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-κB and IκB 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.

Background-Cytochrome P450 (CYP) 2J2 is expressed in the vascular endothelium and metabolizes arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs). The EETs are potent endogenous vasodilators and inhibitors of vascular inflammation. However, it is not known whether genetic polymorphisms of CYP2J2 are associated with increased cardiovascular risks. Methods and Results-All 9 exons of the CYP2J2 gene and its proximal promoter were sequenced in 132 patients to identify potential variants. Functional consequence of a single nucleotide polymorphism (SNP) in the promoter of CYP2J2 was further evaluated by use of transcription factor-binding and reporter assays. A total of 17 polymorphisms were identified. One of the most relevant polymorphisms in terms of frequency and functional importance is located at Ϫ50 (G-50T) in the proximal promoter of CYP2J2. Screening of 289 patients with coronary artery disease and 255 control subjects revealed 77 individuals with the G-50T SNP (17.3% of coronary artery disease patients, 10.6% of control subjects; Pϭ0.026). The association of the G-50T polymorphism remained significant after adjustment for age, gender, and conventional cardiovascular risk factors (OR, 2.23; 95% CI, 1.04 to 4.79). The G-50T mutation resulted in the loss of binding of the Sp1 transcription factor to the CYP2J2 promoter and resulted in a 48.1Ϯ2.4% decrease in CYP2J2 promoter activity (PϽ0.01). Plasma concentrations of stable EET metabolites were significantly lower in individuals with the G-50T SNP. Conclusions-A functionally relevant polymorphism of the CYP2J2 gene is independently associated with an increased risk of coronary artery disease.

The adhesion of circulating leukocytes to the vessel wall is an initiating event in atherogenesis and vascular inflammation (1, 2). Under certain conditions, the "activated" endothelium expresses cell surface adhesion molecules which mediate specific interactions between the endothelium and circulating leukocytes (3, 4). Factors that affect the induction of endothelial cell adhesion molecules such as vascular cell adhesion molecule (VCAM-1) 1 therefore may be important in regulating vascular inflammatory processes. Recent studies suggest that the activation of the pleotropic transcription factor nuclear factor-B (NF-B) is required for the transcriptional induction of endothelial cell adhesion molecules (5).The activation of NF-B involves the degradation of its cytoplasmic inhibitor, IB (6, 7). Presently, five distinct IB proteins have been shown to functionally retain NF-B in the cytoplasm and render it inactive (6). These IB proteins contain ankyrin repeat motifs that mask the nuclear localization sequence of NF-B subunits such as RelA (p65), c-Rel, and RelB (8, 9). Of the different IB proteins, the best described is IB␣. Following cytokine stimulation, IB␣ is phosphorylated by a novel ubiquitinated serine kinase (10). Phosphorylation of IB␣ targets the IB␣ for ubiquitination and rapid degradation by 26 S proteasomes (10, 11). The degradation of IB␣ then allows the unbound NF-B to translocate into the nucleus, where it can transactivate the enhancer elements of many proinflammatory genes (5).Modulation of IB␣ function and expression has been shown to regulate NF-B activation. For example, the phosphorylation of IB␣ is a key regulatory step in the activation of NF-B (11, 12). Indeed, recent studies indicate that salicylates and antioxidants inhibit NF-B and endothelial cell activation by preventing IB␣ phosphorylation and subsequent degradation (13-16). Alternatively, the nuclear accumulation of IB␣ resulting from overexpression of IB␣ or following stimulation with tumor necrosis factor (TNF)-␣ has been shown to displace NF-B from its cognate DNA and terminate NF-B-mediated transcriptional activity (17,18). Finally, some of the anti-inflammatory effects of glucocorticoids may be mediated through their inhibitory effects on NF-B, since glucocorticoids are known to induce IB␣ expression (19,20).We and others have shown that nitric oxide can inhibit NF-B and endothelial cell activation through non-cGMP-dependent mechanisms (21,22). Although NO donors appear to "stabilize" the NF-B-IB␣ heterotrimeric complex (23), the possibility that newly synthesized IB␣ could account for this stabilization has not been excluded. Furthermore, it is not known whether the induction of IB␣ expression by NO donors could actually lead to an increase in cytoplasmic or nuclear IB␣ protein levels. The purpose of this study, therefore, is to determine the mechanism(s) by which NO inhibits of NF-B activation and VCAM-1 expression in terms of its effects on IB␣ phosphorylation, expression, and nuclear accumulation.

Certain statins which are substrates of the CYP3A4 isoform competitively inhibit the metabolic activation of clopidogrel. As a result the relative clopidogrel induced platelet inhibition (P-selectin-expression) is diminished--but still there is a relative clopidogrel effect of more than 80% in the maintenance phase. It may be reasonable to test the therapeutic efficacy of clopidogrel in those patients who require long-term treatment.