Effect of human 15-lipoxygenase-1 metabolites on vascular function in mouse mesenteric arteries and hearts

Abstract

Lipoxygenases regulate vascular function by metabolizing arachidonic acid (AA) to dilator eicosanoids. Previously, we showed that endothelium-targeted adenoviral vector-mediated gene transfer of the human 15-lipoxygenase-1 (h15-LO-1) enhances arterial relaxation through the production of vasodilatory hydroxyepoxyeicosatrienoic acid (HEETA) and trihydroxyeicosatrienoic acid (THETA) metabolites. To further define this function, a transgenic (Tg) mouse line that overexpresses h15-LO-1 was studied. Western blot, immunohistochemistry and RT-PCR results confirmed expression of 15-LO-1 transgene in tissues, especially high quantity in coronary arterial wall, of Tg mice. Reverse-phase HPLC analysis of [(14)C]-AA metabolites in heart tissues revealed enhanced 15-HETE synthesis in Tg vs. WT mice. Among the 15-LO-1 metabolites, 15-HETE, erythro-13-H-14,15-EETA, and 11(R),12(S),15(S)-THETA relaxed the mouse mesenteric arteries to the greatest extent. The presence of h15-LO-1 increased acetylcholine- and AA-mediated relaxation in mesenteric arteries of Tg mice compared to WT mice. 15-LO-1 was most abundant in the heart; therefore, we used the Langendorff heart model to test the hypothesis that elevated 15-LO-1 levels would increase coronary flow following a short ischemia episode. Both peak flow and excess flow of reperfused hearts were significantly elevated in hearts from Tg compared to WT mice being 2.03 and 3.22 times greater, respectively. These results indicate that h15-LO-1-derived metabolites are highly vasoactive and may play a critical role in regulating coronary blood flow.

Keywords: 15-Lipoxygenase; AA; ACh; Coronary flow; EDHFs; Eicosanoids; Endothelium-derived hyperpolarizing factor; HEETAs; HETEs; HPETEs; Indo; Ischemia/reperfusion; LOs; NDGA; Reactive hyperemia; THETAs; Vasodilation; acetylcholine; arachidonic acid; endothelium-derived hyperpolarizing factors; hydroperoxyeicosatetraenoic acids; hydroxy-epoxyeicosatrienoic acids; indomethacin; l-NA; lipoxygenases; monohydroperoxyeicosatetraenoic acids; nitro-l-arginine; nordihydroguaiaretic acid; sEH; soluble epoxide hydrolase; trihydroxyeicosatrienoic acids.

Figure 1

Analysis of h15-LO-1 expression in tissues from WT and h15-LO-1 Tg mice. Panel A shows h15-LO-1 protein expression in aorta, lung, trachea, and heart tissues of WT and h15-LO-1 Tg mice as measured by western immunoblot. 50 μg total protein were loaded per lane. β-Actin was used as a loading control. Representative blots of 3 experiments are shown. Panel B shows h15-LO-1 mRNA expression in aorta, lung, trachea, and heart tissues of WT and h15-LO-1 Tg mice as measured by RT-PCR. Murine s18 was used as a reference gene. Representative gels of three experiments are shown. Panel C shows the expression pattern of h15-LO-1 protein in aorta and heart samples of WT and h15-LO-1 Tg mice that were subjected to immunohistochemical staining with an anti-h15-LO-1 antibody. Control samples were stained with only secondary (goat anti-guinea-pig IgG) antibody. Asterisks are marking the lumens of coronary arteries. Representative areas of 5 slides are shown.

Figure 2

Metabolism of [¹⁴C]-arachidonic acid (AA) by aorta and heart tissues of WT and h15-LO-1 Tg mice. Aortic rings from WT (A) or h15-LO-1 Tg (B) mice and myocardial tissue from WT (C) or h15-LO-1 Tg (D) mice were incubated with [¹⁴C^]-AA in the presence of 10 μM Indo, 30 μM L-NA and 20 μM of A23187. Metabolites were extracted and resolved by RP-HPLC. Migration times of known standards are indicated in each panel. Traces are representative of at least 3 experiments.

Figure 3

Relaxations of U46619-preconstricted mesenteric arteries from WT mice by 15-LO metabolites of arachidonic acid. Panel A: relaxation to erythro- or threo-13-H-14,15-EETA. Panel B: relaxation to trans- or cis-15(S)-H-11,12-EETA. Panel C: relaxation to 11(R),12(S),15(S)-THETA or 15(S)-HETE. Arteries were pretreated with 30 μM L-NA and 10 μM Indo to block the NO and COX pathways. Each value represents the mean±SEM, n=8.

Figure 4

Effect of h15-LO-1 overexpression on vascular relaxation and susceptibility to experimental hypertension. (A) acetylcholine (ACh) and (B) arachidonic acid (AA) relaxations of mouse mesenteric arteries from WT and h15-LO-1 Tg mice. The arteries were pretreated with 30 μM L-NA and 10 μM Indo to block the NO and COX pathways and preconstricted with U46619. Systolic blood pressures of WT and h15-LO-1 Tg male (C) and female (D) mice under basal conditions or L-NAME-induced hypertension. Each value represents the mean±SEM, n=6–8. * p<0.05, ** p<0.01, compared to control.

Figure 5

Reactive hyperemic responses of WT and h15-LO-1 Tg hearts as measured in Langendorff perfusion model. (A) Representative traces of coronary flow are shown with a 40 second period of ischemia. Average peak flow (B) and excess flow (C) values were used for quantitative analysis. (D) Summary of cardiac function (left ventricular developed pressure (LVDP) or maximum and minimum rate of change in left ventricular developed pressure (dP/dt max and dP/dt min)) during pre- and post-ischemic phases of reactive hyperemia. Values represent mean±SEM, n=11. * p<0.01.