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INFLAMMATION AND IMMUNOPHARMACOLOGY

Inhibitors of Poly(ADP-Ribose) Polymerase Modulate Signal Transduction Pathways and the Development of Bleomycin-Induced Lung Injury

Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina Torre Biologica, Policlinico Universitario, Messina, Italy (T.G., E.M., R.D., A.P.C., S.C.); Department of Pharmacy & Pharmacology, University of Bath, Bath, United Kingdom (M.D.T.); and Centre for Experimental Medicine, Nephrology & Critical Care, William Harvey Research Institute, Queen Mary–University of London, London, United Kingdom (C.T.)

Received November 15, 2004; accepted January 7, 2005.

	Abstract

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Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA, plays an important role in the tissue injury associated with inflammation. The aim of our study was to evaluate the therapeutic efficacy of in vivo inhibition of PARP in an experimental model of lung injury caused by bleomycin administration. Mice subjected to intratracheal administration of bleomycin developed significant lung injury and apoptosis (measured by Annexin V coloration). An increase of immunoreactivity to nitrotyrosine and PARP, as well as a significant loss of body weight and mortality, was observed in the lung of bleomycin-treated mice. Administration of the two PARP inhibitors 3-aminobenzamide (3-AB) or 5-aminoisoquinolinone (5-AIQ) significantly reduced the 1) loss of body weight, 2) mortality rate, 3) infiltration of the lung with polymorphonuclear neutrophils (myeloperoxidase activity), 4) edema formation, and 5) histological evidence of lung injury. Administration of 3-AB and 5-AIQ also markedly reduced nitrotyrosine formation and PARP activation. These results demonstrate that treatment with PARP inhibitors reduces the development of inflammation and tissue injury events induced by bleomycin administration in the mice.

The production of reactive oxygen species (ROS) and peroxynitrite contributes to the tissue injury observed during lung fibrosis. ROS and peroxynitrite also cause DNA damage, which results in the activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) (Szabó and Dawson, 1998). Therefore, it has been recently demonstrated that bleomycin (BLEO) administration induced PARP activation in the lung tissues (Genovese et al., 2005). PARP is a ubiquitous, chromatin-bound enzyme that is abundantly present in the nuclei of numerous cell types (Szabó and Dawson, 1998). Continuous or excessive activation of PARP produces a depletion of NAD⁺, which subsequently leads to cellular dysfunction and, ultimately, cell death (Chiarugi, 2002). Chemically distinct inhibitors of PARP activity such as benzamides [e.g., 3-aminobenzamide (3-AB) and nicotinamide] and isoquinolinones can reduce the degree of inflammation, and these investigations have provided the basis for potential clinical applications of PARP inhibitors (Southan and Szabó, 2003).

Therefore, various studies have demonstrated that the chemically distinct PARP inhibitors GPI6150, PJ34, and 3-AB can attenuate PARP activation and provide beneficial actions in vivo during inflammation (Cuzzocrea et al., 2002b). However, in contrast, isoquinolinone derivatives 3-AB and nicotinamide are weak inhibitors of PARP activity that do not readily cross cell membranes (Szabó and Dawson, 1998). Furthermore, although the potency of recently developed PARP inhibitors has improved greatly, most lack good solubility in water, making it difficult to find a biocompatible vehicle for utilization in vivo. Thus, there is still a great need for the development of potent, water-soluble inhibitors of PARP activity. Much effort has been made to develop new PARP inhibitors with better potency, selectivity, and water solubility, and there are now 13 chemical classes of PARP inhibitors (Southan and Szabó, 2003). Twelve years ago, Suto et al. (1991) used a cell-free preparation of PARP (purified 900-fold from calf thymus) to demonstrate that 5-aminoisoquinolinone [5-aminoisoquinolin-1(2H)-one] (5-AIQ) is a water-soluble inhibitor of PARP activity. Because previously published reports of the synthesis of 5-AIQ reported problems of low yield and unreliability (Suto et al., 1991), McDonald et al. (2000) have recently developed a novel and more efficient method for the synthesis of 5-AIQ.

This method leads to a higher yield of 5-AIQ than that previously reported (Wenkert et al., 1964). Suto et al. (1991) and Watson et al. (1998) reported an IC₅₀ of 240 nM when 5-AIQ was evaluated in an in vitro cell-free system consisting of PARP isolated from calf thymus, which is broadly comparable with other potent 5-substituted isoquinolinones. Since 5-AIQ is an analog of the nicotinamide moiety of NAD⁺, it is conceivable that it may also inhibit other ADP-ribosyl transferases. We have previously examined the effect of 5-AIQ on the mono-ADP-ribosylating activity of diphtheria toxin and found that it had an IC₅₀ of approximately 250 µmol/l, indicating a 1000-fold higher selectivity for PARP (McDonald et al., 2000). In addition, when compared with the benzamides 3-AB and nicotinamide and the isoquinolinone 1,5-DHIQ, 5-AIQ is the most potent (water-soluble) inhibitor of PARP we have examined to date (Chatterjee et al., 1999, 2000). Furthermore, we have also previously demonstrated that 5-AIQ can reduce ischemia/reperfusion injury of the heart, intestine, and liver (Mota-Filipe et al., 2002), and 5-AIQ has been shown to provide beneficial effects in rodent models of heart transplantation (Szabó et al., 2002), acute lung injury (Cuzzocrea et al., 2002b), and spinal cord injury (Genovese et al., 2004). Consistent with these findings, the objective of the present study was to investigate the biological effects of pharmacological inhibition of PARP in a mouse model of lung fibrosis with two structurally unrelated inhibitors of PARP, 3-AB as reference inhibitor and 5-AIQ as new synthetic inhibitor, being tested.

	Materials and Methods

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Animals. Adult male CD1 mice (25–30 g; Harlan Nossan, Milan, Italy) were housed in a controlled environment and provided with standard rodent chow and water. Animal care complied with Italian regulations on protection of animals used for experimental and other scientific purposes (D.M. 116192), as well as with the European Economic Community regulations (O.J. of E.C. L 358/1 12/18/1986).

Experimental Groups. Mice were randomly allocated into the following groups: 1) BLEO + vehicle group: mice were subjected to bleomycin-induced lung injury and received the administration of saline (n = 30); 2) 3-AB group: same as the BLEO + vehicle group, but 3-AB at a dose of 10 mg/kg was administered (i.p.) bolus every 24 h starting from day 1 (n = 30); 3) 5-AIQ group: same as the BLEO + vehicle group, but 5-AIQ at a dose of 3 mg/kg was administered (i.p.) bolus every 24 h starting from day 1 (n = 30); 4) sham + saline group: sham-operated group in which identical surgical procedures to the BLEO group was performed, except that the saline was administered instead of bleomycin (n = 30); 5) sham + 3-AB group: identical to sham + saline group except for the administration of 3-AB (i.p.) bolus every 24 h starting from day 1 (n = 30); and 6) sham + 5-AIQ group: identical to sham + saline group except for the administration of 5-AIQ (i.p.) bolus every 24 h starting from day 1.

The dosage of PARP inhibitors regimen has been previously shown to exert anti-inflammatory effects. In particular, we have previously reported that the dose of 5-AIQ reduces the tissue injury caused by ischemia-reperfusion in the liver (dose-response curve study) (Mota-Filipe et al., 2002), as well as lung injury (Cuzzocrea et al., 2002b), and that the dose of 3-AB reduces acute lung inflammation (dose-response curve study) (Cuzzocrea et al., 1998).

Induction of Lung Injury by Bleomycin. Mice received a single intratracheal instillation of 0.9% saline or saline containing bleomycin sulfate (body weight, 1 mg/kg) in a volume of 50 µl and were killed after 15 days by pentobarbitone overdose.

Measurement of Fluid Content in Lung. The wet lung weight was measured after careful excision of extraneous tissues. The lung was exposed for 48 h at 180°C, and the dry weight was measured. Water content was calculated by subtracting dry weight from wet weight.

Histological Examination. Lung biopsies were taken 15 days after injection of bleomycin. Lung biopsies were fixed for 1 week in 10% (w/v) PBS-buffered formaldehyde solution at room temperature, dehydrated using graded ethanol, and embedded in Paraplast (Sherwood Medical, Mahwah, NJ). After embedding in paraffin, the sections were prepared and stained by trichrome stain. All sections were studied using Dialux 22 light microscopy (Leitz, Wetzlar, Germany). The severity of fibrosis was semiquantitatively assessed according to the method proposed by Ashcroft et al. (1988). Briefly, the grade of lung fibrosis was scored on a scale from 0 to 8 by examining section randomly chosen fields per sample at a magnification of 100x. Criteria for grading lung fibrosis were as follows: grade 0, normal lung; grade 1, minimal fibrous thickening of alveolar or bronchiolar walls; grade 3, moderate thickening of walls without obvious damage to lung architecture; grade 5, increased fibrosis with definite damage to lung structure and formation of fibrous bands or small fibrous masses; grade 7, severe distortion of structure and large fibrous areas; and grade 8, total fibrous obliteration of fields.

Immunohistochemical Localization of Nitrotyrosine and PARP. Tyrosine nitration, an index of the nitrosylation of proteins by peroxynitrite and/or ROS, was determined by immunohistochemistry as previously described (Cuzzocrea et al., 2003). At the end of the experiment, the tissues were fixed in 10% (w/v) PBS-buffered formaldehyde, and 8-µm sections were prepared from paraffin-embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60% (v/v) methanol for 30 min. The sections were permeablized with 0.1% (w/v) Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin (DBA, Milan, Italy), respectively. Sections were incubated overnight with anti-nitrotyrosine polyclonal antibody (1:500 in PBS, v/v) or with anti-poly (ADP-ribose) goat polyclonal antibody (1:500 in PBS, v/v). Sections were washed with PBS and incubated with secondary antibody. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex (DBA). To confirm that the immunoreactions for the nitrotyrosine were specific, some sections were also incubated with the primary antibody (anti-nitrotyrosine) in the presence of excess nitrotyrosine (10 mM) to verify the binding specificity. To verify the binding specificity for PAR, some sections were also incubated with only the primary antibody (no secondary) or with only the secondary antibody (no primary). In these situations, no positive staining was found in the sections, indicating that the immunoreactions were positive in all the experiments carried out.

Myeloperoxidase Activity. Myeloperoxidase (MPO) activity, an indicator of polymorphonuclear leukocyte (PMN) accumulation, was determined as previously described (Mullane et al., 1985). At the specified time following injection of bleomycin, lung tissues were obtained and weighed, and each piece was homogenized in a solution containing 0.5% (w/v) hexadecyltrimethyl-ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20,000g at 4°C. An aliquot of the supernatant was then allowed to react with a solution of 1.6 mM tetramethylbenzidine and 0.1 mM hydrogen peroxide. The rate of change in absorbance was measured spectrophotometrically at 650 nm. MPO activity was defined as the quantity of enzyme degrading 1 µmol of peroxide per minute at 37°C and was expressed in milliunits per grams of wet tissue.

Measurement of Cytokines. Portions of lung, collected at 15 days after bleomycin administration, were homogenized in PBS containing 2 mM phenyl-methyl sulfonyl fluoride (Sigma Chemical, Poole, Dorset, UK) as previously described (Diaz-Granados et al., 2000), and tissue levels of TNF- and IL-1 were evaluated. The assay was carried out by using a commercial colorimetric kit (Calbiochem, San Diego, CA), according to the manufacturer's instructions. All cytokine determinations were performed in duplicate serial dilutions.

Annexin V Evaluation. The binding of annexin V-fluorescein isothiocyanate to externalized phosphatidylserine was used is a measurement of apoptotic in lung tissue section with an annexin V-fluorescein isothiocyanate propidium iodide (PI) apoptosis detection kit according to the manufacturer's instructions. Briefly, normal viable cells in culture will stain negative for Annexin V FITC and PI. Cells that are induced to undergo apoptosis will stain positive for Annexin V FITC and negative for PI as early as 1 h after stimulation (Schutte et al., 1998). Annexin V binding assay was used as a tool to measure apoptosis in differentiated neuronal cells. Both cells in later stages of apoptosis and necrotic cells will stain positive for Annexin V FITC and PI. Sections were washed as before, mounted with 90% glycerol in PBS, and observed with a LSM 510 Zeiss laser confocal microscope (Carl Zeiss GmbH, Jena, Germany) equipped with a 40x oil objective.

Preparation of Whole Extracts. All the extraction procedures were performed on ice using ice-cold reagents. Tissues from each mouse were suspended in 6 ml of a high-salt extraction buffer (20 mM pH 7.9 HEPES, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 25% glycerol, 0.5 mM phenylmethylsulphonylfluoride, 1.5 µg/ml soybean trypsin inhibitor, 7 µg/ml pepstatin A, 5 µg/ml leupeptin, 0.1 mM benzamidine, and 0.5 mM dithiothreitol) and homogenized at the highest setting for 2 min in a Polytron PT 3000 tissue homogenizer. The homogenates were chilled on ice for 15 min and then vigorously shaken for a few minutes in the presence of 20 µl of 10% Nonidet P-40. After centrifugation at 13,000g at 4°C for 5 min, the protein concentration in the supernatant was determined by the Bio-Rad (Bio-Rad, Hercules, CA) protein assay kit, and then it was aliquoted and stored at –80°C.

Electrophoretic Mobility-Shift Assay (EMSA). Double-stranded oligonucleotides containing the NF-B recognition sequence (5'-GAT CGA GGG GAC TTT CCC TAG-3') were end-labeled with -[³²P]ATP (MP Biomedicals, Irvine, CA). Aliquots of whole extracts collected 15 days after bleomycin administration (20 µg of protein for each sample) were incubated for 30 min with radiolabeled oligonucleotides (2.5–5.0 x 10⁴ cpm) in 20 µl of reaction buffer containing 2 µg of poly(dI-dC), 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM ethylenediaminotetraacetic acid, 1 mM DL-dithiothreitol, 1 mg/ml bovine serum albumin, and 10% glycerol. The specificity of the DNA/protein binding was determined for NF-B by competition reaction, in which a 50-fold molar excess of unlabeled wild-type, mutant, or Sp-1 oligonucleotide was added to the binding reaction 10 min before the addition of radiolabeled probe. Protein-nucleic acid complexes were resolved by electrophoresis on 4% nondenaturing polyacrylamide gel in 0.5% Tris-borate ethylenediaminotetraacetic acid buffer at 150 V for 2 h at 4°C. The gel was dried and autoradiographed with an intensifying screen at –80°C for 20 h. The relative bands were subsequently quantified by densitometric scanning of the X-ray films with a GS-700 Imaging Densitometer (Bio-Rad) and Molecular Analyst computer program (IBM, White Plains, NY). The time of 15 days after bleomicyn administration was chosen in agreement with other studies (Ortiz et al., 2002).

Western Blot Analysis for IB-. The levels of IB- were quantified in whole extracts 14 days after bleomycin administration, by immunoprecipitation followed by Western blot analysis, according to the manufacturer's instructions (Celbio, Milan, Italy). Briefly, proteins were then transferred onto nitrocellulose membranes, according to the manufacturer's instructions. Briefly, the membranes were saturated by incubation at 4°C overnight with 10% (w/v) nonfat dry milk in PBS and then incubated with anti-IB- (1:1000) for 1 h at room temperature. Membranes were washed three times with 1% (w/v) Triton X-100 in PBS and then incubated with anti-rabbit immunoglobulins coupled to peroxidase (1:1000). The immune complexes were visualized using the enhanced chemiluminescence method (Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK). The relative expressions of the proteins were subsequently quantified by densitometric scanning of the X-ray films with a GS-700 Imaging Densitometer (Bio-Rad) and Molecular Analyst computer program (IBM).

Materials. Unless otherwise stated, all compounds were obtained from Sigma Chemical. All other chemicals were of the highest commercial grade available. All stock solutions were prepared in a 0.9% NaCl nonpyrogenic saline (Baxter, Newbury, Berkshire, UK).

Statistical Evaluation. All values in the figures and text are expressed as mean ± S.E.M. of n observations. For the in vivo studies, n represents the number of animals studied. In the experiments involving histology or immunohistochemistry, the figures shown represent at least three experiments performed on different experimental days. Data sets were examined by one- or two-way analysis of variance, and individual group means were then compared with Student's unpaired t test. A p value of less than 0.05 was considered significant.

	Results

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Effect of PARP Inhibition on the Degree of Bleomycin-Induced Lung Injury. Histological examination of lung sections revealed significant tissue damage (Fig. 1, A and D). Thus, when compared with lung sections taken from saline-treated animals (data not shown), histological examination of lung sections of mice treated with bleomycin were characterized by extensive inflammatory infiltration by neutrophils, lymphocyte, and plasma cells extending through the lung epithelial, fibrosis, and granulomas in the perivascular region (Fig. 1, A and D). Administration of 3-AB or 5-AIQ significantly prevented lung inflammation induced by bleomycin administration (Fig. 1, B and C, respectively; see Fig. 1D for histological score). Furthermore, the injection of bleomycin elicited an inflammatory response characterized by the accumulation of water in lung as an indicator of fluid content (Fig. 2) and neutrophil infiltration in the lung tissues (Fig. 3). The treatment with the two PARP inhibitors significantly reduced the fluid content and the neutrophil infiltration (Figs. 2 and 3).

View larger version (127K): [in this window] [in a new window]   Fig. 1. Effect of 3-AB and 5-AIQ on lung injury and histological score. Lung injury was produced after bleomycin administration and characterized by inflammatory cell infiltration, fibrosis, as well as extensive areas of collagen (A). The treatment with 3-AB (B) or 5-AIQ (C) significantly reduced the extent and severity of the histological signs of colon injury. The histological score (D) was made by two independent observers. Figure is representative of at least three experiments performed on different experimental days. Data are means ± S.E.M. of 30 mice for each group. *, p < 0.01 versus sham; , p < 0.01 versus bleomycin.

View larger version (14K): [in this window] [in a new window]   Fig. 2. The injection of bleomycin elicited an inflammatory response characterized by the accumulation of water in lung as an indicator of edema. The treatment with the two PARP inhibitors 3-AB or 5-AIQ significantly reduced the edema formation. Data are means ± S.E.M. from 10 mice for each group. *, p < 0.01 versus sham; , p < 0.01 versus bleomycin.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Effect of 3-AB and 5-AIQ on myeloperoxidase activity in the lung. MPO activity in the lungs of bleomycin-treated mice was significantly increased in comparison to sham-operated mice. 3-AB and 5-AIQ treatment reduced the bleomycin-induced increase in MPO activity. Data are means ± S.E.M. from 10 mice for each group. *, p < 0.01 versus sham; , p < 0.01 versus bleomycin.

Effects of PARP Inhibition on Changes of Body Weight and Survival Rate. In vehicle-treated mice, the severe lung injury caused by bleomycin administration was associated with a significant loss in body weight (Fig. 4). The treatment with the two PARP inhibitors 5-AIQ or 3-AB significantly reduced the loss in body weight (Fig. 4). The survival of animals was monitored for 15 days. Bleomycin-treated mice, which had received vehicle, developed severe lung injury, and 50% of these animals died within 15 days after bleomycin administration (Fig. 5). In contrast, none of the mice that had been treated with the two PARP inhibitors died (Fig. 5).

View larger version (15K): [in this window] [in a new window]   Fig. 4. Effect of 3-AB and 5-AIQ treatment on body weight. Body weight was recorded immediately before bleomycin administration and daily for the entire experimental period. 3-AB and 5-AIQ treatment significantly prevented the loss of body weight. Data are means ± S.E.M. from 30 mice for each group. *, p < 0.01 versus bleomycin.

View larger version (14K): [in this window] [in a new window]   Fig. 5. Effect of 3-AB and 5-AIQ treatment on bleomycin-induced mortality. Survival is significantly improved in 3-AB- and 5-AIQ-treated mice in comparison to the high mortality rate of the bleomycin-treated mice. Data are means ± S.E.M. from 30 mice for each group. *, p < 0.01 versus bleomycin.

Effect of PARP Inhibition on the Production of TNF- and IL-1 after Bleomycin Administration. To test whether PARP activation may modulate the inflammatory process through the regulation of the secretion of others cytokines, we analyzed the lung levels of proinflammatory cytokines TNF- and IL-1 15 days after bleomycin administration. A substantial increase of TNF- and IL-1 formation was found in lung samples collected from bleomycin-treated mice (Fig. 6). Lung levels of TNF- and IL-1 were significantly reduced in bleomycin-treated mice that had been treated with the two PARP inhibitors (Fig. 6).

View larger version (14K): [in this window] [in a new window]   Fig. 6. Lung TNF- (A) and IL-1 (B) production at 15 days after bleomycin administration. A significant production of cytokines was observed in the lung tissues at 15 days after bleomycin administration. Cytokine levels were significantly reduced in 3-AB and 5-AIQ-treated mice. Data are means ± S.E.M. of 10 mice for each group. *, p < 0.01 versus sham. , p < 0.01 versus bleomycin.

Effects of PARP Inhibition on Bleomycin-Induced Nitrotyrosine Formation and PARP Activation. To determine the localization of "peroxynitrite formation" and/or other nitrogen derivatives produced during colitis, nitrotyrosine, a specific marker of nitrosative stress, was measured by immunohistochemical analysis in the lung. Immunohistochemical analysis of lung sections obtained from mice treated with bleomycin revealed a positive staining for nitrotyrosine manly localized in nuclei of inflammatory cells (Figs. 7A and 9). In contrast, no positive staining for nitrotyrosine was found in the lungs of bleomycin-treated mice that had been treated with 3-AB (Figs. 7B and 9) or with 5-AIQ (Figs. 7C and 9). Immunohistochemical analysis of lung sections obtained from mice treated with bleomycin also revealed a positive staining for PAR manly localized in plasma cell and lymphocytes (Figs. 8A and 9). In contrast, no staining for PAR was found in the lungs of bleomycin-treated mice treated with 3-AB (Figs. 8B and 9) or with 5-AIQ (Figs. 8C and 9). There was no staining for either nitrotyrosine or PAR in lungs obtained from the sham group of mice (data not shown).

View larger version (133K): [in this window] [in a new window]   Fig. 7. Immunohistochemical localization of nitrotyrosine in the lung. After bleomycin injection, positive staining for nitrotyrosine (A) was localized mainly in nuclei of inflammatory cells. There was a marked reduction in the immunostaining in the lungs of bleomycin-treated mice treated with 3-AB (B) and 5-AIQ (C). This figure is representative of at least three experiments performed on different experimental days.

View larger version (17K): [in this window] [in a new window]   Fig. 9. Typical densitometry evaluation. Densitometry analysis of immunocytochemistry photographs (n = 5 photos from each sample collected from all mice in each experimental group) for nitrotyrosine and PAR from lung tissues was assessed. The assay was carried out by using Optilab Graftek software on a Macintosh personal computer (CPU G3–266). Data are expressed as a percentage of total tissue area. N.D., not detectable. *, p < 0.01 versus sham; , p < 0.01 versus bleomycin.

View larger version (129K): [in this window] [in a new window]   Fig. 8. Immunohistochemical localization of PAR in the lung. After bleomycin injection, positive staining for PAR (A) was localized mainly in plasma cell and lymphocytes. There was a marked reduction in the immunostaining in the lungs of bleomycin-treated mice treated with 3-AB (B) and 5-AIQ (C). This figure is representative of at least three experiments performed on different experimental days.

Effect of PARP Inhibition on Apoptosis in Lung Tissue after Bleomycin Administration. To test whether the tissue damage was associated with cell death by apoptosis, we measured Annexin V staining in the lung from bleomycin-treated mice 15 days after bleomycin administration. Almost no apoptotic cells were detectable in the lung tissue of sham-treated mice (data not shown). Fifteen days after bleomycin administration, lung tissues obtained from vehicle-treated mice demonstrated a marked appearance of positive staining to the propidium iodide (Fig. 10A) index of cells in the late stage of apoptosis, as well as some positive staining for the Annexin V FITC index of cells that were induced to undergo apoptosis (Fig. 10B). On the contrary, lung tissue sections from bleomycin-treated mice that had been treated with 3-AB (Fig. 10D) or 5-AIQ (Fig. 10G) demonstrated significantly less presence of cells in the later stages of apoptosis (positive to propidium iodide), and almost no positive staining necrotic for Annexin V FITC was observed (Fig. 10, E and H, respectively).

View larger version (33K): [in this window] [in a new window]   Fig. 10. Effect of PARP inhibitors on apoptosis. Fifteen days after bleomycin administration, lung tissues obtained from vehicle-treated mice demonstrated a marked presence of cells in the later stages of apoptosis (A, positive to propidium iodide) and some positive staining for Annexin V FITC (B, index of cells that are induced to undergo apoptosis). On the contrary, lung tissues section from bleomycin-treated mice, which had been treated with 3-AB (D) or with 5-AIQ (G), demonstrated significantly less presence of cells in the later stages of apoptosis (positive to propidium iodide) and almost no positive staining necrotic for Annexin V FITC (E and H, respectively). C, F, and I, staining combination of A and B, D and E, and G and H, respectively. Figure is representative of at least three experiments performed on different experimental days.

Effect of PARP Inhibitors on IB- Degradation and NF-B Translocation. To investigate the cellular mechanisms by which treatment with 3-AB or 5-AIQ may attenuate bleomycin-induced lung injury, we evaluated IB- degradation and NF-B translocation, one of the major transcription factors involved in the signal transduction of inflammation (Genovese et al., 2004). The appearance of IB- in homogenates of lung tissues was investigated by immunoblot analysis at 15 days after bleomycin administration. A basal level of IB- was detectable in the homogenated lung tissues from sham-treated mice (Fig. 11). IB- levels were substantially reduced in the lung tissues from bleomycin-treated mice (Fig. 11). PARP inhibition prevented such bleomycin-mediated IB- degradation, and the IB- band remained unchanged 15 days after bleomycin administration in both the 3-AB- and 5-AIQ-treated mice (Fig. 11). To detect NF-B/DNA binding activity, whole extracts from lung tissue of each mouse were analyzed by EMSA. A low basal level of NF-B/DNA binding activity was detected in nuclear proteins from tissues of sham-treated mice (Fig. 12). The DNA binding activity significantly increased in whole extracts obtained from lung tissues of vehicle-treated mice 15 days after bleomycin administration (Fig. 12). Treatment of mice with 3-AB or 5-AIQ caused a significant inhibition of bleomycin-induced NF-B/DNA binding activity as revealed by specific EMSA (Fig. 12).

View larger version (26K): [in this window] [in a new window]   Fig. 11. Representative Western blots of IB- degradation (A), as well as the densitometric analysis (B). Western blot analysis shows the effect of 3-AB or 5-AIQ on the degradation of IB- in lung tissue collected 15 days after bleomycin administration. Sham, basal level of IB- band was present in the tissue from sham-treated mice. BLEO, IB- band that disappeared in the lung tissue from bleomycin-treated mice. BLEO + 3-AB, IB- band remained unchanged in the lung tissue from bleomycin-treated mice that received 3-AB. BLEO + 5-AIQ, IB- band remained unchanged in the lung tissue from bleomycin-treated mice that received 5-AIQ. Immunoblotting in A is representative of one lung tissue of 10 analyzed. The results in B are expressed as mean ± S.E.M. from 10 blots. O.D., optical density. *, p < 0.01 versus sham; , P < 0.01 versus spinal cord injury. *, p < 0.01 versus sham; , p < 0.01 versus bleomycin.

View larger version (75K): [in this window] [in a new window]   Fig. 12. Effect of PARP inhibitors on NF-B/DNA binding activity in mice lung. Whole extracts from bleomycin-treated mice (BLEO) or noninflamed (Sham) mice lungs were prepared as described under Materials and Methods and incubated with 32P-labeled NF-B probe. Representative EMSA of NF-B shows the effect of 3-AB (BLEO + 3-AB), as well as the effect of 5-AIQ (BLEO + 5-AIQ) on NF-B/DNA binding activity evaluated in lung tissues 15 days after bleomycin administration. Data illustrated are from a single experiment and are representative of three separate experiments.

The specificity of NF-B/DNA binding complex was demonstrated by the complete displacement of NF-B/DNA binding in the presence of a 50-fold molar excess of unlabeled NF-B probe (wild-type, 50x) in the competition reaction. In contrast, a 50-fold molar excess of unlabeled mutated NF-B probe (Mut., 50x) or Sp-1 oligonucleotide (Sp-1, 50x) had no effect on this DNA-binding activity (data not shown).

	Discussion

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The main findings of the current study are that treatment with 3-AB or 5-AIQ, two structurally unrelated PARP inhibitors, attenuates 1) the degree of PARP activation in the injured lung tissues, 2) the infiltration of the injured lung with neutrophils, 3) cell apoptosis, 4) proinflammatory cytokines, and 5) lung tissue damage. These protective effects were associated with inhibition of DNA binding of the transcription factor NF-B in the inflamed lung. Thus, we propose that the anti-inflammatory activity of PARP inhibitors may be mediated, at least in part, by inhibition of the transcription of certain proinflammatory mediators, which are regulated by NF-B. All of these findings support the view that PARP plays a detrimental role in the development and persistence of inflammation associated with lung fibrosis in the mouse. The pathological features of inflammation and fibrosis are well appreciated, but little is known about their etiology and pathogenesis (American Thoracic Society, 2000).

Bleomycin induction of lung injury in mice is a well established model of interstitial lung disease, resulting in pulmonary fibrosis (Smith et al., 1996). Endotracheal instillation of bleomycin in mice is followed by up-regulated expression of lung cytokines, development of lung inflammation, and accumulation of collagen in the lung (Smith et al., 1996).

Earlier reports (Goodman et al., 1998) point out that the pathogenesis of BLM-induced fibrosis is mediated, at least in part, through the generation of ROS, which causes the peroxidation of membrane lipids and DNA damage. If that perspective is true, then antioxidant therapy may prevent the lung fibrosis caused by BLM and may prevent other diseases related with interstitial pulmonary fibrosis. Because BLM administration results in increased lipid peroxidation and alters activities of antioxidant enzymes in bronchoalveolar lavage fluids and lung tissue (Karam et al., 1998), in previous studies (Venkatesan et al., 1997) some natural or synthetic antioxidants have been used to protect against BLM oxidative lung toxicity both in vivo and in vitro.

Recent studies have also suggested a contributory role for oxidants in gene induction. NF-B is a pleiotropic transcription factor activated by low levels of ROS and inhibited by antioxidants (Cuzzocrea et al., 2004). Consensus binding sequences for NF-B have been identified in the promoter regions of several genes implicated in the pathogenesis of acute and chronic inflammation (Bowie and O'Neill, 2000). In our experimental model of lung fibrosis, in agreement with previous reports (Ortiz et al., 2002), we found that DNA binding activity of NF-B is increased 15 days after bleomycin administration. Therefore, NF-B DNA binding activity is associated with a significant IB- degradation in the lung tissues after bleomycin administration.

Thus, our data support the well established hypothesis that NF-B may represent an important therapeutic target in the treatment of inflammation (Bethea et al., 1998). In this regard, we have recently demonstrated that pyrrolidine dithiocarbamate, an antioxidant reported to be a potent inhibitor of NF-B in vitro, significantly reduced acute lung inflammation by acting as an NF-B inhibitor (Cuzzocrea et al., 2002a). Our present data show that the amelioration of lung damage by pharmacological inhibition of PARP was associated with inhibition of NF-B activation. Although it is difficult to establish the definitive mechanism by which the PARP inhibitors reduce the DNA binding of these nuclear factors in in vivo experiments, our data support the possibility that PARP may be an important modulator of transcription during inflammation. Moreover, in this study, the transient loss of IkB-, which occurs in injured lung tissues from bleomycin-treated mice, was prevented by PARP-inhibitor treatment, which correlated well with the inhibition of NF-B activation, suggesting that PARP inhibitors may also inhibit NF-B activation via stabilization of IB-.

Our results are in agreement with other reports which have clearly demonstrated a role of poly(ADP-ribosyl)ation in signal transduction (Zingarelli et al., 2003). It has been demonstrated that PARP-deficient cells are defective in NF-B-dependent transcriptional activation (Oliver et al., 1999). Similarly, pharmacological inhibitors of PARP abolish mRNA expression of inducible nitric-oxide synthase, interleukin-6, and TNF- in in vitro cultured cells (Hauschildt et al., 1997). Furthermore, Zingarelli et al. (2003) have clearly demonstrated recently that NF-B DNA binding is completely abolished in heart from PARP-deficient mice subjected to ischemia and reperfusion, as well as in the colon from rats subjected to experimental colitis treated with two different PARP inhibitors. The specific mechanism of PARP activation in regulating transcription needs further study. Changes in cellular energetic after PARP activation may interfere with calcium sequestration and biosynthetic processes. Poly(ADP-ribosyl)ation may lead to the relaxation of chromatin with the consequence that genes become more accessible to RNA-polymerase (de Murcia et al., 1988).

Numerous binding sequences of NF-B on various genes with important immunologic functions characterize this transcription factor as a pluripotent factor in the inflammatory response (Xie et al., 1994). Furthermore, the activation of NF-B is a common endpoint of various signal transduction pathways, including the activation of phosphatidylcholine-specific phospholipase C, protein kinase C, protein tyrosine kinases, and mitogen-activated protein kinases and other signaling factors (Novogrodsky et al., 1994). Binding of NF-B to the respective binding sequence on genomic DNA encoding for different proinflammatory genes such as TNF- and IL-1 results in a rapid and effective transcription of these genes (Xie et al., 1994). There is good evidence that TNF- and IL-1 help to propagate the extension of a local or systemic inflammatory process (Wooley et al., 1993). Therefore, it has been demonstrated that TNF- plays a fundamental role in the pathogenesis of bleomycin-induced pulmonary fibrosis (Ortiz et al., 1998). We have previously reported that 5-AIQ inhibits TNF- and IL-1 production in an experimental model of lung inflammation induced by zymosan-activated plasma (Cuzzocrea et al., 2002b). Therefore, the inhibition of the production of TNF- and IL-1 by the two PARP inhibitors 5-AIQ and 3-AB described in the present study is most likely attributed to the inhibitory effect of the activation of NF-B. Another potential mechanism by which PARP inhibition improved lung injury in our experimental model of lung fibrosis is the reduction of neutrophil recruitment into the site of inflammation. Accumulation and activation of inflammatory cells are some of the initial events of tissue injury and are regulated at the transcriptional level. For example, expression of adhesion molecules, such as P-selectin, E-selectin, and intercellular adhesion molecule-1, is regulated by genes responsive to NF-B.

Therefore, we may hypothesize, as previously demonstrated (Cuzzocrea et al., 2002b), that pharmacological inhibition of PARP may also inhibit the recruitment of inflammatory cells at the transcription level. The discovery of the concept that PARP regulates neutrophil trafficking may provide new insights in the interpretation of recent reports demonstrating the protective effect of PARP inhibition in experimental models of shock, ischemia-reperfusion injury, and inflammation. For instance, there is good evidence that PARP activity (including 3-AB and 5-AIQ used in this study) reduces the up-regulation of adhesion molecules (e.g., P-selectin and intercellular adhesion molecule-1) in regional myocardial ischemia and reperfusion of the heart (Zingarelli et al., 1997), the gut (Di Paola et al., 2004), the kidney (Chatterjee et al., 1999), as well as in the inflamed lung (Cuzzocrea et al., 2002b). Therefore, it has been pointed out recently that the ability of PARP inhibitors in reducing infiltration of activated and PAR⁺ PMNs/monocytes into damaged tissues may be independent of PARP activity. Consistent with this is the study by Scott et al. (2003) showing that peroxynitrite-induced oligodendrocyte cell death is PARP-independent.

Extravasated polymorphonuclear leukocytes in the inflammatory sites become activated and produce a variety of inflammatory mediators such as growth factors, chemokines and cytokines, complement components, proteases, nitric oxide, reactive oxygen metabolites, and peroxynitrite, which are important mediators of tissue injury (Cuzzocrea et al., 2001). Prevention of neutrophil-dependent inflammatory pathways is likely to contribute to the improved histological status after inhibition of PARP. Thus, as previously indicated (Mazzon et al., 2002), we propose the following positive feedback cycle: early reactive oxygen species production >> PARP-related endothelial injury >> polymorphonuclear leukocyte infiltration >> more reactive oxygen species production. Inhibition of PARP would intercept this cycle at the level of endothelial injury. This model would explain the reduction of nitrotyrosine staining in lung tissues from bleomycin-treated mice that have been treated with the two PARP inhibitors: reduced neutrophil infiltration leads to reduced reactive oxygen species. Recent studies have demonstrated the induction of apoptosis in different cell line, in response to ROS, peroxynitrite, and nitric oxide (Leist et al., 1997).

It was reported that, in the lungs after bleomycin administration, apoptosis of bronchial and alveolar epithelial cells is observed (Kuwano et al., 1999). Moreover, it was also reported that apoptosis of pneumocytes induced by an agonistic antibody against Fas results in lung fibrosis (Hagimoto et al., 1997). In the present investigation, FR-167653 administration markedly suppressed apoptosis of the lung cells. The role of p38 mitogen-activated protein kinase on the apoptosis of various cell types is controversial, but it is possible that the suppression of apoptosis by FR-167653 is at least explained by inhibiting death signals transduced by the Fas ligand and TNF-, whose expressions enhanced by p38 mitogen-activated protein kinase (Zhang et al., 2000). We demonstrate here that PARP inhibitors inhibit apoptotic cell death in lung tissues tissue from bleomycin-treated mice (as determined by Annexin V coloration). Thus, our in vivo findings support the view that inhibition of PARP directly protects cells by preventing the activation of the apoptosis pathway. In conclusion, our results indicate that PARP inhibitors have strong anti-inflammatory properties resulting in reduced 1) PMN infiltration, 2) activation of NF-B, and 3) degree of tissue injury.

	Acknowledgements

 
We thank Giovanni Pergolizzi and Carmelo La Spada for excellent technical assistance during this study, Caterina Cutrona for secretarial assistance, and Valentina Malvagni for editorial assistance with the manuscript.

	Footnotes

 
doi:10.1124/jpet.104.080705.

ABBREVIATIONS: ROS, reactive oxygen species; PARP, poly(ADP-ribose) polymerase; BLEO, bleomycin; 3-AB, 3-aminobenzamide; GPI6150 1,11b-dihydro-[2H]benzopyrano[4,3,2-de]isoquinolin-3-one; PJ34, dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide hydrochloride; 5-AIQ, 5-aminoisoquinolinone [5-aminoisoquinolin-1(2H)-one]; 1,5-DHIQ, 1,5-dihydroxyisoquinoline (5-hydroxyisoquinolin-1(2H)-one); PBS, phosphate-buffered saline; PAR, poly(ADP-ribose); MPO, myeloperoxidase; BLM, basolateral membrane; PMN, polymorphonuclear leukocyte; TNF, tumor necrosis factor, IL, interleukin; PI, propidium iodide; FITC, fluorescein isothiocyanate; EMSA, electrophoretic mobility shift assay; NF, nuclear factor; FR-167653, 1-[7-(4-fluorophenyl)-1,2,3,4-tetrahydro-8-(4-pyridyl)pyrazolo [5,1-c][1,2,4] triazin-2-yl]-2-phenylethanedione sulfate monohydrate.

Address correspondence to: Prof. Salvatore Cuzzocrea, Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Torre Biologica, Policlinico Universitario, Via C. Valeria–Gazzi, 98100 Messina, Italy. E-mail: salvator{at}unime.it

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