Hydroxyoctadecadienoic acids: novel regulators of macrophage differentiation and atherogenesis

Abstract

Hydroxyoctadecadienoic acids (HODEs) are stable oxidation products of linoleic acid, the generation of which is increased where oxidative stress is increased, such as in diabetes. In early atherosclerosis, 13-HODE is generated in macrophages by 15-lipoxygenase-1. This enhances protective mechanisms through peroxisome proliferator-activated receptor (PPAR)-g activation leading to increased clearance of lipid and lipid-laden cells from the arterial wall. In later atherosclerosis, both 9-HODE and 13-HODE are generated nonenzymatically. At this stage, early protective mechanisms are overwhelmed and pro-inflammatory effects of 9-HODE, acting through the receptor GPR132, and increased apoptosis predominate leading to a fragile, acellular plaque. Increased HODE levels thus contribute to atherosclerosis progression and the risk of clinical events such as myocardial infarction or stroke. Better understanding of the role of HODEs may lead to new pharmacologic approaches to modulate their production or action, and therefore lessen the burden of atherosclerotic disease in high-risk patients.

Introduction

Atherosclerosis is a leading cause of morbidity and premature mortality. Many of the processes involved in atherogenesis are now well understood, but there are considerable gaps in knowledge about how these are regulated. Lipid accumulation in the vascular wall and oxidative stress (both increased in diabetes) lead to the generation of oxidized lipids. The latter are involved in processes which lead to atheromatous plaque formation and rupture. Oxidized derivatives of linoleic acid, 9- and 13-hydroxyoctadecadienoic acid (9-HODE and 13-HODE), are abundant in atherosclerotic lesions and regulate cellular pathways through interaction with recently described cell surface receptors for long-chain fatty acids, and also through the peroxisome proliferator-activated receptor (PPAR) family of transcription factors. In this review, we summarize what is known about the actions of 9-HODE and 13-HODE, and how they may help explain the increased cardiovascular risk in patients with diabetes.

HODEs are stable oxidation products of linoleic acid (LA; C18:2, omega-6). LA is the most abundant fatty acid in atherosclerotic plaques, being seven times more abundant than arachidonic acid (AA). Oxidized lipids accumulate at sites of tissue injury, including atherosclerotic plaque. It was recognized more than 50 years ago that oxidized fatty acids accumulated in low-density lipoprotein (LDL) with age, particularly in individuals susceptible to atherosclerosis [Jira et al. 1998]. HODEs were 20 times more abundant in the LDL of patients with atherosclerosis compared with controls [Jira et al. 1998]. The structure of 9-HODE and 13-HODE, are shown in Figure 1, along with LA and the omega-3 fatty acid α-linolenic acid (ALA; C18:3, omega-3). Although the structures of these fatty acids differ only subtly, there is accumulating evidence that HODEs have distinct biological properties. Accumulation of HODEs in human atherosclerotic lesions was described nearly two decades ago [Kuhn et al. 1992], when they were shown to be components of both the cholesterol ester and phospholipid fractions. LA accounts for 40–45% of the polyunsaturated fatty acid (PUFA) in plaque, and 30% of PUFAs in plaque are oxidized. HODEs are the most abundant oxidation products in plaque, and they are present in all advanced lesions, although the quantity varies from patient to patient [Waddington et al. 2003, 2001].

Figure 1.

Structures of hydroxyoctadecadienoic acids (HODEs) and other C-18 polyunsaturated fatty acids: (A) α-linolenic acid; (B) linoleic acid; (C) 9-HODE; (D) 13-HODE.

The key enzyme for lipid oxidation in macrophages, both in animal models and in humans, is 15-lipoxygenase-1 (15-LOX-1) which is absent from normal vascular intima. With LA as substrate, 15-LOX-1 produces almost exclusively 13-HODE [Jostarndt et al. 2002; Kuhn et al. 1994]. By contrast, nonenzymatic oxidation of LA produces an approximately equal mixture of 9-HODE and 13-HODE [Upston et al. 1997]. Kuhn and colleagues studied progression of HODE deposition in the aorta of rabbits fed a cholesterol-rich diet, and compared this with composition of advanced human lesions [Kuhn et al. 1994]. In early (12-week) rabbit lesions, HODE deposition was predominantly of an enzymatic pattern while nonenzymatic generation of HODEs is the major mechanism in later lesions (26 weeks). Human lesions also showed a nonenzymatic pattern. Brinckmann and Kuhn later confirmed that enzymatic HODE generation predominated in early human lesions [Brinckmann and Kuhn, 1997]. Waddington and colleagues analysed carotid endarterectomy specimens: HODEs were the most abundant oxidised lipid and the pattern of isomers and enantiomers was consistent with nonenzymatic generation [Waddington et al. 2003, 2001]. The HODE content of lesions did not correlate with histopathological parameters studied, and there was no difference between symptomatic and asymptomatic lesions. HODEs have recently been recognized to occur in barley, malt and other plant products. It is not known whether dietary HODE is of any biological importance. Diets providing increased amounts of LA may increase 13-HODE synthesis in the vascular endothelium and thus decrease platelet adhesion and thrombogenicity [Bertomeu et al. 1990].

Whether produced enzymatically or nonenzymatically, the initial step in the synthesis of 13-HODE is formation of 13-hydroperoxy-oxydecadienoic acid (13-HPODE) which is unstable and quickly reduced to 13-HODE. A comparable step is involved in formation of 9-HODE. Recently, Muellner and colleagues reported that the gasotransmitter hydrogen sulphide (H₂S) may be involved in converting HPODEs to HODEs, and was thus postulated to have a potentially atheroprotective effect [Muellner et al. 2009]. For 13-HODE, further reduction to 2,4-dienone, 13-oxooctadecadienoic acid (13-oxo-ODE) is catalysed by the enzyme 13-HODE dehydrogenase. This enzyme, which is expressed in leukocytes, is NAD-dependent and has a narrow range of substrate specificity (but including 9-HODE). The enzyme may play a role in partitioning cells between differentiated and proliferating phenotypes [Bull et al. 1993]. 13-oxo-ODE is a potent ligand for PPAR-γ. Higher HODE concentrations are an indication of oxidative stress in biological fluids and systems. F₂-isoprostanes, more widely used as a marker of oxidative stress, are less abundant (as little as one twentieth) than are HODEs [Yoshida et al. 2008; Carpenter et al. 1995].

Effects of HODEs on monocytes and macrophages

Vessel wall-resident macrophages are derived from circulating monocytes following activation of the latter. Macrophage accumulation in the arterial wall and increased expression of macrophage 15-LOX-1 occur early in atherosclerosis [Wittwer and Hersberger, 2007; Zhao et al. 2004]. Lipoxygenases are nonhaem iron dioxygenases classified according to their positional specificity for AA oxidation. 5-lipoxygenase (5-LOX) and 15-lipoxygenase-1 (15-LOX-1) are involved in atherogenesis, by modulating production of inflammatory mediators, regulating cell trafficking and angiogenesis, and effects on lipid metabolism/transport. The products of 15-LOX-1 from AA are 12-hydroxyeicosatetrae-noic acid (12-HETE) and 15-hydroxyeicosate-traenoic acid (15-HETE). As noted above, the enzyme principally synthesizes 13-HODE from LA. The precise role of 15-LOX-1 in atherosclerosis has been controversial, partly because of the difficulty in extrapolating findings of animal studies to human disease [Wittwer and Hersberger, 2007; Zhao et al. 2004]. In animal studies, disruption of 12/15-LOX (mouse orthologue of 15-LOX-1) decreases lesion progression. Conversely, overexpression of the enzyme is associated with increased lesion size [Wittwer and Hersberger, 2007; Zhao et al. 2004]. Mild enzymatic oxidation of LDL particles leads to their uptake into macrophages through scavenger receptors (scavenger receptor [SR-A] and CD36) while 13-HODE produced by the action of 15-LOX-1 may also increase reverse cholesterol transport through a mechanism involving PPAR-α [Zhao et al. 2004]. Thus, although increased expression of 15-LOX-1 is a feature of early atherosclerosis, the effect of 13-HODE may actually be protective at this stage of the disease.

Accumulation of lipoprotein particles in the sub-endothelial space also occurs early in atherosclerosis. Under conditions of oxidative stress, which prevail in patients prone to atherosclerosis (including those with diabetes), LDL particles, in particular, undergo oxidation (oxLDL). To protect the vasculature from potentially damaging effects of the oxidized lipoproteins, macrophages undergo differentiation allowing them to assimilate extracellular lipid. When this initially adaptive response becomes overwhelmed, the macrophages become lipid-laden (foam cells), develop a pro-inflammatory phenotype, and become prone to apoptosis [Rusinol et al. 2000; Wintergerst et al. 2000]. The most significant components of the scavenger receptor family are CD36 and SR-A. Lipids taken up by macrophages are stored in cytoplasmic droplets as neutral lipids including triglycerides, phospholipids and cholesteryl esters. There is also a process of active cholesterol efflux, following which cholesterol is removed from the circulation by high-density lipoproteins. The major regulators of this latter process are CD36, (ATP)-binding cassette transporter-A1 (ABCA1) and scavenger receptor B1 (SR-B1). CD36 is expressed on macrophages, platelets, smooth muscle cells, endothelial cells and adipocytes. CD36 null mice accumulate less lipid and phospholipid microparticles in their macrophages, have less-advanced atherosclerosis, and also have decreased thrombus formation. As with other genes involved in lipid storage, expression of CD36 in macrophages is regulated by PPAR-γ [Lim et al. 2006; Jostarndt et al. 2004]. There is limited evidence that 13-HODE increases CD36 expression, by activating PPAR-γ [Jostarndt et al. 2002], although other mechanisms may also be involved.

Fatty acid binding protein 4 (FABP4, aP2) has a central role in adipocyte differentiation, during which its expression is regulated by PPAR-γ. FABP4 also regulates lipid accumulation in macrophages, and its expression is again controlled through PPAR-γ [Cabrero et al. 2003]. The latter transcription factor is thought to mediate the enhancing action of oxLDL on FABP4 expression and foam cell formation [Fu et al. 2000, 2002]. Fu and colleagues reported that FABP4 expression in THP1 cells was increased by 9-HODE, 13-HODE, 15-dPGJ₂, and retinoic acid [Fu et al. 2002]. Overexpression of FABP4 led to marked increase in lipid accumulation. [Fu et al. 2006]

Increased susceptibility to apoptosis amongst vascular cells has been well described as a feature of developing atherosclerosis [Wintergerst et al. 2000; Hardwick et al. 1996]. Macrophage apoptosis is at least partly regulated by PPAR-γ [Chinetti et al. 1998]. A pro-apoptotic effect of HODEs has been described in nonvascular cells [Mahipal et al. 2007; Mani et al. 1998]. Two studies have reported that apoptosis of monocytic cells is increased on exposure to HODEs [Hampel et al. 2006; Jostarndt et al. 2002]. Jostarndt and colleagues investigated effects of enzymatically modified LDL on Mono Mac 6 monocytes [Jostarndt et al. 2002]. Oxidized LDL thus produced both induced apoptosis and also increased CD36 expression. The effects of oxLDL were replicated by 13-HODE.

The effect of 9-HODE was not studied. Hampel and colleagues investigated the effects of 9-HODE, 13-HODE and the PPAR-γ agonist ciglitazone on the human U937 line [Hampel et al. 2006]. Both 9-HODE and ciglitazone inhibited cell proliferation. 9-HODE increased the proportion of cells in G_0/1 phase and also enhanced apoptosis. The latter action was not blocked by the PPAR-γ antagonist GW9662. There was no increase in PPAR-γ transcripts but 9-HODE specifically increased PPAR-γ₂ transcripts fourfold. Using a PPRE-luciferase reporter, all three ligands increased PPAR-γ transactivation.

In summary, macrophage accumulation and increased expression of 15-LOX-1 are features of early atherosclerosis and associated with increased generation of 13-HODE. The latter (through PPAR-γ activation) increases CD36 and FABP4 expression and stimulates apoptosis. These processes are protective in early lesions leading to clearance of lipid and debris from the vascular wall, and removal of damaged or lipid-laden cells.

Effects on other vascular cells

The vascular endothelium forms an effective barrier between circulating blood and the subendothelial space. Damage to this barrier leads to activation of endothelial cells, with increased release of inflammatory, chemotactic, and pro-thrombotic mediators. Endothelial damage is also associated with increased permeability of the barrier allowing for migration of monocytes into the subendothelial space and increased deposition of lipoprotein particles in the vascular wall. HODEs have been reported to decrease platelet adhesion to endothelial cells [Haas et al. 1988, 1990]. 13-HODE may also increase synthesis of prostacyclin (PGI₂) [Setty et al. 1987]. PPAR-γ is expressed in endothelial cells, and activation of this by 15-dPGJ₂, 9-HODE or 13-HODE leads to increased secretion of plasminogen activator inhibitor type 1 (PAI-1) [Marx et al. 1999]. Increased levels of this fibrinolytic inhibitor predispose to vascular events and have been proposed to be a link between insulin resistant states and increased vascular risk. PPAR-γ activation in endothelial cells also leads to increased ICAM-1 expression, which, in turn, increases adhesion of monocytes. Endothelial cells have been shown to synthesise 13-HODE [Haas et al. 1990] and 9-HODE [Kaduce et al. 1989]. Synthesis of the latter is inhibited by aspirin and ibuprofen, suggesting that it may be catalysed by cyclo-oxygenase rather than lipoxygenases.

Vascular smooth muscle cells (VSMCs) are also involved in atherogenesis. Following damage to the endothelium, VSMCs proliferate, secrete increased amounts of extracellular matrix, and may differentiate into foam cells. VSMCs synthesize HODEs, predominantly 13-HODE, and this may be catalysed by the prostaglandin synthase complex [Daret et al. 1993]. VSMCs may also respond to HODEs generated by adjacent cells including platelets and macrophages. They respond particularly to 13-HODE [Stoll et al. 1994; Ramboer et al. 1992], with increased intracellular calcium, inositol trispho-sphate, and cyclic guanosine monophosphate (GMP). This release of intracellular messengers contributes to increased expression of VSMC differentiation molecules, increased cell mobility, and release of prostacyclin. Limor and colleagues have shown that VSMC express 12/15-LOX and are thus capable of synthesizing 12-HETE, 15-HETE and 13-HODE [Limor et al. 2008]. As discussed below, macrophages may be partly sequestered in the subendothelial space by downregulation of CCR2 in response to PPAR-γ agonists, while concomitant upregulation of the fractalkine (CXCCL1) receptor CX3CR1 enhances interaction between macrophages and VSMCs [Barlic and Murphy, 2007; Barlic et al. 2006]. VSMCs are responsible for the fibrous cap which protects against plaque rupture and thrombosis. Apoptosis of VSMCs may impair the integrity of the fibrous cap.

Platelets are a rich source of HODEs in vivo, possessing both 15-LOX-1 and prostaglandin synthase enzyme systems. Platelets produce predominantly 13-HODE, with LOX activity being the predominant source in the cytosolic fraction [Truitt et al. 1999; Daret et al. 1989]. In common with 8-epiPGF_2α, another marker of oxidative stress, 13-HODE has potent platelet anti-aggregatory effects [Tloti et al. 1991; Coene et al. 1986]. Conflicting data have been published regarding the effects of 13-HODE on platelet adhesion to endothelial cells with one study reporting no effect when platelet adhesion was studied under flow conditions [de Graaf et al. 1989], while other studies have reported anti-aggregatory actions including decreased thrombin-induced platelet adherence with 13-HODE in vitro [Haas et al. 1990, 1988; Coene et al. 1986]. Damage to the vascular wall may decrease 13-HODE synthesis, and this may increase thrombogenicity [Weber et al. 1990].

Details of the effects of HODEs on nonvascular cells cannot be considered here in detail. They have effects on the functions of polymorphonu-clear leukocytes, and are involved in regulation of bronchial smooth muscle responsiveness. Potentially important roles in regulating reproductive function have also been described. HODEs are generated in gastrointestinal epithelial cells where they act as PPAR-γ agonists. They regulate cell proliferation and inflammation, and this may contribute to protective effects of the molecules in inflammatory bowel disease and large bowel neoplasia.

Mechanisms of action

PPAR nuclear transcription factors are activated by fatty acids and eicosanoids. PPAR-γ is involved in adipocyte differentiation, lipid storage and in regulating insulin sensitivity. It is principally associated with adipose tissue but is also expressed in vascular cells (endothelial, macrophages and VSMCs). PPAR-γ₂, the predominant form in adipose tissue, has an additional 30 amino acids at the N-terminal compared with PPAR-γ₁. PPARs regulate gene expression by forming heterodimers with the retinoic X receptor and binding to PPAR-response elements in the promoter region of target genes. PPAR-γ activation has effects on vascular cells which are protective against atherosclerosis. For example, although PPAR-γ can increase expression of molecules involved in lipid storage (including CD36), the net effect of increased PPAR-γ expression in macrophages is generally to decrease lipid accumulation [Bouhlel et al. 2008]. Furthermore, PPAR-α and PPAR-γ may increase cholesterol efflux by increasing expression of HDL receptor and SR-B1 [Bouhlel et al. 2008]. PPAR-γ activation also decreases levels of pro-inflammatory mediators. Some of the protective effect of PPAR-γ may be through enhancing differentiation of monocytes into alternatively activated M2 macrophages which have anti-inflammatory and other protective properties [Bouhlel et al. 2007]. The level of PPAR-γ expression in plaque correlates with the number of M2 macrophages [Bouhlel et al. 2007].

Of naturally occurring ligands for PPAR-γ, 15-deoxy-Δ^12,14-prostaglandin J₂ (15-dPGj₂) and HODEs are the most significant. There is considerable evidence that HODEs activate PPAR-γ in macrophages leading to increased CD36 expression [Jostarndt et al. 2004; Nagy et al. 1998]. Nagy and colleagues demonstrated the capacity of HODEs to upregulate CD14, CD36 and SR-A (markers for macrophage maturation) [Nagy et al. 1998]. HODEs may also activate PPAR-α [Delerive et al. 2000] and may thus have further beneficial effects on lipid metabolism. In summary, HODEs are potent PPAR-γ agonists and may exert protective effects in atherogenesis by modulating macrophage lipid accumulation and inflammatory mediator generation.

GPR132 is a stress-inducible receptor, expression of which is increased on exposure of cells to DNA-damaging agents. Expression of GPR132 is associated with a block in cell cycle progression in the G2/M phase (G2A =G2 accumulation) [Weng et al. 1998]. GPR132 is highly expressed in macrophages in atherosclerotic plaque. GPR132 is a receptor for oxidized fatty acids with 9-HODE being the most potent ligand [Obinata et al. 2009; Yin et al. 2009]. 13-HODE is a weak ligand, and esterified HODEs do not activate the receptor. A recent study by Hattori and colleagues demonstrated that 9-HODE is a pro-inflammatory mediator in skin and that this action is GPR132-mediated [Hattori et al. 2008]. GPR132 is expressed in macrophages of atherosclerotic plaques, both in humans and in experimental rabbits [Rikitake et al. 2002]. Recent genetic manipulation studies in animals [Bolick et al. 2009, 2007; Parks et al. 2009; Parks et al. 2005] strongly support a role for GPR132 in pathogenesis of atherosclerosis. However, we do not know at present whether GPR132 generally mediates favourable or unfavourable effects in human vascular cells. Another potential mediator of HODE action is the testicular orphan nuclear receptor-4 (TR4), a nuclear transcription factor with involvement in diverse biological processes. In a very recent study, Xie and colleagues showed that TR4^−/− mice had decreased macrophage expression of CD36 and reduced foam cell formation [Xie et al. 2009]. Expression of CD36 was stimulated by 13-HODE partly through the activation of TR4.

Comparison of 9-HODE and 13-HODE

Early in atherosclerosis, 13-HODE is the predominant HODE, while in advanced disease 9-HODE is at least as abundant. The known effects of 9-HODE and 13-HODE are compared in Table 1. Recently, increased 13-HODE levels have been reported in the circulation of patients with essential hypertension, presumably reflecting increased oxidative stress [Wang et al. 2009]. The best evidence that 9-HODE and 13-HODE have differing, indeed opposing effects comes from studies in the skin. Here, 9-HODE has pro-inflammatory actions mediated through GPR132 [Hattori et al. 2008]. By contrast, 13-HODE which accumulates in the cer-amides and phospholipids in the skin [Cho and Ziboh, 1994; Gron et al. 1993a] and has anti-inflammatory actions. 13-HODE also affects proliferation of keratinocytes: levels of PUFAs are decreased in hyperproliferating skin, and the increased proliferation is reversed by topical application of 13-HODE [Miller and Ziboh, 1990]. In psoriasis there is a decrease in 9-HODE, 13-HODE and 15-HETE [Gron et al. 1993b]. The anti-proliferative action of 13-HODE involves down-regulation of the onco-gene activator protein-1 (AP-1).

Table 1.

Actions of HODEs relevant to atherosclerosis.

Effect	Cell type	9-HODE	13-HODE
Present in early lesions	—	—	++
Present in late lesions	—	++	++
Marker of oxidative stress	—	++	++
CD36 expression	Macro	NS	↑
FABP4 expression	Macro	↑↑	↑↑
Lipid accumulation	Macro	↑↑	↑↑
Apoptosis	Macro	↑	↑
Antithrombotic*	Endo	+	++
Differentiation/mobility	VSMC	NS	++
Aggregation	Platelet	NS	↓↓
Inflammation	Skin/colon	↑↑	↓↓

^*However, both 9-HODE and 13-HODE increase PAI-1 expression in endothelial cells [Marx et al. 1999]. Macro, macrophage; Endo, endothelial cell; VSMC, vascular smooth muscle cell; NS, not significant; HODE, hydroxyoctadecadienoic acid.

Conclusions

HODEs are markers of oxidative stress, and components of the oxidized lipid in atherosclerotic plaque. The major effects of HODEs in regulating macrophage functions are summarized in Figure 2. In early atherosclerosis, 13-HODE is generated in macrophages enzymatically by 15-LOX-1. This may be important in activating protective mechanisms which increase clearance of lipid and cellular debris from the vessel wall. Effects of 13-HODE are predominantly mediated through PPAR-γ, although a possible role for the nuclear transcription factor TR4 has been reported. In later atherosclerosis, nonenzymatic oxidation of LA generates a mixture of 9-HODE and 13-HODE. At this stage of the disease, and with the pro-inflammatory actions of 9-HODE acting through the GPR132 receptor, the effects of HODEs may be predominantly harmful rather than beneficial. Increased oxidative stress and enhanced monocyte activation in diabetes may increase generation of HODEs and this could partly explain the susceptibility of diabetic individuals to macrovascular disease. Clearer understanding of the mechanisms involved might lead to development of new atheroprotective agents, and thus help lessen the considerable health and health economic burden of macrovascular disease in high-risk conditions such as diabetes.

Figure 2.

Effects of HODEs on monocytes and macrophages. The contrasting effects of HODEs on the macrophage in early and late atherosclerosis. In early disease, 13-HODE is generated enzymatically by 15-lipoxygenase, and its actions are protective with increased lipid uptake, reverse cholesterol transport and enhanced apoptosis. In later disease, HODEs are synthesized nonenzymatically and pro-inflammatory actions of 9-HODE mediated by the GPR132 receptor contribute to lesion progression.