Organic Nitrates and Nitrate Resistance in Diabetes: The Role of Vascular Dysfunction and Oxidative Stress with Emphasis on Antioxidant Properties of Pentaerithrityl Tetranitrate

Organic nitrates represent a class of drugs which are clinically used for treatment of ischemic symptoms of angina as well as for congestive heart failure based on the idea to overcome the impaired NO bioavailability by “NO” replacement therapy. The present paper is focused on parallels between diabetes mellitus and nitrate tolerance, and aims to discuss the mechanisms underlying nitrate resistance in the setting of diabetes. Since oxidative stress was identified as an important factor in the development of tolerance to organic nitrates, but also represents a hallmark of diabetic complications, this may represent a common principle for both disorders where therapeutic intervention should start. This paper examines the evidence supporting the hypothesis that pentaerithrityl tetranitrate may represent a nitrate for treatment of ischemia in diabetic patients. This evidence is based on the considerations of parallels between diabetes mellitus and nitrate tolerance as well as on preliminary data from experimental diabetes studies

Mitochondrial oxidative stress and nitrate tolerance – comparison of nitroglycerin and pentaerithrityl tetranitrate in Mn-SOD(+/- )mice

BACKGROUND: Chronic therapy with nitroglycerin (GTN) results in a rapid development of nitrate tolerance which is associated with an increased production of reactive oxygen species (ROS). According to recent studies, mitochondrial ROS formation and oxidative inactivation of the organic nitrate bioactivating enzyme mitochondrial aldehyde dehydrogenase (ALDH-2) play an important role for the development of nitrate and cross-tolerance. METHODS: Tolerance was induced by infusion of wild type (WT) and heterozygous manganese superoxide dismutase mice (Mn-SOD(+/-)) with ethanolic solution of GTN (12.5 μg/min/kg for 4 d). For comparison, the tolerance-free pentaerithrityl tetranitrate (PETN, 17.5 μg/min/kg for 4 d) was infused in DMSO. Vascular reactivity was measured by isometric tension studies of isolated aortic rings. ROS formation and aldehyde dehydrogenase (ALDH-2) activity was measured in isolated heart mitochondria. RESULTS: Chronic GTN infusion lead to impaired vascular responses to GTN and acetylcholine (ACh), increased the ROS formation in mitochondria and decreased ALDH-2 activity in Mn-SOD(+/- )mice. In contrast, PETN infusion did not increase mitochondrial ROS formation, did not decrease ALDH-2 activity and accordingly did not lead to tolerance and cross-tolerance in Mn-SOD(+/- )mice. PETN but not GTN increased heme oxygenase-1 mRNA in EA.hy 926 cells and bilirubin efficiently scavenged GTN-derived ROS. CONCLUSION: Chronic GTN infusion stimulates mitochondrial ROS production which is an important mechanism leading to tolerance and cross-tolerance. The tetranitrate PETN is devoid of mitochondrial oxidative stress induction and according to the present animal study as well as numerous previous clinical studies can be used without limitations due to tolerance and cross-tolerance

Einfluss der AMPK-Aktivität auf Endothelfunktion, oxidativen Stress und vaskuläre Inflammation

Die AMPK ist ein ubiquitär exprimiertes, heterotrimeres Enzym, das bei Energiemangel das Überleben der Zelle sichert. Um diese Funktion ausüben zu können fungiert die AMPK als sogenannter „Energie-Sensor“, der durch steigende AMP Mengen aktiviert wird. In diesem Zustand werden ATP verbrauchende Reaktionen inhibiert und gleichzeitig ATP generierende Vorgänge induziert. Im vaskulären System konnte gezeigt werden, dass die endotheliale NOSynthase durch die AMPK aktiviert, die Angiogenese stimuliert, die Endothelzellapoptose und das Wachstum von Gefäßmuskelzellen inhibiert wird. All diese Prozesse sind fundamental in der Entwicklung von kardiovaskulären Krankheiten, was auf eine protektive Funktion der AMPK im vaskulären System hindeutet. In der vorliegenden Arbeit sollten die Effekte der in vivo Modulation der AMPK Aktivität auf Endothelfunktion, oxidativen Stress und Inflammation untersucht werden. Dazu wurden zwei unterschiedliche Mausmodelle genutzt: Einerseits wurde die AMPK Aktivität durch den pharmakologischen AMPK-Aktivator AICAR stimuliert und andererseits die vaskulär vorherrschende AMPK-Isoform durch knock out ausgeschaltet. Zur Induktion von oxidativem Stress wurde ein bereits charakterisiertes Angiotensin II-Modell angewandt. Zur Untersuchung gehörten neben den Superoxid-Messungen auch die Bestimmung der Stickstoffmonoxid-Mengen in Serum und Aortengewebe, die Relaxationsmessungen in isometrischen Tonusstudien sowie HPLC-basierte Assays. Es konnte gezeigt werden, dass durch die Aktivierung der AMPK mittels AICAR die Angiotensin II induzierte Endotheldysfunktion, der oxidative Stress und auch die vaskuläre Inflammation verbessert werden konnte. Weiterhin zeigte sich dass der knock out der vaskulären Isoform (α1) im Angiotensin II Modell eine signifikant verstärkte Endotheldysfunktion, oxidativen Stress und Inflammation nach sich zog. Anhand der erhobenen Daten konnte die NADPH-Oxidase als Hauptquelle des Angiotensin II induzierten oxidativen Stresses identifiziert werden, wobei sich diese Quelle als AMPK sensitiv erwies. Durch die Aktivierung konnte die Aktivität der NADPH-Oxidase verringert und durch die α1AMPK Defizienz signifikant erhöht werden. Auch die mitochondriale Superoxidproduktion konnte durch die Modulation der AMPK Aktivität beeinflusst werden. Die vaskuläre Inflammation, die anhand der Surrogaten VCAM-1, COX-2 und iNOS untersucht wurde, konnte durch Aktivierung der AMPK verringert werden, der knock out der α1AMPK führte so einer sehr starken Expressionssteigerung der induzierbaren NO-Synthase, was in einem starken Anstieg der NO-Produktion und somit der Peroxynitritbildung resultierte.Die dargestellten Daten deuten stark auf eine protektive Funktion der AMPK im vaskulären System hin und sollte als therapeutisches Ziel, nicht nur in Bezug auf diabetische Patienten, in Betracht gezogen werden.The AMP-activated protein kinase (AMPK) is an ubiquitous expressed heterotrimeric enzyme in mammalian cells which allows cellular survival under conditions of decreased energy supply. As a cellular fuel sensor, it is being activated by increases in the cellular AMP : ATP ratio and leads to an inhibition of ATP consuming pathways and activation of ATP generating pathways. In the vasculature, AMPK is known to activate the endothelial NO synthase, stimulates angiogenesis and inhibits the proliferation of vascular smooth muscle cells as well as endothelial cell apoptosis. All these processes play a fundamental role in the development of vascular disease, pointing to a protective role of the AMPK in the vascular system. The aim of this study was to examine the effects of in vivo modulation of the AMPK activity on endothelial function, oxidative stress and vascular inflammation. We examined endothelial function, oxidative stress and vascular inflammation in two different mouse models. On one hand, we activated the AMPK in vivo by daily subcutaneous AICAR injections and on the other hand we inhibited vascular AMPK by knocking out the major vascular isoform, namely the alpha1 subunit of the AMPK. To induce oxidative stress we used the well characteized model of chronic angiotensin II infusion. AMPK activation by AICAR improved angiotensin II induced endothelial dysfunction, decreased oxidative stress and inhibited vascular inflammation. In contrast, deletion of the major vascular Isoform α1AMPK impaired vascular dysfunction induced by low dose angiotensin II infusion, increased the oxidative stress and the vascular inflammatory response. Surprisingly, this was associated with an increase of vascular NO production, which was inactivated byrnconcomitant superoxide production leading to peroxynitrite formation and endothelial dysfunction. The NADPH-oxidase was identified as the major angiotensin II dependent source of reactive oxygen species (ROS) which was AMPK sensitive. Simultaneously, mitochondrial ROS production and xanthine oxidase activity where governed by AMPK activity. Aortic expression of VCAM-1 and COX-2 were analyzed as surrogate markers of vascular inflammation during angiotensin II treatment and were decreased after AMPK activation by AICAR but significantly upregulated after deletion of the α1AMPK. In accordance with these data, the increased NO production in α1AMPK mice was due to an upregulation of iNOS expression in response to angiotensin II treatment. These data demonstrate an essential role of AMPK in the preservation of vascular function. Future clinical studies are warranted to investigate whether pharmacological AMPK activation may be a safe and feasible option for the treatment of vascular disease in humans

Manganese superoxide dismutase and aldehyde dehydrogenase deficiency increase mitochondrial oxidative stress and aggravate age-dependent vascular dysfunction

AimsImbalance 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 resultsAge-dependent effects on vascular function were determined in aortas of C57/Bl6 wild-type (WT), ALDH-22/2, 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 isolatedheart 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-22/2 mice showed a marked vascular dysfunction that was similar in old ALDH-22/2 mice suggesting that ALDH-2 exerts agedependent vasoprotective effects. Aged MnSOD+/2 mice showed the most pronounced phenotype such as severely impaired vasorelaxation, highest levels of mitochondrial ROS formation and mtDNA damage.ConclusionThe correlation between mtROS formation and acetylcholine-dependent relaxation revealed that mitochondrial radical formation significantly contributes to age-dependent endothelial dysfunction