Effects of conjugated linoleic acid isomers on monocyte, macrophage and foam cell phenotype in atherosclerosis

Prostaglandins & other Lipid Mediators 98 (2012) 56–62

Contents lists available at SciVerse ScienceDirect

Prostaglandins and Other Lipid Mediators

Review

Effects of conjugated linoleic acid isomers on monocyte, macrophage and foam cell phenotype in atherosclerosis Declan Mooney ∗ , Cathal McCarthy, Orina Belton School of Biomedical and Biomolecular Science, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland

a r t i c l e

i n f o

Article history: Received 16 August 2011 Received in revised form 19 December 2011 Accepted 20 December 2011 Available online 31 December 2011 Keywords: Conjugated linoleic acid Atherosclerosis Monocyte Macrophage PPAR␥

a b s t r a c t Conjugated linoleic acid (CLA) is a generic term denoting a group of naturally occurring isomers of linoleic acid (18:2, n6) that differ in the position or geometry (i.e. cis or trans) of their double bonds. The predominant isomers in ruminant fats are cis-9,trans-11 CLA (c9,t11-CLA), and trans-10,cis-12 CLA (t10,c12-CLA). The biological activities of CLA have received considerable attention because of its protective effects in cancer, immune function, obesity and atherosclerosis. Importantly, dietary administration of a blend of the two most abundant isomers of CLA, has been shown to inhibit the progression and induce the regression of pre-established atherosclerosis in the ApoE−/− murine model. Studies investigating the mechanisms involved in CLA induced protective effects are continually emerging with results from both in vitro and in vivo models yielding confounding and often inconsistent results depending on both the isomer of CLA and the species under investigation. The purpose of this review is to comprehensively discuss the effects of CLA on monocyte/macrophage function in atherosclerosis. This review also discusses the possible mechanisms through which CLA mediates its atheroprotective effects with a particular emphasis on the migratory capacity of the monocyte and the inflammatory and cholesterol homeostasis of the macrophage. © 2011 Elsevier Inc. All rights reserved.

Contents 1. 2. 3. 4. 5.

6. 7.

Conjugated linoleic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogenesis of atherosclerosis and CLA induced regression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanism of action of CLA isomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CLA modulates functional properties of human monocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect of CLA treatment on macrophage phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. RAW mouse macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. THP-1 macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Conjugated linoleic acids Conjugated linoleic acids (CLAs) are a group of naturally occurring unsaturated fatty acids that differ in the disposition and orientation (cis or trans) of their double bonds. To date, twenty eight CLA isomers have been identified in milk, dairy and beef products. CLA is produced as an intermediate in the bacterial

∗ Corresponding author. Tel.: +353 1 7166748; fax: +353 1 7166701. E-mail address: [email protected] (D. Mooney). 1098-8823/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.prostaglandins.2011.12.006

56 57 58 58 59 59 60 60 61 62

biohydrogenation of the parent compound linoleic acid to stearic acid in ruminant animals [1] where it can be incorporated into tissues or biohydrogenated further to trans-vaccenic acid (TVA). The predominant naturally occurring CLA isomer is cis-9,trans-11 CLA (c9,t11) which accounts for approximately 90% of CLA intake in the diet, with trans-10,cis-12 (t10,c12) accounting for less than 10% [2]. The structure of the two most abundant isomers of CLA and the parent compound, linoleic acid, is shown in Fig. 1. Purification and commercialization of the two most abundant CLA isomers has facilitated detailed investigation of the biological activities of each isomer as well as different isomeric blend

D. Mooney et al. / Prostaglandins & other Lipid Mediators 98 (2012) 56–62

Fig. 1. Structure of the conjugated linoleic isomers cis-9,trans-11 and trans-10,cis-12 and the parent compound linoleic acid [3].

variants. Individual CLA isomers have been shown to mediate differential effects and in certain instances the mixture of a blend of isomers is not predictive. Previous investigations into the biological effects of CLA have identified it as having a potentially diverse range of benefits in health and diseases such as cancer [4–6], obesity [7,8], immune function [9] and atherosclerosis [10–13]. There is now a strong emphasis on identifying either the individual isomer or the optimal isomeric blend responsible for the beneficial effects in each particular disease state. An intense area of current research is in defining the protective pathways and underlying mechanisms through which CLA mediates such a diverse array of beneficial effects. Most of the key molecular and cellular mediators that are involved in each stage of the pathogenesis of atherosclerosis have been identified from the accumulation of LDL following endothelial dysfunction, to monocyte transmigration and differentiation to macrophages, subsequent foam cell formation, smooth muscle cell migration and formation of the fibrous cap. However, to date there have been no defined pathways which would explain how this process could be reversed or indeed if such a pathway exists. The current gold standard for the treatment of atherosclerosis is the HMG-CoA reductase inhibitors, which inhibit endogenous cellular cholesterol synthesis. While these drugs are effective at preventing the progression of the disease they are unable to induce clinically relevant regression. Interest in CLA grew exponentially when it was discovered that administration of a CLA supplemented diet induced regression of pre-established atherosclerosis in rabbits [13,14]. Subsequent studies using other animal models of atherosclerosis, such as the ApoE−/− mouse, have also demonstrated CLA-induced regression of pre-established atherosclerosis [10,11]. However, there are a number of other studies with conflicting results which point to a potential pro-atherogenic role for CLA. The focus of current atherosclerotic studies involving CLA treatment is in identifying the cellular targets and mechanisms through which CLA induces regression. The results of these studies are critical as most patients present clinically with pre-established disease. To date only the effects of CLA on smooth muscle and endothelial cells have been comprehensively examined [15]. The purpose of this review is to summarize and compare the results of in vitro studies that have examined the effects of CLA on the functionality of monocytes, a cell type yet to be reviewed, and macrophages in the context of atherosclerosis. 2. Pathogenesis of atherosclerosis and CLA induced regression Atherosclerosis, the underlying pathology of heart disease, stroke and peripheral vascular disease, is a complex progressive disease with multiple genetic and environmental contributions. It

57

is a chronic inflammatory and cholesterol storage disease characterized by the formation of fibrous lipid laden plaques in the major blood vessels/arteries. The development of atherosclerosis can be divided into three distinct stages: (1) initial endothelial dysfunction and monocyte recruitment, (2) macrophage accumulation and fatty streak formation and (3) plaque development and rupture. Monocytes are involved in the initial stages of atherosclerosis development. Dysfunctional/activated endothelial cells express selectins (E-selectin) and cell adhesion molecules such as VCAM1 and ICAM-1 which are involved in the rolling and firm binding of the monocyte to the endothelial cell layer. Release of monocyte chemoattractant protein 1 (MCP-1) from the activated endothelium and resident inflammatory macrophages in the intima induce the monocyte to undergo transendothelial migration from the lumen into the underlying intima. Upon entering the intima, monocytes undergo differentiation to macrophages in response to macrophage colony stimulating factor (MCSF). Newly formed macrophages express the scavenger receptors, SRA1 and CD36, which mediate the uptake of oxidized low density lipoprotein (oxLDL) into the cell, resulting in a continuous influx of pro-atherogenic oxLDL and the accumulation of cholesterol and lipid droplets in the cytosol. Uptake of oxLDL increases the oxidative stress profile of macrophages leading to increased production of pro-inflammatory mediators such as tumor necrosis factor alpha (TNF), MCP-1 and various interleukins (e.g. IL-6). The release of these mediators continues the recruitment of other monocytes and leukocytes which propagates the development of the disease. Macrophages which contain high concentrations of oxLDL are referred to as foam cells with subendothelial accumulations of foam cells called fatty streaks. The lipid laden foam cells in the fatty streak undergo apoptosis/necrosis and deposit their cholesterol and lipid into a necrotic lipid rich core. Smooth muscle cells (SMCs), resident in the media, migrate to the intima in response to growth factors released from activated macrophages and foam cells. The SMCs encircle the necrotic core and secrete stabilizing extracellular matrix proteins such as collagen and fibrin to form a fibrous cap which is characteristic of an early stage stable plaque. As the disease progresses the continued influx of proinflammatory macrophages will eventually lead to destabilization of the cap through the actions of matrix metalloproteinases (MMPs) which breakdown fibrin and collagen. Eventually this leads to rupture with subsequent thrombus formation clinically manifesting as either a myocardial infarction or stroke. Previously we have shown that an 80:20 isomeric CLA blend induces regression of pre-established atherosclerosis [10,11]. The ApoE−/− animal study design involved administration of a 1% cholesterol diet for 8 weeks to induce atherosclerosis followed by a further 8 weeks of 1% cholesterol alone or 8 weeks of 1% cholesterol diet supplemented with isomeric 1% CLA blend. ApoE−/− mice fed the CLA blend supplemented diet showed a 30% decrease in lesion area. Our group further characterized the potential mechanisms through which CLA may induce regression by identifying the effect of CLA treatment on peroxisome proliferator activated receptor gamma (PPAR␥) expression. It was found that CLA induced the expression of PPAR␥, a nuclear protein previously shown to have anti-inflammatory effects. Our regression study used an 80:20 isomeric blend of the c9,t11 and t10,c12 isomers respectively, other studies using the individual isomers of CLA highlighted the differential effects of each isomer on atherosclerosis development. These studies demonstrated that c9,t11 was the key isomer involved in the impediment of atherosclerosis development with the c9,t11 fed mice exhibiting reduced plasma cholesterol, glucose and reduced lesional area of the aorta as well as increased expression of markers of plaque stability [12]. Evidence identifying a cellular target of CLA was reported by Toomey et al. [10] who showed increased CD68, a macrophage marker, expression in the lesions of

58

D. Mooney et al. / Prostaglandins & other Lipid Mediators 98 (2012) 56–62

ApoE−/− mice fed a high cholesterol diet. In contrast the expression of CD68 was significantly decreased in the lesions of mice fed a CLA supplemented diet suggesting that CLA supplementation decreased the infiltration of macrophages into the atherosclerotic plaque. 3. Mechanism of action of CLA isomers One of the main mechanisms through which CLA isomers mediate their effects has been shown to involve transcription factors referred to as peroxisome proliferator activated receptors (PPARs). PPARs are ligand activated transcription factors that belong to the nuclear receptor superfamily. Since their discovery two decades ago [16] studies examining their functions and mechanistic actions have grown substantially and they are now key therapeutic targets for the treatment of numerous metabolic diseases including diabetes, atherosclerosis and cancer. There are three PPAR isoforms (1) PPAR␣, (2) PPAR␦/␤ and (3) PPAR␥. Upon activation PPARs heterodimerize with the retinoid X receptor (RXR) and bind to PPAR response elements (PPRE) a direct repeat of two hexanucleotide recognition motifs AGGTCA separated by one nucleotide, in the target gene promoter. Unsaturated fatty acids, such as CLA, are ligands for PPARs while saturated fatty acids only induce weak activation [17]. Endogenous ligands for PPARs include eicosanoids and prostaglandins while synthetic ligands include the thiazolidinediones and fibrates. The c9,t11 CLA isomer has also been shown to be a ligand for PPAR␥ [18]. In vitro studies have shown PPARs to have both pro and anti atherogenic effects. The scavenger receptor CD36 contains a PPRE in its promoter region and is thus a transcriptional target for PPAR␥ [19]. Treatment with a PPAR␥ agonist results in increased uptake of oxLDL, however PPAR␥ agonists also increase the activity of the reverse cholesterol transport (RCT) system resulting in increased expression of liver X receptor (LXR) and ATP-binding cassette transporter (ABCA1) respectively which efflux excess cholesterol from the cell [20]. In addition to its role as a PPAR␥ agonist, modulation of cyclooxygenase (COX) activity and expression has also been suggested to underlie some of the beneficial biological effects of CLA. COX enzymes mediate the same rate limiting enzymatic reaction governing the formation of thromboxane, prostaglandins and prostacyclin through the breakdown of arachidonic acid contained in the phospholipids of cell membranes. Products of this reaction have diverse physiological effects and have known roles in inflammation, vascular tone and gastric cytoprotection [21]. There are two isoforms of COX, COX-1 is ubiquitously expressed in tissues and cells throughout the body [22] while COX-2 expression is more stringently regulated and is induced at sites of inflammation in response to cytokine release [23]. COX-2 potentiates the inflammatory response and is thus believed to be pro-atherogenic. The role of COX enzymes in atherosclerosis development has already been well established with evidence demonstrating increased levels of both isoforms and their prostaglandin products in atherosclerotic plaques of human patients [24]. CLA has been shown to act as an inhibitor of the enzymatic activity of COX in vitro [25]. Suppression of COX-2 expression by CLA may be explained by the modulatory effect of CLA on the expression of proteins involved in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF␬B) signaling pathway. 4. CLA modulates functional properties of human monocytes Chemokine mediated transendothelial migration of the monocyte from the lumen, across the activated endothelial cell layer into the underlying intima is a key step in the early development

Table 1 Effects of various CLA isomers on THP-1, human PBMC and porcine PBMC monocytes. Isomer (A) Monocytes • c9,t11 (25 ␮M) t10,c12 (25 ␮M)

• c9,t11 (25 ␮M) t10,c12 (25 ␮M) Blend (80:20) • t10,c12 (100 ␮M) • t10,c12 (10 ␮M)

• t10,c12 (10 ␮M)

Result

Refs.

– c9,t11 ↓ THP-1 monocyte + PBMC migration to MCP-1 (PPAR␥ dependent) – c9,t11 ↓ THP-1 monocyte + PBMC migration to platelet releasate (PPAR␥ independent) – c9,t11 + t10,c12 + Blend CLA isomers ↓ SORLA – Overexpression of SORLA = ↑ THP-1 migration to MCP-1 – ↓ Tissue factor mRNA and antigen following LPS stimulation – ↑ IL-8 production in porcine PBMC with resultant ↑ migration of polymorphonuclear cells – t10,c12 ↑ NF␬B p65 DNA binding activity and TNF␣ (PPAR␥ dependent) in porcine PBMCs – t10,c12 ↓ NF␬B p65 DNA binding activity and TNF␣ in LPS stimulated porcine PBMCs

[26]

[28]

[29] [32]

[33]

of atherosclerosis. The following section discusses the results of studies investigating the effects of CLA on the monocyte migratory phenotype (Table 1). Using the modified Boyden Chamber, an established model to study transendothelial monocyte migration, we previously investigated the effects of CLA on monocyte migration using the THP-1 human monocyte cell line and peripheral blood monocytes (PBMC) isolated from healthy volunteers [26]. Initially THP-1 monocytes were treated with 25 ␮M c9,t11 CLA, t10,c12 CLA and the PPAR␥ agonist troglitazone (5 ␮M). Both c9,t11 CLA and troglitazone significantly inhibited monocyte migration in response to MCP-1 (25 ng/ml) while t10,c12 and the control fatty acid, oleic acid, had no effect. This inhibition was concomitant with an increase in the mRNA expression of PPAR␥. Addition of the PPAR␥ antagonist GW9662 prevented troglitazone mediated inhibition of monocyte migration in response to MCP-1. These studies were extended to PBMCs which were treated with 10 ␮M CLA isomers and similar results were achieved which suggests that inhibition of human monocyte migration to MCP-1 is mediated by the c9,t11 isomer via a PPAR␥ dependent effect. To further expand investigation of CLA inhibitory effects on monocyte migration another chemoattractant, thrombin receptor activating peptide (TRAP) stimulated platelet releasate was investigated. The results showed a divergence in the mechanisms of action of inhibiting migration. c9,t11 CLA significantly reduced monocyte migration but troglitazone had no effect. This suggests that CLA inhibits monocyte migration through at least two differential mechanisms, one that is PPAR␥ dependent and one is PPAR␥ independent depending on the chemoattractant used. Initial transcriptomic studies in our group identified sortilin related receptor (SorLA) as a gene whose expression was significantly decreased in the aorta of our CLA induced model of regression. SorLA expression was localized to the monocyte/macrophage in the aortic vessel wall. Previously this protein had been shown to play a major role in the migration of smooth muscle cells [27]. Initial treatment of THP-1 monocytes with 25 ␮M CLA isomers decreased the expression of SORLA. This decrease in SorLA expression was found to be PPAR␥ dependent through the addition of a PPAR␥ antagonist. Subsequent over-expression of SorLA was found to increase THP-1 monocyte migration in response to MCP-1 [28]. These results indicate that SorLA may be a key gene involved in CLA mediated inhibition of monocyte migration. Tissue factor (TF) is a membrane glycoprotein which is involved in the initial steps of the coagulation cascade. Monocytes are a source of TF and the effects of CLA on its production have been investigated. Treatment of THP-1 monocytes with 100 ␮M of the c9,t11 and t10,c12 isomers showed that t10,c12 inhibits

D. Mooney et al. / Prostaglandins & other Lipid Mediators 98 (2012) 56–62

both the mRNA and antigen expression of monocytic TF following lipopolysaccharide (LPS) stimulation [29]. In contrast c9,t11 had no effect on TF expression. A potential mechanism for this inhibition was elucidated with the addition of the PPAR␣ agonist WY-14643 which also inhibited TF expression. These results suggest that the t10,c12 isomer inhibits TF production through the activation of PPAR␣, a known target for this isomer [30,31]. Other studies have examined the effects of CLA treatment on the inflammatory profile of monocytes [32]. Treatment of porcine PBMCs with 10 ␮M t10,c12 increased the production of the chemokine IL-8 which resulted in increased migration of porcine polymorphonuclear cells. Other studies have shown that treatment of LPS naïve porcine PBMCs with the t10,c12 isomer increased the production of TNF␣ via increased NF␬B p65 DNA binding activity [33] while stimulation of the PBMCs with LPS and subsequent treatment with t10,c12 resulted in an inverse effect on NF␬B p65 DNA binding and decreased TNF␣ production. 5. Effect of CLA treatment on macrophage phenotype Studies examining the effects of conjugated linoleic acid in the macrophage have predominantly focused on two macrophage cell lines (1) RAW 264.7 mouse and (2) human THP-1 macrophages. Treatment of the two cell types with various isomers of CLA and resultant effects on inflammatory profile, anti-oxidant enzyme production and cholesterol homeostasis has resulted in differing and contradictory outcomes that are summarized in Tables 2 and 3. 5.1. RAW mouse macrophages Treatment of RAW macrophages with a 30 ␮M 50:50 blend of c9,t11 and t10,c12 isomers decreased the production of the COX-2 generated product PGE2 as well as nitrite (NO) formation, which are though to be pro-inflammatory. The effects from treatment with this isomeric CLA blend resulted in decreased mRNA expression of both COX-2 and iNOS respectively [34]. Further studies investigating the anti-inflammatory properties of CLA showed that Table 2 Effects of CLA isomers in RAW mouse macrophages. Isomer

Result

(B) RAW mouse macrophages • Blend CLA – ↓ PGE2 + NO production and (30 ␮M) – ↓ COX-2 and iNOS mRNA expression 50:50 (c9,t11:t10,c12) – ↓ NO2 , ↓ iNOS expression, ↓ PGE2 • c9,t11 (200 ␮M) production, ↓ COX 2 promoter activity c9,c11 (200 ␮M) t9,t11 (200 ␮M) – ↓ COX2 mRNA, ↓ TNF␣, ↓ TNF␣ t10,c12 (200 ␮M) mRNA, ↓ IL1b, ↓ IL6 – Effects PPAR␥ dependent – ↓ IL-l receptor antagonist ␣ (IL-1r␣) • t9,t11 (10–100 ␮M) – ↓ IL-1a, IL-1b, IL-6 with LPS stimulation – ↑ TNF␣ and phagocytic activity • t10,c12 (10 ␮M) – GW 9662 (PPAR␥ antagonist) inhibited t10,c12 induced TNF␣ and phagocytosis – ↓ Cholesterol accumulation, ↑ CD36 • t10,c12 (50 ␮M) c9,t11 (50 ␮M) – ↓ Esterified cholesterol – ↑ ABCAl, LXR␣, NPC-1/2 ↑ reverse cholesterol transport – ↑ Cholesterol efflux in the presence of HDL • t9,t11 (100 ␮M) – ↑ ABCG1 promoter activity – t9,t11 induces ABCG1 through SREBP1c – ↑ ADAMTSl, Cat 1, FABP5, IL-1Ra, • t9,t11 (200 ␮M) Lipin 1, RGS1, RhoC – ↓ CycE, ESkK

Refs. [34]

[35]

[36]

[37]

[38]

[39]

[40]

59

Table 3 Effects on CLA isomers on THP-1 macrophage function. Isomer

Result

(C) THP-1 macrophages – ↑ Phagocytosis (COX dependent) • c9,t11 (30 ␮M) t10,c12 (30 ␮M) – ↓ NF␬B, JCOX2 mRNA, ↓ PGE2 – ↓ TXB2 • c9,t11 (30 ␮M) t10,c12 (30 ␮M) – ↓ PGE2 – ↑ Reactive oxygen species (ROS) • c9,t11 (30 ␮M) t10,c12 (30 ␮M) – PPAR␣ antagonist ↓ ROS production – ↑ Increased 8-epi-PGF2␣ (a free radical product of AA oxygenation) • c9,t11 (30 ␮M) – ↑ ROS reactive species t10,c12 – ↑ CD36 • c9,t11 (100 ␮M) t10,c12 (100 ␮M) – c9,t11 ↓ LXR and ABCA1 mRNA – ↑ Total and free cholesterol concentrations – ↑ SREBP1c promoter activity • t9,t11 (100 ␮M) c9,t11 (100 ␮M) – ↑ LXR␣ – ↑ ABCA1, ABCG1 and CETP (PPAR independent effects) • c9,t11 (100 ␮M) – No change in ABCA1, LXR – ↓ Total, free and esterified cholesterol

Refs. [41] [42] [43]

[44] [45]

[46]

[47]

200 ␮M c9,t11 decreased IFN␥ induced NO2 production and mRNA expression of iNOS [35]. PGE2 measured in the supernatant of c9,t11 CLA treated RAW macrophages were decreased with a subsequent reduction in COX-2 promoter activity and mRNA expression. Levels of TNF␣, IL-6 and IL-1␤ production were decreased following treatment with four different isomers: c9,t11, c9,c11, t9,t11, t10,c12 in RAW macrophages. All treatments were thought to mediate their anti-inflammatory effects through a PPAR␥ dependent mechanism as treatment with all four isomers of CLA increased PPAR␥ luciferase activity [35]. The t9,t11 isomer appears to exhibit the most potent anti-inflammatory properties whereby it uniquely activated interleukin 1 receptor antagonist (IL-1Ra) and caused a significant decrease in the production of pro-inflammatory IL6, IL-1␣, IL-1␤ [36]. Transfection of RAW macrophages with an IL-1Ra siRNA abolished t9,t11 isomers ability to increase IL-1Ra levels and subsequently prevent increases in the expression of IL6 and IL-1␣ suggesting that t9,t11 regulates IL-1␤ expression via an alternative mechanism. In contrast to these anti-inflammatory effects discussed one study investigating the effects of the t10,c12 isomer (10 ␮M) on inflammatory profile showed a PPAR␥ dependent increase in TNF␣ production and an increase in phagocytosis activity that was abolished upon addition of a PPAR␥ antagonist GW9662 [37]. The effects of CLA on cholesterol homeostasis and foam cell formation have been thoroughly investigated with the predominant effect of CLA being athero-protective. Pre-treatment of RAW macrophages with either 50 ␮M t10,c12 or c9,t11 decreased foam cell formation in response to acetylated low density lipoprotein (ac-LDL) loading [38]. Gene expression analysis showed a paradoxical increase in the expression of the scavenger receptor CD36, which would suggest that treatment with CLA would increase acLDL uptake and foam cell formation. However analysis of the genes involved in the RCT system (LXR␣, ABCA1, NPC1/2) indicated their expression was similarly increased with CLA treatment. These genes mediate the removal of excess cholesterol from the macrophage to HDL, as determined in this study through the utilization of efflux assays. Ecker et al. showed that the low abundance t9,t11 isomer increased the cholesterol efflux protein ABCG1 through activation of sterol regulatory element binding protein 1c (SREBP-1c) [39]. The most comprehensive study determining the effects of CLA isomers on RAW macrophages has been performed by Lee et al., who performed microarray analysis on RAW macrophages treated with five different isomers: c9,t11, c9,c11, t9,t11, t10,c12, c11,t13. The results were in agreement with

60

D. Mooney et al. / Prostaglandins & other Lipid Mediators 98 (2012) 56–62

previous studies which suggested that CLA increases cholesterol efflux and that the t9,t11 has the most prominent anti-atherogenic effect [40].

5.2. THP-1 macrophages CLA treated THP-1 macrophages have been examined with respect to their inflammatory and cholesterol homeostasis profiles. In contrast to the results obtained using RAW macrophages, the results from THP-1 macrophage studies have been somewhat conflicting. Treatment of THP-1 macrophages with 30 ␮M c9,t11 and t10,c12 isomers resulted in decreased NF␬B, COX-2 mRNA expression and decreased production of PGE2 which was associated with an increase in phagocytosis activity [41]. Upon further examination treatment with these CLA isomers decreased production of the COX-1 generated product TXB2 [42]. The effect of CLA on reactive oxygen species (ROS) generation is widely studied in macrophages. Inflammatory/activated macrophages and dysregulated mitochondrial activity results in increased production of reactive oxygen species. Reactive oxygen species such as highly toxic H2 O2 are formed through the dismutation of superoxide (O2 − ), one of the products generated by the electron transport chain in mitochondria. In the development of atherosclerosis, ROS are involved in the oxygenation of LDL to oxLDL and activation of the endothelial cell layer. In order to inhibit ROS generation cells, including macrophages can express antioxidant enzymes which detoxify cytotoxic H2 O2 to H2 O. To date two studies have investigated the effects of CLA on ROS production in THP-1 macrophages. Both studies determined that treatment with 30 ␮M c9,t11 and t10,c12 isomers increased ROS production [43,44]. Increased ROS generation was associated with an increase in the formation of the free radical arachidonic acid oxygenation product 8-epi-PGF2␣. It was found that this increase was via a

PPAR␣ dependent mechanism as addition of a PPAR␣ antagonist reduced ROS production [43]. Studies examining the effects of CLA on cholesterol homeostasis in the THP-1 macrophage have provided conflicting outcomes. Weldon et al. reported that both the c9,t11 and t10,c12 isomers increase the expression of the scavenger receptor CD36 and show a concomitant decrease in several genes involved in the reverse cholesterol transport system such as ABCA1 and LXR. This resulted in increased foam cell formation with increased intracellular concentrations of both free and esterified cholesterol [45]. In direct contrast, Ecker et al. showed that treatment with CLA isomers, including the t9,t11 isomer, demonstrated inhibition of foam cell formation at varying concentrations and that CLA increased the expression of genes involved in cholesterol efflux such as ABCA1, ABCG1, LXR␣ and CEPT1 via a PPAR independent pathway with the t9,t11 isomer acting as a direct agonist for LXR␣ [39,46]. Salehipour et al. showed that CLA treatment has no effect on the expression of LXR or ABCA1 but were associated with decreased concentrations of intracellular total, free and esterified cholesterol [47]. All three groups used the same concentration of CLA isomers therefore one possible explanation for these differing effects on similar genes is the treatment period with CLA and the use of acLDL to induce foam cell formation. 6. Discussion The effects of CLA isomers on the development, progression and potential resolution of atherosclerosis continue to be thoroughly investigated. In this review we focused on monocyte/macrophage as the cellular target whose phenotype is significantly altered with CLA treatment in atherosclerosis, as shown in Fig. 2. The two most abundant, and thus studied, isomers of CLA with regard to atherosclerosis are the c9,t11 and t10,c12 isomers. Most evidence suggests that the c9,t11 isomer accounts for most of the anti-atherosclerotic effects while the t10,c12 isomer leads to a

Fig. 2. Potential atheroprotective effects of CLA during the early stages of atherosclerosis development. (1) Inhibition of transendothelial monocyte migration from the lumen to the intima. (2) Inhibition of foam cell formation. (3) Reduced expression of pro-inflammatory mediators.

D. Mooney et al. / Prostaglandins & other Lipid Mediators 98 (2012) 56–62

predominantly pro-atherosclerotic effect. In the majority of animal and human studies CLA has been administered as a blend of the two isomers. The reason for inclusion of t10,c12 in the isomeric blend relates to its anti-obesity effects. However in mice, the addition of the t10,c12 isomer does not hinder the ability of CLA to induce regression of atherosclerosis, suggesting the necessity of the t10,c12 isomer in the isomeric blend used in regression studies. Inhibition of monocyte migration by c9,t11 CLA specifically warrants further investigation. Firstly, two distinct mechanisms appear to be involved, the first established mechanism involves activation of PPAR␥ by CLA to inhibit MCP-1 mediated migration. The second mechanism involves a novel PPAR␥ independent migration pathway in response to platelet releasate, which has yet to be fully elucidated. Significantly, the results observed in THP-1 monocytes were replicated in primary human monocytes and presents strong evidence of a potentially beneficial therapeutic effect in preventing early atherosclerosis development. Previous studies investigating inhibition of monocyte migration in animal models such as the LDLR−/− and ApoE−/− mice have demonstrated the profound effects this can have on the development of atherosclerosis irrespective of cholesterol levels. In both studies deletion of MCP-1 at both the gene [48] and protein level [49] resulted in decreased deposition of lipid and decreased macrophage infiltration into the plaque, with smaller and more stable plaque formation, a therapeutically preferable phenotype [50]. In addition to delineating the alternative migration mechanism future work should also examine the effects of CLA on the ability of the monocyte to adhere to activated endothelial cells. Previous investigations have examined the effects of treating endothelial cells with CLA and indeed have shown that treatment results in decreased expression of the pro-inflammatory receptor VCAM-1 [51] concomitant with a decrease in the release of inflammatory mediators such as platelet activating factor [52]. Phenotypically, this results in decreased adherence of monocytes to CLA treated endothelial cells which suggests that CLA is protective in the early stages of atherosclerosis development. The effects of CLA treatment of RAW mouse macrophages and human THP-1 macrophages have shown conflicting results. CLA treatment of RAW macrophages appears to be predominantly atheroprotective with inhibition of foam cell formation, increased expression of genes involved in RCT and decreased production of inflammatory mediators via inhibition of COX-2 and NF␬B, and increased PPAR␥ expression. Similar to RAW macrophage results CLA treatment appears to inhibit COX activity in THP1 macrophages, however CLA treatment does not seem to have equally beneficial effects on the RCT pathway with studies reporting increased expression of the scavenger receptor CD36 and decreased expression of the RCT genes. One potential explanation for these differential effects between the two cell lines may be related to the species of the cell line. It has been well documented that mice are naturally resistant to the development of atherosclerosis, which necessitated the development of the ApoE−/− and LDLR−/− mouse models. This resistance is due to the fact that any cholesterol they engulf is trafficked to high density lipoprotein, while in humans cholesterol is trafficked to LDL. One further explanation is the difference in the expression of key CLA effector proteins. For example RAW macrophages do not express PPAR␣ [35] and as discussed above, it was found that the t10,c12 isomer increased the production of ROS via a PPAR␣ dependent mechanism in THP-1 macrophages. The explanation for differential effects of the CLA isomers has so far predominantly concentrated on their regulation of gene transcription and protein function and indeed from the results discussed above there is a clear isomeric difference. However, a focused biophysical and biochemical approach to identify the

61

structural features of the isomers that account for their specific effects is warranted. This has remained a largely unexamined aspect of previous CLA investigations but recent studies are attempting to corroborate structural isomeric differences e.g. the position of the double bonds, to functional consequences. One example was reported in a recent study by Subbaiah et al., who detailed structural differences between the c9,t11 and t10,c12 isomers with regards to their effect on cholesterol binding which reported increased binding with the t10,c12 isomer [53]. The explanation presented for this isomeric difference was related to the position of the cis double bond which at position c12 in the t10,c12 results in a larger binding pocket for cholesterol than the c9,t11 isomer. Similar future structural studies should be extended to include the low abundance isomers such as the t9,t11 which have shown unique anti-atherosclerotic properties. Dependent on the success of these structural studies the resultant functional data could then be used in the design of new synthetic versions of CLA which would focus solely on the beneficial properties. Another distinctive area of investigation is examination into how fatty acids such as CLA are metabolized and if these metabolites exhibit the same beneficial functions as their parent compounds. Interestingly, in vitro studies suggest that CLA may compete with arachidonic acid for binding to the COX-2 active site. However, in vivo, in the context of atherosclerosis there is no suppression of urinary 2,3-dinor TXB2 or 2,3-dinor-6-keto PGF-1␣, the stable metabolites of TXA2 and PGI2 respectively [10] suggesting an alternative pathway of CLA metabolism. Evidence of these alternative pathways is beginning to emerge with recent investigations using both human smooth muscle and vascular endothelial cells showing that both the c9,t11 and the t10,c12 isomers can undergo ␤ oxidation and are metabolized to C16:2c7,t9 and C16:2t8,c10 respectively [15,54] suggesting that mitochondria and peroxisomes have a role to play in metabolizing CLA isomers. However it is still unclear whether these metabolites are therapeutically relevant and can induce the same anti-atherogenic functions as their parent compounds. This is an area worth exploring as previous investigations into the metabolism of another group of cardioprotective fatty acids, the omega 3 fatty acids, has yielded interesting results which suggest the metabolites of fatty acids are biologically active [55]. It has been shown that n-3 polyunsaturated fatty acids, such as docosahexanoic acid, can be metabolized by aspirin modulated COX-2 activity to generate oxidative metabolites, referred to as electrophilic oxoderivates (EFOX) that are both anti-inflammatory and atheroprotective. Significantly it has also been shown that these oxidative metabolites can induce their effects at nanomolar concentrations in contrast to the parent compounds which are functional at micromolar concentrations [56]. Therefore it is vitally important to establish the pathways and mechanisms that mediate the metabolism of the CLA isomers and to discover if these metabolites are functionally relevant.

7. Conclusion Monocytes, the precursors of inflammatory macrophages, and macrophages which represent the duality of atherosclerosis as both an inflammatory and cholesterol storage disease, are both crucial targets for the regression of this widespread disease. While the results discussed above do not unequivocally present CLA isomers as protective against atherosclerosis they do indicate potentially beneficial effects through inhibition of monocyte migration, inflammatory mediator expression and foam cell formation. Importantly, the results presented provide significant evidence that both monocytes and macrophages are critical cellular targets of CLA and thus provide new avenues of investigation which may elucidate the mechanism of CLA induced regression of atherosclerosis.

62

D. Mooney et al. / Prostaglandins & other Lipid Mediators 98 (2012) 56–62

References [1] Kepler CR, Hirons KP, McNeill JJ, Tove SB. Intermediates and products of the biohydrogenation of linoleic acid by Butyrinvibrio fibrisolvens. J Biol Chem 1966;241(6):1350–4. [2] Loscher CE, Draper E, Leavy O, Kelleher D, Mills KH, Roche HM. Conjugated linoleic acid suppresses NF-kappa B activation and IL-12 production in dendritic cells through ERK-mediated IL-10 induction. J Immunol 2005;175(8):4990–8. [3] Evans M, Brown J, McIntosh M. Isomer-specific effects of conjugated linoleic acid (CLA) on adiposity and lipid metabolism. J Nutr Biochem 2002;13(9):508. [4] Bocca C, Bozzo F, Cannito S, Colombatto S, Miglietta A. CLA reduces breast cancer cell growth and invasion through ERalpha and PI3K/Akt pathways. Chem Biol Interact 2010;183(1):187–93. [5] Kritchevsky D. Antimutagenic and some other effects of conjugated linoleic acid. Br J Nutr 2000;83(5):459–65. [6] Kelley NS, Hubbard NE, Erickson KL. Conjugated linoleic acid isomers and cancer. J Nutr 2007;137(12):2599–607. [7] Navarro V, Fernandez-Quintela A, Churruca I, Portillo MP. The body fatlowering effect of conjugated linoleic acid: a comparison between animal and human studies. J Physiol Biochem 2006;62(2):137–47. [8] Wang YW, Jones PJ. Conjugated linoleic acid and obesity control: efficacy and mechanisms. Int J Obes Relat Metab Disord 2004;28(8):941–55. [9] Reynolds CM, Roche HM. Conjugated linoleic acid and inflammatory cell signalling. Prostaglandins Leukot Essent Fatty Acids 2010;82(4–6):199–204. [10] Toomey S, Harhen B, Roche HM, Fitzgerald D, Belton O. Profound resolution of early atherosclerosis with conjugated linoleic acid. Atherosclerosis 2006;187(1):40–9. [11] Toomey S, Roche H, Fitzgerald D, Belton O. Regression of pre-established atherosclerosis in the apoE−/− mouse by conjugated linoleic acid. Biochem Soc Trans 2003;31(Pt 5):1075–9. [12] Arbones-Mainar JM, Navarro MA, Guzman MA, et al. Selective effect of conjugated linoleic acid isomers on atherosclerotic lesion development in apolipoprotein E knockout mice. Atherosclerosis 2006;189(2):318–27. [13] Kritchevsky D, Tepper SA, Wright S, Czarnecki SK, Wilson TA, Nicolosi RJ. Conjugated linoleic acid isomer effects in atherosclerosis: growth and regression of lesions. Lipids 2004;39(7):611–6. [14] Lee KN, Kritchevsky D, Pariza MW. Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis 1994;108(1):19–25. [15] Eder K, Ringseis R. Metabolism and actions of conjugated linoleic acids on atherosclerosis-related events in vascular endothelial cells and smooth muscle cells. Mol Nutr Food Res 2010;54(1):17–36. [16] Issemann I, Green S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature 1990;347(6294):645–50. [17] Krey G, Braissant O, L’Horset F, et al. Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay. Mol Endocrinol 1997;11(6):779–91. [18] Belury MA. Conjugated linoleic acid is an activator and ligand for peroxisome proliferator-activated receptor-gamma (PPAR␥). Nutrition Res 2002;22(7):817–25. [19] Lim HJ, Lee S, Lee KS, et al. PPARgamma activation induces CD36 expression and stimulates foam cell like changes in rVSMCs. Prostaglandins Other Lipid Mediat 2006;80(3–4):165–74. [20] Moore KJ, Rosen ED, Fitzgerald ML, et al. The role of PPAR-gamma in macrophage differentiation and cholesterol uptake. Nat Med 2001;7(1): 41–7. [21] Simmons DL, Botting RM, Hla T. Cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition. Pharmacol Rev 2004;56(3):387–437. [22] Smith WL. The eicosanoids and their biochemical mechanisms of action. Biochem J 1989;259(2):315–24. [23] Jones DA, Carlton DP, McIntyre TM, Zimmerman GA, Prescott SM. Molecular cloning of human prostaglandin endoperoxide synthase type II and demonstration of expression in response to cytokines. J Biol Chem 1993;268(12): 9049–54. [24] Belton O, Byrne D, Kearney D, Leahy A, Fitzgerald DJ. Cyclooxygenase-1 and -2dependent prostacyclin formation in patients with atherosclerosis. Circulation 2000;102(8):840–5. [25] Coen P, Cummins P, Birney Y, Devery R, Cahill P. Modulation of nitric oxide and 6-keto-prostaglandin F(1alpha) production in bovine aortic endothelial cells by conjugated linoleic acid. Endothelium 2004;11(3–4):211–20. [26] McClelland S, Cox C, O’Connor R, et al. Conjugated linoleic acid suppresses the migratory and inflammatory phenotype of the monocyte/macrophage cell. Atherosclerosis 2010;211(1):96–102. [27] Zhu Y, Bujo H, Yamazaki H, et al. Enhanced expression of the LDL receptor family member LR11 increases migration of smooth muscle cells in vitro. Circulation 2002;105(15):1830–6. [28] McCarthy C, O’Gaora P, James WG, et al. SorLA modulates atheroprotective properties of CLA by regulating monocyte migration. Atherosclerosis 2010;213(2):400–7. [29] Norris LA, Weldon S, Nugent A, Roche HM. LPS induced tissue factor expression in the THP-1 monocyte cell line is attenuated by conjugated linoleic acid. Thromb Res 2006;117(4):475–80.

[30] Moya-Camarena SY, Van den Heuvel JP, Belury MA. Conjugated linoleic acid activates peroxisome proliferator-activated receptor alpha and beta subtypes but does not induce hepatic peroxisome proliferation in Sprague-Dawley rats. Biochim Biophys Acta 1999;1436(3):331–42. [31] Moya-Camarena SY, Vanden Heuvel JP, Blanchard SG, Leesnitzer LA, Belury MA. Conjugated linoleic acid is a potent naturally occurring ligand and activator of PPARalpha. J Lipid Res 1999;40(8):1426–33. [32] Paek J, Kang JH, Kim SS, Son KA, Park MR, Yang MP. Trans-10, cis-12 conjugated linoleic acid directly enhances the chemotactic activity of porcine peripheral blood polymorphonuclear neutrophilic leukocytes by activating F-actin polymerization in vitro. Res Vet Sci 2010;89(2):191–5. [33] Kim DI, Kim KH, Kang JH, et al. Trans-10, cis-12-conjugated linoleic acid modulates NF-kappaB activation and TNF-alpha production in porcine peripheral blood mononuclear cells via a PPARgamma-dependent pathway. Br J Nutr 2011;105(9):1329–36. [34] Iwakiri Y, Sampson DA, Allen KG. Suppression of cyclooxygenase-2 and inducible nitric oxide synthase expression by conjugated linoleic acid in murine macrophages. Prostaglandins Leukot Essent Fatty Acids 2002;67(6):435–43. [35] Yu Y, Correll PH, Vanden Heuvel JP. Conjugated linoleic acid decreases production of pro-inflammatory products in macrophages: evidence for a PPAR gamma-dependent mechanism. Biochim Biophys Acta 2002;1581(3):89–99. [36] Lee Y, Thompson JT, Vanden Heuvel JP. 9E,11E-conjugated linoleic acid increases expression of the endogenous antiinflammatory factor, interleukin-1 receptor antagonist, in RAW 264.7 cells. J Nutr 2009;139(10):1861–6. [37] Song DH, Kang JH, Lee GS, Jeung EB, Yang MP. Upregulation of tumor necrosis factor-alpha expression by trans10-cis12 conjugated linoleic acid enhances phagocytosis of RAW macrophages via a peroxisome proliferator-activated receptor gamma-dependent pathway. Cytokine 2007;37(3):227–35. [38] Ringseis R, Wen G, Saal D, Eder K. Conjugated linoleic acid isomers reduce cholesterol accumulation in acetylated LDL-induced mouse RAW264.7 macrophage-derived foam cells. Lipids 2008;43(10):913–23. [39] Ecker J, Langmann T, Moehle C, Schmitz G. Isomer specific effects of Conjugated Linoleic Acid on macrophage ABCG1 transcription by a SREBP-1c dependent mechanism. Biochem Biophys Res Commun 2007;352(3):805–11. [40] Lee Y, Thompson JT, de Lera AR, Vanden Heuvel JP. Isomer-specific effects of conjugated linoleic acid on gene expression in RAW 264.7. J Nutr Biochem 2009;20(11), 848–59, 59e1–5. [41] Stachowska E, Baskiewicz-Masiuk M, Dziedziejko V, et al. Conjugated linoleic acids can change phagocytosis of human monocytes/macrophages by reduction in Cox-2 expression. Lipids 2007;42(8):707–16. [42] Stachowska E, Dolegowska B, Dziedziejko V, et al. Prostaglandin E2 (PGE2) and thromboxane A2 (TXA2) synthesis is regulated by conjugated linoleic acids (CLA) in human macrophages. J Physiol Pharmacol 2009;60(1):77–85. [43] Stachowska E, Baskiewicz-Masiuk M, Dziedziejko V, et al. Conjugated linoleic acid increases intracellular ROS synthesis and oxygenation of arachidonic acid in macrophages. Nutrition 2008;24(2):187–99. [44] Rybicka M, Stachowska E, Gutowska I, et al. Comparative effects of conjugated linoleic acid (CLA) and linoleic acid (LA) on the oxidoreduction status in THP-1 macrophages. J Agric Food Chem 2011;59(8):4095–103. [45] Weldon S, Mitchell S, Kelleher D, Gibney MJ, Roche HM. Conjugated linoleic acid and atherosclerosis: no effect on molecular markers of cholesterol homeostasis in THP-1 macrophages. Atherosclerosis 2004;174(2):261–73. [46] Ecker J, Liebisch G, Patsch W, Schmitz G. The conjugated linoleic acid isomer trans-9,trans-11 is a dietary occurring agonist of liver X receptor alpha. Biochem Biophys Res Commun 2009;388(4):660–6. [47] Salehipour M, Javadi E, Reza JZ, et al. Polyunsaturated fatty acids and modulation of cholesterol homeostasis in THP-1 macrophage-derived foam cells. Int J Mol Sci 2010;11(11):4660–72. [48] Gu L, Okada Y, Clinton SK, et al. Absence of monocyte chemoattractant protein1 reduces atherosclerosis in low density lipoprotein receptor-deficient mice. Mol Cell 1998;2(2):275–81. [49] Ni W, Egashira K, Kitamoto S, et al. New anti-monocyte chemoattractant protein-1 gene therapy attenuates atherosclerosis in apolipoprotein E-knockout mice. Circulation 2001;103(16):2096–101. [50] Webb NR. Getting to the core of atherosclerosis. Nat Med 2008;14(10):1015–6. [51] Stachowska E, Siennicka A, Baskiewcz-Halasa M, Bober J, Machalinski B, Chlubek D. Conjugated linoleic acid isomers may diminish human macrophages adhesion to endothelial surface. Int J Food Sci Nutr 2012;63(1):30–5. [52] Sneddon AA, McLeod E, Wahle KW, Arthur JR. Cytokine-induced monocyte adhesion to endothelial cells involves platelet-activating factor: suppression by conjugated linoleic acid. Biochim Biophys Acta 2006;1761(7):793–801. [53] Subbaiah PV, Sircar D, Aizezi B, Mintzer E. Differential effects of conjugated linoleic acid isomers on the biophysical and biochemical properties of model membranes. Biochim Biophys Acta 2010;1798(3):506–14. [54] Ringseis R, Muller A, Dusterloh K, Schleser S, Eder K, Steinhart H. Formation of conjugated linoleic acid metabolites in human vascular endothelial cells. Biochim Biophys Acta 2006;1761(3):377–83. [55] Adkins Y, Kelley DS. Mechanisms underlying the cardioprotective effects of omega-3 polyunsaturated fatty acids. J Nutr Biochem 2010;21(9):781–92. [56] Groeger AL, Cipollina C, Cole MP, et al. Cyclooxygenase-2 generates anti-inflammatory mediators from omega-3 fatty acids. Nat Chem Biol 2010;6(6):433–41.