Antibacterial mouthwash alters gut microbiome, reducing nutrient absorption and fat accumulation in Western diet-fed mice.

Prolonged use of antibacterial mouthwash is linked to an increased risk of systemic disease. We aimed to investigate if disturbing the oral microbiota would impact the lower gut microbiome with functional effects in diet-induced obesity. Mice were exposed to oral chlorhexidine and fed a Western diet (WD). Food intake and weight gain were monitored, and metabolic function, blood pressure, and microbiota were analyzed. Chlorhexidine reduced the number of viable bacteria in the mouth and lowered species richness in the gut but with proportional enrichment of some bacteria linked to metabolic pathways. In mice fed a Western diet, chlorhexidine reduced weight gain, body fat, steatosis, and plasma insulin without changing caloric intake, while increasing colon triglycerides and proteins, suggesting reduced absorption of these nutrients. The mechanisms behind these effects as well as the link between the oral microbiome and small intestinal function need to be pinpointed. While the short-term effects of chlorhexidine in this model appear beneficial, potential long-term disruptions in the oral and gut microbiota and possible malabsorption should be considered.

NO-ferroheme is a signaling entity in the vasculature.

Despite wide appreciation of the biological role of nitric oxide (NO) synthase (NOS) signaling, questions remain about the chemical nature of NOS-derived bioactivity. Here we show that NO-like bioactivity can be efficiently transduced by mobile NO-ferroheme species, which can transfer between proteins, partition into a hydrophobic phase and directly activate the sGC-cGMP-PKG pathway without intermediacy of free NO. The NO-ferroheme species (with or without a protein carrier) efficiently relax isolated blood vessels and induce hypotension in rodents, which is greatly potentiated after the blockade of NOS activity. While free NO-induced relaxations are abolished by an NO scavenger and in the presence of red blood cells or blood plasma, a model compound, NO-ferroheme-myoglobin preserves its vasoactivity suggesting the physiological relevance of NO-ferroheme species. We conclude that NO-ferroheme behaves as a signaling entity in the vasculature.

Red blood cells from endothelial nitric oxide synthase-deficient mice induce vascular dysfunction involving oxidative stress and endothelial arginase I.

BACKGROUND & AIMS: Nitric oxide bioactivity (NO) from endothelial NO synthase (eNOS) importantly contributes to the maintenance of vascular homeostasis, and reduced eNOS activity has been associated with cardiovascular disease. Emerging evidence suggests interaction(s) between red blood cells (RBCs) and the endothelium in vascular control; however, the specific role of RBC eNOS is less clear. We aimed to investigate the hypothesis that a lack of RBC eNOS induces endothelial dysfunction. METHODS & RESULTS: RBCs from global eNOS knockout (KO) and wildtype (WT) mice were co-incubated ex vivo overnight with healthy mouse aortic rings, followed by functional and mechanistic analyses of endothelium-dependent and independent relaxations. RBCs from eNOS KO mice induced endothelial dysfunction and vascular oxidative stress, whereas WT RBC did not. No differences were observed for endothelium-independent relaxations. This eNOS KO RBC-induced endothelial dysfunctional phenotype was prevented by concomitant co-incubation with reactive oxygen species scavenger (TEMPOL), arginase inhibitor (nor-NOHA), NO donor (detaNONOate) and NADPH oxidase 4 (NOX4) inhibitor. Moreover, vessels from endothelial cell-specific arginase 1 KO mice were resistant to eNOS KO-RBC-induced endothelial dysfunction. Finally, in mice aortae co-incubated with RBCs from women with preeclampsia, we observed a significant reduction in endothelial function compared to when using RBCs from healthy pregnant women or from women with uncomplicated gestational hypertension. CONCLUSIONS: RBCs from mice lacking eNOS, and patients with preeclampsia, induce endothelial dysfunction in adjacent blood vessels. Thus, RBC-derived NO bioactivity acts to prevent induction of vascular oxidative stress occurring via RBC NOX4-derived ROS in a vascular arginase-dependent manner. Our data highlight the intrinsic protective role of RBC-derived NO bioactivity in preventing the damaging potential of RBCs. This provides novel insight into the functional relationship between RBCs and the vasculature in health and cardiovascular disease, including preeclampsia.

Hemoglobin ß93 Cysteine Is Not Required for Export of Nitric Oxide Bioactivity From the Red Blood Cell.

BACKGROUND: Nitrosation of a conserved cysteine residue at position 93 in the hemoglobin ß chain (ß93C) to form S-nitroso (SNO) hemoglobin (Hb) is claimed to be essential for export of nitric oxide (NO) bioactivity by the red blood cell (RBC) to mediate hypoxic vasodilation and cardioprotection. METHODS: To test this hypothesis, we used RBCs from mice in which the ß93 cysteine had been replaced with alanine (ß93A) in a number of ex vivo and in vivo models suitable for studying export of NO bioactivity. RESULTS: In an ex vivo model of cardiac ischemia/reperfusion injury, perfusion of a mouse heart with control RBCs (ß93C) pretreated with an arginase inhibitor to facilitate export of RBC NO bioactivity improved cardiac recovery after ischemia/reperfusion injury, and the response was similar with ß93A RBCs. Next, when human platelets were coincubated with RBCs and then deoxygenated in the presence of nitrite, export of NO bioactivity was detected as inhibition of ADP-induced platelet activation. This effect was the same in ß93C and ß93A RBCs. Moreover, vascular reactivity was tested in rodent aortas in the presence of RBCs pretreated with S-nitrosocysteine or with hemolysates or purified Hb treated with authentic NO to form nitrosyl(FeII)-Hb, the proposed precursor of SNO-Hb. SNO-RBCs or NO-treated Hb induced vasorelaxation, with no differences between ß93C and ß93A RBCs. Finally, hypoxic microvascular vasodilation was studied in vivo with a murine dorsal skin-fold window model. Exposure to acute systemic hypoxia caused vasodilatation, and the response was similar in ß93C and ß93A mice. CONCLUSIONS: RBCs clearly have the fascinating ability to export NO bioactivity, but this occurs independently of SNO formation at the ß93 cysteine of Hb.

AMP-activated protein kinase activation and NADPH oxidase inhibition by inorganic nitrate and nitrite prevent liver steatosis.

Advanced age and unhealthy dietary habits contribute to the increasing incidence of obesity and type 2 diabetes. These metabolic disorders, which are often accompanied by oxidative stress and compromised nitric oxide (NO) signaling, increase the risk of adverse cardiovascular complications and development of fatty liver disease. Here, we investigated the therapeutic effects of dietary nitrate, which is found in high levels in green leafy vegetables, on liver steatosis associated with metabolic syndrome. Dietary nitrate fuels a nitrate-nitrite-NO signaling pathway, which prevented many features of metabolic syndrome and liver steatosis that developed in mice fed a high-fat diet, with or without combination with an inhibitor of NOS (l-NAME). These favorable effects of nitrate were absent in germ-free mice, demonstrating the central importance of host microbiota in bioactivation of nitrate. In a human liver cell line (HepG2) and in a validated hepatic 3D model with primary human hepatocyte spheroids, nitrite treatment reduced the degree of metabolically induced steatosis (i.e., high glucose, insulin, and free fatty acids), as well as drug-induced steatosis (i.e., amiodarone). Mechanistically, the salutary metabolic effects of nitrate and nitrite can be ascribed to nitrite-derived formation of NO species and activation of soluble guanylyl cyclase, where xanthine oxidoreductase is proposed to mediate the reduction of nitrite. Boosting this nitrate-nitrite-NO pathway results in attenuation of NADPH oxidase-derived oxidative stress and stimulation of AMP-activated protein kinase and downstream signaling pathways regulating lipogenesis, fatty acid oxidation, and glucose homeostasis. These findings may have implications for novel nutrition-based preventive and therapeutic strategies against liver steatosis associated with metabolic dysfunction.

The NO-heme signaling hypothesis.

While the biological role of nitric oxide (NO) synthase (NOS) is appreciated, several fundamental aspects of the NOS/NO-related signaling pathway(s) remain incompletely understood. Canonically, the NOS-derived NO diffuses through the (inter)cellular milieu to bind the prosthetic ferro(Fe2+)-heme group of the soluble guanylyl cyclase (sGC). The formation of ternary NO-ferroheme-sGC complex results in the enzyme activation and accelerated production of the second messenger, cyclic GMP. This paper argues that cells dynamically generate mobile/exchangeable NO-ferroheme species, which activate sGC and regulate the function of some other biomolecules. In contrast to free NO, the mobile NO-ferroheme may ensure safe, efficient and coordinated delivery of the signal within and between cells. The NO-heme signaling may contribute to a number of NOS/NO-related phenomena (e.g. nitrite bioactivity, selective protein S-(N-)nitrosation, endothelium and erythrocyte-dependent vasodilation, some neural and immune NOS functions) and predicts new NO-related discoveries, diagnostics and therapeutics.

Glycyrrhetinic acid reverses the lipopolysaccharide-induced hypocontractility to noradrenaline in rat aorta: implications to septic shock.

Septic shock and associated vascular hyporeactivity to vasoconstrictor agonists remain a major problem of critical care medicine. Here we report that glycyrrhetinic acid (GA), the active component of licorice, effectively restores vascular contractility in the model of lipopolysaccharide (LPS)-treated rat aorta. GA was as effective as the NO synthase inhibitor N(G)-nitroarginine methylester. GA did not affect the vascular NO levels (measured by EPR spin trapping) and relaxations to L-arginine in LPS-treated rings as well as relaxation to S-nitroso-N-acetylpenicillamine in control rings. Thus, GA may represent an interesting alternative to NO synthase inhibitors in sepsis-associated vascular dysfunction.

Heparin-polynitroxides: synthesis and preliminary evaluation as cardiovascular EPR/MR imaging probes and extracellular space-targeted antioxidants.

We report here the synthesis of heparin-polynitroxide derivatives (HPNs) in which nitroxide moieties are linked either to uronic acid or glycosamine residues of the heparin macromolecule. HPNs have low anticoagulant activity, possess superoxide scavenging properties, bind to the vascular endothelium/extra-cellular matrix and can be detected by EPR and MRI techniques. As the vascular wall-targeted redox-active paramagnetic compounds, HPNs may have both diagnostic (molecular MRI) and therapeutic (ecSOD mimics) applications.

Differential effects of heart rate reduction with ivabradine in two models of endothelial dysfunction and oxidative stress.

Heart rate reduction with the I(f)-channel-inhibitor ivabradine is a novel and appealing option in the therapy of patients with ischemic heart disease. The aim of the current study was to determine the effects of ivabradine in two different animal models of vascular disease characterized by increased oxidative stress and endothelial dysfunction. Wistar rats with angiotensin II induced hypertension and ApoE knockout mice were used as animal models of endothelial dysfunction and oxidative stress, with half of the animals receiving ivabradine 10 mg/kg/day in parallel. Ivabradine lead to a sustained 15-20% heart rate reduction, but had no effect on blood pressure. While ivabradine had no effect on endothelial function and vascular reactive oxygen species production in angiotensin II-treated rats, it improved both parameters in ApoE knockout mice. These antioxidative effects were associated with a decreased NADPH oxidase activity and the prevention of eNOS uncoupling. In addition, ivabradine treatment led to an attenuation of angiotensin II signaling and increased the expression of telomere-stabilizing proteins in ApoE knockout mice, which may explain its beneficial effects on the vasculature. The absence of these protective ivabradine effects in angiotensin II-infused rats may relate to the treatment duration or the presence of arterial hypertension.

α1AMP-activated protein kinase preserves endothelial function during chronic angiotensin II treatment by limiting Nox2 upregulation.

OBJECTIVE: Besides its well-described metabolic effects, vascular AMP-activated protein kinase (AMPK) can activate endothelial NO synthase, promotes angiogenesis, and limits endothelial cell apoptosis. The current study was designed to study the effects of α1AMPK deletion during vascular disease in vivo. METHODS AND RESULTS: Chronic angiotensin II infusion at low subpressor doses caused a mild endothelial dysfunction that was significantly aggravated in α1AMPK-knockout mice. Unexpectedly, this endothelial dysfunction was not associated with decreased NO content, because NO levels measured by serum nitrite or electron paramagnetic resonance were even increased. However, because of parallel superoxide production, NO was consumed under production of peroxynitrite in angiotensin II-treated α1AMPK-knockout mice, associated with NADPH oxidase activation and Nox2 upregulation. As Nox2 is also a component of phagocyte NADPH oxidases, we found a vascular upregulation of several proinflammatory markers, including inducible NO synthase, vascular cell adhesion molecule-1, and cyclooxygenase-2. Cotreatment with the NADPH oxidase inhibitor apocynin was able to prevent vascular inflammation and also partially restored endothelial function in α1AMPK-knockout mice. CONCLUSIONS: Our data indicate that in vivo α1AMPK deletion leads to Nox2 upregulation, resulting in endothelial dysfunction and vascular inflammation. This implicates basal AMPK activity as a protective, redox-regulating element in vascular homeostasis.

Spin-labeled heparins as polarizing agents for dynamic nuclear polarization.

A potentially biocompatible class of spin-labeled macromolecules, spin-labeled (SL) heparins, and their use as nuclear magnetic resonance (NMR) signal enhancers are introduced. The signal enhancement is achieved through Overhauser-type dynamic nuclear polarization (DNP). All presented SL-heparins show high (1)H DNP enhancement factors up to E=-110, which validates that effectively more than one hyperfine line can be saturated even for spin-labeled polarizing agents. The parameters for the Overhauser-type DNP are determined and discussed. A striking result is that for spin-labeled heparins, the off-resonant electron paramagnetic resonance (EPR) hyperfine lines contribute a non-negligible part to the total saturation, even in the absence of Heisenberg spin exchange (HSE) and electron spin-nuclear spin relaxation (T(1ne)). As a result, we conclude that one can optimize the use of, for example, biomacromolecules for DNP, for which only small sample amounts are available, by using heterogeneously distributed radicals attached to the molecule.

Inactivation of soluble guanylate cyclase by stoichiometric S-nitrosation.

Dysfunction of vascular nitric oxide (NO)/cGMP signaling is believed to contribute essentially to various cardiovascular disorders. Besides synthesis and/or bioavailability of endothelial NO, impaired function of soluble guanylate cyclase (sGC) may play a key role in vascular dysfunction. Based on the proposal that desensitization of sGC through S-nitrosation contributes to vascular NO resistance ( Proc Natl Acad Sci U S A 104: 12312-12317, 2007 ), we exposed purified sGC to dinitrosyl iron complexes (DNICs), known as potent nitrosating agents. In the presence of 2 mM GSH, DNICs stimulated cGMP formation with EC(50) values of 0.1 to 0.5 microM and with an efficacy of 70 to 80% of maximal activity measured with 10 microM 2,2-diethyl-1-nitroso-oxyhydrazine (DEA/NO). In the absence of GSH, the efficacy of DNICs was markedly reduced, and sGC stimulation was counteracted by the inhibition of both basal and DEA/NO-stimulated cGMP formation at higher DNIC concentrations. Inactivation of sGC was slowly reversed in the presence of 2 mM GSH and associated with stoichiometric S-nitrosation of the protein (2.05 +/- 0.18 mol S-nitrosothiol per mol of 143-kDa heterodimer). S-Nitrosoglutathione and sodium nitroprusside caused partial inhibition of DEA/NO-stimulated sGC that was prevented by GSH, whereas nitroglycerin (0.3 mM) had no effect. Our findings indicate that nitrosation of two cysteine residues in sGC heterodimers results in enzyme inactivation. Protection by physiologically relevant concentrations of GSH (10 microM to 3 mM) suggests that S-nitrosation of sGC may contribute to vascular dysfunction in inflammatory disorders associated with nitrosative and oxidative stress and GSH depletion.

Cyclooxygenase 2-selective and nonselective nonsteroidal anti-inflammatory drugs induce oxidative stress by up-regulating vascular NADPH oxidases.

Cyclooxygenase 2-selective inhibitors (coxibs) and nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with an increase in cardiovascular events. The current study was designed to test the effect of coxibs and nonselective NSAIDs on vascular superoxide and nitric oxide (NO) production. mRNA expression of endothelial NO synthase (eNOS) and of the vascular NADPH oxidases was studied in spontaneously hypertensive rats (SHR) and in human endothelial cells. The expression of Nox1, Nox2, Nox4, and p22phox was increased markedly by the nonselective NSAIDs diclofenac or naproxen and moderately by rofecoxib or celecoxib in the aorta and heart of SHR. The up-regulation of NADPH oxidases by NSAIDs was associated with increased superoxide content in aorta and heart, which could be prevented by the NADPH oxidase inhibitor apocynin. NSAIDs reduced plasma nitrite and diminished the phosphorylation of vasodilator-stimulated phosphoprotein. This demonstrates a reduction in vascular NO production. Aortas from diclofenac-treated SHR showed an enhanced protein nitrotyrosine accumulation, indicative of vascular peroxynitrite formation. Peroxynitrite can uncouple oxygen reduction from NO synthesis in eNOS. Accordingly, the eNOS inhibitor N(G)-nitro-L-arginine methyl ester reduced superoxide content in aortas of NSAID-treated animals, demonstrating eNOS uncoupling under those conditions. Also in human endothelial cells, NSAIDs increased Nox2 expression and diminished production of bioactive NO. In healthy volunteers, NSAID treatment reduced nitroglycerin-induced, NO-mediated vasodilatation of the brachial artery. These results indicate that NSAIDs may increase cardiovascular risk by inducing oxidative stress in the vasculature, with nonselective NSAIDs being even more critical than coxibs in this respect.

Sirolimus-induced vascular dysfunction. Increased mitochondrial and nicotinamide adenosine dinucleotide phosphate oxidase-dependent superoxide production and decreased vascular nitric oxide formation.

OBJECTIVES: This study sought to analyze mechanisms that mediate vascular dysfunction induced by sirolimus. BACKGROUND: Despite excellent antirestenotic capacity, sirolimus-eluting stents have been found to trigger coronary endothelial dysfunction and impaired re-endothelialization. METHODS: To mimic the continuous sirolimus exposure of a stented vessel, Wistar rats underwent drug infusion with an osmotic pump for 7 days. RESULTS: Sirolimus treatment caused a marked degree of endothelial dysfunction as well as a desensitization of the vasculature to the endothelium-independent vasodilator nitroglycerin. Also, sirolimus stimulated intense transmural superoxide formation as detected by dihydroethidine fluorescence in aortae. Increased superoxide production was mediated in part by the vascular nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase as indicated by a marked stimulation of p67(phox)/rac1 NADPH oxidase subunit expression and by increased rac1 membrane association. In addition, superoxide production in rat heart mitochondria was up-regulated by sirolimus, as measured by L012-enhanced chemiluminescence. As a consequence, electron spin resonance measurements showed a 40% reduction in vascular nitric oxide bioavailability, which was further supported by decreased serum nitrite levels. CONCLUSIONS: Sirolimus causes marked vascular dysfunction and nitrate resistance after continuous treatment for 7 days. This impaired vasorelaxation may, in part, be induced by up-regulated mitochondrial superoxide release as well as by an up-regulation of NADPH oxidase-driven superoxide production. Both processes could contribute to endothelial dysfunction observed after coronary vascular interventions with sirolimus-coated stents.

Gp91phox-containing NAD(P)H oxidase increases superoxide formation by doxorubicin and NADPH.

Doxorubicin is a highly effective antineoplastic drug associated with a dose-dependent cardiotoxicity that may result in irreversible cardiomyopathy and heart failure. Gene variants of the superoxide-generating enzyme NAD(P)H oxidase have recently been associated with this phenotype. We investigated the mechanism of this association using lucigenin-enhanced chemiluminescence, spectrophotometry, electrochemical sensor, and electron paramagnetic resonance spectroscopy. Superoxide production was measured in female wild-type and NAD(P)H oxidase-deficient (gp91phox knockout) mice. The magnitude of the increase in superoxide production on the addition of doxorubicin was much higher in hearts of wild-type mice than in enzyme-deficient mice. An increase in superoxide production was observed also on the addition of the NADPH cytochrome P450 reductase. However, doxorubicin reacted with NADPH producing superoxide even in the absence of any enzymatic activity. Taken together, gp91phox-containing NAD(P)H oxidase and NADPH cytochrome P450 reductase can enhance superoxide production caused by the chemical interaction of doxorubicin and NADPH. These findings are in agreement with the recently reported reduced cardiotoxicity following doxorubicin treatment in gp91phox knockout mice and with associations between NAD(P)H oxidase gene variants and sensitivity to doxorubicin.

Deficiency of glutathione peroxidase-1 accelerates the progression of atherosclerosis in apolipoprotein E-deficient mice.

BACKGROUND: We have recently demonstrated that activity of red blood cell glutathione peroxidase-1 is inversely associated with the risk of cardiovascular events in patients with coronary artery disease. The present study analyzed the effect of glutathione peroxidase-1 deficiency on atherogenesis in the apolipoprotein E-deficient mouse. METHODS AND RESULTS: Female apolipoprotein E-deficient mice with and without glutathione peroxidase-1 deficiency were placed on a Western-type diet for another 6, 12, or 24 weeks. After 24 weeks on Western-type diet, double-knockout mice (GPx-1(-/-)ApoE(-/-)) developed significantly more atherosclerosis than control apolipoprotein E-deficient mice. Moreover, glutathione peroxidase-1 deficiency led to modified atherosclerotic lesions with increased cellularity. Functional experiments revealed that glutathione peroxidase-1 deficiency leads to increased reactive oxygen species concentration in the aortic wall as well as increased overall oxidative stress. Peritoneal macrophages from double-knockout mice showed increased in vitro proliferation in response to macrophage-colony-stimulating factor. Also, we found lower levels of bioactive nitric oxide as well as increased tyrosine nitration as a marker of peroxynitrite production. CONCLUSIONS: Deficiency of an antioxidative enzyme accelerates and modifies atherosclerotic lesion progression in apolipoprotein E-deficient mice.

Electron paramagnetic resonance (EPR) spin trapping of biological nitric oxide.

Nitric oxide (NO) is a free radical species with multiple physiological functions. Because of low concentrations and short half-life of NO, its direct measurement in living tissues remains a difficult task. Electron paramagnetic resonance (EPR) spin trapping is probably one of the best suitable platforms for development of new methods for quantification of biological NO. The most reliable EPR-based approaches developed so far are based on the reaction of NO with various iron complexes, both intrinsic and exogenously applied. This review is focused on the current state and perspectives of EPR spin trapping for experimental and clinical NO biology.

Nebivolol inhibits superoxide formation by NADPH oxidase and endothelial dysfunction in angiotensin II-treated rats.

Nebivolol is a beta(1)-receptor antagonist with vasodilator and antioxidant properties. Because the vascular NADPH oxidase is an important superoxide source, we studied the effect of nebivolol on endothelial function and NADPH oxidase activity and expression in the well-characterized model of angiotensin II-induced hypertension. Angiotensin II infusion (1 mg/kg per day for 7 days) caused endothelial dysfunction in male Wistar rats and increased vascular superoxide as detected by lucigenin-derived chemiluminescence, as well as dihydroethidine staining. Vascular NADPH oxidase activity, as well as expression at the mRNA and protein level, were markedly upregulated, as well as NOS III uncoupled, as evidenced by NO synthase III inhibitor experiments and dihydroethidine staining and by markedly decreased hemoglobin-NO concentrations. Treatment with the beta-receptor blocker nebivolol but not metoprolol (10 mg/kg per day for each drug) normalized endothelial function, reduced superoxide formation, increased NO bioavailability, and inhibited upregulation of the activity and expression of the vascular NADPH oxidase, as well as membrane association of NADPH oxidase subunits (Rac1 and p67(phox)). In addition, NOS III uncoupling was prevented. In vitro treatment with nebivolol but not atenolol or metoprolol induced a dissociation of p67(phox) and Rac1, as well as an inhibition of NADPH oxidase activity assessed in heart membranes from angiotensin II-infused animals, as well as in homogenates of Nox1 and cytosolic subunit-transfected and phorbol ester-stimulated HEK293 cells. These findings indicate that nebivolol interferes with the assembly of NADPH oxidase. Thus, inhibitory effects of this beta-blocker on vascular NADPH oxidase may explain, at least in part, its beneficial effect on endothelial function in angiotensin II-induced hypertension.