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Am J Physiol Lung Cell Mol Physiol 293: L619-L629, 2007. First published June 15, 2007; doi:10.1152/ajplung.00037.2007
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Poly(ADP-ribose) polymerase-1 (PARP-1) controls lung cell proliferation and repair after hyperoxia-induced lung damage

Alessandra Pagano,1,2 Isabelle Métrailler-Ruchonnet,1,2 Michel Aurrand-Lions,1 Monica Lucattelli,3 Yves Donati,1,2 and Constance Barazzone Argiroffo1,2

Departments of 1Pathology-Immunology and 2Pediatrics, Medical School, University of Geneva, Switzerland; and 3Department of Physiopathology and Experimental Medicine, University of Siena, Siena, Italy

Submitted 25 January 2007 ; accepted in final form 5 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Oxygen-based therapies expose lung to elevated levels of ROS and induce lung cell damage and inflammation. Injured cells are replaced through increased proliferation and differentiation of epithelial cells and fibroblasts. Failure to modulate these processes leads to excessive cell proliferation, collagen deposition, fibrosis, and chronic lung disease. Poly(ADP-ribose) polymerase-1 (PARP-1) is activated in response to DNA damage and participates in DNA repair, genomic integrity, and cell death. In this study, we evaluated the role of PARP-1 in lung repair during recovery after acute hyperoxia exposure. We exposed PARP-1 –/– and wild-type mice for 64 h to 100% hyperoxia and let them recover in air for 5–21 days. PARP-1-deficient mice exhibited significantly higher lung cell hyperplasia and proliferation than PARP-1 +/+ animals after 5 and 10 days of recovery. This was accompanied by an increased inflammatory response in PARP-1 –/– compared with wild-type animals, characterized by neutrophil infiltration and increased IL-6 levels in bronchoalveolar lavages. These lesions were reversible, since the extent of the hyperplastic regions was reduced after 21 days of recovery and did not result in fibrosis. In vitro, lung primary fibroblasts derived from PARP-1 –/– mice showed a higher proliferative response than PARP-1 +/+ cells during air recovery after hyperoxia-induced growth arrest. Altogether, these results reveal an essential role of PARP-1 in the control of cell repair and tissue remodeling after hyperoxia-induced lung injury.

hyperoxia; poly(ADP-ribose) polymerase-1 knockout mice; cell proliferation; inflammation; p21

OXYGEN IS USED FOR THERAPY of patients suffering from respiratory distress syndrome (IRDS, ARDS). However, breathing oxygen-enriched air exposes the lung to elevated levels of reactive oxygen species (ROS) (41). Hyperoxia-induced lung injury is characterized by extensive parenchymal cell death, which is mediated by both apoptosis and necrosis. The cells of the alveolocapillary barrier are directly damaged, and additional injury occurs by the concomitant inflammatory response resulting in capillary leak and alveolar edema (4). Following oxidative damage, injured and dead cells are replaced through increased proliferation and differentiation of type II epithelial cells and fibroblasts (1, 48). However, failure to modulate these processes leads to excessive cell proliferation, increased collagen deposition, and pulmonary fibrosis, also named chronic lung disease (CLD) (14). Therefore, a regulated cell replacement is necessary for correct repair after acute lung injury.

In response to hyperoxia-induced stress, cells are stopped at specific checkpoints of the cell cycle to prevent DNA mutations. Cell cycle-regulating proteins, such as the tumor suppressor p53 or the cyclin-dependent kinase inhibitor p21, inhibit the progression trough cell cycle and cell proliferation to allow DNA repair (42). When damage is too strong to be repaired, the same molecules can direct the cell toward apoptotic cell death (11, 32). Poly(ADP-ribose) polymerase-1 (PARP-1) is the most abundant nuclear enzyme of the PARP family, which is activated in response to DNA damage and participates in DNA repair, genomic integrity, and cell death. PARP-1 binds rapidly to DNA strand breaks and adds branched poly(ADP-ribose) polymers, using nicotinamide adenine dinucleotide (NAD+) as a substrate, on itself and other nuclear proteins, such as histones, to facilitate the action of DNA repair enzymes (39). PARP-1 is also implicated in cell death. Cleavage of PARP-1 by caspases is a hallmark of apoptosis, whereas excessive DNA damage induces massive PARP activation and necrotic cell death by ATP depletion (20, 44). Inhibition of PARP activity and PARP-1 genetic disruption have been correlated with decreased injury and enhanced cell survival in several experimental mouse models and also with chromosome instability and tumor sensitivity (6, 46, 51, 54). Our group has previously shown that PARP-1 activity is induced in response to acute hyperoxia (100% O2) both in vitro and in vivo and has studied its role in hyperoxia-induced cell death (36).

In the present study, we evaluated the role of PARP-1 in lung repair during recovery after acute hyperoxia exposure, using PARP-1 knockout mice (9, 53). Mice lacking PARP-1 molecule presented excessive lung cell proliferation and hyperplasia, accompanied by a strong inflammatory response, compared with wild-type mice exposed to the same conditions. These in vivo results correlated with a higher proliferative response of primary lung fibroblasts derived from PARP-1 –/– mice during recovery after hyperoxia. Altogether, these data suggest that PARP-1 is a crucial molecule in regulating lung cell proliferation and repair after hyperoxia-induced acute injury.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Oxygen Exposure and Recovery

PARP-1 –/– mice (Sv129 genetic background) and their littermates (PARP-1 +/+) were kindly provided by the laboratory of Z. Q. Wang (Centre International de Recherche sur le Cancer, Lyon, France) and bred in our animal facility. The generation and characterization of these mice have been described in detail previously (53). Absence of PARP-1 gene in mice was confirmed by PCR of genomic DNA according to published conditions, and PARP-1 protein deletion was confirmed by Western blot analysis (36, 46). Twelve- to 14-wk-old PARP-1 +/+ and PARP-1 –/– mice were kept in room air or placed in a sealed Plexiglas chamber and exposed to 100% O2 for 64 h and then allowed to recover in room air for 5, 10, or 21 days. In some experiments (recovery for 5 and 10 days), mice, when removed from hyperoxia, were injected daily with 5-bromo-2'-deoxyuridine (BrdU; 1 mg /mice; Sigma-Aldrich, Buchs, Switzerland) intraperitoneally, according to previously described protocols (3). Animals were then killed, and the right lung was immediately frozen in liquid nitrogen and stored at –80°C for further analysis. The left lung was formalin fixed (4% in 1x PBS) for histological analysis. Alternatively, lungs were instilled intratracheally with OCT (Sakura, Zoeterwoude, The Netherlands), immediately frozen in 2-methylbutane isopentane, and stored at –80°C for immunohistochemistry on cryosections.

As an alternative approach, PARP-1 +/+ mice were treated with the PARP inhibitor PJ34 (Alexis, Lausanne, Switzerland). Briefly, animals were exposed to hyperoxia for 64 h and allowed to recover in air for 10 days. Mice were injected intraperitoneally with either saline solution or PJ34 (10 mg/kg) at time 0, after 48 h of hyperoxia, and at day 1 of air recovery after hyperoxia, according to previous protocols (24, 50). Mice were killed, and lung histology was performed. All study protocols were approved by the local ethical committee on animal experiments (Office Vétérinaire Cantonal of Geneva).

Lung Histology

After fixation, lung was dehydrated in 70% ethanol and embedded in paraffin according to standard procedures. Sections were stained with hematoxylin and eosin (H&E) or Masson's trichrome blue for specific detection of collagen.

Quantification of Hyperplastic Regions

Serial images of lung sections stained with H&E were acquired at a fivefold magnification with a digital camera connected to a microscope (10–15 images per lung, 8 animals per condition; Axiocam, Zeiss, Düsseldorf, Germany). Tissue density of lung sections was measured using MetaMorph 6.1 software (Universal Imaging, Downingtown, PA) and expressed as a percentage of total lung section surface.

Immunohistochemistry

For BrdU labeling, slides were deparaffinized, rehydrated, and treated with 0.5% H2O2 in methanol to inactivate the endogenous peroxidases. Antigen retrieval was made by treating cells with 2 N HCl for 10 min and successively with proteinase K (0.3 mg/ml) for 5 min. Samples were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline (1x TBS) and incubated with a horseradish peroxidase-conjugated anti-BrdU antibody (Roche Biotechnology, Basel, Switzerland). The peroxidase activity was revealed with the addition of the substrate 3,3'-diaminobenzidine tetrahydrocloride (Sigma-Aldrich). Finally, sections were counterstained with hemalun. Normal mouse IgG were used instead of the primary antibody as negative control for nonspecific binding.

For triple staining, lung cryosections (0.4 µm) were fixed in acetone-methanol solution, blocked with 5% goat serum in 1x TBS, and incubated overnight at 4°C with a rabbit polyclonal anti-surfactant protein C antibody (SP-C; Chemicon, Temecula, CA), a mouse monoclonal anti-vimentin antibody (clone V3B4; DakoCytomation, Baar, Switzerland), and a monoclonal rat anti-CD45 antibody (BD Biosciences, San Diego, CA). Alternatively, slides were incubated with anti-SP-C, anti-vimentin, and a monoclonal rat anti-Mac-1 antibody (Caltag Laboratories). After washing, sections were incubated with a goat anti-rabbit-Cy5-coupled antibody (Jackson ImmunoResearch, Suffolk, UK), a goat anti-mouse-Texas red-coupled antibody (Jackson ImmunoResearch), and a goat anti-rat-FITC-coupled antibody (Jackson ImmunoResearch). After washing, slides were mounted using Mowiol. Sections were analyzed with a confocal microscope (LSM 510; Zeiss, Düsseldorf, Germany; Plan-Neofluar x40/1.3 oil objective) connected to a digital camera. Images were taken and analyzed using Image J software.

Lung Hydroxy-Proline Content

Lungs were weighed and homogenized. Hydroxy-proline (HO-proline) content in lung hydrolysates was determined according to methods previously described (10). Data (in µg of HO-proline per lung, n = 7) are expressed as relative increases over respective controls, set as 1 (means ± SD).

Analysis of Bronchoalveolar Lavage Fluids

Bronchoalveolar lavage fluid cell count. Mice were anesthetized, and the bronchoalveolar lavage fluids (BALFs) were obtained by washing intratracheal lung three times with 0.7 ml of physiological solution (0.9% NaCl, 2 ml of total BALFs). Cells were counted using a CASY 1 cell counter (RJF Sales, Scotch Plains, NJ). BALFs were then spun at 400 gfor 5 min, and supernatants were aliquoted and frozen at –80°C.

BALF cell distribution. The BALF cell pellet was resuspended in 1x PBS-1% BSA and spun in a microfuge for cytospin (700 g). BALF cell distribution was quantified on cytospin (x40 magnification) after staining with the Diff-Quick staining set (Dade Behring, Newark, DE). Results are expressed as percentages of leukocyte subsets.

BALF protein content. The BALF protein concentration was determined in BALF supernatants by using Bradford's method (BCA; Bio-Rad Laboratories, Hercules, CA).

Mouse cytokine antibody array. Pools of BALF supernatant from PARP-1 +/+ and PARP-1 –/– animals (n = 7 in each group), exposed to hyperoxia for 64 h and recovered in air for 5 days, were incubated with the RayBio mouse cytokine antibody array membranes (RayBiotech, Norcross, GA) overnight. Cytokines in BALF were then detected according to the manufacturer's instructions.

Transforming growth factor-beta1 assay. Active transforming growth factor-beta1 (TGF-beta1) content was measured on BALF supernatant using ELISA (DuoSet ELISA for mouse TGF-beta1; R&D Systems, Abingdon, UK). Results are expressed as picograms per milliliter (means ± SD).

Analysis of Infiltrating Cells in Total Lung Tissue

Mice were anesthetized with an intraperitoneal injection of pentobarbital (Abbott, Baar, Switzerland), the thoracic cage was opened, and lungs were washed by flushing the right ventricle with saline solution. Lungs were then removed, dissociated by dispase-hyaluronidase digestion (2 mg/ml each; Sigma-Aldrich), and filtered through a 70-µm nylon filter to remove aggregates (25). Single-cell suspensions were washed in 1x PBS and labeled with 7-amino-actinomycin D (7-AAD), biotinylated-anti-CD45 antibody, followed by streptavidin-FITC, and anti-Mac-1-phycoerythrin (PE) antibody. Cells were then analyzed by flow cytometry with a FACSCalibur cell analyzer (BD Biosciences). For analysis, dead (7-AAD+) cells were excluded, and inflammatory (CD45+) cells were gated and analyzed for the presence or absence of the Mac-1 myeloid cell marker to classify myeloid (CD45+, Mac-1+) and lymphoid (CD45+, Mac-1–) cells. To better characterize the myeloid cell subpopulations, we labeled cells with 7-AAD (Molecular Probes), anti-Mac-1-PE, anti-GR1-allophycocyanin (Caltag Laboratories), and anti-F4/80-FITC (Caltag Laboratories). On living (7-AAD negative) cells, total Mac-1+ myeloid cells were gated and analyzed for staining with anti-GR1 and anti-F4/80. This triple staining enables discrimination among granulocytes (Mac-1+, GR1+, F4/80–), inflammatory macrophages (Mac-1+, GR1+, F4/80+), and resident macrophages (Mac-1+, GR1–, F4/80+), as reported previously (16, 23). Results are expressed as percentages of positive cells.

Lung Myeloperoxidase Content

Frozen right lung was homogenized in 0.5% hexadecyltrimethylammonium (in 50 mM KH2PO4, pH 6.0, and 0.5 mM EDTA, 1 ml/100 mg tissue) and centrifuged at 12,000 g for 15 min at 4°C. Supernatant was kept at –20°C until protein determination and myeloperoxidase (MPO) measurement. The enzymatic reaction was measured using 50 µg of protein in a reaction mix containing 94 mM KH2PO4, pH 6.0, and 0.2 mg/ml o-dianisidine. Absorbance was measured at 460 nm at time 0 (basal value) and every each 30 s during the 2 min following addition of H2O2 (final concentration 4 µM). Activity was expressed as the change in optical density per minute per milligram of protein.

Western Blot Analysis of p21 in Total Lung

Thirty micrograms of total lung homogenates were loaded on a gel for SDS-PAGE electrophoresis and blotted to nitrocellulose membranes (Amersham International, Amersham, UK). Membranes were blocked overnight in TBS-T buffer (0.2 M Tris, pH 7.6, 1.5 M NaCl, and 0.1% Tween 20) in 5% milk and incubated with an anti-p21 specific antibody (BD Pharmingen, San Diego, CA). Horseradish peroxidase-conjugated anti-rabbit IgG (Bio-Rad Laboratories) was used as secondary antibody. Bands were visualized with a chemiluminescent substrate (ECL; Amersham International). Anti-actin antibody was used as a control for total protein loading.

Isolation of Primary Lung Fibroblasts

Primary mouse lung fibroblasts were isolated from lung explants of PARP-1 +/+ and PARP-1 –/– mice and grown in Dulbecco's modified Eagle's medium (DMEM; 1,000 mg/l glucose; Sigma-Aldrich), supplemented with 1% penicillin-streptomycin and 10% FCS. Cells were cultured in normoxia (5% CO2-21% O2) or hyperoxia (5% CO2-95% O2) as described previously (36).

Cell Proliferation Assays

Analysis of BrdU incorporation. Cells were exposed to hyperoxia (100% O2) for 64 h. After this time, medium was changed and cells were cultured under normoxic conditions for 1 and 2 days. BrdU was added (20 µM final concentration) for the last 14 h. To measure BrdU incorporation in air and hyperoxia (65 h) conditions, BrdU was added 1 h before cells were harvested. At each time point, cells were fixed, labeled with an anti-BrdU antibody (FITC-conjugated mouse monoclonal anti-BrdU antibody; BD Pharmingen), and stained with propidium iodide for cell cycle analysis, according to published protocols (22). Samples were then analyzed with a FACScan flow cytometer (BD Biosciences Pharmingen, Heidelberg, Germany).

Analysis of [3H]thymidine incorporation. Cells were plated in 96-well plates (5,000 cells/well, 8 wells/sample) and exposed to hyperoxia for 64 h. After that time, medium was changed and cells were incubated at room conditions for 1–3 days. [3H]thymidine (1 µCi/well; Amersham, Arlington Heights, IL) was added for the last 14 h. At each time point, cells were washed and harvested. Cell [3H]thymidine incorporation was measured using a beta counter (1450 Microbeta; Perkin Elmer, Wellesley, MA), and values are expressed as counts per minute (cpm, means ± SD).

Western Blot Analysis of Cleaved Caspase-3 in Primary Lung Fibroblasts

Cells were plated in 100-mm petri dishes (800,000 cells/condition) and exposed to hyperoxia for 64 h. After that time, medium was changed and cells were incubated in air for 1 and 3 days, respectively. At each time point, cells were washed and harvested. Western blot analysis was performed on total protein extracts (70 µg) with anti-cleaved caspase-3 antibody (Cell Signaling, Hitchin, UK). Anti-actin antibody was used for control of protein loading. Thymus extract (25 µg of protein) from a mouse treated with 250 µg of dexamethasone (intraperitoneally) and killed 6 h later was loaded as a positive control.

Statistical Analysis

For all parameters measured, the values for all animals and samples in different experimental conditions were averaged and the SD of the mean was calculated. The significance of differences between the values of the groups was determined with an unpaired-Student's t-test. Where appropriate, two-way ANOVA with Bonferroni posttest was used. Significance levels were set at P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
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PARP-I-Deficient Mice Present Excessive Lung Cell Proliferation During Recovery After Acute Hyperoxia

We exposed PARP-1 +/+ and PARP-1 –/– mice to 100% hyperoxia for 64 h and let them recover in air for 5–21 days before analysis of lung histology. At 5 (not shown) and 10 days of recovery, lung sections from wild-type animals exhibited normal alveolarization with some thickening of alveolar septa. Low inflammatory cell infiltration and no evident collagen deposition, as shown by Masson staining, were observed in these animals (Fig. 1A, a and inset). In contrast, lung sections from PARP-1 –/– mice presented high cell hyperplasia, proliferation, and inflammatory cell infiltration. Collagen deposition was barely detectable in the hyperplastic regions (Fig. 1A, b and inset). The quantification of lung tissue density, as shown in Fig. 1B, revealed similar baseline values in PARP-1 +/+ and PARP-1 –/– mice exposed to normoxia (18.14 ± 1.72% in PARP-1 +/+ vs. 18.35 ± 2.59% in PARP-1 –/–). However, after 10 days of air recovery following hyperoxia exposure, lung sections from PARP-1 –/– mice presented a significantly higher tissue density compared with PARP-1 +/+ mice (25.80 ± 8.66% in PARP-1 +/+ vs. 43.07 ± 13.63% in PARP-1 –/–, P < 0.001), due to the high cellularity and the hyperplastic regions. Interestingly, when wild-type mice were exposed to the same conditions and treated with the potent PARP inhibitor PJ34 (28, 50), we observed a similar phenotype after 10 days of air recovery (Supplemental Fig. S1; supplemental data for this article is available online at the American Journal of Physiology-Lung Cellular and Molecular Physiology website), confirming that cell hyperplasia induced in PARP-1 –/– mice was specific to the absence of PARP-1 protein and its enzymatic activity.


Figure 1
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  		Fig. 1. Poly(ADP-ribose) polymerase-1 (PARP-1) –/– mice present higher extent of lung cell proliferation. A: Masson trichrome staining of lung sections after exposure to 100% hyperoxia followed by recovery in air for 10 and 21 days (H+R 10d, H+R 21d). Representative fields are shown. Magnification x20 in a–d and x40 in insets. B: lung tissue density was quantified on sequential images taken at magnification x5, and values are expressed as percentages of the total lung surface (n = 8 animals for each group). ***P < 0.001, PARP-1 +/+ vs. PARP-1 –/–; {dagger}{dagger}P < 0.01, {dagger}{dagger}{dagger}P < 0.001, air vs. H+R 10d.

 
To analyze whether cellular hyperplasia was followed by fibrosis development, we analyzed histology at later time points after air recovery. After 21 days of air recovery, lung architecture of PARP-1 –/– recovered partially. Less hyperplastic regions were observed, indicating that the proliferative lesions were reversible (Fig. 1A, c and d).

Proliferating cells in lung sections were characterized by immunohistochemistry. After 10 days of air recovery, few BrdU-positive cells were found in PARP-1 +/+ lungs, whereas extensive BrdU incorporation was observed in hyperplastic regions of PARP-1 –/– sections (Fig. 2A). Triple staining of lung sections showed that SP-C (blue staining)- and vimentin (red staining)-positive cells accumulated in these area of intense proliferation (Fig. 2B, compare a vs. e for SP-C staining, and b vs. f for vimentin staining), suggesting that proliferating cells were epithelial type II and mesenchymal cells. A great number of inflammatory CD45-positive cells were recruited into the lesions (Fig. 2B, g). Interestingly, CD45-positive cells appeared closely associated to vimentin-positive structures (see Fig. 2B, h and inset).


Figure 2
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  		Fig. 2. Immunohistochemical characterization of lung hyperplasia. A: lungs from PARP-1 +/+ and PARP-1 –/– mice exposed to H+R 10d were labeled with anti-5-bromo-2'-deoxyuridine (BrdU) antibody; positive nuclei are stained brown (magnification x40). B: triple staining of lung cryosections from PARP-1 +/+ and PARP-1 –/– mice at H+R 10d. Surfactant protein C (SP-C)-positive cells are stained blue (a and e), vimentin-positive cells are red (b and f), and CD45-positive cells are green (c and g); merged images are shown in d and h. Representative fields are shown (magnification x40). Inset in h represents 2x enlargement from original picture.

 
Although strong lung tissue hyperplasia was present, we were not able to detect the presence of myofibroblasts, which are considered the hallmark of fibrotic lesions (38, 52). Indeed, as shown in Fig. 3A, cells positive for {alpha}-smooth actin were clearly evident in the wall of large airways, whereas staining was absent in the hyperplastic regions of PARP-1 –/– lung sections. This result was in agreement with the poor collagen deposition observed in histological preparations (Fig. 1A, detail) and with the similar baseline HO-proline content measured in PARP-1 +/+ and PARP-1 –/– lungs at 10 and 21 days of recovery (Fig. 3B). In summary, these data show that the absence of PARP-1 increases epithelial and fibroblastic cell proliferation and hyperplasia, without myofibroblast differentiation or extensive deposition of mature collagen, after exposure to acute hyperoxia and recovery in air.


Figure 3
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  		Fig. 3. Absence of lung fibrosis in PARP-1 –/– mice after hyperoxia and air recovery. A: anti-{alpha}-smooth muscle actin ({alpha}-SMA) immunostaining on paraffin-embedded sections. Immunolabeled cells are brown, nuclei are counterstained in blue (hemalun). Arrowheads indicate groups of positive cells. Representative fields are shown (magnification x40). B: hydroxy (HO)-proline content was measured in PARP-1 +/+ and PARP-1 –/– lungs exposed to H+R 10d and H+R 21d. Values (measured as µg of HO-proline/lung) are expressed as relative increases over respective controls, set as 1 (means ± SD; n = 8 for each group).

 
Absence of PARP-1 Increases Lung Inflammatory Response During Air Recovery

On the basis of results from immunohistochemical analysis, showing increased amounts of infiltrating inflammatory cells in lung hyperplastic regions, we analyzed the BALF contents of PARP-1 +/+ and PARP-1 –/– mice after 5 days of recovery (H+R 5d). Total protein content and cell number were significantly higher in PARP-1 –/– compared with PARP-1 +/+ BALFs (Fig. 4, A and B). Analysis of BALF cell distribution showed that neutrophil content was significantly increased in PARP-1 –/– compared with PARP-1 +/+ BALFs (Fig. 4C).


Figure 4
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  		Fig. 4. Analysis of inflammatory response in bronchoalveolar lavage fluids (BALFs) from mice exposed to H+R 5d. A: BALF protein content values are expressed as mg/ml (means ± SD, n = 9 for each group). **P < 0.01, PARP-1 +/+ vs. PARP-1 –/–; {dagger}{dagger}P < 0.01, {dagger}{dagger}{dagger}P < 0.001, air vs. H+R 5d. B: BALF cell number. Values (x105) are means ± SD; n = 9. ***P < 0.001, PARP-1 +/+ vs. PARP-1 –/–; {dagger}{dagger}{dagger}P < 0.001, air vs. H+R 5d. C: BALF cell distribution was analyzed on cell cytospins. Values (x105) are means ± SD; n = 9. ***P < 0.001, PARP-1 +/+ vs. PARP-1 –/–.

 
To better characterize the infiltrating cell population, we performed flow cytometry analysis of lung cells, after total lung dissociation, from PARP-1 +/+ and PARP-1 –/– mice exposed to hypoxia and recovery for 5 days. The total CD45+ inflammatory cell population was gated and analyzed for the presence or absence of the myeloid cell marker Mac-1. The proportion of myeloid (Mac-1+, CD45+) cells was significantly increased in lung cell preparations of PARP-1 –/– compared with PARP-1 +/+ mice. Inversely, the percentage of lymphoid (CD45+, Mac-1–) cells was significantly reduced in the same samples (Fig. 5, A and B). These results were confirmed by immunohistochemical analysis showing the accumulation of Mac-1+ cells in the hyperplastic regions of PARP-1 –/– lung sections (Supplementary Fig. S2). Analysis of myeloid cell subsets confirmed a higher accumulation of granulocytes (GR1+, F4/80–) in the lungs of PARP-1 –/– compared with PARP-1 +/+ mice, although values were not significantly different (Fig. 5C). Inflammatory (GR1+, F4/ 80 +) and resident (GR1–, F4/80 +) monocyte/macrophages were also found to be increased in PARP-1 –/– lungs (Fig. 5C). According to these results, MPO activity, a marker for neutrophil infiltration (2), was significantly induced in PARP-1 –/– lung extracts but not in wild-type animals (Fig. 5D).


Figure 5
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  		Fig. 5. Analysis of infiltrating cells in total lungs from mice exposed to H+R 5d. A: cells were labeled with anti-CD45 and anti-Mac-1 antibodies and 7-amino-actinomycin D (7-AAD). Dead cells were excluded based on their positivity for 7-AAD. CD45+ cells were gated and analyzed for the presence or absence of Mac-1. One representative FACS histogram plot, showing Mac-1 labeling for each PARP-1 +/+ and PARP-1 –/– group, is presented. B: quantification of the proportion of lymphoid (CD45+, Mac-1–) and myeloid (CD45+, Mac-1+) cells in total lungs. Values are expressed as percentages of CD45+ cells (means ± SD; n = 5). *P < 0.05, PARP-1 +/+ vs. PARP-1 –/–. C: distribution of myeloid cell subpopulations. Cells were labeled with anti Mac-1, anti-GR1, and anti-F4/80 antibodies. 7-AAD–, Mac-1+ cells were gated and analyzed for the presence of GR1 and F4/80 molecules. Values are expressed as percentages of CD45 + cells. D: myeloperoxidase (MPO) activity in lung tissue. Values are expressed as the change in optical density ({Delta}OD) with absorbance at 460 nm per minute per mg of protein (means ± SD; n = 9). ***P < 0.001, PARP-1 +/+ vs. PARP-1 –/–; {dagger}{dagger}{dagger}P < 0.001, air vs. H+R 5d.

 
The pattern of inflammatory cytokine secretion was analyzed in a pool of BALFs from PARP-1 +/+ and PARP-1 –/– mice (n = 7) exposed to hyperoxia and recovery by using cytokine array blot analysis. As shown in Fig. 6A, inflammatory mediators such as monocyte chemotactic protein (MCP)-1 (Fig. 5A, B7–B8), KC (J5–J6), and LIX (M5–M6) were equally secreted in PARP-1 +/+ and PARP-1 –/– BALFs. However, the proinflammatory IL-6 cytokine was detected only in PARP-1 –/– BALFs, not in PARP-1 +/+ (Fig. 5A, C5–C6). Similarly, spots corresponding to CXCL13/BLC (G1–G2) and to insulin-like growth factor binding protein-5 (IGFBP-5; H3–H4) were stronger in PARP-1 –/– than in PARP-1 +/+ BALFs.


Figure 6
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  		Fig. 6. Cytokine analysis in BALFs from mice exposed to H+R 5d. A: a pool of BALFs from PARP-1 +/+ and PARP-1 –/– mice exposed to H+R 5d was tested using a cytokine antibody array. Spots similarly expressed in the 2 samples are: B7–B8, monocyte chemotactic protein-1; J5–J6, KC; M5–M6, LIX. Spots differentially expressed (circles) are C5–C6, IL-6; G1–G2, CXCL13/BLC; H3–H4, insulin-like growth factor binding protein-5. B: the active form of transforming growth factor-beta1 (TGF-beta1) was quantified in BALFs by performing ELISA. Values are expressed as pg/ml (means ± SD; n = 9). ***P = 0.001, air vs. H + R 5d.

 
We also analyzed BALFs for the presence of the active form of profibrotic TGF-beta1 by ELISA assay. In both PARP-1 +/+ and PARP-1 –/– mouse lungs active TGF-beta1 was produced in significant amounts during recovery after hyperoxia exposure compared with air conditions. However, no significant difference was found between hyperoxia-exposed and air-recovered PARP-1 +/+ and PARP-1 –/– mice (Fig. 6B). In summary, these data show that absence of PARP-1 results in a strong inflammatory response during recovery after hyperoxia with transient tissue hyperplasia but without fibrosis.

PARP-1 Deficiency Does Not Affect p21 Regulation In Vivo

PARP-1 is known to interact with the cell cycle-regulating proteins p53 and p21 (15, 55). Interestingly, the absence of p21 affects lung repair and increases cell proliferation during recovery after hyperoxia exposure in mice (45). We wondered whether PARP-1 could regulate cell proliferation via p21 and analyzed p21 expression during hyperoxia and recovery. Oxygen exposure strongly induced p21 mRNA (not shown) and protein expression, compared with air conditions, in both mouse strains (Fig. 7), as previously described (35). We confirmed that p21 mRNA amounts were downregulated during air recovery (not shown) (45), whereas p21 protein levels remained elevated in both PARP-1 +/+ and PARP-1 –/– lungs (Fig. 7). According to these results, we can conclude that hyperplasia and excessive cell proliferation due to the absence of PARP-1 is not dependent on p21 regulation.


Figure 7
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  		Fig. 7. Analysis of p21 expression. Total lung lysates (30 µg) were analyzed by Western blotting. Actin was used as control of charge. One representative experiment of 3 is shown. Hox 64h, mice exposed to hyperoxia for 64 h.

 
Absence of PARP-1 Affects the Proliferative Response of Primary Lung Fibroblasts After Exposure to Acute Hyperoxia

During acute hyperoxia, cells undergo cell cycle arrest and stop to proliferate to repair DNA damage (13, 32). PARP-1 is known to participate to these events (20). To understand the mechanisms leading to excessive lung cell proliferation following oxygen-induced cell damage, we exposed lung primary fibroblasts derived from PARP-1 +/+ and PARP-1 –/– lungs to hyperoxia for 64 h and tested their ability to escape hyperoxia-induced cell growth inhibition and to proliferate during air recovery after exposure to hyperoxia by measuring BrdU incorporation into duplicating DNA. PARP-1 +/+ and PARP-1 –/– presented the same percentage of BrdU-incorporating cells in air conditions (Fig. 8A). After hyperoxia exposure, both cell strains stopped to synthesize DNA, as shown by the lack of BrdU incorporation (Fig. 8A). During the first 24 h of air recovery, a small proportion of cells started again to incorporate BrdU, although the percentage of BrdU-positive PARP-1 –/– cells was higher than that of PARP-1 +/+ cells (Fig. 8A). This difference was more pronounced after 48 h (Fig. 8A). Similar results were obtained by measuring [3H]thymidine incorporation of cells during recovery. Indeed, although cell proliferation was low during the first 24 h of recovery in both cell types, [3H]thymidine incorporation was significantly higher in recovering PARP-1 –/– than in PARP-1 +/+ cells after 2–3 days of recovery (Fig. 8B). Interestingly, enhanced proliferative response of PARP-1 –/– fibroblasts after hyperoxia correlated with a lower level of active caspase-3 in total lysates of PARP-1 –/– cells. Indeed, hyperoxia induced similar cleavage of caspase-3 in PARP-1 +/+ and PARP-1 –/– fibroblasts, as shown by Western blotting (Fig. 9). However, after 1 day of air recovery, cleaved caspase-3 was downregulated in PARP-1 –/– cells, whereas it was still present in PARP-1 +/+ fibroblasts and was downregulated only after 3 days (Fig. 9). These experiments indicate that the absence of PARP-1 favors reentry of cells in the cell cycle during the recovery phase following hyperoxia-induced arrest and correlates with a decreased level of cleaved caspase-3.


Figure 8
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  		Fig. 8. Analysis of cell proliferation during recovery after hyperoxia exposure. A: BrdU incorporation of primary lung fibroblasts in air, hyperoxia (Hox 64h), and recovery (H+R 1d, H+R 2d) was measured using an anti-BrdU-FITC antibody and analyzed using flow cytometry. Fluorescence intensity is represented by histograms. One representative experiment of 3 is shown. B: the rate of [3H]thymidine incorporation of primary lung fibroblasts during recovery (H+R 1d, 2d, and 3d) was expressed as cpm (mean of quadruplicates ± SD). One representative experiment of 3 is shown. ***P < 0.001, PARP-1 +/+ vs. PARP-1 –/–.

 

Figure 9
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  		Fig. 9. Western blot analysis of cleaved caspase-3 was performed on total cell lysates (70 µg) of PARP-1 +/+ and PARP-1 –/– primary lung fibroblasts exposed for 64 h to hyperoxia (Hox) and allowed to recover in air for 1 (H+R 1d) to 3 days (H+R 3d). Anti-actin antibody was used for control of protein loading. Dex represents thymus extract (25 µg of protein) from a mouse treated with 250 µg of dexamethasone (intraperitoneally) and killed 6 h later, used as a positive control. One representative Western blot is shown.

 

   DISCUSSION
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 ABSTRACT
 MATERIAL AND METHODS
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 DISCUSSION
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 REFERENCES
 
PARP-1 has been defined as the guardian angel of the genome, since it participates in DNA repair after low levels of DNA damage and is involved in cell death when injury is too strong (12, 31). We formerly showed that acute hyperoxia (100% O2 for 72 h) induces strong PARP activity in vitro and in vivo. However, although in vitro epithelial cell necrosis was dependent on PARP activation, absence of PARP-1 or treatment with the PARP inhibitor 3-aminobenzamide (3-AB) was not sufficient to prevent acute hyperoxic injury in vivo (36). In the present study, we showed the essential role of PARP-1 in controlling alveolar cell repair after hyperoxia-induced genotoxic stress.

Breathing enriched oxygen concentration (>90%) induces strong oxidative stress leading to endothelial and epithelial lung cell damage, consequent alveolocapillary barrier disruption, edema, and animal lethality (21, 26). However, when exposed for a shorter time period (48–65 h), mice generally survive, showing increased cell proliferation of type II epithelial cells and fibroblasts and mild inflammation (26, 45, 48). Lung cell proliferation can be assessed by the analysis of BrdU incorporation into dividing DNA or detection of the replication-related molecules pCNA antigen and H3.2 histone. These molecules are induced early during air recovery after hyperoxia (48–72 h of recovery) and are downregulated at later time points (5–10 days of recovery) in mice and rat lungs and in purified type II epithelial cells from recovered animals (8, 26, 45). In the present study we confirmed that wild-type PARP-1 +/+ mice present low amounts of BrdU-positive cells after 5–10 days of air recovery from hyperoxia, indicating that signaling allowing cell repair and proliferation was already induced. However, the absence of PARP-1 promoted lung cell proliferation and hyperplasia, which was exacerbated and maintained after 5–10 days. Cells in the hyperplastic regions of PARP-1 –/– lungs were positive for anti-BrdU staining and were mostly type II epithelial cells and fibroblasts. Interestingly, previous observations on PARP-1 –/– mice older than 6 mo showed that these mice can be affected by extensive epidermal hyperplasia and cell hyperproliferation (53). Indeed, it has been proposed that following an environmental stress, the absence of PARP activity may influence the elimination of cells containing damaged DNA, which could escape growth control. Interestingly, treatment of wild-type mice with the potent PARP inhibitor PJ34 induced lung cell hyperproliferation similar to that in PARP-1 –/– mice, suggesting that excessive lung repair after acute hyperoxia exposure of PARP-1 –/– mice was specific to the absence of PARP activity. Our results add evidence for a PARP-1-dependent control of cell repair in vivo.

PARP-1 is known to exert DNA repair and maintain genomic stability following DNA damage by interacting with cell cycle-regulating molecules such as p53 and p21 (15, 49). p53 and p21 are induced by hyperoxia in vitro and in vivo and are mediators of G1 cell cycle arrest and repair (19, 34, 35). Interestingly, mice lacking p21 that were exposed to acute hyperoxia and subsequent air recovery presented excessive lung remodeling and inflammation similar to that of PARP-1 –/– animals (45). Moreover, inhibition of PARP-1 activity has been correlated with downregulation of p21 and mdm2 expression in vitro (49, 55). We therefore tested whether signaling via p21 was affected by the lack of PARP-1 in vivo. We confirmed that hyperoxia-mediated DNA damage induces the expression of p21 in total lungs (35, 45). However, p21 was similarly regulated during and after hyperoxia in PARP-1 +/+ and PARP-1 –/– lungs, suggesting that PARP-1-dependent cell repair function is not mediated by the p53/p21 signaling pathway in these conditions. Alternatively, PARP-1 may act downstream of p53/p21-mediated signal transduction in our experimental model.

Besides elevated tissue proliferation and hyperplasia, lung lesions in PARP-1 –/– mice were highly reduced at 21 days after recovery, indicating that hyperplasia was reversible and did not convert to fibrosis, with evident collagen deposition. Accordingly, secretion of the active form of TGF-beta1 molecule, which is one of the most important mediators of lung fibrosis (43), was similarly increased in PARP-1 +/+ and PARP-1 –/– mice during recovery. This result is in agreement with a recent publication showing that Bid –/– mice were protected from bleomycin-induced lung fibrosis, although they presented levels of active TGF-beta1 equivalent to those of wild-type mice (7, 27). Therefore, the increase in profibrotic cytokine secretion seems to be insufficient to trigger a fibrotic process. In summary, PARP-1 seems to control the early steps of cell repair after hyperoxia-induced injury and is not implicated in the profibrotic process.

Most of the pathophysiological models in which tissue injury is reduced by the absence or inhibition of PARP-1 activity are characterized by cell death and intense inflammatory response (asthma, LPS-induced acute lung injury, ischemia-reperfusion brain and heart damage) (6, 28, 29). In particular, inhibitors of PARP activity have been successfully used in vivo to downregulate signal transduction and inflammation in bleomycin-induced lung fibrosis or experimental colitis (17, 58). Indeed, PARP-1 has been shown to activate NF-{kappa}B transcription and therefore promote inflammation (37). Apparently in contrast to this literature, the lack of PARP-1 and treatment with a PARP inhibitor correlated, in our model, with excessive lung cell proliferation and inflammation in vivo. We postulate that in our experimental conditions, PARP-1 has rather a role of repair molecule, directly controlling cell proliferation. Increased pulmonary inflammation may be a secondary response to excessive proliferation of lung cells, namely, fibroblasts, and may be unrelated to PARP-1 proinflammatory function. Along this line, we observed that during air recovery after hyperoxia, NF-{kappa}B activity remained low and was similar in PARP-1 +/+ and PARP-1 –/– mouse lungs (not shown). Indeed, no induction, but rather a downregulation, of NF-{kappa}B activity has been observed in hyperoxia-exposed adult mice and has been correlated to protective effects (5, 56).

Lungs of PARP-1 –/– mice exposed to hyperoxia and recovery for 5 days presented a strong infiltration of CD45+, Mac-1+ myeloid cells, particularly neutrophils, as revealed by flow cytometry analysis of cell distribution and by the measure of MPO activity in lung tissue, and correlated with an increased number of neutrophils in BALFs. Accordingly, IL-6 was detected in PARP-1 –/– BALFs, whereas it was undetectable in PARP-1 +/+ BALFs. Interestingly, we also found higher signal for CXCL13/BLC and for IGFBP-5 in PARP-1 –/– BALFs. Interestingly, CXCL13, a chemokine normally implicated in lymphoid organogenesis, has been found to participate with IL-6 in pathogenesis of lymphoproliferative disorders of the lung (18). IGFBP-5 has been shown to be expressed in idiopathic pulmonary fibrosis lung tissues, and recent evidence suggests its implication in pathogenesis of lung fibrosis (57). The relevance of the differential production of these cytokines in PARP-1 –/– and wild-type mice deserves further investigation.

Lack of PARP activity also has been correlated with perturbations of cell cycle during exposure to sublethal DNA damage conditions, leading to absence of DNA synthesis early arrest (22) and escape of the G1 cell checkpoint (47). Our in vitro experiments showed that whereas in basal conditions, PARP-1 +/+ and PARP-1 –/– primary lung fibroblasts have similar rates of growth (not shown), PARP-1 –/– primary lung fibroblasts show a higher proliferative response, measured by BrdU and [3H]thymidine incorporation, during recovery after cell growth arrest in acute hyperoxia. The higher rate of PARP-1 –/– fibroblast proliferation during recovery correlated with less apoptosis in these conditions, as shown by the analysis of cleaved caspase-3, a hallmark of apoptosis signaling (30). In contrast, absence of PARP-1 did not affect cleaved caspase-3 expression during acute hyperoxia, confirming our previous data showing that the treatment with the PARP inhibitor 3-AB did not affect hyperoxia-induced cell apoptosis of primary lung fibroblasts (36). Therefore, PARP-1 seems to have a less important role in hyperoxia-induced cell death in vivo, but rather controls lung repair after hyperoxic stress (36).

It is known that exposure to oxygen induces cell cycle arrest at specific checkpoints to allow DNA damage repair (32). Checkpoints change according to the cell type and the presence or absence of specific cell cycle regulatory molecules (33, 40). Although the murine fibroblastic cell line (L929) arrested at the G2-M checkpoint when exposed to hyperoxia (not shown), we detected cell proliferation arrest (absence of BrdU or [3H]thymidine incorporation) but were unable to show cell cycle arrest at a precise checkpoint in lung primary fibroblasts (not shown). This is probably due to the intrinsic nature of primary cells that are not synchronized in their cell cycle as transformed cell lines.

In summary, our work reveals a crucial role for PARP-1 in regulating lung cell repair after hyperoxia-induced acute injury. These results also underline that lung cell proliferation after a primary insult can increase the inflammatory response, without initiating the fibrotic process, despite increased levels of TGF-beta1. This suggests that fibrosis is the result of dysregulation of multiple checkpoints, one of them being controlled by PARP-1.


   GRANTS
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 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work is supported by the Swiss National Research Foundation Grant 3100A0-109339, the Wölfermann-Naegele Foundation, and the Swiss Society of Pneumology.


   ACKNOWLEDGMENTS
 
We thank Claire Pitteloud, Coralie Reverdin, and Stephanie Carnesecchi for participation in the work; Zhao-Qi Wang for providing PARP-1 –/– mice; Giulio Gabbiani for helpful discussion; Christine Chaponnier, Serena Fineschi, and Fabio Carrozzino for providing reagents and antibodies; Philippe Henchoz for histological preparation; Sergei Startchik for setting up the MetaMorph quantification software; and Dominique Wohlevend for flow cytometry technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Pagano, Centre national de la recherche Scientifique-FRE2737, CISMET, Aix-Marseille Université, 27 bd. Jean Moulin, 13005 Marseille France (e-mail: alessandra.pagano{at}pharmacie.univ-mrs.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.


   REFERENCES
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Adamson IY, Bowden DH. The type 2 cell as progenitor of alveolar epithelial regeneration. A cytodynamic study in mice after exposure to oxygen. Lab Invest 30: 35–42, 1974.[ISI][Medline]
  2. Babior BM. Phagocytes and oxidative stress. Am J Med 109: 33–44, 2000.[CrossRef][ISI][Medline]
  3. Barazzone C, Donati YR, Rochat AF, Vesin C, Kan CD, Pache JC, Piguet PF. Keratinocyte growth factor protects alveolar epithelium and endothelium from oxygen-induced injury in mice. Am J Pathol 154: 1479–1487, 1999.[Abstract/Free Full Text]
  4. Barazzone C, 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.[Free Full Text]
  5. Barazzone-Argiroffo C, Pagano A, Juge C, Metrailler I, Rochat A, Vesin C, Donati Y. Glucocorticoids aggravate hyperoxia-induced lung injury through decreased nuclear factor-{kappa}B activity. Am J Physiol Lung Cell Mol Physiol 284: L197–L204, 2003.[Abstract/Free Full Text]
  6. Boulares AH, Zoltoski AJ, Sherif ZA, Jolly P, Massaro D, Smulson ME. Gene knockout or pharmacological inhibition of poly(ADP-ribose) polymerase-1 prevents lung inflammation in a murine model of asthma. Am J Respir Cell Mol Biol 28: 322–329, 2003.[Abstract/Free Full Text]
  7. Budinger GR, Mutlu GM, Eisenbart J, Fuller AC, Bellmeyer AA, Baker CM, Wilson M, Ridge K, Barrett TA, Lee VY, Chandel NS. Proapoptotic Bid is required for pulmonary fibrosis. Proc Natl Acad Sci USA 103: 4604–4609, 2006.[Abstract/Free Full Text]
  8. Bui KC, Buckley S, Wu F, Uhal B, Joshi I, Liu J, Hussain M, Makhoul I, Warburton D. Induction of A- and D-type cyclins and cdc2 kinase activity during recovery from short-term hyperoxic lung injury. Am J Physiol Lung Cell Mol Physiol 268: L625–L635, 1995.[Abstract/Free Full Text]
  9. Burkart V, Wang ZQ, Radons J, Heller B, Herceg Z, Stingl L, Wagner EF, Kolb H. Mice lacking the poly(ADP-ribose) polymerase gene are resistant to pancreatic beta-cell destruction and diabetes development induced by streptozocin. Nat Med 5: 314–319, 1999.[CrossRef][ISI][Medline]
  10. Cavarra E, Martorana PA, Bartalesi B, Fineschi S, Gambelli F, Lucattelli M, Ortiz L, Lungarella G. Genetic deficiency of {alpha}1-PI in mice influences lung responses to bleomycin. Eur Respir J 17: 474–480, 2001.[Abstract/Free Full Text]
  11. Chen X, Ko LJ, Jayaraman L, Prives C. p53 levels, functional domains, and DNA damage determine the extent of the apoptotic response of tumor cells. Genes Dev 10: 2438–2451, 1996.[Abstract/Free Full Text]
  12. Chiarugi A. Poly(ADP-ribose) polymerase: killer or conspirator? The 'suicide hypothesis' revisited. Trends Pharmacol Sci 23: 122–129, 2002.[CrossRef][Medline]
  13. Clement A, Edeas M, Chadelat K, Brody JS. Inhibition of lung epithelial cell proliferation by hyperoxia. Posttranscriptional regulation of proliferation-related genes. J Clin Invest 90: 1812–1818, 1992.[ISI][Medline]
  14. Coalson J. Animal models of CLD. In: Chronic Lung Disease in Early Infancy, edited by Blande RD and Coalson JJ. New York: Dekker, 2000.
  15. Frouin I, Maga G, Denegri M, Riva F, Savio M, Spadari S, Prosperi E, Scovassi AI. Human proliferating cell nuclear antigen, poly(ADP-ribose) polymerase-1, and p21waf1/cip1. A dynamic exchange of partners. J Biol Chem 278: 39265–39268, 2003.[Abstract/Free Full Text]
  16. Geissmann F, Jung S, Littman DR. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 19: 71–82, 2003.[CrossRef][ISI][Medline]
  17. Genovese T, Cuzzocrea S, Di Paola R, Mazzon E, Mastruzzo C, Catalano P, Sortino M, Crimi N, Caputi AP, Thiemermann C, Vancheri C. Effect of rosiglitazone and 15-deoxy-{Delta}12,14-prostaglandin J2 on bleomycin-induced lung injury. Eur Respir J 25: 225–234, 2005.[Abstract/Free Full Text]
  18. Goya S, Matsuoka H, Mori M, Morishita H, Kida H, Kobashi Y, Kato T, Taguchi Y, Osaki T, Tachibana I, Nishimoto N, Yoshizaki K, Kawase I, Hayashi S. Sustained interleukin-6 signalling leads to the development of lymphoid organ-like structures in the lung. J Pathol 200: 82–87, 2003.[CrossRef][ISI][Medline]
  19. Helt CE, Rancourt RC, Staversky RJ, O'Reilly MA. p53-dependent induction of p21Cip1/WAF1/Sdi1 protects against oxygen-induced toxicity. Toxicol Sci 63: 214–222, 2001.[Abstract/Free Full Text]
  20. Herceg Z, Wang ZQ. Functions of poly(ADP-ribose) polymerase (PARP) in DNA repair, genomic integrity and cell death. Mutat Res 477: 97–110, 2001.[ISI][Medline]
  21. Hokuto I, Perl AK, Whitsett JA. FGF signaling is required for pulmonary homeostasis following hyperoxia. Am J Physiol Lung Cell Mol Physiol 286: L580–L587, 2004.[Abstract/Free Full Text]
  22. Horton JK, Stefanick DF, Naron JM, Kedar PS, Wilson SH. Poly(ADP-ribose) polymerase activity prevents signaling pathways for cell cycle arrest after DNA methylating agent exposure. J Biol Chem 280: 15773–15785, 2005.[Abstract/Free Full Text]
  23. Imhof BA, Aurrand-Lions M. Adhesion mechanisms regulating the migration of monocytes. Nat Rev Immunol 4: 432–444, 2004.[CrossRef][ISI][Medline]
  24. Jagtap P, Soriano FG, Virag L, Liaudet L, Mabley J, Szabo E, Hasko G, Marton A, Lorigados CB, Gallyas F Jr, Sumegi B, Hoyt DG, Baloglu E, VanDuzer J, Salzman AL, Southan GJ, Szabo C. Novel phenanthridinone inhibitors of poly (adenosine 5'-diphosphate-ribose) synthetase: potent cytoprotective and antishock agents. Crit Care Med 30: 1071–1082, 2002.[CrossRef][ISI][Medline]
  25. Lamagna C, Hodivala-Dilke KM, Imhof BA, Aurrand-Lions M. Antibody against junctional adhesion molecule-C inhibits angiogenesis and tumor growth. Cancer Res 65: 5703–5710, 2005.[Abstract/Free Full Text]
  26. Lee J, Reddy R, Barsky L, Weinberg K, Driscoll B. Contribution of proliferation and DNA damage repair to alveolar epithelial type 2 cell recovery from hyperoxia. Am J Physiol Lung Cell Mol Physiol 290: L685–L694, 2006.[Abstract/Free Full Text]
  27. Lee VY, Schroedl C, Brunelle JK, Buccellato LJ, Akinci OI, Kaneto H, Snyder C, Eisenbart J, Budinger GR, Chandel NS. Bleomycin induces alveolar epithelial cell death through JNK-dependent activation of the mitochondrial death pathway. Am J Physiol Lung Cell Mol Physiol 289: L521–L528, 2005.[Abstract/Free Full Text]
  28. Liaudet L, Pacher P, Mabley JG, Virag L, Soriano FG, Hask G, Szab C. Activation of poly(ADP-ribose) polymerase-1 is a central mechanism of lipopolysaccharide-induced acute lung inflammation. Am J Respir Crit Care Med 165: 372–377, 2002.[Abstract/Free Full Text]
  29. Liaudet L, Szabo E, Timashpolsky L, Virag L, Cziraki A, Szabo C. Suppression of poly (ADP-ribose) polymerase activation by 3-aminobenzamide in a rat model of myocardial infarction: long-term morphological and functional consequences. Br J Pharmacol 133: 1424–1430, 2001.[CrossRef][ISI][Medline]
  30. Nagata S. Apoptotic DNA fragmentation. Exp Cell Res 256: 12–18, 2000.[CrossRef][ISI][Medline]
  31. Nicoletti VG, Stella AM. Role of PARP under stress conditions: cell death or protection? Neurochem Res 28: 187–194, 2003.[CrossRef][ISI][Medline]
  32. O'Reilly MA. DNA damage and cell cycle checkpoints in hyperoxic lung injury: braking to facilitate repair. Am J Physiol Lung Cell Mol Physiol 281: L291–L305, 2001.[Abstract/Free Full Text]
  33. O'Reilly MA, Staversky RJ, Finkelstein JN, Keng PC. Activation of the G2 cell cycle checkpoint enhances survival of epithelial cells exposed to hyperoxia. Am J Physiol Lung Cell Mol Physiol 284: L368–L375, 2003.[Abstract/Free Full Text]
  34. O'Reilly MA, Staversky RJ, Stripp BR, Finkelstein JN. Exposure to hyperoxia induces p53 expression in mouse lung epithelium. Am J Respir Cell Mol Biol 18: 43–50, 1998.[Abstract/Free Full Text]
  35. O'Reilly MA, Staversky RJ, Watkins RH, Maniscalco WM. Accumulation of p21Cip1/WAF1 during hyperoxic lung injury in mice. Am J Respir Cell Mol Biol 19: 777–785, 1998.[Abstract/Free Full Text]
  36. Pagano A, Pitteloud C, Reverdin C, Metrailler-Ruchonnet I, Donati Y, Barazzone Argiroffo C. Poly(ADP-ribose)polymerase activation mediates lung epithelial cell death in vitro but is not essential in hyperoxia-induced lung injury. Am J Respir Cell Mol Biol 33: 555–564, 2005.[Abstract/Free Full Text]
  37. Petrilli V, Herceg Z, Hassa PO, Patel NS, Di Paola R, Cortes U, Dugo L, Filipe HM, Thiemermann C, Hottiger MO, Cuzzocrea S, Wang ZQ. Noncleavable poly(ADP-ribose) polymerase-1 regulates the inflammation response in mice. J Clin Invest 114: 1072–1081, 2004.[CrossRef][ISI][Medline]
  38. Phan SH. The myofibroblast in pulmonary fibrosis. Chest 122: 286S–289S, 2002.[CrossRef][ISI][Medline]
  39. Pieper AA, Verma A, Zhang J, Snyder SH. Poly(ADP-ribose) polymerase, nitric oxide and cell death. Trends Pharmacol Sci 20: 171–181, 1999.[CrossRef][Medline]
  40. Rancourt RC, Hayes DD, Chess PR, Keng PC, O'Reilly MA. Growth arrest in G1 protects against oxygen-induced DNA damage and cell death. J Cell Physiol 193: 26–36, 2002.[CrossRef][ISI][Medline]
  41. Ribeiro Carvalho C, de Paula Pinto Schettino G, Maranhão B, Pamplona Bethlem E. Hyperoxia and lung disease. Curr Op Pulm Med 4: 300–304, 1998.[CrossRef]
  42. Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 73: 39–85, 2004.[CrossRef][ISI][Medline]
  43. Sheppard D. Transforming growth factor beta: a central modulator of pulmonary and airway inflammation and fibrosis. Proc Am Thorac Soc 3: 413–417, 2006.[Abstract/Free Full Text]
  44. Soldani C, Scovassi AI. Poly(ADP-ribose) polymerase-1 cleavage during apoptosis: an update. Apoptosis 7: 321–328, 2002.[CrossRef][ISI][Medline]
  45. Staversky RJ, Watkins RH, Wright TW, Hernady E, LoMonaco MB, D'Angio CT, Williams JP, Maniscalco WM, O'Reilly MA. Normal remodeling of the oxygen-injured lung requires the cyclin-dependent kinase inhibitor p21Cip1/WAF1/Sdi1. Am J Pathol 161: 1383–1393, 2002.[Abstract/Free Full Text]
  46. Tong WM, Hande MP, Lansdorp PM, Wang ZQ. DNA strand break-sensing molecule poly(ADP-Ribose) polymerase cooperates with p53 in telomere function, chromosome stability, and tumor suppression. Mol Cell Biol 21: 4046–4054, 2001.[Abstract/Free Full Text]
  47. Trucco C, Oliver FJ, de Murcia G, Menissier-de Murcia J. DNA repair defect in poly(ADP-ribose) polymerase-deficient cell lines. Nucleic Acids Res 26: 2644–2649, 1998.[Abstract/Free Full Text]
  48. Tryka AF, Witschi H, Gosslee DG, McArthur AH, Clapp NK. Patterns of cell proliferation during recovery from oxygen injury. Species differences. Am Rev Respir Dis 133: 1055–1059, 1986.[ISI][Medline]
  49. Vaziri H, West MD, Allsopp RC, Davison TS, Wu YS, Arrowsmith CH, Poirier GG, Benchimol S. ATM-dependent telomere loss in aging human diploid fibroblasts and DNA damage lead to the post-translational activation of p53 protein involving poly(ADP-ribose) polymerase. EMBO J 16: 6018–6033, 1997.[CrossRef][ISI][Medline]
  50. Virag L, Bai P, Bak I, Pacher P, Mabley JG, Liaudet L, Bakondi E, Gergely P, Kollai M, Szabo C. Effects of poly(ADP-ribose) polymerase inhibition on inflammatory cell migration in a murine model of asthma. Med Sci Monit 10: BR77–BR83, 2004.[ISI][Medline]
  51. Virag L, Szabo C. The therapeutic potential of poly(ADP-ribose) polymerase inhibitors. Pharmacol Rev 54: 375–429, 2002.[Abstract/Free Full Text]
  52. Vyalov SL, Gabbiani G, Kapanci Y. Rat alveolar myofibroblasts acquire {alpha}-smooth muscle actin expression during bleomycin-induced pulmonary fibrosis. Am J Pathol 143: 1754–1765, 1993.[Abstract]
  53. Wang ZQ, Auer B, Stingl L, Berghammer H, Haidacher D, Schweiger M, Wagner EF. Mice lacking ADPRT and poly(ADP-ribosyl)ation develop normally but are susceptible to skin disease. Genes Dev 9: 509–520, 1995.[Abstract/Free Full Text]
  54. Wang ZQ, Stingl L, Morrison C, Jantsch M, Los M, Schulze-Osthoff K, Wagner EF. PARP is important for genomic stability but dispensable in apoptosis. Genes Dev 11: 2347–2358, 1997.[Abstract/Free Full Text]
  55. Wieler S, Gagne JP, Vaziri H, Poirier GG, Benchimol S. Poly(ADP-ribose) polymerase-1 is a positive regulator of the p53-mediated G1 arrest response following ionizing radiation. J Biol Chem 278: 18914–18921, 2003.[Abstract/Free Full Text]
  56. Yang G, Abate A, George AG, Weng YH, Dennery PA. Maturational differences in lung NF-{kappa}B activation and their role in tolerance to hyperoxia. J Clin Invest 114: 669–678, 2004.[CrossRef][ISI][Medline]
  57. Yasuoka H, Zhou Z, Pilewski JM, Oury TD, Choi AM, Feghali-Bostwick CA. Insulin-like growth factor-binding protein-5 induces pulmonary fibrosis and triggers mononuclear cellular infiltration. Am J Pathol 169: 1633–1642, 2006.[Abstract/Free Full Text]
  58. Zingarelli B, O'Connor M, Hake PW. Inhibitors of poly (ADP-ribose) polymerase modulate signal transduction pathways in colitis. Eur J Pharmacol 469: 183–194, 2003.[CrossRef][ISI][Medline]



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