Inactivation During Inflammation-Resolution Resolvin

The Journal of Immunology

Resolvin E1 Metabolome in Local Inactivation during Inﬂammation-Resolution1

Song Hong,2,3 Timothy F. Porter,2 Yan Lu,3 Sungwhan F. Oh, Padmini S. Pillai, and Charles N. Serhan4

Resolvin E1 (RvE1; 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid) is a potent anti-inflammatory and proresolving mediator derived from the omega-3 eicosapentaenoic acid. In this study, we report the RvE1 metabolome, namely, the metabolic products derived from RvE1. RvE1 was converted to several novel products by human polymorphonuclear leukocytes and whole blood as well as in murine inflammatory exudates, spleen, kidney, and liver. The potential activity of each of the newly identified products was directly compared with that of RvE1. The new RvE1 products elucidated included 19-hydroxy-RvE1, 20-carboxy-RvE1, and 10,11-dihydro-RvE1. Metabolomic profiles of RvE1 were species-, tissue-, and cell type-specific. Direct comparisons of the bioactions between isolated RvE1 metabolic products indicated that 10,11-dihydro-RvE1, 18-oxo-RvE1, and 20-carboxy-RvE1 displayed reduced bioactivity in vivo. At concentrations as low as 1 nM, RvE1 enhanced macrophage phagocytosis, a proresolving activity that was reduced by metabolic inactivation. These results document novel metabolic products of RvE1 that impact its actions and that both omega-1 hydroxylation and reduction of conjugated double bonds in RvE1 are new pathways of four main routes of RvE1 metabolism in mammalian tissues. Together, these findings indicate that, during inflammation and its controlled resolution, specific tissues inactivate proresolving signals, i.e., RvE1, to permit the coordinated return to homeostasis. Moreover, the RvE1 metabolome may serve as a biomarker of these processes. The Journal of Immunology, 2008, 180: 3512–3519.

esolution of inflammation is an active process (1) con- cursor eicosapentaenoic acid (EPA) and is a potent local mediator trolled in part by the temporal and spatially regulated that i) reduces inflammation in skin and peritonitis (5), ii) is R formation and inactivation of endogenous mediators (2– formed during colitis (6) and dramatically increases survival of 4). The termination of proresolving signals is also required to bring mice with trinitrobenzene sulfonic acid colitis (7), and iii) reduces the inflamed or injured system back to homeostasis. Recent results ocular neovascularization (8) and protects from periodontal in- have revealed several new families of lipid mediators that are both flammation and bone destruction in rabbits (9). RvE1 is a local anti-inflammatory and proresolving that are generated during the acting autacoid that proved to display proresolving actions when by guest on October 1, 2021. Copyright 2008 Pageant Media Ltd. resolution phase of inflammation; these include lipoxins, resolvins, treatments were given either by topical, i.v., or i.p. routes of ad- and protectins (2). For example, resolvin E1 (5S,12R,18R-trihy- ministration (4, 10). Thus, it is critical to gain an appreciation of 5 droxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid or RvE1) is a the routes and pathways involved in metabolic inactivation of local member of the resolvin family that is biosynthesized from the pre- proresolving signals, such as RvE1, during homeostasis and inflammation-resolution.

Along these lines, the inactivation of lipoxin A4 (LXA4) (11) Center for Experimental Therapeutics and Reperfusion Injury, Department of Anes- and RvE1 involves both carbon position and site-speciﬁc dehy- thesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115 drogenation as routes of metabolic inactivation (12). Protection of

http://classic.jimmunol.org Received for publication July 20, 2007. Accepted for publication December 26, 2007. RvE1 from rapid dehydrogenation and/or metabolic inactivation The costs of publication of this article were defrayed in part by the payment of page stabilizes and prolongs its potent in vivo anti-inflammatory actions charges. This article must therefore be hereby marked advertisement in accordance (12). RvE1 is endogenously formed from eicosapentaenoic acid with 18 U.S.C. Section 1734 solely to indicate this fact. via cell-cell interactions when aspirin is administered during in- 1 This work was supported in part by National Institutes of Health Grants DK074448 flammation and/or via cytochrome P450 conversion of its omega-3 and P-50-DE-016191 (to C.N.S.). fatty acid precursor EPA (5, 13). The omega-3 polyunsaturated 2 S.H. and T.F.P. contributed equally to this work and share first authorship.

Downloaded from fatty acids (PUFAs) have long been appreciated for their beneficial 3 Current address: Neuroscience Center, Louisiana State University Health Center, 2020 Gravier Street, Suite D, New Orleans, LA 70112. actions in many human systems, including the immune (14), neural 4 Address correspondence and reprint requests to Dr. Charles N. Serhan, Director, (15, 16) and cardiovascular (17). The molecular mechanisms re- Center for Experimental Therapeutics and Reperfusion Injury, Department of Anes- sponsible, however, for these beneficial actions in human trials thesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital and with supplementation of omega-3 PUFAs remained to be convinc- Harvard Medical School, 75 Francis Street, Boston, MA 02115. E-mail address: [email protected] ingly established (18). In this regard, RvE1 fulfills local mediator 5 Abbreviations used in this paper: RvE1, resolvin E1, 5S,12R,18R-trihydroxy- criteria in that it biosynthesized from precursor EPA and displays 6Z,8E,10E,14Z,16E-eicosapentaenoic acid; LC-UV-MS, liquid chromatography-ul- potent and stereoselective actions both in vitro and in vivo, where traviolet-tandem mass spectrometry; LXA4, lipoxin A4; PMN, polymorphonuclear RvE1 is anti-inflammatory, proresolving, and tissue-protective. leukocyte; PUFA, polyunsaturated fatty acid; Resolvin, resolution phase interaction product; 18-oxo-RvE1; 18-oxo-5S,12R-dihydroxy-6Z,8E,10E,14Z,16E-eicosapenta- For example, RvE1 stops neutrophil infiltration in vivo and trans- enoic acid; EPA, eicosapentaenoic acid; RP-HPLC, reversed phase-HPLC; LTB4, endothelial migration (5), attenuates IL-12 production by dendritic leukotriene B . 4 cells and their mobility toward pathogens (13), and stimulates res- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 olution programs in vivo (10).

www.jimmunol.org The Journal of Immunology 3513

Therefore, it is essential to chart the metabolic conversion of RvE1 and its potential inactivation pathways as well as the bioactivities of RvE1-derived metabolic products. These are needed to appreciate their potential contributions(s) in the local control of inflammation and its resolution. These are also critically required components to fully appreciate the relationship(s) between dietary intake of omega-3 PUFAs and the local biosynthesis of resolvins and protectins in vivo as well as to design RvE1-related therapeutic interventions. In the present report, we investigated the metabolic profiles and pathways for RvE1 in several mammalian tissues and isolated cells. We also determined the anti-inflammatory and proresolving properties of these newly isolated RvE1 metabolic products that together demonstrate the specificity and selectivity of the RvE1 inactivation process. Materials and Methods Materials RvE1 was prepared by total organic synthesis according to published matching criteria (5, 13) and obtained from the Organic Synthesis Core of the National Institutes of Health Center P50-DE016191 (Core Leader Prof. Nicos A. Peta- sis, University of Southern California). Calcium ionophore (A23187) was from Sigma-Aldrich. Radiolabeling of RvE1 was purchased as a custom tri- tiation from American Radiolabeled Chemicals and obtained from catalytic hydrogenation of the provided diacetylenic RvE1 in [3H]gas and isolated in this lab before use via reversed phase-HPLC (RP-HPLC) (as in Ref. 13). Acute inflammation Peritonitis was conducted using 6–8-wk-old male FVB mice (Charles River Laboratories) that were anesthetized with isoflurane. Mice were fed Labora- tory Rodent diet 5001 (LabDiet) containing 0.19% omega-3 fatty acids without additional fatty acid supplementation. RvE1, RvE1-metabolic products, or vehicle was injected into the tail vein (20 ng/mouse). Zymosan A in 1 ml of saline (1 mg/ml) was injected ϳ2 min later into the peritoneum to induce acute inflammation or peritonitis. At 2 h after zymosan administration, the peritoneal lavages were collected and total leukocytes, polymorphonuclear leukocyte (PMN), monocyte, and lymphocyte numbers were enumerated (19), and the lungs, livers, kidneys, and spleens were harvested. Mice were euthanized with isoflurane in accordance with the Harvard Medical Area Standing Committee

by guest on October 1, 2021. Copyright 2008 Pageant Media Ltd. on Animals (Protocol Number 02570). RvE1 metabolism/conversion by cells and tissues Human whole (venous) blood was collected with heparin from deidentiﬁed healthy volunteers (who denied taking medication 2 wk before donation; Brigham and Women’s Hospital Protocol No. 88-02642). Following collec- tion, whole blood (5.0 ml) was immediately incubated with RvE1 (500 ng; 60 min, 37°C). In the meantime, human PMNs (50 ϫ 106 cells) were isolated from the whole blood by Ficoll gradient, then immediately incubated with 500 ng RvE1 and in 0.5 ml PBS ϩ/ϩ with calcium ionophore (A23187, 5 ␮M (pH 7.45), 30 min, 37°C). For incubations with murine resident macrophages, male http://classic.jimmunol.org FVB mice (6–8-wk old) were euthanized with isoﬂurane and the peritonea were lavaged with PBS Ϫ/Ϫ. Harvested cells (7 ϫ 106 cells) were plated on 6-well plates with RPMI 1640 with 10% FBS and incubated at 37°C. After 2 h, the supernatants were removed and adhered cells were washed with PBS ϩ/ϩ two times (70–80% of harvested cells adhered to the plate). Cells were exposed to A23187 (5 ␮M) for 1 min, then 200 ng of RvE1 was added and cells were incubated ((pH 7.45) 37°C). After 2 h, cells were scraped and 2 vol of Ϫ

Downloaded from ice-cold methanol were added; samples were stored at 80°C until C18 solid FIGURE 1. Human PMNs produce novel RvE1-derived metabolites 20- phase extraction. Each type of organ (ϳ200 mg) harvested from mice with peritonitis (2 h) carboxy-RvE1 and 19-hydroxy-RvE1. These new RvE1 products were was gently homogenized on ice (ϳ4°C) then incubated with RvE1 (500 ng) in formed in addition to the recently identified 20-hydroxy-RvE1 (12). PBS ϩ/ϩ (pH 7.45) at 37°C for 60 min. Each incubation was stopped with the Freshly isolated PMNs (50 ϫ 106 cells) were incubated with 500 ng RvE1 addition of 2 vol of ice-cold methanol (19, 20), centrifuged, and the superna- and 0.5 ml PBS ϩ/ϩ with calcium ionophore (A23187, 5 ␮M) ((pH 7.45) tants were extracted using C18 solid phase extraction. 60 min, 37°C). Products were extracted and the metabolic profiles were LC-MS/MS-based mediator lipidomics obtained using an LC-UV-MS/MS with a mobile phase of methanol/water/ acetic acid (v/v/v; 60/40/0.01) (see Materials and Methods). A, Selected Following extraction, isolated materials were taken for analysis of RvE1 ion chromatograms at m/z 379 and 365; MS/MS spectrum of 20-carboxy- and the potential RvE1-derived products using liquid chromatography-UV- RvE1 (B); and MS/MS spectrum of 19-hydroxy-RvE1 (C). tandem mass spectrometry (LC-UV-MS/MS) equipped with a HPLC (P4000) coupled to a photo-diode-array UV detector and an ion trap (LCQ) MS/MS (Thermo Electron) (for further details, see Refs. 5, 21). The mobile phase flowed at 0.2 ml/min using a C18 LC column (Phenomenex Luna 2.1 were extracted as in Ref. 21 and injected into an HPLC-UV (HP1100; mm ϫ 150 mm ϫ 5 ␮m) for these profiles. Agilent) coupled to an ion trap-tandem-mass spectrometer (Q Trap 3200; To monitor endogenous RvE1-related compounds and RvE1-derived Applied Biosystems/Sciex) equipped with a C18 LC column (Agilent metabolites from the time course of acute murine peritonitis, the samples Eclipse Plus, 4.6 mm ϫ 50 mm ϫ 1.8 ␮m) and a mobile phase flow rate 3514 RESOLVIN E1 METABOLOME AND INACTIVATION

FIGURE 2. Human whole blood converts RvE1 to 10,11-dihydro-RvE1. A, Selected ion chromatogram at m/z 351 and m/z 349. B, MS/MS spectrum of 10,11-dihydro-RvE1. Fresh whole human blood (5.0 ml) was incubated with RvE1 (500 ng) ((pH 7.45) 60 min, 37°C). These RvE1 metabolic products were extracted and analyzed using an LC-UV-MS/MS (see Materials and Methods) with mobile phase as methanol/water/acetic acid (v/v/v; 65/35/0.01).

of 0.4 ml/min. The chromatography cycle was 15 min using a mobile phase revealed that ionophore A28137-stimulated PMNs converted of methanol/water/acetic acid (60/40/0.01;v/v/v) that was changed to 80/ RvE1 to novel metabolic products 20-carboxy-RvE1 and 19-hy- 20/0.01 (v/v/v) after 5 min, 95/5/0.01 (v/v/v) after 8 min, and 100/0/0.01 droxy-RvE1, shown in the selected ion chromatograms in Fig. 1A. (v/v/v) to wash the column after 14 min. The metabolic product 20-carboxy-RvE1 was identiﬁed based on Radioactive tracing of RvE1 metabolic proﬁle its MS/MS spectrum at molecular anion m/z 379 (M-H), which

Isolated human PMNs (50 ϫ 106 cells) in 1 ml PBS ϩ/ϩ were exposed to displayed diagnostic fragment ions at m/z 361 (M-H-H2O), 343 zymosan A (1 mg) for 3–5 s and then incubated with 1 ␮g unlabeled RvE1 (M-H-2H2O), 325 (M-H-3H2O), 317 (M-H-H2O-CO2), 297, 275 along with 104 cpm/␮g tritium-labeled RvE1 (6,7,13,14-tetra-tritiated- (293-H2O), 263, 255 (291-2H2O), 237 (293-2H2O), 226 (263- by guest on October 1, 2021. Copyright 2008 Pageant Media Ltd. RvE1) for 40 min ((pH 7.45) 37°C). Extracted materials were subject to 2H O-H), 167 (185-H O), 143 (185 plus 2H-CO ), and 115 (Fig. RP-HPLC equipped with a Luna C18 column (2.1 mm ϫ 150 mm ϫ 5 ␮m; 2 2 2 Phenomenex). The flow rate was set at 0.2 ml/min and collected at 30-s 1B; inset illustrates the main ions). In addition, its UV spectrum ␭ ϭ intervals. Each fraction was mixed with scintillation fluid, and radioactivity displayed max 271 nm, which was indicative of the presence of was counted with a scintillation counter (13). a conjugated triene chromatophore, and its chromatographic reten- Macrophage phagocytosis with RvE1 and its related metabolic tion time was shorter than that of native RvE1 or other RvE1 products metabolites. These properties were consistent with its higher po- larity resulting from the addition of a carboxyl group to the ␻ end The proresolving actions of RvE1 and related products were assessed as in (carbon 20 position) of RvE1. Godson et al. (22). Briefly, exudates from the peritonea of naive mice that http://classic.jimmunol.org were euthanized with isoflurane were collected, and resident macrophages The metabolic product 19-hydroxy-RvE1 was identified based were plated on a 24-well plate (105 cells/well) in PBS ϩ/ϩ and incubated on its physical properties, which included a chromatographic be- for 30 min at 37°C. The compounds to be tested were added to the wells havior with a C18 column of eluting after 20-hydroxy-RvE1 (Fig. at indicated concentrations, and cells were incubated in the dark for 15 min 1A). The UV spectrum with the ␭ ϭ 271 nm obtained for this at 37°C. FITC-labeled zymosan was then added to the wells, which were max incubated again in the dark for 30 min at 37°C. The wells were subse- RvE1-derived product displayed a conjugated triene (Fig. 1C, in- quently aspirated, and extracellular fluorescence was quenched by brief set), and the presence of diagnostic ions in its MS/MS spectrum addition of trypan blue, followed by aspiration and suspension again in was consistent with the structure shown in the inset (Fig. 1C). Downloaded from ϩ ϩ PBS / (pH 7.45). Plates were read using a PerkinElmer Victor (3) plate These ions of diagnostic value present in the MS/MS spectrum reader. (see Fig. 1C, inset) included m/z 365 (M-H), 347 (M-H-H2O), 321

Statistical analysis (320 plus H), 311 (M-H-3H2O), 303 (320-H2O plus H), 291, 283 All results are expressed as mean Ϯ SEM. Statistical signiﬁcance for dif- (320-2H2O-H), 273 (291-H2O), 267 (320-3H2O plus H), 257 (320- ferences between groups was determined using Student’s t test and Fisher’s H2O-CO2-H), 251, 239 (320-2H2O-CO2-H), 221 (320-3H2O-CO2-

protected least signiﬁcant difference. H), 205 (223-H2O), 195, 179 (223-CO2), 170, 161 (223-H2O-

CO2), 123 (141-H2O), and 115. The UV and MS/MS spectrum of Results 20-hydroxy-RvE1 were consistent with the recently reported phys- Human PMN and whole blood generate novel RvE1 metabolic ical properties of this new metabolic product of RvE1 (cf. Ref. 12). products To determine whether RvE1 is converted to metabolic products Because neutrophils play a key role in inflammation and RvE1 is in human blood, we conducted mediator lipidomic LC-UV- biosynthesized via interactions of human endothelial cells and MS/MS analysis with incubations of RvE1 and human blood. One of PMNs (5), we first studied RvE1 conversion with isolated human the new products identified in these incubations was 10,11-dihydro- PMN (Fig. 1). Mediator lipidomic analysis using LC-UV-MS/MS RvE1 (Fig. 2), which eluted slightly later than RvE1 as shown in the The Journal of Immunology 3515 by guest on October 1, 2021. Copyright 2008 Pageant Media Ltd. http://classic.jimmunol.org Downloaded from

FIGURE 3. RvE1 metabolomic proﬁles from human and murine tissues. A, RvE1 (300 ng/ϳ200 mg of tissue) was incubated ((pH 7.45) 60 min, 37°C) with the peritoneal exudates collected from mice with peritonitis 2, 4, 6, or 24 h after i.p. administration of zymosan A (1 mg/mouse). B, Human PMN conversion of [3H]-RvE1: Radioactivity and UV chromatograms showing RvE1 metabolic proﬁle. PMN (50 ϫ 106 cells/ml) were incubated with zymosan 3516 RESOLVIN E1 METABOLOME AND INACTIVATION

selected ion monitoring chromatogram at m/z 351 (M-H) (Fig. 2A). itored levels of endogenous RvE1 and its related precursor and This chromatographic behavior was consistent with its structure hav- metabolic products identiﬁed herein using LC-UV-MS/MS (Fig. ing one fewer double bond than RvE1. Its structure was further sup- 3D). The biosynthetic precursor to RvE1, 18-HEPE, was identiﬁed ported on the basis of the MS/MS spectrum, which contained in exudates in amounts ranging from ϳ50 to 325 pg per mouse in

diagnostic ions at m/z 351 (M-H), 333 (M-H-H2O), 321, 315 both mice with peritonitis and those not exposed to zymosan. The (M-H-2H20), 307 (M-H-CO2), 297 (M-H-3H2O), 293, 289 (M-H- peak level of 18-HEPE was determined to be 2 h after inducing H2O-CO2), 275 (293-H2O), 271 (M-H-2H2O-CO2), 257 (293- inflammation and rapidly declined by 12 h. This time interval co- 2H2O), 253 (M-H-3H2O-CO2), 247 (267-H2O-2H), 235, 231 (293- incides with the period of rapid leukocytic infiltration in to the H2O-CO2), 225, 217 (235-H2O), 213 (231-H2O), 207 (225-H2O), peritoneum (4). These findings suggest that leukocytes appearing 197, 189 (225-2H2O), 179 (197-H2O), 163 (225-H2O-CO2), 145 in exudates in the acute inflammatory phase can rapidly convert (225-2H2O-CO2), 135 (197-H2O-CO2), 123 (125-2H), and 107 18-HEPE in situ. Endogenous RvE1 accumulated at later time ϳ (125-H2O) (Fig. 2B). points, identifiable in trace amounts at 48 h and at 27 pg at 72 h, and other metabolic products were likely below limits of detection Metabolomic profiles of RvE1 with human and mouse tissues in individual exudates. This accumulation of RvE1 at 48 and 72 h To determine the capacity of inflammatory exudates to convert may reflect differences in the cellular exudate composition, as RvE1, we incubated murine inflammatory exudates collected at PMN are the predominant cell type during the initial acute inflam- different time intervals during peritonitis with RvE1. The LC-UV- matory phase of murine peritonitis whereas mononuclear cells are MS/MS analysis indicated that exudates collected at later points the predominant cell type during the later resolution phase. En- during peritonitis converted less RvE1 (Fig. 3A), i.e., the capacity dogenous accumulation of RvE1 at earlier time points in the mu- of inflammatory exudates to metabolize RvE1 apparently declined rine skin air pouch (5), and the temporal differences in the perito- as peritonitis continued from 2–24 h into the resolution phase. neal exudates, likely reflect permeability in the peritoneum vs the During this phase, the total number of cells in exudates, especially skin (cf. Ref. 4, 5). PMN, decreases during resolution and PMN begin to undergo apop- Targeted metabolomic analyses using LC-UV-MS/MS-based tosis (Fig. 3A, left panel). These factors may account, in part, for mediator lipidomics demonstrated that the key products of RvE1 the decreased metabolic capacity to convert RvE1, for example at metabolism in cells and tissues are 20-hydroxy-RvE1, 19-hydroxy- 24 h, in resolving exudates. For the metabolism of tritium-labeled RvE1, 18-oxo-RvE1, 10,11-dihydro-RvE1, and 20-carboxy-RvE1. RvE1 by human PMN, the profile of the radioactive chromatogram These quantitative results for each indicated that omega-1 hy- is plotted as an overlay with the UV chromatogram (Fig. 3B), droxylation and enzymatic reduction of one conjugated carbon- where 20-hydroxy-RvE1 was a major component, thus demon- carbon double-bond in RvE1 are two novel major metabolic path- strating that 20-hydroxy-RvE1 is the major metabolic product in ways that coexist in parallel to the recently delineated pathway, human leukocytes. The radioactive chromatogram represents the namely dehydrogenation of the 18-hydroxy-position of RvE1 (12). complete profile of metabolites from tritium-labeled RvE1 because Specific cytochrome P450 enzymes are known to oxygenate ara- 3 the H in labeled RvE1 (6,7,13,14-tetra-tritiated-RvE1) was re- chidonic acid, leukotriene B4 (LTB4), and prostaglandins to their tained in these products. Also, the use of labeled RvE1 as tracer in respective omega-hydroxy and omega-1-hydroxy metabolites

by guest on October 1, 2021. Copyright 2008 Pageant Media Ltd. these studies provides additional evidence to confirm the conver- (23, 24). Similarly, 20-hydroxy-RvE1 and 19-hydroxy-RvE1 sion of RvE1 to novel compounds. are likely to be P450 products of RvE1, and 20-carboxy-RvE1 To address the pathways involved in metabolic conversion of is likely generated from the further oxidation of the intermedi- RvE1 in different tissues, we determined the quantitative metabo- ate 20-hydroxy-RvE1. lomic profiles of RvE1 in human blood, human PMN, and murine Our earlier results demonstrated that 18-oxo-RvE1 is generated tissues (lung, liver, kidney, or spleen) (Fig. 3C). The major RvE1 from RvE1 via enzymatic dehydrogenation (12). In the present products from human blood were 10,11-dihydro-RvE1, 18-oxo- report, we identified 10,11-dihydro-RvE1 as a key metabolite in RvE1, and 20-hydroxy-RvE1 (Fig. 3C, right panel). However, tissues in vivo. It is likely that RvE1 is converted enzymatically with isolated human PMN, more 20-hydroxy-RvE1 was produced, first to an intermediate 12-oxo-RvE1, then rapidly reduced to http://classic.jimmunol.org followed by 19-hydroxy-RvE1 and 18-oxo-RvE1 (Fig. 3). The 10,11-dihydro-12-oxo-RvE1 that can then be further converted to metabolic products obtained were the same for both murine spleen 10,11-dihydro-RvE1. This may also explain the finding that 10,11- and lung (Fig. 3C, left panel). It is of interest that murine kidney dihydro-RvE1 is a major product in several tissues (Fig. 3). In and liver generated more 18-oxo-RvE1 than other RvE1-derived support of this route of RvE1 metabolism, 10,11-dihydro-12-oxo- metabolic products (Fig. 3C). Along these lines, RvE1 is converted RvE1 (Fig. 4) was also identified from incubations of RvE1 with to 18-oxo-RvE1 in vitro by human recombinant 15-prostaglandin the human THP-1 cell line. To accumulate the transient interme-

Downloaded from dehydrogenase (12). diate, freeze-thaw lysates of THP-1 cells were incubated with To determine the profile and appearance of endogenous RvE1 RvE1 and the cofactor NAD (500 ␮M, 37°C, 30 min) that en- and its metabolic products in inflammatory exudates during the hanced the yield and mass spectral identification (not shown) of established time course of murine peritonitis and its resolution (4), 10,11-dihydro-12-oxo-RvE1. However, we were unable to isolate we obtained peritoneal exudates at specific time intervals and mon- quantities of this intermediate that would permit assessment of the

A (100 ␮g/ml), 1 ␮g unlabeled RvE1, and 104 cpm/␮g tritium-labeled RvE1 (6,7,13,14-tetra-tritiated) in PBS ϩ/ϩ (pH 7.45) for 40 min at 37°C. Extracted materials were separated by RP-HPLC with mobile phase as methanol/water/acetic acid (v/v/v; 55/45/0.01), monitored at 270 nm, and fractions collected at 30-s intervals. C, RvE1 (500 ng) was incubated with: gray bar, human blood (5.0 ml); striped bar, human PMN (50 ϫ 106 cells/ml) ((pH 7.45) 60 min, 37°C), or ϳ200 mg murine tissue (gray bar, lung; black bar, liver; white bar, kidney; or striped bar, spleen) collected at 2 h from mice with peritonitis (after i.p. administration of zymosan A). D, Left panel, Time course of endogenous RvE1 and precursor generation during peritonitis. Inﬂammation was initiated with i.p. administration of zymosan A (1 mg), mice were euthanized, and peritoneal lavages rapidly collected and extracted for LC-UV-MS/MS (see Materials and Methods). Solid line, Total leukocytes; dashed line, 18-HEPE; dotted line, RvE1. Results are representative of n ϭ 3. Right panel, Endogenous RvE1 spectrum obtained from peritonitis at 72 h. The Journal of Immunology 3517

i.v., 20-hydroxy-RvE1 proved to be essentially as potent as RvE1 in reducing infiltration of leukocytes into inflamed peritonea (Fig. 5A) and particularly at stopping PMN infiltration (Fig. 5B). The product 19-hydroxy-RvE1 was significantly less potent than its precursor RvE1. Furthermore, 20-carboxy-RvE1, 18-oxo-RvE1, and 10,11-dihydro-RvE1 were each essentially inactive and did not prevent PMN infiltration. Thus, further metabolism of RvE1 toward the last three metabolites belongs to pathways inactivating RvE1.

RvE1 enhances macrophage phagocytosis: a proresolving action A key and important step in the resolution of acute inﬂammation is the uptake and clearance of apoptotic PMN by macrophages (1,

2). A recently appreciated proresolving action of LXA4 is its potent ability to stimulate the uptake of apoptotic PMN by macro- FIGURE 4. Proposed metabolome for RvE1. Human and murine tissues phages (22, 28) in addition to the now widely documented anti- denotes identiﬁed (ء) convert RvE1 to the illustrated products. The asterisk inﬂammatory actions of LXA (reviewed in Refs. 1, 29–32). proposed intermediates (see text for further details). The stereochemistry of 4 the alcohol at carbon-12 position in the 10,11-dihydro-RvE1 metabolite Recently, LXA4 and RvE1 were each shown to stimulate resolu- remains to be determined. tion in mice as well as stimulate the uptake and clearance of zymosan by macrophages in vivo (10). In this study, we determined whether RvE1 can also stimulate intermediate’s potential biological activity. This pathway of RvE1 isolated macrophages to phagocytize zymosan A. RvE1 proved to further metabolism appears to be similar to the metabolism pro- be a potent agonist of macrophage phagocytosis (Fig. 6). At con-

posed for LTB4 conversion to 10,11-dihydro-LTB4, first identified centrations as low as 0.1 nM, RvE1 enhanced the phagocytic up- in kidney (25). Similar reduction products were identified earlier take of zymosan A (Fig. 6A). We also investigated the time course for prostaglandins (reviewed in Ref. 26) and lipoxins with human of exposure to RvE1 on phagocytosis from 0 to 90 min before monocytes and macrophages (27). Also, when RvE1 was incu- zymosan A (Fig. 6A). The increase in RvE1-stimulated phagocy- bated with isolated murine resident peritoneal macrophages, the tosis was at 15 min. Consistent with our results from the time major product identified using LC-MS/MS proved to be 10,11- course of endogenous production in murine peritonitis (Fig. 3D), dihydro-RvE1, indicating that both human and mouse isolated we found that RvE1’s actions diminished with increasing time of macrophages use this pathway for RvE1 (not shown). The pro- exposure, suggesting that RvE1 is rapidly converted to metabolic posed metabolic routes and pathways in the RvE1 metabolome are products by resident peritoneal macrophages during phagocytosis.

depicted in the scheme shown in Fig. 4. For comparison, the levels obtained with LXA4 were also exam- ined. RvE1 was approximately twice as active in this system as

by guest on October 1, 2021. Copyright 2008 Pageant Media Ltd. Anti-inflammatory activities of RvE1 and related metabolic LXA4 when compared at equal molar concentrations. products Next, we directly compared RvE1 and its metabolic products To establish whether the newly isolated and identified RvE1 me- isolated herein for their ability to stimulate macrophage phagocy- tabolites retain the anti-inflammatory actions of RvE1, each of the tosis. As documented in Fig. 6B (right panel), the 18-oxo-RvE1 new RvE1-derived products was isolated and we compared their metabolite was essentially inactive, whereas a stable analog of actions directly in vivo using acute inflammation, i.e., zymosan RvE1, namely 19-para-fluoro-phenoxy-RvE1, which was designed A-induced peritonitis (where RvE1 has been established to have to resist metabolic inactivation (12), gave potent activity above anti-inflammatory and proresolving actions). When administered that obtained with RvE1 with these cells (Fig. 6B). Of interest, http://classic.jimmunol.org Downloaded from

FIGURE 5. Acute inflammation: comparison of anti-inflammatory actions of RvE1 and RvE1 metabolic products. RvE1 (20 ng/mouse) or the isolated RvE1 metabolic product (as indicated) were injected via tail vein ϳ2 min before administration i.p. of zymosan A (1 mg) to evoke peritonitis. After 2 h, the peritoneal total leukocytes (A) and PMN (B) were enumerated. Results are mean Ϯ SEM for percentage reduction of leukocytes compared with ;significantly different from 0, p Ͻ 0.05 ,ء .zymosan- and vehicle-treated mice (3.9 Ϯ 0.3 ϫ 106 total leukocytes), 2.8 Ϯ 0.2 ϫ 106 PMN, n ϭ 3–6 †, significantly different from values obtained with RvE1, p Ͻ 0.05. 3518 RESOLVIN E1 METABOLOME AND INACTIVATION

FIGURE 6. Proresolving: RvE1 enhances macrophage phagocytosis. A, RvE1 dose response with isolated resident peritoneal macrophages and zymosan A. Murine peritoneal macrophages were isolated (see Materials and Methods), placed in 24-well plates, and exposed to RvE1 at the indicated concentrations for 15 min at 37°C followed by incubation with FITC-labeled zymosan A (30 min, 37°C). After 30 min, wells were aspirated and extracellular fluorescence was quenched by addition of trypan blue (ϳ60 s), followed by aspiration and suspension in PBS ϩ/ϩ. Plates were read using a PerkinElmer Victor3 plate reader, and values represent mean percent increase Ϯ SEM of fluorescence intensity above wells treated with vehicle and FITC-zymosan (n ϭ 4; significantly different from 0, p Ͻ ,ء ;significantly different from 0, p Ͻ 0.05). Inset, Duration of RvE1 exposure and macrophage phagocytosis (n ϭ 3 ,ء 0.05). B, Direct comparison of RvE1, RvE1 metabolic products, and an RvE1 stable analog. Murine resident peritoneal macrophages were incubated with RvE1 or related products. Compounds tested (1 nM) were added to 105 plated resident macrophages in PBS ϩ/ϩ. Compounds were incubated for 15 min .(significantly different from 0, p Ͻ 0.05; †, significantly different from RvE1 p Ͻ 0.05 ,ء ;at 37°C before addition of FITC-labeled zymosan A (n ϭ 3–6

20-carboxy-RvE1 and 20-hydroxy-RvE1 at 1 nM retained the unteers also taking EPA supplements (13). Hence, in humans there same ability as RvE1 to stimulate the phagocytosis of zymosan by are both aspirin-dependent and aspirin-independent routes of RvE1 murine macrophages (results not shown, n ϭ 3). Taken together, biosynthesis. Along these lines, RvE1 is generated de novo in oc- these ﬁndings indicate RvE1 is subject to several routes of metab- ular tissue (8, 36). In addition to aspirin-triggered biosynthesis of

olism (Fig. 4) that yield different compounds, some of which are 15-epi-LXA4 as a local endogenous anti-inﬂammatory mediator, biologically less active than RvE1 whereas others retain function. recent studies by Birnbaum et al. (37–39) demonstrate that statins

Thus, selective blockade of speciﬁc RvE1 metabolism pathways can also initiate the formation of 15-epi-LXA4. Interestingly, com-

by guest on October 1, 2021. Copyright 2008 Pageant Media Ltd. can enhance RvE1 actions. In this regard, the 19-para-fluoro-phe- bination of EPA and statin in a study of Ͼ18,000 patients with a noxy-RvE1 analog reduces the endogenous conversion by both 5-year follow-up demonstrates a significant reduction in coronary omega-1 metabolism as well as the dehydrogenation pathways ini- events (34). Given the potent anti-inflammatory and proresolving tiated at the carbon 18 position. actions documented in the present studies together with earlier in vitro and in vivo results (7, 9), it is likely that RvE1 plays a role Discussion in mediating some of the coronary-protective actions noted for In the present report, we document the RvE1 metabolome and EPA supplementation. bioactivities of the newly identified RvE1-derived metabolic prod- From the results of the present studies, at least four separate ucts. The omega-3 polyunsaturated fatty acids have emerged as an pathways for further metabolism of RvE1 are present in mamma- http://classic.jimmunol.org important approach to reducing the risk of cardiovascular disease lian tissues (Fig. 4). These pathways appear to be species-, organ-, (33, 34). Although it is now clear that omega-3 PUFAs have ben- and cell type-specific. Although 10,11-dihydro-12-oxo-RvE1 eficial actions in human clinical trials, particularly those empha- could not be directly isolated and identified in the tissues studied sizing cardiovascular risk, the mechanisms by which these bene- to date, it is likely to be an intermediate in a 12-oxo-dehydroge- ficial actions occur remained to be established. It is increasingly nation-initiated route of RvE1 further metabolism. Given that the apparent that inflammation plays a key role in the progression of product 10,11-dihydro-RvE1 was essentially biologically inactive

Downloaded from cardiovascular diseases and many other diseases widely occurring compared with RvE1, it is possible that this metabolite of RvE1 in the Western population. Resolution of acute inflammation and may serve as an inactive biomarker of RvE1 transient formation in its potential failure may underlie the mechanisms for presenting vivo. Along these lines, it is of interest to point out that the 20- chronic unresolved inflammation in many diseases (1, 35). hydroxy-RvE1 product of RvE1 made via omega carbon 20 oxi- RvE1 is a potent bioactive product generated from EPA and dation retains some of the activity of RvE1, namely in vivo anti- originally identified in resolving murine exudates. RvE1 proved to inflammatory actions and proresolving actions-accelerating the carry potent stereospecific biological actions in vitro and in com- phagocytic uptake of zymosan. This point demonstrates that not all plex animal disease models (7, 9). The biosynthesis of RvE1 is further metabolites of RvE1 or potentially other resolvins can be initiated by both P450- and aspirin-dependent COX-2-triggered assumed to be biologically inactive. Hence, it is possible that fur- mechanisms. To recapitulate one of several potential routes in- ther metabolites can retain activity and/or possibly possess new volved in the in vivo formation of RvE1 in humans, isolated hu- actions, whereas others are clearly pathways of RvE1 inactivation. man microvascular endothelial cells treated with aspirin in a hy- Omega-1 hydroxylation to 19-hydroxy RvE1 and reduction of a poxic environment convert EPA to intermediates that are further conjugated double bond to 10,11-dihydro-RvE1 are novel meta- transformed by human PMN in coincubations to RvE1 (5). In hu- bolic pathways identified in the present studies that inactivate mans, aspirin increases the plasma levels of RvE1 in healthy vol- RvE1. Adding a p-fluorophenoxy to the 19 position of RvE1, as in The Journal of Immunology 3519

19-p-fluorophenoxy-RvE1, blocks this route of RvE1 inactivation. 15. Salem, N., Jr., B. Litman, H.-Y. Kim, and K. Gawrisch. 2001. Mechanisms of From our earlier report, it is already known that 19-p-fluorophe- action of docosahexaenoic acid in the nervous system. Lipids 36: 945–959. 16. Freeman, M. P. 2000. Omega-3 fatty acids in psychiatry: a review. Ann. Clin. noxy also blocks another metabolic inactivation route, i.e., dehy- Psychiatry 12: 159–165. drogenation of RvE1 to inactive 18-oxo-RvE1 (12). In summation, 17. Holub, D. J., and B. J. Holub. 2004. Omega-3 fatty acids from fish oils and cardiovascular disease. Mol. Cell. Biochem. 263: 217–225. the present results demonstrate and provide additional evidence 18. Weylandt, K. H., J. X. Kang, B. Wiedenmann, and D. C. Baumgart. 2007. Li- that there is efficient endogenous “machinery” that can quench poxins and resolvins in inflammatory bowel disease. Inflamm. Bowel Dis. 13: proresolving signals, such as RvE1, so that the exudates and tis- 797–799. 19. Serhan, C. N., S. Hong, K. Gronert, S. P. Colgan, P. R. Devchand, G. Mirick, and sues can return to homeostasis (1, 2). These results also indicate R.-L. Moussignac. 2002. Resolvins: a family of bioactive products of omega-3 that blocking dehydrogenation of RvE1 and preventing the reduc- fatty acid transformation circuits initiated by aspirin treatment that counter pro- tion of its conjugated double bond by modifying the RvE1 struc- inflammation signals. J. Exp. Med. 196: 1025–1037. 20. Hong, S., K. Gronert, P. Devchand, R.-L. Moussignac, and C. N. Serhan. 2003. ture without attenuating its anti-inflammatory and proresolving Novel docosatrienes and 17S-resolvins generated from docosahexaenoic acid in activities could be one means to develop RvE1-based therapeutics murine brain, human blood and glial cells: autacoids in anti-inflammation. J. Biol. Chem. 278: 14677–14687. that can serve as agonists of resolution. Moreover, identification of 21. Lu, Y., S. Hong, R. Yang, J. Uddin, K. H. Gotlinger, N. A. Petasis, and these further metabolic products in the RvE1 metabolome may be C. N. Serhan. 2007. Identification of endogenous resolvin E1 and other lipid useful in qualifying suitable biomarkers relevant in omega-3 fatty mediators derived from eicosapentaenoic acid via electrospray low energy tandem mass spectrometry: spectra and fragmentation mechanisms. Rapid Commun. acid supplementation studies as well as monitoring their relation to Mass Spectrom. 21: 7–22. the biosynthesis and actions of the E-series resolvins. 22. Godson, C., S. Mitchell, K. Harvey, N. A. Petasis, N. Hogg, and H. R. Brady. 2000. Cutting edge: lipoxins rapidly stimulate nonphlogistic phagocytosis of apoptotic neutrophils by monocyte-derived macrophages. J. Immunol. 164: Acknowledgments 1663–1667. We thank Mary H. Small for assistance with manuscript preparation and 23. Laethem, R. M., M. Balazy, J. R. Falck, C. L. Laethem, and D. R. Koop. 1993. Katherine Gotlinger for expert technical assistance. Formation of 19(S)-, 19(R)-, and 18(R)-hydroxyeicosatetraenoic acids by alcohol-inducible cytochrome P450 2E1. J. Biol. Chem. 268: 12912–12918. 24. Bylund, J., M. Hidestrand, M. Ingelman-Sundberg, and E. H. Oliw. 2000. Iden- Disclosures tification of CYP4F8 in human seminal vesicles as a prominent 19-hydroxylase The lipoxins and resolvins as biotemplates for stable analogs are U.S. patents of prostaglandin endoperoxides. J. Biol. Chem. 275: 21844–21849. 25. Yokomizo, T., T. Izumi, T. Takahashi, T. Kasama, Y. Kobayashi, F. Sato, assigned to Brigham and Women’s Hospital, and Charles N. Serhan is the Y. Taketani, and T. Shimizu. 1993. Enzymatic inactivation of leukotriene B4 by inventor. These analog patents are licensed for clinical development and are a novel enzyme found in the porcine kidney: purification and properties of leu- the subject of consultant agreements for Charles N. Serhan. kotriene B4 12-hydroxydehydrogenase. J. Biol. Chem. 268: 18128–18135. 26. Tai, H. H., C. M. Ensor, M. Tong, H. Zhou, and F. Yan. 2002. Prostaglandin catabolizing enzymes. Prostaglandins Other Lipid Mediat. 68–69: 483–493. References 27. Serhan, C. N., S. Fiore, D. A. Brezinski, and S. Lynch. 1993. Lipoxin A4 me- 1. Serhan, C. N. 2007. Resolution phases of inflammation: novel endogenous anti- tabolism by differentiated HL-60 cells and human monocytes: conversion to inflammatory and pro-resolving lipid mediators and pathways. Annu. Rev. Im- novel 15-oxo and dihydro products. Biochemistry 32: 6313–6319. munol. 25: 101–137. 28. Mitchell, S., G. Thomas, K. Harvey, D. Cottell, K. Reville, G. Berlasconi, 2. Serhan, C. N., and J. Savill. 2005. Resolution of inflammation: the beginning N. A. Petasis, L. Erwig, A. J. Rees, J. Savill, et al. 2002. Lipoxins, aspirin- programs the end. Nat. Immunol. 6: 1191–1197. triggered epi-lipoxins, lipoxin stable analogues, and the resolution of inflamma- 3. Gilroy, D. W., T. Lawrence, M. Perretti, and A. G. Rossi. 2004. Inflammatory tion: stimulation of macrophage phagocytosis of apoptotic neutrophils in vivo. resolution: new opportunities for drug discovery. Nat. Rev. Drug Discov. 3: J. Am. Soc. Nephrol. 13: 2497–2507. 401–416. 29. Jin, S.-W., L. Zhang, Q.-Q. Lian, D. Liu, P. Wu, S.-L. Yao, and D.-Y. Ye. 2007. by guest on October 1, 2021. Copyright 2008 Pageant Media Ltd. 4. Bannenberg, G. L., N. Chiang, A. Ariel, M. Arita, E. Tjonahen, K. H. Gotlinger, Posttreatment with aspirin-triggered lipoxin A4 analog attenuates lipopolysac- S. Hong, and C. N. Serhan. 2005. Molecular circuits of resolution: formation and charide-induced acute lung injury in mice: the role of heme oxygenase-1. Anesth. actions of resolvins and protectins. J. Immunol. 174: 4345–4355. Analg. 104: 369–377. 5. Serhan, C. N., C. B. Clish, J. Brannon, S. P. Colgan, N. Chiang, and K. Gronert. 30. Fiorucci, S., J. L. Wallace, A. Mencarelli, E. Distrutti, G. Rizzo, S. Farneti, 2000. Novel functional sets of lipid-derived mediators with antiinflammatory A. Morelli, J.-L. Tseng, B. Suramanyam, W. J. Guilford, and J. F. Parkinson. ␤ actions generated from omega-3 fatty acids via cyclooxygenase 2-nonsteroidal 2004. A -oxidation-resistant lipoxin A4 analog treats hapten-induced colitis by antiinflammatory drugs and transcellular processing. J. Exp. Med. 192: attenuating inflammation and immune dysfunction. Proc. Natl. Acad. Sci. USA 1197–1204. 101: 15736–15741. 6. Hudert, C. A., K. H. Weylandt, J. Wang, Y. Lu, S. Hong, A. Dignass, 31. Serhan, C. N. 2005. Lipoxins and aspirin-triggered 15-epi-lipoxins are the first C. N. Serhan, and J. X. Kang. 2006. Transgenic mice rich in endogenous n-3 fatty lipid mediators of endogenous anti-inflammation and resolution. Prostaglandins acids are protected from colitis. Proc. Natl. Acad. Sci. USA 103: 11276–11281. Leukotrienes Essent. Fatty Acids 73: 141–162. 7. Arita, M., M. Yoshida, S. Hong, E. Tjonahen, J. N. Glickman, N. A. Petasis, 32. Kowal-Bielecka, O., K. Kowal, O. Distler, and S. Gay. 2007. Mechanisms of http://classic.jimmunol.org R. S. Blumberg, and C. N. Serhan. 2005. Resolvin E1, an endogenous lipid disease: leukotrienes andlipoxins in scleroderma lung disease: insights and po- mediator derived from omega-3 eicosapentaenoic acid, protects against 2,4,6- tential therapeutic implications. Nat. Clin. Pract. Rheumatol. 3: 43–51. trinitrobenzene sulfonic acid-induced colitis. Proc. Natl. Acad. Sci. USA 102: 33. Harris, W. S., K. J. Reid, S. A. Sands, and J. A. Spertus. 2007. Blood omega-3 7671–7676. and trans fatty acids in middle-aged acute coronary syndrome patients. 8. Connor, K. M., J. P. SanGiovanni, C. Lofqvist, C. M. Aderman, J. Chen, Am. J. Cardiol. 99: 154–158. A. Higuchi, S. Hong, E. A. Pravda, S. Majchrzak, D. Carper, et al. 2007. In- 34. Yokoyama, M., H. Origasa, M. Matsuzaki, Y. Matsuzawa, Y. Saito, Y. Ishikawa, creased dietary intake of omega-3-polyunsaturated fatty acids reduces patholog- S. Oikawa, J. Sasaki, H. Hishida, H. Itakura, et al. 2007. Effects of eicosapenta- ical retinal angiogenesis. Nat. Med. 13: 868–873. enoic acid on major coronary events in hypercholesterolaemic patients (JELIS): 9. Hasturk, H., A. Kantarci, T. Ohira, M. Arita, N. Ebrahimi, N. Chiang, a randomised open-label, blinded endpoint analysis. Lancet 369: 1090–1098. Downloaded from N. A. Petasis, B. D. Levy, C. N. Serhan, and T. E. Van Dyke. 2006. RvE1 protects 35. Serhan, C. N., S. D. Brain, C. D. Buckley, D. W. Gilroy, C. Haslett, L. A. J. from local inflammation and osteoclast mediated bone destruction in periodon- O’Neill, M. Perretti, A. G. Rossi, and J. L. Wallace. 2007. Resolution of inflam- titis. FASEB J. 20: 401–403. mation: state of the art, definitions and terms. FASEB J. 21: 325–332. 10. Schwab, J. M., N. Chiang, M. Arita, and C. N. Serhan. 2007. Resolvin E1 and 36. Hong, S., W. C. Gordon, Y. Lu, V. L. Marcheselli, and N. G. Bazan. 2007. protectin D1 activate inflammation-resolution programmes. Nature 447: Lipidomic approach to define pro- and anti-inflammatory mediators in laser-in- 869–874. duce choroidal neovascularization. Invest. Ophthalmol. Visual Sci. 48: 11. Clish, C. B., B. D. Levy, N. Chiang, H.-H. Tai, and C. N. Serhan. 2000. Oxi- e-abstract 6014. doreductases in lipoxin A4 metabolic inactivation. J. Biol. Chem. 275: 37. Birnbaum, Y., Y. Ye, Y. Lin, S. Y. Freeberg, M.-H. Huang, J. R. Perez-Polo, and 25372–25380. B. F. Uretsky. 2007. Aspirin augments 15-epi-lipoxin A4 production by lipopoly- 12. Arita, M., S. Oh, T. Chonan, S. Hong, S. Elangovan, Y.-P. Sun, J. Uddin, saccharide, but blocks the pioglitazone and atorvastatin induction of 15-epi-li- N. A. Petasis, and C. N. Serhan. 2006. Metabolic inactivation of resolvin E1 and poxin A4 in the rat heart. Prostaglandins Other Lipid Mediat. 83: 89–98. stabilization of its anti-inflammatory actions. J. Biol. Chem. 281: 22847–22854. 38. Birnbaum, Y., Y. Ye, Y. Lin, S. Y. Freeberg, S. P. Nishi, J. D. Martinez, M.-H. 13. Arita, M., F. Bianchini, J. Aliberti, A. Sher, N. Chiang, S. Hong, R. Yang, Huang, B. F. Uretsky, and J. R. Perez-Polo. 2006. Augmentation of myocardial N. A. Petasis, and C. N. Serhan. 2005. Stereochemical assignment, anti- production of 15-epi-lipoxin-A4 by pioglitazone and atorvastatin in the rat. Cir- inflammatory properties, and receptor for the omega-3 lipid mediator resolvin E1. culation 114: 929–935. J. Exp. Med. 201: 713–722. 39. Birnbaum, Y., Y. Ye, S. Rosanio, S. Tavackoli, Z. Y. Hu, E. R. Schwarz, and 14. Calder, P. C. 2006. n-3 polyunsaturated fatty acids, inflammation, and inflam- B. F. Uretsky. 2005. Prostaglandins mediate the cardioprotective effects of ator- matory diseases. Am. J. Clin. Nutr. 83: 1505S–1519S. vastatin against ischemia-reperfusion injury. Cardiovasc. Res. 65: 345–355.