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Alpha-linolenic acid (ALA), docosahexaenoic acid (DHA), rumenic acid (RmA), and punicic acid (PunA) are claimed to influence several physiological functions including insulin sensitivity, lipid metabolism and inflammatory processes. In this double-blind randomized controlled trial, we investigated the combined effect of ALA, DHA, RmA and PunA on subjects at risk of developing metabolic syndrome. Twenty-four women and men were randomly assigned to two groups. Each day, they consumed two eggs enriched with oleic acid (control group) or enriched with ALA, DHA, RmA, and PunA (test group) for 3 months. The waist circumference decreased significantly (-3.17 cm, p &lt, 0.001) in the test group. There were no major changes in plasma insulin and blood glucose in the two groups. The dietary treatments had no significant effect on endothelial function as measured by peripheral arterial tonometry, although erythrocyte nitrosylated hemoglobin concentrations tended to decrease. The high consumption of eggs induced significant elevations in plasma low-density lipoprotein (LDL)- and high-density lipoprotein (HDL)-cholesterol (p &lt, 0.001), which did not result in any change in the LDL/HDL ratio in both groups. These results indicate that consumption of eggs enriched with ALA, DHA, RmA and PunA resulted in favorable changes in abdominal obesity without affecting other factors of the metabolic syndrome.

Fe-based materials are currently considered for manufacturing biodegradable coronary stents. Here we show that Fe has a strong potential to generate hydroxyl radicals (HO·) during corrosion. This HO· generation, but not corrosion, can be inhibited by catalase. Oxidative stress was observed (increased HO-1 expression) in aortic rings after direct exposure to Fe, but not in the presence of catalase or after indirect exposure. This oxidative stress response induced an uncoupling of eNOS in, and a consequent reduced NO production by endothelial cells exposed to Fe. In isolated rat aortic rings NO production was also reduced by HO· generated during Fe corrosion, as indicated by the protective role of catalase. Finally, all these mechanisms contributed to impaired endothelium- dependent relaxation in aortic rings caused by HO· generated during the direct contact with Fe. This deleterious impact of Fe corrosion on the endothelial function should be integrated when considering the use of biodegradable Fe-based alloys for vascular implants.

Objective— Members of the microRNA (miR)-199a family, namely miR-199a-5p and miR-199a-3p, have been recently identified as potential regulators of cardiac homeostasis. Also, upregulation of miR-199a expression in cardiomyocytes was reported to influence endothelial cells. Whether miR-199a is expressed by endothelial cells and, if so, whether it directly regulates endothelial function remains unknown. We investigate the implication of miR-199a products on endothelial function by focusing on the NOS (nitric oxide synthase)/NO pathway. Approach and Results— Bovine aortic endothelial cells were transfected with specific miRNA inhibitors (locked-nucleic acids), and potential molecular targets identified with prediction algorithms were evaluated by Western blot or immunofluorescence. Ex vivo experiments were performed with mice treated with antagomiRs targeting miR-199a-3p or -5p. Isolated vessels and blood were used for electron paramagnetic resonance or myograph experiments. eNOS (endothelial NO synthase) activity (through phosphorylations Ser1177/Thr495) is increased by miR-199a-3p/-5p inhibition through an upregulation of the PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B) and calcineurin pathways. SOD1 (superoxide dismutase 1) and PRDX1 (peroxiredoxin 1) upregulation was also observed in locked-nucleic acid–treated cells. Moreover, miR-199a-5p controls angiogenesis and VEGFA (vascular endothelial growth factor A) production and upregulation of NO-dependent relaxation were observed in vessels from antagomiR-treated mice. This was correlated with increased circulated hemoglobin-NO levels and decreased superoxide production. Angiotensin infusion for 2 weeks also revealed an upregulation of miR-199a-3p/-5p in vascular tissues. Conclusions— Our study reveals that miR-199a-3p and miR-199a-5p participate in a redundant network of regulation of the NOS/NO pathway in the endothelium. We highlighted that inhibition of miR-199a-3p and -5p independently increases NO bioavailability by promoting eNOS activity and reducing its degradation, thereby supporting VEGF-induced endothelial tubulogenesis and modulating vessel contractile tone.

ObjectiveTo investigate the beneficial role of prebiotics on endothelial dysfunction, an early key marker of cardiovascular diseases, in an original mouse model linking steatosis and endothelial dysfunction.DesignWe examined the contribution of the gut microbiota to vascular dysfunction observed in apolipoprotein E knockout (Apoe−/−) mice fed an n-3 polyunsaturated fatty acid (PUFA)-depleted diet for 12 weeks with or without inulin-type fructans (ITFs) supplementation for the last 15 days. Mesenteric and carotid arteries were isolated to evaluate endothelium-dependent relaxation ex vivo. Caecal microbiota composition (Illumina Sequencing of the 16S rRNA gene) and key pathways/mediators involved in the control of vascular function, including bile acid (BA) profiling, gut and liver key gene expression, nitric oxide and gut hormones production were also assessed.ResultsITF supplementation totally reverses endothelial dysfunction in mesenteric and carotid arteries of n-3 PUFA-depleted Apoe−/− mice via activation of the nitric oxide (NO) synthase/NO pathway. Gut microbiota changes induced by prebiotic treatment consist in increased NO-producing bacteria, replenishment of abundance in Akkermansia and decreased abundance in bacterial taxa involved in secondary BA synthesis. Changes in gut and liver gene expression also occur upon ITFs suggesting increased glucagon-like peptide 1 production and BA turnover as drivers of endothelium function preservation.ConclusionsWe demonstrate for the first time that ITF improve endothelial dysfunction, implicating a short-term adaptation of both gut microbiota and key gut peptides. If confirmed in humans, prebiotics could be proposed as a novel approach in the prevention of metabolic disorders-related cardiovascular diseases.

Photosensitizers (PS) are ideally devoid of any activity in the absence of photoactivation, and rely on molecular oxygen for the formation of singlet oxygen ((1)O2) to produce cellular damage. Off-targets and tumor hypoxia therefore represent obstacles for the use of PS for cancer photodynamic therapy. Herein, we describe the characterization of OR141, a benzophenazine compound identified through a phenotypic screening for its capacity to be strictly activated by light and to kill a large variety of tumor cells under both normoxia and hypoxia. This new class of PS unraveled an unsuspected common mechanism of action for PS that involves the combined inhibition of the mammalian target of rapamycin (mTOR) signaling pathway and proteasomal deubiquitinases (DUBs) USP14 and UCH37. Oxidation of mTOR and other endoplasmic reticulum (ER)-associated proteins drives the early formation of high molecular weight (MW) complexes of multimeric proteins, the concomitant blockade of DUBs preventing their degradation and precipitating cell death. Furthermore, we validated the antitumor effects of OR141 in vivo and documented its highly selective accumulation in the ER, further increasing the ER stress resulting from (1)O2 generation upon light activation.

The data presented in this article are associated with the research article entitled “Heme-Nitrosylated Hemoglobin and Oxidative Stress in Women Consuming Combined Contraceptives. Clinical Application of the EPR Spectroscopy” (Lobysheva et al., 2017 [1]), and describe the characteristics of redox status in blood, as well as biochemical and clinical parameters of young female subjects consuming (or not) contraceptive pills (CP). Erythrocyte concentration of reduced thiols reflecting erythrocyte redox capacity was measured before and after sample deproteinization by electron paramagnetic resonance spectroscopy (EPR) using a nitroxide biradical spin probe specifically interacting with reduced thiols; additional data were obtained by a colorimetric method using Ellman׳s reagents in the same samples. The products of nitric oxide oxidation, nitrite and total NOx (in presence of nitrate reductase) were measured in the plasma of study subjects by a colorimetric assay based on the detection of red-violet colored azo dye after reaction of nitrite with the Griess reagent. Biochemical and clinical parameters reflective of cardiovascular risk factors (diastolic blood pressure, C-reactive protein, triglycerides and homocysteine concentrations in venous blood) were compared in subgroups of consumers of CP containing ethinyl estradiol and different types of synthetic progestogens. Parameters reflective of the integrity of the vasculature, - erythrocyte concentration of heme-nitrosylated hemoglobin (5-coordinate α-heme-FeII-NO, HbNO) measured directly by the EPR subtraction method; index of reactive hyperemia response (FRHI) measured by digital pulse tonometry using EndoPAT; oxidative vascular stress measured as total plasma peroxide concentration were compared in subgroups of young women taking CP containing ethinyl estradiol at different concentrations and for various durations. Keywords: Endothelial function, Vascular oxidative status, Contraceptives, Heme-nitrosylated hemoglobin, Electron paramagnetic resonance

The water channels, aquaporins (AQPs) are key mediators of transcellular fluid transport. However, their expression and role in cardiac tissue is poorly characterized. Particularly, AQP1 was suggested to transport other molecules (nitric oxide (NO), hydrogen peroxide (H2O2)) with potential major bearing on cardiovascular physiology. We therefore examined the expression of all AQPs and the phenotype of AQP1 knockout mice (vs. wild-type littermates) under implanted telemetry in vivo, as well as endothelium-dependent relaxation in isolated aortas and resistance vessels ex vivo. Four aquaporins were expressed in wild-type heart tissue (AQP1, AQP7, AQP4, AQP8) and two aquaporins in aortic and mesenteric vessels (AQP1-AQP7). AQP1 was expressed in endothelial as well as cardiac and vascular muscle cells and co-segregated with caveolin-1. AQP1 knockout (KO) mice exhibited a prominent microcardia and decreased myocyte transverse dimensions despite no change in capillary density. Both male and female AQP1 KO mice had lower mean BP, which was not attributable to altered water balance or autonomic dysfunction (from baroreflex and frequency analysis of BP and HR variability). NO-dependent BP variability was unperturbed. Accordingly, endothelium-derived hyperpolarizing factor (EDH(F)) or NO-dependent relaxation were unchanged in aorta or resistance vessels ex vivo. However, AQP1 KO mesenteric vessels exhibited an increase in endothelial prostanoids-dependent relaxation, together with increased expression of COX-2. This enhanced relaxation was abrogated by COX inhibition. We conclude that AQP1 does not regulate the endothelial EDH or NO-dependent relaxation ex vivo or in vivo, but its deletion decreases baseline BP together with increased prostanoids-dependent relaxation in resistance vessels. Strikingly, this was associated with microcardia, unrelated to perturbed angiogenesis. This may raise interest for new inhibitors of AQP1 and their use to treat hypertrophic cardiac remodeling.

Introduction Based on their good mechanical properties, bioresorbable Fe-based materials have been proposed during the last decade as candidate alloys for coronary stents. Previous investigations on the biocompatibility of metallic materials often overlooked the complexity of the biodegradation phenomenon. Objective We document, for the first time, the generation of reactive oxygen species (ROS) during Fe-based materials corrosion and their deleterious impacts on endothelial function. Method ROS generation was evaluated by the terephtalic acid (TA) hydroxylation assay. The endothelium of rat aortic rings was directly exposed (6 h) by inserting an iron wire into the lumen in the presence/absence of catalase. Indirect exposure to iron wire was included to assess the potential role of solubilized iron ions. Nitric oxide (NO) production by rings was measured by EPR spin-trapping, and endothelium-dependent relaxation was assessed with the organ-bath method. Induction of oxidative stress in rings was assessed via the mRNA expression of the oxidative stress response gene heme oxygenase-1 (HO-1). Results Iron exhibited a strong potential to generate ROS in the TA assay. ROS generation, but not corrosion, was inhibited by catalase. Direct contact with iron significantly impaired endothelium-dependent relaxation of aortic rings. Direct contact with the material in the presence of catalase, or indirect contact did not affect endothelium function. NO production by isolated rat aortic rings was inhibited by ROS generated by the corrosion of iron, as indicated by the protective role of catalase. Expression of HO-1 was increased after direct exposure to iron, not to indirect exposure or in the presence of catalase. Conclusion The demonstration of ROS production during corrosion, induction of oxidative stress, inhibition of NO production and consequent endothelium dysfunction raise concern about the biocompatibility of biodegradable Fe-based alloys for vascular implants.

An increased risk of venous thromboembolism was identified in young women consuming combined contraceptive pills (CP) suggesting a disturbance of vascular homeostasis but the impact of CP on endothelial function and redox status of the vasculature was not thoroughly analyzed. We measured the bioavailability of nitric oxide (NO), a main mediator of vascular homeostasis in a cohort of young female subjects (n=114) and compared the results in users or not of CPs containing ethinyl estradiol and synthetic progestogens. Vascular NO availability was measured by quantification of the heme-nitrosylated hemoglobin (5-coordinate-α-HbNO) concentrations in venous erythrocytes using Electron Paramagnetic Resonance spectroscopy (EPR). Vascular oxidative status was assessed by measurement of peroxides in plasma, and of the thiol redox state in erythrocytes. In addition, endothelial function was assessed by digital reactive hyperemia pulse tonometry using EndoPAT. We observed that the HbNO level was significantly lower in erythrocytes of subjects consuming CPs versus controls (162±8 and 217±12 nmol/L ) . This correlated with significantly increased levels of plasma peroxides (1.8±0.1 mmol/L versus 0.8±0.1 mmol/L in controls) and decreased concentrations of erythrocyte reduced thiols (by 12%). Interestingly, the level of oxidized ceruloplasmin-Cu(II) was also significantly higher in the group consuming CPs. The EndoPAT index showed a trend towards impairment in CP users, and was significantly lower in subjects that consumed CPs containing drospirenone, and had lowest erythrocyte HbNO levels. Conclusion This cross-sectional cohort study demonstrates that a decrease of HbNO measured by quantitative EPR in human venous erythrocytes is correlated with the development of endothelial dysfunction under CPs consumption, in parallel with increased vascular oxidative stress.

Reduced bioavailability of nitric oxide is a hallmark of endothelial dysfunction in metabolic and cardiovascular diseases, but its quantification in circulating blood remains a challenge.NO can form iron-nitrosyl complexes with hemoglobin(5-coordinate-α-HbNO) in erythrocytes(RBCs).We hypothesized that this complex reflects bioavailability of vascular NO and endothelial function in vivo.We developed a modified subtraction method using EPR to quantify it in RBCs from mouse, rat and human venous blood. NO could be supplied from vascular or erythrocytic NOS.We detected eNOS proteins in rodent and human RBC.To test eNOS functionality, we measured nitrite/ nitrate production and HbNO formation in human RBCs and from eNOS(+/+) and eNOS(-/-)mice in vitro and its sensitivity to NOS or arginase inhibitors. Nitrite and HbNO signals increased after arginase inhibition and were abrogated upon NOS inhibition in human and eNOS(+/+) but insensitive to these modulators in eNOS(-/-)RBCs. We found that upon exposure to exogenous NO-donor, the formation of HbNO was higher in hypoxic conditions(1%O2:0.018+/–0.002 compared to room air 0.0036+/–0.0004 μmol HbNO/μmolNO-donor).Accordingly, the stability of pre-formed HbNO was higher under hypoxia(17% degradation after 30 min vs 49% at room air), and preserved at 21% of O2 by incubation with catalase(CAT:2μM HbNO vs 0.5μM HbNO in untreated controls).Conversely, CAT inhibition increased ROS formation in RBCs, measured by FACS analysis of DCFDA fluorescence. This suggested that HbNO formation is sensitive to oxidative degradation, possibly by H2O2. We compared circulating HbNO levels in venous RBCs from normal volunteers or patients with cardiovascular diseases and found decreased HbNO in patients(0.141+/–11μM vs 0.22+/–12μM in volunteers; N=38 and48).We conclude that HbNO reflects exposure of RBCs to NO in vivo and is sensitive to oxidative degradation by H2O2.HbNO could be developed as a biomarker of NO bioavailability and/or oxidative stress ex vivo. The author hereby declares no conflict of interest

Endothelial dysfunction is considered to be an early event in atherosclerosis and plays a pivotal role in the development, progression and clinical complications of atherosclerosis. Previous studies have shown the beneficial effects of combined inhibition of thromboxane synthase and antagonism of thromboxane receptors by BM-573 on atherosclerosis; however our knowledge about the beneficial effects of BM-573 on endothelial function and increased blood pressure related to early stage of atherosclerosis is limited. In the present study, we investigated the effects of short-term (3uM, 1 hour) and chronic (10mg/L, 8 weeks) treatments with BM-573 on vasodilatory function, nitric oxide (NO) bioavailability, oxidative stress and systolic blood pressure in 15 weeks old apolipoprotein E-deficient (ApoE-KO) mice. ApoE-KO mice showed a reduced endothelium-derived relaxation. In addition, NO bioavailability was reduced and oxidative stress and blood pressure were increased in ApoE-KO mice versus wild-type mice. BM-573 treatments were able to improve the relaxation profile in ApoE-KO mice. Short-term effects of BM-573 were mainly mediated by an increased phosphorylation of both eNOS and Akt, whereas BM-573 in vivo treatment also reduced oxidative stress and restored NO bioavailability. In addition, chronic administration of BM-573 reduced systolic blood pressure in ApoE-KO mice. In conclusion, pharmacological modulation of TxA2 biosynthesis and biological activities by dual TP antagonism/TxAS inhibition with BM-573, already known to prevent plaque formation, has the potential to correct vasodilatory dysfunction at the early stages of atherosclerosis.

AIMS: In endothelial cells, caveolin-1 (cav-1) is known to negatively modulate the activation of endothelial nitric oxide synthase, a key regulator of blood pressure (BP). However, the impact of genetic alteration of cav-1 on vascular nitric oxide (NO) production and BP homeostasis in vivo is unknown. METHODS AND RESULTS: We used spectral analysis of systolic blood pressure (SBP) variability in mice chronically equipped with telemetry implants to identify frequency ranges (0.05-0.4 Hz; very low frequency, VLF) specifically responding to NO, independently of changes in absolute BP or systemic neurohormone levels. VLF variability was inversely correlated to aortic vasodilator-stimulated Ser(239) phosphoprotein (VASP) phosphorylation, reflecting NO bioactivity. We show that mice deficient in cav-1 have decreased VLF variability paralleled with enhanced systemic and vascular production of NO at unchanged mean SBP levels. Conversely, VLF variability was increased upon acute injection of mice, with a peptide containing the caveolin-scaffolding domain (CSD; residues 82-101) fused to an internalization sequence of antennapedia that decreased vascular and circulating NO in vivo. CONCLUSION: These data highlight the functional importance of cav-1 for the production of bioactive NO in conduit arteries and its control of central BP variability. Given the impact of the latter on target organ damage, this raises the interest for genetic, pharmacological, or molecular interventions that modulate cav-1 expression in diseases with NO-dependent endothelial dysfunction.

The biology of NO (nitric oxide) is poorly explained by the activity of the free radical NO (·NO) itself. Although·NO acts in an autocrine and paracrine manner, it is also in chemical equilibrium with other NO species that constitute stable stores of NO bioactivity. Among these species, S-nitrosylated hemoglobin ( S-nitrosohemoglobin; SNO-Hb) is an evolved transducer of NO bioactivity that acts in a responsive and exquisitely regulated manner to control cardiopulmonary and vascular homeostasis. In SNO-Hb, O2sensing is dynamically coupled to formation and release of vasodilating SNOs, endowing the red blood cell (RBC) with the capacity to regulate its own principal function, O2delivery, via regulation of blood flow. Analogous, physiological actions of RBC SNO-Hb also contribute to central nervous responses to blood hypoxia, the uptake of O2from the lung to blood, and baroreceptor-mediated control of the systemic flow of blood. Dysregulation of the formation, export, or actions of RBC-derived SNOs has been implicated in human diseases including sepsis, sickle cell anemia, pulmonary arterial hypertension, and diabetes mellitus. Delivery of SNOs by the RBC can be harnessed for therapeutic gain, and early results support the logic of this approach in the treatment of diseases as varied as cancer and neonatal pulmonary hypertension.

This study investigated the consequences of deletion of the long isoform of myosin light chain kinase (MLCK210) on the cardiovascular changes induced by the bacterial endotoxin lipopolysaccharide (LPS) and cecal ligation puncture using MLCK210 −/− mice. Here, we provide evidence that deletion of MLCK210 enhanced survival after intraperitoneal injection of LPS or cecal ligation puncture. LPS-induced vascular hyporeactivity to vasoconstrictor agents was completely prevented in aorta from MLCK210 −/− mice. This was associated with a decreased up-regulation of nuclear facor-κB expression and activity, inducible nitric-oxide synthase, and level of oxidative stress in the vascular media. Furthermore, LPS-induced increase of nitric oxide production in the circulation and tissues (including heart, liver, and lung) that was correlated with an increased expression of inducible nitric-oxide synthase was also reduced in MLCK210 −/− mice. These data demonstrate a role for MLCK210 in endotoxin shock injury associated with oxidative and nitrosative stresses and vascular hyporeactivity.

SCOPE Western diets are characterized by low intake of n-3 PUFA compensated by constant amounts of n-6 PUFA. Reduced intake of n-3 PUFA is associated with increased cardiovascular risk, as observed in nonalcoholic fatty liver disease patients. The study aimed to evaluating the impact of dietary n-3 PUFA depletion on endothelial function, an early key event of cardiovascular diseases. METHODS AND RESULTS C57Bl/6J or apolipoprotein E knock-out (apoE-/- ) were fed control (CT) or n-3 PUFA-depleted diets (DEF) for 12 wks. Mice fed n-3 DEF diet developed a hepatic steatosis, linked to changes in hepatic expression of genes controlled by Sterol Regulatory Element Binding Protein-1 and -2. Vascular function was assessed on second- and third-order mesenteric arteries and n-3 PUFA-depleted apoE-/- mice presented endothelial dysfunction characterized by decreased vasorelaxation in response of acetylcholine. The presence of a nitric oxide synthase (NOS) inhibitor blunted the relaxation in each groups and heme-nitrosylated hemoglobin blood (Hb-NO) level was significantly lower in n-3 PUFA-depleted apoE-/- mice. CONCLUSION Twelve weeks of n-3 DEF diet promote steatosis and accelerate the process of endothelial dysfunction in apoE-/- mice by a mechanism involving the NOS/NO pathway. We propose n-3 PUFA-depleted apoE-/- mice as a new model to study endothelial dysfunction related to hepatic steatosis independently of obesity.

An enhanced endothelial formation of nitric oxide (NO) by red wine polyphenolic compounds (RWPs) has been involved in the protective effect of chronic intake of red wine on coronary diseases. However, the mechanism underlying the activation of endothelial NO synthase (eNOS) remains unclear. In the presence of indomethacin and charybdotoxin plus apamin to prevent the formation of prostanoids and endothelium-derived hyperpolarizing factor, respectively, RWPs caused pronounced endothelium-dependent relaxations in porcine coronary arteries. Relaxations to RWPs were abolished by N(omega)-nitro-L-arginine (L-NA, a competitive inhibitor of NO synthase) and the membrane permeant analog of superoxide dismutase (SOD), MnTMPyP, and reduced by polyethylene glycol-SOD (PEG-SOD), PEG-catalase and inhibitors of PI3-kinase (wortmannin and LY294002). RWPs caused the L-NA-sensitive formation of NO, as assessed by electron spin resonance spectroscopy and the formation of cyclic guanosine monophosphate in coronary artery endothelial cells; these responses were reduced by MnTMPyP, PEG-catalase, and inhibitors of PI3-kinase. RWPs caused the sustained phosphorylation of Akt and eNOS at Ser1177 in endothelial cells, which were abolished by MnTMPyP and inhibitors of PI3-kinase. These data demonstrate that RWPs induce the redox-sensitive activation of the PI3-kinase/Akt pathway in endothelial cells which, in turn, causes phosphorylation of eNOS, resulting in an increased formation of NO.

The ability of various nitric oxide (NO) donors to induce long-lasting inhibition of contraction in isolated arteries was compared. All the studied compounds elicited a relaxant effect in rat aortic rings precontracted with norepinephrine (NE). Almost maximal relaxation was obtained with 1 μM of each compound. The S -nitrosating agents S- nitrosoglutathione (GSNO), S -nitroso- N -acetylpenicillamine, S -nitroso- N -acetylcysteine, and sodium nitroprusside (1 μM) produced a decrease of the maximal effect of NE that persisted after removal of the drug. This hyporesponsiveness to NE was associated with a relaxant effect of N- acetylcysteine, a low-molecular weight thiol that can displace NO from cysteine-NO bonds. Such modifications of contraction were not observed in aortic rings previously exposed to 1 μM S- nitrosocysteine, glyceryl trinitrate, 3-morpholinosydnonimine, or 2-( N , N -diethylamino)-diazenolate-2-oxide (DEA-NO). The same differential effects of GSNO and DEA-NO on contraction were also observed in porcine coronary arteries. Rat aortic rings previously exposed to 100 μM GSNO, but not to 100 μM DEA-NO, displayed a persistent increase in NO content (determined by NO spin trapping) and cysteine-NO residues (determined by immunostaining with an anti-cysteine-NO antiserum). The GSNO-induced increase in cysteine-NO residues in aortic tissue was prevented by the thiolmodifying agent p -hydroxymercuribenzoic acid. This study shows that in isolated arteries, the effects of S- nitrosating agents differed from those of other NO-donating agents. S- Nitrosating agents induced a persistent inhibition of contraction, which was attributed to the formation of releasable NO stores by S- nitrosation of tissue thiols. These differential effects of NO donors may be important for orientating their therapeutic indications.

S-Nitrosation of cysteine residues plays an important role in nitric oxide (NO) signaling and transport. The aim of the present study was to investigate the role of S-nitrosothiols as a storage form of NO, which may account for the long-lasting effects in the vasculature. Rat aorta exposed to S-nitrosoglutathione (GSNO) displayed, even after washout of the drug, a persistent increase in cysteine-NO residues (detected by immunostaining using an antiserum that selectively recognized S-nitrosoproteins) and in NO content (detected by NO spin-trapping), a persistent attenuation of the effect of vasoconstrictors, and a relaxant response upon addition of low molecular weight (LMW) thiols. Rat mesenteric and porcine coronary artery exposed in vitro to GSNO, as well as aorta and mesenteric arteries removed from rats treated in vivo with GSNO, displayed similar modifications of contraction. In isolated aorta exposed to GSNO, the decrease of the contractile response and the relaxant effect of LMW thiols were both blunted by NO scavengers (oxyhemoglobin or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) or by a cyclic GMP-dependent protein kinase inhibitor (Rp-8-bromoguanosine-3',5'-cyclic monophosphorothioate). In these arteries, mercuric chloride (which cleaves the cysteine-NO bond) exerted a transient relaxation, completely abolished the one of LMW thiols, and blunted the increase in cysteine-NO residues and NO content. Together, these data support the idea that S-nitrosation of cysteine residues is involved in long-lasting effects of NO on arterial tone. They suggest that S-nitrosation of tissue thiols is a mechanism of formation of local NO stores from which biologically active NO can subsequently be released.

Iodine deficiency (ID) induces microvascular changes in the thyroid gland via a TSH-independent reactive oxygen species-hypoxia inducible factor (HIF)-1α-vascular endothelial growth factor (VEGF) pathway. The involvement of nitric oxide (NO) in this pathway and the role of calcium (Ca(2+)) and of ryanodine receptors (RYRs) in NO synthase 3 (NOS3) activation were investigated in a murine model of goitrogenesis and in three in vitro models of ID including primary cultures of human thyrocytes. ID activated NOS3 and the production of NO in thyrocytes in vitro and increased the thyroid blood flow in vivo. Using bevacizumab (a blocking antibody against VEGF-A) in mice, it appeared that NOS3 is activated upstream of VEGF-A. L-nitro arginine methyl ester (L-NAME, a NOS inhibitor) blocked the ID-induced increase in thyroid blood flow in vivo and NO production in vitro, as well as ID-induced VEGF-A mRNA and HIF-1α expression in vitro, while S-nitroso-acetyl-penicillamine (SNAP, a NO donor) did the opposite. Ca(2+) is involved in this pathway as intracellular Ca(2+) flux increased after ID, and thapsigargin activated NOS3 and increased VEGF-A mRNA expression. Two of the three known mammalian RYR isoforms (RYR1 and RYR2) were shown to be expressed in thyrocytes. RYR inhibition using ryanodine at 10 μ M decreased ID-induced NOS3 activation, HIF-1α and VEGF-A expression, while RYR activation with ryanodine at 1nM increased NOS3 activation and VEGF-A mRNA expression. In conclusion, during the early phase of TSH-independent ID-induced microvascular activation, ID sequentially activates RYRs and NOS3, thereby supporting ID-induced activation of the NO/HIF-1α/VEGF-A pathway in thyrocytes.

Background— β1-2-adrenergic receptors (AR) are key regulators of cardiac contractility and remodeling in response to catecholamines. β3-AR expression is enhanced in diseased human myocardium, but its impact on remodeling is unknown. Methods and Results— Mice with cardiac myocyte-specific expression of human β3-AR (β3-TG) and wild-type (WT) littermates were used to compare myocardial remodeling in response to isoproterenol (Iso) or Angiotensin II (Ang II). β3-TG and WT had similar morphometric and hemodynamic parameters at baseline. β3-AR colocalized with caveolin-3, endothelial nitric oxide synthase (NOS) and neuronal NOS in adult transgenic myocytes, which constitutively produced more cyclic GMP, detected with a new transgenic FRET sensor. Iso and Ang II produced hypertrophy and fibrosis in WT mice, but not in β3-TG mice, which also had less re-expression of fetal genes and transforming growth factor β1. Protection from Iso-induced hypertrophy was reversed by nonspecific NOS inhibition at low dose Iso, and by preferential neuronal NOS inhibition at high-dose Iso. Adenoviral overexpression of β3-AR in isolated cardiac myocytes also increased NO production and attenuated hypertrophy to Iso and phenylephrine. Hypertrophy was restored on NOS or protein kinase G inhibition. Mechanistically, β3-AR overexpression inhibited phenylephrine-induced nuclear factor of activated T-cell activation. Conclusions— Cardiac-specific overexpression of β3-AR does not affect cardiac morphology at baseline but inhibits the hypertrophic response to neurohormonal stimulation in vivo and in vitro, through a NOS-mediated mechanism. Activation of the cardiac β3-AR pathway may provide future therapeutic avenues for the modulation of hypertrophic remodeling.

Impaired nitric oxide (NO)-dependent endothelial function is associated with the development of cardiovascular diseases. We hypothesized that erythrocyte levels of nitrosylated hemoglobin (HbNO-heme) may reflect vascular endothelial function in vivo. We developed a modified subtraction method using Electron Paramagnetic Resonance (EPR) spectroscopy to identify the 5-coordinate α-HbNO (HbNO) concentration in human erythrocytes and examined its correlation with endothelial function assessed by peripheral arterial tonometry (PAT). Changes in digital pulse amplitude were measured by PAT during reactive hyperemia following brachial arterial occlusion in a group of healthy volunteers (50 subjects). Erythrocyte HbNO levels were measured at baseline and at the peak of hyperemia. We digitally subtracted an individual model EPR signal of erythrocyte free radicals from the whole EPR spectrum to unmask and quantitate the HbNO EPR signals. RESULTS: Mean erythrocyte HbNO concentration at baseline was 219+/-12 nmol/L (n = 50). HbNO levels and reactive hyperemia (RH) indexes were higher in female (free of contraceptive pills) than male subjects. We observed a dynamic increase of HbNO levels in erythrocytes isolated at 1-2 min of post-occlusion hyperemia (120+/-8% of basal levels); post-occlusion HbNO levels were correlated with basal levels. Both basal and post-occlusion HbNO levels were significantly correlated with reactive hyperemia (RH) indexes (r = 0.58; P

458 Implication of microRNA 199a3p and 199a5p in vascular function : modulation of the eNOS/NO pathway {#article-title-2} Introduction: MiR 199a3p and 199a5p have been mainly implicated in proliferation, cell survival and remodeling in context of cancerogenesis but more recent data proposed a role in cardiovascular functions. Objectives: We seek to assess how miR199a3p and miR199a5p modulate endothelial function by identifying their molecular targets in endothelial cells. Methods: Mice were treated with miR199a3p/5p inhibitors and after 30 days, the contractile profile and endothelial function were evaluated ex vivo in the aorta. Cultured endothelial cells were transfected with miR199a3p/5p inhibitors or a scramble sequence. NO production, expression and/or activity of eNOS and its major regulators were measured by EPR and Western blot respectively. Implication of Akt pathway was analysed using LY294002 treatment. Angiogenesis was investigated in 2D-culture in matrigel support. Results: Vessels from mice treated with antagomirs against miR199a3p or 5p showed a larger NO-dependent relaxation compared to controls. Circulating HbNO measured by EPR in venous blood collected from these mice was significantly increased compared to controls suggesting a role of both miRs in the control of the NOS/ NO pathway. Repression of miR 199a3p/5p improved eNOS activity through an increased eNOS phosphorylation at serine 1177 and a decreased phosphorylation status of threonine 495. The eNOS allosteric regulator, cav-1, was not modulated by LNA treatments. However, both treatments promoted Akt activation and calcineurin expression. Furthermore, addition of LY294002 inhibited the increase of eNOS phosphorylation at serine 1177 induced by LNA treatment. Interestingly, repression of miR199a5p also promoted SOD expression, suggesting that miR199a5p also improved NO bioavailability. In addition, inhibition of miR199a5p upregulated VEGF production and promoted tubes formation in endothelial cells. Conclusion: Our results demonstrate that miR199a3p/5p modulate the NOS/NO pathway in the endothelium by promoting NO production and repressing NO degradation. These results also suggest a strong implication of the Akt pathway in this modulation. Interestingly, miR199a5p shows additional control of angiogenesis. # 459 Role of endothelial cell adenosine deaminase acting on RNA-2 in ischemic/inflammatory disease in vivo {#article-title-3} Background: RNA binding proteins (RBPs) are key players in posttranscriptional regulation of mRNAs. Adenosine deaminase acting on RNA-2 (ADAR2) enzyme binds to double-stranded RNAs and controls brain development. The role of ADAR2 in endothelial cell function has not been described so far. Methods and Results: ADAR2 is expressed in human and murine endothelial cells and is 2-fold induced by hypoxia or hind limb ischemia in mice (P

Objective— We analyzed the role of caveolin-1 (Cav-1) in the cross-talk between NADPH oxidase and endothelial nitric oxide synthase (eNOS) signaling in endothelial caveolae. Methods and Results— In intact endothelial cells, angiotensin II (AII) concurrently increased NO and O 2 −· production (to 158±12% and 209±5% of control). NO production was sensitive to inhibition of NADPH oxidase and small interfering RNA downregulation of nonreceptor tyrosine kinase cAbl. Reciprocally, N-nitro- l -arginine methyl ester, a NOS inhibitor, partly inhibited O 2 −· stimulated by AII (by 47±11%), indicating eNOS uncoupling, as confirmed by increased eNOS monomer/dimer ratio (by 35%). In endothelial cell fractions separated by isopycnic ultracentrifugation, AII promoted colocalization of cAbl and the NADPH oxidase subunit p47phox with eNOS to Cav-1-enriched fractions, as confirmed by proximity ligation assay. Downregulation of Cav-1 by small interfering RNA (to 50%), although it preserved eNOS confinement, inhibited AII-stimulated p47phox translocation and NADPH oxidase activity in Cav-1-enriched fractions and reversed eNOS uncoupling. AII infusion produced hypertension and decreased blood hemoglobin-NO in Cav-1 +/+ mice but not in heterozygote Cav-1 +/− mice with similar Cav-1 reduction. Conclusion— Cav-1 critically regulates reactive oxygen species–dependent eNOS activation but also eNOS uncoupling in response to AII, underlining the possibility to treat endothelial dysfunction by modulating Cav-1 abundance.

ObjectivesThe aim of this study was to investigate whether nebivolol has added effects on left ventricular (LV) dysfunction and remodeling early after myocardial infarction (MI) beyond its β1-receptor–blocking properties.BackgroundNebivolol is a third-generation selective β1-adrenoreceptor antagonist that stimulates endothelial cell nitric oxide (NO) production and prevents vascular reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. Both endothelial NO synthase–derived NO production and NADPH oxidase activation are critical modulators of LV dysfunction early after MI.MethodsMice with extensive anterior MI (n = 90) were randomized to treatment with nebivolol (10 mg/kg/day), metoprolol-succinate (20 mg/kg/day), or placebo for 30 days starting on day 1 after surgery.ResultsInfarct size was similar among the groups. Both β1-adrenergic receptor antagonists caused a similar decrease in heart rate. Nebivolol therapy improved endothelium-dependent vasorelaxation and increased early endothelial progenitor cells 4 weeks after MI compared with metoprolol and placebo. Nebivolol, but not metoprolol, inhibited cardiac NADPH oxidase activation after MI, as detected by electron spin resonance spectroscopy analysis. Importantly, nebivolol, but not metoprolol, improved LV dysfunction 4 weeks after MI (LV ejection fraction: nebivolol vs. metoprolol vs. placebo: 32 ± 4% vs. 17 ± 6% vs. 19 ± 4%; nebivolol vs. metoprolol: p < 0.05) and was associated with improved survival 4 weeks post-MI compared with placebo. Nebivolol had a significantly more pronounced inhibitory effect on cardiomyocyte hypertrophy after MI compared with metoprolol.ConclusionsNebivolol improves LV dysfunction and survival early after MI likely beyond the effects provided by conventional β1-receptor blockade. Nebivolol induced effects on NO-mediated endothelial function, early endothelial progenitor cells and inhibition of myocardial NADPH oxidase likely contribute to these beneficial effects of nebivolol early after MI.

The biological status of nitrite recently evolved from an inactive end product of nitric oxide (NO) metabolism to a major intravascular and tissue storage of NO. Several enzymes and proteins may indeed work as nitrite reductases. The endothelial NO synthase (eNOS) is proposed to be one of them, particularly when oxygen is lacking. Here, we examined whether the lack of caveolin, a scaffold protein known to limit eNOS activity under basal conditions and to be down-regulated in tumor vessels, could favor the reconversion of nitrite into NO and thereby promote angiogenesis. We found that nitrite-rich serum from caveolin-deficient mice and exogenous nitrite exert proangiogenic effects on aortic explants cultured in a three-dimensional collagen matrix. We identified a higher intrinsic capacity of caveolin-deficient vessels and endothelial cells to convert nitrite into bioactive NO. These effects did occur under moderate hypoxia and were abolished on exposure to a NO scavenger. Evidence for eNOS acting as a nitrite reductase derived from the failure to reproduce the proangiogenic effects of nitrite on eNOS-deficient aorta rings and endothelial cells. Finally, in a mouse tumor model, we documented the higher nitrite content in hypoxic tumors and identified inducible NO synthase as the major source of nitrite. Altogether, these data identify the lack of caveolin observed in the tumor vasculature as a favorable ground for nitrite-driven formation of endothelial tubes in the hypoxic tumor microenvironment. This work also strengthens the therapeutic value of the modulation of caveolin expression to interfere with tumor angiogenesis. (Mol Cancer Res 2009;7(7):1056–63)

Rationale: Nutritional disorders are often associated with a high risk to develop cardiovascular diseases, endothelial dysfunction being an early key marker. Changing the gut microbiota by prebiotics counteracts metabolic diseases. The work tests the hypothesis of a potential impact of prebiotics on the endothelial dysfunction induced by a nutritional depletion in n-3 polyunsaturated fatty acids (PUFA) in ApoE-/- mice. Methods: C57Bl/6J (WT) and ApoE-/- (KO) mice were fed a n-3 PUFA depleted-diet (DEF) for 12 weeks. For the last 15 days, WT and KO mice were or not supplemented with prebiotics (PRE). The vascular function was evaluated in mesenteric and carotid arteries. Circulating HbNO adducts were assessed by EPR. Results: The n-3 PUFA depletion induced an endothelial dysfunction in KO-DEF mesenteric arteries, as attested by a significant decrease of endothelial-dependent relaxation compared to WT DEF. Interestingly, PRE treatment in this group was able to improve the endothelial function by restoring the endothelial-dependent relaxation. PRE treatment in KO-DEF mice also lead to a vessel enlargement, to an increased intima-media thickness in the 2nd order mesenteric arteries, to an increased basal tone and a better KCl-induced contractibility of mesenteric arteries, compared to non-supplemented KO DEF mice. More, circulating HbNO was increased in KO-DEF-PRE mice and an improvement of endothelial function was also observed in carotid arteries. Conclusion: Our results demonstrated an outward remodeling and an improvement of the endothelial dysfunction in mesenteric arteries following prebiotic supplementation, probably due to activation of NO/NOS pathway. Data are in progress to evaluate how changes in the gut microbiota composition and function act on vascular function. This supports a novel therapeutic role for dietary prebiotics in the control of cardiovascular disease risk

Background— The therapeutic effects of nonspecific β-blockers are limited by vasoconstriction, thus justifying the interest in molecules with ancillary vasodilating properties. Nebivolol is a selective β 1 -adrenoreceptor antagonist that releases nitric oxide (NO) through incompletely characterized mechanisms. We identified endothelial β 3 -adrenoreceptors in human coronary microarteries that mediate endothelium- and NO-dependent relaxation and hypothesized that nebivolol activates these β 3 -adrenoreceptors. Methods and Results— Nebivolol dose-dependently relaxed rodent coronary resistance microarteries studied by videomicroscopy (10 μmol/L, −86±6% of prostaglandin F2α contraction); this was sensitive to NO synthase (NOS) inhibition, unaffected by the β 1-2 -blocker nadolol, and prevented by the β 1-2-3 -blocker bupranolol ( P 3 -adrenoreceptor–deficient mice. Nebivolol (10 μmol/L) also relaxed human coronary microvessels (−71±5% of KCl contraction); this was dependent on a functional endothelium and NO synthase but insensitive to β 1-2 -blockade (all P 3 -adrenoreceptor– or endothelial NOS–deficient mice. In cultured endothelial cells, 10 μmol/L nebivolol increased NO release by 200% as measured by electron paramagnetic spin trapping, which was also reversed by NOS inhibition. In parallel, endothelial NOS was dephosphorylated on threonine 495 , and fura-2 calcium fluorescence increased by 91.8±23.7%; this effect was unaffected by β 1-2 -blockade but abrogated by β 1-2-3 -blockade (all P Conclusions— Nebivolol dilates human and rodent coronary resistance microarteries through an agonist effect on endothelial β 3 -adrenoreceptors to release NO and promote neoangiogenesis. These properties may prove particularly beneficial for the treatment of ischemic and cardiac failure diseases through preservation of coronary reserve.

S-Nitrosating nitric oxide (NO) donors like S-nitrosoglutathione (GSNO) induce a persistent inhibition of vascular tone, through the formation of releasable NO stores. In this study, we investigate whether GSNO also induces NO stores-related effects in vessels exhibiting tolerance to glyceryl-trinitrate. Rat aortic rings treated with glyceryl-trinitrate (100 microM for 1 h) exhibited increased level of superoxide and a decrease in NO elevation and relaxation induced by subsequent addition of glyceryl-trinitrate. In glyceryl-trinitrate-treated rings as in controls, pre-exposure to GSNO (1 microM for 30 min) induced a persistent hyporesponsiveness to noradrenaline and a relaxant response to N-acetylcysteine (a low molecular weight thiol which can displace NO from NO stores), both of which being inhibited by guanylyl-cyclase or cyclic GMP-dependent protein kinase inhibitors. These data indicate that GSNO can promote the formation of releasable NO stores in arteries exhibiting increased superoxide level and tolerance to glyceryl-trinitrate. Formation of releasable NO stores is of potential interest to restore the protective effect of NO in organic nitrate-tolerant blood vessels.

Beneficial effects of lactobacilli have been reported in experimental colitis. On the other hand, despite the controversial role of nitric oxide (NO) in the inflammatory gut process, a protective action of exogenous NO in inflammation has been suggested. Consequently, this study aimed to determine the effect of (i) sodium nitroprusside (SNP), a NO donor and (ii) treatment with Lactobacillus farciminis, which produces NO in vitro, on trinitrobenzene sulphonic acid (TNBS)-induced colitis in rats and to evaluate the role of exogenous NO in this effect.Rats were divided into three groups receiving one of the following: (i) a continuous intracolonic (IC) infusion of SNP for 4 days, (ii) L. farciminis orally for 19 days, or (iii) saline. On day 1 and day 15, respectively, TNBS and saline were administrated IC, followed by a continuous IC infusion of saline or haemoglobin, a NO scavenger. At the end of treatments, the following parameters were evaluated: macroscopic damage of colonic mucosa, myeloperoxidase and nitric oxide synthase activities and colonic luminal NO production.In colitic rats, SNP and L. farciminis treatment significantly (P0.05) reduced macroscopic damage scores, myeloperoxidase and nitric oxide synthase activities compared to controls. Haemoglobin infusion abolished the anti-inflammatory effect of both NO donor treatments, but had no effect per se on colitis.NO released intraluminally by SNP infusion or by L. farciminis given orally improves TNBS-induced colitis in rats. These results indicate a protective role of NO donation in colonic inflammation and show for the first time a mechanism involving NO delivery by a bacterial strain reducing an experimental colitis.

Various oxime derivatives were evaluated as nitric oxide (NO) donors in arteries. Relaxation of rat aortic rings was used for bioassay of NO production, and electron paramagnetic resonance spectroscopy for demonstration of NO elevation. In rings with or without endothelium or adventitia, hydroxyguanidine and hydroxyurea were almost inactive, whereas formamidoxime, acetaldoxime, acetone oxime, acetohydroxamic acid and formaldoxime elicited relaxation. Active compounds increased NO levels in endothelium-denuded rings. Formaldoxime was the most potent agent for both relaxation and NO elevation in aortic rings, and it also increased NO in human aortic smooth muscle cells. In endothelium-denuded rings, relaxation was inhibited by a NO scavenger (2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide) and by inhibitors of soluble guanylyl-cyclase (1H[1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one) or cyclic GMP-dependent protein kinases (Rp-8-bromo cyclic GMP monophosphorothioate). Neither Nω-nitro- l -arginine methylester (a NO synthases inhibitor) nor proadifen (a cytochrome P450 inhibitor) decreased the effect of oxime derivatives. However, 7-ethoxyresorufin (7-ER, an inhibitor of P4501A1 which can also inhibit various NADPH-dependent reductases) abolished the relaxant effect of these compounds, without affecting the one of glyceryl trinitrate (GTN) or 2-(N,N-diethylamino)-diazenolate-2-oxide. 7-ER also abolished formaldoxime-induced NO increase in aortic rings. In rings tolerant to GTN, formaldoxime-induced relaxation and NO elevation were not different from those obtained in control rings. In conclusion, some oxime derivatives release NO by 7-ER-sensitive pathways in aortic smooth muscle, thus eliciting vasorelaxation. Pathways of NO formation are likely distinct from NO synthases and from those responsible for GTN biotransformation. Oxime derivatives could be useful for NO delivery in arteries in which endothelial NO synthase activity is impaired.

Background: The human heart contains β3-adrenoreceptors (β3AR) which are coupled to nitric oxide (NO) synthesis; however, their role in cardiac remodeling is unclear Methods: Mice with cardiomyocyte-specific overexpression of human β3AR (TG) & wild-type littermates (WT) were treated with isoproterenol (Iso30mg/kg/d), angiotensin II (AngII 2mg/kg/d), or saline by osmotic minipump, with/without L-NAME (2mg/mL). In vitro hypertrophic response, signaling & NO production (EPR) were analyzed in neonatal rat ventricular myocytes infected with an adenovirus expressing the human β3AR (hβ3-NRVM). Subcellular localization of β3AR was studied in adult ventricular myocytes using the proximity ligation assay (PLA). Results: PLA showed colocalization of the β3AR & eNOS with caveolin-3, which co-segregated with AMP-activated protein kinase (AMPK) & eNOS after sucrose gradient fractionation. Following 10 day Iso or AngII, cardiac myocyte hypertrophy & an upregulation of βMHC mRNA expression were observed in WT (μm2: 682±18 Sal vs 914±54 Iso; p

International audience; Nitric Oxide (NO) and Reactive oxygen species (ROS) are endogenous regulators of angiogenesis-related events as endothelial cell proliferation and survival, but NO/ROS defect or unbalance contribute to cancers. We recently designed a novel photoactive inhibitor of NO-Synthases (NOS) called NS1, which binds their NADPH site in vitro. Here, we show that NS1 inhibited NO formed in aortic rings. NS1-induced NO decrease led to an inhibition of angiogenesis in a model of VEGF-induced endothelial tubes formation. Beside this effect, NS1 reduced ROS levels in endothelial and melanoma A375 cells and in aorta. In metastatic melanoma cells, NS1 first induced a strong decrease of VEGF and blocked melanoma cell cycle at G2/M. NS1 decreased NOX(4) and ROS levels that could lead to a specific proliferation arrest and cell death. In contrast, NS1 did not perturb melanocytes growth. Altogether, NS1 revealed a possible cross-talk between eNOS- and NOX(4) -associated pathways in melanoma cells via VEGF, Erk and Akt modulation by NS1 that could be targeted to stop proliferation. NS1 thus constitutes a promising tool that modulates NO and redox stresses by targeting and directly inhibiting eNOS and, at least indirectly, NADPH oxidase(s), with great potential to control angiogenesis.