Published online before print May 27, 2008, doi: 10.1161/CIRCULATIONAHA.107.743997
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(Circulation. 2008;117:2977-2985.)
© 2008 American Heart Association, Inc.
Molecular Cardiology |
Reduction in Myocardial Ischemia/Reperfusion Injury in Group X Secretory Phospholipase A2–Deficient Mice
From the Department of Internal Medicine II (D.F., Y.S., T.K., T.Y., T.N., J.O., M.K., K. Kawabata, K. Kugiyama) and Laboratory Animal Support Section (H.T.), Center for Life Science Research, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi; and Shionogi Research Laboratories, Shionogi and Co Ltd, Osaka (Y.I., N.S., Y.Y., K.H.), Japan.
Correspondence to Kiyotaka Kugiyama, MD, PhD, Department of Internal Medicine II, University of Yamanashi, Faculty of Medicine, 1110 Shimokato, Chuo City, Yamanashi, 409-3898 Japan. E-mail kugiyama{at}yamanashi.ac.jp
Received October 4, 2007; accepted April 11, 2008.
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Abstract |
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Background— Group X secretory phospholipase A2 (sPLA2-X) has the most potent hydrolyzing activity toward phosphatidylcholine and elicits a marked release of arachidonic acid among several types of sPLA2. sPLA2-X is expressed in neutrophils, but its pathogenic role remains unclear.
Methods and Results— We generated mice that lack sPLA2-X and studied their response to myocardial ischemia/reperfusion. The sPLA2-X–/– mice had a significant reduction in myocardial infarct size and a decrease in myocardial myeloperoxidase activity compared with sPLA2-X+/+ mice. Myocardial infarct size was also significantly reduced in lethally irradiated sPLA2-X+/+ mice reconstituted with sPLA2-X–/– bone marrow compared with sPLA2-X+/+ bone marrow. The extent of myocardial ischemia/reperfusion injury was comparable between sPLA2-X–/– and sPLA2-X+/+ mice in Langendorff experiments using isolated hearts and blood-free perfusion buffer, supporting a potential role of sPLA2-X in blood in myocardial ischemia/reperfusion injury. In the infarcted myocardium of sPLA2-X+/+ mice, sPLA2-X was released from neutrophils but not myocardial tissues and platelets and was undetectable in the peripheral serum. The sPLA2-X–/– mice had lower accumulation of neutrophils in ischemic myocardium, and the isolated sPLA2-X–/– neutrophils had lower release of arachidonic acid and attenuated cytotoxic activities including respiratory burst compared with sPLA2-X+/+ neutrophils. The attenuated functions of sPLA2-X–/– neutrophils were reversible by the exogenous addition of sPLA2-X protein. Furthermore, administration of a sPLA2 inhibitor reduced myocardial infarct size and suppressed the cytotoxic activity of sPLA2-X+/+ neutrophils.
Conclusions— Myocardial ischemia/reperfusion injury was attenuated in sPLA2-X–/– mice partly through the suppression of neutrophil cytotoxic activities.
Key Words: myocardial infarction • polymorphonuclear leukocytes • secretory phospholipase A2
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Introduction |
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Phospholipases A2 (PLA2s) are ubiquitous enzymes that hydrolyze the sn-2-acyl bond of phospholipids of cell membrane and lipoproteins and yield free fatty acids and lysophospholipids, precursors of various bioactive lipid mediators.1–4 Among free fatty acids, arachidonic acid (AA) is known as a precursor of eicosanoids that include prostaglandins, thromboxanes, and leukotrienes.1–4 At present, a number of PLA2s have been identified and classified into different families on the basis of their biochemical features and primary structures. Among them, secretory PLA2s (sPLA2s) are the secreted enzymes that possess several characteristic features, including a low molecular mass (13 to 18 kDa) and an absolute catalytic requirement for millimolar concentrations of Ca2+.1–7 On the basis of the primary structures, human sPLA2s are now classified into 9 different groups (IB, IIA, IID, IIE, IIF, III, V, X, and XII).1–7 sPLA2s exhibit tissue- and species-specific expression, which suggests that their cellular functions may differ.1–3,6,8,9 The recently cloned group X sPLA2 (sPLA2-X), one of sPLA2s, possesses several characteristics distinct from other sPLA2s.1–3,7,10–12 Among the human sPLA2s, sPLA2-X has the most potent hydrolyzing activity toward phosphatidylcholine and elicits a marked release of AA from various intact cell membranes independently of cytosolic PLA2 (cPLA2).10–12 sPLA2-X is expressed in various inflammatory cells including neutrophils.11,13 However, the pathogenic role of sPLA2-X in neutrophil-related tissue injury remains unclear.
Clinical Perspective p 2985
We have previously shown that plasma levels of sPLA2-IIA are a strong predictor of coronary artery disease.14 During myocardial ischemia/reperfusion, neutrophils migrate through the endothelium of myocardial vascular beds to extravascular space where the activated neutrophils release a barrage of cytotoxic products, such as reactive oxygen species and proteolytic enzymes, leading to myocardial injury.15,16 AA and its lipid metabolites mediated by PLA2 in neutrophils themselves have been shown to contribute to the activation of neutrophils during myocardial ischemia/reperfusion.17–19 Furthermore, the secreted sPLA2-X is capable of exerting cytotoxic activities on neighboring cells.20 In the present study, we generated mice that lack sPLA2-X (sPLA2-X–/– mice) and studied their response to myocardial ischemia/reperfusion to investigate the potential role of sPLA2-X in myocardial ischemia/reperfusion injury.
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Methods |
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Materials
LY374388 ([3-aminooxalyl-1-benzyl-2-ethyl-6-methyl-1H-indol- 4-yloxy]-acetic acid methyl ester) is a prodrug of LY329722 (sodium [3-aminooxalyl-1-benzyl-2-ethyl-6-methyl-1H-indol-4-yloxy]-acetic acid), an inhibitor of sPLA2 activity. For details, see the online-only Data Supplement.
Mice
The experimental protocol was approved by the University of Yamanashi Animal Care and Use Committee, and procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (1996). All sPLA2-X–/– mice analyzed were male and from F13 to F15, and the littermate sPLA2-X+/+ male mice were used as controls. For details, see the online-only Data Supplement.
Construction of Targeting Vector and Generation of sPLA2-X–Deficient Mice
The mouse sPLA2-X gene was cloned from a 129 SVJ genomic library (Stratagene) with the use of oligo DNA, with 5'-tacacctgtcactctcctatgtgtg-3' of mouse sPLA2-X as a probe. The 2.8-kb XbaI-AccII and 3.0-kb PstI-PstI genomic fragments derived from the isolated clone were used for the construction of the targeting vector (Figure I in the online-only Data Supplement). Please refer to the online-only Data Supplement.
Bone Marrow Transplantation Experiment
The sPLA2-X+/+ mice (7 to 8 weeks of age) were lethally irradiated with 8 Gy. Immediately after irradiation, 5x106 bone marrow cells from sPLA2-X+/+ mice or sPLA2-X–/– mice were injected into the irradiated sPLA2-X+/+ mice through the tail vein. The resulting chimeric mice were subjected to the myocardial ischemia/reperfusion experiments in vivo at 6 weeks after the transplantation. For details, see the online-only Data Supplement.
Measurements of mRNA and Protein Expression Levels
Total RNA was extracted from tissues and cells with a Qiagen RNeasy kit and DNase I (Qiagen, Tokyo, Japan). The polymerase chain reaction primers used are listed in Table I in the online-only Data Supplement. For details, see the online-only Data Supplement.
Enzyme-Linked Immunosorbent Assay for sPLA2-X and Immunostaining of Mouse Hearts
sPLA2-X concentrations were measured as described previously.11 In this assay, the purified recombinant mouse sPLA2-X was used as a standard protein that was detectable in the range of 1 pg/mL to 20 ng/mL. The immunostaining was performed by the ABC technique (Vectastain ABC kit, Vector Laboratories, Burlingame, Calif) and a sequential double-labeling method with the use of anti-human sPLA2-X polyclonal antibody and anti-mouse neutrophil polyclonal antibody (Cedarlane, Ontario, Canada).11 For immunofluorescence, a part of the myocardial sections was incubated with the primary antibodies followed by the secondary antibodies. The primary antibodies included anti-
-actinin antibody (Sigma, Tokyo, Japan), anti-human sPLA2-X polyclonal antibody, and anti-mouse neutrophil polyclonal antibody. The secondary antibodies for the immunofluorescence study included Alexa Fluor 488, Alexa Fluor 546, and Alexa Fluor 647 (Invitrogen, Carlsbad, Calif). For details, see the online-only Data Supplement.
Myocardial Ischemia and Reperfusion In Vivo and Assessment of Area at Risk and Infarct Size
The sPLA2-X+/+ mice, the sPLA2-X–/– mice, and the chimeric mice were subjected to 1 hour of myocardial ischemia and 24 hours of reperfusion. When the effect of LY374388, a prodrug of LY329722 (an inhibitor of sPLA2 activity), on myocardial infarct size was examined in the sPLA2-X+/+ mice, LY374388 or vehicle was administered once intraperitoneally at a dose of 100 mg/kg 1 hour before the ischemia or at the beginning of the reperfusion. For details, see the online-only Data Supplement.
In Vivo Transthoracic Echocardiography
M-mode echocardiography was performed at baseline and after 1 hour of ischemia followed by 24 hours of reperfusion. For details, see the online-only Data Supplement.
Langendorff Experiment
The exercised hearts were mounted on a Langendorff perfusion system at 37°C and perfused with blood-free Krebs-Henseleit buffer. The hearts were subjected to no-flow global normothermic ischemia followed by reperfusion. Parameters of cardiac function were monitored at baseline and during ischemia/reperfusion. Finally, infarct size was measured. For details, see the online-only Data Supplement.
Measurement of Myeloperoxidase Activity and Neutrophil Counts in Infarcted Hearts
The myeloperoxidase assay procedures are similar to methods described in our previous report.21 Please refer to the online-only Data Supplement.
Preparations of Neutrophils and Platelets and Assays of Neutrophil Functions
Neutrophils were isolated from the peritoneal fluid and the bone marrow. Neutrophil functions, including migration, respiratory burst, elastase release, and AA release, were measured. For details, see the online-only Data Supplement.
Preparation and Culture of Mouse Cardiomyocytes and Effects of Recombinant Mouse sPLA2-X Protein on Cultured Mouse Myocardial Cells
Primary cultures of adult mouse cardiomyocytes were prepared by collagenase digestion from the ventricles of 10- to 14-week-old sPLA2-X+/+ mice. Please refer to the online-only Data Supplement.
Statistical Analysis
Results are expressed as mean±SEM. Mean values were compared between 2 groups with an unpaired t test; comparisons of means before and after ischemia/reperfusion were made with the paired t test. The comparison among 
3 groups was performed by 1-way ANOVA with the Scheffé F procedure for post hoc analysis. P<0.05 was considered statistically significant.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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Generation and Characterization of sPLA2-X–/– Mice
We generated mice that lacked sPLA2-X on a C57BL/6J background (Figure I in the online-only Data Supplement). Homozygous sPLA2-X–/– mice were born at the expected Mendelian frequency. The sPLA2-X–/– mice were viable, fertile, and appeared healthy. Necropsy and microscopic examination of major tissues revealed no significant pathology in the sPLA2-X–/– mice. No significant difference was found in body weight, heart size, heart rate, blood pressure, complete blood count, and percentages of neutrophils in peripheral blood and bone marrow between the sPLA2-X+/+ and sPLA2-X–/– mice (Table II in the online-only Data Supplement). Expression levels of mRNA for cPLA2, sPLA2-V, and PLA2-VI in either the myocardium or isolated neutrophils were similar between the sPLA2-X+/+ and sPLA2-X–/– mice (Figure II in the online-only Data Supplement). The sPLA2-IIA is naturally disrupted by a frameshift mutation in C57BL/6J background mice.8
Myocardial Infarct Size and Echocardiography
In ischemia/reperfusion experiments in vivo, the infarct size/area at risk and the infarct size/left ventricular (LV) mass were significantly smaller in the sPLA2-X–/– mice than in the sPLA2-X+/+ mice (Figure 1A). Echocardiographic parameters at baseline were comparable between the 2 genotypes of mice. Fractional shortening (FS) after myocardial ischemia/reperfusion injury decreased less in the sPLA2-X–/– mice than in the sPLA2-X+/+ mice (Figure 1B). The myocardial infarct size was also significantly reduced in the lethally irradiated sPLA2-X+/+ mice reconstituted with sPLA2-X–/– bone marrow compared with the irradiated sPLA2-X+/+ mice reconstituted with sPLA2-X+/+ bone marrow (Figure 1C). However, in Langendorff experiments using isolated hearts and blood-free perfusion buffer, the infarct size/LV mass and the LV functional parameters after ischemia/reperfusion were comparable between the 2 genotypes of mice (Figure 1D and Table III in the online-only Data Supplement).
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P<0.05 compared with the respective baselines. *P<0.01. C, Bone marrow transplant experiments. The irradiated recipient sPLA2-X+/+ mice were transplanted with bone marrow cells from sPLA2-X+/+ mice (+/+
+/+, black bars) or sPLA2-X–/– mice (–/–
Expression and Serum Concentrations of sPLA2-X in sPLA2-X+/+ Mice
sPLA2-X mRNA and protein were expressed in isolated neutrophils, whereas they were not detectable in the sham-operated hearts of sPLA2-X+/+ mice (Figure 2A). Immunoblot analysis and enzyme-linked immunosorbent assay showed that sPLA2-X was also detectable in the extracellular supernatant of N-formyl-methionyl-leucyl-phenylalanine (fMLP)–stimulated neutrophils but neither in the supernatant of adenosine diphosphate–stimulated platelets (<1 pg/mL) nor in the circulating peripheral serum after myocardial ischemia/reperfusion (<1 pg/mL) in sPLA2-X+/+ mice (Figure 2B and 2C). Both immunohistochemical light microscopy and confocal fluorescence microscopy showed that the immunoreactivity of sPLA2-X was detected in the neutrophils in the ischemic myocardium and in the myocardial area where neutrophils accumulated (Figure 3).
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Myeloperoxidase Activity and Number of Neutrophils in Ischemic Myocardium
Increase in myeloperoxidase activity in ischemic myocardium was significantly less in sPLA2-X–/– mice than in sPLA2-X+/+ mice (Figure 4A). The number of neutrophils in the ischemic myocardium was also less in sPLA2-X–/– mice than in sPLA2-X+/+ mice (Figure 4B).
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Functions of Isolated Neutrophils
Neutrophils isolated from sPLA2-X–/– mice had a lower respiratory burst and reduced migration in response to C5a or fMLP and less elastase release in response to serum-opsonized zymosan (OZ) than neutrophils from sPLA2-X+/+ mice (Figures 5, 6A, and 6
B). The respiratory burst and the migration in response to A23187 (2 µmol/L) were also reduced in sPLA2-X–/– neutrophils compared with sPLA2-X+/+ neutrophils (respiratory burst: 0.51±0.1 versus 1.1±0.1 counts [x106], respectively; n=5 in each experiment; P=0.02; migration: 18±0.2 versus 38±0.2 neutrophils per high-power field, respectively; n=5 in each experiment; P=0.01). However, neutrophil function in response to AA (1 µmol/L) and phorbol-12-myristate-13-acetate (PMA) (10 µmol/L) was comparable between sPLA2-X–/– and sPLA2-X+/+ mice (Figures 5 and 6A). The attenuated function of sPLA2-X–/– neutrophils was reversed by the addition of mouse sPLA2-X protein (0.1 to 1 ng/mL) (Figures 5 and 6A), which alone at these doses did not induce a respiratory burst or migration. The addition of LY329722, an inhibitor of sPLA2 activity, suppressed sPLA2-X+/+ neutrophil responses to C5a, fMLP, or OZ (Figures 5, 6A, and 6B). Neutrophils isolated from bone marrow in sPLA2-X–/– mice also had a suppressed respiratory burst in response to fMLP compared with those from sPLA2-X+/+ mice (2.2±0.1 versus 4.8±0.2 counts [x105], respectively; n=5 in each experiment; P=0.01). Extracellular release of AA in response to C5a or fMLP was lower in sPLA2-X–/– neutrophils than in sPLA2-X+/+ neutrophils (Figure 6C). LY329722 suppressed the AA release from sPLA2-X+/+ neutrophils (Figure 6C). Expression levels of mRNA of NADPH oxidase p47phox and p67phox, elastase, CD11b/18, and cyclooxygenase-2 were comparable between sPLA2-X–/– and sPLA2-X+/+ neutrophils (Figure III in the online-only Data Supplement).
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Cytotoxic Effects of Recombinant Mouse sPLA2-X Protein on Cultured Mouse Myocardial Cells
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay showed that incubation with sPLA2-X protein dose-dependently injured cultured cardiomyocytes, in association with the increase in lactate dehydrogenase (LDH) concentration in the culture medium and the release of AA from the cardiomyocytes (Figure 7A to 7C). Furthermore, sPLA2-X protein aggravated the hypoxic injury of the cultured cardiomyocytes in the lower concentrations of sPLA2-X protein (Figure 7D). The cytotoxic effect of sPLA2-X was comparable between the cultured myocardial cells from sPLA2-receptor–/– mice and the littermate sPLA2-receptor+/+ mice (Figure 7A and 7B). Moreover, LY329722, an inhibitor of sPLA2 activity, suppressed the cytotoxic effect of sPLA2-X protein (Figure 7D).
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Effects of LY374388 in sPLA2-X+/+ Mice
Both the infarct size/LV mass and the infarct size/area at risk were significantly smaller in the mice treated with LY374388, a prodrug of LY329722, either 1 hour before myocardial ischemia or at the time of reperfusion compared with vehicle-treated mice (Figure 8A). In addition, echocardiography showed that LVFS decreased less in LY374388-treated than in vehicle-treated mice (Figure 8B). Myeloperoxidase activity and number of neutrophils in ischemic myocardium were decreased in LY374388-treated more than in vehicle-treated mice (Figure 4).
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Discussion |
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The present study demonstrated that myocardial ischemia/reperfusion injury was reduced in sPLA2-X–/– mice. Moreover, myocardial ischemia/reperfusion injury was also reduced in the irradiated sPLA2-X+/+ mice reconstituted with bone marrow from sPLA2-X–/– mice. The extent of myocardial ischemia/reperfusion injury was comparable between sPLA2-X–/– and sPLA2-X+/+ mice in Langendorff experiments using isolated hearts and blood-free perfusion buffer, supporting the concept that a deficiency in sPLA2-X in blood constituents may contribute to the reduction in myocardial ischemia/reperfusion injury in sPLA2-X–/– mice. The present study showed that sPLA2-X was expressed in neutrophils in the ischemic myocardium of sPLA2-X+/+ mice and that sPLA2-X was not expressed in either the sham-operated myocardial tissues or platelets and was undetectable in the circulating peripheral serum in sPLA2-X+/+ mice. Furthermore, the sPLA2-X–/– mice had a lower accumulation of neutrophils in ischemic myocardium, and the sPLA2-X–/– neutrophils had a reduction in cytotoxic activities such as respiratory burst and elastase release. Taken together, the suppression of the migratory and cytotoxic actions of neutrophils may account for the reduction in myocardial ischemia/reperfusion injury in sPLA2-X–/– mice. Moreover, the present study showed that the exogenous administration of sPLA2-X protein caused cell injury in the cultured cardiomyocytes, suggesting that sPLA2-X secreted from the activated neutrophils also might participate in myocardial ischemia/reperfusion injury in a paracrine manner.20 Therefore, sPLA2-X in neutrophils plays an important role in the pathogenesis of myocardial ischemia/reperfusion injury. Various types of PLA2 have long been implicated to have a crucial role in degradation of membrane phospholipids during myocardial ischemia/reperfusion injury,17–19,22 but the precise subtype of PLA2 in these phenomena remains undetermined.23 The present study using sPLA2-X–deficient mice reveals a specific role of sPLA2-X in myocardial ischemia/reperfusion injury. It has been shown that sPLA2-X is also expressed in macrophages.24 Although the recruited macrophages within the ischemic myocardium mainly contribute to the healing process of the infarcted myocardium,25 it remains to be determined whether the functions of sPLA2-X–/– monocytes/macrophages may also be altered.
The intracellular pathways by which sPLA2s are involved in the cell activations are complex and are interrelated with multiple intracellular signals1–3,7,11–13; therefore, the precise mechanisms by which the neutrophil activities in response to the external stimuli, eg, C5a, fMLP, OZ, or A23187, were suppressed in the sPLA2-X–/– mice remain unclear. However, the present study showed that the neutrophil activities in response to PMA or AA, which are capable of directly stimulating respiratory burst and migration,26,27 were not suppressed in sPLA2-X–/– neutrophils. Furthermore, expression levels of NADPH oxidase p47phox and p67phox, elastase, CD11b/18, and cyclooxygenase-2 were comparable between sPLA2-X–/– and sPLA2-X+/+ neutrophils. These results suggest that the machinery for respiratory burst and migration seems to be preserved in sPLA2-X–/– neutrophils.
sPLA2-X in neutrophils is confined in intracellular granules13 that secrete their protein contents in response to external stimuli in a cell surface receptor–dependent or –independent manner. The secreted sPLA2-X releases AA from the outer surface of the plasma membrane of the activated neutrophils. In addition, sPLA2-X prior secretion produces AA in the intracellular space in the stimulated mammalian cells.28 A number of reports have shown that AA and its lipid metabolites, known as eicosanoids, in turn participate in the further activation of neutrophils after the external stimuli.13,29 This positive feedback mechanism may amplify the sequence of cytotoxic activation of neutrophils through free radicals and proteolytic enzymes. These activation mechanisms may be weakened in sPLA2-X–/– neutrophils.
The present study showed that LY329722 and its prodrug, LY374388, inhibitors of sPLA2 activity, were capable of reducing myocardial ischemia/reperfusion injury as well as activities of neutrophils. However, these inhibitors are not specific inhibitors of sPLA2-X, but they also inhibit other sPLA2s such as sPLA2-V. Because other sPLA2s as well as sPLA2-X can also be involved in myocardial ischemia/reperfusion injury, the inhibition of sPLA2-X and other sPLA2s might be therapeutically useful for acute myocardial infarction. sPLA2-X can exert various biological responses by binding to the cell surface sPLA2-receptor independently of its enzymatic activity.24,30 However, the enzymatic activity seemed to be required for sPLA2-X–related myocardial injury and activities of neutrophils on the basis of present experiments using sPLA2-receptor–/– cardiomyocytes or LY329722, an inhibitor of sPLA2 activity. Although no previous report indicates species-associated difference in the expression profiles of sPLA2-X between humans and mice, sPLA2-IIA is expressed in myocardium of humans but not C57BL/6J mice.8,9 The species difference in the expression profiles and the activities of some sPLA2s should be noted for the translation studies.2,3,5 Previous animal studies showed that the anti-neutrophil interventions did not consistently reduce myocardial infarct size.31 Furthermore, previous clinical trials32,33 showed that monoclonal antibodies to CD11/CD18 failed to reduce myocardial infarct size in patients with acute myocardial infarction. However, the mechanisms of neutrophil activation are very complex, involving multiple signaling pathways, and these negative studies do not necessarily deny the possibility of the translation of the present data to humans because the effects of the anti-neutrophil interventions used in the previous negative studies on the sPLA2-X secretion in human neutrophils remain unknown.
The cytotoxic activities of neutrophils play a critical role in ischemia/reperfusion syndromes in other organs as well as myocardium. Therefore, the inhibition of sPLA2-X may be also useful for ischemic tissue injury and inflammation. In conclusion, myocardial ischemia/reperfusion injury was attenuated in sPLA2-X–/– mice partly through the suppression of neutrophil cytotoxic activities.
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Acknowledgments |
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We gratefully acknowledge the technical assistance of M. Hayashi and A. Watanabe.
Sources of Funding
This study was supported by grants-in-aid for (B)(2)-15390244 and (B)-19390209 and Priority Areas (C) Medical Genome Science 15012222 from the Ministry of Education, Culture, Sports, Science, and Technology; and Health and Labor Sciences Research Grants for Comprehensive Research on Aging and Health (H15-Choju-012), Tokyo, Japan.
Disclosures
Yoshikazu Ishimoto, Noriko Suzuki, Yasunori Yokota, and Kohji Hanasaki are employees of Shionogi Research Laboratories, Shionogi and Co Ltd (Osaka, Japan). They contributed to the development of sPLA2-X–/– mice and sPLA2-receptor–/– mice, but they did not contribute to data on the effects of sPLA2 inhibitors (LY329722 and its prodrug, LY374388). The rights of these sPLA2 inhibitors gifted from Shionogi have now been out-licensed to another company. The other authors report no conflicts.
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The online-only Data Supplement, which contains expanded Methods, tables, and figures, is available with this article at http://circ.ahajournals.org/ cgi/content/full/CIRCULATIONAHA.107.743997/DC1.
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