Superoxid als Signalmolekül für die Endothelfunktion

This work was initiated by the observation that treatment with PN inhibits PGIS with a concomitant appearance of a protein band that positively reacts with an antibody against NT. This study has confirmed tyrosine nitration of PGIS as the underlying mechanism of enzyme inhibition and has provided evidence for a broad physiological and pathophysiological significance of this new posttranslational protein modification. Our results focus on superoxide as a messenger, which combines with NO to exert tyrosine nitration. It turned out that the important process of endothelial activation is primarily based on superoxide generation, which can be mediated by xanthine oxidase (endotoxin), NADPH-oxidase (hyperglycemia/ diabetes), mitochondria (aging) or NO-synthase - oxidase (still under debate, e.g. aging). The results of this study are discussed under the aspects of1.) chemistry and biochemistry of PGIS nitration2.) superoxide as a messenger and3.) endothelial cell activation type IThe most important results are summarized as follows:1. Nitration of prostacyclin synthase· For this study two reliable and sensitive methods for the detection of nitrated PGIS and nitration in general have been established: The immunoprecipitation of nitrated PGIS and the total hydrolysis of microsomes and homogenates followed by HPLC analysis.· Both methods in combination with MALDI-TOF mass spectroscopy, confirmed PGIS nitration by peroxynitrite.· The applied methods confirm a high proteolytic stability of PGIS· Increased PGIS nitration was observed in different model systems, like hypoxia-reoxygenation, hyperglycemia and endotoxemia.2. Superoxide as a messenger· Our results demonstrate that superoxide in combination with NO generates peroxynitrite, which serves as a highly reactive derivate of the superoxide radical in the cellular system.· In the very early stage of inflammation in coronary arteries, xanthine oxidase was identified as major superoxide source.· In the aging process, mitochondria mainly contributed to the superoxide generation, partially caused by the reduction of functionally Mn-SOD· The superoxide generation under hyperglycaemic conditions was caused by the induction of NADPH-oxidase and further via the activation of the PKC cascade.3. Endothelial cell activation Type I· This work confirms, that different endogenous superoxide sources can lead into Phase 1 of endothelial cell activation (ECA-I) and therefore modulate endothelial function.· Endothelial properties are inverted within the first hour of superoxide formation, without affecting gene or protein expression.· The reaction of superoxide with NO leads to increased peroxynitrite generation, which nitrates and inactivates PGIS, thus strongly attenuating the two important signalling molecules NO and prostacyclin, which are essential for vascular homeostasis. In contrast, the substrate of PGIS, PGH2, with its vasoconstricting properties accumulates and therefore the balance of endothelial mediators is shifted towards vasoconstriction

Acetaminophen inhibits prostanoid synthesis by scavenging the PGHS-activator peroxynitrite

The primary pharmacological target of acetaminophen is prostaglandin endoperoxide H2 synthase (PGHS). The enzymatic catalytic mechanism is radical-based, initiated, and maintained by the persistent presence of peroxides, particularly peroxynitrite, which is termed "peroxide tone". Whereas the prevailing concept assumes a direct reduction of the active, oxidized enzyme by acetaminophen, here we show that acetaminophen is a potent scavenger of peroxynitrite (peroxynitrite-mediated phenol nitration, IC50≈72µM; Sin-1-mediated DHR123 oxidation, IC50≈11µM) and thus inhibits PGHS by eliminating the peroxide tone. Nanomolar concentrations of peroxynitrite increased the activity of isolated PGHS and prostacyclin formation by aortic endothelial cells. This elevated activity was efficiently inhibited by pharmacologically relevant concentrations of acetaminophen (IC50{approx}10µM for 6-keto-PGF1α) and other free radical scavengers. However, when the peroxide tone was provided by H2O2 or tert-butyl-OOH, acetaminophen had only negligible inhibitory effects. Our concept could help to explain the efficacy of acetaminophen to inhibit PGHS in cell types with moderate oxidant formation. However, high levels of peroxynitrite or other peroxides such as lipid peroxides formed at inflammatory sites might overwhelm the ability of acetaminophen to decrease PGHS activation. The concept presented herein provides a molecular basis to explain the excellent analgesic and antipyretic properties of acetaminophen together with its minimal anti-inflammatory effects. Schildknecht, S., Daiber, A., Ghisla, S., Cohen, R. A., Bachschmid, M. M. Acetaminophen inhibits prostanoid synthesis by scavenging the PGHS-activator peroxynitrite

MRI of atherosclerosis and fatty liver disease in cholesterol fed rabbits

Abstract Background The globally rising obesity epidemic is associated with a broad spectrum of diseases including atherosclerosis and non-alcoholic fatty liver (NAFL) disease. In the past, research focused on the vasculature or liver, but chronic systemic effects and inter-organ communication may promote the development of NAFL. Here, we investigated the impact of confined vascular endothelial injury, which produces highly inflamed aortic plaques that are susceptible to rupture, on the progression of NAFL in cholesterol fed rabbits. Methods Aortic atherosclerotic inflammation (plaque Gd-enhancement), plaque size (vessel wall area), and composition, were measured with in vivo magnetic resonance imaging (MRI) in rabbits fed normal chow or a 1% cholesterol-enriched atherogenic diet. Liver fat was quantified with magnetic resonance spectroscopy (MRS) over 3 months. Blood biomarkers were monitored in the animals, with follow-up by histology. Results Cholesterol-fed rabbits with and without injury developed hypercholesterolemia, NAFL, and atherosclerotic plaques in the aorta. Compared with rabbits fed cholesterol diet alone, rabbits with injury and cholesterol diets exhibited larger, and more highly inflamed plaques by MRI (P < 0.05) and aggravated liver steatosis by MRS (P < 0.05). Moreover, after sacrifice, damaged (ballooning) hepatocytes and extensive liver fibrosis were observed by histology. Elevated plasma gamma-glutamyl transferase (GGT; P = 0.014) and the ratio of liver enzymes aspartate and alanine aminotransferases (AST/ALT; P = 0.033) indicated the progression of steatosis to non-alcoholic steatohepatitis (NASH). Conclusions Localized regions of highly inflamed aortic atherosclerotic plaques in cholesterol-fed rabbits may contribute to progression of fatty liver disease to NASH with fibrosis

Regulation of cell physiology and pathology by protein S-glutathionylation:lessons learned from the cardiovascular system

Significance: Reactive oxygen and nitrogen species contributing to homeostatic regulation and the pathogenesis of various cardiovascular diseases, including atherosclerosis, hypertension, endothelial dysfunction, and cardiac hypertrophy, is well established. The ability of oxidant species to mediate such effects is in part dependent on their ability to induce specific modifications on particular amino acids, which alter protein function leading to changes in cell signaling and function. The thiol containing amino acids, methionine and cysteine, are the only oxidized amino acids that undergo reduction by cellular enzymes and are, therefore, prime candidates in regulating physiological signaling. Various reports illustrate the significance of reversible oxidative modifications on cysteine thiols and their importance in modulating cardiovascular function and physiology. Recent Advances: The use of mass spectrometry, novel labeling techniques, and live cell imaging illustrate the emerging importance of reversible thiol modifications in cellular redox signaling and have advanced our analytical abilities. Critical Issues: Distinguishing redox signaling from oxidative stress remains unclear. S-nitrosylation as a precursor of S-glutathionylation is controversial and needs further clarification. Subcellular distribution of glutathione (GSH) may play an important role in local regulation, and targeted tools need to be developed. Furthermore, cellular redundancies of thiol metabolism complicate analysis and interpretation. Future Directions: The development of novel pharmacological analogs that specifically target subcellular compartments of GSH to promote or prevent local protein S-glutathionylation as well as the establishment of conditional gene ablation and transgenic animal models are needed. Antioxid. Redox Signal. 16, 524–542

Endogenous peroxynitrite modulates PGHS-1–dependent thromboxane A2 formation and aggregation in human platelets

Aggregation of activated platelets is considerably mediated by the autocrine action of thromboxane A2 (TxA2) which is formed in a prostaglandin endoperoxide H2 synthase-1 (PGHS-1 or COX-1)-dependent manner. The activity of PGHS-1 can be stimulated by peroxides, an effect termed "peroxide tone", that renders PGHS-1 the key regulatory enzyme in the formation of TxA2. Activated platelets release nitric oxide (*NO) and superoxide (O*2) but their interactions with the prostanoid pathway have been controversially discussed in platelet physiology and pathophysiology. The current study demonstrates that endogenously formed peroxynitrite at nanomolar concentrations, originating from the interaction of *NO and *O2, potently activated PGHS-1, which parallels TxA2 formation and aggregation in human platelets. Inhibition of the endogenous formation of either *NO or O*2 resulted in a concentration-dependent decline of PGHS-1 activity, TxA2 release, and aggregation. The concept of peroxynitrite as modulator of TxA2 formation and aggregation explains the interaction of *NO and O*2 with the PGHS pathway and suggests a mechanism by which antioxidants can regulate PGHS-1-dependent platelet aggregation. This may provide a molecular explanation for the clinically observed hyperreactivity of platelets in high-risk patients and serve as a basis for novel therapeutic interventions

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

Autocatalytic nitration of prostaglandin endoperoxide synthase-2 by nitrite inhibits prostanoid formation in rat alveolar macrophages

Aims: Prostaglandin endoperoxide H2 synthase (PGHS) is a well-known target for peroxynitrite-mediated nitration. In several experimental macrophage models, however, the relatively late onset of nitration failed to coincide with the early peak of endogenous peroxynitrite formation. In the present work,weaimed to identify an alternative, peroxynitrite-independent mechanism, responsible for the observed nitration and inactivation of PGHS-2 in an inflammatory cell model. Results: In primary rat alveolar macrophages stimulated with lipopolysaccharide (LPS), PGHS-2 activity was suppressed after 12 h, although the prostaglandin endoperoxide H2 synthase (PGHS-2) protein was still present. This coincided with a nitration of the enzyme. Coincubation with a nitric oxide synthase-2 (NOS - 2) inhibitor preserved PGHS-2 nitration and at the same time restored thromboxane A 2 (TxA 2) synthesis in the cells. Formation of reactive oxygen species (ROS) was maximal at 4 h and then returned to baseline levels. Nitrite (NO 2 -) production occurred later than ROS generation. This rendered generation of peroxynitrite and the nitration of PGHS-2 unlikely. We found that the nitrating agent was formed from NO2-, independent from superoxide (•O 2 - ). Purified PGHS-2 treated with NO 2 - was selectively nitrated on the active site Tyr371, as identified by mass spectrometry (MS). Exposure to peroxynitrite resulted in the nitration not only of Tyr 371, but also of other tyrosines (Tyr). Innovation and Conclusion: The data presented here point to an autocatalytic nitration of PGHS-2 byNO 2 - , catalyzed by the enzyme's endogenous peroxidase activity and indicate a potential involvement of this mechanism in the termination of prostanoid formation under inflammatory conditions. © 2012 Mary Ann Liebert, Inc