Heart rate reduction with the I(f)-channel-inhibitor ivabradine is a novel and appealing option in the therapy of patients with ischemic heart disease. The aim of the current study was to determine the effects of ivabradine in two different animal models of vascular disease characterized by increased oxidative stress and endothelial dysfunction. Wistar rats with angiotensin II induced hypertension and ApoE knockout mice were used as animal models of endothelial dysfunction and oxidative stress, with half of the animals receiving ivabradine 10 mg/kg/day in parallel. Ivabradine lead to a sustained 15–20% heart rate reduction, but had no effect on blood pressure. While ivabradine had no effect on endothelial function and vascular reactive oxygen species production in angiotensin II-treated rats, it improved both parameters in ApoE knockout mice. These antioxidative effects were associated with a decreased NADPH oxidase activity and the prevention of eNOS uncoupling. In addition, ivabradine treatment led to an attenuation of angiotensin II signaling and increased the expression of telomere-stabilizing proteins in ApoE knockout mice, which may explain its beneficial effects on the vasculature. The absence of these protective ivabradine effects in angiotensin II-infused rats may relate to the treatment duration or the presence of arterial hypertension.

Objective-Besides its well-described metabolic effects, vascular AMP-activated protein kinase (AMPK) can activate endothelial NO synthase, promotes angiogenesis, and limits endothelial cell apoptosis. The current study was designed to study the effects of alpha 1AMPK deletion during vascular disease in vivo.Methods and Results-Chronic angiotensin II infusion at low subpressor doses caused a mild endothelial dysfunction that was significantly aggravated in alpha 1AMPK-knockout mice. Unexpectedly, this endothelial dysfunction was not associated with decreased NO content, because NO levels measured by serum nitrite or electron paramagnetic resonance were even increased. However, because of parallel superoxide production, NO was consumed under production of peroxynitrite in angiotensin II-treated alpha 1AMPK-knockout mice, associated with NADPH oxidase activation and Nox2 upregulation. As Nox2 is also a component of phagocyte NADPH oxidases, we found a vascular upregulation of several proinflammatory markers, including inducible NO synthase, vascular cell adhesion molecule-1, and cyclooxygenase-2. Cotreatment with the NADPH oxidase inhibitor apocynin was able to prevent vascular inflammation and also partially restored endothelial function in alpha 1AMPK-knockout mice.Conclusion-Our data indicate that in vivo alpha 1AMPK deletion leads to Nox2 upregulation, resulting in endothelial dysfunction and vascular inflammation. This implicates basal AMPK activity as a protective, redox-regulating element in vascular homeostasis. (Arterioscler Thromb Vasc Biol. 2011;31:560-566.)

Heme oxygenase-1 (HO-1) is highly protective in various pathophysiological states such as cardiovascular and neurodegenerative diseases. HO-1-derived bilirubin is an efficient scavenger of reactive oxygen and nitrogen species (RONS). It remains to determine whether conversion of biliverdin to bilirubin is an essential step for HO-1-conferred protection of endothelial cells. RONS scavenging activities of biliverdin versus bilirubin were assessed by different RONS generating systems and detection techniques. We also silenced the biliverdin reductase (BVR) or HO-1 gene in cultured primary human endothelial cells (HUVECs) and measured the effect on RONS formation upon stimulation with lipopolysaccharide (LPS). In addition, effects of bilirubin and biliverdin on expression of GTP-cyclohydrolase were assessed in an endothelial cell line (EA.hy 926). HO-1- and BVR-silenced cells have increased levels of oxidative stress and bilirubin but not biliverdin increased expression of the protective protein GTP-cyclohydrolase. Moreover, protection by hemin-induced HO-1 expression or biliverdin-triggered bilirubin formation was impaired upon silencing of the HO-1 or BVR gene, respectively. Since bilirubin significantly scavenged RONS but chronic treatment was even more protective our observations support direct and indirect antioxidant properties of BVR and bilirubin and an important role for BVR and bilirubin in HO-1 conferred protection of endothelial cells.

Background: AMP-activated protein kinase (AMPK) plays a pivotal role in cellular adaptation to decreased energy supply. In the vasculature, AMPK activates endothelial NO synthase, promotes angiogen...

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.

Objective: Pentaerithrityl tetranitrate (PETN)-treatment is devoid of nitrate tolerance which has been attributed to the induction of the antioxidant enzyme heme oxygenase-1 (HO-1). With the present study we tested, whether chronic treatment with PETN can improve angiotensin-II (AT-II)-induced vascular oxidative stress and dysfunction. Methods and Results: In contrast to isosorbide mononitrate (ISMN, 75mg/kg/d/7d), co-treatment with PETN (15mg/kg/d/7d) improved impaired endothelial and smooth muscle function and normalized vascular and cardiac ROS production (mitochondria, NADPH oxidase activity and uncoupled endothelial nitric oxide synthase [eNOS]) as assessed by dihydroethidine (DHE)-staining, lucigenin-enhanced chemiluminescence and quantitative HPLC of DHE-oxidation products in AT-II (1mg/kg/d/7d) treated rats. The antioxidant features of PETN were recapulated in spontaneously hypertensive rats. In addition to increase in HO-1 protein expression, PETN but not ISMN augmented aortic protein levels of the tetrahydrobiopterin-synthesizing enzymes GTP-cyclohydrolase-I and dihydrofolate reductase in AT-II-treated rats, thereby preventing eNOS uncoupling. In AT-II-treated (0.1mg/kg/d/7d) HO-1+/− mice, PETN failed to be antioxidant, whereas HO-1 induction by hemin (25mg/kg) mimicked the PETN effect in AT-II-treated HO-1+/+ mice. Conclusions: PETN improves AT-II-induced vascular oxidative stress and dysfunction via induction of HO-1. Thus, PETN is the first organic nitrate to be beneficial in an experimental model of arterial hypertension.

OBJECTIVES:This study sought to analyze mechanisms that mediate vascular dysfunction induced by sirolimus.BACKGROUND:Despite excellent antirestenotic capacity, sirolimus-eluting stents have been found to trigger coronary endothelial dysfunction and impaired re-endothelialization.METHODS:To mimic the continuous sirolimus exposure of a stented vessel, Wistar rats underwent drug infusion with an osmotic pump for 7 days.RESULTS:Sirolimus treatment caused a marked degree of endothelial dysfunction as well as a desensitization of the vasculature to the endothelium-independent vasodilator nitroglycerin. Also, sirolimus stimulated intense transmural superoxide formation as detected by dihydroethidine fluorescence in aortae. Increased superoxide production was mediated in part by the vascular nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase as indicated by a marked stimulation of p67(phox)/rac1 NADPH oxidase subunit expression and by increased rac1 membrane association. In addition, superoxide production in rat heart mitochondria was up-regulated by sirolimus, as measured by L012-enhanced chemiluminescence. As a consequence, electron spin resonance measurements showed a 40% reduction in vascular nitric oxide bioavailability, which was further supported by decreased serum nitrite levels.CONCLUSIONS:Sirolimus causes marked vascular dysfunction and nitrate resistance after continuous treatment for 7 days. This impaired vasorelaxation may, in part, be induced by up-regulated mitochondrial superoxide release as well as by an up-regulation of NADPH oxidase-driven superoxide production. Both processes could contribute to endothelial dysfunction observed after coronary vascular interventions with sirolimus-coated stents.

Several enzymatic sources of reactive oxygen species (ROS) were described as potential reasons of eNOS uncoupling in diabetes mellitus. In the present study, we investigated the effects of AT1-receptor blockade with chronic telmisartan (25 mg/kg/day, 6.5 weeks) therapy on expression of the BH4-synthesizing enzyme GTP-cyclohydrolase I (GCH-I), eNOS uncoupling, and endothelial dysfunction in streptozotocin (STZ, 60 mg/kg iv, 7 weeks)-induced diabetes mellitus (type I). Telmisartan therapy did not modify blood glucose and body weight. Aortas from diabetic animals had vascular dysfunction as revealed by isometric tension studies (acetylcholine and nitroglycerin potency). Vascular and cardiac ROS produced by NADPH oxidase, mitochondria, eNOS, and xanthine oxidase were increased in the diabetic group as was the expression of NADPH oxidase subunits at the protein level. The expression of GCH-I and the phosphorylation of eNOS at Ser1177 was decreased by STZ treatment. Therapy with telmisartan normalized these parameters. The present study demonstrates for the first time that AT1-receptor blockade by telmisartan prevents downregulation of the BH4 synthase GCH-I and thereby eNOS uncoupling in experimental diabetes. In addition, telmisartan inhibits activation of superoxide sources like NADPH oxidase, mitochondria, and xanthine oxidase. These effects may explain the beneficial effects of telmisartan on endothelial dysfunction in diabetes.

AIMS:Imbalance between pro- and antioxidant species (e.g. during aging) plays a crucial role for vascular function and is associated with oxidative gene regulation and modification. Vascular aging is associated with progressive deterioration of vascular homeostasis leading to reduced relaxation, hypertrophy, and a higher risk of thrombotic events. These effects can be explained by a reduction in free bioavailable nitric oxide that is inactivated by an age-dependent increase in superoxide formation. In the present study, mitochondria as a source of reactive oxygen species (ROS) and the contribution of manganese superoxide dismutase (MnSOD, SOD-2) and aldehyde dehydrogenase (ALDH-2) were investigated.METHODS AND RESULTS:Age-dependent effects on vascular function were determined in aortas of C57/Bl6 wild-type (WT), ALDH-2(-/-), MnSOD(+/+), and MnSOD(+/-) mice by isometric tension measurements in organ chambers. Mitochondrial ROS formation was measured by luminol (L-012)-enhanced chemiluminescence and 2-hydroxyethidium formation with an HPLC-based assay in isolated heart mitochondria. ROS-mediated mitochondrial DNA (mtDNA) damage was detected by a novel and modified version of the fluorescent-detection alkaline DNA unwinding (FADU) assay. Endothelial dysfunction was observed in aged C57/Bl6 WT mice in parallel to increased mitochondrial ROS formation and oxidative mtDNA damage. In contrast, middle-aged ALDH-2(-/-) mice showed a marked vascular dysfunction that was similar in old ALDH-2(-/-) mice suggesting that ALDH-2 exerts age-dependent vasoprotective effects. Aged MnSOD(+/-) mice showed the most pronounced phenotype such as severely impaired vasorelaxation, highest levels of mitochondrial ROS formation and mtDNA damage.CONCLUSION:The correlation between mtROS formation and acetylcholine-dependent relaxation revealed that mitochondrial radical formation significantly contributes to age-dependent endothelial dysfunction.

Chronic nitroglycerin treatment results in development of nitrate tolerance associated with endothelial dysfunction (ED). We sought to clarify how mitochondria- and NADPH oxidase (Nox)-derived reactive oxygen species (ROS) contribute to nitrate tolerance and nitroglycerin-induced ED. Nitrate tolerance was induced by nitroglycerin infusion in male Wistar rats (100 microg/h/4 day) and in C57/Bl6, p47(phox/) and gp91(phox/) mice (50 microg/h/4 day). Protein and mRNA expression of Nox subunits were unaltered by chronic nitroglycerin treatment. Oxidative stress was determined in vascular rings and mitochondrial fractions of nitroglycerin-treated animals by L-012 enhanced chemiluminescence, revealing a dominant role of mitochondria for nitrate tolerance development. Isometric tension studies revealed that genetic deletion or inhibition (apocynin, 0.35 mg/h/4 day) of Nox improved ED, whereas nitrate tolerance was unaltered. Vice versa, nitrate tolerance was attenuated by co-treatment with the respiratory chain complex I inhibitor rotenone (100 microg/h/4 day) or the mitochondrial permeability transition pore blocker cyclosporine A (50 microg/h/4 day). Both compounds improved ED, suggesting a link between mitochondrial and Nox-derived ROS. Mitochondrial respiratory chain-derived ROS are critical for the development of nitrate tolerance, whereas Nox-derived ROS mediate nitrate tolerance-associated ED. This suggests a crosstalk between mitochondrial and Nox-derived ROS with distinct mechanistic effects and sites for pharmacological intervention.

Cyclooxygenase 2-selective inhibitors (coxibs) and nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with an increase in cardiovascular events. The current study was designed to test the effect of coxibs and nonselective NSAIDs on vascular superoxide and nitric oxide (NO) production. mRNA expression of endothelial NO synthase (eNOS) and of the vascular NADPH oxidases was studied in spontaneously hypertensive rats (SHR) and in human endothelial cells. The expression of Nox1, Nox2, Nox4, and p22phox was increased markedly by the nonselective NSAIDs diclofenac or naproxen and moderately by rofecoxib or celecoxib in the aorta and heart of SHR. The up-regulation of NADPH oxidases by NSAIDs was associated with increased superoxide content in aorta and heart, which could be prevented by the NADPH oxidase inhibitor apocynin. NSAIDs reduced plasma nitrite and diminished the phosphorylation of vasodilator-stimulated phosphoprotein. This demonstrates a reduction in vascular NO production. Aortas from diclofenac-treated SHR showed an enhanced protein nitrotyrosine accumulation, indicative of vascular peroxynitrite formation. Peroxynitrite can uncouple oxygen reduction from NO synthesis in eNOS. Accordingly, the eNOS inhibitor NG-nitro-l-arginine methyl ester reduced superoxide content in aortas of NSAID-treated animals, demonstrating eNOS uncoupling under those conditions. Also in human endothelial cells, NSAIDs increased Nox2 expression and diminished production of bioactive NO. In healthy volunteers, NSAID treatment reduced nitroglycerin-induced, NO-mediated vasodilatation of the brachial artery. These results indicate that NSAIDs may increase cardiovascular risk by inducing oxidative stress in the vasculature, with nonselective NSAIDs being even more critical than coxibs in this respect.

Objective— Nitrate tolerance is likely attributable to an increased production of reactive oxygen species (ROS) leading to an inhibition of the mitochondrial aldehyde dehydrogenase (ALDH-2), representing the nitroglycerin (GTN) and pentaerythrityl tetranitrate (PETN) bioactivating enzyme, and to impaired nitric oxide bioactivity and signaling. We tested whether differences in their capacity to induce heme oxygenase-1 (HO-1) might explain why PETN and not GTN therapy is devoid of nitrate and cross-tolerance. Methods and Results— Wistar rats were treated with PETN or GTN (10.5 or 6.6 μg/kg/min for 4 days). In contrast to GTN, PETN did not induce nitrate tolerance or cross-tolerance as assessed by isometric tension recordings in isolated aortic rings. Vascular protein and mRNA expression of HO-1 and ferritin were increased in response to PETN but not GTN. In contrast to GTN therapy, NO signaling, ROS formation, and the activity of ALDH-2 (as assessed by an high-performance liquid chromatography–based method) were not significantly influenced by PETN. Inhibition of HO-1 expression by apigenin induced “tolerance” to PETN whereas HO-1 gene induction by hemin prevented tolerance in GTN treated rats. Conclusions— HO-1 expression and activity appear to play a key role in the development of nitrate tolerance and might represent an intrinsic antioxidative mechanism of therapeutic interest.

Aims Protein kinase C (PKC) plays an important rote in the regulation of angiogenesis. However, downstream targets of PKC in endothelial cells are poorly defined.Methods and results mRNA expression of vascular endothelial growth factor (VEGF) was analysed by quantitative real-time RT-PCR in human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells. siRNA was used to knockdown PKC isoforms and VEGF. Matrigel tube formation assay was used to analyse the angiogenic activity of endothelial cells. Phorbol-12-myristate-13-acetate (PMA) enhanced the ability of HUVEC to organize into tubular networks when plated on Matrigel, a phenomenon that could be prevented by PKC inhibitors. PMA markedly increased the expression of VEGF in HUVEC and EA.hy 926 cells. The enhancement in VEGF expression was prevented by PKC inhibitors and by an inhibitor of the Erk1/2 pathway. PMA-induced tube formation was reduced by inhibition of the VEGF receptor kinase, or by VEGF knockdown. PMA led to an activation of PKC isoforms alpha,delta and epsilon in HUVEC. Knockdown of PKC alpha diminished PMA-induced VEGF expression and angiogenesis. Also endothelial progenitor cells isolated from human peripheral blood showed enhanced VEGF expression and improved angiogenic activity in response to PKC activation. Moreover, incubation of HUVEC with VEGF led to PKC alpha activation and PKC-dependent VEGF upregulation.Conclusions PKC alpha activation promotes angiogenic activity of human endothelial cells. This is likely to be largely mediated by induction of VEGF. VEGF enhances its own expression via a PKC a-dependent positive feedback mechanism.