8-11-14-eicosatrienoic-acid and Hyperhomocysteinemia

Hyperhomocysteinemia is a risk factor of atherogenesis. Soluble epoxide hydrolase (sEH) is a major enzyme that hydrolyzes epoxyeicosatrienoic acids and attenuates their cardiovascular protective effects. Whether homocysteine (Hcy) regulates sEH and the underlying mechanism remains elusive.. To elucidate the mechanism by which Hcy regulates sEH expression and endothelial activation in vitro and in vivo.. Hcy treatment in cultured human endothelial cells dose-dependently and time-dependently upregulated sEH mRNA and protein. Hcy increased the expression of adhesion molecules, which was markedly reversed by inhibiting sEH activity. Hcy-induced sEH upregulation is associated with activation of activating transcription factor-6 (ATF6). Bioinformatics analysis revealed a putative ATF6-binding motif in the promoter region of the sEH gene, which was found at a methylation site. Site-directed mutagenesis and chromatin immunoprecipitation assays demonstrated that Hcy treatment or ATF6 overexpression promoted ATF6 binding to the promoter of sEH and increased its activity. Results of methylation-specific polymerase chain reaction revealed that the ATF6 binding site on the sEH promoter was partially methylated and was demethylated with Hcy. SiRNA knockdown of ATF6α or SP1 blocked and ATF6 overexpression and DNA methyltransferase inhibitor mimicked the effect of homocysteine on sEH upregulation. In vivo, immunofluorescence assay revealed elevated expression of sEH and adhesion molecules in the aortic intima of mice with mild hyperhomocysteinemia, which was attenuated by sEH deletion or inhibition.. ATF6 activation and DNA demethylation may coordinately contribute to Hcy-induced sEH expression and endothelial activation. Inhibition of sEH may be a therapeutic approach for treating Hcy-induced cardiovascular diseases.

Topics: 8,11,14-Eicosatrienoic Acid; Activating Transcription Factor 6; Animals; Aorta; Base Sequence; DNA Methylation; Endothelial Cells; Epoxide Hydrolases; Gene Expression Regulation, Enzymologic; Homocysteine; Human Umbilical Vein Endothelial Cells; Humans; Hyperhomocysteinemia; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Promoter Regions, Genetic; Pyrenes; RNA, Small Interfering; Solubility; Up-Regulation

Hyperhomocysteinemia (HHcy) is associated with atherosclerotic events involving the modulation of arachidonic acid (AA) metabolism and the activation of matrix metalloproteinase-9 (MMP-9). Cytochrome P450 (CYP) epoxygenase-2J2 (CYP2J2) is abundant in the heart endothelium, and its AA metabolites epoxyeicosatrienoic acids (EETs) mitigates inflammation through NF-kappabeta. However, the underlying molecular mechanisms for MMP-9 regulation by CYP2J2 in HHcy remain obscure. We sought to determine the molecular mechanisms by which P450 epoxygenase gene transfection or EETs supplementation attenuate homocysteine (Hcy)-induced MMP-9 activation. CYP2J2 was over-expressed in mouse aortic endothelial cells (MAECs) by transfection with the pcDNA3.1/CYP2J2 vector. The effects of P450 epoxygenase transfection or exogenous supplementation of EETs on NF-kappabeta-mediated MMP-9 regulation were evaluated using Western blot, in-gel gelatin zymography, electromobility shift assay, immunocytochemistry. The result suggested that Hcy downregulated CYP2J2 protein expression and dephosphorylated PI3K-dependent AKT signal. Hcy induced the nuclear translocation of NF-kappabeta via downregulation of IKbetaalpha (endogenous cytoplasmic inhibitor of NF-kappabeta). Hcy induced MMP-9 activation by increasing NF-kappabeta-DNA binding. Moreover, P450 epoxygenase transfection or exogenous addition of 8,9-EET phosphorylated the AKT and attenuated Hcy-induced MMP-9 activation. This occurred, in part, by the inhibition of NF-kappabeta nuclear translocation, NF-kappabeta-DNA binding and activation of IKbetaalpha. The study unequivocally suggested the pivotal role of EETs in the modulation of Hcy/MMP-9 signal.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Cells, Cultured; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Enzyme Activation; Enzyme Induction; Homocysteine; Hyperhomocysteinemia; I-kappa B Proteins; Matrix Metalloproteinase 9; Mice; NF-KappaB Inhibitor alpha; Oxygenases; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Transcription Factor RelA; Transfection