European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS).

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.The EU-ROS consortium (COST Action BM1203) was supported by the European Cooperation in Science and Technology (COST). The present overview represents the final Action dissemination summarizing the major achievements of COST Action BM1203 (EU-ROS) as well as research news and personal views of its members. Some authors were also supported by COST Actions BM1005 (ENOG) and BM1307 (PROTEOSTASIS), as well as funding from the European Commission FP7 and H2020 programmes, and several national funding agencies

Mitochondrial Oxidative Stress, Mitochondrial DNA Damage and Their Role in Age-Related Vascular Dysfunction

The prevalence of cardiovascular diseases is significantly increased in the older population. Risk factors and predictors of future cardiovascular events such as hypertension, atherosclerosis, or diabetes are observed with higher frequency in elderly individuals. A major determinant of vascular aging is endothelial dysfunction, characterized by impaired endothelium-dependent signaling processes. Increased production of reactive oxygen species (ROS) leads to oxidative stress, loss of nitric oxide (•NO) signaling, loss of endothelial barrier function and infiltration of leukocytes to the vascular wall, explaining the low-grade inflammation characteristic for the aged vasculature. We here discuss the importance of different sources of ROS for vascular aging and their contribution to the increased cardiovascular risk in the elderly population with special emphasis on mitochondrial ROS formation and oxidative damage of mitochondrial DNA. Also the interaction (crosstalk) of mitochondria with nicotinamide adenosine dinucleotide phosphate (NADPH) oxidases is highlighted. Current concepts of vascular aging, consequences for the development of cardiovascular events and the particular role of ROS are evaluated on the basis of cell culture experiments, animal studies and clinical trials. Present data point to a more important role of oxidative stress for the maximal healthspan (healthy aging) than for the maximal lifespan

Redox regulation of genome stability by effects on gene expression, epigenetic pathways and DNA damage/repair

Reactive oxygen and nitrogen species (e.g. H2O2, nitric oxide) confer redox regulation of essential cellular signaling pathways such as cell differentiation, proliferation, migration and apoptosis. In addition, classical regulation of gene expression or activity, including gene transcription to RNA followed by translation to the protein level, by transcription factors (e.g. NF-κB, HIF-1α) and mRNA binding proteins (e.g. GAPDH, HuR) is subject to redox regulation. This review will give an update of recent discoveries in this field, and specifically highlight the impact of reactive oxygen and nitrogen species on DNA repair systems that contribute to genomic stability. Emphasis will be placed on the emerging role of redox mechanisms regulating epigenetic pathways (e.g. miRNA, DNA methylation and histone modifications). By providing clinical correlations we discuss how oxidative stress can impact on gene regulation/activity and vise versa, how epigenetic processes, other gene regulatory mechanisms and DNA repair can influence the cellular redox state and contribute or prevent development or progression of disease. Keywords: Oxidative stress, Redox signaling, Gene regulation, Epigenetics, DNA repai

Formation of 2-nitrophenol from salicylaldehyde as a suitable test for low peroxynitrite fluxes

There has been some dispute regarding reaction products formed at physiological peroxynitrite fluxes in the nanomolar range with phenolic molecules, when used to predict the behavior of protein-bound aromatic amino acids like tyrosine. Previous data showed that at nanomolar fluxes of peroxynitrite, nitration of these phenolic compounds was outcompeted by dimerization (e.g. biphenols or dityrosine). Using 3-morpholino sydnonimine (Sin-1), we created low fluxes of peroxynitrite in our reaction set-up to demonstrate that salicylaldehyde displays unique features in the detection of physiological fluxes of peroxynitrite, yielding detectable nitration but only minor dimerization products. By means of HPLC analysis and detection at 380 nm we could identify the expected nitration products 3- and 5-nitrosalicylaldehyde, but also novel nitrated products. Using mass spectrometry, we also identified 2-nitrophenol and a not fully characterized nitrated dimerization product. The formation of 2-nitrophenol could proceed either by primary generation of a phenoxy radical, followed by addition of the NO2-radical to the various resonance structures, or by addition of the peroxynitrite anion to the polarized carbonyl group with subsequent fragmentation of the adduct (as seen with carbon dioxide). Interestingly, we observed almost no 3- and 5-nitrosalicylic acid products and only minor dimerization reaction. Our results disagree with the previous general assumption that nitration of low molecular weight phenolic compounds is always outcompeted by dimerization at nanomolar peroxynitrite fluxes and highlight unique features of salicylaldehyde as a probe for physiological concentrations of peroxynitrite

Heparan Sulfate-Editing Extracellular Sulfatases Enhance Vascular Endothelial Growth Factor Bioavailability for Ischemic Heart Repair

Korf-Klingebiel M, Reboll MR, Grote K, et al. Heparan Sulfate-Editing Extracellular Sulfatases Enhance Vascular Endothelial Growth Factor Bioavailability for Ischemic Heart Repair. Circulation research. 2019;125(9):787-801.RATIONALE: Mechanistic insight into the inflammatory response after acute myocardial infarction (MI) may inform new molecularly-targeted treatment strategies to prevent chronic heart failure.; OBJECTIVE: We identified the sulfatase SULF2 in an in silico secretome analysis in bone marrow cells from patients with acute MI and detected increased sulfatase activity in myocardial autopsy samples. SULF2 (Sulf2 in mice) and its isoform SULF1 (Sulf1) act as endo-sulfatases removing 6 O sulfate groups from heparan sulfate (HS) in the extracellular space, thus eliminating docking sites for HS binding proteins. We hypothesized that the Sulfs have a role in tissue repair after MI.; METHODS AND RESULTS: Both Sulfs were dynamically upregulated after coronary artery ligation in mice, attaining peak expression and activity levels during the first week after injury. Sulf2 was expressed by monocytes and macrophages, Sulf1 by endothelial cells and fibroblasts. Infarct border-zone capillarization was impaired, scar size increased, and cardiac dysfunction more pronounced in mice with a genetic deletion of either Sulf1 or Sulf2. Studies in bone marrow-chimeric Sulf-deficient mice and Sulf-deficient cardiac endothelial cells established that inflammatory cell-derived Sulf2 and endothelial cell-autonomous Sulf1 promote angiogenesis. Mechanistically, both Sulfs reduced HS sulfation in the infarcted myocardium, thereby diminishing vascular endothelial growth factor A (Vegfa) interaction with HS. Along this line, both Sulfs rendered infarcted mouse heart explants responsive to the angiogenic effects of HS-binding Vegfa164 but did not modulate the angiogenic effects of non-HS-binding Vegfa120. Treating wild-type mice systemically with the small molecule HS antagonist surfen (bis 2 methyl-4-amino-quinolyl-6-carbamide, 1 mg/kg/day) for 7 days after MI released Vegfa from HS, enhanced infarct border-zone capillarization, and exerted sustained beneficial effects on cardiac function and survival.; CONCLUSIONS: These findings establish HS-editing Sulfs as critical inducers of postinfarction angiogenesis and identify HS sulfation as a therapeutic target for ischemic tissue repair

Nitroglycerin induces DNA damage and vascular cell death in the setting of nitrate tolerance

Nitroglycerin (GTN) and other organic nitrates are widely used vasodilators. Their side effects are development of nitrate tolerance and endothelial dysfunction. Given the potential of GTN to induce nitro-oxidative stress, we investigated the interaction between nitro-oxidative DNA damage and vascular dysfunction in experimental nitrate tolerance. Cultured endothelial hybridoma cells (EA.hy 926) and Wistar rats were treated with GTN (ex vivo: 10–1000 µM; in vivo: 10, 20 and 50 mg/kg/day for 3 days, s.c.). The level of DNA strand breaks, 8-oxoguanine and O 6-methylguanine DNA adducts was determined by Comet assay, dot blot and immunohistochemistry. Vascular function was determined by isometric tension recording. DNA adducts and strand breaks were induced by GTN in cells in vitro in a concentration-dependent manner. GTN in vivo administration leads to endothelial dysfunction, nitrate tolerance, aortic and cardiac oxidative stress, formation of DNA adducts, stabilization of p53 and apoptotic death of vascular cells in a dose-dependent fashion. Mice lacking O 6-methylguanine-DNA methyltransferase displayed more vascular O 6-methylguanine adducts and oxidative stress under GTN therapy than wild-type mice. Although we were not able to prove a causal role of DNA damage in the etiology of nitrate tolerance, the finding of GTN-induced DNA damage such as the mutagenic and toxic adduct O 6-methylguanine, and cell death supports the notion that GTN based therapy may provoke adverse side effects, including endothelial function. Further studies are warranted to clarify whether GTN pro-apoptotic effects are related to an impaired recovery of patients upon myocardial infarction

CD40L contributes to angiotensin II-induced pro-thrombotic state, vascular inflammation, oxidative stress and endothelial dysfunction

CD40 ligand (CD40L) is involved in the vascular infiltration of immune cells and pathogenesis of atherosclerosis. Additionally, T cell CD40L release causes platelet, dendritic cell and monocyte activation in thrombosis. However, the role of CD40L in angiotensin II (ATII)-driven vascular dysfunction and hypertension remains incompletely understood. We tested the hypothesis that CD40L contributes to ATII-driven vascular inflammation by promoting platelet-leukocyte activation, vascular infiltration of immune cells and by amplifying oxidative stress. C57BL/6 and CD40L(-/-) mice were infused with ATII (1 mg/kg/day for 7 days) using osmotic minipumps. Vascular function was recorded by isometric tension studies, and reactive oxygen species (ROS) were monitored in blood and heart by optical methods. Western blot, immunohistochemistry, FACS analysis and real-time RT-PCR were used to analyze immune cell distribution, pro-inflammatory cytokines, NAPDH oxidase subunits, T cell transcription factors and other genes of interest. ATII-treated CD40L(-/-) mice showed improved endothelial function, suppression of blood platelet-monocyte interaction (FACS), platelet thrombin generation (calibrated automated thrombography) and coagulation (bleeding time), as well as decreased oxidative stress in the aorta, heart and blood compared to wild-type mice. Moreover, ATII-treated CD40L(-/-) mice displayed decreased levels of TH1 cytokines released by splenic CD4( ) T cells (ELISA) and lower expression levels of NOX-2, T-bet and P-selectin as well as diminished immune cell infiltration in aortic tissue compared to controls. Our results demonstrate that many ATII-induced effects on vascular dysfunction, such as vascular inflammation, oxidative stress and a pro-thrombotic state, are mediated at least in part via CD40L