Human Umbilical Vein Endothelial Cells Express P450 2C8 mRNA: Cloning of Endothelial P450 Epoxygenase

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Abstract
Human umbilical vein endothelial cells (EC) metabolize arachidonic acid (AA) through three major pathways—cyclooxygenase, lipoxygenase and cytochrome P450 (P450) isozymes. Previously, we have shown that pathophysiological concentrations of native low density lipoprotein (n-LDL) increase EC P450-dependent epoxyeicosatrienoic acid (EET) production. The present study was designed to identify putative P450 isozymes involved in EC epoxidation of AA. Reverse transcription—polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) were employed to detect P450 2 family cDNA from EC mRNA. Degenerate primers complimenting 2 homologous regions from 6 different P450 2 families were designed to capture a 440 bp cDNA fragment corresponding to the heme binding region of P450 2 isozymes. RT-PCR of EC total RNA with these primers amplified a 440 bp fragment. After gel purification, the fragment was cloned, sequenced and found to share a high degree of identity with human liver P450 2C8 and 2C9. New primers were designed based on the nucleotide sequences of the 440 bp fragment and a third homologous upstream region from the 440 bp fragment. RT-PCR was used to capture 5′ sequences upstream from the 440 bp fragment and lock-docking 3′-RACE was used to capture sequences downstream from the 440 bp fragment. These steps successfully expanded the number of nucleotides captured and sequenced to 1.4 kb. The partial clone was a homologous to human liver P450 2C8, sharing a >99.8% identity, and >86.1% with P450 2C9. When sequence analysis of independently cloned 440 bp fragments was performed, no other P450 2 isozymes were detected. To determine if P450 2C isozyme family members were capable of AA epoxidation, ECV-304 cells, which usually generate low levels of EETs were transfected with pcDNA3 plasmids containing DNA encoding functional P450 2C9. Transient P450 2C9 transfection of this eternal endothelial cell line increased EET production levels up to 4-fold, which was similar in magnitude to EET production by EC exposed to native low density lipoprotein (n-LDL). These data indicate that EC epoxidize AA by a P450 2C8 isozyme.