Cytochrome P450 Isoform Expression in Human Vascular Endothelial Cells
Epoxy derivatives of arachidonic acid may act as important autocrine and paracrine mediators of endothelial function including regulation of vascular tone and control of inflammation. To identify potential candidates for catalyzing the synthesis of these and further arachidonic acid metabolites, we studied human vascular endothelial cells for the expression of individual cytochrome P450 isoforms belonging to the CYP families 1, 2, 3 and 4. An RT-PCR screening performed with subfamily- and isoform-specific primer pairs revealed mRNAs for the P450 forms 1A1, 1B1, 2C8, 2E1, 2J2, 3A7, 4A11 and 4F2. The identity of the RT-PCR products was confirmed by DNA sequencing. In addition, P450 1A2 mRNA was detected after induction with β-naphthoflavone which also enhanced the expresion of P450s 1A1 and 1B1. P450s 2B6 and 3A4 were not detectable. Similar P450 isoform patterns were obtained analyzing primary human endothelial cells originating from aorta, coronary arteries, dermal microvessels and umbilical veins, as well as an immortalized human endothelial cell line (HMEC-1). Further studies with HMEC-1 cells showed the expression of all human members of the P450 2C subfamily (2C8, 2C9, 2C18 and 2C19). We next used gaschromatography-mass spectrometry to identify the regioisomeric epoxeicosatrienoic acids produced by HMEC-1 cells. Among the P450 forms detected by the RT-PCR screening, P450 2C8 and 2J2 are the leading candidates for producing vasoactive epoxyeicosatrienoic acids. Using recombinant human P450 1A1, we then found that this P450 form catalyzes the formation of various regioisomeric hydroxy derivatives of arachidonic acid. We conclude that P450 1A1 known primarily for its role in polycyclic aromatic hydrocarbon metabolism, may interfere with endothelial arachidonic acid metabolism, particularly after its induction by drugs and xenobiotics. Furthermore, P450s 4A11 and 4F2 probably contribute to the degradation of lipid mediators of inflammation.