Cytochrome P450 Epoxygenase Metabolism Of Arachidonic Acid Attenuates Endothelial Cell Apoptosis Induced By LPS

Background:The epoxyeicosatrienoic acids (EETs) are cytochrome P450 (CYP) epoxygenase arachidonic acid (AA) metabolites. AA is present in vivo in the form esterified to glycerophospholipids inside of cell membrane. Activation of phospholipases (e.g. cytosolic phospholipase A2) releases free AA from the phospholipid (PL) pools and makes it available for oxidative metabolism by several enzyme systems. The PGHSs and LOXs have been extensively studied, and the"third pathway"of AA metabolism was found, wherein multiple cytochrome P450 epoxygenases metabolize AA to four epoxyeicosatrienoic acid regiosomes: 5,6-EET, 8,9-EET, 11,12-EET and 14,15-EET. EETs have vasodilatory properties similar to that of nitrogen monoxidum(NO)and prostacyclin(PGI2). Early studies have demonstrated that EETs could make smooth muscle cell (SMC) hyperpolarized through opening Ca2+-sensitive potassium channel(KCa2+), so EETs was proposed to be endothelial derived hyperpolarizing factors (EDHFs). EETs have recently been shown to play important nonvasodilatory roles within the vasculature, for example, physiological concentrations (100nM) of EETs or overexpression of human CYP2J2 decreased cytokine-induced endothelial cell adhesion molecule expression. In recent years, mang studies have already shown that EETs could inhibit LLCPKcl4 cell apoptosis induced by H2O2, etoposide etc. However, it remains elucidated whether EETs can protect bovine aortic endothelial cells (BAECs) against apoptosis induced by LPS. In this study, therefore, we investigated the effects of endogenous and exogenous EETs on apoptosis of BAECs induced by LPS and the relevant signaling mechanisms involved.Methods: CYP expoxygenase genes, CYP2C11OR, CYP2J2 and CYPF87V were transfected into cultured BAECs for 1 weeks using recombinant adeno-associated viral vector (rAAV), or EETs were added into the culture medium directly. BAECs were treated with LPS (100 ng/ml) or pretreated with mitogenactivated protein kinase kinase inhibitor PD98059, mitogenactivated protein kinase inhibitor Apigenin, phosphatidylinositol-3 (PI-3) kinase inhibitor LY294002 and inhibitor of AKT. Apoptosis was detected by morphological observations (Acridine Orange/Ethidium Bromide staining), flow cytometry assay and Caspase3 activity assay. As well the level of bcl-2, PI-3K, phospho-Akt, mitogen-activated protein kinase (MAPK) phospho-P38 and the phospho-ERK1/2 were probed using corresponding antibodies by Western blots. Results: LPS treatment significantly induced BAEC apoptosis, but incubation with 8,9-, 11,12-, and 14,15-EET markedly attenuated LPS-induced BAEC apoptosis (37.03%±3.014%, 33.45%±7.918% and 35.75%±7.858%, respectively) compared with vehicle control (47.33%±3.154%). Also CYP epoxygenase genes attenuated LPS-induced BAEC apoptosis and significantly reduced the ratios of apoptic BAECs. Moreover, addition of CYP epoxygenase inhibitor 17-ODYA reversed antiapoptic effects of epoxygenase gene overexpressions. Furthermore, in BAECs pretreated with EET or transfected with cytochrome P450 epoxygenase, the expression of Bcl-2, PI-3K, phosphorylation of ERK1/2, phosphorylation of Akt were significantly increased compared with that of controls, in contrast, phosphorylation of P38 were less. The antiapoptotic effects of EETs were partially blocked by mitogen activated protein kinase kinase inhibitor PD98059, mitogenactivated protein kinase inhibitor Apigenin, PI-3K inhibitor LY294002 and inhibitor of Akt.Conclusion: LPS induced apoptosis of the cultured endothelial cells and EETs as well as forced overexpression of epoxygenase genes can inhibits LPS-induced endothelial apoptosis partially by activation of MAPK and PI-3K/Akt pathways, which may reduce degradation of bcl-2 and activation of caspase-3. These results suggest that CYP epoxygenases may protect cardiovascular system from inflammatory injury and atherosclerosis.