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GW0742, A HIGH-AFFINITY PPAR -β/δ AGONIST, INHIBITS ACUTE LUNG INJURY IN MICE

Di Paola, Rosanna*; Crisafulli, Concetta; Mazzon, Emanuela*; Esposito, Emanuela*; Paterniti, Irene; Galuppo, Maria; Genovese, Tiziana*†; Thiemermann, Christoph; Cuzzocrea, Salvatore*†

doi: 10.1097/SHK.0b013e3181b8f2fb
Basic Science Aspects
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Several lines of evidence suggest a biological role for peroxisome proliferator-activated receptor (PPAR) β/δ in the pathogenesis of a number of diseases. The aim of this study was to investigate the effects of a high-affinity PPAR-β/δ agonist, GW0742, in a mouse model of carrageenan (CAR)-induced pleurisy. Injection of CAR into the pleural cavity of mice elicited an acute inflammatory response characterized by accumulation of fluid containing a large number of neutrophils (polymorphonuclear leukocytes) in the pleural cavity, infiltration of polymorphonuclear leukocytes in lung tissues and subsequent lipid peroxidation, and increased production of nitrite/nitrate, TNF-α, and IL-1β. Furthermore, CAR induced lung apoptosis (Bax and Bcl-2 expression), and nitrotyrosine formation was determined by immunohistochemical analysis of lung tissues. Administration of GW0742 (0.3 mg/kg, i.p. bolus) 30 min before and 30 min after a challenge with CAR caused a reduction in all the parameters of inflammation measured. Thus, based on these findings, we propose that a PPAR-β/δ agonist such as GW0742 may be useful in the treatment of various inflammatory diseases.

*IRCCS Centro Neurolesi "Bonino-Pulejo"; Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Italy; and The William Harvey Research Institute, Centre for Experimental Medicine, Nephrology and Critical Care, St Bartholomew's and The Royal London School of Medicine and Dentistry, London, United Kingdom

Received 27 Feb 2009; first review completed 19 Mar 2009; accepted in final form 15 Jul 2009

Address reprint requests to Salvatore Cuzzocrea, MD, PhD, Dipartimento Clinico e Sperimentale di Medicina e Farmacologia, Torre Biologica, Policlinico Universitario, 98123 Messina, Italy. E-mail: salvator@unime.it.

This research was supported by PRIN 2007.

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INTRODUCTION

The inflammatory process is invariably characterized by a production of prostaglandins, leukotrienes, histamine, bradykinin, platelet-activating factor, and by the release of chemicals from tissues and migrating cells. Carrageenan (CAR)-induced local inflammation is commonly used to evaluate anti-inflammatory effects of nonsteroidal drugs. Therefore, CAR-induced local inflammation (pleurisy) is a useful model to assess the contribution of mediators involved in cellular alterations during the inflammatory process.

In particular, the initial phase of acute inflammation (0-1 h), which is not inhibited by nonsteroidal anti-inflammatory drugs such as indomethacin or aspirin, has been attributed to the release of histamine, 5-hydroxytryptamine and bradykinin, followed by a late phase (1-6 h) mainly sustained by prostaglandin release and attributed to the induction of inducible cyclo-oxygenase in the tissue (1). It seems that the onset of the CAR-induced acute inflammation has been linked to neutrophil infiltration and the production of neutrophil-derived free radicals such as hydrogen peroxide, superoxide, and hydroxyl radical, as well as the release of other neutrophil-derived mediators.

Peroxisome proliferator activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily, which includes the classical steroid, thyroid, and retinoid hormone receptors, as well as many orphan receptors. So far, 3 PPAR isotypes have been identified and are commonly designated PPAR-α, PPAR-β/δ, and PPAR-γ (2). Peroxisome proliferator activated receptors have been implicated in the pathogenesis of a number of diseases, including diabetes mellitus, obesity, atherosclerosis, and neurological diseases (3, 4), and therefore represent an important pharmacological target. Both PPAR-α and PPAR-γ have been well characterized for their roles in lipid and glucose metabolism using specific marketed drugs such as the thiazolidindiones, ligands of PPAR-γ prescribed for the treatment of type-2 diabetes, and the fibrates, PPAR-α ligands prescribed for their lipid-modulating properties (5, 6). In contrast, the biological role and function of PPAR-β/δ (NUC-1) remains relatively unclear.

Peroxisome proliferator activated receptor β/δ is ubiquitously expressed in a variety of tissues, including liver, adipose tissue, skeletal muscle, kidney, cerebellum, thalamus, cerebellar cortex, and intestine. Physiologically, PPAR-β/δ has been associated with adipocyte precursor cell proliferation (7), oligodendrocyte differentiation (8), and cholesterol homeostasis. Compared with PPAR-α and PPAR-γ, PPAR-β/δ is the least understood member of the PPAR family (9). Peroxisome proliferator activated receptor β/δ is ubiquitously expressed in most adult tissues and very early during embryogenesis. The multiplicity of phenotypes induced by PPAR-β/δ gene disruption in the mouse reflects the importance of this nuclear receptor in development, and early functional studies indicate PPAR-β/δ involvement in epidermal differentiation (10), maturation, and skin wound healing (11). Evidence has also suggested that activation of PPAR-β/δ promotes fatty acid catabolism in several tissues such as skeletal muscle and adipose (12). More recent studies indicate a potential role of PPAR-β/δ in regulating glucose metabolism and insulin sensitivity (13). These actions could explain the apparent beneficial effects of synthetic PPAR-β/δ agonists on circulating lipids, insulin resistance, and obesity that have been reported in some animal models (13, 14). Preclinical, in vivo studies using high-affinity PPAR-β/δ agonists have demonstrated efficacy in models of diabetes and obesity β-oxidation, suggesting that modulation of the δ-isoform may have a role in treating these diseases and metabolic syndrome. The use of selective PPAR-β/δ agonists in preclinical studies suggests that this subtype also possesses anti-inflammatory properties. In vivo data suggest that ligands to the β/δ isoform have activity in a number of disease models that are partly driven by the inflammatory response (15).

On the contrary, the role of the PPAR-β/δ receptor in conditions associated with experimental acute lung inflammation have, however, not yet been fully investigated. The present study was designed to gain a better understanding of the possible influence of PPAR-β/δ using the high-affinity PPAR-β/δ agonist GW0742 in a rodent model of lung injury.

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MATERIALS AND METHODS

Animals

Male Adult 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 was in compliance with Italian regulations on protection of animals used for experimental and other scientific purpose (D.M. 116192) as well as with the European Economic Community regulations (O.J. of E.C. L 358/1 12/18/1986).

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Carrageenan-induced pleurisy

Mice were anesthetized with isoflurane and submitted to a skin incision at the level of the left sixth intercostal space. The underlying muscle was dissected, and saline (0.1 mL) or saline containing 2% λ-CAR (0.1 mL) was injected into the pleural cavity as previously described (16). The inflammatory cells (∼70% of macrophages) in the pleural exudate were suspended in phosphate buffer saline (PBS) and counted with an optical microscope in a Burker chamber after vital Trypan Blue staining.

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Experimental groups

Mice were randomly allocated into the following groups: 1) for the CAR + vehicle group, mice were subjected to CAR-induced pleurisy and received the vehicle for GW0742 (10% dimethyl sulfoxide [v/v], i.p. bolus) 30 min before and 30 min after CAR administration (n = 10); 2) for the CAR + GW0742 group, mice were subjected to CAR-induced pleurisy and received GW0742 (0.3 mg/kg, i.p., bolus) 30 min before and 30 min after CAR administration (n = 10); 3) for the sham + saline group, identical surgical procedures to the CAR group were performed except that saline was administered instead of CAR (n = 10); 4) for the sham + GW0742 group, mice received GW0742 (0.3 mg/kg, i.p., bolus) 30 min before and 30 min after administration of saline (n = 10).

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Histological examination

Lung tissue samples were taken 4 h after injection of CAR. Lung tissue samples 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). Sections were then deparaffinized with xylene and stained with hematoxylin and eosin. All sections were studied using an Axiovision Ziess (Milan, Italy) microscope. The following morphological criteria were used for scoring: 0, normal lung; grade 1, minimal edema or infiltration of alveolar or bronchiolar walls; grade 3, moderate edema and inflammatory cell infiltration without obvious damage to lung architecture; and grade 4, severe inflammatory cell infiltration with obvious damage to lung architecture. All the histological studies were performed in a blinded fashion.

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Localization of Fasl, Bax, Bcl-2, nitrotyrosine, TNF-α, iNOS, and intercellular adhesion molecule 1 by immunohistochemistry

The tissues were fixed in 10% PBS-buffered formaldehyde, and 8-μm sections were prepared from paraffin-embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% H2O2 in 60% methanol for 30 min. The sections were permeabilized with 0.1% Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating the section in 2% normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with avidin and biotin (DBA, Milan, Italy). Sections were incubated overnight with polyclonal rabbit antinitrotyrosine (1:500 in PBS), anti-Bax (1:500 in PBS v/v; Santa Cruz Biotechnology, Santa Cruz, Calif), anti-TNF-α (1:100 in PBS, v/v; Santa Cruz Biotechnology), anti-Bcl-2 (1:100 v/v; Santa Cruz Biotechnology), or anti-iNOS (1:500 in PBS, v/v; Santa Cruz Biotechnology), or intercellular adhesion molecule (ICAM) 1 (1:500; Santa Cruz Biotechnology) antibody. Specific labeling was detected with a biotin-conjugated goat anti-rabbit, donkey anti-goat, or goat anti-mouse immunoglobulin (Ig) G and avidin-biotin peroxidase complex (DBA). To verify the binding specificity for Fasl, Bax, Bcl-2, TNF-α, or anti-iNOS, some sections were also incubated with primary antibody only (no secondary antibody) or with secondary antibody only (no primary antibody). In these situations, no positive staining was found in the sections, indicating that the immunoreactions were positive in all the experiments performed. To confirm that the immunoreactions for the nitrotyrosine were specific, some sections were also incubated with the primary antibody (antinitrotyrosine) in the presence of excess nitrotyrosine (10 mM) to verify the binding specificity.

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Determination of myeloperoxidase activity

Myeloperoxidase (MPO) activity, an indicator of polymorphonuclear leukocyte (PMN) accumulation, was determined as previously described (17). Lung tissues were obtained and weighed 4 h after intrapleural injection of CAR. Each piece of tissue was homogenized in a solution containing 0.5% hexadecyltrimethylammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20,000 g at 4°C. An aliquot of the supernatant was then allowed to react with a solution of tetramethylbenzidine (1.6 mM) and 0.1 mM H2O2. The rate of change in absorbance was measured spectrophotometrically at 650 nm. Myeloperoxidase activity was defined as the quantity of enzyme degrading 1 μmol of peroxide per minute at 37°C and was expressed in units per gram weight of wet tissue.

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Malondialdehyde measurement

Malondialdehyde (MDA) levels in the lung tissue were determined as an indicator of lipid peroxidation as previously described (18). Lung tissue collected at the specified time was homogenized in 1.15% (w/v) KCl solution. A 100-μL aliquot of the homogenate was added to a reaction mixture containing 200 μL of 8.1% (w/v) sodium dodecyl sulfate, 1.5 mL of 20% (v/v) acetic acid (pH 3.5), 1.5 mL of 0.8% (w/v) thiobarbituric acid, and 700 μL distilled water. Samples were then boiled for 1 h at 95°C and centrifuged at 3,000 g for 10 min. The absorbance of the supernatant was measured using spectrophotometry at 650 nm.

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Measurement of cytokines

TNF-α and IL-1β levels were evaluated in the exudate 4 h after the induction of pleurisy by CAR injection as previously described (19). The assay was performed using a colorimetric commercial enzyme-linked immunosorbent assay kit (Calbiochem-Novabiochem Corporation, Milan, Italy).

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Western blot analysis for IκB-α, NF-κB p65, PPAR-β/δ, iNOS, Bax, and Bcl-2

Cytosolic and nuclear extracts were prepared as previously described (20) with slight modifications. Briefly, lung tissues from each mouse were suspended in extraction buffer A containing 0.2 mM PMSF, 0,15 μM pepstatin A, 20 μM leupeptin, 1 mM sodium orthovanadate, homogenized at the highest setting for 2 min, and centrifuged at 1,000 x g for 10 min at 4° C. Supernatants represented the cytosolic fraction. The pellets, containing enriched nuclei, were re-suspended in Buffer B containing 1% Triton X-100, 150 mM NaCl, 10 mM TRIS-HCl pH 7.4, 1 mM EGTA, 1 mM EDTA, 0.2 mM phenylmethanesulfonyl fluoride, 20 μm leupeptin, 0.2 mM sodium orthovanadate. After centrifugation for 30 min at 15,000 g at 4°C, the supernatants containing the nuclear protein were stored at −80 for further analysis. The levels of IκB-α, iNOS, Bax, and Bcl-2 were quantified in cytosolic fraction from lung tissue collected at 4 h after CAR administration, whereas NF-κB p65 and PPAR-β/δ levels were quantified in nuclear fraction. The filters were blocked with 1× PBS, 5% (w/v) nonfat dried milk (PM) for 40 min at room temperature and subsequently probed with specific Abs IκB-α (Santa Cruz Biotechnology; 1:1,000), anti-iNOS (1:1,000; Trasduction, Milan, Italy), anti-Bax (1:500; Santa Cruz Biotechnology), anti-Bcl-2 (1:500; Santa Cruz Biotechnology), anti-NF-κB p65 (1:1,000; Santa Cruz Biotechnology), or anti-PPAR-β/δ (1:1000; Santa Cruz Biotechnology) antibody in 1× PBS, 5% w/v nonfat dried milk, 0.1% Tween-20 (PMT) at 4°C overnight. Membranes were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2,000; Jackson ImmunoResearch, West Grove, Pa) for 1 h at room temperature.

To ascertain that blots were loaded with equal amounts of proteic lysates, they were also incubated in the presence of the antibody against α-tubulin protein (1:10,000; Sigma-Aldrich Corp., Milan, Italy). The relative expression of the protein bands of IκB-α (∼37 kDa), NF-κB p65 (65 kDa), Bax (∼23 kDa), iNOS (∼130 kDa), and PPAR-β/δ (∼60 kDa) were quantified by densitometric scanning of the x-ray films with GS-700 Imaging Densitometer (GS-700; Bio-Rad Laboratories, Milan, Italy) and a computer program (Molecular Analyst; IBM, Armonk, NY) and standardized for densitometric analysis to β-actin or lamin levels.

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Measurement of nitrite/nitrate

Total nitrite in pleural exudates, an indicator of NOS, was measured as previously described (21). Briefly, the nitrate in the sample was first reduced to nitrite by incubation with nitrate reductase (670 mU/mL) and nicotinamide adenine dinucleotide phosphate reduced form (160 μM) at room temperature for 3 h. The total nitrite concentration in the samples was then measured using the Griess reaction by adding 100 μL of Griess reagent 0.1% (w/v) naphthylethylendiamide dihydrochloride in H2O and 1% (w/v) sulfanilamide in 5% (v/v) concentrated H3PO4 vol. 1:1 to the 100-μL sample. The optical density at 550 nm was measured using an enzyme-linked immunosorbent assay microplate reader (SLT-Lab Instruments, Salzburg, Austria). Nitrite concentrations were calculated by comparison with optical density at 550 nm of standard solutions of sodium nitrite prepared in H2O.

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Materials

Unless otherwise stated, all compounds were obtained from Sigma-Aldrich Company. Secondary and nonspecific IgG antibodies for immunohistochemical analysis were from Vector Laboratories Inc. (Burlingame, Calif).

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Data analysis

All values in the figures and text are expressed as mean ± SEM 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 are representative of at least three experiments performed on different experimental days. The results were analyzed by one-way ANOVA, followed by a Bonferroni post hoc test for multiple comparisons. A P value of less than 0.05 was considered significant.

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RESULTS

Effects of GW0742 on carrageenan-induced pleurisy

When compared with lung sections taken from saline-treated animals (sham group; Fig. 1A), histological examination of lung sections taken from mice treated with CAR revealed significant tissue damage and edema (Fig. 1, B and D; see histological score), as well as infiltration of PMNs within the tissues (MPO activity; Fig. 1E). GW0742 (0.3 mg/kg) reduced the degree of lung injury (Fig. 1, C and D; see histological score). Furthermore, injection of CAR elicited an acute inflammatory response characterized by the accumulation of fluid (edema) in the pleural cavity (Table 1) containing large amounts of PMNs (Table 1). Treatment with GW0742 attenuated CAR-induced edema formation and PMN infiltration (Table 1).

Table 1

Table 1

Fig. 1

Fig. 1

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Effects of GW0742 on CAR-induced NO production

No positive staining for iNOS was observed in the lung tissues obtained from the sham group (data not shown). Immunohistochemical analysis of lung sections obtained from CAR-treated mice revealed positive staining for iNOS (Fig. 2, A, A1, and C; see densitometry analysis). In contrast, no staining for iNOS was found in the lungs of CAR-treated mice that had been treated with GW0742 (Fig. 2, B and C; see densitometry analysis). A significant increase in iNOS expression 4 h after CAR injection was also detected in lungs obtained from mice subjected to CAR-induced pleurisy, as assayed by Western blot analysis (Fig. 2, D and D1; see densitometry analysis). GW0742 (0.3 mg/kg) treatment significantly attenuated this iNOS expression (Fig. 2D; see densitometry analysis). NO levels were also significantly increased in the exudate obtained from mice administered CAR (Fig. 2E). Treatment of mice with GW0742 significantly reduced NO exudate levels (Fig. 2E).

Fig. 2

Fig. 2

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Effects of GW0742 on CAR-induced nitrotyrosine formation and lipid peroxidation

Immunohistochemical analysis of lung sections obtained from mice treated with CAR revealed positive staining for nitrotyrosine (Fig. 3, B, B1, and E; see densitometry analysis). In contrast, no positive staining for nitrotyrosine was found in the lungs of CAR-treated mice, which had been treated with GW0742 (Fig. 3C). There was no staining for nitrotyrosine (Fig. 3, A and E; see densitometry analysis) in lungs obtained from the sham group of mice. In addition, at 4 h after CAR-induced pleurisy, MDA levels were also measured in the lungs as an indicator of lipid peroxidation. As shown in Figure 3D, MDA levels were significantly increased in the lungs of CAR-treated mice. Lipid peroxidation was significantly attenuated by the intraperitoneal injection of GW0742 (Fig. 3D).

Fig. 3

Fig. 3

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Effects of GW0742 on the release of proinflammatory cytokines and FAS-ligand expression induced by CAR

When compared with sham animals, injection of CAR resulted in an increase in the levels of TNF-α in the pleural exudates (Table 1). The release of TNF-α was significantly attenuated by treatment with GW0742 (Table 1).

We also evaluated the TNF-α protein expression in the lung tissues by immunohistochemical assay. No positive staining for TNF-α was observed in the lung tissues collected from sham mice (Fig. 4A). On the contrary, tissue sections obtained from mice 4 h after CAR administration showed positive staining for TNF-α localized in the infiltrated inflammatory cells, pneumocytes, an in the vascular wall (Fig. 4, B, B1, and G; see densitometry analysis). In contrast, no positive staining for TNF-α was found in the lungs of CAR-treated mice that had been treated with GW0742 (Fig. 4, C and G).

Fig. 4

Fig. 4

Immunohistochemical analysis of lung sections obtained from mice treated with CAR revealed positive staining for Fas-ligand (Fig. 4, E, E1, and H). In contrast, no positive staining for nitrotyrosine was found in the lungs of CAR-treated mice that had been treated with GW0742 (Fig. 4, F and H; see densitometry analysis). There was no staining for nitrotyrosine (Fig. 4D) in lungs obtained from the sham group of mice.

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Effects of GW0742 on infiltration of neutrophils

No positive staining for ICAM-1 was found in lung sections from saline-treated mice (Fig. 5A). Acute lung inflammation (4 h after CAR administration) induced positive staining for ICAM-1 localized in the vessels in lung tissue sections (Fig. 5, B and D; see densitometry analysis) mainly localized in the vascular endothelium (Fig. 5B1). GW0742 treatment abolished the increased expression of ICAM-1 induced by CAR (Fig. 4, C and D; see densitometry analysis). This expression of adhesion molecules seemed to correlate with an influx of leukocytes into the lung tissue; thus, we investigated the effect of GW0742 on neutrophil infiltration by measurement of MPO activity. Myeloperoxidase activity was significantly elevated at 4 h after CAR administration in CAR group (Fig. 5E). Treatment with PPAR-β/δ attenuated neutrophil infiltration into the lung tissue (Fig. 5E).

Fig. 5

Fig. 5

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Effect of GW0742 on IκB-α degradation and NF-κB p65 activation

We evaluated IκB-α degradation and nuclear NF-κB p65 by Western blot analysis to investigate the cellular mechanisms by which treatment with GW0742 may attenuate the development of CAR-induced pleurisy.

A basal level of IκB-α was detected in the lung tissues of sham animals (Fig. 6, A and A1; see densitometry analysis), whereas in CAR-treated mice, IκB-α levels were substantially reduced (Fig. 6, A and A1; see densitometry analysis). GW0742 prevented CAR-induced IκB-α degradation. The IκB-α levels observed in these animals were similar to those of the sham group (Fig. 6, A and A1; see densitometry analysis).

Fig. 6

Fig. 6

In addition, CAR administration caused a significant increase in the NF-κB p65 levels in the nuclear fractions from lung tissues (Fig. 6, B and B1; see densitometry analysis) compared with sham-treated mice (Fig. 6, B and B1; see densitometry analysis). GW0742 treatment significantly reduced the levels of NF-κB p65 as shown (Fig. 6, B and B1; see densitometry analysis).

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Effect of GW0742 on PPAR-β/δ expression

The basal level of PPAR-β/δ was detected in the nuclear fraction of lung protein from the sham group (Fig. 7, A and A1; see densitometry analysis). Peroxisome proliferator-activated receptor β/δ expression was significantly decreased in lung from CAR group, whereas the treatment with GW0742 restored its expression (Fig. 7, A and A1; see densitometry analysis). Blots were probed with antilamin antibody to demonstrate enriched nuclear fractions (Fig. 7, A and A1; see densitometry analysis).

Fig. 7

Fig. 7

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Western blot analysis and immunohistochemistry for Bax and Bcl-2

The presence of Bax in lung homogenates was investigated by Western blot 4 h after CAR administration. A basal level of Bax was detected in lung tissues obtained from sham-treated animals (Fig. 8, A and A1; see densitometry analysis). Bax levels were substantially increased in the lung tissues from CAR-treated mice (Fig. 8, A and A1; see densitometry analysis). On the contrary, GW0742 treatment prevented the CAR-induced Bax expression (Fig. 8, A and A1; see densitometry analysis).

Fig. 8

Fig. 8

To detect Bcl-2 expression, whole extracts from lung tissues of mice were also analyzed by Western blot analysis. A basal level of Bcl-2 expression was detected in lung tissues from sham-treated mice (Fig. 8, B and B1; see densitometry analysis). At 4 h after CAR administration, Bcl-2 expression was significantly reduced (Fig. 8, B and B1; see densitometry analysis). Treatment of mice with GW0742 significantly attenuated CAR-induced inhibition of Bcl-2 expression (Fig. 8, B and B1; see densitometry analysis).

Lung samples were also collected 4 h after CAR administration to determine the immunohistological staining for Bax and Bcl-2. Lung tissues taken from sham-treated mice did not stain for Bax (Fig. 9A), whereas lung sections obtained from CAR-treated mice exhibited positive staining for Bax (Fig. 9, B, B1, and G; see densitometry analysis). GW0742 treatment reduced the degree of positive staining for Bax in the lung of mice subjected to CAR-induced pleurisy (Fig. 9, C and G; see densitometry analysis).

Fig. 9

Fig. 9

In addition, lung sections from sham-treated mice demonstrated positive staining for Bcl-2 (Fig. 9, D and D1), whereas in CAR-treated mice, Bcl-2 staining was significantly reduced (Fig. 9, E and H; see densitometry analysis). GW0742 treatment significantly attenuated the loss of positive staining for Bcl-2 in mice subjected to CAR-induced pleurisy (9, F, F1, and H; see densitometry analysis).

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DISCUSSION

This study provides the first evidence that GW0742 attenuates 1) the development of CAR-induced pleurisy, 2) the infiltration of the lung with PMNs, 3) the degree of lipid peroxidation in the lung, 4) NF-κB activation, 5) the nitration of tyrosine residues, 6) iNOS expression, 7) proinflammatory cytokine production, 8) Fas-ligand expression, 9) Bax and Bcl-2 expression, and 10) the degree of lung injury caused by injection of CAR. All of these findings support the view that GW0742 attenuates the degree of acute inflammation in the mouse. What, then, is the mechanism by which GW0742 reduces acute inflammation?

Peroxisome proliferator-activated receptors are members of the nuclear hormone receptor superfamily. These receptors, which exist in three different isoforms (PPAR-α, PPAR-γ, and PPAR-β/δ), have been detected in various species with a tissue-specific differential expression (22). Peroxisome proliferator-activated receptors regulate a variety of physiological processes, including adipogenesis, glucose metabolism, and placental function (23, 24). Therefore, only recent studies have implicated the various PPAR isoforms in inflammation (22). In particular, both PPAR-α and PPAR-γ receptors have been reported to regulate the inflammatory response (25); however, the extent of this regulation and, indeed, its direction are still controversial. Recently, it has been demonstrated that the PPAR-α receptor may exert anti-inflammatory properties (26).

Proof of a role of PPAR-β/δ in the regulation of acute lung injury is of special interest because several transcription factors important to the regulation of acute inflammation serve as substrates for PPARs (16, 26). Among these is the transcription factor NF-κB, whose function is strikingly altered by PPAR activation. Nuclear factor-κB plays a central role in the regulation of many genes responsible for the generation of mediators or proteins in inflammation. These include the genes for TNF-α, IL-1β, iNOS, and cyclo-oxygenase 2 (27). The extent to which PPAR-β/δ activates or blocks NF-κB signaling remains unclear.

We report here that CAR caused a significant reduction of the basal level of IκB-α in the lung tissues at 4 h, whereas treatment with the PPAR-β/δ agonist GW0742 significantly inhibited IκB-α degradation. Moreover, we also demonstrate that GW0742 inhibited NF-κB translocation. Taken together, the balance between proinflammatory and prosurvival roles of NF-κB may depend on the phosphorylation status of p65, and PPAR-β/δ may play a central role in this process.

There is good evidence that TNF-α and IL-1β help propagate the extension of a local or systemic inflammatory process (28). Various studies have clearly reported that inhibition of TNF-α formation significantly prevents the development of the inflammatory process (29). This study demonstrates that GW0742 attenuates the TNF-α and IL-1β production in the pleural exudates of CAR-treated mice. Therefore, the inhibition of the production of TNF-α and IL-1β by GW0742 described in the present study is most likely attributed to the inhibitory effect of activated NF-κB (30). Indeed, the expression of TNF-α, IL-1, and IL-6 was significantly reduced in the bronchoalveolar lavage fluid of LPS-treated mice (31).

The release of inflammatory mediators and particularly chemokines seems to be associated with the activation of Fas in the lung (32). It has recently been delineated that Fas activation in vivo resulted in acute lung epithelial injury and lung inflammation (32). In this regard, it has been found that CAR injection promotes Fas-ligand expression, whereas treatment with GW0742 visibly and significantly reduced Fas-ligand expression.

Furthermore, the absence of an increased expression of the adhesion molecule in the lung from CAR mice treated with GW0742 correlated with the reduction of leukocyte infiltration as assessed by the specific granulocyte enzyme MPO and with the attenuation of the lung tissue damage as evaluated by histological examination. Activation and accumulation of leukocytes is one of the initial events of tissue injury due to the release of oxygen free radicals, arachidonic acid metabolites, and lysosomal proteases (33). These results demonstrate that GW0742 may interrupt the interaction neutrophils and endothelial cells at the late firm adhesion phase mediated by ICAM. Inhibition of MPO activity by GW0742 occurs at concentrations normally required in vivo for specific PPAR-β/δ activation (34, 35). This demonstrates that the anti-inflammatory effect of GW0742 occurs at concentrations that do not activate PPAR-α or PPAR-γ. GW0742 represents a highly specific (murine EC50s: 28 nM for PPAR-β/δ; 8,900 nM for PPAR-α, and >10,000 nM for PPAR-γ), high-affinity PPAR-β/δ agonist with an acceptable pharmacokinetic profile and activity in vivo. Lee et al. (36) showed that PPARδ regulates inflammation via an unconventional ligand-dependent transcriptional pathway, in which its association and disassociation with inflammatory repressors were determined by ligand binding.

Enhanced formation of NO by iNOS may contribute to the inflammatory process (37). This study demonstrates that GW0742 attenuates the expression of iNOS in the lung in CAR-treated mice. This reduction in the expression of iNOS by GW0742 may contribute to the attenuation of nitrotyrosine formation and lipid peroxidation in the lung in CAR-treated animals. Nitrotyrosine formation, along with its detection by immunostaining, was initially proposed as a relatively specific marker for the detection of the endogenous formation "footprint" of peroxynitrite. There is, however, recent evidence that certain other reactions can also induce tyrosine nitration, for example, reaction of nitrite with hypoclorous acid and the reaction of MPO with hydrogen peroxide can lead to the formation of nitrotyrosine (38). Increased nitrotyrosine staining is therefore considered as an indicator of "increased nitrosative stress," rather than a specific marker of the generation of peroxynitrite.

Generation of free radicals and NO by activated macrophages have also been implicated in causing oligodendrocyte apoptosis.

We identified proapoptotic transcriptional changes, including the up-regulation of proapoptotic Bax and down-regulation of anti-apoptotic Bcl-2, using Western blot assay and by immunohistochemical staining. We report in the present study for the first time that treatment with GW0742 in acute lung injury documents features of apoptotic cell death after CAR administration, suggesting that protection from apoptosis may be a prerequisite for anti-inflammatory approaches. In particular, we demonstrated that treatment with GW0742 lowers the signal for Bax in the treated group when compared with lung sections obtained from CAR-treated mice, whereas on the contrary, the signal is highly expressed for Bcl-2 in GW0742-treated mice than in CAR-treated mice. This demonstrates that GW0742, by inhibiting NF-κB, prevents the loss of the antiapoptotic pathway and reduces the proapoptotic pathway activation with a mechanism still yet to be discovered. Taken together, the results of the present study enhance our understanding of the role of PPAR-β/δ in the pathophysiology of acute inflammation. Our results imply that PPAR-β/δ agonists such as GW0742 may be useful in the therapy of inflammation.

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ACKNOWLEDGMENTS

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

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Keywords:

Carrageenan; pleurisy; PMNs; lipid peroxidation; cytokines; apoptosis

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