Pentaerythritol tetranitrate improves angiotensin II-induced vascular dysfunction via induction of heme oxygenase-1

Abstract

The organic nitrate pentaerythritol tetranitrate is devoid of nitrate tolerance, which has been attributed to the induction of the antioxidant enzyme heme oxygenase (HO)-1. With the present study, we tested whether chronic treatment with pentaerythritol tetranitrate can improve angiotensin II-induced vascular oxidative stress and dysfunction. In contrast to isosorbide-5 mononitrate (75 mg/kg per day for 7 days), treatment with pentaerythritol tetranitrate (15 mg/kg per day for 7 days) improved the impaired endothelial and smooth muscle function and normalized vascular and cardiac reactive oxygen species production (mitochondria, NADPH oxidase activity, and uncoupled endothelial NO synthase), as assessed by dihydroethidine staining, lucigenin-enhanced chemiluminescence, and quantification of dihydroethidine oxidation products in angiotensin II (1 mg/kg per day for 7 days)-treated rats. The antioxidant features of pentaerythritol tetranitrate were recapitulated in spontaneously hypertensive rats. In addition to an increase in HO-1 protein expression, pentaerythritol tetranitrate but not isosorbide-5 mononitrate normalized vascular reactive oxygen species formation and augmented aortic protein levels of the tetrahydrobiopterin-synthesizing enzymes GTP-cyclohydrolase I and dihydrofolate reductase in angiotensin II-treated rats, thereby preventing endothelial NO synthase uncoupling. Haploinsufficiency of HO-1 completely abolished the beneficial effects of pentaerythritol tetranitrate in angiotensin II-treated mice, whereas HO-1 induction by hemin (25 mg/kg) mimicked the effect of pentaerythritol tetranitrate. Improvement of vascular function in this particular model of arterial hypertension by pentaerythritol tetranitrate largely depends on the induction of the antioxidant enzyme HO-1 and identifies pentaerythritol tetranitrate, in contrast to isosorbide-5 mononitrate, as an organic nitrate able to improve rather than to worsen endothelial function.

Figure 1

Effects of in vivo PETN (15mg/kg/d) and ISMN (75mg/kg/d) treatment on the concentration response relationship to acetylcholine (ACh) (A) and nitroglycerin (GTN) (B) in aortic rings from angiotensin-II-infused (AT-II, 1mg/kg/d for 7d) rats. (C) The effect of sepiapterin (100 μM), a BH₄ precursor, and PEG-SOD (100 U/ml) pretreatment of aortic rings from AT-II-infused rats for 1 h was determined in separate experiments. Data are the mean±SEM of n=36–57 aorta from 10–15 rats per group (A and B) and n=6–8 from 3 rats per group (C). P < 0.05: * vs. Ctr/DMSO; # vs. AT-II+PETN; $ vs. AT-II/BH₄. The statistics were based on 1-way-ANOVA comparison of pD₂-values and efficacies (see table S1) but also on comparisons of all concentrations in all groups by 2-way-ANOVA analysis (for sake of clarity significance is not shown for all data points).

Figure 2

Effects of in vivo PETN and ISMN treatment on vascular superoxide levels (A, B) in angiotensin-II-infused (AT-II) rats. (A) Transverse aortic cryo-sections were labeled with dihydroethidine (DHE, 1μM), which produces red fluorescence when oxidized by reactive oxygen species. “E” indicates the endothelium; lamina autofluorescence is green. Pictures shown are representative for at least 6 animals/group. E means endothelium. (B) Densitometric quantification of the dihydroethidine-derived reactive oxygen species signal throughout the vessel wall (left panel) and lucigenin (5μM) enhanced chemiluminescence (ECL) in intact aortic ring segments (right panel). The data are mean±SEM of n=18–19 experiments with tissue from at least 10 animals/group. P < 0.05: * vs. Ctr/DMSO; # vs. AT-II+PETN. C, control; A, angiotensin-II-treated; P, angiotensin-II- and PETN-treated; I, angiotensin-II- and ISMN-treated.

Figure 3

Effects of in vivo PETN and ISMN treatment on mitochondrial reactive oxygen species formation (A, B), NADPH oxidase activity (A, C) and eNOS-dependent reactive oxygen species formation (uncoupling) (D, E) in angiotensin-II-infused (AT-II) rats. (A) Reactive oxygen species formation in isolated cardiac mitochondria was measured by L-012 (100μM) enhanced chemiluminescence (ECL, left panel) and reactive oxygen species production (NADPH oxidase activity) in membrane fractions from hearts was determined by lucigenin (5μM) enhanced chemiluminescence (ECL, right panel). (B, C) Reactive oxygen species formation in isolated cardiac mitochondria and NADPH oxidase activity in membraneous fractions was also quantified by 2-hydroxyethidium levels. The inserts show representative HPLC chromatograms. E+ means ethidium, 2-HE means 2-hydroxyethidium. The data are mean±SEM of n=31–43 (mitochondria and NADPH oxidase activity) experiments with tissue from at least 10 animals/group. The HPLC data are mean±SEM of n=9 (mitochondria), n=15 (NADPH oxidase activity) experiments with tissue from 3–5 animals/group. (D, E) Fluorescence microscopy revealed reactive oxygen species formation by red staining (upper column). To determine eNOS-dependent reactive oxygen species formation, vessels were pre-incubated with the NOS inhibitor L-NAME (lower column). Densitometric data are presented by bar graphs (solid, w/o L-NAME and open, with L-NAME). Pictures and data shown are representative for at least 4 animals/group. For methodological details see “Figure S4” in the online data supplement available at http://hyper.ahajournals.org. P < 0.05: * vs. Ctr/DMSO; # vs. AT-II+PETN. C, control; A, angiotensin-II-treated; P, angiotensin-II- and PETN-treated; I, angiotensin-II- and ISMN-treated.

Figure 4

Effects of in vivo PETN and ISMN treatment on vascular heme oxygenase-1 (HO-1, mRNA and protein), eNOS, GTP-cyclohydrolase-I (GCH-I) and dihydrofolate reductase (DHFR) expression in aortic tissue from hypertensive rats. Expression of HO-1 mRNA (A) as well as HO-1 (B), eNOS (C), GCH-I (D) and DHFR (E) protein were assessed by RT-PCR and Western blot analysis, respectively. Representative blots are shown at the bottom of the densitometric bar graphs. The data are mean ± SEM of aortic rings from 6–8 animals/group. P < 0.05: * vs. Ctr/DMSO; # vs. AT-II+PETN. C, control; A, angiotensin-II-treated; P, angiotensin-II- and PETN-treated; I, angiotensin-II- and ISMN-treated.

Figure 5

Effects of HO-1 deficiency versus HO-1 induction on vascular improvement by pentaerithrityl tetranitrate (PETN). (A, B) PETN treatment (75mg/kg/d for 4d) had no effect on PETN potency (PETN-induced relaxation) in aorta from control mice (HO-1^+/+) but caused nitrate tolerance in aorta from mice with partial HO-1 deficiency (HO-1^+/−). In accordance, cardiac mitochondrial ROS formation (L-012 ECL) was increased in PETN-treated HO-1^+/− mice. P < 0.05: * vs. HO-1^+/+/DMSO; # vs. HO-1^+/−/DMSO. S, solvent; P, PETN-treated. (C, D) Hemin (25mg/kg i.p.)-triggered HO-1 induction improved high dose AT-II (1mg/kg/d for 7d)-induced endothelial dysfunction (ACh-response) in aorta and NADPH oxidase activity in heart (lucigenin ECL) from control mice (HO-1^+/+). P < 0.05: * vs. AT-II-treated HO-1^+/+/DMSO. (E, F) PETN (75mg/kg/d for 7d) failed to prevent endothelial dysfunction (ACh-response) induced by low dose AT-II (0.1mg/kg/d for 7d) in aorta from HO-1^+/− mice. In accordance, PETN did not improve NADPH oxidase activity (2-HE formation by HPLC analysis) in cardiac samples from AT-II-treated HO-1^+/− mice. P < 0.05: * vs. AT-II-treated HO-1^+/−/DMSO. All data are mean ± SEM of aortic rings and hearts from 4–5 animals/group.