Classes of Lipid Mediators and Their Effects on Vascular Inflammation in Atherosclerosis

It is commonly believed that the inactivation of inflammation is mainly due to the decay or cessation of inducers. In reality, in connection with the development of atherosclerosis, spontaneous decay of inducers is not observed. It is now known that lipid mediators originating from polyunsaturated fatty acids (PUFAs), which are important constituents of all cell membranes, can act in the inflamed tissue and bring it to resolution. In fact, PUFAs, such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), are precursors to both pro-inflammatory and anti-inflammatory compounds. In this review, we describe the lipid mediators of vascular inflammation and resolution, and their biochemical activity. In addition, we highlight data from the literature that often show a worsening of atherosclerotic disease in subjects deficient in lipid mediators of inflammation resolution, and we also report on the anti-proteasic and anti-thrombotic properties of these same lipid mediators. It should be noted that despite promising data observed in both animal and in vitro studies, contradictory clinical results have been observed for omega-3 PUFAs. Many further studies will be required in order to clarify the observed conflicts, although lifestyle habits such as smoking or other biochemical factors may often influence the normal synthesis of lipid mediators of inflammation resolution.


Introduction
Atherosclerosis is a lipoprotein-driven, non-resolving chronic inflammatory disease that could cause heart attacks [1,2] and cardiovascular disease [3]. Atherosclerosis begins with the retention of apolipoprotein B-containing lipoproteins within the subendothelial intima of arteries, which leads to the activation of endothelial cells, recruitment of leukocytes, and accumulation of cells, extracellular matrix, and lipids [4].
Macrophages, the major leukocyte type ingesting retained lipoproteins in the subendothelial region, in some cases modified by oxidation [5], become foam cells that help to repair and to clear the tissue from the necrotic cells. However, the prolonged pathogenesis of atherosclerosis is marked by endothelial cell dysfunction, followed by the deposition of lipid-laden macrophages and VSMC proliferation, leading to the occlusion of the arterial lumen. A build-up of fatty deposits inside the arteries (atherosclerosis) and an increased risk of blood clots are usually associated with cardiovascular diseases, a general term for conditions affecting the heart or blood vessels. In fact, in several cardiovascular diseases, a non-resolved acute inflammation [3] could cause chronic fibrotic disease [6,7]. Sometimes, plaque necrosis further increases the inflammatory response [8] and could break down the integrity of the fibrous cap, triggering acute occlusive thrombosis and tissue infarction [8,9].
In the past, it was assumed that the reduction or elimination of vascular inflammation was principally caused by the decay or cessation of lipid inducers; therefore, the use of LDL-lowering therapy has been revealed to be very useful. Data from basic and clinical was principally caused by the decay or cessation of lipid inducers; therefore, the use of LDL-lowering therapy has been revealed to be very useful. Data from basic and clinical studies indicates that anti-inflammatory treatment could represent an important approach to prevent coronary and cerebral atherosclerotic events. However, despite the availability of this safe therapy, the incidence of cardiovascular disease has been increasing [10].
In recent years, the importance of lipid mediators in resolving inflammation has been spotlighted [11][12][13][14][15]. Lipid mediators originate from PUFAs, which represent an important constituent of all the cell membranes in the body. In particular, AA, EPA, and DHA are precursors of several lipid mediators that display pro-or anti-inflammatory properties based on the PUFA, enzyme, and receptor to which they are related. Originally considered only as membrane and energy storage molecules, lipid mediators are now recognized as powerful signaling molecules that regulate a multitude of cellular responses, including growth, apoptosis, and inflammation/infection [16].
However, defects in the resolution of inflammation by lipid mediators have emerged as key causal factors of atherosclerosis [6,17,18]. Specifically, here, we will discuss the role of lipids derived from AA (prostanoids, leukotrienes, epoxyeicosatrienoic acids, and lipoxins), EPA (E-series resolvins), and DHA (D-series resolvins, protectins, and maresins) in relation to cardiovascular inflammation, with the goals of investigating obstacles in the path of vascular inflammation resolution; and identifying novel therapeutic targets in cardiovascular diseases.
Lipid mediators are grouped into three classes based on their structure and function [19].
LXA4 and LXB4 are produced principally in human mucosal tissues, in blood vessels, and in platelets [22], and are present in all tissues via transcellular interactions between platelets, neutrophils, and resident cells [41]. LXs were first isolated and identified as inhibitors of polymorphonuclear neutrophil infiltration and as stimulators of the nonphlogistic recruitment of macrophages [42,43]. LXA4 regulates cellular functions through the activation of specific receptors (LXA4 receptor/formyl peptide receptor 2 and G-proteincoupled receptor 32) expressed by neutrophils and monocytes [44].

Molecular and Biochemical Characteristic of EETs
EETs are metabolites of AA by cytochrome CYP epoxygenases [45,46] that show beneficial effects in cardiovascular diseases attributed to anti-inflammation, vasodilation, and natriuresis [47].

Role of Prostanoids, LTs, HETEs, LXs, EETs in Atherosclerotic Progression
Prostanoids: Studies performed in mice through genetically manipulated enzymes and receptors highlighted the many physiological and pathological functions of prostanoids in atherosclerosis (see Table 1). Table 1. Studies of Class 1,2,3 lipid mediators in animal models.

Studies in Animal Models
Lipid Mediators Effects Ref.
COX-1 and -2 are mediators of pain, fever, and inflammation and the primary targets of nonsteroidal anti-inflammatory drugs (NSAIDs). However, studies in mice using COX-2 knockout or COX-2 derived PGI2 receptor deletion showed elevated blood pressure and thrombogenesis [82]. Moreover, COX-2-selective drugs such as coxibs, rofecoxib, celecoxib, and lumiracoxib NSAIDs, inhibit cyclooxygenase-2 (COX-2), thereby suppressing the biosynthesis of PGI2, and increasing the risk of thrombotic events such as in myocardial infarction and stroke [83,84].
LTs: Besides their principal role in asthma and in inflammation, LTs can also increase cardiovascular risk, being important mediators of the inflammatory process in cardiovascular pathologies [64,65]. In particular, cysteinyl-LTs seem to be a potent coronary artery vasoconstrictor that stimulates the proliferation of arterial smooth muscle cells and increases the P-selectin surface expression of endothelial cells [36,64]. Increased levels of LTs are present in patients with myocardial infarction (MI), stroke, atherosclerosis, and aortic aneurysms; and the urinary excretion of LTE4 has been reported to be increased in patients with cardiac ischemia [64,85]. Interestingly, two human genetic studies showed a relationship between polymorphism of the 5-LOX pathway and relative risk for MI, stroke, and atherosclerosis, focusing attention on LTs in the pathogenesis of these diseases [86].
Studies of 15-lipoxygenating LOX-isoforms (ALOX15, ALOX15B) indicate that their regulation of vascular tone plays an important role in the development of hypertension [66]. It has been reported that ALOX15-deficient mice exhibited higher resistance towards L-NAME-and high-salt-induced hypertension than wild type controls, although systolic blood pressures did not differ [52]. In support of this, ALOX15-deficient mice treated with wild-type peritoneal macrophages, which are a major source of ALOX15 in mice, lost their resistance toward L-NAME-induced hypertension. Analyzing human atherosclerotic plaques revealed a significant increase in 12/15-LOX expression [93]. It was also reported that ALOX5 and ALOX15B were expressed at high levels in advanced atherosclerotic lesions [94]. However, the role of 12/15-LOX in human atherosclerotic lesion development is still not yet clear. Other studies showed an increased 12/15-LOX expression with decreased lesion severity in human clinical samples. Specifically, a polymorphism in 12/15-LOX promoter (a C to T substitution at position −292), which promotes its increased expression and activity, was found to be associated with a reduction in the risk of atherosclerosis [95].
EETs: EETs are expressed in the heart, especially in the endothelium, and possess beneficial effects against inflammation, fibrosis, and apoptosis, which could combat cardiovascular diseases [96]. Some studies showed that EETs mediate the vasodilation of vascular smooth muscle by activating Ca 2+ -activated K + channels [97], and that its vasodilator effect is independent of nitric oxide (NO) in response to bradykinin [98]. In animal models of hypertension, EETs synthesis was inhibited [99], while it was upregulated by soluble epoxide hydrolase (sEH) inhibitors or by CYP gene overexpression, which in turn decreased blood pressure [100,101]. Interestingly, in Ang II-induced hypertension, sEH expression was upregulated, while EETs were downregulated [53].
Other studies showed that sEH inhibition reduced cholesterol and low-density lipoprotein (LDL) levels, while it increased high-density lipoprotein (HDL) levels [102], preventing atherosclerosis in several models, including ApoE −/− mice and high-fat diet (HFD)induced models [103]. Lower EET levels were observed in obstructed CAD patients compared with patients with no apparent CAD [67]. Furthermore, some authors provided evidence that treatments with sEH inhibitors could reduce the infarct size and chronic cardiac remodeling post MI [104] and that the elevation of EETs might represent a promising therapeutic strategy combating chronic cardiac remodeling post MI and heart failure [105,106]. Already in 2006, Xu et al. reported that treatment with sEH inhibitors prevented the development of cardiac hypertrophy after 3 weeks of the transverse aortic constriction in mice, and reversed the development of cardiac hypertrophy caused by chronic pressure overload [107].
Moreover, Althurwi et al. demonstrated that the elevation of EET levels by the overexpression of YP2J2 prevented the initiation of cardiac hypertrophy through the NF-κBmediated mechanism [108,109]. In particular, in vitro studies showed that the overexpression of CYP2J2 or the increasing levels of 14,15-EET (see structure in Figure 1) ameliorated oxidative-stress-reducing apoptosis and inflammation [110]. However, despite the beneficial effect of EETs as anti-inflammatory mediators, because of their instability, it was impossible to develop corresponding exogenous drugs; several efforts with unremarkable results have been made to increase the level of endogenous EETs, including gene therapy, and EET analogs. Only sEH inhibitor and CYP2J2 gene upregulation seem to provide promising data.
LXs: LXA4 and LXB4 exert potent anti-inflammatory and resolution actions [111,112]. We will discuss these, together with the lipid mediators, in Class 3.
LPA: LPA belongs to the glyceryl-based lysophospholipid family [124] and duced externally to cells, specifically by LPC or autotaxin (ATX; lysophosphol [125], and is degraded by lipid phosphatases [126]. SP1: SP1 is structurally similar to LPA, with the one difference of having a sph backbone rather than glycerol. Sphingolipids are essential structural constituen plasma membrane of eukaryotic organisms and some bacteria, where they are hi riched along with cholesterol in microdomains or lipid rafts. In response to a wide stressful stimuli, membrane sphingomyelin are rapidly metabolized to the b sphingolipid intermediate, ceramide, and subsequently to sphingosine phosphory two sphingosine kinases, resulting in the formation of SP1 [127].

Role of PAF, LPA, and SP1 in Atherosclerotic Progression
PAF: Experiments on animal models showed that PAF plays an important ro pathogenesis of cardiovascular disorders, and some studies performed in humans elevated PAF levels in patients, with increased risk of ischemic events in those w nary artery disease [68][69][70] (see Table 2). In particular, PAFs seems to stimulate
LPA: LPA belongs to the glyceryl-based lysophospholipid family [124] and is produced externally to cells, specifically by LPC or autotaxin (ATX; lysophospholipase D) [125], and is degraded by lipid phosphatases [126]. SP1: SP1 is structurally similar to LPA, with the one difference of having a sphingosine backbone rather than glycerol. Sphingolipids are essential structural constituents of the plasma membrane of eukaryotic organisms and some bacteria, where they are highly enriched along with cholesterol in microdomains or lipid rafts. In response to a wide range of stressful stimuli, membrane sphingomyelin are rapidly metabolized to the bioactive sphingolipid intermediate, ceramide, and subsequently to sphingosine phosphorylation by two sphingosine kinases, resulting in the formation of SP1 [127].

Role of PAF, LPA, and SP1 in Atherosclerotic Progression
PAF: Experiments on animal models showed that PAF plays an important role in the pathogenesis of cardiovascular disorders, and some studies performed in humans reported elevated PAF levels in patients, with increased risk of ischemic events in those with coronary artery disease [68][69][70] (see Table 2). In particular, PAFs seems to stimulate reactive oxygen and nitrogen species, thus triggering the oxidative and nitrosative stress pathways, inducing vasoconstriction [128]. Various substances such as thrombin, angiotensin II, vasopressin, histamine, leukotrienes, and hydrogen peroxide cause the production of PAF in endothelial cells [129]. Other studies suggest that PAF plays a role in the evolution of myocardial injury observed during reperfusion [54] (see Table 1). In conclusion, the effects of PAF are closely associated with atherosclerosis, through various mechanisms such as inflammation, endothelial dysfunction, oxidative and nitrosative stress, and platelet reactivity.
LPA: LPA exerts several effects on vasculature [130,131] resulting in vasoconstriction and increased blood pressure [132]. This happens through the activation of at least six known G-protein-coupled LPA receptors (LPARs) [133] and also peroxisome proliferatoractivated receptor γ (PPARγ) [134], and P2Y10 [135]. Compared to normal arteries, individual LPARs are differentially expressed in human atherosclerotic lesions suggesting a distinct role in the initiation and progression of atherosclerosis [136]. Pathological conditions that involve platelet activation, such as inflammation and atherosclerosis, seem to increase LPA production [137]. LPA is elevated in the serum of patients with acute heart attacks [71] and with acute coronary syndrome [72] and is found accumulated in atherosclerotic plaques [73].
In the vasculature, platelets are one of the major sources of LPA production [138], and LPA in turn can contribute to thrombosis, a critical pathogenic condition of vascular diseases. LPA seems also to increase the permeability of the endothelium. In fact in HU-VECs LPA via LPAR6-Gα13-RhoA-ROCK pathway induces actin stress fiber formation and increases cell permeability [139,140]. Finally, LPA promotes the progression of atherosclerosis via its various effects on inflammatory cells; for example, promoting inflammatory cell migration [141], cytokine secretion [142], and macrophage transformation [143]. LPA also regulates macrophage-related processes, ox-LDL uptake [143], and matrix metalloproteinase (MMP) secretion [144]. MMPs, particularly MMP9, are principle mediators of extracellular matrix production and degradation, which contribute to atherosclerotic plaque instability [145]. Key proteins in LPA homeostasis are increasingly dysregulated in the plaque during atherogenesis, favoring intracellular LPA production. This might at least partly explain the observed progressive accumulation of this thrombogenic proinflammatory lipid in human and mouse plaques. Thus, intervention in enzymatic LPA production may be an attractive measure to lower intraplaque LPA content, thereby reducing plaque progression and thrombogenicity [55].
SP1: S1P has emerged as a potent signaling molecule that regulates diverse cellular processes including cell proliferation, survival, differentiation, and migration [146], maintaining vascular integrity. S1P stimulates COX 2, suggesting the existence of a functional crosstalk between the sphingolipid and eicosanoid pathways [147]. Moreover, many of the cardioprotective functions of HDL, such as vasodilation, angiogenesis, and the endothelial barrier function, may be attributable to its S1P cargo [148]. In a mouse model of acute myocardial infarction, treatment with an engineered S1P chaperone (ApoM-Fc) attenuated myocardial damage after ischemia/reperfusion injury [56].
Additionally, ceramides, that belong to sphingolipids, are grouped in a class of bioactive lipids which are present during vascular inflammation [149], but they have opposing effects on vascular endothelium compared to S1P [150,151]. Ceramide properties mainly involve the regulation of apoptosis, and their signaling is potentially linked to atherosclerosis progression and pathology. Growing evidence indicates that circulating ceramides may predict acute cardiovascular risk, and may potentially be more predictive than LDL cholesterol [152].

Molecular and Biochemical Characteristics of Rvs, PDs and MaRs
Rvs: Rvs are divided into two groups: E-series derived from eicosapentaenoic acid and RvD-series from docosahexaenoic acid [155]. The biosynthesis of RvEs is initiated by vascular endothelial cells that convert EPA into 18R-hydroperoxyeicoapentaenoic acid and 18S-hydroperoxyeicoapentaenoic acid (18-HEPE), and proceeds via a transcellular biosynthesis via acetylated COX-2 and cytochrome P450 [156]. These intermediates are rapidly taken up by human neutrophils and metabolized to RvE1 and RvE2 by 5-lipoxygenase. 8-HEPE can directly be transformed into RvE3 through the action of 12/15-LOX by leukocytes [157].
In addition, another EPA-derived E-series resolvin, RvE4, is produced by human neutrophils and macrophages by 5-LOX, and successively by a second enzymatic lipoxygenation, to yield a hydroperoxyl group at the carbon C-5 position [158]. RvE4 activates G-protein-coupled receptors such as Chemokine-like receptor 1 (ChemR23) and antagonizes the BLT1 receptor, which usually mediates the proinflammatory actions of LTB4 [159][160][161]. Upon selective binding to the receptors, RvE1 attenuates nuclear factor-kappaB signaling and the production of pro-inflammatory cytokines, including tumor necrosis factor alpha. RvDs target G-protein-coupled receptor 32, LxA4 receptor/formyl peptide receptor 2 receptors, Formyl-Peptide Receptor 2/3/LXA4 Receptor, and CB2 receptors that are expressed on platelets and polymorphonuclear neutrophils. The activation of CB2 leads to the inhibition of P-selectin expression, thus decreasing polymorphonuclear neutrophil chemotaxis [44].
PD: Protectins are biosynthesized via a lipoxygenase-mediated pathway from Docosahexaenoic acid and it is converted into an intermediate that contains 17S-hydroxyperoxide, and successively, by leukocytes, into 10,17 Dihydroxy-docosahexaenoic acid, known as PD1 [162,163] or neuroprotection [164], for its protective role in the nervous and immune systems [165,166]. PD1 is also produced by human peripheral blood lymphocytes with a T-helper 2 phenotype; it reduces tumor necrosis factor alpha and interferon gamma secretion, blocks T-cell migration, and promotes T-cell apoptosis [167].

Molecular and Biochemical Characteristics of Rvs, PDs and MaRs
Rvs: Rvs are divided into two groups: E-series derived from eicosapentaenoic acid and RvD-series from docosahexaenoic acid [155]. The biosynthesis of RvEs is initiated by vascular endothelial cells that convert EPA into 18R-hydroperoxyeicoapentaenoic acid and 18S-hydroperoxyeicoapentaenoic acid (18-HEPE), and proceeds via a transcellular biosynthesis via acetylated COX-2 and cytochrome P450 [156]. These intermediates are rapidly taken up by human neutrophils and metabolized to RvE1 and RvE2 by 5-lipoxygenase. 8-HEPE can directly be transformed into RvE3 through the action of 12/15-LOX by leukocytes [157].
In addition, another EPA-derived E-series resolvin, RvE4, is produced by human neutrophils and macrophages by 5-LOX, and successively by a second enzymatic lipoxygenation, to yield a hydroperoxyl group at the carbon C-5 position [158]. RvE4 activates G-protein-coupled receptors such as Chemokine-like receptor 1 (ChemR23) and antagonizes the BLT1 receptor, which usually mediates the proinflammatory actions of LTB4 [159][160][161]. Upon selective binding to the receptors, RvE1 attenuates nuclear factor-kappaB signaling and the production of pro-inflammatory cytokines, including tumor necrosis factor alpha. RvDs target G-protein-coupled receptor 32, LxA4 receptor/formyl peptide receptor 2 receptors, Formyl-Peptide Receptor 2/3/LXA4 Receptor, and CB2 receptors that are expressed on platelets and polymorphonuclear neutrophils. The activation of CB2 leads to the inhibition of P-selectin expression, thus decreasing polymorphonuclear neutrophil chemotaxis [44].
PD: Protectins are biosynthesized via a lipoxygenase-mediated pathway from Docosahexaenoic acid and it is converted into an intermediate that contains 17S-hydroxyperoxide, and successively, by leukocytes, into 10,17 Dihydroxy-docosahexaenoic acid, known as PD1 [162,163] or neuroprotection [164], for its protective role in the nervous and immune systems [165,166]. PD1 is also produced by human peripheral blood lymphocytes with a T-helper 2 phenotype; it reduces tumor necrosis factor alpha and interferon gamma secretion, blocks T-cell migration, and promotes T-cell apoptosis [167].
MaRs: Maresins form the third-largest family of pro-resolving lipid mediators (PRLMs) derived from DHA20. The biosynthesis of maresins occurs primarily in M2 macrophages and is initiated by a reaction involving human macrophage 12-lipoxygenase (12-LOX), which is a key enzyme in the synthesis of maresins [169,171].

Role of PRLMs in Atherosclerotic Progression
PRLMs such as RvE and RvD, PD, MaR, and LX induce a biochemical transition from inflammation to resolution, taking the place of prostaglandins and leukotrienes in exudates at the tissue level [8,169,172,173]. RvE1-E4 are more prominently involved in reducing inflammation by increasing the efferocytosis of apoptotic cells by macrophages, blocking leukocyte recruitment into inflamed tissues (Figure 4).
MaRs: Maresins form the third-largest family of pro-resolving lipid m (PRLMs) derived from DHA20. The biosynthesis of maresins occurs primarily in rophages and is initiated by a reaction involving human macrophage 12-lipox (12-LOX), which is a key enzyme in the synthesis of maresins [169,171].

Role of PRLMs in Atherosclerotic Progression
PRLMs such as RvE and RvD, PD, MaR, and LX induce a biochemical transit inflammation to resolution, taking the place of prostaglandins and leukotrienes in at the tissue level [8,169,172,173]. RvE1-E4 are more prominently involved in red flammation by increasing the efferocytosis of apoptotic cells by macrophages, leukocyte recruitment into inflamed tissues (Figure 4). Increased plasma levels of RvE1 have been observed in individuals taking a eicosapentaenoic acid, resulting in the amelioration of the clinical signs of inflam [74,75]. D-series have been shown to reduce inflammation by decreasing platelet-le adhesion [174].
Protectins reduce polymorphonuclear neutrophil transmigration through en cells and enhance the clearance (efferocytosis) of apoptotic polymorphonuclear neu by human macrophages [170].
Maresins were identified as molecules produced by macrophages with hom functions in resolving inflammation. In fact, the macrophage phagocytosis of a cells triggers the biosynthesis of maresin-1 [175]. Maresin-1 stimulates efferocyto human cells and also has regenerative functions [176]. Finally, LXA4 and B4 exe anti-inflammatory and resolution actions [110].
Temporal changes in the expression, activity, and localization of enzymes su and 15-lipoxygenases (LOXs) appear to activate the "switch" responsible for the re actions [173,177,178]. Consequently, PRLMs coordinate crosstalk between leukoc local cell populations, promoting an M1-M2 phenotypic transition in macrophage is central to tissue repair. PRLMs further promote resolution by positive-feedbac on LOX activity and their receptor expression in leukocytes [177][178][179][180]. Data from and rabbit models showed that pro-resolving lipid mediators decrease atheropro Increased plasma levels of RvE1 have been observed in individuals taking aspirin or eicosapentaenoic acid, resulting in the amelioration of the clinical signs of inflammation [74,75]. D-series have been shown to reduce inflammation by decreasing platelet-leukocyte adhesion [174].
Protectins reduce polymorphonuclear neutrophil transmigration through endothelial cells and enhance the clearance (efferocytosis) of apoptotic polymorphonuclear neutrophils by human macrophages [170].
Maresins were identified as molecules produced by macrophages with homeostatic functions in resolving inflammation. In fact, the macrophage phagocytosis of apoptotic cells triggers the biosynthesis of maresin-1 [175]. Maresin-1 stimulates efferocytosis with human cells and also has regenerative functions [176]. Finally, LXA4 and B4 exert potent anti-inflammatory and resolution actions [110].
Temporal changes in the expression, activity, and localization of enzymes such as 4and 15-lipoxygenases (LOXs) appear to activate the "switch" responsible for the resolution actions [173,177,178]. Consequently, PRLMs coordinate crosstalk between leukocytes and local cell populations, promoting an M1-M2 phenotypic transition in macrophages, which is central to tissue repair. PRLMs further promote resolution by positive-feedback effects on LOX activity and their receptor expression in leukocytes [177][178][179][180]. Data from murine and rabbit models showed that pro-resolving lipid mediators decrease atheroprogression and promote plaque stability [18,172,180,181].
Alternatively a relative "resolution deficit" may contribute to disease progression or limit clinical responses to existing therapies and would therefore suggest opportunities for monitoring and/or treatments that incorporate lipid mediators [74,179,[181][182][183]. In fact, circulating levels of PRLMs, in particular 15-epi-lipoxin A4 , were significantly lower in individuals with peripheral arterial disease and correlated inversely with disease severity [74,76](see Table 2).
In situations such as restenosis, and other pathological reactions secondary to interventions, characterized by high levels of IL-6 and other inflammatory cytokines, augmented levels of PRLMs could limit harmful arterial reactions and restenosis. An imbalance between proinflammatory and resolving factors may decide the evolution of atherosclerotic disease [3].
There is also favorable data regarding the antithrombotic activity of Maresin 1. In fact, it promotes the hemostatic function of human platelets, but suppresses the inflammatory activity, suggesting its important role in the resolution of thrombotic events [184]. At sites of thrombosis or injury, platelet-neutrophil interaction gives rise to the biosynthesis of PRLMs such as MaR1 which induce resolution [185].
The activity of RvS in thrombosis is directed towards reducing platelet activation [180,186]. RvDs and Lxs also activate macrophages for the phagocytosis of the clot and apoptotic cells [187]. In addition, myocardial ischemia/reperfusion studies in the mouse animal model have established a protective effect of PRLMs with respect to tissue damage and the reduction of ROS [57][58][59] (see Table 1).
Although until recently lipid mediators have been overlooked as resolvers of inflammation, they are now being reevaluated because of their activities and biosynthesis in the vascular system. In fact, in vitro, RvD1, RvD2, MaR1, LXA4, and protectin D1 reduce the production of inflammatory cytokines, adhesion molecules, and the adhesive effect of leukocytes to endothelial cells during an inflammatory event [6,[188][189][190]. These resolving effects result from the ability of these molecules to reduce NF-kB, NO, prostacyclin synthesis, and ROS production [191,192].
Reduced monocyte adhesion to VSMCs and the reduced expression of adhesion molecules have been also observed in vitro studies with inflammatory cytokines after treatment with RvD1, RvD2, and MaR1. Another resolving effect by LXA4, RvD1, RvD2, and RvE1 is to attenuate VSMC migration [193,194].

Protective Function of ω-3 Fatty Acid Diet in Atherosclerotic Progression
Individuals with a high consumption of fish oils show a greater proportion of EPA and DHA-containing phospholipids in particular cell types, compared to individuals consuming plant oil supplements [195]. EPA provides precursors for the production of the anti-inflammatory mediator family of series 3 prostanoids. The synthesis of prostanoids is catalyzed by cyclooxygenase enzymes. The cleavage of EPA by phospholipase A 2 allows EPA to be converted to anti-inflammatory mediators [196].
Human studies have shown the limited endogenous synthesis of docosahexaenoic acid (DHA) from ω-3 fatty acid precursor α-linolenic acid (ALA), therefore supporting the necessity of a diet containing ω -3 PUFAs for optimal health and development [197,198]. Circulating levels of the RvD1 or its precursor DHA are reduced in individuals with symptomatic carotid atherosclerosis [199].
Adults receiving EPA+DHA therapy demonstrated significantly greater reductions in circulating levels of proinflammatory cytokines, compared with those receiving placebo therapy [200]. EPA+DHA therapy may be an effective low-risk dietary intervention for assuaging the harmful effects of inflammation.
A large prospective randomized clinical trial of 11,324 patients with a recent myocardial infarction showed a relatively reduced risk of death in response to long chain polyunsaturated fatty acid dietary supplementation [201] (see Table 3).  In another clinical trial, 0.8-1.2 g omega-3 FA supplementation has shown a positive effect in reducing the incidence of major adverse cardiovascular events (MACE), cardiovascular death, and myocardial infarction [202]. A lower rate of myocardial infarction was also observed in people consuming an Inuit diet, characterized by the high consumption of marine mammals and fish [207]. Meta-analyses in adults have shown that the Mediterranean diet, enriched in ω-3 PUFA and oleic acid, exerts a protective effect against cardiovascular diseases [208][209][210][211]. This beneficial effect could be due to their anti-arrhythmic, anti-inflammatory, anti-thrombotic, and hypolipidemic effects, improving vascular function [212,213].
PUFA administration seems to be also protective against aneurysm, a condition characterized by chronic inflammation of the arterial wall that gradually loses its structural integrity by an incessant protease activity. In fact, in animal studies, PUFAs reduced aortic wall inflammation and impairment of the matrix [184]. A recently published trial by Bhatt et al., involving 8179 participants and a 5 year follow-up, reported that patients who received 4 g/d ω-3 PUFA showed a 30% reduction in cardiovascular events, compared with the control group [203]. The JELIS trial, conducted in Japan, recruited 18,645 patients with a total cholesterol of 6.5 mmol/L or greater, and demonstrated a 19% relative reduction in major coronary events after a 1800 mg dose of EPA daily [204].
However, some conflicting data exists [214][215][216]. Other clinical trials on PUFA administration have reported contradictory findings with regard to cardiovascular death and/or major cardiovascular events [205,214,217,218]. Recent studies by Xie et al. suggest that the population most at risk of suffering major cardiovascular events and deaths could benefit more by taking a higher dose of ω-3PUFA for a more prolonged intervention period (daily dose × intervention period > 8 g/day). Earlier treatment schedules of only 1 g/d for 1-year ω-3 PUFA supplementation showed no benefits regarding sudden cardiac death, and in fact revealed a small increase in major cardiovascular and cerebrovascular events [206]. Furthermore, a large-scale randomized controlled trial of the effects of marine ω-3 fatty acid supplementation on cardiovascular outcomes has reported increased risks of atrial fibrillation (AF), in particular in trials testing >1 g/d. [205]. Recent meta-analyses reflected these discrepancies [219,220].
Therefore, the potential reasons for disparate findings may be dose-related. In addition, isomeric forms of the mediators may show different capabilities or conflicting biological effects. More advanced age or smoking habits may also affect the biosynthesis of lipid mediators [221].  In another clinical trial, 0.8-1.2 g omega-3 FA supplementation has shown a positive effect in reducing the incidence of major adverse cardiovascular events (MACE), cardiovascular death, and myocardial infarction [202]. A lower rate of myocardial infarction was also observed in people consuming an Inuit diet, characterized by the high consumption of marine mammals and fish [207]. Meta-analyses in adults have shown that the Mediterranean diet, enriched in ω-3 PUFA and oleic acid, exerts a protective effect against cardiovascular diseases [208][209][210][211]. This beneficial effect could be due to their anti-arrhythmic, anti-inflammatory, anti-thrombotic, and hypolipidemic effects, improving vascular function [212,213].
PUFA administration seems to be also protective against aneurysm, a condition characterized by chronic inflammation of the arterial wall that gradually loses its structural integrity by an incessant protease activity. In fact, in animal studies, PUFAs reduced aortic wall inflammation and impairment of the matrix [184]. A recently published trial by Bhatt et al., involving 8179 participants and a 5 year follow-up, reported that patients who received 4 g/d ω-3 PUFA showed a 30% reduction in cardiovascular events, compared with the control group [203]. The JELIS trial, conducted in Japan, recruited 18,645 patients with a total cholesterol of 6.5 mmol/L or greater, and demonstrated a 19% relative reduction in major coronary events after a 1800 mg dose of EPA daily [204].
However, some conflicting data exists [214][215][216]. Other clinical trials on PUFA administration have reported contradictory findings with regard to cardiovascular death and/or major cardiovascular events [205,214,217,218]. Recent studies by Xie et al. suggest that the population most at risk of suffering major cardiovascular events and deaths could benefit more by taking a higher dose of ω-3PUFA for a more prolonged intervention period (daily dose × intervention period > 8 g/day). Earlier treatment schedules of only 1 g/d for 1-year ω-3 PUFA supplementation showed no benefits regarding sudden cardiac death, and in fact revealed a small increase in major cardiovascular and cerebrovascular events [206]. Furthermore, a large-scale randomized controlled trial of the effects of marine ω-3 fatty acid supplementation on cardiovascular outcomes has reported increased risks of atrial fibrillation (AF), in particular in trials testing >1 g/d. [205]. Recent meta-analyses reflected these discrepancies [219,220]. Therefore, the potential reasons for disparate findings may be dose-related. In addition, isomeric forms of the mediators may show different capabilities or conflicting biological effects. More advanced age or smoking habits may also affect the biosynthesis of lipid mediators [221].
In conclusion, further studies on pro-resolving lipid-mediator biosynthesis and pathways and on diets, with related doses and periods, are needed to understand the best formulations for an effective resolution in relation to cardiovascular disease. Understanding these variables will be necessary for the development of effective drug therapies.