Anti-inflammatory properties of cytochrome P450 epoxygenase-derived eicosanoids

Abstract

The epoxyeicosatrienoic acids (EETs) are products of cytochrome P450 epoxygenases that have vasodilatory properties similar to that of endothelium-derived hyperpolarizing factor. The cytochrome P450 isoform CYP2J2 was cloned and identified as a potential source of EETs in human endothelial cells. Physiological concentrations of EETs or overexpression of CYP2J2 decreased cytokine-induced endothelial cell adhesion molecule expression, and EETs prevented leukocyte adhesion to the vascular wall by a mechanism involving inhibition of transcription factor NF-kappaB and IkappaB kinase. The inhibitory effects of EETs were independent of their membrane-hyperpolarizing effects, suggesting that these molecules play an important nonvasodilatory role in vascular inflammation.

Fig. 1

(A) Immunohistochemical localization of CYP2J2 in human coronary artery (arrows). Shown are photomicrographs of adjacent sections of one large (top panels; bar, 250 µm) and one small (bottom panels; bar, 100 µm) human coronary artery stained with anti-human CYP2J2 or preimmune IgG (11). (B) Reversed-phase HPLC chromatogram of metabolites generated during incubation of transfected endothelial cells with radiolabeled arachidonic acid (15). Endothelial cells were transfected with either empty vector (pcDNA3.1) or CYP2J2-containing expression vector (pcDNA3.1/CYP2J2). The retention times of authentic standards are indicated by bars above the respective peaks. (Top) Cells transfected with pcDNA3.1; (bottom) cells transfected with pcDNA3.1/CYP2J2.

Fig. 2

Cell-surface enzyme immunoassay. (A) The effects of cytochrome P450 epoxygenase-derived eicosanoids (100 nM) on endothelial cell VCAM-1 expression [measured in optical density (OD) units] in response to TNF-α (10 ng/ml), IL-1α (10 ng/ml), or LPS (10 ng/ml). (B) The effects of [11,12]-EET (100 nM) on unstimulated (control) or TNF-α (10 ng/ml)–stimulated VCAM-1, E-selectin, and ICAM-1 expression. The differences between treatment with TNF-α alone and in the presence of EETs were statistically significant (*P < 0.05, **P < 0.01). (C) Effect of [11,12]-EET (100 nM) or [14,15]-EET (100 nM) on bovine endothelial cells transiently transfected with a κB heterologous promoter construct (pκB.Luc). The κB promoter activity was determined relative to basal activity (*P < 0.01 versus TNF-α). (D) Effect of transfection with empty vector (pcDNA3.1) or CYP2J2 cDNA subcloned into pcDNA3.1 on TNF-α–induced VCAM-1 or heterologous κB promoter activity in the presence or absence of SKF-525A (100 µM) (*P < 0.01 versus TNF-α alone; **P < 0.05 versus TNF-α + CYP2J2).

Fig. 3

(A) Immunofluorescence studies showing the subcellular localization of NF-κB subunit Rel A in unstimulated endothelial cells (control) and endothelial cells stimulated with TNF-α (10 ng/ml, 15 min) in the presence or absence of [11,12]-EET (100 nM). Experiments were performed three times with similar results. Bar, 10 µm. (B) The fate of IκB-α after treatment with TNF-α (10 ng/ml, 15 min) with or without [11,12]-EET (100 nM), [14,15]-EET (100 nM), or 26S proteasome inhibitor, MG132 (10 µM). NS, non-specific bands. (C) IKK activity in unstimulated endothelial cells and endothelial cells stimulated with TNF-α (10 ng/ml, 15 min) with and without [11,12]-EET (100 nM), [5,6]-EET (100 nM), or [8,9]-EET (100 nM). In some experiments, [11,12]-EET (100 nM) was added directly to the IKK assay (in vitro). As a control for nonspecific phosphorylation, the IκB-α mutant (Ser³², Ser³⁶→T ) was used as substrate. The assay was performed three times with similar results.

Fig. 4

(A) Expression of endothelial VCAM-1 (arrows) in the murine carotid artery 5 hours after an intraperitoneal injection of saline (control) or TNF-α (10 µg/kg) in the presence or absence of continuous intra-arterial infusion of [11,12]-EET or [14,15]-EET (both at 100 ng/kg per minute for 5 hours) (25). Bar, 250 µm. Effect of EETs on TNF-α–induced U937 mononuclear cell (B) adhesion and (C) rolling in the murine carotid artery. The number of adherent and rolling cells per mm² of endothelium was measured after infusion of U937-labeled cells (20). Mononuclear cell adhesion or rolling was not detected in control mice. Mice were treated with TNF-α alone (□) and in the presence of [11,12]-EET (○), [14,15]-EET (∆), or a mAb (MK2) directed against VCAM-1 (■) (23). For mononuclear cell adhesion, threeseparate experiments yielded similar results with less than 10% variation (*P < 0.05 versus TNF-α alone).