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.

Sodium Nitrite Attenuates Reduced Activity of Vascular Matrix Metalloproteinase-2 and Vascular Hyper-Reactivity and Increased Systolic Blood Pressure Induced by the Placental Ischemia Model of Preeclampsia in Anesthetized Rats.

Preeclampsia is a maternal hypertension disorder associated with vascular dysfunction and fetal and placental growth restrictions. Placental ischemia is suggested as the primary trigger of preeclampsia-associated impairments of both endothelium-derived nitric oxide (NO) and the vascular activity of extracellular matrix metalloproteinase-2 (MMP-2). Reduced uteroplacental perfusion pressure (RUPP) is a placental ischemia model of preeclampsia. Reduction of sodium nitrite to NO may occur during ischemic conditions. However, sodium nitrite effects in the RUPP model of preeclampsia have not yet been investigated. Pregnant rats were divided into four groups: normotensive pregnant rats (Norm-Preg), pregnant rats treated with sodium nitrite (Preg + Nitrite), preeclamptic rats (RUPP), and preeclamptic rats treated with sodium nitrite (RUPP + Nitrite). Maternal blood pressure and fetal and placental parameters were recorded. Vascular function, circulating NO metabolites, and the gelatinolytic activity of vascular MMP-2 were also examined. Sodium nitrite attenuates increased blood pressure, prevents fetal and placental weight loss, counteracts vascular hyper-reactivity, and partially restores NO metabolites and MMP-2 activity. In conclusion, sodium nitrite reduction to NO may occur during RUPP-induced placental ischemia, thereby attenuating increased blood pressure, fetal and placental growth restriction, and vascular hyper-reactivity associated with preeclampsia and possibly restoring NO and MMP-2 activity, which underlie the blood pressure-lowering effects.

Cardiovascular characterization of the novel organic mononitrate NDIBP in rats.

Organic nitrates are widely used to restore endogenous nitric oxide (NO) levels reduced by endothelial nitric oxide synthase dysfunction. However, these drugs are associated with undesirable side effects, including tolerance. This study aims to investigate the cardiovascular effects of the new organic nitrate 1,3-diisobutoxypropan-2-yl nitrate (NDIBP). Specifically, we assessed its effects on blood pressure, vascular reactivity, acute toxicity, and the ability to induce tolerance. In vitro and ex vivo techniques showed that NDIBP released NO both in a cell-free system and in isolated mesenteric arteries preparations through a process catalyzed by xanthine oxidoreductase. NDIBP also evoked endothelium-independent vasorelaxation, which was significantly attenuated by 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO, 300 µM), a nitric oxide scavenger; 1-H-[1,2,4] oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 10 µM), a soluble guanylyl cyclase inhibitor; tetraethylammonium (TEA, 3 mM), a potassium channel blocker; febuxostat (500 nM), a xanthine oxidase inhibitor; and proadifen (10 µM), an inhibitor of cytochrome P450 enzyme. Furthermore, this organic nitrate did not induce tolerance in isolated vessels and presented low toxicity following acute oral administration. In vivo changes on cardiovascular parameters were assessed using normotensive and renovascular hypertensive rats. NDIBP evoked a reduction of blood pressure that was significantly higher in hypertensive animals. Our results suggest that NDIBP acts as a NO donor, inducing blood pressure reduction without having the undesirable effects of tolerance. Those effects seem to be mediated by activation of NO-sGC-cGMP pathway and positive modulation of K+ channels in vascular smooth muscle.

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 new organic nitrate 2-nitrate-1,3-diocthanoxypropan (NDOP) induces nitric oxide production and vasorelaxation via activation of inward-rectifier potassium channels (KIR).

INTRODUCTION: Cardiovascular diseases are coupled to decreased nitric oxide (NO) bioavailability, and there is a constant search for novel and better NO-donors. Here we synthesized and characterized the cardiovascular effects of the new organic nitrate 2-nitrate-1,3-dioctanoxypropan (NDOP). METHODS: A combination of in vitro and in vivo experiments was performed in C57BL/6 mice and Wistar rats. Thus, the ability of NDOP in donating NO in a cell-free system and in vascular smooth muscles cells (VSMC) and its ability to induce vasorelaxation in aortic rings from mice were evaluated. In addition, changes in blood pressure and heart rate to different doses of NDOP were evaluated in conscious rats. Finally, acute pre-clinical toxicity to oral administration of NDOP was assessed in mice. RESULTS: In cell-free system, NDOP increased NO levels, which was dependent on xanthine oxidoreductase (XOR). NDOP also increased NO levels in VSMC, which was not influenced by endothelial NO synthase. Furthermore, incubation with the XOR inhibitor febuxostat blunted the vasorelaxation in aortic ring preparations. In conscious rats, NDOP elicited dose-dependent reduction in blood pressure accompanied with increased heart rate. In vessel preparations, NDOP (10-8-10-3 mol/L) induced endothelium-independent vasorelaxation, which was inhibited by the NO scavengers 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and hydroxocobalamin or by inhibition of soluble guanylyl cyclase using H- [1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one. To investigate if NDOP acts through potassium channels, selective blockers were used. Inhibition of BKCa, Kv or KATP subtypes of potassium channels had no effect, but inhibition of inward-rectifier potassium channels (KIR) significantly reduced NDOP-mediated vasorelaxation. Lastly, NDOP showed low toxicity (LD50 ~5000 mg/kg). CONCLUSION: Bioactivation of NDOP involves functional XOR, and this new organic nitrate elicits vasorelaxation via NO-cGMP-PKG signaling and activation of KIR channels. Future studies should further characterize the underlying mechanism and evaluate the therapeutic benefits of chronic NDOP treatment in relevant cardiovascular disease models.

The Impact of Bariatric Surgery on Pelvic Floor Dysfunction: A Systematic Review.

OBJECTIVE: To determine the effect of bariatric surgery (BS) on the prevalence of pelvic floor dysfunctions (PFD), specifically on urinary incontinence (UI), pelvic organ prolapse (POP), and fecal incontinence (FI). DATA SOURCES: A systematic review (PROSPERO registration no. CRD42017068452) with a literature search was performed using the PubMed, Scopus, and SciELO databases for all publications related to BS and PFD, with no language restrictions, from inception to September 2018. METHODS OF STUDY SELECTION: Two authors screened for study eligibility and extracted data. Only prospective cohorts assessing women with morbid obesity and the prevalence of PFD before and after BS in multiple academic and private centers were included. UI, POP, and FI were defined according to the International Urogynecological Association/International Continence Society joint consensus, and diagnosis was made based on self-report or questionnaires. TABULATION, INTEGRATION, AND RESULTS: Our search strategy retrieved 957 results. Of those, 28 studies were included for full analysis, and 20 studies (n = 3684 patients) were selected for final analysis. The main reasons for exclusion were missing data before and after BS (n = 7) and combined data of men and women who underwent BS (n = 1). Laparoscopic Roux-en-Y gastric bypass was the most common surgical technique. Pooled analysis (16 studies) showed that women had a mean body mass index reduction of 12.90 kg/m2 after treatment (95% confidence interval [CI], -14.82 to -10.97; p < .0001). The relative risk reduction was 67% (n = 19; odds ratio [OR], 0.33; 95% CI, 0.26-0.41; p < .0001) for UI, 52% (n = 5; OR, 0.48; 95% CI, 0.22-1.07; p = .07) for POP, and 20% (n = 9; OR, 0.80; 95% CI, 0.53-1.21; p = .29) for FI. Funnel plots for UI and FI did not suggest any publication bias. With regard to the standardized questionnaires for PFD, the International Consultation on Incontinence-Short Form, Pelvic Floor Impact Questionnaire-7 and its subscale Colorectal-Anal Impact Questionnaire-7, Pelvic Floor Distress Inventory-20, and its subscale Urinary Distress Inventory-6 showed statistically significantly lower scores. Sexual function, represented by the Pelvic Organ Prolapse/Urinary Incontinence Sexual Questionnaire-12, showed no statistically significant improvement after surgery. CONCLUSION: BS has a significant impact on reducing UI, but FI and POP, in obese women.

Cathepsin L promotes Vascular Intimal Hyperplasia after Arterial Injury.

The inflammatory pathways that drive the development of intimal hyperplasia (IH) following arterial injury are not fully understood. We hypothesized that the lysosomal cysteine protease cathepsin L activates processes leading to IH after arterial injury. Using a mouse model of wire-induced carotid artery injury we showed that cathepsin L activity peaks at day 7 and remains elevated to 28 days. The genetic deletion of cathepsin L prevented IH and monocyte recruitment in the carotid wall. The injury-induced increases in cathepsin L mRNA and activity were mitigated in mice with myeloid-specific deletion of toll like receptor 4 (TLR4) or myeloid differentiation primary response gene 88 (MyD88). We further discovered that a HIV-protease inhibitor saquinavir (SQV), which is known to block recombinant mouse cathepsin L activity in vitro, prevented IH after arterial injury. SQV also suppressed LPS (TLR4 agonist) induced monocyte adhesion to endothelial monolayers. These findings establish cathepsin L as a critical regulator of the inflammation that leads to IH and that the TLR4- MyD88 pathway in myeloid lineages regulates cathepsin L expression in the vessel wall following wire injury. The FDA approved drug, SQV blocks IH though mechanisms that may include the suppression of cathepsin L.

Inducible Nitric Oxide Synthase Polymorphisms and Nitric Oxide Levels in Individuals with Chronic Periodontitis.

This study aimed to investigate whether the -1026(A>C)(rs2779249) and +2087(A>G)(2297518) polymorphisms in the NOS2 gene were associated with chronic periodontitis (CP) and with salivary levels of nitrite (NO2-) and/or nitrate + nitrite (NOx). A group of 113 mixed-race patients were subjected to periodontal, genetic, and biochemical evaluations (65 CP/48 periodontally healthy subjects). DNA was extracted from oral epithelial cells and used for genotyping by polymerase chain reaction (real-time). Salivary NOx concentrations were determined using an ozone-based chemiluminescence assay. Association of CP with alleles and genotypes of the -1026(A>C) polymorphism was found (X² test, p = 0.0075; 0.0308), but this was not maintained after multiple logistic regression, performed to estimate the effect of covariates and polymorphisms in CP. This analysis demonstrated, after correction for multiple comparisons, that only the female gender was significantly associated with CP. Polymorphisms analyzed as haplotypes were not associated with CP. NOx levels were significantly higher in the control group of heterozygous individuals for both polymorphisms. In conclusion, the female gender was significantly associated with CP, and higher levels of salivary NOx were found in control subjects and associated with the heterozygous state of the NOS2 polymorphisms, reinforcing the potential of NO metabolites as markers of periodontitis status.

The HIV Protease Inhibitor Saquinavir Inhibits HMGB1-Driven Inflammation by Targeting the Interaction of Cathepsin V with TLR4/MyD88.

Extracellular high-mobility group box 1 (HMGB1) (disulfide form), via activation of toll-like receptor 4 (TLR4)-dependent signaling, is a strong driver of pathologic inflammation in both acute and chronic conditions. Identification of selective inhibitors of HMGB1-TLR4 signaling could offer novel therapies that selectively target proximal endogenous activators of inflammation. A cell-based screening strategy led us to identify first generation HIV-protease inhibitors (PI) as potential inhibitors of HMGB1-TLR4 driven cytokine production. Here we report that the first-generation HIV-PI saquinavir (SQV), as well as a newly identified mammalian protease inhibitor STO33438 (334), potently block disulfide HMGB1-induced TLR4 activation, as assayed by the production of TNF-α by human monocyte-derived macrophages (THP-1). We further report on the identification of mammalian cathepsin V, a protease, as a novel target of these inhibitors. Cellular as well as recombinant protein studies show that the mechanism of action involves a direct interaction between cathepsin V with TLR4 and its adaptor protein MyD88. Treatment with SQV, 334 or the known cathepsin inhibitor SID26681509 (SID) significantly improved survival in murine models of sepsis and reduced liver damage following warm liver ischemia/reperfusion (I/R) models, both characterized by strong HMGB1-TLR4 driven pathology. The current study demonstrates a novel role for cathepsin V in TLR4 signaling and implicates cathepsin V as a novel target for first-generation HIV-PI compounds. The identification of cathepsin V as a target to block HMGB1-TLR4-driven inflammation could allow for a rapid transition of the discovery from the bench to the bedside. Disulfide HMGB1 drives pathologic inflammation in many models by activating signaling through TLR4. Cell-based screening identified the mammalian protease cathepsin V as a novel therapeutic target to inhibit TLR4-mediated inflammation induced by extracellular HMGB1 (disulfide form). We identified two protease inhibitors (PIs) that block cathepsin V and thereby inhibit disulfide HMGB1-induced TLR4 activation: saquinavir (SQV), a first-generation PI targeting viral HIV protease and STO33438 (334), targeting mammalian proteases. We discovered that cathepsin V binds TLR4 under basal and HMGB1-stimulated conditions, but dissociates in the presence of SQV over time. Thus cathepsin V is a novel target for first-generation HIV PIs and represents a potential therapeutic target of pathologic inflammation.

The antihypertensive effects of sodium nitrite are not associated with circulating angiotensin converting enzyme inhibition.

Nitrite-derived nitric oxide (NO) formation exerts antihypertensive effects. Because NO inhibits angiotensin converting enzyme (ACE) activity, we carried a comprehensive series of experiments in rats to test the hypothesis that sodium nitrite exerts antihypertensive effects by inhibiting ACE. We examined whether sodium nitrite (15 mg/kg; or vehicle; by gavage): (I) attenuates the pressor responses to angiotensin I at doses of 0.03, 0.1, 0.3, 1, 3, and 10 µg/kg intravenously; (II) attenuates the acute hypertension induced by L-NAME (100 mg/kg; or vehicle; by gavage); (III) attenuates the chronic hypertension induced by L-NAME (1 g/L in drinking water; or vehicle) administered for 6 weeks; (IV) attenuates the hypertension in the 2 kidney-1 clip (2K1C) chronic hypertension model. Blood samples were collected at the end of each study and plasma angiotensin converting enzyme (ACE) activity was measured with a fluorimetric assay using Hippuryl-His-Leu as substrate. ACE inhibitors were used as positive controls. Plasma nitrite concentrations were measured by ozone-based reductive chemiluminescence. The in vitro effects of sodium nitrite (0, 1, 3, 10, 30, 100 µmol/L) on plasma ACE activity were also determined. We found that sodium nitrite did not affect the pressor responses to angiotensin I. Moreover, while sodium nitrite exerted significant antihypertensive effects in acute and chronic hypertension models, no significant effects on plasma ACE activity were found. In vitro experiments showed no effects of sodium nitrite on plasma ACE activity. This is the first study to demonstrate that the acute and chronic antihypertensive effects of sodium nitrite are not associated with significant inhibition of circulating ACE activity.

The Nitrate-Nitrite-Nitric Oxide Pathway: Potential Role in Mitigating Oxidative Stress in Hypertensive Disorders of Pregnancy.

Hypertensive diseases of pregnancy (HDPs) represent a global clinical challenge, affecting 5-10% of women and leading to complications for both maternal well-being and fetal development. At the heart of these complications is endothelial dysfunction, with oxidative stress emerging as a pivotal causative factor. The reduction in nitric oxide (NO) bioavailability is a vital indicator of this dysfunction, culminating in blood pressure dysregulation. In the therapeutic context, although antihypertensive medications are commonly used, they come with inherent concerns related to maternal-fetal safety, and a percentage of women do not respond to these therapies. Therefore, alternative strategies that directly address the pathophysiology of HDPs are required. This article focuses on the potential of the nitrate-nitrite-NO pathway, abundantly present in dark leafy greens and beetroot, as an alternative approach to treating HDPs. The objective of this review is to discuss the prospective antioxidant role of nitrate. We hope our discussion paves the way for using nitrate to improve endothelial dysfunction and control oxidative stress, offering a potential therapy for managing HDPs.

Tamoxifen and its metabolites cause acute vasorelaxation of aortic rings by inducing vasodilator prostanoid synthesis.

The vascular effects of tamoxifen (Tam) and its metabolites are poorly known. We compared the vasorelaxation induced by Tam and its metabolites (N-desmethyl-Tam, 4-hydroxy-Tam, and endoxifen) in aortic rings from rats using standardized organ bath procedures, and we investigated the mechanisms involved in this effect. Tam and its metabolite-induced vasorelaxation in a concentration-dependent manner. Although 4-hydroxy-Tam and Tam had similar potency (pD2 = 8.5 ± 0.1 vs. 8.8 ± 0.1, respectively) and maximum effect (Emax = 88.5% ± 1.3% vs. 92.6% ± 1.3%, respectively), N-desmethyl-Tam and endoxifen were more potent and showed higher Emax than Tam did (pD2 = 9.0 ± 0.1 and 8.9 ± 0.1; Emax = 101.1% ± 1.8% and 101.0% ± 1.8% for N-desmethyl-Tam and endoxifen, respectively). Although preincubation of aortic rings with the estrogen receptor antagonist ICI 182780 or with the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester hydrochloride induced no changes in the vasorelaxation induced by Tam or 4-hydroxy-Tam, both drugs significantly reduced Emax in response to N-desmethyl-Tam or to endoxifen. Inhibition of cyclooxygenase with indomethacin or the incubation with the prostaglandin D2 and E2 receptor antagonist AH6809 reduced the vasorelaxation-induced Tam and its metabolites by approximately 50%. Preincubation with Nω-nitro-L-arginine methyl ester hydrochloride combined with indomethacin abolished the vasorelaxation-induced Tam and its metabolites. These results show that Tam and its metabolites cause acute vasorelaxation by inducing vasodilator prostanoids synthesis.

Hydrogen sulfide improves neutrophil migration and survival in sepsis via K+ATP channel activation.

RATIONALE: Recovering the neutrophil migration to the infectious focus improves survival in severe sepsis. Recently, we demonstrated that the cystathionine gamma-lyase (CSE)/hydrogen sulfide (H(2)S) pathway increased neutrophil recruitment to inflammatory focus during sterile inflammation. OBJECTIVES: To evaluate if H(2)S administration increases neutrophil migration to infectious focus and survival of mice. METHODS: Sepsis was induced by cecal ligation and puncture (CLP). MEASUREMENTS AND MAIN RESULTS: The pretreatments of mice with H(2)S donors (NaHS or Lawesson's reagent) improved leukocyte rolling/adhesion in the mesenteric microcirculation as well as neutrophil migration. Consequently, bacteremia levels were reduced, hypotension and lung lesions were prevented, and the survival rate increased from approximately 13% to approximately 80%. Even when treatment was delayed (6 h after CLP), a highly significant reduction in mortality compared with untreated mice was observed. Moreover, H(2)S pretreatment prevented the down-regulation of CXCR2 and l-selectin and the up-regulation of CD11b and G protein-coupled receptor kinase 2 in neutrophils during sepsis. H(2)S also prevented the reduction of intercellular adhesion molecule-1 expression in the endothelium of the mesenteric microcirculation in severe sepsis. Confirming the critical role of H(2)S on sepsis outcome, pretreatment with dl-propargylglycine (a CSE inhibitor) inhibited neutrophil migration to the infectious focus, enhanced lung lesions, and induced high mortality in mice subjected to nonsevere sepsis (from 0 to approximately 80%). The beneficial effects of H(2)S were blocked by glibenclamide (a ATP-dependent K(+) channel blocker). CONCLUSIONS: These results showed that H(2)S restores neutrophil migration to the infectious focus and improves survival outcome in severe sepsis by an ATP-dependent K(+) channel-dependent mechanism.

Hemodynamic effects of inducible nitric oxide synthase inhibition combined with sildenafil during acute pulmonary embolism.

While endogenous nitric oxide (NO) may be relevant to the beneficial hemodynamic effects produced by sildenafil during acute pulmonary embolism (APE), huge amounts of inducible NO synthase (iNOS)-derived NO may contribute to lung injury. We hypothesized that iNOS inhibition with S-methylisothiourea could attenuate APE-induced increases in oxidative stress and pulmonary hypertension and, therefore, could improve the beneficial hemodynamic and antioxidant effects produced by sildenafil during APE. Hemodynamic evaluations were performed in non-embolized dogs treated with saline (n=4), S-methylisothiourea (0.01 mg/kg followed by 0.5 mg/kg/h, n=4), sildenafil (0.3 mg/kg, n=4), or S-methylisothiourea followed by sildenafil (n=4), and in dogs that received the same drugs and were embolized with silicon microspheres (n=8 for each group). Plasma nitrite/nitrate (NOx) and thiobarbituric acid reactive substances (TBARS) concentrations were determined by Griess and a fluorometric assay, respectively. APE increased mean pulmonary arterial pressure (MPAP) and pulmonary vascular resistance index (PVRI) by 25±1.7 mm Hg and by 941±34 dyn s cm(-5) m(-2), respectively. S-methylisothiourea neither attenuated APE-induced pulmonary hypertension, nor enhanced the beneficial hemodynamic effects produced by sildenafil after APE (>50% reduction in pulmonary vascular resistance). While sildenafil produced no change in plasma NOx concentrations, S-methylisothiourea alone or combined with sildenafil blunted APE-induced increases in NOx concentrations. Both drugs, either alone or combined, produced antioxidant effects. In conclusion, although iNOS-derived NO may play a key role in APE-induced oxidative stress, our results suggest that the iNOS inhibitor S-methylisothiourea neither attenuates APE-induced pulmonary hypertension, nor enhances the beneficial hemodynamic effects produced by sildenafil.

Dietary nitrite extends lifespan and prevents age-related locomotor decline in the fruit fly.

Aging is associated with decreased nitric oxide (NO) bioavailability and signalling. Boosting of a dietary nitrate-nitrite-NO pathway e.g. by ingestion of leafy green vegetables, improves cardiometabolic function, mitochondrial efficiency and reduces oxidative stress in humans and rodents, making dietary nitrate and nitrite an appealing intervention to address age-related disorders. On the other hand, these anions have long been implicated in detrimental health effects of our diet, particularly in formation of carcinogenic nitrosamines. The aim of this study was to assess whether inorganic nitrite affects lifespan in Drosophila melanogaster and investigate possible mechanisms underlying any such effect. In a survival assay, female flies fed a nitrite supplemented diet showed lifespan extension by 9 and 15% with 0.1 and 1 µM nitrite respectively, with no impact of nitrite on reproductive output. Interestingly, nitrite could also protect female flies from age-dependent locomotor decline, indicating a protective effect on healthspan. NO generation from nitrite involved Drosophila commensal bacteria and was indicated by a fluorescent probe as well as direct measurements of NO gas formation with chemiluminescence. Nutrient sensing pathways such as TOR and sirtuins, have been strongly implicated in lifespan extension. In aged flies, nitrite supplementation significantly downregulated dTOR and upregulated dSir2 gene expression. Total triglycerides and glucose were decreased, a described downstream effect of both TOR and sirtuin pathways. In conclusion, we demonstrate that very low doses of dietary nitrite extend lifespan and favour healthspan in female flies. We propose modulation of nutrient sensing pathways as driving mechanisms for such effects.

Nitric oxide-dependent biodegradation of graphene oxide reduces inflammation in the gastrointestinal tract.

Understanding the biological fate of graphene-based materials such as graphene oxide (GO) is crucial to assess adverse effects following intentional or inadvertent exposure. Here we provide first evidence of biodegradation of GO in the gastrointestinal tract using zebrafish as a model. Raman mapping was deployed to assess biodegradation. The degradation was blocked upon knockdown of nos2a encoding the inducible nitric oxide synthase (iNOS) or by pharmacological inhibition of NOS using l-NAME, demonstrating that the process was nitric oxide (NO)-dependent. NO-dependent degradation of GO was further confirmed in vitro by combining a superoxide-generating system, xanthine/xanthine oxidase (X/XO), with an NO donor (PAPA NONOate), or by simultaneously producing superoxide and NO by decomposition of SIN-1. Finally, by using the transgenic strain Tg(mpx:eGFP) to visualize the movement of neutrophils, we could show that inhibition of the degradation of GO resulted in increased neutrophil infiltration into the gastrointestinal tract, indicative of inflammation.

Resveratrol improves endothelial cell markers impaired by plasma incubation from women who subsequently develop preeclampsia.

In this study, we demonstrated that plasma collected from women who subsequently developed preeclampsia caused increased heme oxygenase-1 (HO-1) production and decreased levels of nitric oxide (NO) markers in endothelial cells (HUVECs). Conversely, no changes in HO-1 or NO markers were found when HUVECs were treated with plasma from women who remained healthy throughout pregnancy. These alterations in HO-1 and NO markers were prevented by cotreatment with the polyphenol resveratrol, which also improved GSH levels. In addition, we evaluated changes induced by plasma incubation in the expression of genes and their related pathways associated with antioxidant defenses, such as Nrf2, ARE activity, and GSR. Collectively, our findings suggest that even before the appearance of clinical symptoms of preeclampsia, plasma from affected women is able to induce modifications in endothelial cells with respect to HO-1 production and NO markers. We believe that this in vitro strategy may offer an attractive alternative to the exploitation of candidate markers or screening molecules, such as resveratrol, for the prevention and management of preeclampsia.