Vascular aging : Chronic oxidative stress and impairment of redox signaling—consequences for vascular homeostasis and disease
2013-02, Bachschmid, Markus, Schildknecht, Stefan, Matsui, Reiko, Zee, Rebecca, Häussler, Dagmar, Cohen, Richard, Pimental, David, van der Loo, Bernd
Characteristic morphological and molecular alterations such as vessel wall thickening and reduction of nitric oxide occur in the aging vasculature leading to the gradual loss of vascular homeostasis. Consequently, the risk of developing acute and chronic cardiovascular diseases increases with age. Current research of the underlying molecular mechanisms of endothelial function demonstrates a duality of reactive oxygen and nitrogen species in contributing to vascular homeostasis or leading to detrimental effects when formed in excess. Furthermore, changes in function and redox status of vascular smooth muscle cells contribute to age-related vascular remodeling. The age-dependent increase in free radical formation causes deterioration of the nitric oxide signaling cascade, alters and activates prostaglandin metabolism, and promotes novel oxidative posttranslational protein modifications that interfere with vascular and cell signaling pathways. As a result, vascular dysfunction manifests. Compensatory mechanisms are initially activated to cope with age-induced oxidative stress, but become futile, which results in irreversible oxidative modifications of biological macromolecules. These findings support the ‘free radical theory of aging’ but also show that reactive oxygen and nitrogen species are essential signaling molecules, regulating vascular homeostasis.
Age-associated cellular relocation of Sod 1 as a self-defense is a futile mechanism to prevent vascular aging
2006-06-09, van der Loo, Bernd, Bachschmid, Markus, Skepper, Jeremy N., Labugger, Ralf, Schildknecht, Stefan, Hahn, Raphael, Müssig, Elisabeth, Gygi, Daniel, Lüscher, Thomas F.
Vascular aging is characterized by the presence of chronic oxidative stress. Although cytosolic Sod 1 has a key role in the detoxification of superoxide ((*)O(2)(-)), little is known about its importance in vascular aging. We found that inhibition of Sod 1 had no effect on (*)O2- generation. Furthermore, its expression decreased in an age-dependent manner. Interestingly, Sod 1 loses its membrane-association and is also lost from the caveolae with increasing age. Instead, a relocation of Sod 1 to the mitochondria takes place, presumably in an attempt to maintain mitochondrial integrity and to counter-balance age-associated oxidative stress. Unlike Sod 2, which is constitutively expressed in mitochondria to control (*)O2- radical fluxes, Sod 1 is not inactivated by peroxynitrite and is not nitrated as a function of age. These novel insights into oxidative stress-associated vascular aging and the understanding about how redox-systems are regulated in old age may identify new targets to ameliorate aging as the greatest cardiovascular risk factor.
Redox regulation of vascular prostanoid synthesis by the nitric oxide-superoxide system
2005-12-09, Bachschmid, Markus, Schildknecht, Stefan, Ullrich, Volker
Oxygen is involved in cell signaling through oxygenases and oxidases and this applies especially for the vascular system. Nitric oxide (*NO) and epoxyarachidonic acids are P450-dependent monooxygenase products and prostacyclin is formed via cyclooxygenase and a heme-thiolate isomerase. The corresponding vasorelaxant mechanisms are counteracted by superoxide which not only traps *NO but through the resulting peroxynitrite blocks prostacyclin synthase by nitration of an active site tyrosine residue. In a model of septic shock, this leads to vessel constriction by activation of the thromboxane A2-prostaglandin endoperoxide H2 receptor. This sequence of events is part of endothelial dysfunction in which the activated vascular smooth muscle counteracts and regenerates vessel tone by cyclooxygenase-2-dependent prostacyclin synthesis. Peroxynitrite was found to activate cyclooxygenases by providing the peroxide tone at nanomolar concentrations. Such new insights into the control of vascular function have allowed us to postulate a concept of redox regulation in which a progressive increase of superoxide production by NADPH-oxidase, mitochondria, xanthine oxidase, and even uncoupled NO-synthase triggers a network of signals originating from an interaction of *NO with superoxide.
Endotoxin elicits nitric oxide release in rat but prostacyclin synthesis in human and bovine vascular smooth muscle cells
2005-02-04, Schildknecht, Stefan, Bachschmid, Markus, Weber, Klaus, Maass, Dierk, Ullrich, Volker
Lipopolysaccharide (LPS) exposure to cells and tissues can mimic the biochemical events leading to septic shock. Previous data demonstrated a massive upregulation of prostaglandin endoperoxide H2 synthase (PGHS-2), but not NO synthase-2 (NOS-2) in bovine smooth muscle cells (SMC) between 2 and 12 h of LPS exposure. This caused an abundant release of prostacyclin (PGI2) by constitutive PGI2-synthase as a counterregulation to a dysfunctional endothelium. We here report that human as well as bovine SMC mainly respond by the induction of PGHS-2 and the subsequent release of PGI2, whereas rat SMC exhibited a distinct induction of NOS-2 and released significantly higher amounts of *NO compared with cattle and human. The induction of either PGHS-2 or NOS-2 in the three different species investigated seems to be mutually exclusive in the time window of 2-24 h. This finding should be considered in the setup of experimental models for the investigation of septic shock.
Protein Tyrosine Nitration and Thiol Oxidation by Peroxynitrite—Strategies to Prevent These Oxidative Modifications
2013, Daiber, Andreas, Daub, Steffen, Bachschmid, Markus, Schildknecht, Stefan, Oelze, Matthias, Steven, Sebastian, Schmidt, Patrick, Megner, Alexandra, Wada, Masayuki, Tanabe, Tadashi, Münzel, Thomas, Bottari, Serge, Ullrich, Volker
The reaction product of nitric oxide and superoxide, peroxynitrite, is a potent biological oxidant. The most important oxidative protein modifications described for peroxynitrite are cysteine-thiol oxidation and tyrosine nitration. We have previously demonstrated that intrinsic heme-thiolate (P450)-dependent enzymatic catalysis increases the nitration of tyrosine 430 in prostacyclin synthase and results in loss of activity which contributes to endothelial dysfunction. We here report the sensitive peroxynitrite-dependent nitration of an over-expressed and partially purified human prostacyclin synthase (3.3 μM) with an EC50 value of 5 μM. Microsomal thiols in these preparations effectively compete for peroxynitrite and block the nitration of other proteins up to 50 μM peroxynitrite. Purified, recombinant PGIS showed a half-maximal nitration by 10 μM 3-morpholino sydnonimine (Sin-1) which increased in the presence of bicarbonate, and was only marginally induced by freely diffusing NO2-radicals generated by a peroxidase/nitrite/hydrogen peroxide system. Based on these observations, we would like to emphasize that prostacyclin synthase is among the most efficiently and sensitively nitrated proteins investigated by us so far. In the second part of the study, we identified two classes of peroxynitrite scavengers, blocking either peroxynitrite anion-mediated thiol oxidations or phenol/tyrosine nitrations by free radical mechanisms. Dithiopurines and dithiopyrimidines were highly effective in inhibiting both reaction types which could make this class of compounds interesting therapeutic tools. In the present work, we highlighted the impact of experimental conditions on the outcome of peroxynitrite-mediated nitrations. The limitations identified in this work need to be considered in the assessment of experimental data involving peroxynitrite.
Autocatalytic tyrosine nitration of prostaglandin endoperoxide synthase-2 in LPS-stimulated RAW 264.7 macrophages
2006-02-03, Schildknecht, Stefan, Heinz, Kathrin, Daiber, Andreas, Hamacher, Jürg, Kavaklí, Cengiz, Ullrich, Volker, Bachschmid, Markus
In the literature, biological tyrosine nitrations have been reported to depend not only on peroxynitrite but also on nitrite/hydrogen peroxide linked to catalysis by myeloperoxidase. In endotoxin-stimulated RAW 264.7 macrophages, we have detected a major nitrotyrosine positive protein band around 72 kDa and identified it as prostaglandin endoperoxide synthase-2 (PGHS-2). Isolated PGHS-2 in absence of its substrate arachidonate was not only tyrosine-nitrated with peroxynitrite, but also with nitrite/hydrogen peroxide in complete absence of myeloperoxidase. Our data favor an autocatalytic activation of nitrite by PGHS-2 with a subsequent nitration of the essential tyrosine residue in the cyclooxygenase domain. Under inflammatory conditions, nitrite formed via NO-synthase-2 may therefore act as an endogenous regulator for PGHS-2 in stimulated macrophages. Nitration of PGHS-2 by the autocatalytic activation of nitrite further depends on the intracellular concentration of arachidonate since arachidonate reacted competitively with nitrite and could prevent PGHS-2 from nitration when excessively present.
Redox regulation : a new challenge for pharmacology
2005-09-15, Frein, Daniel, Schildknecht, Stefan, Bachschmid, Markus, Ullrich, Volker
Redox signaling is evolving as a new field of biochemical and pharmacological research. Unlike oxidative stress which is characterized by a macroscopic shift in cellular redox potentials and usually accompanied by oxygen radical induced damage, redox regulation involves subtle and more chemically defined oxidations of short duration. Most important is the reductive component as a necessary part of a reversible regulatory process. Examples of redox regulation occur during early stages of the immune response, in hypoxia or in endothelial dysfunction. Persistent oxidative events together with a decline in the cellular reduction potential lead to oxidative stress as is seen in the pathophysiology of sepsis, reperfusion damage, atherosclerosis and diabetes. Oxidative signals involve superoxide and nitric oxide as the main players which form a system of oxidizing, nitrating or nitrosating species leading to posttranslational modifications of proteins. Modern techniques of immunohistochemistry and mass spectrometry allow a correlation of protein modification, e.g., disulfide, S-oxide, S-nitroso or nitrotyrosine formation, with enzyme activities and cellular responses. In this commentary, examples of the control of prostanoid synthesis by the NO/O2- system are described. Redox regulation represents an interesting challenge for the development of drugs that modulate the oxidative trigger mechanisms or enforce the reductive pathways.
Role of reduced lipoic acid in the redox regulation of mitochondrial aldehyde dehydrogenase (ALDH-2) activity : Implications for mitochondrial oxidative stress and nitrate tolerance
2007-01-05, Wenzel, Philip, Hink, Ulrich, Oelze, Matthias, Schuppan, Swaantje, Schaeuble, Karin, Schildknecht, Stefan, Ho, Kwok K., Weiner, Henry, Bachschmid, Markus, Münzel, Thomas, Daiber, Andreas
Chronic therapy with nitroglycerin results in a rapid development of nitrate tolerance, which is associated with an increased production of reactive oxygen species. We have recently shown that mitochondria are an important source of nitroglycerin-induced oxidants and that the nitroglycerin-bioactivating mitochondrial aldehyde dehydrogenase is oxidatively inactivated in the setting of tolerance. Here we investigated the effect of various oxidants on aldehyde dehydrogenase activity and its restoration by dihydrolipoic acid. In vivo tolerance in Wistar rats was induced by infusion of nitroglycerin (6.6 microg/kg/min, 4 days). Vascular reactivity was measured by isometric tension studies of isolated aortic rings in response to nitroglycerin. Chronic nitroglycerin infusion lead to impaired vascular responses to nitroglycerin and decreased dehydrogenase activity, which was corrected by dihydrolipoic acid co-incubation. Superoxide, peroxynitrite, and nitroglycerin itself were highly efficient in inhibiting mitochondrial and yeast aldehyde dehydrogenase activity, which was restored by dithiol compounds such as dihydrolipoic acid and dithiothreitol. Hydrogen peroxide and nitric oxide were rather insensitive inhibitors. Our observations indicate that mitochondrial oxidative stress (especially superoxide and peroxynitrite) in response to organic nitrate treatment may inactivate aldehyde dehydrogenase thereby leading to nitrate tolerance. Glutathionylation obviously amplifies oxidative inactivation of the enzyme providing another regulatory pathway. Furthermore, the present data demonstrate that the mitochondrial dithiol compound dihydrolipoic acid restores mitochondrial aldehyde dehydrogenase activity via reduction of a disulfide at the active site and thereby improves nitrate tolerance.
Hydralazine is a powerful inhibitor of peroxynitrite formation as a possible explanation for its beneficial effects on prognosis in patients with congestive heart failure
2005-12-30, Daiber, Andreas, Oelze, Matthias, Coldewey, Meike, Kaiser, K., Huth, C., Schildknecht, Stefan, Bachschmid, Markus, Nazirisadeh, Y., Ullrich, Volker, Mülsch, Alexander, Münzel, Thomas, Tsilimingas, N.
The hemodynamic and anti-ischemic effects of nitroglycerin (GTN) are rapidly blunted as a result of the development of nitrate tolerance. Hydralazine has been shown to prevent tolerance in experimental and clinical studies, all of which may be at least in part secondary to antioxidant properties of this compound. The antioxidant effects of hydralazine were tested in cell free systems, cultured smooth muscle cells, isolated mitochondria, and isolated vessels. Inhibitory effects on the formation of superoxide and/or peroxynitrite formation were tested using lucigenin and L-012 enhanced chemiluminescence as well as DHE-fluorescence. The peroxynitrite scavenging properties were also assessed by inhibition of nitration of phenol. Prevention of impairment of NO downstream signaling and GTN bioactivation was determined by measurement of P-VASP (surrogate parameter for the activity of the cGMP-dependent kinase-I, cGK-I) and mitochondrial aldehyde dehydrogenase (ALDH-2) activity. Hydralazine dose-dependently decreased the chemiluminescence signal induced by peroxynitrite from SIN-1 and by superoxide from HX/XO in a cell free system, by superoxide in smooth muscle cells and mitochondria acutely challenged with GTN. Moreover, hydralazine inhibited the peroxynitrite-mediated nitration of phenols as well as proteins in smooth muscle cells in a dose-dependent fashion. Finally, hydralazine normalized impaired cGK-I activity as well as impaired vascular ALDH-2 activity. Our results indicate that hydralazine is a highly potent radical scavenger. Thus, the combination with isosorbide dinitrate (ISDN) will favorably influence the nitroso-redox balance in the cardiovascular system in patients with congestive heart failure and may explain at least in part the improvement of prognosis in patients with chronic congestive heart failure.
Peroxynitrite provides the peroxide tone for PGHS-2-dependent prostacyclin synthesis in vascular smooth muscle cells
2005-07, Schildknecht, Stefan, Bachschmid, Markus, Ullrich, Volker
Endotoxin-treated vascular smooth muscle cells (VSMCs) were recently shown to release high amounts of prostacyclin (PGI2) dependent on the induction of prostaglandin endoperoxide synthase-2 (PGHS-2). In contrast to endothelial PGI2-synthase, for which nitration and inhibition by peroxynitrite was reported, addition of SIN-1 as a peroxynitrite-generating system did not cause inhibition but rather doubled PGI2 release by VSMC. The hypothesis of peroxynitrite supplementing an unsaturated peroxide tone for PGHS-2 was supported by H2O2 exerting the same effect. Studies performed with purified PGHS-2 revealed maximal elevation of enzyme activity in the presence of equimolar concentrations of *NO and *O2-, which together form peroxynitrite, while excessive production of either one radical was inhibitory. Most importantly, 6-keto-PGF1alpha formation by intact VSMC depended on a nearly equimolar generation of *NO and *O2- for providing the endogenous peroxide tone. These findings, together with the observation that an excess of exogenously added *NO, as well as uric acid as a scavenger of peroxynitrite potently reduced PGI2 release, underlined the role of peroxynitrite as the dominating and rate-limiting intracellular mediator of peroxide tone in VSMC. The results allow us to postulate a new cross-talk between the *NO and the prostanoid pathways with a crucial role for peroxynitrite in providing the peroxide tone for a continuous activation of PGHS-2.