The Global Burden of Disease Study identified cardiovascular risk factors as leading causes of global deaths and life-years lost. Endothelial dysfunction is a pathomechanism associated with these risk factors and stressors, and is an early predictor of atherosclerosis. Oxidative stress triggers endothelial dysfunction, a hallmark of cardiovascular diseases. Endothelial dysfunction is largely based on impaired endothelial nitric oxide synthase (eNOS) function and activity or down-stream signalling of nitric oxide. Molecules affecting eNOS functionality, eNOS protein itself as well as components of the eNOS down-stream signalling cascade are attractive therapeutic targets for vascular integrity and homeostasis. Potential strategies for the pharmacological exploitation of these targets are highlighted in the present work. Recent advances and future therapeutic strategies for the treatment of cardiovascular and other diseases should be directed against such targets, including targets so far not considered sufficiently as well as lifestyle changes.

Oral treatment of apolipoprotein E-knockout (ApoE-KO) mice with the putative sirtuin 1 (SIRT1) activator resveratrol led to a reduction of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in the heart. In contrast, the SIRT1 inhibitor EX527 enhanced the superoxide production in isolated human polymorphonuclear granulocytes. In human monocytic THP-1 cells, phorbol ester-stimulated superoxide production was enhanced by inhibitors of histone deacetylases (HDACs; including quisinostat, trichostatin A (TSA), PCI34051, and tubastatin A) and decreased by inhibitors of histone acetyltransferases [such as garcinol, curcumin, and histone acetyltransferase (HAT) Inhibitor II]. These results indicate that protein acetylation and deacetylation may represent crucial mechanisms regulating NADPH oxidase-mediated superoxide production. In cell-free systems, incubation of recombinant Rac1 with SIRT1 resulted in decreased Rac1 acetylation. Mass spectrometry analyses identified lysine 166 (K166) in Rac1 as a residue targeted by SIRT1. Deacetylation of Rac1 by SIRT1 markedly reduced the interaction of Rac1 with p67phox in in vitro assays. Computational modeling analyses revealed that K166 deacetylation of Rac1 led to a 5-fold reduction in its binding affinity to guanosine-5'-triphosphate, and a 21-fold decrease in its binding potential to p67phox. The latter is crucial for Rac1-mediated recruitment of p67phox to the membrane and for p67phox activation. In conclusion, both SIRT1 and non-sirtuin deacetylases play a role in regulating NADPH oxidase activity. Rac1 can be directly deacetylated by SIRT1 in a cell-free system, leading to an inhibition of Rac1-p67phox interaction. The downstream targets of non-sirtuin deacetylases are still unknown. The in vivo significance of these findings needs to be investigated in future studies.

stress-induced Regulation expression ER stress and Ca -disturbance Abstract Paraoxonase-2 (PON2) is a ubiquitously expressed anti-oxidative protein, which is largely found in the endoplasmic reticulum (ER). Addressing the cytoprotective functions of PON2, we observed that PON2 overexpression provided significant resistance to ER stress-induced caspase-3 activation when the ER stress was induced by interference with protein modification (by tunicamycin or dithiotreitol), but not when ER stress was induced by disturbance of Ca 2+ homeostasis (by thapsigargin or A23187). When analyzing the underlying molecular events, we found an activation of the PON2 promoter in response to all tested ER stress-inducing stimuli. However, only tunicamycin and dithiotreitol resulted in increased PON2 mRNA and protein levels. In contrast, when ER stress was caused by thapsigargin or A23187, we observed a Ca 2+ -dependent active degradation of PON2 mRNA, elicited by its 5’untranslated region. In addition, thapsigargin and A23187 also induced PON2 protein degradation by a Ca 2+ -dependent calpain-mediated mechanism. Thus, we provide evidence that independent mechanisms mediate the degradation of PON2 mRNA and protein after disturbance of Ca 2+ homeostasis. Further, because Ca 2+ -disturbance induces ER stress, but abrogates the otherwise protective function of PON2 against ER stress-induced apoptosis, we propose that the underlying cause of ER stress determines the efficacy of putative cellular defence mechanisms. of 78kDa; PON2, paraoxonase-2; ROS, reactive oxygen species; UPR, unfolded protein response pathway; UTR, untranslated region. and PON2 overexpression endothelial cells significantly more to stress-induced apoptosis caspase-3 activation), after down-regulation of PON2 as to whether PON2 was generally protective against ER stress-induced apoptosis. We here report that the protective and anti-apoptotic effect of PON2 was not a general phenomenon; it was absent, when the ER stress was induced by Ca 2+ -deregulation. The current study was designed to uncover the underlying molecular events leading to a loss of protection by PON2 under specific ER stress conditions. caspase-dependent death Ca 2+ -dependent anti-apoptotic phosphatidylinositol 3-kinase (data not shown); (3) PON2 protein degradation occurs independent of the mRNA’s 5’UTR, because PON2-GFP protein is degraded after Ca 2+ -disturbance although the PON2-GFP mRNA lacks any UTR; (4) We show that calpain proteases mediate Ca 2+ -stimulated PON2 protein decay. Taken together, our findings suggest that UPR-induced activation of the PON2 promoter is a relatively non-specific effect independent of the inducing stimulus. In contrast, the subsequent regulation of PON2 mRNA and protein stability seems to be specific for the stress-inducing mechanism(s). Increases in cytosolic Ca 2+ -concentrations lead to rapid degradation of both PON2 mRNA and protein by separate, unrelated mechanisms. We thus demonstrate that the mechanisms causing ER stress are of major importance for PON2 regulation and its protective function against ER stress-induced caspase-3 activation.

Ethnopharmacological relevance: Red fruit (Pandanus conoideus Lam) oil (RFO) is utilized by inhabitants of the Papua Island to treat diseases such as infections, cancer, and cardiovascular disease, but the mechanism of action is unknown. Aim of the study: We have recently shown that RFO stimulates nitric oxide (NO) production in endothelial cells. The present study was conducted to investigate the molecular mechanism of endothelial NO synthase (eNOS) activation by RFO. Materials and methods: NO production by endothelial cells was determined with electron paramagnetic resonance. The vascular function of isolated mouse aorta was examined using a wire myograph system. Phosphorylation of eNOS was studied with Western blot analyses. Results: RFO induced concentration-dependent vasodilation in isolated mouse aorta. The vasodilator effect of RFO was lost in endothelium-denuded aorta and in aorta from mice deficient in eNOS. Treatment of human EA.hy 926 endothelial cells with RFO led to an enhancement of eNOS phosphorylation at serine 1177 and NO production. The RFO-induced eNOS phosphorylation and NO production were reduced by inhibitors of Akt or AMPK, but not by an inhibitor of CaMKII. The effects of RFO were decreased by pharmacological inhibition of PI3K, indicating an involvement of the PI3K-Akt pathway. Moreover, acetone-soluble fractions and oily fractions of RFO showed higher efficacies than the RFO polar fraction in activating eNOS. Conclusions: RFO contains highly active compounds that enhance NO production through Akt- or AMPK-mediated eNOS phosphorylation. The increase in endothelial NO production is likely to represent one of the molecular mechanisms responsible for the therapeutic effects of RFO.

Krankenhäuser setzen verstärkt auch auf Ärzte aus dem Ausland. Doch wie lässt sich eine Vergleichbarkeit der Abschlüsse und Qualifikationen gewährleisten? Ein Kooperationsstudiengang zwischen einer Uniklinik in Ho-Chi-Minh-Stadt (Vietnam) und der Universitätsmedizin Mainz zeigt, wie es gehen könnte.

Red fruit oil (RFO) is used in traditional medicine for the treatment of a number of diseases. However, evidence for the biological effects and action mechanisms is still lacking. In the present study, we show for the first time that RFO stimulated the phosphorylation of the endothelial nitric oxide synthase (eNOS) and enhanced the NO production in human endothelial cells. In isolated mouse aorta, RFO induced a vasodilation, with a significant effect evident at a concentration as low as 1:100,000 dilution. The RFO-induced vasodilation could be completely prevented by eNOS inhibition, indicating that RFO contains highly potent substances stimulating eNOS activity. In addition, RFO reduced oxidative stress in endothelial cells, as demonstrated with three different methods. In the comet assay, RFO significantly decreased oxidative DNA damage induced by genotoxicants. These results suggest that enhanced endothelial NO production and reduced oxidative stress may represent part of the molecular mechanisms responsible for the therapeutic effects of RFO.

| Under physiological conditions, nitric oxide (NO) is produced in the vasculature mainly by the endothelial nitric oxide synthase (eNOS). Endothelial NO relaxes blood vessels, inhibits platelet activity, and protects against atherosclerosis. Under pathological conditions such as hypertension, diabetes, and hypercholesterolemia, eNOS may become uncoupled. Uncoupled eNOS generates superoxide at the expense of NO and contributes substantially to oxidative stress and endothelial dysfunction. Major mechanisms of eNOS uncoupling include deficiency of the eNOS cofactor tetrahydrobiopterin, deficiency of the eNOS substrate Larginine, and eNOS S-glutathionylation. Reversal of eNOS uncoupling may represent a feasible strategy for the prevention and treatment of cardiovascular diseases.

Under physiological conditions, nitric oxide (NO) is produced in the vasculature mainly by the endothelial NO synthase (eNOS). Experiments using gene-disrupted mice have demonstrated that eNOS has antihypertensive, antithrombotic, and antiatherosclerotic effects. Recent studies show that eNOS is expressed not only in the endothelium but also in the perivascular adipose tissue (PVAT). Resveratrol prevents eNOS uncoupling and upregulates eNOS expression and activity. These effects of resveratrol are well established for the eNOS enzyme in the endothelium. Interestingly, resveratrol also improves PVAT function. However, a causal role for eNOS in the effects of resveratrol on PVAT function has not yet been verified and needs to be studied in the future.

Background and Purpose We have recently shown that a reduced function of endothelial nitric oxide synthase (eNOS) in the perivascular adipose tissue (PVAT) contributes crucially to obesity‐induced vascular dysfunction in mice. The current study was conducted to test the hypothesis that vascular dysfunction in obesity can be reversed by in vivo improvement of PVAT eNOS activity. Experimental Approach Male C57BL/6J mice were fed a high‐fat diet (HFD) for 22 weeks to induce obesity. During the last 4 weeks of HFD feeding, the obese mice were treated p.o. with the standardized Crataegus extract WS® 1442, which has been shown previously to improve eNOS activity. Key Results Diet‐induced obesity in mice markedly reduced the vasodilator response of thoracic aorta to acetylcholine in wire myograph experiments. Strikingly, this vascular dysfunction was only evident in PVAT‐containing aorta but not in PVAT‐free aorta. In vivo treatment of obese mice with WS® 1442 had no effect on body weight or epididymal fat mass, but completely restored the vascular function of PVAT‐containing aorta. Feeding a HFD led to a reduced phosphorylation and an enhanced acetylation of PVAT eNOS, both effects were reversed by WS® 1442 treatment. Conclusion and Implications PVAT plays a key role in vascular dysfunction in diet‐induced obese mice. Not obesity itself, but a PVAT dysfunction is responsible for obesity‐induced vascular disorders. Improving PVAT function by pharmacological means (e.g. with WS® 1442) can ameliorate vascular function even without reducing body weight or fat mass. Linked Articles This article is part of a themed section on Molecular Mechanisms Regulating Perivascular Adipose Tissue – Potential Pharmacological Targets? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.20/issuetoc

Major reactive oxygen species (ROS)–producing systems in vascular wall include NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase, xanthine oxidase, the mitochondrial electron transport chain, and uncoupled endothelial nitric oxide (NO) synthase. ROS at moderate concentrations have important signaling roles under physiological conditions. Excessive or sustained ROS production, however, when exceeding the available antioxidant defense systems, leads to oxidative stress. Animal studies have provided compelling evidence demonstrating the roles of vascular oxidative stress and NO in atherosclerosis. All established cardiovascular risk factors such as hypercholesterolemia, hypertension, diabetes mellitus, and smoking enhance ROS generation and decrease endothelial NO production. Key molecular events in atherogenesis such as oxidative modification of lipoproteins and phospholipids, endothelial cell activation, and macrophage infiltration/activation are facilitated by vascular oxidative stress and inhibited by endothelial NO. Atherosclerosis develops preferentially in vascular regions with disturbed blood flow (arches, branches, and bifurcations). The fact that these sites are associated with enhanced oxidative stress and reduced endothelial NO production is a further indication for the roles of ROS and NO in atherosclerosis. Therefore, prevention of vascular oxidative stress and improvement of endothelial NO production represent reasonable therapeutic strategies in addition to the treatment of established risk factors (hypercholesterolemia, hypertension, and diabetes mellitus).

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.

Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid with anti-inflammatory, antiviral and anti-cancer properties. Beneficial cardiovascular effects such as increased nitric oxide (NO) production through enhancement of endothelial NO synthase (eNOS) activity and upregulation of eNOS expression have been demonstrated for this compound. In the present study, immortalized human EA.hy 926 endothelial cells were incubated for up to 1 h with 1–100 µM BA and with the phosphatidylinositol-3-kinase (PI3K) inhibitors LY294002 and wortmannin, or the estrogen receptor (ER) antagonist ICI 182,780. Phosphorylation status of eNOS and total eNOS protein were analyzed by Western blotting using a serine 1177 phosphosite-specific antibody. Bioactive NO production was assessed by determination of cGMP content in rat lung fibroblasts (RFL-6) reporter cells. Short-term incubation of EA.hy 926 cells with BA resulted in eNOS phosphorylation at the serine 1177 residue in a concentration- and time-dependent manner with a half-maximal effective concentration of 0.57 µM. This was associated with an enhanced production of NO. BA-induced eNOS phosphorylation and NO production was completely blocked by pretreatment with ICI 182,780, and was attenuated by pretreatment with the PI3K inhibitors wortmannin and LY294002. These results indicate that fast non-genomic effects of ER with downstream signaling through the PI3K/Akt pathway and consecutive eNOS phosphorylation at serine 1177 are involved in BA-induced eNOS activation.

The antioxidant effects of resveratrol (3,5,4'‐trihydroxy‐trans‐stilbene) contribute substantially to the health benefits of this compound. Resveratrol has been shown to be a scavenger of a number of free radicals. However, the direct scavenging activities of resveratrol are relatively poor. The antioxidant properties of resveratrol in vivo are more likely to be attributable to its effect as a gene regulator. Resveratrol inhibits NADPH oxidase‐mediated production of ROS by down‐regulating the expression and activity of the oxidase. This polyphenolic compound reduces mitochondrial superoxide generation by stimulating mitochondria biogenesis. Resveratrol prevents superoxide production from uncoupled endothelial nitric oxide synthase by up‐regulating the tetrahydrobiopterin‐synthesizing enzyme GTP cyclohydrolase I. In addition, resveratrol increases the expression of various antioxidant enzymes. Some of the gene‐regulating effects of resveratrol are mediated by the histone/protein deacetylase sirtuin 1 or by the nuclear factor‐E2‐related factor‐2. In this review article, we have also summarized the cardiovascular effects of resveratrol observed in clinical trials.Linked ArticlesThis article is part of a themed section on Redox Biology and Oxidative Stress in Health and Disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.12/issuetoc

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 l -arginine deficiency and eNOS uncoupling in PVAT. The combination therapy with l -arginine and arginase inhibitors may represent a novel therapeutic strategy for obesity-induced vascular disease.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin best characterized for its survival and differentiative effects on neurons. Recent studies demonstrated that BDNF and its receptors are also expressed in the peripheral vasculature, where it stimulates angiogenesis and promotes the survival of endothelial cells. This study was designed to investigate the angiogenic effects of BDNF and its expressional regulation by tumor necrosis factor (TNF-alpha) and protein kinase C (PKC) in endothelial cells. In the Matrigel angiogenesis assay, BDNF-stimulated vascular tube formation of human umbilical vein endothelial cells (HUVEC) was completely blocked by an inhibition of the TrkB receptor, but only partially inhibited by the inhibition of the p75(NTR) signaling. Treatment of HUVEC and HUVEC-derived EA.hy 926 cells with TNF-alpha resulted in a downregulation of BONE expression, which could be prevented by the TNFR1 antagonist WP9QY. BDNF downregulation by TNF-alpha was associated with decreased angiogenic activity of HUVEC. The effect of TNF-alpha on BDNF expression could not be abolished by the inhibition of PKC. Treatment of HUVEC and EA.hy 926 cells with PKC-activating phorbol esters (phorbol-12-myristate-13-acetate, PMA or phorbol-12,13-dibutyrate) resulted in a downregulation of BDNF expression, whereas the inactive 4 alpha-phorbol-12,13-didecanoate was without effect. PMA had no significant effect on BDNF mRNA stability and the downregulation of BDNF mRNA expression by PKC activation was likely a transcriptional event. BDNF downregulation by PMA could be prevented by PKC inhibitors Go 6983 and rottlerin, but not by Go 6976. Thus, a Go 6983/rottlerin-sensitive PKC isoform is likely to be responsible for PMA-induced BDNF downregulation. (C) 2015 S. Karger AG, Basel

Objective To find out whether dexamethasone induces an uncoupling of the endothelial nitric oxide synthase (eNOS). Methods & Results A major cause of eNOS uncoupling is a deficiency of its cofactor tetrahydrobiopterin (BH4). Treatment of human EA.hy 926 endothelial cells with dexamethasone decreased mRNA and protein expression of both BH4-synthesizing enzymes: GTP cyclohydrolase I and dihydrofolate reductase. Consistently, a concentration- and time-dependent reduction of BH4, dihydrobiopterin (BH2) as well as BH4: BH2 ratio was observed in dexamethasone-treated cells. Surprisingly, no evidence for eNOS uncoupling was found. We then analyzed the expression and phosphorylation of the eNOS enzyme. Dexamethasone treatment led to a down-regulation of eNOS protein and a reduction of eNOS phosphorylation at serine 1177. A reduction of eNOS expression may lead to a relatively normal BH4: eNOS molar ratio in dexamethasone-treated cells. Because the BH4-eNOS stoichiometry rather than the absolute BH4 amount is the key determinant of eNOS functionality (i.e., coupled or uncoupled), the down-regulation of eNOS may represent an explanation for the absence of eNOS uncoupling. Phosphorylation of eNOS at serine 1177 is needed for both the NO-producing activity of the coupled eNOS and the superoxide-producing activity of the uncoupled eNOS. Thus, a reduction of serine 1177 phosphorylation may render a potentially uncoupled eNOS hardly detectable. Conclusions Although dexamethasone reduces BH4 levels in endothelial cells, eNOS uncoupling is not evident. The reduction of NO production in dexamethasone-treated endothelial cells is mainly attributable to reduced eNOS expression and decreased eNOS phosphorylation at serine 1177.

Artichoke (Cynara scolymus L.) is one of the world’s oldest medicinal plants with multiple health benefits. We have previously shown that artichoke leaf extracts and artichoke flavonoids upregulate the gene expression of endothelial-type nitric oxide synthase (eNOS) in human endothelial cells. Whereas NO produced by the eNOS is a vasoprotective molecule, NO derived from the inducible iNOS plays a pro-inflammatory role in the vasculature. The present study was aimed to investigate the effects of artichoke on iNOS expression in human coronary artery smooth muscle cells (HCASMC). Incubation of HCASMC with a cytokine mixture led to an induction of iNOS mRNA expression. This iNOS induction was concentration- and time-dependently inhibited by an artichoke leaf extract (1–100 µg/mL, 6 h or 24 h). Consistently, the artichoke leaf extract also reduced cytokine-induced iNOS promoter activation and iNOS protein expression. In addition, treatment of HCASMC with four well-known artichoke compounds (cynarin > cyanidin > luteolin ≈ cynaroside) led to a downregulation iNOS mRNA and protein expression, with cynarin being the most potent one. In conclusion, artichoke contains both eNOS-upregulating and iNOS-downregulating compounds. Such compounds may contribute to the beneficial effects of artichoke and may per se have therapeutic potentials.