Biological Assessment of the NO-Dependent Endothelial Function.

Nitric oxide (NO) is implicated in numerous physiological processes, including vascular homeostasis. Reduced NO bioavailability is a hallmark of endothelial dysfunction, a prequel to many cardiovascular diseases. Biomarkers of an early NO-dependent endothelial dysfunction obtained from routine venous blood sampling would be of great interest but are currently lacking. The direct measurement of circulating NO remains a challenge due by its high reactivity and short half-life. The current techniques measure stable products from the NO signaling pathway or metabolic end products of NO that do not accurately represent its bioavailability and, therefore, endothelial function per se. In this review, we will concentrate on an original technique of low temperature electron paramagnetic resonance spectroscopy capable to directly measure the 5-α-coordinated heme nitrosyl-hemoglobin in the T (tense) state (5-α-nitrosyl-hemoglobin or HbNO) obtained from fresh venous human erythrocytes. In humans, HbNO reflects the bioavailability of NO formed in the vasculature from vascular endothelial NOS or exogenous NO donors with minor contribution from erythrocyte NOS. The HbNO signal is directly correlated with the vascular endothelial function and inversely correlated with vascular oxidative stress. Pilot studies support the validity of HbNO measurements both for the detection of endothelial dysfunction in asymptomatic subjects and for the monitoring of such dysfunction in patients with known cardiovascular disease. The impact of therapies or the severity of diseases such as COVID-19 infection involving the endothelium could also be monitored and their incumbent risk of complications better predicted through serial measurements of HbNO.

Oxidative stress-induced endothelial dysfunction and decreased vascular nitric oxide in COVID-19 patients.

BACKGROUND: SARS-CoV-2 targets endothelial cells through the angiotensin-converting enzyme 2 receptor. The resulting endothelial injury induces widespread thrombosis and microangiopathy. Nevertheless, early specific markers of endothelial dysfunction and vascular redox status in COVID-19 patients are currently missing. METHODS: Observational study including ICU and non-ICU adult COVID-19 patients admitted in hospital for acute respiratory failure, compared with control subjects matched for cardiovascular risk factors similar to ICU COVID-19 patients, and ICU septic shock patients unrelated to COVID-19. FINDINGS: Early SARS-CoV-2 infection was associated with an imbalance between an exacerbated oxidative stress (plasma peroxides levels in ICU patients vs. controls: 1456.0 ± 400.2 vs 436 ± 272.1 mmol/L; P < 0.05) and a reduced nitric oxide bioavailability proportional to disease severity (5-α-nitrosyl-hemoglobin, HbNO in ICU patients vs. controls: 116.1 ± 62.1 vs. 163.3 ± 46.7 nmol/L; P < 0.05). HbNO levels correlated with oxygenation parameters (PaO2/FiO2 ratio) in COVID-19 patients (R2 = 0.13; P < 0.05). Plasma levels of angiotensin II, aldosterone, renin or serum level of TREM-1 ruled out any hyper-activation of the renin-angiotensin-aldosterone system or leucocyte respiratory burst in ICU COVID-19 patients, contrary to septic patients. INTERPRETATION: Endothelial oxidative stress with ensuing decreased NO bioavailability appears as a likely pathogenic factor of endothelial dysfunction in ICU COVID-19 patients. A correlation between NO bioavailability and oxygenation parameters is observed in hospitalized COVID-19 patients. These results highlight an urgent need for oriented research leading to a better understanding of the specific endothelial oxidative stress that occurs during SARS-CoV-2. FUNDING: Stated in the acknowledgments section.

A Three-Month Consumption of Eggs Enriched with ω-3, ω-5 and ω-7 Polyunsaturated Fatty Acids Significantly Decreases the Waist Circumference of Subjects at Risk of Developing Metabolic Syndrome: A Double-Blind Randomized Controlled Trial.

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 < 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 < 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.

Redox regulation of nitrosyl-hemoglobin in human erythrocytes.

Oxidative stress perturbs vascular homeostasis leading to endothelial dysfunction and cardiovascular diseases. Vascular reactive oxygen species (ROS) reduce nitric oxide (NO) bioactivity, a hallmark of cardiovascular and metabolic diseases. We measured steady-state vascular NO levels through the quantification of heme nitrosylated hemoglobin (5-coordinate-α-HbNO) in venous erythrocytes of healthy human subjects using electron paramagnetic resonance (EPR) spectroscopy. To examine how ROS may influence HbNO complex formation and stability, we identified the pro- and anti-oxidant enzymatic sources in human erythrocytes and their relative impact on intracellular redox state and steady-state HbNO levels. We demonstrated that pro-oxidant enzymes such as NADPH oxidases are expressed and produce a significant amount of ROS at the membrane of healthy erythrocytes. In addition, the steady-state levels of HbNO were preserved when NOX (e.g. NOX1 and NOX2) activity was inhibited. We next evaluated the impact of selective antioxidant enzymatic systems on HbNO stability. Peroxiredoxin 2 and catalase, in particular, played an important role in endogenous and exogenous H2O2 degradation, respectively. Accordingly, inhibitors of peroxiredoxin 2 and catalase significantly decreased erythrocyte HbNO concentration. Conversely, steady-state levels of HbNO were preserved upon supplying erythrocytes with exogenous catalase. These findings support HbNO measurements as indicators of vascular oxidant stress and of NO bioavailability and potentially, as useful biomarkers of early endothelial dysfunction.

MicroRNA-199a-3p and MicroRNA-199a-5p Take Part to a Redundant Network of Regulation of the NOS (NO Synthase)/NO Pathway in the Endothelium.

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.

Nitrosyl-hemoglobin formation in rodent and human venous erythrocytes reflects NO formation from the vasculature in vivo.

Reduced bioavailability of nitric oxide (NO) is a major feature of endothelial dysfunction characteristic of cardiovascular and metabolic diseases but the short half-life of NO precludes its easy quantification in circulating blood for early diagnosis. In erythrocytes, NO can react with hemoglobin to form an iron-nitrosyl complex (5-coordinate-α-HbNO) directly quantifiable by Electron Paramagnetic Resonance spectroscopy (EPR) in mouse, rat and human venous blood ex vivo. However, the sources of the nitrosylating species in vivo and optimal conditions of HbNO preservation for diagnostic use in human erythrocytes are unknown. Using EPR spectroscopy, we found that HbNO stability was significantly higher under hypoxia (equivalent to venous pO2; 12.0±0.2% degradation of HbNO at 30 minutes) than at room air (47.7±0.2% degradation) in intact erythrocytes; at 20°C (15.2±0.3% degradation after 30 min versus 29.6±0.1% at 37°C) and under acidic pH (31.7±0.8% versus 62.2±0.4% degradation after 30 min at physiological pH) at 50% of haematocrit. We next examined the relative contribution of NO synthase (NOS) from the vasculature or in erythrocytes themselves as a source of nitrosylating NO. We detected a NOS activity (and eNOS expression) in human red blood cells (RBC), and in RBCs from eNOS(+/+) (but not eNOS(-/-)) mice, as measured by HbNO formation and nitrite/nitrate accumulation. NO formation was increased after inhibition of arginase but abrogated upon NOS inhibition in human RBC and in RBCs from eNOS(+/+) (but not eNOS(-/-)) mice. However, the HbNO signal from freshly drawn venous RBCs was minimally sensitive to the inhibitors ex vivo, while it was enhanced upon caveolin-1 deletion in vivo, suggesting a minor contribution of erythrocyte NOS to HbNO complex formation compared with vascular endothelial NOS or other paracrine NO sources. We conclude that HbNO formation in rodent and human venous erythrocytes is mainly influenced by vascular NO sources despite the erythrocyte NOS activity, so that its measurement by EPR could serve as a surrogate for NO-dependent endothelial function.

Targeting the gut microbiota with inulin-type fructans: preclinical demonstration of a novel approach in the management of endothelial dysfunction.

OBJECTIVE: To 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. DESIGN: We 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. RESULTS: ITF 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. CONCLUSIONS: We 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.

Clinical and biochemical data of endothelial function in Women Consuming Combined Contraceptives.

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.

Heme-nitrosylated hemoglobin and oxidative stress in women consuming combined contraceptives. Clinical application of the EPR spectroscopy.

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±0.1mmol/L versus 0.8±0.1mmol/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.

Nutritional depletion in n-3 PUFA in apoE knock-out mice: A new model of endothelial dysfunction associated with fatty liver disease.

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.

Effects of BM-573 on Endothelial Dependent Relaxation and Increased Blood Pressure at Early Stages of Atherosclerosis.

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 (3 µM, 1 hour) and chronic (10 mg/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.

Regulation of NADPH-dependent Nitric Oxide and reactive oxygen species signalling in endothelial and melanoma cells by a photoactive NADPH analogue.

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.

Enhanced expression of ß3-adrenoceptors in cardiac myocytes attenuates neurohormone-induced hypertrophic remodeling through nitric oxide synthase.

BACKGROUND: ß1-2-adrenergic receptors (AR) are key regulators of cardiac contractility and remodeling in response to catecholamines. ß3-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 ß3-AR (ß3-TG) and wild-type (WT) littermates were used to compare myocardial remodeling in response to isoproterenol (Iso) or Angiotensin II (Ang II). ß3-TG and WT had similar morphometric and hemodynamic parameters at baseline. ß3-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 ß3-TG mice, which also had less re-expression of fetal genes and transforming growth factor ß1. 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 ß3-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, ß3-AR overexpression inhibited phenylephrine-induced nuclear factor of activated T-cell activation. CONCLUSIONS: Cardiac-specific overexpression of ß3-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 ß3-AR pathway may provide future therapeutic avenues for the modulation of hypertrophic remodeling.

Nitrosylated hemoglobin levels in human venous erythrocytes correlate with vascular endothelial function measured by digital reactive hyperemia.

UNLABELLED: 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<0.0001 for basal HbNO). CONCLUSION: The study demonstrates quantitative measurements of 5-coordinate α-HbNO in human venous erythrocytes, its dynamic physiologic regulation and correlation with endothelial function measured by tonometry during hyperemia. This opens the way to further understanding of in vivo determinants of NO bioavailability in human circulation.

Moderate caveolin-1 downregulation prevents NADPH oxidase-dependent endothelial nitric oxide synthase uncoupling by angiotensin II in endothelial cells.

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. CONCLUSIONS: 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.

Nebivolol exerts beneficial effects on endothelial function, early endothelial progenitor cells, myocardial neovascularization, and left ventricular dysfunction early after myocardial infarction beyond conventional ß1-blockade.

OBJECTIVES: The 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 ß1-receptor-blocking properties. BACKGROUND: Nebivolol is a third-generation selective ß1-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. METHODS: Mice 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. RESULTS: Infarct size was similar among the groups. Both ß1-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. CONCLUSIONS: Nebivolol improves LV dysfunction and survival early after MI likely beyond the effects provided by conventional ß1-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.

Vascular caveolin deficiency supports the angiogenic effects of nitrite, a major end product of nitric oxide metabolism in tumors.

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.

Control of blood pressure variability in caveolin-1-deficient mice: role of nitric oxide identified in vivo through spectral analysis.

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.

Interaction of reactive oxygen and nitrogen species with albumin- and methemoglobin-bound dinitrosyl-iron complexes.

Destructive effect of superoxide anions O2- derived from KO(2) or xanthine-xanthine oxidase system on dinitrosyl-iron complexes bound with bovine albumin or methemoglobin (DNIC-BSA or DNIC-MetHb) was demonstrated. The sensitivity of DNIC-BSA synthesized by the addition of DNIC with cysteine, thiosulfate or phosphate (DNIC-BSA-1, DNIC-BSA-2 or DNIC-BSA-3, respectively) to destructive action of O2- decreased in row: DNIC-BSA-1>DNIC-BSA-3>DNIC-BSA-2. The estimated rate constant for the reaction between O2- and DNIC-BSA-3 was equal to approximately 10(7)M(-1)s(-1). However, hydrogen peroxide and tert-butyl hydrogenperoxide (t-BOOH) did not induce any noticeable degradation of DNIC-BSA-3 even when used at concentrations exceeding by one order of magnitude those of the complex. As to their action on DNIC-MetHb both hydrogen peroxide and t-BOOH-induced rapid degradation of the complex. Both agents could induce the process due to the effect of alkylperoxyl or protein-derived free radicals formed at the interaction of the agents with ferri-heme groups of MetHb. Peroxynitrite (ONOO(-)) could also initiate protein-bound DNIC degradation more efficiently in the reaction with DNIC-BSA-3. Higher resistance of DNIC-MetHb to peroxynitrite was most probably due to the protective action of heme groups on ONOO(-). However, the analysis allows to suggest that the interaction of protein-bound DNICs with O2- is the only factor responsible for the degradation of the complexes in cells and tissues.

Transport and peripheral bioactivities of nitrogen oxides carried by red blood cell hemoglobin: role in oxygen delivery.

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, O(2) sensing 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, O(2) delivery, via regulation of blood flow. Analogous, physiological actions of RBC SNO-Hb also contribute to central nervous responses to blood hypoxia, the uptake of O(2) from 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.