1. Introduction
Cytochrome P450 (CYP) is a superfamily of membrane-bound, NADPH-dependent heme-containing monooxygenases involved in the oxidation of both xenobiotics and endogenous compounds. CYPs insert an oxygen atom from molecular oxygen into their product while reducing the second atom to water. CYP2J2 is the only member of the human CYP2J sub-family and one of the major arachidonic acid (AA) epoxygenases. The CYP2J2 gene contains nine exons and eight introns spanning approximately 40.3 kilobases (kb), including about 6 kb of a 5′-flanking region and about 1 kb of 3′-untranslated region [
1] (GenBank accession number AF272142). This gene encodes for 502 amino acids, which translate to a protein product approximately 58 kDa. CYP2J2 is primarily expressed in the heart, and to a lesser degree, in the liver, kidney, skeletal muscle, lung, brain, pancreas, and gastrointestinal tract [
1,
2,
3]. Protein expression of CYP2J2 in the human heart is highly variable in contrast to reportedly stable expression in the liver [
2]. Similar to many CYP isozymes, CYP2J2 is polymorphically expressed throughout the population. However, the identified polymorphisms are relatively rare and seem to be ethnic-specific, with the exception of
CYP2J2*7. This SNP (rs890293) is associated with a G > T substitution in the promoter region (−50 bp), which results in reduced binding of transcription factor Sp1 [
1].
CYP2J2*7 was discovered in different ethnic groups with an allelic frequency ranging from 1.1–17% [
1,
4]. In Caucasian populations, studies show that the
CYP2J2*7 allele results in 40% lower protein expression, without significant changes in enzyme activity using ebastine or astemizole as substrates [
5]. The presence and different allelic frequencies of
CYP2J2*7 among various ethnic groups can, therefore, alter the risk of developing cardiovascular disease (CVD) as summarized in
Table 1. The different findings in
Table 1 and the reasons for the conflicting results are further discussed in
Section 3.1.1.
Several xenobiotics, including ritonavir, astemizole, ebastine, terfenadine, amiodarone, diclofenac, bufurarol, dasatinib, nilotinib, and sorafenib, were identified as CPY2J2 substrates [
2,
6,
7,
8,
9,
10,
11,
12]. However, apart from ebastine, the contribution of CYP2J2 to drug clearance is not significant, because most substrates are also metabolized by CYP3A4. As a result, most of the research on CYP2J2 is focused on its ability to oxidize AA to four bioactive regioisomers of
cis-epoxyeicosatrienoic acids (EETs) in vivo [
2,
13].
Arachidonic acid is a 20 carbon ω-6 polyunsaturated fatty acid with four
bis-allylic
cis-double bonds. AA is predominantly esterified at the
sn-2 position in phospholipids with greater amounts in phosphatidylcholine [
28]. Concentrations of free AA are variable, ranging between 2.7–50 μM in human plasma [
29,
30]. Most AA circulates bound to proteins, including albumin, fatty acid binding protein (FABP), and low-density lipoprotein [
31,
32,
33]. Concentrations of esterified AA were determined to be approximately 5 mM per volume (the per volume calculation, used by the authors, was performed by estimating 30 μg of AA in a billion platelets in total volume of 20 μL), 3 pmol/1 million leukocytes, and 15 μM in resting human platelets, leukocytes, and islets of Langerhans, respectively [
34,
35,
36]. Augmented AA esterified in the membrane increases membrane permeability in adult Wistar rat heart mitochondria and increases membrane fluidity in fresh rat aorta myocytes [
37,
38]. The addition of AA to diet improved cognitive function in healthy, elderly men with low serum AA and synaptic plasticity in aged rats [
39,
40]. AA has also been reported to modulate ion channels, and an increase in intracellular free AA can trigger apoptosis [
41,
42]. Lastly, AA is a substrate for cyclooxygenases, lipoxygenases, and CYP metabolic pathways. Through the CYP pathway, AA can be metabolized to 19-hydroxyeicosatetraenoic acid (19-HETE) and 20-HETE by ω−hydroxylases, primarily the CYP4A and CYP4F families. Most pertinent to this review, AA is biotransformed to the bioactive epoxyeicosatrienoic acids (EETs) by CYP epoxygenases, especially CYP2J2 (
Scheme 1).
CYP epoxygenases, mainly the CYP2C sub-family and CYP2J2, are reported to form exclusively one or more of the four possible
cis-EETs. Several other isozymes including CYP3A4, CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP2E1, and CYP4X1 [
43] have also been reported to catalyze the formation of EETs to some extent. Once formed, EETs can be hydrolyzed to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH) [
44], incorporated back into the phospholipid membrane via acyl-CoA dependent mechanism [
45], or bound to FABP to maintain intracellular levels of EETs and prevent hydrolysis by sEH resulting in prolonging duration of action of EETs (
Scheme 1) [
46]. EETs function both as autocrine and paracrine mediators in the cardiovascular system, kidney, and pancreas. In the cardiovascular system, EETs have been shown to promote angiogenesis, promote hyperpolarization of vascular smooth muscle leading to modulation of vascular tone, and possess anti-inflammatory properties [
47,
48,
49,
50].
EETs are putatively believed to bind to and stimulate a receptor(s) activating signaling cascades in order to exert such a range of physiological effects. EETs have been shown to activate various cascades including the MAPK-associated pathways, such as JNK/c-Jun [
51] and the PI3K/Akt [
52]. There have been several attempts to identify the endogenous EET receptor to date, with a few studies showing EET activation of PPARα [
53,
54] and therefore control over PPARα regulated genes. Recent work by Park et al. identified the G-protein coupled receptor GPR40, also known as free fatty acid receptor 1 (FFA1), as a possible target for EETs in the vascular system [
55]. Specifically, they were able to show that EETs can alter the Ca
2+ flux in HEK293 cells expressing GPR40 and thus mediate the relaxation of bovine arteries and affect the whole cell potassium currents of HUVEC cells expressing GPR40. Additionally, the effects of EETs in these systems were mitigated by the addition of GPR40 antagonists or calcium-chelating agents [
55]. GPR40 is expressed primarily in the pancreas and the brain, at both the mRNA and protein levels [
56,
57], as well as in the liver, heart, and skeletal muscle at an mRNA level [
56,
58]. While this finding is exciting and the first report identifying an “EET receptor” more work is needed to determine if the GPR40 is also the receptor for EETs in cardiomyocytes. Most of the work describing GPR40 is focused on its role in the pancreas and the brain. Future efforts should focus on whether EETs bind to GPR40 and exert the EET-mediated cardioprotection, particularly as protein levels of GPR40 are largely unknown in the various sections of the heart.
While the regulatory mechanisms governing many of the hepatic CYP isoforms are well-studied, there is a paucity of information regarding the regulation of CYP2J2 gene expression. Overexpression of CYP2J2 in experimental animals has been reported to be protective in several disease states, including CVD, whereas CYP2J2 SNPs are associated with risk of incident CVD in humans. In the following sections, we focus on how CVD affects regulation of CYP2J2 in the heart, kidney, and pancreas. We will also present genetic associations of CYP2J2 polymorphisms with related outcomes in humans.
2. CYP2J2 Expression and Regulation in the Heart
Several studies reported the robust expression of CYP2J2 in the heart [
2,
59,
60,
61]. In non-diseased human hearts, the CYP2J2 protein is observed in the cardiomyocytes and the endothelium of blood vessels via immunohistochemical staining [
59]. Although CYP2C9 is highly expressed in endothelial cells compared with CYP2J2, CYP2C9 could only be found in the aorta and coronary arteries of non-diseased human hearts [
59,
62]. In a model of primary human ventricular myocytes, mRNA expression suggests that CYP2J2 is the dominant CYP epoxygenase in ventricular myocytes, and canonical xenobiotic inducers of CYP enzymes have little to no effect on
CYP2J2 expression [
61]. Combined with the primarily extrahepatic expression pattern, this suggests an important endogenous function for CYP2J2.
Few studies have focused on CYP2J2 regulatory mechanisms in cell lines. In HepG2 cells, CYP2J2 is responsive to, and can be upregulated via, the c-Jun/Nrf2 pathway, while treating ventricular myocytes with butylated hydroxyanisole resulted in only a modest and insignificant increase in gene expression [
61,
63,
64].
While typical CYP inducers seem to have little to no regulatory effect, studies have shown that disease states can alter CYP2J2 expression. Bystrom and colleagues demonstrated that
CYP2J2 expression can be induced in human peripheral blood mononuclear cells in response to bacterial lipopolysaccharide [
65]. In human cardiomyocytes, our group has shown that expression can be upregulated by reactive oxygen species either directly or by treating with doxorubicin [
66]. Studies performed in human first-trimester trophoblast-derived cell lines showed that angiotensin-II and hypoxic condition did not alter CYP2J2 expression; however, tumor necrosis-α (TNF-α), which is elevated in preeclampsia, increased protein expression [
67] (
Table 2). The involvement of several factors that govern
CYP2J2 expression hint at tissue-dependent variation, but it is important to note that these are in vitro experiments using different cell types and treating with relatively high concentrations of effectors. More robust studies to determine the regulation of this enzyme in healthy and diseased cell lines and tissues will be more informative.
4. Protective Role of CYP2J2 in the Kidney
A limited number of studies reported on CYP2J2 expression in the kidney. CYP2J2 protein was found in the human kidney at moderate levels, while its transcript was barely detectable [
2]. To date, Wu’s study is the sole report on CYP2J2 protein expression in human kidney. An animal study by Chen et al. demonstrated that CYP2J2 expression could be detected in the kidneys of transgenic mice overexpressing CYP2J2 in the endothelium [
82]. This suggests that human CYP2J2 may be expressed in human renal endothelium; however, more focused studies are needed to confirm this finding at the mRNA, protein, and activity levels.
Endothelial-specific overexpression of CYP2J2 in streptozotocin-induced diabetic mice attenuated renal damage by minimizing the excretion of albumin and scarring of the glomeruli. In addition, endothelial-specific overexpression of CYP2J2 mitigated the activation of the TGF-β/Smad signaling pathway, which was altered in diabetic mice. In order to confirm the mechanism of diabetic nephropathy observed in the mouse model, this group treated human renal proximal tubular cells with TGF-β1. They found that TGF-β1 inhibited E-cadherin expression while activating the Smad pathway, which further induced renal tubular fibrosis and induced tubular epithelial-mesenchymal transdifferentiation, which was then prevented by the addition of exogenous EETs [
82]. In a chronic kidney disease mouse model, increasing EET levels, by chemical inhibition of sEH, led to a decrease in both TGF-β1 and p-Smad3 and induction of PPAR-γ activity [
102]. This study and a study by Kawai et al. [
103] suggested that EETs acted as a PPAR-γ agonist, which led to a decrease in expression of TGF-β1 and p-Smad3 and therefore, attenuated renal damage.
CYP2J2 gene therapy improved systolic blood pressure by increasing the expression of atrial natriuretic peptide in spontaneously hypertensive rats [
76]. In monocrotaline-induced pulmonary hypertensive Sprague–Dawley rats, CYP2J2 gene therapy also attenuated development and vascular remodeling associated with pulmonary hypertension [
77]. In a rat model of chronic kidney failure, the overexpression of CYP2J2 via a recombinant adeno-associated viral vector was able to protect further injury to the remaining kidney by inhibiting apoptosis and fibrosis [
78]. Another study on CYP2J2 gene delivery via a recombinant adeno-associated virus in mice suppressed adventitial remodeling and inflammation and hypertension induced by angiotensin-II [
104]. Overexpression of aortic CYP2J2 via a recombinant adeno-associated virus in angiotensin-II induced abdominal aortic aneurysm apolipoprotein E-deficient mice led to higher EET levels, which activated PPAR and inhibited inflammatory responses [
79].
Total plasma EET levels in 10 patients with renovascular disease were significantly lower than 10 normotensive patients [
105]. Due to EETs (especially 11,12-EET) functioning as the endothelial-derived hyperpolarizing factor in modulating vascular tone, alteration of EET levels in renovascular disease can be associated with an increase in blood pressure. Therapy using an EET analog or sEH inhibitor might attenuate renal injury and improve blood pressure. A few studies on EET analogs in rat models seemed to ameliorate cisplatin-induced nephrotoxicity and renal injury associated with radiation [
106,
107]. EET analogs also exhibited a protective effect against renal fibrosis by reducing the endothelial-to-mesenchymal transition in a renal fibrosis mouse model [
108]. The administration of sEH inhibitors in angiotensin-II-induced hypertensive rats exhibited improvement in vascular function, blood pressure, and attenuation in renal injury [
109,
110]. However, a study in a 5/6-nephroectomy mouse model failed to show the protective effect of sEH inhibition [
111]. This last study infers that a relatively proper functioning kidney is required to get the protective effect of sEH inhibition.
Carrying a
CYP2J2*7 allele was not associated with increased risk of hypertension in African-American subjects [
22,
23]. An association between
CYP2J2*7 and an increased risk of hypertension was found in a Caucasian population in Tennessee, in a Chinese Han population, in a Russian population, and in Saudi Arabian population but not in middle-aged Swedes and South Indian populations (
Table 1) [
17,
23,
24,
25,
26,
27]. The CC genotype of another SNP (rs2280275) has been suggested to be a genetic marker for risk of essential hypertension in an Uygur population but not in a Han population [
112]. The
CYP2J2*7 SNP has also been reported to affect the renal function and the risk of adverse events associated with tacrolimus and mycophenolate sodium in kidney transplant patients in Brazil [
113]. Although not conclusive, CYP2J2 genetic variation may reduce EET levels, which could potentially lead to hypertension.
5. CYP2J2, EETs, and Risk of Diabetes
Diabetes is characterized by dysfunctions in the body’s ability to produce (type I) or respond to (type II) the hormone insulin [
114]. The resulting consequences for the body are that it ultimately becomes hyperglycemic due to chronically high blood glucose levels. As of 2014, over 422 million people live with diabetes worldwide, and as of 2015, 30.3 million people in the United States alone have diabetes [
115,
116]. In adults, 90% of diabetics have type II diabetes. Diabetes is a chronic disorder associated with long-term complications including CVD, retinopathy, neuropathy, and kidney disease [
115,
116].
Animal studies have consistently, and overwhelmingly, shown a protective role for EETs, and thus CYP2J2, in the etiology and progression of diabetes [
80,
81,
117]. Ma and colleagues demonstrated that mice carrying the transgene for cardiac-specific overexpression of human CYP2J2 showed improved glucose and insulin plasma levels, as well as improved glucose tolerance and uptake compared to their wild-type counterparts when challenged with a high fat diet and streptozotocin exposure [
80]. This study also went on to demonstrate that cardiac CYP2J2 overexpressing mice were protected from the cardiovascular consequences of diabetes, in particular myocardial hypertrophy [
80]. They attributed these effects to the activation of the PPAR-γ and MAPK pathways, along with higher atrial natriuretic peptide (ANP) production [
80]. This was followed by a study, which demonstrated that CYP2J2 overexpression resulted in attenuated inflammatory responses in isolated hepatocytes and in diabetic mice [
81]. Inflammatory pathways leading to elevated cytokine levels are thought to be an important factor in the development of type II diabetes [
81,
117,
118]. In addition, Li et al. also showed that CYP2J2 expression activated the PPAR-γ pathway, which they reasoned could lead to decreased dyslipidemia in their animal models by increasing adipogenesis [
81]. Together, these studies demonstrate a clear role for CYP2J2-mediated production of EETs in the prevention of diabetes and its cardiovascular consequences. Efforts to increase production, or decrease the metabolism of EETs are therefore potential therapeutic strategies to treat diabetes.
CYP2J2 in the Pancreas
The pancreas has a central role in the etiology of diabetes and therefore, the consequent CVD. The pancreas, specifically the β cells in the Islets of Langerhans, is responsible for the metered release of insulin into the bloodstream due to elevated blood glucose levels. As glucose levels rise, increased glycolysis in these cells cause a rise in intracellular ATP levels, the downstream effect of which is that intracellular Ca
2+ concentrations rise and insulin is released into the bloodstream [
114,
119]. Diabetes, both type I and type II, is characterized by issues involving the health of the β cells. Type I diabetes is typically characterized by autoimmune destruction of these cells and thus, the reduced ability of the pancreas to produce insulin in response to glucose in the blood. Type II diabetes is putatively due to the increasing resistance of peripheral tissues to respond to the insulin produced by the pancreas but may also be due to the increased resistance of the β cells to high glucose levels in the bloodstream and apoptotic β-cell death [
114,
120]. Together, these two causes result in reduced insulin release into the bloodstream, as well as decreased response by peripheral tissues to the insulin signal.
Evidence supporting a protective role of CYP2J2 and EETs in the pancreas is limited to a few studies. In 1997, Zeldin et al. reported findings from a study where the authors detected CYP2J2 in human pancreatic tissue, as well as the rat homolog, CYP2J3, in rat pancreatic tissue. Immunoblots using CYP2J specific rabbit anti-human antibodies showed that CYP2J2 expression in the human pancreas is localized to the cells in the Islet of Langerhans [
121]. CYP2J2 is proposed to be one of the primary epoxygenases in the human pancreas due to the significant correlation obtained between CYP2J2 protein and extracted total EETs [
121]. The effect of CYP2J2 overexpression on β-cell health has not been studied; however, previous reports showed that raising EET levels can reduce Islet β-cell apoptosis. Luo et al. showed that impairment of sEH has positive outcomes in a diabetic mouse model [
122]. In addition to reducing β-cell death, sEH knockout or inhibition improved glucose tolerance and insulin secretion [
122].
Improvements in blood glucose level, insulin levels, and inflammation markers were observed in diet-induced obese mice that express endothelial-specific CYP2J2 [
83]. CYP2J2 gene therapy in diabetic mice significantly improves metabolic function and insulin sensitivity of diabetic mice by altering the expression of enzymes involved in maintaining glucose homeostasis [
81]. In a separate study, CYP2J2 gene introduction, along with the administration of an sEH inhibitor, in mice fed with a high fat diet suggests that higher EET levels promote better metabolic function, insulin sensitivity, and reduce inflammation associated with a high fat diet [
117]. Further mechanistic studies on how EETs improved metabolic function in mice fed with a high fat diet are needed and will provide insights into therapeutic strategies for obesity-induced metabolic diseases. Perhaps therapy using EET analogs or sEH inhibitors to maintain EET levels will be useful in preventing extensive damage associated with diabetes. Treatment with an sEH inhibitor was shown to prevent diabetic retinopathy in diabetic mice [
123]. A dual sEH inhibitor and PPAR-γ agonist are able to attenuate renal injury in metabolic syndrome rat model [
124], which is very promising for future therapies involving EETs and maintaining EET levels in the pancreas.