In mouse arteries, Alox15 [leukocyte-type 12/15-lipoxygenase (LO)] is assumed to regulate vascular function by metabolizing arachidonic acid (AA) to dilator eicosanoids that mediate the endothelium-dependent relaxations to AA and acetylcholine (ACh). We used Alox15(-/-) mice, made by targeted disruption of the Alox15 gene, to characterize its role in the regulation of blood pressure and vascular tone. Systolic blood pressures did not differ between wild-type (WT) and Alox15(-/-) mice between 8-12 wk of age, but Alox15(-/-) mice exhibited resistance toward both N(G)-nitro-L-arginine-methyl ester (L-NAME)- and deoxycorticosterone acetate (DOCA)/high-salt-induced hypertension. ACh relaxed mesenteric arteries and abdominal aortas of WT and Alox15(-/-) mice to an identical extent. The LO inhibitor nordihydroguaiaretic acid attenuated the ACh relaxations by 35% in arteries from both WT and Alox15(-/-) mice. Reverse-phase HPLC analysis of [(14)C]AA metabolites in aorta and peritoneal macrophages (PM) revealed differences. Unlike PM, aorta tissue did not produce detectable amounts of 15-hydroxyeicosatetraenoic acid. Although Alox15 mRNA was detected in aorta, high-resolution gel electrophoresis with immunodetection revealed no Alox15 protein expression. Unlike aorta, Alox15 protein was detected in PM, intestine, fat, lung, spleen, and skin from WT, but not Alox15(-/-), mice. Injection of WT PM, a primary source of Alox15 protein, into Alox15(-/-) mice abolished their resistance toward L-NAME-induced hypertension. On the other hand, WT mice acquired resistance to L-NAME-induced hypertension after depletion of macrophages by clodronate injection. These studies indicate that Alox15 is involved in development of experimental hypertension by altering macrophage functions but not via synthesis of the vasoactive LO metabolites in mouse arteries.

Siangjong L, Gauthier KM, Pfister SL, Smyth EM, Campbell WB. Endothelial 12(S)-HETE vasorelaxation is mediated by thromboxane receptor inhibition in mouse mesenteric arteries. Am J Physiol Heart Circ Physiol 304: H382-H392, 2013. First published November 30, 2012; doi:10.1152/ajpheart.00690.2012.-Arachidonic acid (AA) metabolites mediate endothelium-dependent relaxation in many vascular beds. Previously, we identified the major AA 12/15-lipoxygenase (12/15-LO) metabolite of mouse arteries as 12-hydroxyeicosatetraenoic acid (12-HETE). The goal was to determine the stereospecific configuration of mouse vascular 12-HETE and characterize the role of 12-HETE stereoisomers in the regulation of vascular tone. Using normal, reverse phase, and chiral HPLC, the stereospecific configuration was identified as 12(S)-HETE. 12(S)-HETE relaxed U46619-, carbocyclic thromboxane A2-, PGF2␣-, and 8-iso PGF2␣-preconstricted mesenteric arteries, but not phenylephrine-preconstricted arteries. 12(R)-HETE was more potent than 12(S)-HETE in relaxing U46619-preconstricted mouse arteries (maximum relaxations ϭ 91.4 Ϯ 2.7% and 71.8 Ϯ 5.9%, respectively). Neither 12-HETE isomer caused constriction. Pretreatment with 12(S)-or 12(R)-HETE (1 M) inhibited constrictions to U46619 but not phenylephrine. To investigate the role of thromboxane A2 (TP) receptors in 12-HETE vascular actions, [3 H]SQ29548 radioligand binding studies were performed in mouse platelets. U46619, 12(R)-HETE, and 12(S)-HETE displaced [3 H]SQ29548 binding with IC50s of 0.07, 0.32, and 1.73 M, respectively. Both 12(S)-and 12(R)-HETE inhibited intracellular calcium increases induced by U46619 (10 nM) in HEK293 cells overexpressing TP ␣ receptor (65.5% and 45.1%, respectively) and coexpressing prostacyclin (IP) and TP ␣ receptors (58.0% and 27.1%, respectively). The LO inhibitor NDGA (10 M) reduced AA relaxations in arteries preconstricted with U46619 but not phenylephrine. These results indicate that exogenous and endogenous 12(S)-HETE relax mouse mesenteric arteries that are preconstricted with thromboxane agonists. These 12(S)-HETE relaxations are mediated by TP receptor competitive inhibition and inhibition of TP agonistinduced increases in intracellular calcium.12-HETE; arachidonic acid; lipoxygenase; thromboxane receptors; vasorelaxation THE DYNAMIC REGULATION of vascular relaxation and constriction is extremely important for the regulation of tissue blood flow. Disruption of this regulation occurs in many cardiovascular diseases. The vascular endothelium plays a central role in the regulation of vascular tone by producing a variety of dilator and constrictor substances. Three major dilator mediators are nitric oxide (NO), prostaglandin I 2 (PGI 2 ), and a group of compounds called endothelium-derived hyperpolarizing factors (EDHFs) (9,13,19). PGI 2 , an arachidonic acid (AA) cyclooxygenase (COX) metabolite, was the first endothelial relaxing factor discovered (19). In addition to PGI 2 , AA is also metabolized by cytochrome P450 (CYP450) to epoxyeicosatrienoic acids (EETs...

Aim 12/15-lipoxygenase (12/15-LO) metabolizes arachidonic acid (AA) into several vasoactive eicosanoids. In mouse arteries, we previously characterized the enzyme’s 15-LO metabolites 12(S)-hydroxyeicosatetraenoic acid (HETE), 15-HETE, hydroxyepoxyeicosatrienoic acids (HEETAs) and 11,12,15-trihydroxyeicosatrienoic acids (11,12,15-THETAs) as endothelium-derived relaxing factors. However, the observed 12-LO metabolites remained uncharacterized. The purpose of this study was to determine the structure and biological functions of eicosanoids generated by the enzyme’s 12-LO activity. Methods Metabolites extracted from aortas of C57BL/6 male mice were separated using a series of reverse and normal phase chromatographic steps and identified as hepoxilin A3, trioxilin A3 and trioxilin C3 by mass-spectrometry. Activities of these natural compounds were tested on isometric tension and intracellular calcium release. The role of thromboxane (TP) receptor was determined in HEK293 cells overexpressing TPα receptor (TPα-HEK). Results All identified vascular 12-LO metabolites were biologically active. In mouse mesenteric arteries, trioxilin A3, C3 and hepoxilin A3 (3μM) relaxed arteries constricted with the thromboxane mimetic, U46619 (maximum relaxations of 78.9±3.2, 29.7±4.6, 82.2±5.0 and 88.0±2.4% respectively), but not phenylephrine-constricted arteries. In TPα-HEK cells, trioxilin A3, C3 and hepoxilin A3 (10μM) inhibited U46619 (10nM)-induced increases in intracellular calcium by 53.0±7.2%, 32.8±5.0% and 37.9±13.5%, respectively. In contrast, trioxilin B3 and hepoxilin B3 were not synthesized in arteries and exhibited little biological activity. Conclusion Trioxilin A3 and C3 and hepoxilin A3 are endogenous vascular relaxing factors. They are not endothelium-derived hyperpolarizing factors but mediate vascular relaxation by inhibiting TP agonist induced increases in intracellular calcium. Thus, they regulate vascular homeostasis by acting as endogenous TP antagonists.

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 [14C]-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 expression was most abundant in 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.