Inside epoxyeicosatrienoic acids and cardiovascular disease

Abstract

EPOXYEICOSATRIENOIC ACIDS: SYNTHESIS AND METABOLISM

Arachidonic acid (AA) is a polyunsaturated omega-6 fatty acid which is released from the sn2 position of membrane phospholipids by the activity of phospholipases (PLs) and among them it is noteworthy the role of cytosolic(c)PLA₂. Free AA can be metabolized to eicosanoids through three major pathways: (i) the cyclooxygenase (COX) pathway, which generates prostanoids; (ii) the lipoxygenase (LOX) pathway, which generates leukotrienes and hydroxyeicosatetraenoic acids (HETEs); (iii) the cytochrome P450 (CYP) pathway, which includes CYP epoxygenase and CYP ω-hydroxylase enzymes (Wang and Dubois, 2012; Oni-Orisan et al., 2014). CYP epoxygenases, such as members of the CYP2C and CYP2J families, metabolize AA to four biologically active epoxyeicosatrienoic acids (EETs; 5, 6-EET, 8, 9-EET, 11, 12-EET, and 14, 15-EET; Figure ​Figure11). Among the members of the CYP2C and CYP2J families of CYP enzymes, CYP2J2, CYP2C8, and CYP2C9 are the predominant epoxygenase isoforms that convert AA into EETs. CYP ω-hydroxylases convert AA to HETEs. CYP4A and CYP4F enzymes mainly catalyze the ω-hydroxylation of AA to 20-HETE (Powell et al., 1998; Figure ​Figure11). In addition, CYP1A1, CYP1B1, and CYP2E1 were reported to catalyze the formation of different regioisomers of HETEs.

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FIGURE 1

The cascade of arachidonic acid (AA). AA is a polyunsaturated omega-6 fatty acid which is released from the sn2 position of membrane phospholipids by the activity of cPLA₂. Free AA can be metabolized to eicosanoids through three major pathways: the cyclooxygenase (COX) pathway, the lipoxygenase (LOX) pathway, and the cytochrome P450 (CYP) pathway. In the CYP pathway, AA is converted to epoxyeicosatrienoic acids (EETs) and 20-HETE by CYP epoxygenases and CYP ω-hydroxylases, respectively. All EETs are then further metabolized by soluble epoxide hydrolase (sEH) forming the less active dihydroxyeicosatrienoic acids (DHETs). Modified from Imig and Hammock (2009).

EETs are synthesized by cells which express CYP epoxygenase activity. The CYP epoxygenase inserts an oxygen atom on a carbon attached to one of the double bonds of AA, and the double bond is reduced as the epoxide forms (Spector, 2009). Each CYP epoxygenase produces several regioisomers, with one form usually predominating. Each regioisomer contains two R/S enantiomeric forms in different proportions (Spector, 2009). Because the regioisomers have a number of similar metabolic and functional properties, all the EETs can be generally considered as a single class of compounds, even if some qualitative differences in the actions of the various regioisomers exist (Spector, 2009). All EETs are then further metabolized by soluble epoxide hydrolase (sEH, EC 3.3.2.7–11; Wang and Dubois, 2012; Bellien and Joannides, 2013), which acts by opening epoxides to diols by the addition of water, forming the less active dihydroxyeicosatrienoic acids (DHETs). This conversion of EET to DHET by sEH attenuates most biological effects of EETs, making sEH a target for increasing and prolonging the actions of EETs (Spector, 2009). In addition, EETs undergo β-oxidation, forming 16-carbon epoxy-derivatives that accumulate in the extracellular fluid, and they can be chain-elongated to form 22-carbon derivatives that are incorporated into phospholipids. EETs are also readily incorporated into cellular membranes via esterification to phospholipids for subsequent release by phospholipases (Spector, 2009). Endothelial cells are a major site of EET incorporation, primarily acylated at the sn2 position of phospholipids and capable of being released by PLA₂ (Weintraub et al., 1999).

BIOLOGICAL EFFECTS OF EETs

Epoxygenase enzymes are localized in endothelial and vascular smooth muscle cells, and also in astrocytes and cardiomyocytes (Imig, 2012; Wang and Dubois, 2012; Oni-Orisan et al., 2014). While 20-HETE is a vasoconstrictor, EETs are vasodilators, except in the pulmonary bed (Bellien and Joannides, 2013). The mechanisms of EET-induced vasodilatation are complex, but increasing evidences have suggested the involvement of endothelial and smooth muscle membrane receptors (Bellien et al., 2011; Chen et al., 2011). In fact, EETs are able to diffuse from the endothelial cells to activate large conductance calcium-activated potassium (BKCa) channels located on the smooth muscle cells, causing their hyperpolarization and relaxation. Then, the endothelial hyperpolarization is transmitted to the smooth muscle through the gap junctions, or the accumulation of K⁺ released from the endothelial KCa channels into the myoendothelial space induces smooth muscle hyperpolarization by the activation of inward rectifying potassium channels and/or Na⁺/K⁺-ATPase (Archer et al., 2003; Huang et al., 2005). The potent vasodilatory effects of EETs are more pronounced in the presence of inhibition of prostacyclin and nitric oxide biosynthesis (Deng et al., 2010). For all of these reasons, CYP-derived EETs are considered as one of the primary endothelium-derived hyperpolarizing factors (EDHFs; Bellien and Joannides, 2013).

Recently, in addition to maintain vascular tone, it has been established a further role of CYP epoxygenase-derived EETs in the maintenance of cardiovascular homeostasis (Figure ​Figure22). In fact, EETs induce vasodilation and exert anti-inflammatory effects in blood vessels in an autocrine manner (Fleming, 2008), limiting leukocyte adhesion and transmigration across the endothelium, inhibiting platelet aggregation, promoting fibrinolysis, and reducing vascular smooth muscle cell proliferation (Figure ​Figure22; Fleming, 2008; Deng et al., 2010; Bellien and Joannides, 2013). EETs can protect the myocardium and brain from ischemia, attenuate hypertension-induced renal damage, and reduce cigarette smoke-induced lung inflammation (Figure ​Figure22; Imig and Hammock, 2009; Deng et al., 2010; Wang and Dubois, 2012). EETs control these different functions by inducing endothelial cell proliferation, survival, and stimulating renal epithelial cell proliferation and survival through multiple signaling pathways (Xu et al., 2011). EETs play a role in the regulation of intracellular Ca²⁺ levels and endoplasmic reticulum homeostasis, through the expression of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a) which transfers Ca²⁺ from the cytosol of the cardiomyocyte to the lumen of the sarcoplasmic reticulum during muscle relaxation (Wang et al., 2014).

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FIGURE 2

Biological effects of EETs.

In addition, EETs play important roles in the regulation of angiogenesis and in the control of metabolic processes (Figure ​Figure22; Fleming, 2007; Lorthioir et al., 2012). Moreover, recent emerging evidences have indicated that EETs are involved in cancer biology; in fact, it has been shown that EETs are critical for primary tumor growth and metastasis, by directly promoting cancer cell proliferation, survival, migration and invasion (Figure ​Figure22; Jiang et al., 2007; Panigrahy et al., 2012; Wang and Dubois, 2012).

EETs IN CARDIOVASCULAR DISEASE

PHARMACOLOGICAL STRATEGIES TO MODULATE CYP-DERIVED EPOXYEICOSATRIENOIC ACIDS PATHWAY

The generation of transgenic mice with endothelial expression of the human CYP2J2 and CYP2C8 epoxygenases, thus leading to increased endothelial EET biosynthesis, has proved that endothelial CYP epoxygenases regulate blood pressure. In fact, these mice exhibit enhanced afferent arteriolar dilation, lower blood pressure and attenuated hypertension-induced renal injury compared to wild-type (Lee et al., 2010). These findings suggest the potential therapeutic utility of antihypertensive strategies that may increase CYP-derived EETs.

Different pharmacological strategies have been developing to increase EET availability: (i) the administration of EET analogs; (ii) the inhibition of EET catabolism by sEH inhibitors.

Epoxyeicosatrienoic acid analogs (designed to resist metabolism and improve their solubility) have been synthesized and have permit to determine EET structure function relationships and define the physiological roles of each EET regioisomer in the cardiovascular system (Sudhahar et al., 2010). Recently, it has been selected a 11,12-EET analog capable to lower blood pressure in spontaneously hypertensive rats (Sudhahar et al., 2010), supporting the use of EETs analog in the treatment of hypertension.

sEH inhibitors are efficacious antihypertensive agents in the Ang II-dependent animal model, a model of human essential hypertension (Imig et al., 2002; Bellien et al., 2011; Imig, 2012). In addition to their efficacy in lowering blood pressure, sEH inhibitors improve endothelial function and reduce hypertension-induced renal injury and cardiac hypertrophy/dysfunction. Moreover, an increasing number of studies have also shown the beneficial effects of sEH inhibitors in other cardiovascular disorders, including ischemia–reperfusion, heart failure, and atherosclerosis (Zhang et al., 2009; Bellien et al., 2011; Merabet et al., 2012).

Among numerous synthesized sEH inhibitors, AR9281 [1-(1-acetyl-piperidin-4-yl)-3-adamantan-1-yl-urea; Imig and Hammock, 2009; Anandan et al., 2011] was selected for phase 1 clinical trial. AR9281 showed a safety profile and it directly and dose-dependently inhibited blood sEH activity, in healthy subjects (Chen et al., 2012). Its efficacy in patients with hypertension and type 2 diabetes has been evaluated in phase II clinical trials, which results are not yet published, even if the trial was completed in 2009 (http://clinicaltrials.gov/show/{"type":"clinical-trial","attrs":{"text":"NCT00847899","term_id":"NCT00847899"}}NCT00847899).

CONCLUSION

Epoxyeicosatrienoic acids have many biological functions which contribute importantly to vascular physiology and to maintain cardiovascular homeostasis (Archer et al., 2003; Imig, 2012; Bellien and Joannides, 2013; Oni-Orisan et al., 2014). Thus, the increase in EET availability is emerged as a new therapeutic opportunity in the clinical management of patients at high cardiovascular risk. However, the use of EETs analogs and sEH inhibitors is limited due to the recent evidences that EETs also promote tumor growth and metastasis (Figure ​Figure22; Michaelis et al., 2005; Jiang et al., 2007; Panigrahy et al., 2011, 2012; Bellien and Joannides, 2013).

Appropriate clinical studies should be performed to characterize the safety profile of these novel classes of drugs before they can be considered for the treatment of cardiovascular disease in humans.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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