Sven Wind

Publications of Sven Wind

• Oxidative stress and endothelial dysfunction in aortas of aged spontaneously hypertensive rats by NOX1/2 is reversed by NADPH oxidase inhibition.

  Authors: Sven Wind, Knut Beuerlein, Melanie E Armitage, Ashraf Taye, Arun H S Kumar, Daniel Janowitz, Christina Neff, Ajay M Shah, Kirstin Wingler, Harald H H W Schmidt

  Hypertension. 09/2010; 56(3):490-7.

  Arterial hypertension is associated with increased levels of reactive oxygen species, which may scavenge endothelium-derived NO and thereby diminish its vasorelaxant effects. However, theArterial hypertension is associated with increased levels of reactive oxygen species, which may scavenge endothelium-derived NO and thereby diminish its vasorelaxant effects. However, the quantitatively relevant source of reactive oxygen species is unclear. Thus, this potential pathomechanism is not yet pharmacologically targetable. Several enzymatic sources of reactive oxygen species have been suggested: uncoupled endothelial NO synthase, xanthine oxidase, and NADPH oxidases. Here we show that increased reactive oxygen species formation in aortas of 12- to 14-month-old spontaneously hypertensive rats versus age-matched Wistar Kyoto rats is inhibited by the specific NADPH oxidase inhibitor VAS2870 but neither by the xanthine oxidase inhibitor oxypurinol nor the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester. NADPH oxidase activity, as well as protein expression of its catalytic subunits, NOX1 and NOX2, was increased in the aortas of spontaneously hypertensive rats, whereas the expression of NOX4 protein, the most abundant NOX isoform, was not significantly changed. Impaired acetylcholine-induced relaxation of spontaneously hypertensive rat aortas was significantly improved by VAS2870. In conclusion, NOX1 and NOX2 but not NOX4 proteins are increased in aged spontaneously hypertensive rat aortas. Importantly, these NOX isoforms, in particular, ectopic expression of NOX1 in endothelial cells, appear to affect vascular function in an NADPH oxidase inhibitor-reversible manner. NADPH oxidases may, thus, be a novel target for the treatment of systemic hypertension.

• Apocynin is not an inhibitor of vascular NADPH oxidases but an antioxidant.

  Authors: Sabine Heumüller, Sven Wind, Eduardo Barbosa-Sicard, Harald H H W Schmidt, Rudi Busse, Katrin Schröder, Ralf P Brandes

  Hypertension. 03/2008; 51(2):211-7.

  A large body of literature suggest that vascular reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases are important sources of reactive oxygen species. Many studies, however, reliedA large body of literature suggest that vascular reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases are important sources of reactive oxygen species. Many studies, however, relied on data obtained with the inhibitor apocynin (4'-hydroxy-3'methoxyacetophenone). Because the mode of action of apocynin, however, is elusive, we determined its mechanism of inhibition on vascular NADPH oxidases. In HEK293 cells overexpressing NADPH oxidase isoforms (Nox1, Nox2, or Nox4), apocynin failed to inhibit superoxide anion generation detected by lucigenin chemiluminescence. In contrast, apocynin interfered with the detection of reactive oxygen species in assay systems selective for hydrogen peroxide or hydroxyl radicals. Importantly, apocynin interfered directly with the detection of peroxides but not superoxide, if generated by xanthine/xanthine oxidase or nonenzymatic systems. In leukocytes, apocynin is a prodrug that is activated by myeloperoxidase, a process that results in the formation of apocynin dimers. Endothelial cells and smooth muscle cells failed to form these dimers and, therefore, are not able to activate apocynin. Dimer formation was, however, observed in Nox-overexpressing HEK293 cells when myeloperoxidase was supplemented. As a consequence, apocynin should only inhibit NADPH oxidase in leukocytes, whereas in vascular cells, the compound could act as an antioxidant. Indeed, in vascular smooth muscle cells, the activation of the redox-sensitive kinases p38-mitogen-activate protein kinase, Akt, and extracellular signal-regulated kinase 1/2 by hydrogen peroxide and by the intracellular radical generator menadione was prevented in the presence of apocynin. These observations indicate that apocynin predominantly acts as an antioxidant in endothelial cells and vascular smooth muscle cells and should not be used as an NADPH oxidase inhibitor in vascular systems.

• Pharmacological and biochemical studies on the contribution of NADPH oxidases to oxidative stress in the aorta of spontaneously hypertensive rats

  Authors: Sven Wind

  Vaskulärer oxidativer Stress führt zu einer Beeinträchtigung der endothelabhängigen Gefäßrelaxation. Stickstoffmonoxid scheint unter Bedingungen, wie sie bei Diabetes mellitus, HypercholesterolämieVaskulärer oxidativer Stress führt zu einer Beeinträchtigung der endothelabhängigen Gefäßrelaxation. Stickstoffmonoxid scheint unter Bedingungen, wie sie bei Diabetes mellitus, Hypercholesterolämie oder arteriellem Bluthochdruck vorliegen, von Superoxid und anderen reaktiven Sauerstoffspezies (ROS) inaktiviert zu werden. Als Hauptquellen für ROS in Blutgefässen sind NADPH Oxidasen identifiziert worden. Die vorliegende Arbeit untersucht die Rolle von NADPH Oxidasen in Aorten von 12 bis 14 Monate alten spontan-hypertensiven Ratten (SHR) im Vergleich zu gleichaltrigen Wistar Kyoto Ratten (WKY). Die in-situ Detektion von ROS mittels DHE Fluoreszenz zeigte, dass die ROS Produktion in Aorten von SHR stark erhöht ist. Die NADPH Oxidase-Inhibitoren Apocynin und die neue Substanz VAS2870, nicht aber der eNOS-Hemmstoff L-NAME oder der Xanthinoxidase-Inhibitor Oxypurinol unterdrücken diese ROS Bildung, was auf NADPH Oxidasen als Hauptquelle für oxidativen Stress hinweist. Mit diesem Befund einhergehend zeigten SHR Aorten eine erhöhte NADPH Oxidase Aktivität. NADPH Oxidasen sind Multimer-Komplexe, die aus verschiedenen zytosolischen und zwei membranständigen Untereinheiten, p22phox und einer katalytischen Nox Isoform, bestehen. In Rattenaorten sind die Isoformen Nox1, Nox2 und Nox4 exprimiert, wie mittels RT-PCR in Aortenhomogenaten von SHR und WKY bestätigt wurde. Die immunhistochemische Analyse der Gefässe deutete eine primäre Lokalisation von Nox1 in der Media an, während Nox2 in Adventitia und Intima und Nox4 in der gesamten Aortenwand gefunden wurden. Im Vergleich zu WKY, konnten erhöhte Nox1 und Nox2 Proteinspiegel im SHR-Aortenhomogenat mittels quantitativer Western Blot Analyse nachgewiesen werden, während die Nox4 Expression nicht signifikant verändert war. Weiterhin war die Nox1 Expression im Endothel der SHR Aorten hochreguliert, wie Kolokalisierungsexperimente von Nox1 mit dem glatten Muskelzell-Marker α-SM-actin und dem Endothelzell-spezifischen Marker RECA-1 zeigten. Zusammenfassend weisen diese Daten darauf hin, dass Nox1 und Nox2-basierte NADPH Oxidasen den oxidativen Stress in der Aortenwand gealteter SHR verursachen. Um zu untersuchen, ob der durch NADPH Oxidasen hervorgerufene oxidative Stress mit einer endothelialen Dysfunktion der Aorten zusammenhängt, wurde die Azetylcholin-induzierte, endothelabhängige Gefässdilatation bestimmt. In SHR-Aorten war die Relaxation im Vergleich zur WKY Kontrolle beeinträchtigt, konnte aber in Anwesenheit von Apocynin und VAS2870 signifikant verbessert werden. Diese, durch die Inhibitoren bewirkte Verbesserung der Relaxation war in WKY Aorten weniger ausgeprägt, was auf die funktionelle Relevanz der erhöhten NADPH Oxidase Aktivität hindeutet. Daraus lassen sich folgende Schlussfolgerungen ableiten: Nox1 und Nox2- basierte NADPH Oxidasen wurden als Hauptquellen für oxidativen Stress in SHR Aorten identifiziert und tragen damit signifikant zur endothelialen Dysfunktion bei. Deshalb stellt die Hemmung dieser Enzyme einen vielversprechenden, therapeutischem Ansatz dar. Der neuartige NADPH Oxidase Hemmstoff VAS2870 konnte den oxidativen Stress in SHR Aorten wirksam reduzieren und damit deren endotheliale Funktion verbessern. Somit verdeutlicht die vorliegende Arbeit das Potential von VAS2870 als experimentelles „Tool“ in der Pharmakologie von NADPH Oxidasen. Darüberhinaus stellt VAS2870 auch eine interessante Modellsubstanz für die Behandlung kardiovaskulärer und anderer mit oxidativem Stress assoziierten Erkrankungen dar. Vascular oxidative stress is associated with a dysfunction of endothelium-dependent relaxation. Inactivation of nitric oxide by superoxide and other reactive oxygen species (ROS) appears to occur under conditions such as diabetes mellitus, hypercholesterolemia or arterial hypertension. NADPH oxidases have been identified as major source of ROS in blood vessels. The present study investigates the role of NADPH oxidases in aortae of 12-14 month old spontaneously hypertensive rats (SHR) in comparison to age-matched Wistar Kyoto rats (WKY). ROS production was largely increased in aortae of SHR as measured by in situ detection of DHE fluorescence. Suppression of ROS formation by the NADPH oxidase inhibitors, apocynin and the novel compound VAS2870, but not by the eNOS inhibitor L-NAME or the xanthine oxidase inhibitor oxypurinol, suggested NADPH oxidases as a major source of oxidative stress. In line with these findings, NADPH oxidase activity in aortic homogenates of SHR was significantly elevated. NADPH oxidases are multimeric complexes consisting of several cytosolic and two membranic subunits, namely p22phox and a catalytic Nox isoform. In rat aortae, the isoforms Nox1, Nox2 and Nox4 are expressed, which was confirmed by RT-PCR in aortic homogenates of SHR and WKY. Immunohistochemical analysis of the vessels demonstrated a predominant location of Nox1 in the aortic media. Nox2 was localized in the adventitia as well as the intima and Nox4 was located throughout the aortic wall. Quantitative Western blot analysis revealed increased levels of Nox1 and Nox2 protein in SHR aortic homogenates, while Nox4 expression was not significantly changed. Furthermore, Nox1 protein appeared to be upregulated especially in the intima of SHR aortae as indicated by colocalization of Nox1 immunofluorescence with the VSMC marker α-SM-actin and the endothelial cell marker RECA-1. These data suggest that Nox1 and Nox2-based NADPH oxidases are mainly responsible for oxidative stress in the aortic wall of aged SHR. Acetylcholine-induced endothelial-dependent relaxation was determined to investigate whether the NADPH oxidase-produced oxidative stress is linked to endothelial dysfunction. Vasorelaxation was impaired in aortic rings of SHR in comparison to the WKY controls. However, it was significantly improved in the presence of apocynin and VAS2870 in SHR and to a smaller extent in WKY, indicating the functional relevance of the enhanced NADPH oxidase activity. In conclusion, Nox1 and Nox2 comprising NADPH oxidases were identified as major sources of oxidative stress in SHR aortae and thus contributed significantly to endothelial dysfunction. Targeting these enzymes therefore promises to be of immense therapeutic relevance. The novel NADPH oxidase inhibitor VAS2870 was shown to effectively suppress the oxidative stress and to improve the endothelial function in SHR. In consequence, the present study highlights VAS2870 as an experimental tool in the pharmacology of NADPH oxidases and – furthermore - as an interesting model compound for the treatment of cardiovascular and other diseases associated with oxidative stress.