemer G. Role of the mitochondrial perme- ability transition pore in apoptosis. Biosci Rep 17: 67–76, 1997.

25. Hogg N, Browning J, Howard T, Winterford C, Fitzpatrick D, and Gobe G. Apoptosis in vascular endothelial cells caused by serum depri- vation, oxidative stress and transforming growth factor-beta. Endothelium 7: 35–49, 1999.

26. Kazzaz JA, Xu J, Palaia TA, Mantell L, Fein AM, and Horowitz S.

Cellular oxygen toxicity. Oxidant injury without apoptosis. J Biol Chem 271: 15182–15186, 1996.

27. Kistler GS, Caldwell PR, and Weibel ER. Development of fine struc- tural damage to alveolar and capillary lining cells in oxygen-poisoned rat lungs. J Cell Biol 32: 605–628, 1967.

28. Koyama S, Sato E, Nomura H, Kubo K, Miura M, Yamashita T, Nagai S, and Izumi T. Bradykinin stimulates type II alveolar cells to release neutrophil and monocyte chemotactic activity and inflammatory cytokines. Am J Pathol 153: 1885–1893, 1998.

29. Kuida K, Zheng TS, Na S, Kuan C, Yang D, Karasuyama H, Rakic P, and Flavell RA. Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature 384: 368–372, 1996.

30. Martinou JC and Green DR. Breaking the mitochondrial barrier. Nat Rev Mol Cell Biol 2: 63–67, 2001.

31. Mason RJ and Williams MC. Type II alveolar cell. Defender of the alveolus. Am Rev Respir Dis 115: 81–91, 1977.

32. Massaro G and Massaro D. Pulmonary granular pneumocytes: loss of mitochondrial granules during hyperoxia. J Cell Biol 59: 246–250, 1973.

33. Matthew E, Pun R, Simonich M, Iwamoto H, and Dedman J. Cyclo- sporin A protects lung function from hyperoxic damage. Am J Physiol Lung Cell Mol Physiol 276: L786–L795, 1999.

34. Nakatsuka H, Ohta S, Tanaka J, Toku K, Kumon Y, Maeda N, Sakanaka M, and Sakaki S. Release of cytochrome c from mitochondria to cytosol in gerbil hippocampal CA1 neurons after transient forebrain ischemia. Brain Res 849: 216–219, 1999.

35. Narula J, Pandey P, Arbustini E, Haider N, Narula N, Kolodgie FD, Dal Bello B, Semigran MJ, Bielsa-Masdeu A, Dec GW, Israels S, Ballester M, Virmani R, Saxena S, and Kharbanda S. Apoptosis in heart failure: release of cytochrome c from mitochondria and activation of caspase-3 in human cardiomyopathy. Proc Natl Acad Sci USA 96: 8144–8149, 1999.

36. O’Reilly MA, Staversky RJ, Huyck HL, Watkins RH, LoMonaco MB, D’Angio CT, Baggs RB, Maniscalco WM, and Pryhuber GS. Bcl-2 family gene expression during severe hyperoxia induced lung injury. Lab Invest 80: 1845–1854, 2000.

37. Petit PX, Goubern M, Diolez P, Susin SA, Zamzami N, and Kroemer G. Disruption of the outer mitochondrial membrane as a result of large amplitude swelling: the impact of irreversible permeability transition.

FEBS Lett 426: 111–116, 1998.

38. Petronilli V, Nicolli A, Costantini P, Colonna R, and Bernardi P.

Regulation of the permeability transition pore, a voltage-dependent mito- chondrial channel inhibited by cyclosporin A. Biochim Biophys Acta 1187:

255–259, 1994.

39. Ruffolo SC, Breckenridge DG, Nguyen M, Goping IS, Gross A, Korsmeyer SJ, Li H, Yuan J, and Shore GC. BID-dependent and BID-independent pathways for BAX insertion into mitochondria. Cell Death Differ 7: 1101–1108, 2000.

40. Sanchez Mejia RO and Friedlander RM. Caspases in Huntington’s disease. Neuroscientist 7: 480–489, 2001.

41. Sullivan PG, Keller JN, Bussen WL, and Scheff SW. Cytochrome c release and caspase activation after traumatic brain injury. Brain Res 949:

88–96, 2002.

42. Tokieda K, Iwamoto HS, Bachurski C, Wert SE, Hull WM, Ikeda K, and Whitsett JA. Surfactant protein-B-deficient mice are susceptible to hyperoxic lung injury. Am J Respir Cell Mol Biol 21: 463–472, 1999.

43. Tsan MF. Superoxide dismutase and pulmonary oxygen toxicity: lessons from transgenic and knockout mice. Int J Mol Med 7: 13–19, 2001.

44. Wang X, Ryter SW, Dai C, Tang ZL, Watkins SC, Yin XM, Song R, and Choi AM. Necrotic cell death in response to oxidant stress involves the activation of the apoptogenic caspase-8/bid pathway. J Biol Chem 278:

29184–29191, 2003.

45. Wispe JR, Warner BB, Clark JC, Dey CR, Neuman J, Glasser SW, Crapo JD, Chang LY, and Whitsett JA. Human Mn-superoxide dis- mutase in pulmonary epithelial cells of transgenic mice confers protection from oxygen injury. J Biol Chem 267: 23937–23941, 1992.

46. Yoshimoto T, Kristian T, Hu B, Ouyang YB, and Siesjo BK. Effect of NXY-059 on secondary mitochondrial dysfunction after transient focal ischemia: comparison with cyclosporin A. Brain Res 932: 99–109, 2002.

47. Zamzami N and Kroemer G. The mitochondrion in apoptosis: how Pandora’s box opens. Nat Rev Mol Cell Biol 2: 67–71, 2001.

48. Zamzami N, Marchetti P, Castedo M, Hirsch T, Susin SA, Masse B, and Kroemer G. Inhibitors of permeability transition interfere with the disruption of the mitochondrial transmembrane potential during apoptosis.

FEBS Lett 384: 53–57, 1996.

49. Zhang X, Shan P, Sasidhar M, Chupp GL, Flavell RA, Choi AM, and Lee PJ. Reactive oxygen species and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase mediate hyperoxia-induced cell death in lung epithelium. Am J Respir Cell Mol Biol 28: 305–315, 2003.

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