FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by WAYMAN, N. S.
Right arrow Articles by THIEMERMANN, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by WAYMAN, N. S.
Right arrow Articles by THIEMERMANN, C.
(The FASEB Journal. 2002;16:1027-1040.)
© 2002 FASEB

Ligands of the peroxisome proliferator-activated receptors (PPAR-{gamma} and PPAR-{alpha}) reduce myocardial infarct size

NICOLE S. WAYMAN, YOSHIYUKI HATTORI*, MICHELLE C. MCDONALD, HELDER MOTA-FILIPE{dagger}, SALVATORE CUZZOCREA{ddagger}, BARBARA PISANO§, PRABAL K. CHATTERJEE and CHRISTOPH THIEMERMANN1

Department of Experimental Medicine and Nephrology, William Harvey Research Institute, St. Bartholomew’s and The Royal London School of Medicine and Dentistry, London EC1M 6BQ, UK;
* Department of Endocrinology, Dokkyo University School of Medicine, Mibu, Japan;
{dagger} Laboratory of Pharmacology, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal;
{ddagger} Institute of Pharmacology, School of Medicine, University of Messina, Messina 98123, Italy; and
§ Department of Experimental Pharmacology, University of Naples, Federico 11, Italy

1Correspondence: Department of Experimental Medicine and Nephrology, William Harvey Research Institute, St. Bartholomew’s and The Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK. E-mail: c.thiemermann{at}mds.qmw.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study was designed to investigate the effects of various chemically distinct activators of PPAR-{gamma} and PPAR-{alpha} in a rat model of acute myocardial infarction. Using Northern blot analysis and RT-PCR in samples of rat heart, we document the expression of the mRNA for PPAR-{gamma} (isoform 1 but not isoform 2) as well as PPAR-ß and PPAR-{alpha} in freshly isolated cardiac myocytes and cardiac fibroblasts and in the left and right ventricles of the heart. Using a rat model of regional myocardial ischemia and reperfusion (in vivo), we have discovered that various chemically distinct ligands of PPAR-{gamma} (including the TZDs rosiglitazone, ciglitazone, and pioglitazone, as well as the cyclopentanone prostaglandins 15D-PGJ2 and PGA1) cause a substantial reduction of myocardial infarct size in the rat. We demonstrate that two distinct ligands of PPAR-{alpha} (including clofibrate and WY 14643) also cause a substantial reduction of myocardial infarct size in the rat. The most pronounced reduction in infarct size was observed with the endogenous PPAR-{gamma} ligand, 15-deoxy{Delta}12,14-prostagalndin J2 (15D-PGJ2). The mechanisms of the cardioprotective effects of 15D-PGJ2 may include 1) activation of PPAR-{alpha}, 2) activation of PPAR-{gamma}, 3) expression of HO-1, and 4) inhibition of the activation of NF-{kappa}B in the ischemic-reperfused heart. Inhibition by 15D-PGJ2 of the activation of NF-{kappa}B in turn results in a reduction of the 1) expression of inducible nitric oxide synthase and the nitration of proteins by peroxynitrite, 2) formation of the chemokine MCP-1, and 3) expression of the adhesion molecule ICAM-1. We speculate that ligands of PPAR-{gamma} and PPAR-{alpha} may be useful in the therapy of conditions associated with ischemia-reperfusion of the heart and other organs. Our findings also imply that TZDs and fibrates may help protect the heart against ischemia-reperfusion injury. This beneficial effect of 15D-PGJ2 was associated with a reduction in the expression of the 1) adhesion molecules ICAM-1 and P-selectin, 2) chemokine macrophage chemotactic protein 1, and 3) inducible isoform of nitric oxide synthase. 15D-PGJ2 reduced the nitration of proteins (immunohistological analysis of nitrotyrosine formation) caused by ischemia-reperfusion, likely due to the generation of peroxynitrite. Not all of the effects of 15D-PGJ2, however, are due to the activation of PPAR-{gamma}. For instance, exposure of rat cardiac myocytes to 15D-PGJ2, but not to rosiglitazone, results in an up-regulation of the expression of the mRNA for heme-oxygenase-1 (HO-1). Taken together, these results provide convincing evidence that several, chemically distinct ligands of PPAR-{gamma} reduce the tissue necrosis associated with acute myocardial infarction.—Wayman, N. S., Hattori, Y., McDonald, M. C., Mota-Filipe, H., Cuzzocrea, S., Pisano, B., Chatterjee, P. K., Thiemermann, C. Ligands of the peroxisome proliferator-activated receptors (PPAR-{gamma} and PPAR-{alpha}) reduce myocardial infarct size.


Key Words: ischemia-reperfusion • infarction • PPAR • heart • adhesion molecules • inducible nitric oxide synthase • reperfusion injury


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors that are related to retinoid, steroid, and thyroid hormone receptors (1) . All members of this superfamily have a similar structure: the amino-terminal region allows ligand-independent activation, confers constitutive activity on the receptor, and is negatively regulated by phosphorylation. This region is followed by a DNA binding domain (two zinc finger motifs separated by a linker region) and the carboxyl-terminal ligand binding domain (2) . The PPAR subfamily is composed of three members: PPAR-{alpha}, PPAR-ß, and PPAR-{gamma} (3) . The name PPAR is derived from the fact that activation by xenobiotics of PPAR-{alpha} results in peroxisome proliferation in rodent hepatocytes. Activation of PPAR-ß or PPAR-{gamma}, however, does not elicit this response (3) . Most tissues in humans (and rodents) have all three receptor subtypes, although there is considerable variability in the relative expression. There are two known isoforms of PPAR-{gamma}: PPAR-{gamma}1 and PPAR-{gamma}2. PPAR-{gamma}1 is the major isoform, accounting for ~85% of PPARs in adipose tissue. The isoforms are generated from the same gene by mRNA splicing and differ in their amino-terminal end, with PPAR-{gamma}2 having an additional 30 amino acids (4) .

PPARs regulate gene expression by binding as heterodimers with retinoid X receptors (RXRs) to specific PPAR response elements (PPRE) in the promotor regions of specific target genes. RXRs are also members of the nuclear hormone receptor superfamily activated by binding of 9-cis retinoic acid (4) . In the absence of a ligand, high-affinity complexes are formed between the PPAR-RXR heterodimer and nuclear receptor corepressor proteins, preventing transcriptional activation by sequestration of the nuclear receptor heterodimer from the promotor. Binding of a ligand to the heterodimer results in the release of the corepressor from the complex, which in turn results in the binding of the activated heterodimer to the response element in the promotor region of the relevant target genes, resulting in either activation or suppression of a specific gene (4) .

The recent development of a novel class of insulin-sensitizing drugs, the thiazolidinediones (TZDs), represents a significant advance in anti-diabetic therapy. Type 2 diabetes, also known as non-insulin-dependent diabetes mellitus, is a chronic disease that affects 5–10% of adults over the age of 30 in most populations. Type 2 diabetes is characterized by resistance of peripheral tissues to the effects of insulin that is manifested as a reduction in insulin-stimulated glucose uptake in skeletal muscle and adipose tissue, defective insulin-dependent suppression of hepatic glucose output, and reduced insulin secretion of pancreatic beta cells. There is now good evidence that the beneficial effects of TZDs are due to the activation of PPAR-{gamma} (5) . For instance, the synthetic TZDs were the first class of compounds to be identified as PPAR-{gamma} ligands. The insulin sensitizer rosiglitazone is the most potent and selective PPAR-{gamma} agonist (3 , 6 , 7) , and there is a good correlation between the potency of the TZDs as PPAR-{gamma} agonist in vitro and their efficacy at lowering glucose levels in vivo (8) . There is less information about endogenous ligand(s) for PPAR-{gamma}. However, the cyclopentanone prostaglandin 15-deoxy{Delta}12,14 PGJ2 (15D-PGJ2), which is the metabolite of the prostaglandin D2, has been suggested to function as an endogenous ligand for PPAR-{gamma} (9) . Another cyclopentanone prostaglandin, PGA1, also binds to and activates PPAR-{gamma} (10) . There is recent evidence that PPAR-{gamma} agonists may have a therapeutic role in conditions associated with inflammation (11) , but the effects of PPAR-{gamma} agonists in conditions associated with ischemia-reperfusion of the heart (or any other organ) have not yet been investigated.

This study investigates the effects of various PPAR-{gamma} and PPAR-{alpha} agonists on infarct size in a rodent model of regional myocardial ischemia and reperfusion. We investigated the effects of 1) the PPAR-{gamma} ligands rosiglitazone, ciglitazone, pioglitazone (all of which are TZDs), 15-deoxy{Delta}12,14-prostaglandin J2 (15D-PGJ2), and prostaglandin A1 (PGA1) and 2) The PPAR-{alpha} ligands clofibrate and WY 14643 on infarct size caused by regional myocardial ischemia and reperfusion in the anesthetized rat. Having discovered that the cyclopentanone prostaglandin 15D-PGJ2 causes the most pronounced reduction in infarct size, we carried out studies aimed at elucidating the mechanisms of the cardioprotective effect of 15D-PGJ2. In these studies, we investigated the effect of 15D-PGJ2 on the ischemia-reperfusion-induced 1) expression of the mRNAs of inducible nitric oxide synthase (iNOS), macrophage chemotactic protein 1 (MCP-1) (by Northern blot analysis), 2) up-regulation of intercellular adhesion molecule 1 (ICAM-1) (by immunofluorescence), 3) the up-regulation of the adhesion molecule P-selectin, 4) the nitration of tyrosine residues, an indicator of the formation of peroxynitrite (by immunohistochemistry), 5) expression of heme-oxygenase-1 (HO-1) mRNA, and 6) activation of the transcription factor nuclear factor kappa B (NF-{kappa}B) by measuring the degradation of I{kappa}B-{alpha} by Western blot analysis. To elucidate whether the expression of HO-1 caused by 15D-PGJ2 contributes to the cardioprotective effects of the cyclopentanone prostaglandin, we investigated whether an inhibitor of HO-1 activity (Zinc protoporphyrin IX) attenuates the cardioprotective effects of 15D-PGJ2.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The protocol was approved by the Institutional Animal Research Committee and the care of the animals was in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85–23, revised 1996).

Coronary artery ligation in the rat in vivo
The method of coronary artery occlusion and reperfusion in the anesthetized rat was performed as described previously (12 , 13) . Briefly, 176 male Wistar rats (200–250 g, Tuck, Rayleigh, Essex, UK) were anesthetized with thiopentone sodium (Intraval®, 120 mg/kg intraperitoneal; Rhone-Merrieux, Essex, UK). The rats were tracheotomized, intubated, and ventilated with a Harvard ventilator (30% inspiratory oxygen concentration, 70 strokes/min, tidal volume: 8–10 ml/kg). Body temperature was maintained at 38 ± 1°C. The right carotid artery was cannulated and connected to a pressure transducer (Spectramed, P23XL) to monitor mean arterial blood pressure (MAP). The right jugular vein was cannulated for the administration of drugs. Subsequently, a lateral thoracotomy was performed and the heart was suspended in a temporary pericardial cradle. A snare occluder was placed around the left anterior descending coronary artery (LAD). After completion of the surgical procedure, the animals were allowed to stabilize for 30 min before LAD ligation. The coronary artery was occluded at time 0 by tightening of the occluder. This was associated with the typical hemodynamic (fall in MAP) changes in myocardial ischemia. After 25 min of acute myocardial ischemia, the occluder was reopened to allow the reperfusion for 2 h. Heart rate and MAP were continuously recorded on a 4-channel Grass 7D polygraph recorder (Grass, MA). After the 2 h reperfusion period, 1 ml blood was taken from the carotid artery and centrifuged at 6000 rpm for 3 min. The plasma was transferred to a microcentrifuge tube and frozen at -20°C until it was sent on dry ice to an independent laboratory for the analysis of plasma troponin T (TnT) levels. The coronary artery was reoccluded and Evans blue dye (1 ml of 2% w/v) was injected into the left ventricle via the right carotid artery cannula to distinguish between perfused and nonperfused (AR) sections of the heart. The Evans blue solution stains the perfused myocardium and the occluded vascular bed remains uncolored. The animals were killed with an overdose of anesthetic. The heart was excised and sectioned into slices of 3–4 mm; the right ventricular wall was removed and the area at risk (pink) was separated from the nonischemic (blue) area. The area at risk was cut into small pieces and incubated with p-nitroblue tetrazolium (NBT, 0.5 mg/ml) for 40 min at 37°C. In the presence of intact dehydrogenase enzyme systems (viable myocardium), NBT forms a dark blue formazan; areas of necrosis lack dehydrogenase activity and therefore fail to stain (14) . Pieces were separated according to staining and weighed to determine the infarct size as a percentage of the weight of the area at risk, or AR. Table 1 illustrates the experimental groups studied.


View this table:
[in this window]
[in a new window]
  		Table 1. Experimental groups studied

Determination of myocardial infarct size by measurement of troponin T
An electrochemiluminescence immunoassay for in vitro quantitative determination of TnT in human serum or plasma was performed using the standard sandwich antibody principle. The primary incubation used a biotinylated monoclonal TnT specific antibody and a monoclonal TnT-specific antibody labeled with ruthenium complex (Roche, Basel, Switzerland), which reacted to form a sandwich complex. Streptavidin-coated microparticles (Roche, Basel, Switzerland) were added before the second incubation phase, binding the complex to the solid phase via the biotin-streptavidin interaction. The reaction mixture was then aspirated into the measuring cell where the microparticles were magnetically captured onto the surface of the electrode. ProCell was used to remove any unbound substance and a voltage was applied to the electrode, inducing chemiluminescent emission, which was measured by a photomultiplier. The results were extrapolated from a previously constructed calibration curve.

RT-PCR for PPAR isoforms and HO-1
The animals were killed after the experimental procedure; heart tissues were removed, immediately frozen in liquid nitrogen, and stored at -70°C until RNA extraction. Cultured neonatal rat cardiac myocytes and fibroblasts were prepared as described previously (15) . Total RNA was extracted from the tissues and cultured cardiac ventricular cells by the guanidinium isothiocyanate/acid phenol method (16) . Reverse transcription-polymerase chain reaction (RT-PCR) was performed by standard methods as described (19) . The first strand of cDNA was synthesized from 1 µg of RNA by use of random primers and M-MLV reverse transcriptase (Promega, Madison, WI), followed by PCR amplification with synthetic gene-specific primers derived from the published sequences for rat PPAR{alpha} (accession number: M88592), PPARß/{delta} (U40064), PPAR{gamma}1 (AF156665), and PPAR{gamma}2 (AF156666). PCR amplification was performed with a DNA PCR kit (Applied BioSystems, Foster City, CA) according to the following schedule: denaturation, annealing, and elongation at 95, 55, and 72°C for 30 s, 30 s, and 1 min, respectively, for 30 cycles. Parallel amplification of rat glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was performed for reference using primers as described (20) . Possible contamination of any PCR component was excluded by carrying out a PCR reaction with these components in the absence of RT product in each set of experiments (negative control). To verify that amplification did not proceed from residual genomic DNA, a control PCR reaction was carried out for each RNA using RNA as the template. PCR products were electrophoresed on a 2% agarose gel containing ethidium bromide and visualized by UV-induced fluorescence.

Immunofluorescent localization of nitrotyrosine
The animals were killed after the experimental procedure; heart tissues were removed, immediately frozen in liquid nitrogen, and stored at -70°C. Tyrosine nitration, an index of the nitrosylation of proteins by peroxynitrite and/or oxygen-derived free radicals, was determined by immunofluorescence as described previously (17) . After reperfusion, tissues were fixed in 10% buffered formalin and 8 µm sections were prepared from paraffin embedded tissues. Sections were incubated overnight with anti-nitrotyrosine rabbit polyclonal antibody (1:500 in PBS, v/v). Sections were washed with PBS and incubated with secondary antibody TRITC-conjugated anti-rabbit (Jackson, West Grove, PA) for 2 h at room temperature. Sections were washed as before, mounted with 90% v/v glycerol in PBS, and observed with a Nikon RCM8000 confocal microscope equipped with a 40x oil objective.

Immunofluorescent analysis of P-selectin and ICAM-1
The animals were killed after the experimental procedure and the heart tissues were removed, immediately frozen in liquid nitrogen, and stored at -70°C. Localization of P-selectin and ICAM-1 was detected by immunofluorescence as described previously (18) . Sections of the heart were fixed in 10% buffered formalin and permeabilized with 0.1% Triton X-100 in PBS for 20 min and incubated in 2% normal rat serum (for P-selectin evaluation) or hamster serum (for ICAM-1) for 2 h in order to minimize nonspecific adsorption. Sections were incubated overnight with rabbit anti-human polyclonal antibody directed toward P-selectin (CD62P), which reacts with rat and with mouse anti-rat antibody directed at ICAM-1 (CD54) (1:500 in PBS, v/v) (DBA, Milan, Italy). Sections were washed with PBS and incubated with secondary antibody (TRITC-conjugated anti-rabbit and with FITC-conjugated anti-mouse (Jackson, West Grove, PA) or TRITC-conjugated anti-goat antibody (1:80 in PBS, v/v) for 2 h at room temperature. Sections were washed as before, mounted with 90% v/v glycerol in PBS, and observed with a Nikon RCM8000 confocal microscope equipped with a 40x oil objective.

Cell culture
Cell lines
Human atrial myoblasts (Girardi cells) were obtained from the European Collection of Cell Cultures (ACACC; Salisbury, Wiltshire, UK) and grown to confluence in culture flasks containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with L-glutamine (3.5 mM) and 10% fetal calf serum (FCS). Cells were passaged every 2 days, removed by treatment with trypsin/EDTA, then cultured in 96-well plates until they reached confluence. This cell line derives from a biopsy specimen of the right auricular appendage of an adult human heart.

Primary cultures of neonatal ventricular myocytes and nonmyocytes were prepared according to the procedure described by (15) . Apical halves of cardiac ventricles from 1- to 2-day-old Wistar rats were separated and minced in a chilled balanced salt solution (116 mM NaCl, 20 mM HEPES, 12.5 mM NaH2PO4, 5.6 mM glucose, 5.4 mM KCl, and 0.8 mM MgSO4, pH 7.35). Ventricular myocytes were dispersed in the balanced salt solution containing 0.06% collagenase type II (Worthington Biochemical Corp., Freehold, NJ) with agitation for 6 min at 37°C. The digestion steps were repeated five to seven times until the tissue was completely digested. The cells were combined, centrifuged and resuspended in chilled FCS (Life Technologies, Grand Island, NY). To segregate myocytes from nonmyocytes, a discontinuous gradient of Percoll (Sigma Chemical Co., St. Louis, MO) consisting of 40.5% and 58.5% was prepared in balanced salt solution and ventricular cells were suspended in the layer of 58.5% Percoll. After centrifugation at 3000 rpm for 30 min, the upper layer consisted of a mixed population of nonmyocyte cell types and the lower layer consisted almost exclusively of cardiac myocytes. Myocytes and nonmyocytes were washed twice by centrifugation and resuspension to remove all traces of Percoll.

After myocytes were incubated twice on uncoated 10 cm culture dishes for 30 min to remove any remaining nonmyocytes, the unattached viable cells (purified myocytes) were plated at a density of 4 x 105 cells/well onto gelatin-coated 6-well tissue culture plates and cultured in DMEM (Life Technologies) supplemented with 10% FCS and antibiotics (50 U/ml penicillin and 50 µg/ml streptomycin; ICN Biomedical, Aurora, OH) at 37°C for 48 h in humidified air with 5% CO2.

Nonmyocytes were resuspended in DMEM with 10% FCS and plated onto uncoated 10 cm culture dishes for 30 min. Then nonadherent cells and debris were washed away and fresh medium was added. Cells were allowed to grow to confluence, trypsinized, and passaged 1:3. This procedure yielded cultures of cells comprised almost exclusively of fibroblasts (by the first passage) (see ref 16 ). Nonmyocytes at the second or third passage were plated onto 6-well plates and allowed to grow to confluence (2.5x105 cells/well).

After incubation in DMEM with FCS, myocytes and nonmyocytes were maintained in serum-free DMEM for 12 h. The cultured cells were replaced with fresh serum-free DMEM containing 1 mg/ml BSA (Seikagaku Corp., Tokyo, Japan) and the plates were incubated for 6 to 48 h. After incubation, the cells were submitted for RNA extraction.

Northern blot analysis
RNA was extracted from the cells by a modified guanidinium isothiocyanate phenol method (19) . Probes were obtained by RT-PCR using specific primers for iNOS and MCP-1 (20 , 21) , labeled with [{alpha}-32P] dCTP by random priming, and used for Northern blot analysis of mRNA expression. Total RNA (20 µg per lane) was subjected to electrophoresis on a 1.2% agarose gel containing formaldehyde and transferred to nitrocellulose filters. The filters were prehybridized at 68°C for 15 min and hybridized with the 32P-labeled iNOS cDNA probe in rapid hybridization solution (QUIKHYB; Stratagene, La Jolla, CA) at 68°C for 1 h. The hybridized filters were washed twice for 15 min at room temperature with 2x SSC/0.1% sodium dodecyl sulfate (SDS), then twice for 30 min at 60°C with 0.1x SSC/0.1% SDS. The filters were exposed to an imaging plate (Fuji Photo Film Co., Tokyo, Japan) at room temperature for 6 h and analyzed using a FUJIX bioimaging analyzer (BAS2000II, Fuji Photo Film Co.). The filters were stripped and reprobed for the presence of GAPDH mRNA.

Preparation of cytosolic fractions and Western blot analysis for I{kappa}B{alpha}
The animals were killed after the experimental procedure and the heart tissues were removed, immediately frozen in liquid nitrogen and stored at -70°C until protein extraction. All extraction procedures were performed on ice with ice-cold reagents. Tissues were washed twice in PBS (ICN Biomedical, Milan, Italy) and cytosolic extracts were prepared by homogenizing the tissues in an extraction buffer with the following composition: 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM ethylene glycol-bis[ß-aminoethyl ether]-N,N,N'N'-tetraacetic acid (EGTA), 1 mM DL-dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 15 µg/ml soybean trypsin inhibitor, 3 µg/ml pepstatin A, 2 µg/ml leupeptin, 40 µM benzamidine (all from Sigma, Milan, Italy), then incubated on ice for 15 min. After centrifugation at 13,000 g at 4°C for 5 min, the supernatant was aliquoted and stored at -80°C. Protein concentration was determined by the Bio-Rad protein assay kit (Bio-Rad, Milan, Italy). Immunoblotting analysis of I{kappa}B{alpha} proteins was performed on cytosolic fractions. Protein concentration was determined and equivalent amounts (50 µg) for each sample were mixed with gel loading buffer (50 mM Tris, 8% (w/v) SDS, 10% (w/v) glycerol, 10% (v/v) 2-mercaptoethanol, 0.008% (w/v) bromphenol blue) in a ratio of 1:1, boiled for 3 min, and electrophoresed on a 12% (w/v) discontinuous polyacrylamide minigel (Novex). The proteins were transferred onto nitrocellulose membranes, saturated by incubation for 3 h at room temperature with 10% (w/v) nonfat dry milk in PBS-0.1% Triton X-100, and incubated with anti-I{kappa}B-{alpha} antibody (1:500) (sc-371, Santa Cruz Biotechnology, Milan, Italy) overnight at 4°C. The membranes were washed three times with 0.5% Triton X-100 in PBS and incubated with anti-rabbit immunoglobulins coupled to peroxidase (1:1000) (Vector, Burlingame, CA). The immunocomplexes were visualized by the ECL chemiluminescence method (Amersham, Milan, Italy). The relative expression of the proteins was quantified by densitometric scanning of the X-ray films with GS-700 Imaging Densitometer (Bio-Rad, Milan, Italy) and a computer program (Molecular Analyst, IBM).

Materials
Unless otherwise stated, all compounds were obtained from Sigma-Aldrich Company Ltd. (Poole, Dorset, UK). Thiopentone sodium (Intraval Sodium®) was obtained from Rhône Mérieux Ltd. (Harlow, Essex, UK). A polyclonal antibody to iNOS was purchased from Affiniti Research Products Ltd. (Exeter, England). The cell culture reagents were supplied by Life Technologies (Paisley, Scotland, UK). All other chemicals were of the highest commercial grade available. All stock solutions were prepared in nonpyrogenic saline (0.9% NaCl; Baxter Healthcare Ltd., Thetford, Norfolk, UK).

Statistical evaluation
All data are presented as means ± SE of n observations, where n represents the number of animals or blood samples studied. For repeated measurements (hemodynamics), a 2-factorial analysis of variance (ANOVA) was performed. Data without repeated measurements (multiple organ injury/failure) was analyzed by 1-factorial ANOVA, followed by a Bonferroni test for multiple comparisons. A P value of less than 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of PPAR isoforms in the rat heart
We examined the gene expression of PPAR isoforms in the rat heart tissues (LV and RV), cultured ventricular cells (neonatal rat ventricular myocytes, and nonmyocytes, which have been shown to be predominantly fibroblasts (5) . As shown in Fig. 1 , products of expected size were obtained for the PPAR isoforms (523 bp for PPAR{alpha}, 496 bp for PPARß, and 614 bp for PPAR{gamma}). The PCR product specific for PPAR{gamma}2, which differs from PPAR{gamma}1 by a sequence of 30 additional NH2-terminal amino acids, was not detected in the tissues or cultured cells from heart but was detected in fat tissue (data not shown).



View larger version (85K):
[in this window]
[in a new window]
  		Figure 1. PPAR-{gamma} isotype expression in the heart. RT-PCR analysis of the PPAR isotype (PPAR-{alpha}, PPAR-ß, PPAR-{gamma}1 and PPAR-{gamma}2) expression in the left ventricle (LV), right ventricle (RV), cardiac myocytes (CM), and cardiac fibroblasts (CF). Parallel amplification of rat housekeeping gene glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was performed as a positive control.

Effects of the thiazolidinediones rosiglitazone, ciglitazone, or pioglitazone on myocardial infarct size in the rat
The mean values for the AR were similar in all animal groups studied and ranged from 48 ± 3 to 52 ± 3% (P>0.05, Fig. 2 3 4 ). In rats that received the vehicle for the thiazolidinediones (10% DMSO, 1 ml/kg intravenous (i.v.) 30 min before the occlusion of the LAD), occlusion of the LAD for 25 min followed by reperfusion for 2 h resulted in an infarct size of 47 ± 3% (n=11) of the AR. When compared with vehicle, administration of rosiglitazone (0.3, 1 or 3 mg/kg i.v. bolus administration 30 min before the onset of myocardial ischemia) caused a dose-related reduction in myocardial infarct size (Fig. 2) . A maximum reduction of infarct size of 45% was achieved with the highest dose of rosiglitazone used. Compared with vehicle, administration of ciglitazone (0.3 mg/kg i.v. bolus administration 30 min before the onset of myocardial ischemia) caused a significant reduction in infarct size of ~45% whereas the higher dose of this compound was not effective (Fig. 3 ). When compared with vehicle, administration of pioglitazone (0.3 or 1 mg/kg i.v. bolus administration 30 min before the onset of myocardial ischemia) also caused a significant reduction in myocardial infarct size (Fig. 4 ). A maximum reduction of infarct size of 25% was achieved with the highest dose of pioglitazone used. Sham operation alone did not result in a significant degree of infarction in any of the animal groups studied (<1% of the AR) (Figs. 2 3 4) . Coronary artery occlusion and reperfusion caused a progressive fall in mean arterial blood pressure (from a baseline MAP of 118±8 mm/Hg to 82±7 mm/Hg at the end of the 2 h reperfusion period) compared with sham-operated animals. None of the thiazolidinediones had any significant effect on blood pressure (data not shown).



View larger version (27K):
[in this window]
[in a new window]
  		Figure 2. A) Area at risk (AR) and B) infarct size (IS) in rats subjected to regional myocardial ischemia (25 min) and reperfusion (2 h). Different groups of animals were subjected to the surgical procedure alone (no left anterior descending coronary artery occlusion, sham n=8) or to left anterior descending (LAD) coronary artery occlusion and reperfusion and treated with either vehicle (10% DMSO control, n=11) or rosiglitazone (0.3, n=6; 1, n=6; or 3, n=5 mg/kg i.v. bolus, 30 min before LAD occlusion). *P < 0.05 when compared to 10% DMSO control.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 3. A) Area at risk (AR) and B) infarct size (IS) in rats subjected to regional myocardial ischemia (25 min) and reperfusion (2 h). Different groups of animals were subjected to the surgical procedure alone (no left anterior descending coronary artery occlusion, sham n=8) or to left anterior descending (LAD) coronary artery occlusion and reperfusion and treated with either vehicle (10% DMSO control, n=11) or ciglitazone (0.3, n=6; or 1, n=4 mg/kg i.v. bolus, 30 min before LAD occlusion). *P < 0.05 when compared to 10% DMSO control.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 4. A) Area at risk (AR) and B) infarct size (IS) in rats subjected to regional myocardial ischemia (25 min) and reperfusion (2 h). Different groups of animals were subjected to the surgical procedure alone (no left anterior descending coronary artery occlusion, sham n=8) or to left anterior descending (LAD) coronary artery occlusion and reperfusion and treated with either vehicle (10% DMSO control, n=11) or pioglitazone (0.3, n=5; or 1, n=5 mg/kg i.v. bolus, 30 min before LAD occlusion). *P < 0.05 when compared to 10% DMSO control.

Effects of the cyclopentanone prostaglandins 15D-PGJ2 and PGA1 on myocardial infarct size in the rat
Mean values for the AR were similar in all animal groups studied and ranged from 45 ± 32 to 52 ± 2% (P>0.05, Fig. 5 ). In rats that received the vehicle for the cyclopentanone prostaglandins (10% DMSO, 1 ml/kg i.v. 30 min before the occlusion of the LAD), occlusion of the LAD for 25 min followed by reperfusion for 2 h resulted in an infarct size of 47 ± 4% (n=8) of the AR. Compared with vehicle, administration of 15D-PGJ2 (0.1 or 0.3 mg/kg i.v. bolus administration 30 min before the onset of myocardial ischemia) caused a dose-related reduction in myocardial infarct size (Fig. 5B ). A maximum reduction of infarct size of 85% was achieved with the highest dose of 15D-PGJ. Administration of PGA1 (0.3 mg/kg i.v. bolus administration 30 min before the onset of myocardial ischemia) caused a significant reduction in infarct size of ~45% when compared with vehicle (Fig. 5C ). Sham operation alone did not result in a significant degree of infarction in any of the animal groups studied (<1% of the AR) (Fig. 5) . Coronary artery occlusion and reperfusion caused a progressive fall in mean arterial blood pressure (from a baseline MAP of 112±10 mm/Hg to 88±7 mm/Hg at the end of the 2 h reperfusion period) compared with sham-operated animals. 15D-PGJ2 and PGA1 did not confer any significant effect on blood pressure (data not shown).



View larger version (25K):
[in this window]
[in a new window]
  		Figure 5. A) Area at risk (AR) and B) infarct size (IS) in rats subjected to regional myocardial ischemia (25 min) and reperfusion (2 h). Different groups of animals were subjected to the surgical procedure alone (no left anterior descending coronary artery occlusion, sham n=7) or to left anterior descending (LAD) coronary artery occlusion and reperfusion and treated with either vehicle (10% DMSO control, n=8) or 15d-PGJ2 (0.1, n=6; or 0.3, n=7 mg/kg i.v. bolus, 30 min before LAD occlusion). *P < 0.05 when compared to 10% DMSO control.

Effects of the cyclopentanone prostaglandin 15D-PGJ2 on the release into the plasma of TnT
In rats that received vehicle (10% DMSO), occlusion of the LAD for 25 min followed by reperfusion for 2 h resulted in a significant increase in the plasma levels of TnT, which was largely attenuated by pretreatment of rats with 15D-PGJ2 (0.3 mg/kg i.v.; Fig. 6 ).



View larger version (12K):
[in this window]
[in a new window]
  		Figure 6. Plasma TnT levels caused by LAD occlusion (25 min) and reperfusion (2 h). Different groups of animals were subjected to the surgical procedure alone (no left anterior descending coronary artery occlusion, sham n=7) or to left anterior descending (LAD) coronary artery occlusion and reperfusion and treated with either vehicle (10% DMSO control, n=8) or 15d-PGJ2 (0.3, n=7 mg/kg i.v. bolus, 30 min before LAD occlusion). *P < 0.05 when compared to 10% DMSO control.

Effects of the cyclopentanone prostaglandin 15D-PGJ2 on expression of the mRNAs for iNOS caused by ischemia-reperfusion
To elucidate whether regional myocardial ischemia and reperfusion leads to the expression of iNOS, we investigated the expression of iNOS mRNA (by Northern blot analysis) in biopsies obtained from the AR (e.g., the ischemic-reperfused myocardium) of the left ventricle. In rats that received vehicle (10% DMSO), occlusion of the LAD for 25 min followed by reperfusion for 2 h resulted in a significant increase in iNOS mRNA expression (Fig. 7 ), which was significantly attenuated by pretreatment of rats with 15D-PGJ2 (0.3 mg/kg i.v.; Fig. 7 ).



View larger version (32K):
[in this window]
[in a new window]
  		Figure 7. Inducible nitric oxide synthase (iNOS) mRNA expression in the area at risk of the left ventricle after ischemia (25 min) and reperfusion (2 h). Different groups of animals were subjected to the surgical procedure alone (no left anterior descending coronary artery occlusion, sham n=3) or to left anterior descending (LAD) coronary artery occlusion and reperfusion and treated with either vehicle (10% DMSO control, n=3) or 15d-PGJ2 (0.3, n=3 mg/kg i.v. bolus, 30 min before LAD occlusion). *P < 0.05 when compared to 10% DMSO control.

Effects of 15D-PGJ2 on the nitration of proteins (nitrotyrosine staining) caused by regional myocardial ischemia and reperfusion
At the end of the reperfusion period, heart sections were taken to determine the staining for nitrotyrosine residues within protein by immunofluorescence. Sections of heart from sham-treated rats did not reveal any immunofluorescence for nitrotyrosine (Fig. 8 A) within the normal architecture. A positive staining for nitrotyrosine (Fig. 8B ) was found in the area at risk of hearts subjected to ischemia and reperfusion (n=7). In contrast, no immunofluorescence for nitrotyrosine was observed in hearts obtained from rats that had been pretreated with 15D-PGJ2 and subsequently subjected to ischemia and reperfusion (Fig. 8C ). To confirm that the staining (immunoreaction) for nitrotyrosine was 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.



View larger version (51K):
[in this window]
[in a new window]
  		Figure 8. Immunofluorescent staining for nitrotyrosine in sham treated animals (A). B) Control animals treated with 10% DMSO and C) animals treated with 15d-PGJ2. Immunofluorescent staining for intercellular cell adhesion molecule 1 (ICAM-1) in E) sham animals, F) control animals treated with 10% DMSO, and G) animals treated with 15d-PGJ2. Immunofluorescent staining for P-selectin in E) sham animals, F) control animals treated with 10% DMSO, and G) animals treated with 15d-PGJ2. Different groups of animals were subjected to the surgical procedure alone (no left anterior descending coronary artery occlusion, sham n=5) or to left anterior descending (LAD) coronary artery occlusion and reperfusion and treated with either vehicle (10% DMSO 1 ml/kg, control, n=5) or 15d-PGJ2 (0.3, n=5 mg/kg i.v. bolus, 30 min before LAD occlusion).

Effects of the cyclopentanone prostaglandin 15D-PGJ2 on expression of the mRNAs for MCP-1 caused by ischemia-reperfusion
To elucidate whether regional myocardial ischemia and reperfusion leads to the expression of the chemokine MCP-1, we investigated the expression of MCP-1 mRNA (by Northern blot analysis) in biopsies obtained from the AR (e.g., the ischemic-reperfused myocardium) of the left ventricle. In rats that received vehicle (10% DMSO), occlusion of the LAD for 25 min followed by reperfusion for 2 h resulted in a significant increase in MCP-1 mRNA expression (n=7, Fig. 9 ), which was attenuated by pretreatment of rats with 15D-PGJ2 (0.3 mg/kg i.v.; n=6, Fig. 9 ).



View larger version (35K):
[in this window]
[in a new window]
  		Figure 9. MCP-1 mRNA expression in the area at risk of the left ventricle after ischemia (25 min) and reperfusion (2 h). Different groups of animals were subjected to the surgical procedure alone (no left anterior descending coronary artery occlusion, sham n=3) or to left anterior descending (LAD) coronary artery occlusion and reperfusion and treated with either vehicle (10% DMSO control, n=3) or 15d-PGJ2 (0.3, n=3 mg/kg i.v. bolus, 30 min before LAD occlusion).

Effects of 15D-PGJ2 on the expression of adhesion molecules (ICAM-1, P-selectin)
To elucidate whether regional myocardial ischemia and reperfusion leads to the expression of the adhesion molecules ICAM-1 and/or P-selectin, we investigated the expression of these proteins by immunofluorescence. Staining of heart sections obtained from sham-operated rats with an anti-ICAM-1 antibody showed specific fluorescence along the blood vessels, demonstrating that ICAM-1 is constitutively expressed (Fig. 8D ). In contrast, no positive staining for P-selectin was found in heart tissue sections from sham-operated rats (Fig. 8G ). In hearts subjected to ischemia and reperfusion (control), staining intensity for ICAM-1 on the endothelium in blood vessels located in the AR had substantially increased (Fig. 8E ). These vessels also exhibited positive staining for P-selectin (Fig. 8H ). A reduced staining for ICAM-1 and no staining for P-selectin were found in the AR of hearts obtained from rats pretreated with 15D-PGJ2 and subsequently subjected to ischemia and reperfusion (Fig. 8F, I , respectively). To verify the binding specificity for ICAM-1 or P-selectin, some sections were incubated with only the primary antibody (no secondary) or only the secondary antibody (no primary). In these experiments, no positive staining was found in the sections, indicating that the immunoreaction was positive in all the experiments carried out.

Effect of 15D-PGJ2 on I{kappa}B-{alpha} degradation
To elucidate whether regional myocardial ischemia and reperfusion leads to the degradation of I{kappa}B{alpha} (and hence the activation of NF-{kappa}B), we investigated the cytosolic levels of I{kappa}B{alpha} by immunoblotting analysis. A basal level of I{kappa}B{alpha} was detectable in the cytosolic fraction of biopsies obtained from the AR of sham-operated rats (Fig. 10 ), whereas in hearts subjected to ischemia and reperfusion, I{kappa}B-{alpha} levels were significantly reduced (Fig. 10) . 15D-PGJ2 pretreatment significantly prevented I{kappa}B{alpha} degradation after ischemia and reperfusion (Fig. 10) .



View larger version (48K):
[in this window]
[in a new window]
  		Figure 10. Western blot analysis showing the effect of 15-deoxy-{Delta}12,14-PGJ2 on I{kappa}B-{alpha} degradation in rat heart tissues (A) and related densitometric analysis (B). Sham: basal level of I{kappa}B{alpha} in cytosolic fractions of biopsies obtained from the area at risk of sham-operated rats. I/R: in the ischemic-reperfused myocardium, the intensity of the band corresponding to I{kappa}B-{alpha} protein is significantly reduced compared to the sham-operated group. 15d-PGJ2: Degradation of I{kappa}B-{alpha} caused by ischemia and reperfusion is significantly attenuated by pretreatment of rats with 15d-PGJ2. Each immunoblotting is from a single experiment and is representative of three separate experiments. Densitometry results are expressed as mean ± SE of three separate experiments. **P < 0.01 vs. sham-operated group, P < 0.001 vs. ischemia-reperfusion group.

Effects of 15D-PGJ2 on the expression of heme-oxygenase-1 (HO-1) mRNA in human cardiac myoblasts: a comparison with rosiglitazone
To elucidate whether 15D-PGJ2 or rosiglitazone cause the expression of the cardioprotective enzyme HO-1, we investigated the effects of 15D-PGJ2 and rosiglitazone on the expression of the mRNA of HO-1 and GAPDH (housekeeping gene). Compared to cardiac myoblasts that had not been exposed to the cyclopentanone prostaglandin, 15D-PGJ2 caused a concentration-dependent increase in the mRNA for HO-1. In contrast, rosiglitazone had no effect on the expression of the mRNA for HO-1 (Fig. 11 ).



View larger version (42K):
[in this window]
[in a new window]
  		Figure 11. The expression of heme-oxygenase-1 (HO-1) in human cardiac myoblasts. 15d-PGJ2 but not rosiglitazone produces a concentration-dependent increase in the levels of hemoxygenase in human cardiac myoblasts. Concentrations of 15d-PGJ2 and rosiglitazone used were 1, 2.5, and 10 µM. Parallel amplification of rat housekeeping gene GAPDH was performed as a positive control.

Role of HO-1 in the cardioprotective effects of 15D-PGJ2
To investigate whether expression of HO-1 contributes to the cardioprotective effects of 15D-PGJ2, we investigated whether zinc protoporphyrin IX (ZnPP IX), an inhibitor of HO-1, attenuates the cardioprotective effects afforded by 15D-PGJ2. Mean values for the AR in this separate study were similar in all animal groups and ranged from 49 ± 2 to 58 ± 2% (P>0.05, Fig. 12 ). In rats that received the vehicle for the cyclopentanone prostaglandin (10% DMSO), occlusion of the LAD for 25 min followed by reperfusion for 2 h resulted in an infarct size of 54 ± 5% (n=6) of the AR. Pretreatment of rats with ZnPP IX did not affect the infarct size in vehicle-treated rats (Fig. 12) . When compared with vehicle, administration of 15D-PGJ2 (0.3 mg/kg i.v. bolus administration 30 min before the onset of myocardial ischemia) caused a significant reduction in myocardial infarct size (Fig. 12) . Pretreatment of rats with ZnPP IX abolished the cardioprotective effects of 15D-PGJ2 (Fig. 12) . Sham operation alone did not result in a significant degree of infarction in any of the animal groups studied (<1% of the AR) (Fig. 12) .



View larger version (38K):
[in this window]
[in a new window]
  		Figure 12. A) Area at risk (AR) and B) infarct size (IS) in rats subjected to regional myocardial ischemia (25 min) and reperfusion (2 h). Different groups of animals were subjected to the surgical procedure alone (no left anterior descending coronary artery occlusion, sham n=12) or to left anterior descending (LAD) coronary artery occlusion and reperfusion and treated with either vehicle (10% DMSO control, n=6) or ZnPP IX (0.3 mg/kg, n=7) or 15d-PGJ2 (0.3 mg/kg, n=5) or ZnPP IX 0.3 mg/kg i.v. bolus, 35 min before LAD occlusion plus 15d-PGJ2 0.3 mg/kg i.v. bolus, 30 min before LAD occlusion, n=10). *P < 0.05 when compared to 10% DMSO control. +P < 0.05 when compared to ZnPP IX alone. #P < 0.05 when compared to ZnPP IX plus 15d-PGJ2.

Effects of the PPAR-{alpha} agonists clofibrate and WY 14643 on myocardial infarct size in the rat
Mean values for the AR were similar in all animal groups studied and ranged from 48 ± 4 to 56 ± 2% (P>0.05, Fig. 13 14 ). In rats that received vehicle for the PPAR-{alpha} agonists (10% DMSO, 1 ml/kg i.v. 30 min before the occlusion of the LAD), occlusion of the LAD for 25 min followed by reperfusion for 2 h resulted in an infarct size of 54 ± 5% (n=6) of the AR. Administration of clofibrate (0.3 or 1 mg/kg i.v. bolus administration 30 min before the onset of myocardial ischemia) caused a significant reduction in myocardial infarct size compared with vehicle (Figs. 13 , 14) . A maximum reduction of infarct size of 41% was achieved with 0.3 mg/kg of the PPAR-{alpha} agonist. Compared with vehicle, administration of WY 14643 (0.3 or 1 mg/kg i.v. bolus) caused a dose-related and significant reduction in infarct size of ~44% at the higher dose (Figs. 13 , 14) . Thus, both PPAR-{alpha} agonists caused a significant reduction in myocardial infarct size in the rat in vivo (Figs. 13 , 14) . Coronary artery occlusion and reperfusion caused a progressive fall in mean arterial blood pressure (from a baseline MAP of 146±7 mm/Hg to 99±5 mm/Hg at the end of the 2 h reperfusion period compared with sham-operated animals. Neither clofibrate nor WY 14643 conferred any significant effect on blood pressure (data not shown).



View larger version (30K):
[in this window]
[in a new window]
  		Figure 13. A) Area at risk (AR) and B) infarct size (IS) in rats subjected to regional myocardial ischemia (25 min) and reperfusion (2 h). Different groups of animals were subjected to the surgical procedure alone (no left anterior descending coronary artery occlusion, sham n=12) or to left anterior descending (LAD) coronary artery occlusion and reperfusion and treated with either vehicle (10% DMSO control, n=6) or clofibrate (0.3, n=8; or 1, n=9 mg/kg i.v. bolus, 30 min before LAD occlusion). *P < 0.05 when compared to 10% DMSO control.



View larger version (29K):
[in this window]
[in a new window]
  		Figure 14. A) Area at risk (AR) and B) infarct size (IS) in rats subjected to regional myocardial ischemia (25 min) and reperfusion (2 h). Different groups of animals were subjected to the surgical procedure alone (no left anterior descending coronary artery occlusion, sham n=12) or to left anterior descending (LAD) coronary artery occlusion and reperfusion and treated with either vehicle (10% DMSO control, n=6) or WY 14643 (0.3, n=3; or 1, n=6 mg/kg i.v. bolus, 30 min before LAD occlusion). *P < 0.05 when compared to 10% DMSO control.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We report here that pretreatment of rats 30 min before LAD occlusion with the TZDs rosiglitazone, ciglitazone, and pioglitazone caused a substantial reduction in the infarct size caused by regional myocardial ischemia and reperfusion. The maximal reductions in infarct size afforded by these TZDs (rosiglitazone: ~45%; ciglitazone: ~45% and pioglitazone: ~25%) appear to correlate positively with their potency as PPAR-{gamma} agonists in vitro (8) . These findings suggest that the reduction in myocardial infarct size afforded by these three TZDs is due at least in part to their ability to activate PPAR-{gamma}. There is good evidence that the PGD2 metabolite 15D-PGJ2 is a potent PPAR-{gamma} agonist in vitro and that 15D-PGJ2 may serve as an endogenous PPAR-{gamma} ligand (9) . We discovered that pretreatment of rats with a low dose of 15D-PGJ2 (0.3 mg/kg i.v.) caused a very substantial (~85%) reduction in the infarct size caused by regional ischemia and reperfusion. The cyclopentanone prostaglandin PGA1 is also a PPAR-{gamma} agonist, but is less potent than 15D-PGJ2 (10) . We have discovered that PGA1 (0.3 mg/kg i.v.) also causes a significant reduction in myocardial infarct size. Taken together, these findings support the view that various chemically distinct agonists of PPAR-{gamma} reduce myocardial infarct size in the rat in vivo.

When myocardial infarct size was determined by macromolecular staining with NBT, 15D-PGJ2 afforded the most pronounced reduction in myocardial infarct size. As the reduction in infarct size caused by 15D-PGJ2 was ~85% and hence greater than any other xenobiotic we had used, we wanted to confirm the degree of reduction in infarct size caused by this cyclopentanone prostaglandin by another parameter. There is now good evidence that myocardial cell necrosis leads to the release of cardiac TnT and that TnT is specific for myocardial tissue injury (23) . In humans, serum levels of cardiac TnT rise significantly 3–4 h after the occurrence of cardiac symptoms and can remain elevated for up to 14 days (24) . We document in our study that 25 min of occlusion of the LAD, followed by 2 h of reperfusion, results in a significant increase in the plasma levels of TnT, indicating the development of myocardial cell necrosis. We report that 15D-PGJ2 attenuated the rise in the plasma levels of TnT caused by myocardial ischemia-reperfusion by 70%. This finding clearly confirms that 15D-PGJ2 causes a marked reduction in myocardial infarct size in the rat.

What, then, is the mechanism(s) by which 15D-PGJ2 elicits its cardioprotective effects in vivo? Although 15D-PGJ2 is a potent PPAR-{gamma} agonist, it has been reported that 15D-PGJ2 (unlike the TZDs) also activates PPAR-{alpha}. To elucidate whether activation of PPAR-{alpha} can, in principle, protect the heart against injury, we investigated the effects of two distinct PPAR-{alpha} ligands, namely, clofibrate and WY 14643, on the infarct size caused by regional myocardial ischemia and reperfusion in the rat. We demonstrate for the first time that clofibrate and WY 14643 both cause a significant reduction in myocardial infarct size. The degree of reduction in infarct size afforded by these PPAR-{alpha} agonists (~45%) is similar to the degree of protection afforded by the PPAR-{gamma} agonists studied (~45%). Thus, we propose that 1) activation of either PPAR-{alpha} or PPAR-{gamma} can protect the heart against ischemia-reperfusion injury and 2) activation of both PPAR-{alpha} and PPAR-{gamma} contributes to the substantial cardioprotective effects of 15D-PGJ2. As the cardioprotective effects of 15D-PGJ2 were quite substantial, it is possible that other, non-PPAR-related effects of this cyclopentanone prostaglandin contribute to the observed cardioprotective effects. For instance, there is evidence that 15D-PGJ2 inhibits the activation of the transcription factor NF-{kappa}B by preventing the phosphorylation of IK kinase (IKK) and thus preventing the degradation of I{kappa}B (25). We demonstrate here that regional myocardial ischemia and reperfusion lead to the degradation of I{kappa}B-{alpha} and hence activation of NF-{kappa}B. Pretreatment of rats with 15D-PGJ2 attenuated the degradation of I{kappa}B-{alpha} caused by regional myocardial ischemia and reperfusion. Thus, we propose that the dose of 15D-PGJ2 used in this study was sufficient to inhibit the activation of NF-{kappa}B caused by ischemia and reperfusion in the heart.

As there is limited evidence that agents that inhibit the activation of NF-{kappa}B also reduce myocardial tissue injury (26) , it is possible that any inhibition of the activation of this transcription factor by 15D-PGJ2 may contribute to the cardioprotective effects of this cyclopentanone prostaglandin. Activation of NF-{kappa}B results in the transcription of many proinflammatory genes, including the ones for tumor necrosis factor {alpha}, interleukin 1ß, ICAM-1, P-selectin, iNOS, and MCP-1, to name but a few (27 28 29) . We report here that regional myocardial ischemia and reperfusion in the rat results in expression of the mRNAs for iNOS, MCP-1 (determined by Northern blot analysis), and the proteins of P-selectin and ICAM-1 (by immunofluorescence). We found that pretreatment of rats with 15D-PGJ2 attenuated the expression of iNOS, MCP-1 (mRNA), P-selectin, and ICAM-1 (protein). These findings suggest that 1) regional myocardial ischemia and reperfusion results in the activation of NF-{kappa}B and subsequent expression of proinflammatory genes and 2) 15D-PGJ2 attenuates the activation of NF-{kappa}B and ultimately the expression of downstream target genes.

Upon its detection by immunofluorescence, nitrotyrosine formation was initially proposed as a relatively specific marker for detection of the endogenous formation of peroxynitrite (30) . There is, however, recent evidence that other reactions can also induce tyrosine nitration: for instance, the reaction of nitrite with hypochlorous acid and the reaction of myeloperoxidase with hydrogen peroxide can lead to the formation of nitrotyrosine (31) . Increased nitrotyrosine staining is therefore considered an indication of ‘increased nitrosative stress’ rather than a specific marker of the generation of peroxynitrite. We report here that regional myocardial ischemia and reperfusion lead to a substantial increase in the immunofluorescence for nitrotyrosine, which was abolished by 15D-PGJ2. Thus, we propose that reduction of the expression of iNOS protein and activity caused by 15D-PGJ2 contributes to reduction by this agent of the formation of peroxynitrite in the rat heart.

A variety of environmental stresses (including ischemia-reperfusion) lead to expression of heme-oxygenase-1 (HO-1), which results in the formation of the antioxidant bilirubin and carbon monoxide. There is substantial evidence that this up-regulation of HO-1 protects many tissues and organs, including the heart, against subsequent injury by ischemia-reperfusion (32 , 33) . We report here that 15D-PGJ2 causes expression of the cardioprotective protein HO-1 in human cardiac myoblasts in a concentration-dependent fashion. The specific PPAR-{gamma} agonist rosiglitazone did not cause the expression of HO-1 in these cells. These findings suggest that 1) the expression of HO-1 caused by 15D-PGJ2 in human cardiac myoblasts is independent of the activation of PPAR-{gamma}, and 2) up-regulation of HO-1 by 15D-PGJ2 may contribute to the cardioprotective effects of this cyclopentanone prostaglandin in vivo. To test the latter hypothesis, we investigated the effects of ZnPP IX, an inhibitor of HO-1 activity in our model of regional myocardial ischemia and reperfusion in the rat. We demonstrate that pretreatment of rats with ZnPP IX alone does not affect the infarct size caused by ischemia and reperfusion of the heart, suggesting that the expression of endogenous HO-1 afforded by ischemia and reperfusion itself (if any) is not sufficient to protect the heart against ischemia-reperfusion injury. In contrast, ZnPP IX attenuated the cardioprotective effects of 15D-PGJ2, suggesting that 1) 15D-PGJ2 causes the expression of HO-1 protein and activity in the heart and 2) this enhanced expression of HO-1 also contributes to the cardioprotective effects of this cyclopentanone prostaglandin.

In conclusion, this study provides the first evidence that various chemically distinct ligands of PPAR-{gamma} (including the TZDs rosiglitazone, ciglitazone, and pioglitazone as well as the cyclopentanone prostaglandins 15D-PGJ2 and PGA1) cause a substantial reduction of myocardial infarct size in the rat. We also demonstrate that two distinct ligands of PPAR-{alpha} (including clofibrate and WY 14643) also cause a substantial reduction of myocardial infarct size in the rat. The most pronounced reduction in infarct size was observed with 15D-PGJ2. The mechanisms of the cardioprotective effects of 15D-PGJ2 may include 1) activation of PPAR-{alpha}, 2) activation of PPAR-{gamma}, 3) expression of HO-1, and 4) inhibition by of the activation of NF-{kappa}B in the ischemic-reperfused heart. The inhibition by 15D-PGJ2 of the activation of NF-{kappa}B, in turn, results in a reduction of 1) the expression of iNOS and the nitration of proteins by peroxynitrite, 2) the formation of the chemokine MCP-1, and 3) the expression of the adhesion molecule ICAM-1. We speculate that ligands of PPAR-{gamma} and PPAR-{alpha} may be useful in the therapy of conditions associated with ischemia-reperfusion of the heart (e.g., myocardial infarction, heart transplantation, bypass surgery) and other organs (11) . Our findings also imply that TZDs may be useful in reducing ischemic injury in patients with diabetes mellitus, which have a high incidence of coronary heart disease and peripheral vascular disease. While this study was under review, Ti-Yue and colleagues reported that rosiglitazone causes a significant reduction in myocardial infarct size in the rat (34) . Finally, our findings imply that fibrates may protect the heart against ischemia-reperfusion injury.


   ACKNOWLEDGMENTS
 
Part of this work was awarded the Menarini-Academy Cardiovascular Research Award for Basic Science in April 2001 and the Poster Award of the Federation of European Pharmacological Societies in July 2001.

Received for publication October 2, 2001. Revision received March 7, 2002.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Evans, R. M. (1988) The steroid and thyroid hormone receptor superfamily. Science 240,889-895[Abstract/Free Full Text]
  2. Moras, D., Gronemeyer, H. (1998) The nuclear receptor ligand-binding domain: structure and function. Curr. Opin. Cell Biol. 10,384-391[CrossRef][Medline]
  3. Murphy, G. J., Holder, J. C. (2000) PPAR-gamma agonists: therapeutic role in diabetes, inflammation and cancer. Trends Pharmacol. Sci. 21,469-474[CrossRef][Medline]
  4. Desvergne, B., Wahli, W. (1999) Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocr. Rev. 20,649-688[Abstract/Free Full Text]
  5. Spiegelman, B. M. (1998) PPAR-gamma: adipogenic regulator and thiazolidinedione receptor. Diabetes 47,507-514[Abstract]
  6. Lehmann, J. M., Lenhard, J. M., Oliver, B. B., Ringold, G. M., Kliewer, S. A. (1997) Peroxisome proliferator-activated receptors alpha and gamma are activated by indomethacin and other nonsteroidal anti-inflammatory drugs. J. Biol. Chem. 272,3406-3410[Abstract/Free Full Text]
  7. Young, P. W., Buckle, D. R., Cantello, B. C., Chapman, H., Clapham, J. C., Coyle, P. J., Haigh, D., Hindley, R. M., Holder, J. C., Kallender, H., Latter, A. J., Lawrie, K. W., Mossakowska, D., Murphy, G. J., Roxbee, C. L., Smith, S. A. (1998) Identification of high-affinity binding sites for the insulin sensitizer rosiglitazone (BRL-49653) in rodent and human adipocytes using a radioiodinated ligand for peroxisomal proliferator-activated receptor gamma. J. Pharmacol. Exp. Ther. 284,751-759[Abstract/Free Full Text]
  8. Willson, T. M., Brown, P. J., Sternbach, D. D., Henke, B. R. (2000) The PPARs: from orphan receptors to drug discovery. J. Med. Chem. 43,527-550[CrossRef][Medline]
  9. Ricote, M., Li, A. C., Willson, T. M., Kelly, C. J., Glass, C. K. (1998) The peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation. Nature (London) 391,79-82[CrossRef][Medline]
  10. Bishop-Bailey, D. (2000) Peroxisome proliferator-activated receptors in the cardiovascular system. Br. J. Pharmacol. 129,823-834[CrossRef][Medline]
  11. Nakajima, A., Wada, K., Miki, H., Kubota, N., Nakajima, N., Terauchi, Y., Ohnishi, S., Saubermann, L. J., Kadowaki, T., Blumberg, R. S., Nagai, R., Matsuhashi, N. (2001) Endogenous PPAR gamma mediates anti-inflammatory activity in murine ischemia-reperfusion injury. Gastroenterology 120,460-469[CrossRef][Medline]
  12. Wayman, N. S., McDonald, M. C., Threadgill, M. D., Thiemermann, C. (2001) 5-Amino isoquinoline, a potent inhibitor of poly (adenosine 5'-diphosphate ribose) polymerase, reduces myocardial infarct size. Eur. J. Pharmacol. 430,93-100[CrossRef][Medline]
  13. Thiemermann, C., Zacharowski, K. (2000) Selective activation of EP3 receptors reduces myocardial infarct size: a novel insight into the cardioprotective effects of prostaglandins. Pharmacol. Ther. 87,61-67[CrossRef][Medline]
  14. Nachlas, M.M., Shnitka, T. K. (1963) Macroscopic identification of early myocardial infarcts by alterations in dehydrogenase activity. Am. J. Pathol. 42,379-405[Medline]
  15. Horio, T., Nishikimi, T., Yoshihara, F., Nagaya, N., Matsuo, H., Takishita, S., Kangawa, K. (1998) Production and secretion of adrenomedullin in cultured rat cardiac myocytes and nonmyocytes: stimulation b