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Prepublished online May 17, 2002; doi:10.1182/blood-2002-01-0316

Oxidized omega-3 fatty acids in fish oil inhibit leukocyte-endothelial interactions through activation of PPAR α Sanjeev Sethi, Ouliana Ziouzenkova, Heyu Ni, Denisa D Wagner, Jorge Plutzky and Tanya N Mayadas

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From bloodjournal.hematologylibrary.org by guestMay on June 2013. For use only. Blood First Edition Paper, prepublished online 17,1,2002; DOIpersonal 10.1182/blood-2002-01-0316

Oxidized omega-3 fatty acids in fish oil inhibit leukocyte-endothelial interactions through activation of PPARα

Short title: Oxidized n-3 fatty acids inhibit inflammation via PPARα

Sanjeev Sethi∗#, Ouliana Ziouzenkova†§, Heyu Ni‡§, Denisa D. Wagner‡, Jorge Plutzky†, Tanya N. Mayadas∗ ∗Vascular Research Division, Department of Pathology, †Division of Vascular Medicine, Department of Medicine, Brigham and Women’s Hospital and ‡Center for Blood Research and Department of Pathology, Harvard Medical School, Boston, MA 02115, USA

§ these authors contributed equally to this work #Current address: Department of Pathology, Univ. of Iowa Hospitals and Clinics, Iowa City, IA 52242, USA

This research was supported by National Heart, Lung and Blood Institute (T.N.M., D.D.W.), a research award from the American Diabetes Association (J.P.), the LeDucq Center for Cardiovascular Research (J.P.) and the American Heart Association (S.S). HN is a research fellow of the Heart and Stroke Foundation of Canada.

Corresponding author: Tanya N. Mayadas, Dept. of Pathology, Brigham and Women's Hospital, 221 Longwood Ave., Rm. 404, Boston, MA 02130. Tel: 617-278-0194; Fax: 617-732-5933; e-mail: [email protected]

1 Copyright 2002 American Society of Hematology


Word count: 142 words in the abstract, 3716 words in the text Appropriate scientific heading: Hemostasis, Thrombosis, and Vascular Biology

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ABSTRACT Omega-3 fatty acids which are abundant in fish oil improve the prognosis of several chronic inflammatory diseases although the mechanism for such effects remain unclear. These fatty acids, such as eicosapentanoic acid (EPA), are highly polyunsaturated and readily undergo oxidation. We show that oxidized, but not native unoxidized EPA significantly inhibited human neutrophil and monocyte adhesion to endothelial cells in vitro by inhibiting endothelial adhesion receptor expression. In transcriptional co-activation assays, oxidized EPA potently activated the peroxisome proliferatoractivated receptor (PPAR)α, a member of the nuclear receptor family. In vivo, oxidized, but not native, EPA markedly reduced leukocyte rolling and adhesion to venular endothelium of LPS-treated mice. This occurred via a PPARα-dependent mechanism since oxidized EPA had no such effect in LPS-treated PPARα-deficient mice. Therefore, the beneficial effects of omega-3 fatty acids may be explained by a PPARα-mediated anti-inflammatory effect of oxidized EPA.

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INTRODUCTION Consumption of omega-3 fatty acids in fish oil has been reported to improve the prognosis of several chronic inflammatory diseases characterized by leukocyte accumulation, including atherosclerosis, systemic lupus erythematosus, psoriasis, inflammatory bowel disease and rheumatoid arthritis1, 2, 3, 4. Fish oil is also recommended for treatment of IgA nephropathy, the most common form of primary renal disease worldwide 5, 6. Several studies suggest that omega-3 fatty acid supplementation may reduce the inflammatory response by attenuating leukocyte adhesion to the vessel wall 7, 8, 9. However the primary mechanism for the anti-inflammatory effects of fish oil remain unclear 10. Omega-3 fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are highly polyunsaturated and readily undergo oxidation 11. This suggests the possibility that oxidized omega-3 fatty acids may be an important component of the observed anti-inflammatory effects of fish oil. Indeed, Sethi et al., 1996 showed that oxidized EPA and DHA are more potent than native fatty acids in reducing RNA levels of leukocyte adhesion receptors and the adhesion of leukocyte cell lines to endothelial cells in vitro 12. Polyunsaturated fatty acids may exert their effects in cells by activation of Peroxisome Proliferator-Activated Receptors (PPAR). PPARs are ligandactivated transcription factors that regulate genes important in cell differentiation and various metabolic processes. Known PPAR isoforms include: PPARγ, important in lipoprotein metabolism, adipogenesis and insulin sensitivity; PPARα, implicated in fatty acid metabolism; and PPARδ, about which the least is known 13, 14. Synthetic PPARα ligands such as fibrates, e.g. fenofibric acid, are used in patients to lower triglyceride-rich lipoproteins. Synthetic PPARγ ligands such as thiazolidinediones, e.g. rosiglitazone, are insulin sensitizing drugs used in the treatment of diabetes. Polyunsaturated fatty acids and eicosanoids are known natural ligands of PPARα, γ and δ although they are required in relatively high concentrations (approx.100µM) for PPAR activation and are not 4


selective for PPAR subtypes 15, 16. On the other hand, derivatives of fatty acids further activate PPAR and can exhibit selectivity for PPARα or PPARγ in in vitro assays. For example, a cyclooxygenase product of arachidonic acid, 15d-delta12,14-PGJ2 is a selective PPARγ ligand whereas its lipoxygenase product 8(S)hydroxy-(5Z,9E,11Z,14Z)-eicosatetraenoic acid (HETE) is a selective PPARα agonist 17. Recently, both PPARα and PPARγ were identified in endothelial cells 14. Synthetic PPARα agonists decrease the transcription of endothelial VCAM-1 and the adhesion of HL60 cells to cytokine-activated endothelial cells 18. Other studies suggest that PPARγ agonists may also inhibit cytokine-induced VCAM-1 expression 19, 20 although such effects were not seen in vivo 21.

The aim of this study was to determine the effects of oxidized versus native omega-3 fatty acid, EPA, on the interaction of human blood leukocytes with endothelial cells in vitro, to examine if PPARs are activated by oxidized EPA and are therefore potential targets of oxidized EPA mediated effects, and to determine the relevance of the paradigm established in vitro, in LPS-mediated inflammation in vivo. Our results show that oxidized EPA inhibits the adhesion of freshly isolated human peripheral blood neutrophils and monocytes to an activated endothelial cell monolayer under static and physiological flow conditions, by reducing the surface expression of leukocyte adhesion receptors. We demonstrate that oxidized EPA potently activates PPARα to levels seen with fenofibric acid, a well characterized synthetic PPARα agonist. Finally, we demonstrate that oxidized EPA, but not native EPA, significantly decreases leukocyte adhesion to the inflamed endothelium in vivo and that activation of PPARα is essential for this effect.

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METHODS Preparation of fatty acids EPA was purchased specifically from Cayman Chemicals. It was relatively unoxidized as assessed by the thiobarbituric acid (TBA)-reactive substances (TBARs) assay using malondialdhyde (MDA) as a standard; this assay is a measure of fatty acid oxidation 22. EPA was dissolved in 100% ethanol and stored as a 100mM stock under nitrogen to insure minimal oxidation. 3.3mM of EPA was made up in PBS and 5µM cupric sulfate and 300µM ascorbic acid were sequentially added and are referred to as oxidizing reagents. The sample was incubated at 37°C for 12 hours. Extensive oxidation was observed as assessed by TBARs. For treatment of cells, oxidized EPA, or native EPA was diluted in media containing 20%FCS to final concentrations between 10-100µM as indicated in the Figures/Figure legends. The vehicle control (veh) for oxidized EPA was media containing PBS plus oxidizing agents.

Adhesion assay Human umbilical vein endothelial cells (HUVEC) monolayers in 96 well plates were pre-treated for 1 hour with vehicle control, native (100µM) or oxidized EPA (10, 50, 100µM) in standard growth medium. After 1 hour, the HUVEC were washed, the medium was replaced and the cells were stimulated with LPS (50ng/ml) (Sigma), hIL-1β (10U/ml) or hTNFα (10ng/ml) for 5 hours. Leukocytes

were

fluorescently

labeled

with

the

fluorescent

probe

bis-carboxyethyl-

carboxyfluorescein, acetoxymethyl ester (BCECF-AM) (Molecular Probes, Eugene, OR), added to wells (6x104/well) and incubated for 10 minutes at 37°C. The wells were washed, filled with media, the plates were sealed, inverted and spun at 100 x g for 5 minutes to remove non-adherent leukocytes. Adherence of labeled leukocytes was determined in a plate reader. Control studies indicated that fluorescence was a linear function of leukocytes in the range of 3000 to 60,000 6


cells/microplate well. Based on the standard curve obtained, the results are reported as the number of neutrophils or monocytes adherent to the underlying endothelium.

Parallel plate flow assay Confluent HUVEC plated on a coverslip were treated as described for the adhesion assay. The HUVECs were placed in a parallel plate flow chamber under defined laminar flow and neutrophils (106/ml) were applied at fluid shear stress varying from 2 to 0.25 dynes/cm2 as previously described 23. Leukocyte behavior was recorded on videotape and analyzed. The number of rolling and adherent

neutrophils cells were determined in a minimum of 5 high power fields (20X magnification) (20 seconds/field) at each shear stress and averaged.

Fluorescent immunoassay HUVEC monolayers in 96 well plates were treated as indicated above. The cells were washed with RPMI with 1% FBS and placed on ice. Primary monoclonal antibodies to ICAM-1, E-selectin, VCAM-1, endoglin and HLA class I were diluted in RPMI 1% FBS and added to each well for 1 hour on ice. The cells were washed three times with RPMI 1% FBS and FITC F(ab)2 goat anti-mouse IgG was added to each well. After incubation for 1 hour on ice the cells were washed twice with DPBS containing 20% FBS (to quench non-specific binding) and twice with DPBS. The cells were solubilized with lysis buffer and the fluorescence was read in a fluorescence microplate reader.

Transcriptional coactivation assay Transient transfection was carried out in primary bovine aortic endothelial cells using the Superfect method (Qiagen, Germany) according to manufacturer’s protocols. Briefly, cells were plated in 24well plates at 2.3x104 cells per well in DMEM containing 5% fetal calf serum. After 16 hours of 7


growth, cells were washed with HBSS and transfected with medium containing Superfect/DNA complexes (ratio 5.5/1) for 4 hours. Reporter construct pUASx4-TK-luc and chimeric human PPARα–LBD constructs have been described previously 24. PCMX-ß-galactosidase expression vector was used as a transfection control. Transfected cells were left overnight in medium, followed by treatment with the indicated compounds for 10 hours. Luciferase activity was assayed in a lysis buffer (Pharmingen) using a reporter assay system (Pharmingen) according to manufacturer’s instructions. ß-galactosidase activity was monitored at 540nm using chlorophenyl red b-D galactopyranoside (Boehringer Mannheim) as a substrate. Roziglitazone and fenofibric acid were used at concentrations of 1µM and 100µM respectively.

Intravital microscopy of mesenteric venules 4-week old 129SV wild type and PPARα -/- mice 25(inbred 129SV strain, Jackson laboratories) were injected (intra-peritoneal) with 500µl of PBS with oxidation reagents (Vehicle), 500µl of 3.3mM oxidized EPA (to achieve approximately 275µM concentration in the blood assuming 3 mls of blood volume and 3 mls of extravascular fluid and equal distribution between these two compartments), or with 500µl of 3.3mM native unoxidized EPA in PBS. One hour later the mice were injected with LPS (1µg in 100µl PBS). Five hours later mice were anesthetized and the mesentery was gently exteriorized and prepared for intravital microscopy as previously described 26. Rolling leukocytes were quantitated in venules of 25-35µm diameter by counting the number of cells passing a given plane perpendicular to the vessel axis in three minutes, and the number per minute was calculated. Centerline erthrocyte velocity (Vrbc) was measured using an optical Doppler velocimeter and venular shear rate was calculated as previously described 26. A minimum of three venules were studied per animal. Leukocytes adherent to the vessel wall were counted per mm2 of venular area at the end of

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every 3 minutes. At the end of the experimental procedure, peripheral blood was collected from the retroorbital plexus and total leukocyte counts were determined 26.

Statistical Analysis Data are presented as average±SEM. Statistical significance was assessed by unparied Student’s t test.

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RESULTS

Generation of oxidized EPA. Unoxidized EPA was diluted in media and subjected to oxidation by the addition of cupric sulfate and ascorbic acid as described in Methods. Gas Chromatography Mass Spectrometry (GCMS) of the samples revealed a single peak in the unoxidized EPA sample which was consistent with native EPA while oxidized EPA had less than 1% of the native EPA and a number of additional peaks which likely correspond to EPA oxidation products (data not shown). The extent of EPA oxidation in unoxidized or oxidized EPA is quantitated by measuring the amount of aldehydes present as previously reported12. The aldehyde content of 100µM unoxidized EPA was less than 0.02µM malondialdehye (MDA). Oxidized EPA was 1.78µM MDA in 100µM EPA12 which is in the range seen in plasma of humans (1.52 to 3.45 µM MDA) and animals (1.93µM MDA) fed fish oil diets27, 28, 29, 30, 31. Unoxidized EPA diluted in PBS to 100µM and exposed to air (but not additional oxidizing

agents) for 2 days at 37°C also showed significant oxidation (1.44µM MDA) which highlights the propensity for this highly polyunsaturated fatty acid to oxidize.

Oxidized EPA inhibits neutrophil and monocyte interaction with the endothelium under static adhesion, and fluid shear stress conditions In static adhesion assays, pretreatment of human umbilical vein endothelial cells (HUVECs) with oxidized EPA for 1hr prior to LPS stimulation significantly inhibited the adhesion of peripheral blood human neutrophils and monocytes while incubation with unoxidized, native EPA had little effect (Fig. 1A,B). The inhibition of LPS-induced adhesion was dose-dependent; 100µM of oxidized EPA reduced leukocyte adhesion to levels close to those seen for unstimulated HUVEC. Oxidized EPA similarly inhibited neutrophil adhesion to TNFα or IL-1β activated HUVECs (Fig.1C). The effect of 10


oxidized EPA on leukocyte rolling and firm adhesion to HUVECs under fluid shear stress conditions was assessed using a parallel plate flow chamber 23. HUVECs stimulated with LPS for 5hrs supported significant neutrophil rolling and adhesion under flow (0.5 dynes/cm2). Treatment of HUVEC with oxidized EPA for 1hr prior to LPS stimulation significantly inhibited both rolling (Fig. 2A) and adhesion (Fig. 2B) while native EPA had no effect.

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Oxidized EPA inhibits cytokine-induced surface expression of leukocyte adhesion receptors on endothelial cells LPS-stimulation of HUVECs induces expression of adhesion receptors, such as E-selectin, and VCAM-1 and ICAM-1, which support leukocyte tethering and adhesion respectively. Treatment with 12


oxidized EPA resulted in a dose-dependent reduction in LPS-stimulated surface expression of ICAM1, VCAM-1, and E-selectin whereas treatment with native EPA did not (Table 1). Oxidized EPA had no significant effect on the expression of endoglin, a membrane protein that binds TGFβ, and HLA class I molecules which are constitutively expressed.

PPARα is activated in endothelial cells treated with oxidized EPA We tested the possibility that oxidized EPA exerts its effects on endothelial cells through PPAR receptors, hypothesizing that oxidation of EPA might increase its ability to activate PPAR as compared to native EPA. Using two hybrid transcriptional co-activation assays in bovine aortic endothelial cell (BAEC) 24, we found that both oxidized and native EPA can modestly activate 13


PPARγ in a dose-independent manner (Fig. 3A). In contrast, oxidized EPA potently activates PPARα in a dose-dependent manner from 5 to 30 µM and was approximately 2-2.5 fold stronger than native EPA at equivalent concentrations. Interestingly, the activity by oxidized EPA was at least as potent as fenofibric acid, a well-documented synthetic PPARα ligand (Fig. 3B): The estimated half maximal activity for oxidized EPA was 10µM as compared to an EC50 of fenofibric acid which is 30µM. Analysis of the effects of 100µM doses of oxidized EPA on PPARγ and PPARα activation was not possible because unlike HUVECs which were viable and responsive to cytokines at this dose of EPA, this concentration was toxic to the transfected BAEC likely because the transfection protocol and/or the expression of high levels of the LBD construct is somewhat toxic to the cells: Untransfected 14


BAEC treated with 100µM EPA remained viable. Thus oxidized EPA potently activates PPARα in endothelial cells which may explain the observed reduction in LPS-induced endothelial cell receptor expression and leukocyte adhesion.

Oxidized EPA reduces leukocyte-endothelial interactions in vivo through activation of PPARα Next, we determined if oxidized versus native EPA reduced leukocyte adhesion during inflammation in vivo and if so, whether this occurred through a PPARα-dependent mechanism. Wild-type mice and mice deficient in PPARα were given an intraperitoneal injection of oxidized EPA, native EPA or vehicle alone. After 1hr, mice were injected intraperitoneally with LPS and 5hrs later underwent surgery and intravital microscopy of their mesenteric venules. In wild-type mice, LPS induced significant rolling and firm adhesion of leukocytes to the endothelium (Fig. 4). Similar levels of rolling and adhesion were observed in LPS-treated PPARα-deficient mice suggesting that PPARα does not play a role in LPS-induced leukocyte adhesion to the endothelium. LPS-injected wild-type mice pretreated with oxidized EPA, but not native EPA, had markedly reduced leukocyte rolling (75%) and adhesion (75%) to inflamed venular endothelium compared to LPS-injected wild type mice pretreated with vehicle alone. Indeed, levels of rolling and adhesion in the LPS and oxidized EPA treated mice approached those seen in mice injected with vehicle alone (Fig. 4). In contrast, LPStreated PPARα-deficient mice pretreated with native or oxidized EPA demonstrated no decrease in leukocyte rolling and adhesion compared to wild-type or PPARα-deficient mice treated with LPS alone. Peripheral blood leukocyte counts and venular shear rates were comparable between all the different groups of mice (data not shown). Together, these data indicate that oxidized EPA inhibits leukocyte-endothelial interactions in vivo and that this occurs through a PPARα-dependent mechanism. The much stronger inhibitory effect of oxidized EPA versus native EPA on leukocyte

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adhesion in wild-type animals correlated well with its more potent activation of PPARα in endothelial cell cultures compared to native EPA (Fig. 3B), a difference not seen for PPARγ activation (Fig. 3A).

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17


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DISCUSSION Our data suggests that the beneficial effects of fish oil in chronic inflammatory diseases may be due to the oxidative modification of omega-3 fatty acids and its subsequent inhibition of leukocyte adhesion receptor expression and leukocyte-endothelial interactions. Oxidized EPA significantly inhibited LPSinduced leukocyte rolling and adhesion, which are processes mediated by leukocyte adhesion receptors of the selectin, and integrin and IgG superfamily respectively 32. Native EPA had no effect on leukocyte recruitment in these models. Our proposed mechanism of omega-3 fatty acid actions may also contribute to the decreased atherosclerosis among populations with high intake of fish rich in omega-3 fatty acids 4. The amount of oxidized EPA that yielded a decrease in leukocyteendothelial interactions in vivo was approximately 275µM in circulation which may be achieved in humans with fish oil supplementation (700µM total EPA)28 or salmon-rich containing diets 33, 34. Oxidation of EPA leads to the generation of a mixture of aldehydes, peroxides and other oxidation products which are probably responsible for the observed anti-inflammatory effect 12. Although the structure(s) of the specific aldehyde/peroxide(s) that is responsible for the antiinflammatory activity is not currently known, these products are undoubtedly present in humans consuming these fatty acids for the following reasons: Highly polyunsaturated long chained EPA is readily oxidized at room temperature even in the absence of exogenous oxidizing reagents. More importantly, in vivo, a large increase in tissue and plasma accumulation of fatty acid oxidation products is noted in subjects consuming fish oil 7, 27, 28, 35 even after addition of anti-oxidant supplements to the diet 27, 28 which suggests extensive oxidation of omega-3 fatty acids such as EPA in vivo. During inflammation, increased oxidation of dietary omega-3 fatty acids may occur due to increased oxidative stress, and the expression of enzymes (e.g. cyclooxygenase and lipoxygenase) capable of oxidizing polyunsaturated fatty acids. On the other hand, fish oil consumption does not lead to an increase in oxidation of plasma proteins 28 which is important since protein oxidation can 19


contribute to the development of diseases such as atherosclerosis 36, 37. Endothelial cells have also been identified as a source of the omega-3 fatty acid DHA 38, 39, which suggests the intriguing possibility that during inflammation, this fatty acid could be locally oxidized and serve as a potent natural anti-inflammatory mechanism for limiting the inflammatory response. Our study provides a mechanism by which oxidized EPA inhibits leukocyte-endothelial interactions. Both oxidized and native EPA modestly activated PPARγ, but oxidized EPA potently activated PPARα in endothelial cells, and to a much greater extent than native EPA. Therefore, oxidation of the omega-3 fatty acid converts it into a stronger PPARα agonist. A recent study suggests that oxidized LDL and not native LDL dose-dependently activates PPARα in endothelial cells and that the oxidized phospholipid component is responsible for this effect 40. In particular, oxidized metabolites of linoleic acid, 9- and 13-hydroxyoctadecadienoic acid (9-HODE and 13HODE) were potent PPARα ligands in endothelial cells 40. This, along with our study, suggests the possibility that oxidation of lipids may be one of the first steps involved in the generation of potent PPARα agonists in endothelial cells. Although our studies indicate that native EPA activates PPARα about half as well as oxidized EPA it had no effect on leukocyte-endothelial interactions in vitro or in vivo. The reasons for this may include the following. The co-transcriptional GAL4 ligand binding domain (LBD) assay is a valuable method of isolating one aspect of ligand-PPAR interaction, namely the affinity of LBD for ligand. However, this assay excludes other cellular events that impact on PPAR-induced cellular transcriptional responses such as phosphorylation of PPARα 41 or PPARγ 42 and the interaction between coactivators and corepressors with PPAR 43, all of which may differentially effect the ability of oxidized versus native EPA to modulate PPARα mediated responses. For example, “nonproportional” responses have been seen for PPARγ and the putative ligand 15dPGJ2. This prostaglandin demonstrates much less PPARγ LBD affinity than the potent thiazolidinedione class of 20


PPARγ agonists 24 but has comparable biological effects to these drugs in vitro 24, 44. Another explanation for the differential results of oxidized EPA and EPA in the in vivo and transfection assays is that PPARα activation by EPA seen in the co-transcritional assays might remain beneath a threshold required for seeing a functional effect. These limitations of the co-transfection assay emphasize the importance of obtaining evidence for PPARα activation in in vivo settings. Indeed, we show that oxidized EPA significantly inhibited leukocyte-endothelial interactions in the complex environment of LPS-induced inflammation in mice in vivo, a response that was absent in PPARα deficient mice. Together our data demonstrate that oxidized EPA can both activate a PPARα LBD in vitro, and exert potent anti-leukocyte adhesive effects in vivo which are entirely PPARα dependent. On the other hand, LPS treatment of wild-type and PPARα deficient mice led to significant leukocyte rolling and adhesion which was comparable in both genotypes suggesting that PPARα does not play a role in LPS induced leukocyte-endothelial cell interactions. To our knowledge, ours is the first demonstration that a PPAR agonist (oxidized EPA) has potent inhibitory effects on neutrophilendothelial interactions in vivo 18, 19, 20. Our studies also indicate that unlike the in vitro studies, which report that PPARα activation leads to the downregulation of select leukocyte adhesion receptors (VCAM-1 but

not ICAM-1 or E-selectin) 18, in vivo, PPARα is responsible for

downregulating a spectrum of adhesion molecules required for leukocyte rolling and adhesion. The downregulatory effects of oxidized EPA mediated PPARα activation is likely through an inhibition of LPS or cytokine induced activation of the transcription factor NFκB which stimulates the transcription of several cytokine inducible adhesion receptors including E-selectin and VCAM-1 (Fig 5). Evidence for this comes from studies demonstrating that ligand-activated PPARα can inhibit NFκB activity through as yet partially unknown mechanism(s) (i.e. direct interactions with the p65 subunit of NFκB, or increased synthesis of the NFκB inhibitor, IκBα have been implicated 45, 46, 47), and our previous data showing that oxidized EPA inhibits NFκB activation 12. 21


The oxidized EPA mediated activation of PPARα, may be relevant in biological responses beyond those that were studied here. It may accelerate fatty acid metabolism which has implications for obesity, a known risk factor for cardiovascular disease and diabetes. In addition both fish oil and the PPARα agonist, fenofibrate have cholesterol and triglyceride lowering effects suggesting that oxidized EPA induced PPARα activation may contribute to the lipid lowering effect of fish oil. Although, a recent study in PPARα-deficient mice suggested PPARα and fish oil affect lipoprotein metabolism through different mechanisms 48. In summary, oxidized EPA is a potent inhibitor of leukocyte interactions with the endothelium compared to native EPA, both in vitro and in an in vivo context of inflammation. Oxidized EPA’s effects are mediated through activation of PPARα which is responsible for the downregulation of leukocyte adhesion receptor expression required for leukocyte-endothelial interactions. We propose that oxidation of EPA and its activation of PPARα is the underlying mechanism for the beneficial effects of fish oil. Finally, given the potency of oxidized EPA in activating PPARα, and that its effects were comparable to those seen with the PPARα agonist, fenofibrate, our studies also suggest that oxidized omega-3 fatty acids may represent a class of naturally occurring, low toxicity, PPARα agonists with potent anti-inflammatory properties.

Acknowledgements The authors would like to thank Ms. Kay Case for providing HUVECs, and Drs. F.W. Luscinskas and Y.C. Lym for their help with the parallel plate flow chamber.

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28


ICAM-1 VCAM-1 E-selectin Endoglin HLA Class I

Veh.

Veh.

54±5 6±4 nd 911±65 76±7

+LPS 272±40 211±17 248±11 736±107 79±9

EPA (100µ M) +LPS 373±36 312±38 328±75 907±45 151±16

oxEPA (10µ M) +LPS 265±71 198±16 191±27 781±48 67±11

oxEPA (50µ M) +LPS 182±50 92±26 138±36 840±38 121±31

oxEPA (100µ M) +LPS 61±15* 29±10† 53±6† 743±16 109±25

Table 1 Effect of oxidized EPA on expression of endothelial adhesion molecules. HUVECs were pre-treated for 1 hour with vehicle (Veh), native (EPA) or oxidized EPA (oxEPA) prior to stimulation with LPS for 5 hours. Surface expression of adhesion molecules was quantitated by fluorescent immunoassay using appropriate monoclonal antibodies. nd= none detected. *p

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