Basal and inducible anti-inflammatory epoxygenase activity in endothelial cells

Abstract

The roles of CYP lipid-metabolizing pathways in endothelial cells are poorly understood. Human endothelial cells expressed CYP2J2 and soluble epoxide hydrolase (sEH) mRNA and protein. The TLR-4 agonist LPS (1 μg/ml; 24 h) induced CYP2J2 but not sEH mRNA and protein. LC-MS/MS analysis of the stable commonly used human endothelial cell line EA.Hy926 showed active epoxygenase and epoxide hydrolase activity: with arachidonic acid (stable epoxide products 5,6-DHET, and 14,15-DHET), linoleic acid (9,10-EPOME and 12,13-EPOME and their stable epoxide hydrolase products 9,10-DHOME and 12,13-DHOME), docosahexaenoic acid (stable epoxide hydrolase product 19,20-DiHDPA) and eicosapentaenoic acid (stable epoxide hydrolase product 17,18-DHET) being formed. Inhibition of epoxygenases using either SKF525A or MS-PPOH induced TNFα release, but did not affect LPS, IL-1β, or phorbol-12-myristate-13-acetate (PMA)-induced TNFα release. In contrast, inhibition of soluble epoxide hydrolase by AUDA or TPPU inhibited basal, LPS, IL-1β and PMA induced TNFα release, and LPS-induced NFκB p65 nuclear translocation. In conclusion, human endothelial cells contain a TLR-4 regulated epoxygenase CYP2J2 and metabolize linoleic acid>eicosapentaenoic acid > arachidonic acid>docosahexaenoic acid to products with anti-inflammatory activity.

Keywords: Endothelial; Epoxygenase; Soluble epoxide hydrolase; TNFα.

Supplementary Figure 1

Supplementary figure.

Supplementary Figure 2

Supplementary figure.

Fig. 1

Expression and regulation of CYP2J2 and sEH by LPS in human endothelial cells. (A) qRT-PCR (4 h) and (B) immunofluorescent analysis (24 h) of CYP2J2 and sEH expression in EA.Hy926 treated with LPS (1 μg/ml). qRT-PCR data represent relative expression compared to GADPH as mean ± SEM from n = 4 separate experiments. (C) qRT-PCR (4 h) of human blood out growth endothelial cells and (D) immunofluorescent analysis (24 h) of CYP2J2 in human primary aortic endothelial cells treated with LPS (1 μg/ml). qRT-PCR data represent relative expression compared to GADPH as mean ± SEM from n = 3 separate donors. ^∗ indicates p < 0.05 by one-sample t-test between control and LPS treatment. Immunofluorescent micrographs are representative of n = 3 experiments. As a negative control, in some experiments primary antibody was omitted (2^° Ab) which showed no specific staining.

Fig. 2

Oxylipin product profile of control and LPS stimulated EA.Hy926 endothelial cells. LC/MS/MS analysis of the (A) epoxygenase, (B) lipoxygenase and (C) cyclo-oxygenase products release (pg/ml) from EA.Hy926 untreated or treated with LPS (1 μg/ml) for 24 h. Data represents the mean ± SEM from n = 3. ^∗ indicates p < 0.05 for each analyte by unpaired t-test between control and LPS treatment.

Fig. 3

Epoxygenase inhibition induces TNFα release from EAHy926 endothelial cells. (A) Fold TNFα release (7 h) from EA.Hy926 treated with vehicle control (0.01% DMSO), or epoxygenase inhibitor, MS-PPOH (10 μM) or SKF525A (10 μM). Data represents mean ± SEM from n = 7 replicates from 4 separate experiments. ^∗ indicates p < 0.05 by one-sample t-test between control and epoxygenase inhibitors. (B) Fold TNFα release (7 h) from EA.Hy926 treated with vehicle control (0.01% DMSO), 5 nM PMA, 1 μg/ml LPS or 10 ng/ml IL-1β in the presence of absence of 10 μM MS-PPOH. Data represents mean ± SEM from n = 5 replicates from 4 separate experiments. ^∗ indicates p < 0.05 by one-sample t-test between control and inflammatory stimuli inhibitors. ^† indicates p < 0.05 by one-sample t-test between treatment in presence or absence of MS-PPOH.

Fig. 4

sEH inhibition limits basal and inflammation induced TNFα release from EAHy926 endothelial cells. (A) TNFα release (fold from control; 7 h) from EA.Hy926 treated with vehicle control (0.01% DMSO), 5 nM PMA, 1 μg/ml LPS or 10 ng/ml IL-1β in the presence of absence of the sEH inhibitor AUDA (10 μM). Data represents mean ± SEM from n = 8 replicates from 4 separate experiments. ^∗ indicates p < 0.05 by paired t-test between treatment in presence or absence of AUDA. (B) EA.Hy926 were treated with LPS (1 μg/ml; 2 h) to induce p65 nuclear localization. Scale bar 50 μ. LPS induced p65 nuclear localization is absent or strongly diminished in cells treated with sEH inhibitors TPPU (1 μM; middle panels) or AUDA (10 μM; lower panels). 2nd Ab control shows immunofluorescence levels when primary antibody was omitted. Each panel was enhanced by 20% brightness and 20% contrast in PowerPoint to aid visualization. Data is representative of n = 3 experiments.