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.

induced obesity, namely uncoupling of the endothelial nitric oxide (NO) synthase (eNOS) in PVAT. Materials and MethodsMaterials and Methods are available in the online-only Data Supplement.© 2015 American Heart Association, Inc. Objective-The present study was conducted to investigate the contribution of perivascular adipose tissue (PVAT) to vascular dysfunction in a mouse model of diet-induced obesity. Approach and Results-Obesity was induced in male C57BL/6J mice with a high-fat diet for 20 weeks, and vascular function was studied with myograph. In PVAT-free aortas isolated from obese mice, the endothelium-dependent, nitric oxide-mediated vasodilator response to acetylcholine remained normal. In contrast, a clear reduction in the vasodilator response to acetylcholine was observed in aortas from obese mice when PVAT was left in place. Adipocytes in PVAT were clearly positive in endothelial nitric oxide synthase (eNOS) staining, and PVAT nitric oxide production was significantly reduced in obese mice. High-fat diet had no effect on eNOS expression but led to eNOS uncoupling, evidenced by diminished superoxide production in PVAT after eNOS inhibition. As mechanisms for eNOS uncoupling, arginase induction and l-arginine deficiency were observed in PVAT. Obesity-induced vascular dysfunction could be reversed by ex vivo l-arginine treatment and arginase inhibition. Conclusions-Diet-induced obesity leads to Results Role of PVAT in Vascular Dysfunction in Diet-Induced Obese MiceMale C57BL/6J mice were fed with either HFD or normal control diet (NCD) for 20 weeks starting at the age of 8 weeks.The HFD-treated mice developed obesity (Table). Vasodilator response to acetylcholine was studied using aortas with or without PVAT in a wire myograph system. In aortas without PVAT, no significant differences in endothelium-dependent, NO-mediated vasodilator response to acetylcholine were found between NCD and HFD groups ( Figure 1A). In contrast, a clear reduction in the vasodilator response to acetylcholine was observed in the aorta of obese animals compared with lean controls when PVAT was left intact ( Figure 1B). The acetylcholine-induced vasodilation in the mouse aorta was NO-dependent because it could be completely blocked by the NO synthase inhibitor N G -nitro-l-arginine methyl ester (l-NAME; Figure 1C). In endothelium-denuded aortic rings from control mice, acetylcholine induced a significant vasodilation if PVAT was left intact ( Figure I in the online-only Data Supplement). The PVAT-mediated vasodilation was preventable with l-NAME, indicating that PVAT-derived NO contributes to acetylcholine-induced vasodilation under normal conditions. Reduced NO Production in PVAT of Diet-Induced Obese MiceTo directly access NO production from PVAT, time-lapse fluorescence imaging was performed with aorta sections prepared from NCD or HFD-fed mice stained with the fluorescent NO probe 4,5-diaminofluorescein diacetate. As shown in Figure 1D, basal NO production could be detected in PVAT. The PVAT NO production was enhanced by ace...

ObjectiveIn diabetes, vascular dysfunction is characterized by impaired endothelial function due to increased oxidative stress. Empagliflozin, as a selective sodium-glucose co-transporter 2 inhibitor (SGLT2i), offers a novel approach for the treatment of type 2 diabetes by enhancing urinary glucose excretion. The aim of the present study was to test whether treatment with empagliflozin improves endothelial dysfunction in type I diabetic rats via reduction of glucotoxicity and associated vascular oxidative stress.MethodsType I diabetes in Wistar rats was induced by an intravenous injection of streptozotocin (60 mg/kg). One week after injection empagliflozin (10 and 30 mg/kg/d) was administered via drinking water for 7 weeks. Vascular function was assessed by isometric tension recording, oxidative stress parameters by chemiluminescence and fluorescence techniques, protein expression by Western blot, mRNA expression by RT-PCR, and islet function by insulin ELISA in serum and immunohistochemical staining of pancreatic tissue. Advanced glycation end products (AGE) signaling was assessed by dot blot analysis and mRNA expression of the AGE-receptor (RAGE).ResultsTreatment with empagliflozin reduced blood glucose levels, normalized endothelial function (aortic rings) and reduced oxidative stress in aortic vessels (dihydroethidium staining) and in blood (phorbol ester/zymosan A-stimulated chemiluminescence) of diabetic rats. Additionally, the pro-inflammatory phenotype and glucotoxicity (AGE/RAGE signaling) in diabetic animals was reversed by SGLT2i therapy.ConclusionsEmpagliflozin improves hyperglycemia and prevents the development of endothelial dysfunction, reduces oxidative stress and improves the metabolic situation in type 1 diabetic rats. These preclinical observations illustrate the therapeutic potential of this new class of antidiabetic drugs.

Aims: Oxidative stress is involved in the development of cardiovascular disease. There is a growing body of evidence for a crosstalk between different enzymatic sources of oxidative stress. With the present study, we sought to determine the underlying crosstalk mechanisms, the role of the mitochondrial permeability transition pore (mPTP), and its link to endothelial dysfunction. Results: NADPH oxidase (Nox) activation (oxidative burst and translocation of cytosolic Nox subunits) was observed in response to mitochondrial reactive oxygen species (mtROS) formation in human leukocytes. In vitro, mtROS-induced Nox activation was prevented by inhibitors of the mPTP, protein kinase C, tyrosine kinase cSrc, Nox itself, or an intracellular calcium chelator and was absent in leukocytes with p47phox deficiency (regulates Nox2) or with cyclophilin D deficiency (regulates mPTP). In contrast, the crosstalk in leukocytes was amplified by mitochondrial superoxide dismutase (type 2) (MnSOD +/ -) deficiency. In vivo, increases in blood pressure, degree of endothelial dysfunction, endothelial nitric oxide synthase (eNOS) dysregulation/uncoupling (e.g., eNOS S-glutathionylation) or Nox activity, p47phox phosphorylation in response to angiotensin-II (AT-II) in vivo treatment, or the aging process were more pronounced in MnSOD +/ -mice as compared with untreated controls and improved by mPTP inhibition by cyclophilin D deficiency or sanglifehrin A therapy. Innovation: These results provide new mechanistic insights into what extent mtROS trigger Nox activation in phagocytes and cardiovascular tissue, leading to endothelial dysfunction. Conclusions: Our data show that mtROS trigger the activation of phagocytic and cardiovascular NADPH oxidases, which may have fundamental implications for immune cell activation and development of AT-II-induced hypertension. Antioxid. Redox Signal. 20, 247-266.

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.