nitratedfattyacids:endogenousanti-inflammatory ...mercuryms chromatography system equipped with a...

Nitrated Fatty Acids: Endogenous Anti-inflammatorySignaling Mediators*□S

Received for publication, April 7, 2006, and in revised form, July 21, 2006 Published, JBC Papers in Press, August 3, 2006, DOI 10.1074/jbc.M603357200

Taixing Cui‡1,2, Francisco J. Schopfer§2,3, Jifeng Zhang‡, Kai Chen‡, Tomonaga Ichikawa‡, Paul R. S. Baker§4,Carlos Batthyany§, Balu K. Chacko¶3, Xu Feng¶, Rakesh P. Patel¶, Anupam Agarwal�, Bruce A. Freeman§5,and Yuqing E. Chen‡6

From the ‡Cardiovascular Center, University of Michigan Medical Center, Ann Arbor, Michigan 48109, the §Department ofPharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, the ¶Department of Pathologyand �Division of Nephrology, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294

Nitroalkene derivatives of linoleic acid (LNO2) and oleic acid(OA-NO2) are present; however, their biological functionsremain to be fully defined. Herein, we report that LNO2 andOA-NO2 inhibit lipopolysaccharide-induced secretion of proin-flammatory cytokines in macrophages independent of nitricoxide formation, peroxisome proliferator-activated receptor-�activation, or induction of heme oxygenase-1 expression. Theelectrophilic nature of fatty acid nitroalkene derivativesresulted in alkylationof recombinantNF-�Bp65protein in vitroand a similar reaction with p65 in intact macrophages. Thenitroalkylation of p65 by fatty acid nitroalkene derivativesinhibited DNA binding activity and repressed NF-�B-depend-ent target gene expression. Moreover, nitroalkenes inhibitedendothelial tumor necrosis factor-�-induced vascular cell adhe-sion molecule 1 expression andmonocyte rolling and adhesion.These observations indicate that nitroalkenes such as LNO2 andOA-NO2, derived from reactions of unsaturated fatty acids andoxides of nitrogen, are a class of endogenous anti-inflammatorymediators.

Reactive oxygen species and NO-derived oxidizing, nitrosat-ing and nitrating products mediate diverse cell signaling andpathologic processes in cardiovascular, pulmonary, and neuro-degenerative diseases (1, 2). These reactive inflammatorymedi-ators chemically modify carbohydrates, DNA bases, aminoacids, and unsaturated fatty acids to form oxidized, nitrosatedand nitrated derivatives. For example, accumulation of inflam-matory-induced protein tyrosine nitration products represents

a shift from the physiological signal-transducing actions of NO toan oxidative, nitrative, and potentially pathogenic pathway (1).Recently, it has been reported that nitration products of

unsaturated fatty acids (nitroalkenes) are formed via NO-de-pendent oxidative reactions (3–5). These derivatives were ini-tially viewed to be, like nitrotyrosine, a “footprint” of NO-de-pendent redox reactions (3, 6).More recently, we have observedthat nitrolinoleate (LNO2)7 mediates pluripotent cell signalingactions, since it induces relaxation of phenylephrine-precon-stricted rat aortic rings, inhibits thrombin-induced Ca2� eleva-tions and aggregation of human platelets, and attenuateshuman neutrophil superoxide generation, degranulation, andintegrin expression. These cell responses are mediated by bothcGMP- and cAMP-dependent and -independent mechanisms(7–9).LNO2 positional isomers, including 9-, 10-, 12-, and 13-ni-

tro-9,12-cis-octadecadienoic acids, have been identified as freeacids in human plasma and red blood cells and as esterifiedcomponents of plasma lipoproteins and red blood cell mem-branes (10). In addition, plasma and red cell free and esterifiednitrooleate (OA-NO2, isomers 9- and 10-nitro-9-cis-octadece-noic acid) was also identified in healthy human blood (11).Current knowledge indicates that enzymatically oxidized

unsaturated fatty acid-derived products, such as prostaglan-dins, thromboxanes, leukotrienes, epoxyeicosatrienoic acids,hydroxyeicosatetraenoic acids, lipoxins, and resolvins serve aslipid mediators or autacoids. These signaling mediators actwithin a local microenvironment to orchestrate both physio-logical and pathological events, including platelet aggregation,constriction of vascular smoothmuscle, neonatal development,wound healing, and resolution of inflammation (12, 13). In thiscontext, endogenous nitrated fatty acid derivatives, such as

* This work was supported by National Institutes of Health Grants HL068878,HL075397, and S06GM08248 (to Y. E. C.), HL70146 (to R. P. P.), and HL58115and HL64937 (to B. A. F.). The costs of publication of this article weredefrayed in part by the payment of page charges. This article must there-fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec-tion 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table S1, Fig. 1, and Movie 1.

1 Supported by American Diabetes Association Grant JFA 7-05-JF-12.2 These authors contributed equally to this work.3 Supported by the postdoctoral fellowships from the American Heart Asso-

ciation Southeast Affiliate.4 Supported by National Institutes of Health Cardiovascular Hypertension

Training Grant T32HL07457.5 To whom correspondence may be addressed. Tel.: 412-648-9319; Fax: 412-

648-2229; E-mail: [email protected] To whom correspondence may be addressed. Tel.: 734-763-7838; Fax: 734-

936-2641; E-mail: [email protected].

7 The abbreviations used are: LNO2, nitrolinoleate (positional isomers 10-ni-tro-9-cis,12-cis-octadecadienoic acid (C10) and 12-nitro-9-cis,12-cis-octa-decadienoic acid (C12)); OA-NO2, esterified nitrooleate (9- and 10-nitro-9-cis-octadecenoic acid (C9 and C10)); LPS, lipopolysaccharide; PMA, phorbol12-myristate 13-acetate; DETA-NONOate, 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene; cPTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazo-line-1-oxyl 3-oxide; PPAR�, peroxisome proliferator-activated receptor;HO-1, heme oxygenase 1; VCAM-1, vascular cell adhesion molecule 1; IL-6,interleukin 6; TNF�, tumor necrosis factor �; MCP-1, monocyte chemoat-tractant protein 1; ESI, electrospray ionization; MS, mass spectrometry;DTPA, diethylenetriaminepentaacetate; OA, oleic acid; LA, linoleic acid;HUVEC, human umbilical vein endothelial cells; FBS, fetal bovine serum;ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 15d-PGJ2, 15-deoxy-�12,14-prostaglandin J2.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 47, pp. 35686 –35698, November 24, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

35686 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 47 • NOVEMBER 24, 2006

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OA-NO2 and LNO2, represent an emerging class of NO andfatty acid-derived signaling molecules (14). At present, the cellsignalingmechanisms anddetailed structure-function relation-ships of this family of fatty acid derivatives are an object ofinterest. Recently, nitrated unsaturated fatty acids were shownto be potent electrophiles that mediate reversible nitroalkyla-tion reactions with both glutathione and the Cys and His resi-dues of proteins. This occurs both in vitro and in vivo and isviewed to transduce redox- and NO-dependent cell signalingby inducing a covalent, thiol-reversible post-translationalmod-ification that regulates protein structure, function, and subcel-lular distribution (15).Herein, we report the effects of LNO2 and OA-NO2 on

inflammatory responses in vascular cells, includingmonocytes/macrophages and endothelial cells. These data indicate that thenitroalkene derivatives of linoleic acid and oleic acid (LNO2 andOA-NO2) are a novel class of endogenous, electrophilic medi-ators in the vasculature that can exert adaptive anti-inflamma-tory signaling reactions via the post-translational modificationof transcriptional regulatory proteins.

EXPERIMENTAL PROCEDURES

Materials

LNO2 and OA-NO2 were synthesized, purified, and quanti-tated as previously described (10, 11). Briefly, OA-NO2 andLNO2 were synthesized using a nitrosenylation reaction. OA-NO2 and LNO2 were purified by preparative TLC developedtwice using silica HF plates and a solvent system consisting ofhexane/ether/acetic acid (70:30:1, v/v/v). The regions corre-sponding to OA-NO2 and LNO2 were scraped and extracted.Stock concentrations of OA-NO2 and LNO2 were quantitatedby chemiluminescent nitrogen analysis, using caffeine as astandard, and confirmed spectrophotometrically using anextinction coefficient (E268) of 8.22mM�1 cm�1 for OA-NO2 in100 mM phosphate buffer at pH 7.4. Lipopolysaccharide (LPS),phorbol 12-myristate 13-acetate (PMA), zinc protoporphryin-IX, 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (DETA-NONOate), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazo-line-1-oxyl 3-oxide (cPTIO), sodium nitrite (NaNO2),diethylenetriaminepentaacetate (DTPA), and Na2HPO4 werefrom Sigma. Rosiglitazone, 15-deoxy-�12,14-PGJ2, Wy14643,andGW501516were fromCaymanChemical (AnnArbor,MI).Streptavidin-coated plates were from Pierce. RecombinantNF-�B p65 protein and TransAMTM NF-�B p65 Chemi kitwere from Active Motif (Carlsbad, CA).

Fatty Acid Biotinylation

The synthesis of biotinylatedOA, LA,OA-NO2, and LNO2wasperformed as described for the synthesis of biotinylated (15S)-hy-droxyeicosatetraenoic acids (16). Briefly, 1-methylpiperidine (5.3�l, 48�mol)wasaddedtoasolutionofeitherOA,LA,OA-NO2,orLNO2 (15 mg in 1.5 ml of methylene chloride). After cooling themixture to �78 °C, isobutylchloroformate (6.2 �l, 48 �mol) wasadded.The reactionmixturewasmaintained at�78 °C for 45minand then warmed to �20 °C. After 35 min, a heated (80 °C) solu-tion of biotin hydrazide (48.6 mg, 188 �mol) in dimeth-ylformamide (2 ml) was added. The mixture was immediatelycooled andmaintained at �20 °C for 40 min and then warmed to

room temperature. After extraction with methylene chloride, theproductswere chromatographed and purified by preparativeTLCdeveloped twice using silicaHFplates, using a solvent systemcon-sisting of methylene chloride/methanol (80/20, v/v). The regionscorresponding to the biotinylated OA, LA, OA-NO2, and LNO2were scraped and extracted. Stock concentrations of biotinylatedOA, LA,OA-NO2, and LNO2were quantitated by chemilumines-cent nitrogen analysis using caffeine as a standard. The character-ization of the biotinylatedOA, LA,OA-NO2, and LNO2was doneby direct infusion into a ESI MS (ion trap) at both positive andnegative mode or by ESI-liquid chromatography-MS/MS in thenegative ion mode, using multiple reaction monitoring and anenhanced product information scanmode on a 4000 hybrid triplequadrupole/linear ion trapmass spectrometer (Q-Trap) (AppliedBiosystems, Foster City, CA). The ESI HPLC analysis wasperformed by C-18 reverse phase chromatography using aMercuryMS chromatography system equipped with a LunaC18 column (Phenomenex, Belmont, CA) equilibrated in70% A (aqueous formic acid (0.01%)) and 30% B (0.01% for-mic acid in acetonitrile). One minute after injection, a 2-mingradient was initiated to reach a composition of 60% A andthen to 7% A over an additional 4 min, held for 1 min at 7% A,and then back to 70% A and re-equilibrated. The retentiontimes (min) and specific multiple reaction monitorings usedfor each species were as follows: biotinylated LA 7.50, 519/476; biotinylated OA 7.94, 521/478; biotinylated LNO2 6.90,564/517; biotinylated OA-NO2 7.30, 566/519.

Cell Culture

Bone marrow cells were isolated from 8–12-week-old hemeoxygenase-1 (HO-1) knockout mice or wild-typemice as previ-ously (17). Briefly, bone marrow macrophages were preparedby culturing isolated bone marrow cells in �-minimal essentialmedium (Invitrogen) containing 10% heat-inactivated fetalbovine serum (FBS) in the presence of 0.1 volume of culturesupernatant from macrophage colony-stimulating factor-pro-ducing cells for 2 days. Human umbilical vein endothelial cells(HUVEC)were purchased fromBioWhittaker (SanDiego, CA).The cells were cultured in endothelial cell growth medium-2(BioWhittaker), containing 5% fetal calf serum, human basicfibroblast growth factor, insulin-like growth factor, human epi-

SCHEME 1. Structures of LNO2 and OA-NO2. Structures of LNO2 and OA-NO2used in this investigation are identical to those detected in the healthyhuman circulation (10, 11)

Nitroalkenes Inhibit Inflammation

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thelial growth factor, vascular endo-thelial growth factor, 50 �g/ml gen-tamicin, 50 ng/ml amphotericin-B,hydrocortisone, and ascorbic acid.Early passages (passages 3–5) ofHUVEC were used for all experi-ments. THP-1 cells (a humanmonocyte cell line from ATCC,Manassas, VA; catalog numberTIB-202TM) were cultured in RPMI 1640(ATCC, catalog number 30-2001)supplemented with 10% FBS.THP-1 monocytes were differenti-ated into macrophages with PMA(0.1 �M) for 5 days. RAW264.7 cells(amurinemacrophage cell line fromATCC, catalog no. TIB-71TM) werecultured in Dulbecco’s modifiedEagle’s medium (Invitrogen) sup-plemented with 10% FBS. Humanperipheral blood mononuclear cellswere purchased from Cambrex BioScience (East Rutherford, NJ; cata-log number CC-2702) and culturedwith RPMI 1640 supplementedwith10% FBS. The nonadherent cellswere removed with PBS beforetreatment. CV-1 cells (Africangreen monkey kidney fibroblast cellline from ATCC; catalog numberCCL-70TM) were cultured in Dul-becco’s modified Eagle’s medium/F-12 supplemented with 10% FBS.

Cytokine Assay

Macrophages in fresh 1% FBS or1% delipidated FBS (Cocalico Bio-logicals; catalog numberCBX4234)-containing culture medium weretreated as indicated. Medium wascollected from triplicate wells18–20 h after treatment. The con-centrations of human and mouseTNF�, IL-6, and MCP-1 releasedfrom cells into medium was meas-ured by enzyme-linked immunosor-bent assay (ELISA) kits using proto-cols supplied by themanufacturer (R&DSystems,Minneapolis,MN). The half-maximal concentration for LNO2 and OA-NO2inhibition of LPS-induced cytokine synthesis (IC50) was calcu-lated using software obtained from GraphPad Software, Inc.

Quantitative Real Time Reverse Transcription-PCR

Total RNA from cell pellets was extracted using RNeasy kits(Qiagen Inc., Valencia, CA), and reverse transcription reactions(Advantage RT for PCR kit; Clontech) were performed with0.5–1 �g of DNase I (Qiagen)-treated RNA. Quantitative realtime reverse transcription-PCRs were carried out using the

LightCycler thermocycler and the SYBR green I kits (RocheApplied Science) according to the manufacturer’s recommen-dations. Cycle numbers obtained at the log-linear phase of thereaction were plotted against a standard curve prepared withserially diluted control samples. Expression levels of targetgenes were normalized by concurrent measurement of glycer-aldehyde-3-phosphate dehydrogenase (GAPDH)mRNA levels.

Transfection and Reporter Gene Assay

CV-1 cells at �85% confluence in 24-well plates were tran-siently co-transfected using Lipofectamine 2000 (Invitrogen)

FIGURE 1. Nitrated fatty acids inhibit LPS-induced inflammatory cytokine secretion in macrophages.A–C, LNO2 and OA-NO2 inhibited LPS-induced inflammatory cytokine secretion in THP-1 and RAW264.7 mac-rophages and human peripheral blood mononuclear cells (hPBMC) in either lipid-rich or delipidated serum.Cells were stimulated as indicated overnight in fresh culture medium with 1% lipid-rich normal serum (L-se-rum) (A and B) or with 1% delipidated serum (D-serum). The secretion of proinflammatory cytokines wasassessed by ELISA. Values are expressed as mean � S.D. (n � 6). *, p � 0.05 versus LPS alone. ND, nondetectable.




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with a plasmid containing the luciferase gene under the controlof three tandem PPAR-response elements (3� PPRE TK-lucif-erase) in pGL3 basic vector (Promega, Madison, WI) andhuman PPAR�1 in pcDNA3.1 vector (Invitrogen), respectively.Green fluorescent protein expression plasmid was co-trans-fected as an internal control for transfection efficiency. Twen-ty-four hours after transfection, cells were cultured for 4 h inOpti-MEM I (Invitrogen) and then treated with various stimulias indicated for an additional 16–20 h. RAW264.7 cells weretransiently co-transfected with NF-�B-luciferase reporter (NF-�B-luc) (Stratagene, La Jolla, CA) and green fluorescent proteinexpression plasmids. Twenty-four hours after transfection,cells were pretreated with nitrated fatty acids or control fattyacids in 1% FBS Eagle’s minimal essential medium for 16–20 hand then stimulated with LPS (1 �g/ml) for 6 h. Each transfec-tion was performed in triplicate on at least 3 occasions.Reporter luciferase assay kits (Promega) were used to measurethe luciferase activity of cells with a luminometer, according tothe manufacturer’s instructions (Victor II; PerkinElmer LifeSciences). Luciferase activity was normalized by green fluores-cent protein units.

NF-�B Activity Assay in Vitro

Partially purified human recombinant p65 protein (470units/ml) was incubated in phosphate buffer 100 mM contain-ing 100 �M DTPA (pH 7.4) with different concentrations ofLNO2, OA-NO2, LA, or OA for 1 h at room temperature. Theremaining p65 activity was measured using the TransAMTM

NF-�B p65 Chemi kit from Active Motif following the manu-facturer’s instructions. A modification was introduced into theprotocol by avoiding any addition of dithiothreitol to sampleand buffer preparations. The amount of p65 added to each wellwas 10 ng. The p65 activity was not affected by native fatty acids(LA andOA) or vehicle. Thus, the activity of p65 incubatedwith1 �M of corresponding native fatty acids was considered maxi-mal (100%).

Analysis of p65 Alkylation byNitrated Fatty Acids

Partially purified human recom-binant p65 protein (470 units/ml)was incubated in 100mM phosphatebuffer containing 100 �M DTPA(pH 7.4) with different concentra-tions of biotinylated LNO2,OA-NO2, LA, or OA for 1 h at roomtemperature. The reaction mixture(25 ng of p65) was added to strepta-vidin-coated plates that were previ-ously blocked for 1 h with TweenTris-buffered saline (TTBS: 0.5%Tween 20, 30 mM Tris, 150 mMNaCl) containing 3% albumin, incu-bated for 1 h at room temperature,and washed three times using TTBScontaining 1% albumin. The plateswere then incubated with anti-p65in 1% albumin containing TTBS for1 h at room temperature, washed

three times, incubated with goat anti-rabbit IgG for 1 h at roomtemperature, and then washed four times. Color developmentwas followed at 650 nm using tetramethyl benzidine assubstrate.

Immunoprecipitation and Immunoblotting Analysis

Cells treated under different experimental conditions werewashed quickly with ice-cold PBS containing 1 mM Na3VO4,frozen in liquid nitrogen, scraped off, and lysed inNonidet P-40lysis buffer (1% Nonidet P-40, 25 mM HEPES (pH 7.5), 50 mMNaCl, 50 mM NaF, 5 mM EDTA, 10 nM okadaic acid, 1 mMsodium orthoavanadate, 1 mM phenylmethylsulfonyl fluoride,and 10 �M aprotinin) for 10–15 min on ice. Insoluble materialwas removed by centrifugation at 14,000 � g for 20 min at 4 °C.Protein concentrationwasmeasured in the cleared supernatantvia Coomassie dye binding (Bio-Rad). The cell lysates (200 �g)were precipitated overnight with 50 �l of UltraLink Immobi-lized NeutrAvidin Plus (Pierce), or 50 �l of a 50% slurry ofprotein G-Sepharose 4 Fast Flow (Sigma) was added to 1 ml ofcell lysate with equal amounts of protein and incubated at 4 °Cfor 1 h with gentle shaking. The precleared lysates (200 �g)were incubated with anti-p65 with constant agitation at 4 °Covernight and then further incubated with protein G-Sepha-rose 4 Fast Flow for 1 h. These precipitates were washed fourtimes with Nonidet P-40 lysis buffer. The whole cell lysates (20�g) or precipitates were subjected to SDS-PAGE and electro-transferred onto Hybond-ECL nitrocellulose membrane.Immunoblotting was done using anti-p65 (sc-372), anti-vascu-lar cell adhesion molecule-1 (anti-VCAM-1; sc-13160), anti-actin (sc-1616) (Santa Cruz Biotechnology, Inc., Santa Cruz,CA), and anti-HO-1 (SPA-895) (Stressgen Biotechnologies,Inc., San Diego, CA).

Analysis of THP-1 Monocyte Adhesion and Rolling on HUVEC

HUVECwere grown until 95% confluent in 48-well fibronec-tin-coated plates (for static adhesion analyses) or 25-mmdishes

FIGURE 1—continued




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(for laminar flow analyses) and then incubated for 2 h with orwithout LNO2. Subsequently, HUVEC were activated withTNF� (2 ng/ml) for 16 h.Static Adhesion of THP-1 Cells on HUVEC—Fluorescently

labeled monocytes treated with Cell Tracker Green (MolecularProbes, Eugene, OR) were added at a leukocyte/endothelialratio of 10:1. After a 30-min incubation at 37 °C, unboundmonocytes were removed by washing (nonadherent fraction).Both fractions (adherent and nonadherent cells) were lysedusing 1 M NaOH (20 min), and fluorescence was measured atexcitation � 480 nm and emission � 515 nm in a PerkinElmerLife Sciences fluorescent plate reader.Adhesion and Rolling of THP-1 Monocyte Cell on HUVEC in

Laminar Flow—THP-1 cell adhesion to and rolling on HUVECduring flow were determined using a Glycotech flow chambersystem (Rockville, MD) at flow rates corresponding to a wallshear rate of 0.5–1.5 dynes/cm2. Fluorescently labeled THP-1cells were viewed on a Leica inverted fluorescence microscopeequipped with differential interference contrast optics and aHamamatsu Orca ER digital CCD camera (Compix Inc., Cran-berry Township, PA). Real time images of each field were cap-tured at 30 frames/s for 5 min using the automated image cap-ture feature of Simple PCI software (Compix). Resulting timelapse images were analyzed by themotion tracking and analysisfeature of Simple PCI software to calculate rolling velocities andthe number of firmly bound cells. Cells not moving for 5 s ormore were considered firmly bound cells and were counted.The rolling velocities for THP-1 cells were calculated usingSimple PCI software.

Statistics

Values are expressed as mean � S.D. throughout. The datawere analyzed using analysis of variance with the Newman-Keuls test unless specified. Values of p � 0.05 were consideredto be statistically significant.

RESULTS

LNO2 and OA-NO2 Inhibit LPS-induced Secretion of Proin-flammatory Cytokines in Macrophages—To explore the phys-iological actions of LNO2 and OA-NO2, we investigated theimpact of synthetic LNO2 and OA-NO2 (Scheme 1), identi-cal to that detected in the healthy human circulation (10, 11),on inflammatory responses of monocytes/macrophages. Theconcentration-dependent effects of LNO2 and OA-NO2 onLPS-induced proinflammatory cytokine secretion by macro-phages were first defined. LNO2 and OA-NO2 dose-depend-ently inhibited the LPS-induced secretion of proinflamma-tory cytokines, including IL-6, TNF�, and MCP-1, in THP1and RAW264.7 macrophages in either lipid-rich serum(L-serum) or delipidated serum (D-serum). In THP-1 mac-rophages, the half-maximal concentration for LNO2 inhibi-tion of LPS-induced proinflammatory cytokine synthesis(LNO2 IC50) was 1.2–1.9 �M (L-serum) and 1.2–1.8 �M(D-serum) for IL-6, 0.4–0.8 �M (L-serum) and 0.5–0.8 �M(D-serum) for TNF�, and 0.5–0.6 �M (L-serum) and 0.5–0.7�M (D-serum) for MCP-1 (Fig. 1A). The half-maximal con-centration for OA-NO2 inhibition of LPS-induced proin-flammatory cytokine synthesis (OA-NO2 IC50) was 1.1–1.7

�M (L-serum) and 1.0–1.9 �M (D-serum) for IL-6, 0.3–0.9�M (L-serum) and 0.5–0.8 �M (D-serum) for TNF�, and0.4–0.6 �M (L-serum) and 0.5–0.8 �M (D-serum) forMCP-1(Fig. 1A). In RAW264.7 cells, the LNO2 IC50 was 1.1–1.7 �M(L-serum) and 1.3–1.8 �M (D-serum) for IL-6, 1.4–1.8 �M

(L-serum) and 1.2–1.8 �M (D-serum) for TNF�, and 1.1–1.7�M (L-serum) and 1.1–1.8 �M (D-serum) for MCP-1 (Fig.1B). The OA-NO2 IC50 was 1.8–2.5 �M (L-serum) and 1.9–2.4 �M (D-serum) for IL-6, 1.7–2.2 �M (L-serum) and 1.4–1.9 �M (D-serum) for TNF�, and 1.2–1.9 �M (L-serum) and1.3–2.2 �M (D-serum) for MCP-1 (Fig. 1B). Similarresponses were observed for the IC50 values of LNO2 andOA-NO2 in the inhibition of LPS-induced proinflammatorycytokine synthesis in primary cultures of human macro-phages: LNO2 IC50, 0.5–0.7 �M (D-serum) for IL-6, 0.4–0.6�M (D-serum) for TNF�, and 0.6–0.8 �M (D-serum) forMCP-1; OA-NO2 IC50, 0.7–0.8 �M (D-serum) for IL-6, 0.5–0.7 �M (D-serum) for TNF�, and 0.6–0.8 �M (D-serum) forMCP-1 (Fig. 1C). In addition, there were no statistically sig-nificant differences in the anti-inflammatory effects of thesenitroalkenes in the presence or absence of the lipidic com-ponents of serum. The native fatty acid precursors of LNO2and OA-NO2, LA or OA, also displayed no inhibitory effectstoward LPS-induced proinflammatory cytokine secretion(Fig. 1). These results affirm that the anti-inflammatoryactions of LNO2 andOA-NO2 are direct and not mediated byhydrophobic interactions or via reaction products generatedby the interaction of nitroalkenes with the lipophilic compo-nents of serum or macrophage membranes. In some controlexperiments, it was observed that the native fatty acids LAand OA slightly inhibited LPS-induced proinflammatorycytokine synthesis (10–20% inhibition) at concentrationsgreater than 5 �M (not shown). The subsequent nitration of LAand OA added to cultured cells remains a subject of furtherinvestigation. Higher concentrations of LNO2 (�5 �M) andOA-NO2 (�5�M) did not induce cytotoxic effects in our exper-imental conditions, as evidenced by an absence of alterations incellularmorphology and no detectable release of cellular lactatedehydrogenase (not shown). Collectively, our results indicatethat LNO2 andOA-NO2 exert strong anti-inflammatory effectsin macrophages. The close correspondence between IC50 valuesfor suppressing LPS-induced cytokine expression and nitroalkeneconcentration suggests a commonmechanism of action.The Inhibition of Proinflammatory Cytokine Secretion by

LNO2 and OA-NO2 in Macrophages Is NO-independent—Ni-trated fatty acids can undergo Nef-like decay reactions in aque-ous milieu that yield NO (18). The partition coefficient ofLNO2 is �1,500:1 (hydrophobic versus aqueous compart-ments). Thus, only 1 in 1,500 molecules of LNO2 areexpected to decay to yield NO in serum lipoprotein-contain-ing media and cell models, yielding at the most femtomolarconcentrations of NO. OA-NO2 is relatively more stable inaqueous milieu and only minimally decays to release NO(not shown). Furthermore, it has been reported that veryhigh concentrations of the NO donor DETA-NONOate(0.1–1.0 mM) are required to inhibit LPS-induced expressionof TNF� and IL-1 in human alveolar macrophages (19). Sim-




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ilarly, a 0.2–2.0 mM concentration of the NO donor SNAP isneeded to attenuate cytokine-induced expression of induci-ble NO synthase in the NR8383 rat alveolar macrophage cellline (19). In addition, a 200–1000 �M concentration of theNO donor SNAP is needed to attenuate cytokine-inducedexpression of inducible NO synthase in the NR8383 rat alve-olar macrophage cell line (20). Thus, it is unlikely that theanti-inflammatory effects of LNO2 and OA-NO2 are due toNO release from nitroalkenes being employed at a maximalconcentration of �5 �M. Nevertheless, we investigated thepotential involvement of NO or NO-derived species inLNO2- and OA-NO2-mediated anti-inflammatory signalingactions by comparing with NO donors, nitrite (NO2

�), andNO scavengers. As shown in Fig. 2, LPS-induced inflamma-tory cytokine production was not affected by the NO donorDETA-NONOate (100 �M) or nitrite (50 �M), the primaryend product of NO oxidation, at concentrations far exceed-ing those observed in vivo (21). In addition, the NO scav-enger cPTIO (100 �M) also had negligible effects on theinhibitory actions of LNO2 or OA-NO2 toward LPS-inducedcytokine expression (Fig. 2). These results indicate thatthe cytokine-suppressive actions of LNO2 and OA-NO2 areNO-independent.PPAR� Does Not Mediate the LNO2- and OA-NO2-mediated

Inhibition of Proinflammatory Cytokine Secretion in Macro-phages—We have demonstrated that LNO2 and OA-NO2 arepotent PPAR� ligands (11, 22). This PPAR� ligand activity isspecific for LNO2 orOA-NO2 andnotmediated by LNO2decayproducts, NO donors S-nitrosoglutathione (�100�M) or spermine-NONOate ((Z)-1-{N-[3-Aminopropyl]-N-[4-(3-aminopropylam-monio)-butyl]-amino}-diazen-1-ium-1,2-diolate) (�100 �M), LA,oxidizedLA,orOA(11,22).BecausePPAR�playsacritical role intheregulation of inflammatory responses (23), the role of PPAR� innitroalkene-induced inhibition of cytokine expression was inves-tigated in macrophages. In RAW264.7 cells, the relative expressionlevelsofPPARsweremeasuredbyquantitative real timereverse tran-scription-PCR. RAW264.7 cells expressed low levels of PPAR�1 andPPAR� and undetectable levels of PPAR� and PPAR�2 (Fig. 3A).LPS-induced secretion of inflammatory cytokines was not inhibitedby the high affinity PPAR� ligand rosiglitazone (2.5�M, a concentra-tion thatmaximally activatedPPAR�), butwas significantly inhibitedby 15-deoxy-�12,14-prostaglandin J2 (15d-PGJ2; 2.5�M, a concentra-tion that weakly activates PPAR�). The action of 15d-PGJ2, a lowabundance and low affinity PPAR� ligand, was comparable with in-hibitory effects of nitroalkenes, whereas the selective PPAR� ligand(GW501516) and the PPAR� ligand (Wy14643) showed no effect(Fig. 3B). Moreover, PMA-induced secretion of TNF� and MCP-1was inhibitedbyLNO2 andOA-NO2, aswell as 15d-PGJ2, butnot byrosiglitazone (Fig. 3B), indicating that nitroalkenes inhibited the se-cretion of inflammatory cytokines in macrophages via PPAR�-independent pathways. These observations are also consistent withprevious reports demonstrating that PPAR�-independent pathwayscontribute to anti-inflammatory effects of somePPAR� ligands (24).HO-1 Is Not Essential for the Inhibitory Actions of LNO2 and

OA-NO2 on Macrophage Cytokine Secretion—In inflammatoryconditions, tissues undergo protective responses to maintainfunctionality and viability. A central event in the mediation of

protective responses is expression of the inducible enzymeHO-1,which catalyzes the degradation of heme and yields biliv-erdin, carbonmonoxide, and iron (25). The electrophilic deriv-ative of arachidonic acid, 15d-PGJ2 inducesHO-1 expression invarious cell types, including macrophages, via mechanismsindependent of PPAR� activation (26, 27). Inmurine J774mac-rophages, the induction of HO-1 appears to be essential for theanti-inflammatory actions of 15d-PGJ2 (26). Accordingly, weexaminedwhetherHO-1 is a signaling intermediate of the cyto-kine-suppressive actions of nitroalkenes. LNO2 and OA-NO2in a concentration- and time-dependent manner up-regulatedHO-1 expression in RAW264.7 cells (Fig. 4A). The inhibitoryaction of nitroalkenes toward LPS-dependent responses wasnot reversible by inhibition of HO-1 catalytic activity by zincprotoporphryin-IX (Fig. 4B). To further confirm this observa-

FIGURE 2. The inhibitory effect of LNO2 and OA-NO2 on LPS-inducedproinflammatory cytokine secretion is NO-independent. Cells were stim-ulated overnight as indicated in fresh culture medium with 1% lipid-rich nor-mal serum. DETA-NO represents DETA-NONOate. The secretion of proinflam-matory cytokines was assessed by ELISA. Values are expressed as mean � S.D.(n � 6). *, p � 0.05 versus LPS alone. ND, nondetectable.




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tion, bone marrow-derived macrophages from HO-1 wild type(HO-1�/�) and knock-out (HO-1�/�) mice were utilized tofurther probe the mechanism of nitroalkene suppression ofLPS-induced expression of IL-6, TNF�, and MCP-1. The nullexpression ofHO-1 inHO-1�/� bonemarrowmacrophagewasconfirmed by Western blot (Fig. 4C). HO-1 deficiency did notinfluence the inhibitory effects of nitroalkenes toward LPS-in-duced secretion of cytokines in bone marrowmacrophage (Fig.4D), indicating that HO-1 does not transduce the suppressionof LPS-induced cytokine expression by nitroalkenes.LNO2 and OA-NO2 Inhibit NF-�B Signaling—The alkenyl

nitro configuration of nitrated fatty acids indicates electro-philic reactivity of the �-carbon adjacent to the nitro-bonded carbon, facilitating Michael addition reactions withbiological nucleophiles such as protein Cys and His residues.This electrophilic property of nitroalkenes was first sug-gested by the biological detection of �,�-nitrohydroxy fatty

acid derivatives (11). Also, syn-thetic nitroalkenes generated sim-ilar �,�-nitrohydroxy derivativesunder aqueous conditions uponreaction with the low levels ofhydroxide ion present at physio-logical pH (18). Appreciating thatnitroalkenes are electrophilic, acandidate mechanism underlyingLNO2 and OA-NO2 control ofinflammatory cascades in macro-phages can thus include theadduction (e.g. nitroalkylation) ofkey regulatory proteins. Thisnitroalkene-mediated post-trans-lational modification of proteins,such as transcription factors,would be expected to alter proteinactivity, function, and anatomicdistribution (15).The transcription factorNF-�B, a

dimer of members of the Rel familyof proteins, plays a pivotal role inorchestrating inflammatory re-sponses via the regulation of genesthat encode proinflammatory cyto-kines. This includes IL-6, TNF�,and MCP-1 and adhesion mole-cules, including intercellular adhe-sion molecule-1 and VCAM-1 (28).In resting cells, NF-�B is typicallysequestered in the cytoplasm byassociation with an inhibitory pro-tein, I�B. In response to variousstimuli, I�B kinase is activated andphosphorylates I�B on two serineresidues, resulting in I�B ubiquiti-nation, degradation by the protea-some, and facilitation of NF-�Bmigration into the nucleus tomediate the expression of inflam-matory response genes (28). The

most predominant form of NF-�B is a heterodimer of p65and p50 subunits (28). The DNA binding activities of the p65and p50 subunits of NF-�B are already appreciated to beinhibited by alkylation of a highly conserved Cys residuelocated in the DNA-binding domain (Cys62 in p50, Cys38 inp65). This alkylation reaction can be mediated by electro-philes, including 15d-PGJ2, sesquiterpene lactone, ethylpyruvate, and N-ethyl maleimide, leading to suppression ofthe transactivation of NF-�B target genes (29–32). Theseobservations underscore a unique mechanism of NF-�Binhibition based on the post-translational modification ofp65 and/or p50 through covalent alkylation of critical cys-teine residues in their DNA binding domains by a Michaeladdition reaction.Therefore, we tested whether LNO2 and OA-NO2 inhibit

NF-�B DNA binding by alkylation of p65/Cys38 and/or p50/Cys62, thus inhibiting LPS-induced inflammatory responses.

FIGURE 3. Nitrated fatty acids inhibit secretion of proinflammatory cytokines in macrophages via PPAR�-independent pathways. A, relative expression levels of PPARs in RAW264.7 cells. Expression of PPARs was analyzedby real time PCR. Expression of PPARs in WAT and liver was used as positive control. B, comparison of PPAR ligandsand nitrated lipids on the induced secretion of proinflammatory cytokines in RAW264.7 cells. Inset, the activity ofPPAR� induced by various PPAR� ligands was measured using a CV-1 reporter assay (f, OA-NO2; �, LNO2; Œ,rosiglitazone (Rosi); F, 15d-PGJ2; E, vehicle). Proinflammatory cytokine production was measured by ELISA. Theconcentrations used in B were: LPS, 1 �g/ml; PMA, 0.1 �M; OA-NO2, 2.5 �M; LNO2, 2.5 �M; Rosiglitazone, 3 �M;15d-PGJ2, 3 �M; GW501516, 0.5 �M; Wy14643, 100 �M. Values are expressed as mean � S.D. (n � 6). *, p � 0.05 versusLPS alone. WAT, white adipose tissue of C57BL/6J mice; Liver, liver of C57BL/6J mice.




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Consistent with this concept, NF-�B activation in LPS-treated RAW264.7 macrophages was inhibited by LNO2 andOA-NO2 but not by LA and OA (Fig. 5A). Additionally,LNO2 and OA-NO2 inhibited DNA binding of highly puri-fied p65 in vitro in a dose-dependent manner (0.01–1 �M). Incontrast, the native fatty acids LA and OA, even at highconcentrations (1 �M), had no effect on the DNA binding

activity of p65 (Fig. 5B). To explore the interaction betweenfatty acid nitroalkene derivatives and p65 protein further,the possible covalent alkylation of highly purified p65 pro-tein in vitro was studied using biotinylated LA, OA, LNO2,and OA-NO2. The biotinylated fatty acids were synthesized,purified by thin layer chromatography, and structurallycharacterized by electrospray ionization mass spectrometry.

FIGURE 4. HO-1 is not essential for nitrated fatty acid-mediated anti-inflammatory signaling. A, time course and dose responses of nitrated fattyacid-induced HO-1 expression in RAW264.7 cells. B, effect of HO-1 inhibitor on nitrated fatty acid-mediated anti-inflammatory effect. Values are expressed asmean � S.D. (n � 4). *, p � 0.05 versus LPS alone. C, expression of HO-1 in bone marrow-derived macrophages of HO-1 wild-type (WT) and knock-out (KO) mice.D, the anti-inflammatory effects of LNO2 and OA-NO2 in macrophages differentiated from bone marrow cells of HO-1 WT and KO mice. Values are expressed asmean � S.D. (n � 4). *, p � 0.05 versus LPS alone. HO-1 expression was examined by Western blot analysis, and proinflammatory cytokine production wasmeasured by ELISA as described under “Experimental Procedures.”




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Biotinylated fatty acids had the followingm/z in the negativeion mode: 519, LA; 521, OA; 564, LNO2; 566, OA-NO2, withnitrated fatty acid derivatives presenting the characteristicneutral loss of 47 corresponding to the nitro group (Fig. 5C).These structures were confirmed by respective C–N bondfragmentations that are depicted in Fig. 5C (a). The additionof biotinylated LNO2 and OA-NO2 to purified p65 in vitroresulted in a dose-dependent association with p65. Asexpected, biotinylated native fatty acids (LA and OA) did notassociate with p65 (Fig. 5D). To demonstrate the nature ofthis interaction and the relevance of this reaction in an intactcell model, RAW264.7 cells were treated with biotinylatedLA, OA, LNO2 andOA-NO2. There was a specific and robustcovalent nitroalkylation of p65 (Fig. 5E) and, to a lesserextent, p50 by fatty acid nitroalkene derivatives (not shown).The corresponding biotinylated native fatty acids did notreact with p65. The addition of biotinylated LA and LNO2 toLPS-elicited RAW264.7 cells resulted in a significant inhibi-tion of MCP-1 release in response to biotinylated LNO2 (p �0.05 compared with control) (Fig. 5F). The inhibition ofMCP-1 expression and release in response to LNO2 wasgreater than for biotinylated LNO2. There was no impact ofbiotinylated LA on MCP-1 release. In aggregate, the regula-tion of NF-�B signaling activity, via nitroalkene alkylationand consequent inhibition of p65, mediates suppression ofmacrophage cytokine expression and release. These resultsindicate that LNO2 and OA-NO2 can act endogenously asadaptive inflammatory mediators by inhibiting diverseNF-�B-mediated proinflammatory responses.LNO2 and OA-NO2 Inhibit Monocyte Adhesion to Endothe-

lial Cells—To explore more physiological functional aspects ofLNO2- and OA-NO2-mediated anti-inflammatory actions,the influence of nitroalkenes on an initial step of acute andchronic inflammation was addressed by analyzing the adhe-sion of monocytes to endothelial cells (33, 34)). As a conse-quence of endothelial cell activation by various inflamma-tory stimuli, monocytes adhere to activated endotheliumfirst by rolling and then by firm adhesion to migrate ulti-mately into the intima, where they differentiate into macro-phages and regulate the differentiation and function of vas-cular and nonvascular cells through the expression andsecretion of cytokines, reactive species, and other chemicalmediators. The transendothelial migration of monocytes islargely dependent on a class of adhesion molecules, includ-

ing intercellular adhesion molecule-1, VCAM-1, E-selectin,and P-selectin. VCAM-1 is a central mediator of the selectiverecruitment of monocytes and lymphocytes to atheroscle-rotic lesions (34). Since VCAM-1 is an NF-�B target gene(28), we hypothesized that LNO2 and OA-NO2 inhibit theinteraction of monocytes with endothelium by down-regu-lation of VCAM-1. In this regard, LNO2 and OA-NO2 dose-dependently inhibited VCAM-1 expression induced by LPSor TNF� in both THP-1 monocytes and HUVEC, whereasnative fatty acids (LA and OA) had no effect (Fig. 6, A and B).The inhibition of VCAM-1 expression by nitroalkenesoccurred at concentrations as low as 0.625 �M, well withinthe ranges of physiological LNO2 and OA-NO2 levels in thehealthy human circulation (10, 11). In THP-1monocytes, theNO donor DETA-NONOate (100 �M) had no effect on theLPS-induced VCAM-1 expression (Fig. 6A), suggesting thatNO is not mediating the inhibitory effect of nitroalkenestoward VCAM-1 expression. In endothelial cells, NO inhibitsNF-�B activity and VCAM-1 expression, but only at very highconcentrations (34). Multiple lines of reasoning indicate thatnitroalkenes do not inhibit TNF�-induced VCAM-1 expres-sion via NO release. First, whereas LNO2 is competent torelease NO in an aqueous milieu, this species is not expected todecay to yield NO in cell models due to hydrophobic stabiliza-tion, and if it did so would at best yield femtomolar levels of NO(18). Second, OA-NO2 is much more stable in an aqueousmilieu and only minimally decays to release NO within thepresent experimental time frames. Finally, in a recent report(35), the inhibition of VCAM-1 expression by the NO donorS-nitrosoglutathione only occurred at concentrations of 100�M. Since much lower concentrations of nitroalkenes (�2.5�M) inhibited VCAM-1 expression in the present report, it isviewed that any NO derived from LNO2 or OA-NO2 decay wasnot reaching a level that would mediate a significant impact onVCAM-1 expression.To evaluate the functional significance of nitroalkene-medi-

ated down-regulation of VCAM-1 expression, the effect ofLNO2 on the adhesion of THP-1monocytes to TNF�-activatedHUVECwas examined under both static and laminar flow con-ditions. LNO2 treatment of TNF�-activated endothelial cellsfully inhibited the static adherence of 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein-labeled monocytes (Fig. 7). Similarresponses were obtained under more physiological flow-medi-ated conditions (i.e. cells were cultured with a flow force rang-

FIGURE 5. Nitrated fatty acids inhibit NF-�B activation by nitroalkylation of p65. A, LNO2 and OA-NO2 inhibit LPS-induced NF-�B activation in RAW264.7cells. Cells were pretreated with LNO2 (2.5 �M) and OA-NO2 (2.5 �M) overnight and then stimulated with LPS (1 �g/ml) for 6 h. NF-�B activity was assessed asdescribed under “Experimental Procedures.” Values are expressed as mean � S.D. (n � 4). The data were analyzed using analysis of variance with theNewman-Keuls test. B, LNO2 and OA-NO2 directly inhibited NF-�B activity in vitro. Purified p65 was incubated with LNO2, OA-NO2, LA, and OA at differentconcentrations (0.01–1 �M) and then subjected to p65 activity assay as described under “Experimental Procedures.” Native fatty acids LA and OA at 0.01–1 �M

had no effect on the DNA binding activity of p65. Therefore, the activity of p65 treated with LA (1 �M) or OA (1 �M) was used as a control and set to 100%. Valuesare expressed as mean � S.D. (n � 4). The data were analyzed using analysis of variance with the Newman-Keuls test. C, structural characterization andchromatographic properties of biotinylated OA, LA, OA-NO2, and LNO2. a, structural characterization of biotinylated products by direct infusion into a ESI MS(ion trap) in negative ion mode. Main fragments are identified. Biotinylated lipids were prepared and characterized as described under “Experimental Proce-dures.” b, the corresponding elution profiles and retention times for the different biotinylated native and nitrated fatty acids are shown. D, p65 nitroalkylationby biotinylated LNO2 and OA-NO2 (B-LNO2 and B-OA-NO2, respectively) but not by native biotinylated fatty acids. Alkylation of p65 by nitroalkenes was assessedas described under “Experimental Procedures.” E, LNO2 and OA-NO2 covalently bind to p65 in vivo. RAW264.7 cells were treated with 0.1 �M biotinylated fattyacids and their corresponding nitro-derivatives, as indicated, for �1.5 h. Cell lysates were then treated with anti-p65 antibody or NeutroAvidin Plus followedby immunoblotting with anti-p65 antibody. F, biotinylated LNO2 inhibits LPS-induced inflammatory cytokine secretion in RAW264.7 macrophages. Cells werestimulated as indicated overnight, and the secretion of proinflammatory cytokines was assessed by ELISA. Values are expressed as mean � S.D. (n � 3). *, p �0.05 versus LPS alone.




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ing from 0.5 to 1.5 dynes/cm2). The numbers of THP-1 mono-cytes rolling on endothelium were also decreasedsignificantly by LNO2 treatment (Fig. 7). These results indi-cate that endogenous and inflammatory induced fatty acidnitration products serve as adaptive mediators by inhibitingmonocyte-macrophage and endothelial cell functions.

DISCUSSION

The observation that NO reacts with peroxyl radical inter-mediates of oxidizing unsaturated fatty acids, thus inhibitingthe autocatalytic progression of peroxidation reactions, led to asearch for novel reaction products (3, 4). In concert with thisapparent antioxidant action of NO, it was also observed thatunsaturated fatty acids reacting with NO and NO-derived spe-cies yielded nitrated derivatives (5). Although the detailedstructural nature of all nitrated fatty acid derivatives presentclinically under basal and inflammatory states remains to bedefined, a significant proportion of these species are present inhuman blood as nitroalkenes because of nitro group bonding toolefinic carbons of fatty acids (10, 11). Due to (a) nitroalkenedecay during hydrolysis of complex lipids, (b) the diversity ofcomplex lipid classes that contain esterified nitrated fatty acids,and (c) the recent report of abundant, reversible protein alkyla-tion by electrophilic fatty acid nitroalkene derivatives (15), nettissue concentrations of esterified and protein-adducted poolsof nitroalkenes remain to be defined.Current data reveal that nitrated fatty acids serve as media-

tors of diverse physiological and pathophysiological cell signal-ing processes, including vascular cell and inflammatory signal-ing. This notion was first supported by an appreciation thatsynthetic LNO2 activates guanylate cyclase in cultured vascularsmooth muscle cells and vessel segments, inducing relaxationof vessel segments, in an NO-dependent manner (7). Subse-quent analysis revealed a hydrophobically regulated Nef-likereaction of nitroalkenes that producesNO in low yields (18, 36).Also, LNO2 inhibits platelet aggregation and leukocyte func-tion via both cGMP- and cAMP-dependent and independentmechanisms, suggesting that nitro-fatty acids display reactivi-ties beyond serving as reserves for NO (7–9).More recently, intact nitroalkenes, rather than their aqueous

and aerobic decay products, have been observed to activatePPAR� at physiological concentrations, thus representing a sig-nificant component of endogenous ligand activity for PPAR�(11, 22). In addition to regulating many aspects of basal metab-olism, PPAR�-regulated gene products play criticalmodulatoryroles in pathophysiological processes, such as metabolic syn-drome (a condition characterized by multiple related clinicaldisorders, including insulin resistance, obesity, hyperlipidemia,hypertension, and heart disease), diabetes, and cardiovasculardisease (23, 24, 39). Thus, LNO2 and OA-NO2 are expected toexert diverse PPAR�-dependent biological effects.

From a broader perspective, nitroalkenes represent a class ofpluripotent cell signaling and inflammatory mediators thattransduce the signaling actions of NO via multiple reactionmechanisms. Of note, LNO2 decays in aqueous milieu with ahalf-life of �30 min, whereas OA-NO2 is relatively stable, withonly minimal decay occurring over 2 h (11). In the presence ofother lipids or amphiphiles at levels above the critical micellarconcentration, nitroalkenes are stabilized and become highlyresistant to NO release via the Nef-like reaction. This propertymay account for their accumulation to detectable levels in thevascular compartment. Thus, any additional signaling actionsof individual nitrated fatty acid regio- and stereo-isomers will

FIGURE 6. Nitrated fatty acids inhibit the LPS- and TNF-� induced expres-sion of VCAM-1 in THP-1 monocytes and human endothelial cells,respectively. A, LNO2 and OA-NO2 inhibited LPS-induced expression ofVCAM-1 in THP-1 monocytes. B, LNO2 and OA-NO2 inhibited TNF�-inducedexpression of VCAM-1 in human endothelial cells. VCAM-1 expression wasexamined by Western blot analysis as described under “ExperimentalProcedures.”




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be regulated by biodistribution, pharmacokinetics, and specificsecondary reactivities.Nitrated fatty acids have been reported in a variety of tissues,

with these derivatives including nitro-oleate, nitrolinoleate,nitro-arachidonate, and cholesteryl nitrolinoleate (6, 14, 36–38).WithacombinationofHPLCseparationsandESI-MS-MS/MS,alldetectable unsaturated fatty acids in healthy human serum andurine display some component of nitrated and �,�-nitrohydroxyderivatives (11). This latter derivative, formed by reaction withhydroxide at neutral pH, was the first indication that nitroalkenesmight act biologically as electrophiles.The electrophilic nature of the �-carbon to the nitroalkene

bond of nitrated unsaturated fatty acids facilitates reactionwithprotein thiol and histidine residues (15). At physiologically rel-evant concentrations, nitroalkenes inhibit GAPDH (IC50 of 3�M) in a thiol-reversible manner. Six GAPDH residues aremodified by nitroalkenes in vitro, including the catalyticallycritical Cys149. This adduction of GAPDH and other biomol-ecules by nitroalkenes significantly increases hydrophobicityand facilitates translocation to membranes. Moreover, adductsbetweenOA-NO2 and bothGAPDHand glutathione have beenidentified in vivo in human red blood cells (15). The post-trans-lational modification of other proteins by oxidized, electro-philic lipids has also been reported. For example, 15d-PGJ2 and4-hydroxy-2-nonenal form adducts with proteins that containnucleophilic centers (40). The electrophilic 4-hydroxy-2-non-enal reacts with sulfhydryl groups (41), the imidazole moiety of

histidine (42), and the �-amino oflysine (43). Covalent modificationby electrophilic lipids has beenshown to alter the structure andactivities of a variety of proteins (i.e.GAPDH (15, 44), cathepsin B (45),Keap1 (46), and insulin (42)). More-over, electrophiles, including 15d-PGJ2, sesquiterpene lactone, ethylpyruvate, and N-ethyl maleimide,mediate the suppression of thetransactivation of NF-�B targetgenes (29–32).The inhibition of TNF� and LPS-

induced monocyte cytokine expres-sion by nitrated fatty acids displaysan IC50 that falls consistently withinthe clinically relevant range of 500nM. Nitrated fatty acids displayapparent affinity for p65 nitroalky-lation, in that potential alternativecellular targets well exceed 20 mMconcentration in cells. Thesepotential alternative targets mini-mally include low molecularweight thiols and proteins con-taining cysteines and histidines.The determinants of target moleculereactivity with nitro-fatty acids willinclude compartmentalization, stericrestriction, the local ionic microenvi-

ronment, diffusional limitations, the rate constant for nitroalkene-nucleophile reaction, and finally, nitroalkene concentration.TheregulationofNF-�B-dependent signalingby itsp65subunit

is complex and also can include phosphorylation and acetylationin the course of cytoplasmic-to-nuclear trafficking. Althoughthese regulatory steps have been extensively described in the liter-ature, their extents and relative contributions have yet to be eluci-dated. Inaddition, the threshold forp65activationbyvariouspost-translational modifications (i.e. nitroalkylation, phosphorylation,and acetylation) in the context of mol % extents of target residuemodification remains undefined, as with various other proteinkinase-mediated signaling events.The biotinylation of electrophilic molecules has aided in the

identification of potential cellular targets, due to an ability toenrich modified proteins and enhance their sensitivity fordetection via secondary labeling strategies. In this regard, bio-tinylated nitro-fatty acid derivatives retain the electrophiliccharacter of the nitroalkene moiety. A limitation may be thatthe biotinylation of a small molecule, such as a fatty acid caninfluence distribution and in turn may influence reactivity dueto changes in size, charge, and hydrophobic characteristicsinduced by derivatization. If biotinylated nitro-fatty acidsinduced biological responses akin to nonmodified nitrated fattyacids, it can be assumed that the same signaling mediators andpathways would be targeted. To test this hypothesis, the biotin-ylated derivatives of linoleic and nitro-linoleic acidwere used inprobing effects on MCP-1 release by LPS-elicited RAW264.7

FIGURE 7. LNO2 inhibits THP-1 monocyte adhesion and rolling on human endothelial cells. THP-1 mono-cyte adhesion and rolling on HUVEC was analyzed as described under “Experimental Procedures.” All values areexpressed as mean � S.D. (n � 4) and were representative of five independently performed experiments. Thedata were analyzed using Student’s paired t test. *, p � 0.05 versus TNF� (�); #, p � 0.05 versus TNF� (�). ND,nondetectable.




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cells. Biotinylated nitro-linoleic acid induced a significant inhi-bition of cytokine release by LPS-elicited RAW264.7 cells at alower potency than the corresponding nonbiotinylated nitro-fatty acid. This indicates that a similar signaling target is beingnitroalkylated (p65, Fig. 5F).Herein, we have extended our understanding of the physiolog-

ical signaling capabilities of LNO2 andOA-NO2by reporting theirpotent and broad regulation of vascular inflammatory responsesvia post-translational modification of susceptible transcriptionfactors, such as p65. We have demonstrated that LNO2 and OA-NO2, at clinically relevant concentrations, inhibit LPS-inducedsecretion of proinflammatory cytokines in macrophages andendothelial cells. Also, TNF�-induced VCAM-1 expression andmonocyte rolling and adhesion are inhibited by these fatty acidnitroalkene derivatives. These particular anti-inflammatoryactions of LNO2 andOA-NO2 are independent of NO formation,PPAR� activation, and induction of HO-1 expression. Rather, theelectrophilic nature of fatty acid nitroalkenes facilitates the alkyla-tion of the recombinant NF-�B p65 subunit in vitro and the cor-responding alkylation of p65 in vivo, thus repressing NF-�B-de-pendent target gene expression. This inhibitory nitroalkylation ofa key transcriptional regulatory factor in turn inhibits the down-stream expression and action of critical components in the prop-agation of inflammatory responses, namely proinflammatorycytokine release and integrin expression. In aggregate, theseresults reveal that nitroalkenes, such as LNO2 and OA-NO2, reg-ulate the initiation andprogressionof inflammatoryprocesses andrepresent a distinct class of endogenous cell signaling mediators.

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Agarwal, Bruce A. Freeman and Yuqing E. ChenR. S. Baker, Carlos Batthyany, Balu K. Chacko, Xu Feng, Rakesh P. Patel, Anupam

Taixing Cui, Francisco J. Schopfer, Jifeng Zhang, Kai Chen, Tomonaga Ichikawa, PaulNitrated Fatty Acids: Endogenous Anti-inflammatory Signaling Mediators

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