Irina Lobysheva

Endothelial beta3-adrenoreceptors mediate nitric oxide-dependent vasorelaxation of coronary microvessels in response to the third-generation beta-blocker nebivolol.

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 β1-adrenoreceptor antagonist that releases nitric oxide (NO) through incompletely characterized mechanisms. We identified endothelial β3-adrenoreceptors in human coronary microarteries that mediate endothelium- and NO-dependent relaxation and hypothesized that nebivolol activates these β3-adrenoreceptors. Methods and Results—Nebivolol dose-dependently relaxed rodent coronary resistance microarteries studied by videomicroscopy (10 &mgr;mol/L, −86±6% of prostaglandin F2α contraction); this was sensitive to NO synthase (NOS) inhibition, unaffected by the β1-2-blocker nadolol, and prevented by the β1-2-3-blocker bupranolol (P<0.05; n=3 to 8). Importantly, nebivolol failed to relax microarteries from β3-adrenoreceptor–deficient mice. Nebivolol (10 &mgr;mol/L) also relaxed human coronary microvessels (−71±5% of KCl contraction); this was dependent on a functional endothelium and NO synthase but insensitive to β1-2-blockade (all P<0.05). In a mouse aortic ring assay of neoangiogenesis, nebivolol induced neocapillary tube formation in rings from wild-type but not β3-adrenoreceptor– or endothelial NOS–deficient mice. In cultured endothelial cells, 10 &mgr;mol/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 threonine495, and fura-2 calcium fluorescence increased by 91.8±23.7%; this effect was unaffected by β1-2-blockade but abrogated by β1-2-3-blockade (all P<0.05). Conclusions—Nebivolol dilates human and rodent coronary resistance microarteries through an agonist effect on endothelial β3-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.

Nebivolol exerts beneficial effects on endothelial function, early endothelial progenitor cells, myocardial neovascularization, and left ventricular dysfunction early after myocardial infarction beyond conventional β1-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 β₁-receptor-blocking properties. BACKGROUND Nebivolol 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. 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 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. CONCLUSIONS Nebivolol 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.

Enhanced Expression of β3-Adrenoceptors in Cardiac Myocytes Attenuates Neurohormone-Induced Hypertrophic Remodeling Through Nitric Oxide Synthase

Background— &bgr;1-2-adrenergic receptors (AR) are key regulators of cardiac contractility and remodeling in response to catecholamines. &bgr;3-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 &bgr;3-AR (&bgr;3-TG) and wild-type (WT) littermates were used to compare myocardial remodeling in response to isoproterenol (Iso) or Angiotensin II (Ang II). &bgr;3-TG and WT had similar morphometric and hemodynamic parameters at baseline. &bgr;3-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 &bgr;3-TG mice, which also had less re-expression of fetal genes and transforming growth factor &bgr;1. 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 &bgr;3-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, &bgr;3-AR overexpression inhibited phenylephrine-induced nuclear factor of activated T-cell activation. Conclusions— Cardiac-specific overexpression of &bgr;3-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 &bgr;3-AR pathway may provide future therapeutic avenues for the modulation of hypertrophic remodeling.

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.

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 O2 −· 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 O2 −· 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.

Genetic deletion of aquaporin-1 results in microcardia and low blood pressure in mouse with intact nitric oxide-dependent relaxation, but enhanced prostanoids-dependent relaxation

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.

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.

Nitrosylated Hemoglobin Levels in Human Venous Erythrocytes Correlate with Vascular Endothelial Function Measured by Digital Reactive Hyperemia

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.

Involvement of Nitric Oxide in Iodine Deficiency-Induced Microvascular Remodeling in the Thyroid Gland: Role of Nitric Oxide Synthase 3 and Ryanodine Receptors

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 3 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-nitroarginine methyl ester (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, whereas S-nitroso-acetyl-penicillamine (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 3 known mammalian RYR isoforms (RYR1 and RYR2) were shown to be expressed in thyrocytes. RYR inhibition using ryanodine at 10μM decreased ID-induced NOS3 activation, HIF-1α, and VEGF-A expression, whereas 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.

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. (Mol Cancer Res 2009;7(7):1056–63)