Pentaerythritol Tetranitrate Improves Angiotensin II–Induced Vascular Dysfunction via Induction of Heme Oxygenase-1

Abstract

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 GTPcyclohydrolase 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. (Hypertension. 2010;55:897-904.)Key Words: pentaerythritol tetranitrate Ⅲ isosorbide-5-mononitrate Ⅲ angiotensin-II Ⅲ SHR Ⅲ endothelial dysfunction Ⅲ vascular oxidative stress B oth arterial hypertension and coronary artery disease are associated with an activation of the circulating and local renin-angiotensin system and increased oxidative stress within the vascular wall. 1,2 Angiotensin-II (AT-II) treatment has been shown to cause endothelial dysfunction, which is at least in part mediated by increased vascular reactive oxygen species (ROS) levels. 3,4 ROS sources involved may include the NADPH oxidases, 3 an uncoupled endothelial NO synthase (NOS; eNOS), 4 and mitochondrial superoxide sources. 5 The crucial role of the NADPH oxidase as an important superoxide source was further substantiated by the demonstration that NADPH oxidase 1 overexpression in transgenic mice potentiates AT-II-induced hypertension, 6 whereas blood pressure responses to AT-II were reduced in NADPH oxidase 1-deficient mice. 7 Increased vascular ROS production and endothelial dysfunction ...

Endothelial Dysfunction in Tristetraprolin-deficient Mice Is Not Caused by Enhanced Tumor Necrosis Factor-α Expression

“…Determination of Vascular RONS Formation-Vascular RONS formation was determined by dihydroethidine (1 mol/liter)-dependent fluorescence microtopography in aortic cryosections (36,37).…”

Taking up the cudgels for the traditional reactive oxygen and nitrogen species detection assays and their use in the cardiovascular system

“…We also established that increased cardiac membrane NADPH oxidase activity in diabetic animals was normalized by the organic nitrate pentaerithrityl tetranitrate with antioxidant properties, which was positively correlated with reductions in cardiac 3-nitrotyrosine and malondialdehyde content [64]. In hypertensive mice cardiac membrane NADPH oxidase activity was increased as measured by NADPHstimulated lucigenin ECL signals and confirmed by HPLC-based quantification of 2-hydroxyethidium in the same membrane fractions [23]. These are just a few examples from our previous studies performed within the last 2 decades.…”

“…The DHE cryo staining assay yielded reliable increases in signal intensity in tissues from numerous animal disease models but also human samples: diabetes mellitus in rat [36,37] To determine eNOSdependent ROS formation, vessels were preincubated with the NOS inhibitor L-NAME (500 µM, lower panel), embedded in Tissue Tek resin, frozen, cryo-sectioned, and stained with DHE (1 µM) [23]. It should be noted that DHE does not react with "accumulated" ROS (most likely superoxide) in the cryo-sections, which would have been decomposed during storage, but DHE is oxidized by de novo formed ROS coming from uncoupled eNOS or NADPH oxidase after freezing and thawing.…”

“…A fixed area was used for densitometric quantification and the procedure is shown for one representative endothelial cell layer of AT-II treatment group. The method of densitometric quantification of endothelial DHE staining was adopted from a published protocol [68](A-C) Stainings were selected from unused pictures and graphs were drafted de novo from original data published in Schuhmacher et al, Hypertension 2010 [23]. Aortic endothelial DHE staining correlated well with endothelial dysfunction measured by ACh-dependent relaxation using isometric tension recording [62] (D), impaired calcium ionophore-stimulated tolerance in rat [38], mouse [39], rabbits [40] and human [41]; isosorbide-5-mononitrate-induced endothelial dysfunction [26]; cyclooxygenase inhibitor-induced NADPH oxidase activation in rat [42]; atherosclerosis in Watanabe Heritable Hyperlipidemic (WHHL) rabbits [40,43], ApoE knockout [44] and high fat diet fed mice (Steven et al, in revision in Cardiovasc.…”

“…These are only selected references since in total we have successfully used the DHE (cryo) staining assay in more than 70 original publications with all kinds of pharmacological interventions of genetic manipulation of redox pathways (for summary see [1,10,34]. The DHE (cryo) staining intensity always nicely correlated with the severity of the respective phenotype, was reduced by pharmacological therapy [22,23,36,43,45,48,53] or genetic deletion of the Nox subunit p47 phox [54] and aggravated by genetic deletion of protective antioxidant enzymes such as heme oxygenase-1, manganese superoxide dismutase or glutathione peroxidase-1 [49,55,56] and eliminated by ex vivo incubation with specific superoxide quenchers such as PEG-SOD [53]. It would be a quite surprising coincidence if in all these models, interventions and studies DHE (cryo) staining always would have identified the diseased group where higher oxidative stress levels were shown by other widely accepted methods for ROS and RNS detection, in particular since in all these models reduction in superoxide leels went along with an increase in vascular…”