Elevated endothelial nitric oxide bioactivity and resistance to angiotensin-dependent hypertension in 12/15-lipoxygenase knockout mice

Abstract

12/15-Lipoxygenase (12/15-LOX) plays a pathogenic role in atherosclerosis. To characterize whether 12/15-LOX also contributes to endothelial dysfunction and hypertension, regulation of vessel tone and angiotensin II (ang II) responses were characterized in mice deficient in 12/15-LOX. There was a twofold increase in the magnitude of l-nitroarginine-methyl ester-inhibitable, acetylcholine-dependent relaxation or phenylephrine-dependent constriction in aortic rings isolated from 12/15-LOX(-/-) mice. Plasma NO metabolites and aortic endothelial NO synthase (eNOS) expression were also elevated twofold. Angiotensin II failed to vasoconstrict 12/15-LOX(-/-) aortic rings in the absence of L-nitroarginine-methyl ester, and ang II impaired acetylcholine-induced relaxation in wild-type, but not 12/15-LOX(-/-) rings. In vivo, 12/15-LOX(-/-) mice had similar basal systolic blood pressure measurements to wild type, however, blood pressure elevations in response to ang II infusion (1.1 mg/kg/day) were significantly attenuated (maximal pressure, 143.4 +/- 4 mmHg versus 122.1 +/- 5.3 mmHg for wild type and 12/15-LOX(-/-), respectively). In contrast, vascular hypertrophic responses to ang II, and ang II type 1 receptor (AT1-R) expression were similar in both strains. This study shows that 12/15-LOX(-/-) mice have increased NO biosynthesis and impaired ang II-dependent vascular responses in vitro and in vivo, suggesting that 12/15-LOX signaling contributes to impaired NO bioactivity in vascular disease in vivo.

Figure 1

Detection of 12/15-LOX products in peritoneal lavage and aortae of 12/15-LOX^−/− and wild-type mice. Arachidonate, linoleate, and their oxidized products were analyzed in peritoneal lavage and aorta from 12/15-LOX^−/− and wild-type mice using LC/ESI/MS/MS after reduction, extraction, and saponification, as described in Materials and Methods. Results are expressed as ratio of oxidized lipid product:precursor fatty acid. HETE, hydroxyeicosatetraenoic acid; HODE, hydroxyoctadecadienoic acid.

Figure 2

12/15-LOX^−/− aortic rings show alterations in phenylephrine constriction and ACh relaxation. Aortic ring functional responses were determined as described in Materials and Methods. A: Constriction dose response to phenylephrine in wild-type or 12/15-LOX^−/− rings (n = 8 to 9). B: ACh relaxation dose-response curves in wild-type or 12/15-LOX^−/− rings (n = 8 to 9). C: Dose-response curves to SNP in wild-type or 12/15-LOX^−/− rings (n = 8 to 9). D: Dose-response curves to DETA NONOate in wild-type or 12/15-LOX^−/− rings (n = 3). ★, P < 0.05 compared to wild-type group, using two-way analysis of variance to isolate differences between groups. Data are expressed as mean ± SEM.

Figure 3

NO bioactivity is higher in 12/15-LOX^−/− than wild-type aortic rings, but is not modulated by selective iNOS or nNOS inhibitors. Aortic ring functional responses were determined as described in Materials and Methods. A: Constriction dose response to phenylephrine in wild-type or 12/15-LOX^−/− rings in the presence or absence of 300 μmol/L L-NAME (n = 6 to 9). B: Relaxation dose response to ACh in phenylephrine-preconstricted wild-type or 12/15-LOX^−/− rings in the presence or absence of 300 μmol/L L-NAME (n = 6 to 9). C: Effect of selective nNOS (1 μmol/L, N^ω-propyl-l-arginine, NPA) or iNOS (10 μmol/L, 1400W) inhibition on PE constriction of 12/15-LOX^−/− aortic rings. D: Effect of selective nNOS (1 μmol/L, NPA) or iNOS (10 μmol/L, 1400W) inhibition on ACh relaxation of 12/15-LOX^−/− aortic rings. ★, P < 0.05 compared to wild-type group; ⋆, P < 0.05 compared to 12/15-LOX^−/− group using two-way analysis of variance to isolate differences between groups; †, P < 0.05 compared to L-NAME-treated wild-type group using two-way analysis of variance with Bonferroni’s posttest to isolate differences between concentrations. Data are expressed as mean ± SEM.

Figure 4

12/15-LOX^−/− mice have elevated eNOS and plasma NOx in vivo. Aortic sections from wild-type and 12/15-LOX^−/− mice were sectioned and stained for eNOS, as described in Materials and Methods. Representative sections are shown for each condition. A and B: Wild type; C and D: 12/15-LOX^−/−; E and F: isotype control antibody. A, C, and E: eNOS fluorescence; B, D, and F: corresponding phase contrast images. G: Pixel intensity was determined after fluorescence staining of aortic sections as described in Materials and Methods (n = 3 separate aortae; ★, P < 0.05, unpaired Student’s t-test). H: NOx levels in plasma from wild-type or 12/15-LOX^−/− mice were determined as described in Materials and Methods (n = 6; ★, P < 0.05, unpaired Student’s t-test).

Figure 5

12/15-LOX^−/− aortic rings lack functional responses to ang II. A: Ang II constriction dose-response curves were constructed using aortic rings from wild-type and 12/15-LOX^−/− mice (n = 5 to 8). B: Ang II constriction dose-response curves using aortic rings from wild-type and 12/15-LOX^−/− mice were repeated in the presence of 300 μmol/L L-NAME (n = 5 to 8). ⋆, P < 0.05 compared to 12/15-LOX^−/− group; unpaired Student’s t-test. C: ACh dose-response curves were constructed for wild-type aortic rings, with or without preincubation in 0.1 μmol/L ang II for 30 minutes (n = 5 to 9). D: ACh dose-response curves were constructed for 12/15-LOX^−/− aortic rings, with or without preincubation in 0.1 μmol/L ang II for 30 minutes (n = 5 to 9). ★, P < 0.05 compared to wild-type group using two-way analysis of variance to isolate differences. Data are expressed as mean ± SEM.

Figure 6

Ang II-dependent elevations in systolic blood pressure are attenuated in 12/15-LOX^−/− mice, but vascular hypertrophy is unaltered. Ang II (1.1 mg/kg/day) was infused into male 10- to 12-week-old wild-type and 12/15-LOX^−/− mice by osmotic minipump as described in Materials and Methods. A: Systolic blood pressure (blood pressure) was monitored daily for 2 days before implantation and 7 days after implantation via tail cuff plethysmography. ○, Ang II-infused 12/15-LOX^−/−; □, ang II-infused wild-type mice (n = 5 to 9 animals per group, mean ± SEM; ★, P < 0.01 compared to day 0; using analysis of variance test with Dunnett’s posthoc test to isolate differences). B: Heart and body weight was recorded and compared before and after ang II infusion (n = 6 to 11, mean ± SEM; ★, P < 0.05, compared to wild-type group; ⋆, P < 0.05 compared to 12/15-LOX^−/− group; unpaired Student’s t-test). C: Medial area was determined as described in Materials and Methods. For each aorta, three sections were analyzed and averaged (n = 5, mean ± SEM; ★, P < 0.05, compared to wild-type group; ⋆, P < 0.05 compared to 12/15-LOX^−/− group; unpaired Student’s t-test).

Figure 7

L-NAME induced hypertension in wild-type and 12/15-LOX^−/− mice. L-NAME (100 mg.kg⁻¹.day⁻¹) was administered in drinking water to male 10- to 12-week-old wild-type and 12/15-LOX^−/− mice, and blood pressure was monitored daily using tail-cuff plethysmography, as described in Materials and Methods (n = 5 animals per group, mean ± SEM; ★, P < 0.01 compared to baseline; using analysis of variance test with Dunnett’s posthoc test to isolate differences).

Figure 8

AT1-R is down-regulated after ang II infusion in both wild-type and 12/15-LOX^−/− mice. Aortic sections from wild-type and 12/15-LOX^−/− mice before or after ang II infusion were sectioned and stained for AT1-R, as described in Materials and Methods. Representative sections are shown for each condition. A: Wild type; B: 12/15-LOX^−/−; C: wild type + ang II; D: 12/15-LOX^−/− + ang II; E: isotype control antibody. F–J: Phase-contrast images for A–E, respectively. K: Pixel intensity was determined after fluorescence staining of aortic sections as described in Materials and Methods (n = 4 separate animals, with each three separate sections per aorta analyzed in triplicate for each section; ★, P < 0.05, unpaired Student’s t-test).