Abstract 650: Adenoviral Expression of 15-Lipoxygenase-1 in Rabbit Aortic Endothelium: Role in Arachidonic Acid-Induced Relaxation
Endothelium-dependent vasorelaxation of rabbit aorta is mediated by arachidonic acid (AA) metabolites from cyclooxygenase (COX) and 15-lipoxygenase (15-LO) pathways or by endothelial nitric-oxide synthase (eNOS) pathway. The major 15-LO-1 metabolites of AA, 11,12,15-trihydroxyeicosatrienoic acid (THETA) and 15-hydroxy-11,12-epoxyeicosatrienoic acid (HEETA), caused concentration-dependent relaxation. We tested the hypothesis that over-expression of 15-LO-1 in rabbit aorta increases vascular relaxation mediated by HEETA and THETA. Aorta from 6 weeks old rabbits and cultured rabbit aortic endothelial cells (RAECs) were treated with adenoviruses containing human 15-LO-1 (Ad-15-LO-1) or β-galactosidase (Ad-β-Gal). When transduced with 3 X 108 pfu of Ad-15-LO-1, an increased expression of a 75kD protein in RAEC (13 ± 1%) and rabbit aorta (158 ± 5%) was observed by immunoblotting with an anti-human 15-LO-1 antibody. Immunohistochemical studies on Ad-15-LO-1-transduced rabbit aorta showed the presence of 15-LO-1 in ECs. When Ad-15-LO-1-transduced RAEC and rabbit aorta were incubated with [14C]AA and analyzed by high performance liquid chromatography, there was increased production of HEETA and THETA compared with that of Ad-β-Gal or control RAEC and rabbit aorta. Liquid chromatography tandem-mass spectometric analysis was performed to identify 11,12,15-THETA (Major ions MS2; 197,167,139,127,113,71 m/z) and 11,14,15-THETA (Major ion MS2; 235,167,85,56 m/z) in Ad-15-LO-1 transduced aorta incubated with AA. In presence of COX and eNOS inhibitors, Ad-15-LO-1-treated aortic rings showed enhanced relaxation to AA (max 31.7 ± 3.2 %) as compared to Ad-β-Gal-treated (max 12.7 ± 3.2 %) or control non-treated rings (max 13.1 ± 1.6%) (p <0.01). In conclusion, these data indicate that over-expression of 15-LO-1 in the rabbit aortic vascular endothelium significantly increases the production of the vasodilatory HEETA and THETA and enhances the relaxations to AA. This confirms the role of HEETA and THETA as endothelium-derived relaxing factors.