Date Published: March 22, 2017
Publisher: Public Library of Science
Author(s): Ahmad Hanif, Matthew L. Edin, Darryl C. Zeldin, Christophe Morisseau, John R. Falck, Mohammed A. Nayeem, John Calvert.
Arachidonic acid is metabolized to epoxyeicosatrienoic acids (EETs) by cytochrome (CYP) P450 epoxygenases, and to ω-terminal hydroxyeicosatetraenoic acids (HETEs) by ω-hydroxylases. EETs and HETEs often have opposite biologic effects; EETs are vasodilatory and protect against ischemia/reperfusion injury, while ω-terminal HETEs are vasoconstrictive and cause vascular dysfunction. Other oxylipins, such as epoxyoctadecaenoic acids (EpOMEs), hydroxyoctadecadienoic acids (HODEs), and prostanoids also have varied vascular effects. Post-ischemic vasodilation in the heart, known as coronary reactive hyperemia (CRH), protects against potential damage to the heart muscle caused by ischemia. The relationship among CRH response to ischemia, in mice with altered levels of CYP2J epoxygenases has not yet been investigated. Therefore, we evaluated the effect of endothelial overexpression of the human cytochrome P450 epoxygenase CYP2J2 in mice (Tie2-CYP2J2 Tr) on oxylipin profiles and CRH. Additionally, we evaluated the effect of pharmacologic inhibition of CYP-epoxygenases and inhibition of ω-hydroxylases on CRH. We hypothesized that CRH would be enhanced in isolated mouse hearts with vascular endothelial overexpression of human CYP2J2 through modulation of oxylipin profiles. Similarly, we expected that inhibition of CYP-epoxygenases would reduce CRH, whereas inhibition of ω-hydroxylases would enhance CRH. Compared to WT mice, Tie2-CYP2J2 Tr mice had enhanced CRH, including repayment volume, repayment duration, and repayment/debt ratio (P < 0.05). Similarly, inhibition of ω-hydroxylases increased repayment volume and repayment duration, in Tie2-CYP2J2 Tr compared to WT mice (P < 0.05). Endothelial overexpression of CYP2J2 significantly changed oxylipin profiles, including increased EETs (P < 0.05), increased EpOMEs (P < 0.05), and decreased 8-iso-PGF2α (P < 0.05). Inhibition of CYP epoxygenases with MS-PPOH attenuated CRH (P < 0.05). Ischemia caused a decrease in mid-chain HETEs (5-, 11-, 12-, 15-HETEs P < 0.05) and HODEs (P < 0.05). These data demonstrate that vascular endothelial overexpression of CYP2J2, through changing the oxylipin profiles, enhances CRH. Inhibition of CYP epoxygenases decreases CRH, whereas inhibition of ω-hydroxylases enhances CRH.
Arachidonic acid (AA) can be metabolized to epoxyeicosatrienoic acids (EETs) by cytochrome P450 (CYP) epoxygenases, primarily of the CYP2C and CYP2J subfamilies. In a parallel pathway, CYP ω-hydroxylases, such as CYP4A, hydroxylate AA to ω-terminal HETEs (hydroxyeicosatetraenoic acids), including the potent vasoconstrictor 20-HETE . CYP epoxygenases can generate four distinct EET regioisomers: 5,6-, 8,9-, 11,12- and 14,15-EET. EETs are involved in numerous biological functions, including hyperpolarization and relaxation of vascular smooth muscle cells [2, 3]. Mouse cardiomyocytes with increased EET generation are protected against ischemia/reperfusion injury [4, 5]. EETs have short half lives, mainly due to their conversion to dihydroxyeicosatrienoic acids (DHETs)  by soluble epoxide hydrolase (sEH). Different strategies have been used to experimentally overcome this shortcoming to assess the beneficial effects of EETs, including endothelial overexpression of CYP epoxygenases, such as CYP2J2 [7, 8]. While humans have just one CYP2J enzyme, CYP2J2, mice express 7 functional CYP2J proteins . CYP2J2 is expressed in different vascular tissues, including the heart muscle and coronary arteries [4, 9]. Isolated aortic endothelial cells from Tie2-CYP2J2 Tr mice had 30% higher production of 11,12- and 14,15-EETs into culture medium . In male mice, endothelial overexpression of human CYP2J2 (Tie2-CYP2J2 Tr) enhanced blood flow and suppressed inflammation to protect against experimental cerebral ischemia .
While CYP2J2 overexpression has shown beneficial effects on vasodilation, inflammation, and contractile recovery from cardiac ischemia, the relevance of CYP2J2 metabolism in CRH had not been previously explored. The signaling mechanisms involved in CRH may be substantially different from the mechanisms which improve cardiac function or chronic hypertension in other models. Herein, we investigated the role of endothelial-specific CYP2J2 overexpression and the accompanying oxylipin changes in the modulation of CRH using isolated WT and Tie2-CYP2J2 Tr mouse hearts. Our data demonstrated that: 1) Endothelial overexpression of CYP2J2 was associated with changes in some oxylipin profiles, including increase in EETs, EpOMEs, and some mid-chain HETEs; 2) Brief ischemia caused changes in some oxylipin profiles, including decrease in mid-chain HETEs, HODEs, and prostanoids; 3) Endothelial overexpression of CYP2J2 increased CRH; 4) Inhibition of CYP epoxygenases (by MS-PPOH) attenuated CRH in WT and Tie2-CYP2J2 Tr mice; and 5) Inhibition of ω-hydroxylases (by DDMS) enhanced CRH in WT and Tie2-CYP2J2 Tr mice.