CYP2J2-mediated metabolism of arachidonic acid in heart: A review of its kinetics, inhibition and role in heart rhythm control.

Cytochrome P450 2 J2 (CYP2J2) is primarily expressed extrahepatically and is the predominant epoxygenase in human cardiac tissues. This highlights its key role in the metabolism of endogenous substrates. Significant scientific interest lies in cardiac CYP2J2 metabolism of arachidonic acid (AA), an omega-6 polyunsaturated fatty acid, to regioisomeric bioactive epoxyeicosatrienoic acid (EET) metabolites that show cardioprotective effects including regulation of cardiac electrophysiology. From an in vitro perspective, the accurate characterization of the kinetics of CYP2J2 metabolism of AA including its inhibition and inactivation by drugs could be useful in facilitating in vitro-in vivo extrapolations to predict drug-AA interactions in drug discovery and development. In this review, background information on the structure, regulation and expression of CYP2J2 in human heart is presented alongside AA and EETs as its endogenous substrate and metabolites. The in vitro and in vivo implications of the kinetics of this endogenous metabolic pathway as well as its perturbation via inhibition and inactivation by drugs are elaborated. Additionally, the role of CYP2J2-mediated metabolism of AA to EETs in cardiac electrophysiology will be expounded.

Investigating the association between CYP2J2 inhibitors and QT prolongation: a literature review.

Drug withdrawal post-marketing due to cardiotoxicity is a major concern for drug developers, regulatory agencies, and patients. One common mechanism of cardiotoxicity is through inhibition of cardiac ion channels, leading to prolongation of the QT interval and sometimes fatal arrythmias. Recently, oxylipin signaling compounds have been shown to bind to and alter ion channel function, and disruption in their cardiac levels may contribute to QT prolongation. Cytochrome P450 2J2 (CYP2J2) is the predominant CYP isoform expressed in cardiomyocytes, where it oxidizes arachidonic acid to cardioprotective epoxyeicosatrienoic acids (EETs). In addition to roles in vasodilation and angiogenesis, EETs bind to and activate various ion channels. CYP2J2 inhibition can lower EET levels and decrease their ability to preserve cardiac rhythm. In this review, we investigated the ability of known CYP inhibitors to cause QT prolongation using Certara's Drug Interaction Database. We discovered that among the multiple CYP isozymes, CYP2J2 inhibitors were more likely to also be QT-prolonging drugs (by approximately 2-fold). We explored potential binding interactions between these inhibitors and CYP2J2 using molecular docking and identified four amino acid residues (Phe61, Ala223, Asn231, and Leu402) predicted to interact with QT-prolonging drugs. The four residues are located near the opening of egress channel 2, highlighting the potential importance of this channel in CYP2J2 binding and inhibition. These findings suggest that if a drug inhibits CYP2J2 and interacts with one of these four residues, then it may have a higher risk of QT prolongation and more preclinical studies are warranted to assess cardiovascular safety.

Involvement of multiple cytochrome P450 isoenzymes in drug interactions between ritonavir and direct oral anticoagulants.

Herein, we aimed to determine the significance of drug interactions (DIs) between ritonavir and direct oral anticoagulants (DOACs) and identify the involved cytochrome P450 (CYP) isoenzymes. Using an in vitro cocktail method with human liver microsomes (HLM), we observed that ritonavir strongly inhibited CYPs in the following order: CYP3A, CYP2C8, CYP2D6, CYP2C9, CYP2C19, CYP2B6, and CYP2J2 (IC50: 0.023-6.79 µM). The degree of CYP2J2 inhibition was inconclusive, given the substantial discrepancy between the HLM and human expression system. Selective inhibition of CYP3A decreased the O-demethylation of apixaban by only 13.4%, and the involvement of multiple CYP isoenzymes was suggested, all of which were inhibited by ritonavir. Multiple CYP isoenzymes contributed also to the metabolism of rivaroxaban. Replacement of the incubation medium with phosphate buffer instead of HEPES enhanced apixaban hydroxylation. On surveying the FDA Adverse Event Reporting System, we detected that the signal of the proportional reporting ratio of "death" and found increase for "hemoglobin decreased" (12.5-fold) and "procedural hemorrhage" (201.9-fold) on administering apixaban with ritonavir; these were far less significant for other CYP3A inhibitors. Overall, these findings suggest that co-administration of ritonavir-boosted drugs with DOACs may induce serious DIs owing to the simultaneous inhibition of multiple CYP isoenzymes.

Influence of Genetic Polymorphisms on the Short-Term Response to Ranibizumab in Patients With Neovascular Age-Related Macular Degeneration.

Purpose: To determine whether genetic risk single nucleotide polymorphisms (SNPs) for age-related macular degeneration (AMD) influence short-term response to intravitreal ranibizumab treatment. Methods: Forty-four treatment-naive AMD patients were included in a prospective observational study. They underwent three monthly injections of intravitreal ranibizumab for neovascular AMD. After an initial clinical examination (baseline measurement), a follow-up visit was performed to determine treatment response one month after the third injection (treatment evaluation). Patients were evaluated based on ophthalmoscopy, fluorescein angiography, optical coherence tomography (OCT), and OCT angiography. Peripheral venous blood was collected for DNA analysis at baseline visit. Patients were genotyped for single-nucleotide polymorphisms within AMD-relevant genes and classified on good or poor responders based on visual acuity, central retinal thickness, intraretinal fluid, and subretinal fluid. Results: One hundred ten AMD-associated SNPs have been analyzed. Six were found to be relevant when associated to ranibizumab treatment response. The genetic variants rs890293 (CYP2J2), rs11200638 (HTRA1), rs405509 (APOE), rs9513070 (FLT1), and rs8135665 (SLC16A8) predisposed patients to a good response, whereas rs3093077 (CRP) was associated with a poor response. FTL1, SLC16A8, and APOE were the SNPs that showed significance (P < 0.05) but did not pass Bonferroni correction. Conclusions: This is the first study that links novel polymorphisms in genes such as CRP, SCL16A8, or CYP2J2 to treatment response to ranibizumab therapy. On the other hand, HTRA1, FLT1, and APOE are linked to a good ranibizumab response. These SNPs may be good candidates for short-term treatment response biomarkers in AMD patients. However, further studies will be necessary to confirm our findings.

Altered distribution of fatty acid exerting lipid metabolism and transport at the maternal-fetal interface in fetal growth restriction.

INTRODUCTION: Fetal growth restriction (FGR) is a common complication of pregnancy. Lipid metabolism and distribution may contribute to the progression of FGR. However, the metabolism-related mechanisms of FGR remain unclear. The aim of this study was to identify metabolic profiles associated with FGR, as well as probable genes and signaling pathways. METHODS: Metabolomic profiles at the maternal-fetal interface (including the placenta, maternal and fetal serum) from pregnant women with (n = 35) and without (n = 35) FGR were analyzed by gas chromatography-mass spectrometry (GC-MS). Combined with differentially expressed genes (DEGs) from the GSE35574 dataset, analysis was performed for differential metabolites, and identified by the Metabo Analyst dataset. Finally, the pathology and screened DEGs were further identified. RESULTS: The results showed that fatty acids (FAs) accumulated in the placenta and decreased in fetal blood in FGR cases compared to controls. The linoleic acid metabolism was the focus of placental differential metabolites and genes enrichment analysis. In this pathway, phosphatidylcholine can interact with PLA2G2A and PLA2G4C, and 12(13)-EpOME can interact with CYP2J2. PLA2G2A and CYP2J2 were elevated, and PLA2G4C was decreased in the FGR placenta. DISCUSSION: In conclusion, accumulation of FAs in the placental ischemic environments, may involve linoleic acid metabolism, which may be regulated by PLA2G2A, CYP2J2, and PLA2G4C. This study may contribute to understanding the underlying metabolic and molecular mechanisms of FGR.

Glabridin plays dual action to intensify anti-metastatic potential of paclitaxel via impeding CYP2C8 in liver and CYP2J2/EETs in tumor of an orthotopic mouse model of breast cancer.

In spite of unprecedented advances in modern cancer therapy, there is still a dearth of targeted therapy to circumvent triple-negative breast cancer (TNBC). Paclitaxel is the front-line therapy against TNBC, but the main constraints of its treatment are dose-related adverse effects and emerging chemoresistance. In this context, glabridin (phytoconstituent from Glycyrrhiza glabra) is reported to hit multiple signalling pathways at the in-vitro level, but hardly any information is known at the in-vivo level. We aimed here to elucidate glabridin potential with an underlying mechanism in combination with a low dose of paclitaxel using a highly aggressive mouse mammary carcinoma model. Glabridin potentiated the anti-metastatic efficacy of paclitaxel by substantially curtailing tumor burden and diminishing lung nodule formation. Moreover, glabridin remarkably attenuated epithelial-mesenchymal transition (EMT) traits of hostile cancer cells via up-regulating (E-cadherin & occludin) and down-regulating (Vimentin & Zeb1) vital EMT markers. Besides, glabridin amplified apoptotic induction effect of paclitaxel in tumor tissue by declining or elevating pro-apoptotic (Procaspase-9 or Cleaved Caspase-9 & Bax) and reducing anti-apoptotic (Bcl-2) markers. Additionally, concomitant treatment of glabridin and paclitaxel predominantly lessened CYP2J2 expression with marked lowering of epoxyeicosatrienoic acid (EET)'s levels in tumor tissue to reinforce the anti-tumor impact. Simultaneous administration of glabridin with paclitaxel notably enhanced plasma exposure and delayed clearance of paclitaxel, which was mainly arbitrated by CYP2C8-mediated slowdown of paclitaxel metabolism in the liver. The fact of intense CYP2C8 inhibitory action of glabridin was also ascertained using human liver microsomes. Concisely, glabridin plays a dual role in boosting anti-metastatic activity by augmenting paclitaxel exposure via CYP2C8 inhibition-mediated delaying paclitaxel metabolism and limiting tumorigenesis via CYP2J2 inhibition-mediated restricting EETs level. Considering the safety, reported protective efficacy, and the current study results of boosted anti-metastatic effects, further investigations are warranted as a promising neoadjuvant therapy for crux paclitaxel chemoresistance and cancer recurrence.

CYP2J deficiency leads to cardiac injury and presents dual regulatory effects on cardiac function in rats.

Cytochrome P450 2J2 (CYP2J2) enzyme is widely expressed in aortic endothelial cells and cardiac myocytes and affects cardiac function, but the underlying mechanism is still unclear. Based on CYP2J knockout (KO) rats, we have directly studied the metabolic regulation of CYP2J on cardiac function during aging. The results showed that CYP2J deficiency significantly reduced the content of epoxyeicosatrienoic acids (EETs) in plasma, aggravated myocarditis, myocardial hypertrophy, as well as fibrosis, and inhibited the mitochondrial energy metabolism signal network Pgc-1α/Ampk/Sirt1. With the increase of age, the levels of 11,12-EET and 14,15-EET in plasma of KO rats decreased significantly, and the heart injury was more serious. Interestingly, we found that after CYP2J deletion, the heart initiated a self-protection mechanism by upregulating cardiac mechanism factors Myh7, Dsp, Tnni3, Tnni2, and Scn5a, as well as mitochondrial fusion factors Mfn2 and Opa1. However, this protective effect disappeared with aging. In conclusion, CYP2J deficiency not only reduces the amount of EETs, but also plays a dual regulatory role in cardiac function.

Genetic variants in epoxyeicosatrienoic acid processing and degradation pathways are associated with gestational diabetes mellitus.

AIM: To explore the genetic effects of CYP2C8, CYP2C9, CYP2J2, and EPHX2, the key genes involved in epoxyeicosatrienoic acid processing and degradation pathways in gestational diabetes mellitus (GDM) and metabolic traits in Chinese pregnant women. METHODS: A total of 2548 unrelated pregnant women were included, of which 938 had GDM and 1610 were considered as controls. Common variants were genotyped using the Infinium Asian Screening Array. Association studies of single nucleotide polymorphisms (SNPs) with GDM and related traits were performed using logistic regression and multivariable linear regression analyses. A genetic risk score (GRS) model based on 12 independent target SNPs associated with GDM was constructed. Logistic regression was used to estimate odds ratios and 95% confidence intervals, adjusting for potential confounders including age, pre-pregnancy body mass index, history of polycystic ovarian syndrome, history of GDM, and family history of diabetes, with GRS entered both as a continuous variable and categorized groups. The relationship between GRS and quantitative traits was also evaluated. RESULTS: The 12 SNPs in CYP2C8, CYP2C9, CYP2J2, and EPHX2 were significantly associated with GDM after adjusting for covariates (all P < 0.05). The GRS generated from these SNPs significantly correlated with GDM. Furthermore, a significant interaction between CYP2J2 and CYP2C8 in GDM (PInteraction = 0.014, ORInteraction= 0.61, 95%CI 0.41-0.90) was observed. CONCLUSION: We found significant associations between GDM susceptibility and 12 SNPs of the four genes involved in epoxyeicosatrienoic acid processing and degradation pathways in a Chinese population. Subjects with a higher GRS showed higher GDM susceptibility with higher fasting plasma glucose and area under the curve of glucose and poorer ß-cell function.

Role of cytochrome P450-epoxygenase and soluble epoxide hydrolase in the regulation of vascular response.

The role of cytochrome P450-epoxygenase has been seen in cardiovascular physiology and pathophysiology. The aberration in CYP450-epoxygenase genes occur due to genetic polymorphisms, aging, or environmental factors, that increase susceptibility to cardiovascular diseases (CVDs). The actual role played by the CYP450-epoxygenases is the metabolism of arachidonic acid (AA) and linoleic acid (LA) into epoxyeicosatrienoic acids (EETs) and epoxyoctadecaenoic acid (EpOMEs) metabolites (oxylipins) and others, which is involved in vasodilation and myocardial-protection. But the genetic polymorphisms in CYP450-epoxygenases lose their beneficial cardiovascular effects of oxylipins, and the soluble epoxide hydrolase (sEH) antagonizes beneficial oxylipins into diols. Like sEH converts EETs into dihydroxyeicosatrienoic acid (DHETs), EpOMEs into dihydroxyoctadecaenoic acid (DiHOMEs), and reverses its beneficial effects, and the sEH gene (Ephx2) polymorphisms cause the enzyme to become overactive and convert epoxy-fatty acids into diols, making them vulnerable to CVDs, including hypertension. Other, enzymes like ω-hydroxylases (CYP450-4A11 & CYP450-4F2)-derived oxylipins from AA, ω-terminal-hydroxyeicosatetraenoic acids (19-, 20-HETE), lipoxygenase-derived oxylipins, mid-chain hydroxyeicosatetraenoic acids (5-, 11-, 12-, 15-HETEs), and the cyclooxygenase-derived prostanoids (prostaglandins: PGD2, PGF2α; thromboxane: Txs, oxylipins) are involved in vasoconstriction, hypertension, inflammation, and cardiac toxicity. Also, there are significant interactions were seen between adenosine receptors [adenosine A2A receptor (A2AAR) and adenosine A1 receptor (A1AR)] with CYP450-epoxygenases, ω-hydroxylases, sEH, and their derived oxylipins in the regulation of the cardiovascular response. Moreover, polymorphisms exist in CYP450-epoxygenases, ω-hydroxylases, sEH, and the adenosine receptor genes in populations associated with CVDs. This chapter will discuss the role of oxylipins' interactions with adenosine receptors in cardiovascular function/dysfunction in mice and humans.

Effects of genetic polymorphisms of CYP2J2, CYP2C9, CYP2C19, CYP4F2, CYP4F3 and CYP4A11 enzymes in preeclampsia and gestational hypertension.

INTRODUCTION: The aim of this study was to investigate the effects of cytochrome P450 (CYP) 2J2, CYP2C9, CYP2C19 and CYP4F2, CYP4F3 and CYP4A11 genetic polymorphisms in preeclampsia and gestational hypertension (GHT) patients in a sample of Turkish population. MATERIALS-METHODS: Patients (n = 168; 110 GHT and 58 preeclampsia) and healthy pregnant women (n = 155, controls) participated in the study. For genotyping, polymerase chain reaction (PCR) and restriction analysis (RFLP) were used. Substance levels were measured using LC-MS. RESULTS: Plasma DHET levels in GHT and preeclampsia patients were significantly lower than those in the control group (62.7%, 66.3% vs.100.0%, respectively, p < 0.0001). An increase in CYP2J2*7 allele frequency was observed in the preeclampsia group, as compared to GHT group (12.1% vs. 4.5%; odds ratio, O.R. = 2.88, p < 0.01). The frequencies of CYP2C19*2 and*17 alleles were higher in GHT group as compared to the control group (17.7% vs. 11.6%, O.R. = 1.99, p < 0.01; and 28.6% vs.18.4%, O.R. = 2.03, p < 0.01, respectively). An increased frequency of CYP4F3 rs3794987 G allele was found in GHT group as compared to the control group (48.0% vs. 38.0%; O.R. = 1.53, p < 0.01). DISCUSSION: DHET plasma levels were significantly reduced in hypertensive pregnant groups as compared to the control group. The allele frequency distributions for CYP2J2*7, CYP2C19 *2, *17 and CYP4F3 rs3794987 were significantly different in hypertensive pregnant patients as compared to the healthy control subjects. Our results may suggest that investigated genetic polymorphisms may be useful in diagnosis and clinical management of GHT and preeclampsia patients.

Investigating the relevance of CYP2J2 inhibition for drugs known to cause intermediate to high risk torsades de pointes.

Cardiac cytochrome P450 2J2 (CYP2J2) metabolizes endogenous polyunsaturated fatty acid, arachidonic acid (AA), to bioactive regioisomeric epoxyeicosatrienoic acid (EET) metabolites. This endogenous metabolic pathway has been postulated to play a homeostatic role in cardiac electrophysiology. However, it is unknown if drugs that cause intermediate to high risk torsades de pointes (TdP) exhibit inhibitory effects against CYP2J2 metabolism of AA to EETs. In this study, we demonstrated that 11 out of 16 drugs screened with intermediate to high risk of TdP as defined by the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative are concurrently reversible inhibitors of CYP2J2 metabolism of AA, with unbound inhibitory constant (Ki,AA,u) values ranging widely from 0.132 to 19.9 µM. To understand the physiological relevancy of Ki,AA,u, the in vivo unbound drug concentration within human heart tissue (Cu,heart) was calculated via experimental determination of in vitro unbound partition coefficient (Kpuu) for 10 CYP2J2 inhibitors using AC16 human ventricular cardiomyocytes as well as literature-derived values of fraction unbound in plasma (fu,p) and plasma drug concentrations in clinical scenarios leading to TdP. Notably, all CYP2J2 inhibitors screened belonging to the high TdP risk category, namely vandetanib and bepridil, exhibited highest Kpuu values of 18.2 ± 1.39 and 7.48 ± 1.16 respectively although no clear relationship between Cu,heart and risk of TdP could eventually be determined. R values based on basic models of reversible inhibition as per FDA guidelines were calculated using unbound plasma drug concentrations (Cu,plasma) and adapted using Cu,heart which suggested that 4 out of 10 CYP2J2 inhibitors with intermediate to high risk of TdP demonstrate greatest potential for clinically relevant in vivo cardiac drug-AA interactions. Our results shed novel insights on the relevance of CYP2J2 inhibition in drugs with risk of TdP. Further studies ascertaining the role of CYP2J2 metabolism of AA in cardiac electrophysiology, characterizing inherent cardiac ion channel activities of drugs with risk of TdP as well as in vivo evidence of drug-AA interactions will be required prior to determining if CYP2J2 inhibition could be an alternative mechanism contributing to drug-induced TdP.

11,12-EET suppressed LPS induced TF expression and thrombus formation by accelerating mRNA degradation rate via strengthening PI3K-Akt signaling pathway and inhibiting p38-TTP pathway.

Epoxyeicosatrienoic acids (EETs), which are synthesized from arachidonic acid by cytochrome P450 epoxygenases, function primarily as autocrine and paracrine effectors in the cardiovascular system. So far, most research has focused on the vasodilatory, anti-inflammatory, anti-apoptotic and mitogenic properties of EETs in the systemic circulation. However, whether EETs could suppress tissue factor (TF) expression and prevent thrombus formation remains unknown. Here we utilized in vivo and in vitro models to investigate the effects and underlying mechanisms of exogenously EETs on LPS induced TF expression and inferior vein cava ligation induced thrombosis. We observed that the thrombus formation rate and the size of the thrombus were greatly reduced in 11,12-EET treated mice，accompanied by decreased TF and inflammatory cytokines expression. Further in vitro studies showed that by enhancing p38 MAPK activation and subsequent tristetraprolin (TTP) phosphorylation, LPS strengthened the stability of TF mRNA and induced increased TF expression. However, by strengthening PI3K-dependent Akt phosphorylation, which acted as a negative regulator of p38-TTP signaling pathway，11,12-EET reduced LPS-induced TF expression in monocytes. In addition, 11,12-EET inhibited LPS-induced NF-κB nuclear translocation by activating the PI3K/Akt pathway. Further study indicated that the inhibitory effect of 11,12-EET on TF expression was mediated by antagonizing LPS-induced activation of thromboxane prostanoid receptor. In conclusion, our study demonstrated that 11,12-EET prevented thrombosis by reducing TF expression and targeting the CYP2J2 epoxygenase pathway may represent a novel approach to mitigate thrombosis related diseases.

MiR-3646 accelerates inflammatory response of Ang II-induced hVSMCs via CYP2J2/EETs axis in hypertension model.

BACKGROUND: Inflammatory response of human vascular smooth muscle cells (hVSMCs) is a driving factor in hypertension progression. It has been reported that miR-3646 was significantly up-regulated in serum samples from patients with coronary artery disease and acute myocardial infarction mice. However, its role and underlying molecular mechanism related to inflammatory response of angiotensin II (Ang II)-induced hVSMCs remain unclear. OBJECTIVE: We aimed to explore the potential molecular mechanisms related to inflammatory response of angiotensin II (Ang II)-induced hVSMCs. METHODS: Ang II-induced hypertension model was established after hVSMCs treated with 1 µM Ang II at 24 h. The interaction between microRNA 3646 (miR-3646) and cytochrome P450 2J2 (CYP2J2) was assessed by dual-luciferase reporter gene assay. MTS assay, Lipid Peroxidation MDA Assay Kit, ELISA, Western blot, and qRT-PCR were performed to examine viability, malondialdehyde (MDA) level, inflammatory cytokine levels, and the level of genes and proteins. RESULTS: Our findings illustrated that miR-3646 was up-regulated but CYP2J2 was down-regulated in Ang II-induced hVSMCs. Mechanically, miR-3646 negatively targeted to CYP2J2 in Ang II-induced hVSMCs. These findings indicated that miR-3646 regulated inflammatory response of Ang II-induced hVSMCs via targeting CYP2J2. Moreover, functional researches showed that CYP2J2 overexpression alleviated inflammatory response of Ang II-induced hVSMCs via epoxyeicosatrienoic acids/peroxisome proliferator-activated receptor-γ (EETs/PPARγ) axis, and miR-3646 aggravated inflammatory response of Ang II-induced hVSMCs via mediating CYP2J2/EETs axis. CONCLUSION: MiR-3646 accelerated inflammatory response of Ang II-induced hVSMCs via CYP2J2/EETs axis. Our findings illustrated the specific molecular mechanism of miR-3646 regulating hypertension.

CYP2C8 and CYP2J2 gene variations increase the risk of hypertensive intracerebral hemorrhage.

PURPOSE: Many studies have shown that cytochrome P450 (CYP) gene polymorphisms are usually associated with an increased risk of cardiovascular and cerebrovascular diseases. To explore the association of CYP2C8 and CYP2J2 gene polymorphisms with hypertensive intracerebral hemorrhage (HICH) in the Han Chinese population. METHODS: Forty HICH patients and 40 control subjects were recruited for this study. Two single nucleotide polymorphisms (SNP) (rs1058932, rs2275622) in the CYP2C8 gene and two SNPs (rs2271800, rs1155002) in the CYP2J2 gene were selected for genotyping by direct sequencing. Statistical analysis was applied to examine the effect of genetic variation on HICH. RESULTS: We found that variant alleles of CYP2C8 rs1058932 (A) and rs2275622 (C) were both significantly associated with HICH, especially in females. We also found significant associations of CYP2C8 rs1058932 (A) and rs2275622 (C) variant alleles with poor outcomes in HICH patients, especially in males. CONCLUSIONS: CYP2C8 gene polymorphisms might increase the risk of HICH in the Han Chinese population and might lead to poor outcomes. This finding adds to the body of literature supporting novel therapeutic strategies for HICH.

Endothelial CYP2J2 overexpression restores the BRB via METTL3-mediated ANXA1 upregulation.

Blood-retinal barrier (BRB) breakdown is responsible for multiple ocular diseases, such as diabetic retinopathy, age-related macular degeneration, and retinal vascular occlusive diseases. Increased vascular permeability contributes to vasogenic edema and tissue damage, with consequent adverse effects on vision. Herein, we found that endothelial CYP2J2 overexpression maintained BRB integrity after ischemia-reperfusion injury and consequently protected against retinal ganglion cell loss. Oxidative stress repressed endothelial ANXA1 expression in vivo and in vitro. CYP2J2 upregulated methyltransferase-like 3 (METTL3) expression and hence promoted ANXA1 translation via ANXA1 m6 A modification in endothelium under oxidative stress. CYP2J2 maintained the distribution of endothelial tight junctions and adherens junctions in an ANXA1-dependent manner. Endothelial ANXA1 plays an indispensable role in vascular homeostasis and stabilization during development. Endothelial ANXA1 deletion disrupted retinal vascular perfusion as well as BRB integrity. CYP2J2 metabolites restored BRB integrity in the presence of ANXA1. Our findings identified the CYP2J2-METTL3-ANXA1 pathway as a potential therapeutic target for relieving BRB impairments.

Targeted Metabolomics Study of Human Plasma Revealed Activation of the Cytochrome P450 Epoxygenase/Epoxide Hydrolase Axis in Patients with IgA Nephropathy.

IgA nephropathy (IgAN) is the most common primary glomerulonephritis and a leading cause of chronic kidney disease. The pathogenic mechanism of IgAN remains largely unknown and thus a specific therapeutic target is lacking. Here, we reported that the cytochrome P450 (CYP) epoxygenase/epoxide hydrolase (EH) axis was activated in the patients and is likely a therapeutic target for IgAN. Specifically, quantitative profiling of the plasma from IgAN patients and healthy controls revealed significant changes in plasma levels of CYP/EH-mediated lipid epoxides and diols. Subsequently, CYP2C8, CYP2C18, CYP2J2, EPHX1, and EPHX2 were found to be significantly increased in whole blood cells at mRNA levels from the IgAN patients when compared with those of healthy controls. Immunohistochemical analysis showed that all five CYPs and two EHs were upregulated in the kidney tissue from IgAN patients when compared with normative renal tissue, but the expression locations of the proteins were different with most of them. Treatment of HK-2 cells with IgA1 increased cell viability, compressed cell apoptosis, and increased the protein levels of CYP2C9, EPHX1, and EPHX2. All the results agreed that CYPs/EHs axis is likely the prophylactic and therapeutic target for IgAN, providing IgAN patients with a new intervention strategy.

Cardiac Disease Alters Myocardial Tissue Levels of Epoxyeicosatrienoic Acids and Key Proteins Involved in Their Biosynthesis and Degradation.

CYP2J2 is the main epoxygenase in the heart that is responsible for oxidizing arachidonic acid to cis-epoxyeicosatrienoic acids (EETs). Once formed, EETs can then be hydrolyzed by soluble epoxide hydrolase (sEH, encoded by EPHX2) or re-esterified back to the membrane. EETs have several cardioprotective properties and higher levels are usually associated with better cardiac outcomes/prognosis. This study investigates how cardiovascular disease (CVD) can influence total EET levels by altering protein expression and activity of enzymes involved in their biosynthesis and degradation. Diseased ventricular cardiac tissues were collected from patients receiving Left Ventricular Assist Device (LVAD) or heart transplants and compared to ventricular tissue from controls free of CVD. EETs, and enzymes involved in EETs biosynthesis and degradation, were measured using mass spectrometric assays. Terfenadine hydroxylation was used to probe CYP2J2 activity. Significantly higher cis- and trans-EET levels were observed in control cardiac tissue (n = 17) relative to diseased tissue (n = 24). Control cardiac tissue had higher CYP2J2 protein levels, which resulted in higher rate of terfenadine hydroxylation, compared to diseased cardiac tissues. In addition, levels of both NADPH-Cytochrome P450 oxidoreductase (POR) and sEH proteins were significantly higher in control versus diseased cardiac tissue. Overall, alterations in protein and activity of enzymes involved in the biosynthesis and degradation of EETs provide a mechanistic understanding for decreased EET levels in diseased tissues.

Cardiomyocyte-specific CYP2J2 and its therapeutic implications.

INTRODUCTION: Cytochrome P450s (CYPs) are a superfamily of monooxygenases with diverse biological roles. CYP2J2 is an isozyme highly expressed in the heart where it metabolizes endogenous substrates such as N-3/N-6 polyunsaturated fatty acids (PUFA) to produce lipid mediators involved in homeostasis and cardioprotective responses. Expanding our knowledge of the role CYP2J2 has within the heart is important for understanding its impact on cardiac health and disease. AREAS COVERED: The objective of this review was to assess the state of knowledge regarding cardiac CYP2J2. A literature search was conducted using PubMed-MEDLINE (from 2022 and earlier) to evaluate relevant studies regarding CYP2J2-mediated cardioprotection, small molecule modulators, effects of CYP2J2 substrates toward biologically relevant effects and implications of CYP2J2 polymorphisms and sexual dimorphism in the heart. EXPERT OPINION: Cardiac CYP2J2-mediated metabolism of endogenous and exogenous substrates have been shown to impact cardiac function. Identifying individual factors, like sex and age, that affect CYP2J2 require further elucidation to better understand CYP2J2's clinical relevance. Resolving the biological targets and activities of CYP2J2-derived PUFA metabolites will be necessary to safely target CYP2J2 and design novel analogues. Targeting CYP2J2 for therapeutic aims offers a potential novel approach to regulating cardiac homeostasis, drug metabolism and cardioprotection.

Inhibition of Cytochrome P450 2J2-Mediated Metabolism of Rivaroxaban and Arachidonic Acid by Ibrutinib and Osimertinib.

Covalent tyrosine kinase inhibitors (TKIs) ibrutinib and osimertinib are associated with cardiac arrhythmia. The interactions between these TKIs with CYP2J2 that is highly expressed in the human heart are unknown. In vitro metabolism experiments were performed to characterize CYP2J2-mediated metabolism of ibrutinib and osimertinib. Unbound distribution coefficient (Kpuu) for both TKIs was determined in AC16 cardiomyocytes. In vitro reversible and time-dependent CYP2J2 inhibition experiments were conducted with exogenous and endogenous substrates, namely rivaroxaban and arachidonic acid (AA), respectively, where kinetic parameters were estimated via one-site and multisite kinetic modeling. Ibrutinib was efficiently metabolized by CYP2J2 to a hydroxylated metabolite, M35, following substrate inhibition kinetics. Osimertinib is not a substrate of CYP2J2. Both TKIs depicted Kpuu values above 1 and equipotently inhibited CYP2J2-mediated hydroxylation of rivaroxaban in a concentration-dependent manner without time-dependency. The mode of reversible inhibition of CYP2J2-mediated metabolism of rivaroxaban and AA by osimertinib was described by Michaelis-Menten kinetics, whereas a two-site kinetic model recapitulated the atypical inhibitory kinetics of ibrutinib, assuming multiple substrate-binding domains within the CYP2J2 active site. The inhibition of ibrutinib and osimertinib on cardiac AA metabolism could be clinically significant considering the preferable distribution of both TKIs to cardiomyocytes with R cut-off values of 1.160 and 1.026, respectively. The dysregulation of CYP2J2-mediated metabolism of AA to cardioprotective epoxyeicosatrienoic acids by ibrutinib and osimertinib serves as a novel mechanism for TKI-induced cardiac arrhythmia. Mechanistic characterization of CYP2J2-mediated typical and atypical enzyme kinetics further illuminates the unique catalytic properties of CYP2J2. SIGNIFICANCE STATEMENT: We reported for the first time that ibrutinib is efficiently metabolized by CYP2J2. By using rivaroxaban and arachidonic acid (AA) as substrates, we characterized the typical and atypical inhibition kinetics of CYP2J2 by ibrutinib and osimertinib. The inhibition of both drugs on cardiac AA metabolism could be clinically significant considering their preferable distribution to cardiomyocytes. Our findings serve as a novel mechanism for drug-induced cardiac arrhythmia and shed insights into the multisite interactions between CYP2J2 and ligands.

Cytochrome P450 2J Genes Are Expressed in Dogs, Cats, and Pigs, and Encode Functional Drug-Metabolizing Enzymes.

Cytochrome P450s (P450s) have been identified and analyzed in dogs and pigs, species that are often used in preclinical drug studies. Moreover, P450s are clinically important for drug therapy not only in humans, but also in species under veterinary care, including dogs and cats. In the present study, seven P450s homologous to human CYP2J2, namely, dog CYP2J2; cat CYP2J2; and pig CYP2J33, CYP2J35, CYP2J91, and CYP2J93, were newly identified and characterized, along with pig CYP2J34 previously identified. The cDNAs of these CYP2Js contain open reading frames of 502 amino acids, except for CYP2J35 (498 amino acids), and share high sequence identity (77%-80%) with human CYP2J2. Phylogenetic analysis revealed that dog and cat CYP2J2 were closely related, whereas pig CYP2Js formed a cluster. All seven CYP2J genes contain nine coding exons and are located in corresponding genomic regions, with the pig CYP2J genes forming a gene cluster. These CYP2J2 mRNAs were predominantly expressed in the small intestine with additional expression in the kidney and brain for dog CYP2J2 and pig CYP2J91 mRNAs, respectively. All seven CYP2Js metabolized human CYP2J2 substrates terfenadine, ebastine, and astemizole, indicating that they are functional enzymes. Dog CYP2J2 and pig CYP2J34 and CYP2J35 efficiently catalyzed ebastine primary hydroxylation and secondary carebastine formation at low substrate concentrations, just as human CYP2J2 does. Velocity-versus-substate plots exhibited sigmoidal relationships for dog CYP2J2, cat CYP2J2, and pig CYP2J33, indicating allosteric interactions. These results suggest that dog, cat, and pig CYP2Js have similar functional characteristics to human CYP2J2, with slight differences in ebastine and astemizole oxidations. SIGNIFICANCE STATEMENT: Dog CYP2J2; cat CYP2J2; and pig CYP2J33, CYP2J34, CYP2J35, CYP2J91, and CYP2J93, homologous to human CYP2J2, were identified and characterized by sequence, phylogenetic, and genomic structure analyses. Intestinal expression patterns of CYP2J mRNAs were characteristic in dogs, cats, and pigs. Dog, cat, and pig CYP2Js likely play roles as drug-metabolizing enzymes in the small intestine, similar to human CYP2J2.