Restore Nitric Oxide Bioavailability Research Articles

Dapagliflozin prevents oxidative stress-induced endothelial dysfunction via sirtuin 1 activation

Recent studies have demonstrated that dapagliflozin, a sodium-glucose cotransporter type 2 (SGLT2) inhibitor, prevents endothelial dysfunction; however, direct effects of dapagliflozin on the endothelium under oxidative stress and the underlying mechanism of action are not completely understood. This study aimed to define the role and related mechanisms of dapagliflozin in hydrogen peroxide (H2O2)-induced endothelial dysfunction. The endothelium-dependent vasorelaxation effect of dapagliflozin was assessed in an organ bath study. Endothelial dysfunction was assessed using protein expression level and phosphorylation of endothelial nitric oxide synthase (eNOS), nitric oxide (NO), reactive oxygen species (ROS), senescence-associated beta-galactosidase (SA-β-gal) activity, and senescence marker proteins (p21, p53). Co-immunoprecipitation and protein acetylation were performed to detect protein interactions. Dapagliflozin exerted a direct vasorelaxant effect in the aortic rings of C57BL/6 J mice. Furthermore, there was a significant improvement in endothelium-dependent vasorelaxation in dapagliflozin-treated diabetic mice compared to vehicle controls. Moreover, intracellular ROS levels and ONOO- levels, increased by H2O2, were reduced by dapagliflozin. Importantly, dapagliflozin inhibited H2O2-induced senescence in the human umbilical vein endothelial cells (HUVECs), as indicated by reduced SA‐β‐gal, p21, and p53. Mechanistically, dapagliflozin reversed the H2O2-mediated inhibition of eNOS serine phosphorylation and sirtuin 1 (SIRT1) expression in endothelial cells. In particular, SIRT1-mediated eNOS deacetylation is reportedly involved in dapagliflozin-enhanced eNOS activity. These findings indicate that dapagliflozin ameliorates endothelial dysfunction by restoring eNOS activity, restoring NO bioavailability, and reducing ROS generation via SIRT1 activation in oxidative stress-stimulated endothelial cells.

Combination Sodium Nitrite and Hydralazine Therapy Attenuates Heart Failure With Preserved Ejection Fraction Severity in a "2-Hit" Murine Model.

Background Recent studies have suggested that cardiac nitrosative stress mediated by pathological overproduction of nitric oxide (NO) via inducible NO synthase (iNOS) contributes to the pathogenesis of heart failure with preserved ejection fraction (HFpEF). Other studies have suggested that endothelial NO synthase (eNOS) dysfunction and attenuated NO bioavailability contribute to HFpEF morbidity and mortality. We sought to further investigate dysregulated NO signaling and to examine the effects of a NO-based dual therapy (sodium nitrite+hydralazine) following the onset of HFpEF using a "2-hit" murine model. Methods and Results Nine-week-old male C57BL/6 N mice (n=15 per group) were treated concurrently with high-fat diet and N(ω)-nitro-L-arginine methyl ester (L-NAME)(0.5 g/L per day) via drinking water for 10 weeks. At week 5, mice were randomized into either vehicle (normal saline) or combination treatment with sodium nitrite (75 mg/L in the drinking water) and hydralazine (2.0 mg/kg IP, BID). Cardiac structure and function were monitored with echocardiography and invasive hemodynamic measurements. Cardiac mitochondrial respiration, aortic vascular function, and exercise performance were also evaluated. Circulating and myocardial nitrite were measured to determine the bioavailability of NO. Circulating markers of oxidative or nitrosative stress as well as systemic inflammation were also determined. Severe HFpEF was evident by significantly elevated E/E', LVEDP, and Tau in mice treated with L-NAME and HFD, which was associated with impaired NO bioavailability, mitochondrial respiration, aortic vascular function, and exercise capacity. Treatment with sodium nitrite and hydralazine restored NO bioavailability, reduced oxidative and nitrosative stress, preserved endothelial function and mitochondrial respiration, limited the fibrotic response, and improved exercise capacity, ultimately attenuating the severity of "two-hit" HFpEF. Conclusions Our data demonstrate that nitrite, a well-established biomarker of NO bioavailability and a physiological source of NO, is significantly reduced in the heart and circulation in the "2-hit" mouse HFpEF model. Furthermore, sodium nitrite+hydralazine combined therapy significantly attenuated the severity of HFpEF in the "2-hit" cardiometabolic HFpEF. These data suggest that supplementing NO-based therapeutics with a potent antioxidant and vasodilator agent may result in synergistic benefits for the treatment of HFpEF.

Late‐Life Treatment with the Senolytic ABT‐263 Reverses Aortic Stiffening and Improves Endothelial Function with Aging

Advancing age is the primary risk factor for the development of cardiovascular diseases (CVD), driven largely by age-related arterial dysfunction. Two key manifestations of arterial dysfunction with advancing age are aortic stiffening and vascular endothelial dysfunction. Excessive reactive oxygen species (ROS)-induced oxidative stress is a major macro-mechanistic process causing arterial dysfunction during aging; however, the upstream mechanistic event(s) driving oxidative stress are incompletely understood. A rapidly emerging candidate is cellular senescence, a state of irreversible cell-cycle arrest that can be cleared with senolytics. Purpose We tested the hypotheses that late-life treatment with the senolytic ABT-263 (ABT) would: 1) reverse age-related aortic stiffening by reducing aortic intrinsic mechanical wall stiffness (elastic modulus [EM]); and 2) increase endothelial function by increasing nitric oxide (NO) bioavailability and reducing oxidative stress. Methods Old (27 mo) male C57BL6/N mice were treated with vehicle ([V]; 10% EtOH, 30% PEG400 and 60% Phosal 50 PG; n = 7) or ABT (50 mg/kg/day in [V]; n = 6) by oral gavage using a 1 week on – 2 weeks off – 1 week on dosing paradigm. A cohort of young adult mice (6 mo; n = 5) served as a young control (YC) reference group. Aortic pulse wave velocity (PWV), an in vivo measure of aortic stiffness, was measured pre- and post-treatment. Aortic elastic modulus was assessed by performing stress-strain testing in excised aortic rings. Endothelial function was assessed via ex vivo carotid artery endothelial-dependent dilation (EDD) with increasing concentrations of acetylcholine (ACh). NO bioavailability (ACh in the presence of the NO-synthase inhibitor, L-NAME) and the role of excessive ROS in regulating EDD (ACh with the addition of the ROS scavenger, TEMPOL) were assessed as potential mechanisms. Results Aortic stiffness. ABT reversed aortic PWV in old mice (pre: 456 ± 7 vs post: 375 ± 11 cm/sec, P = 0.0003), to levels of YC (348 ± 13 cm/sec; P = 0.14 vs. ABT), whereas no effect was observed in the [V]-treated group. Reduced aortic PWV with ABT was accompanied by lower aortic EM (V: 2738 ± 152 kPa, ABT: 2228 ± 155 kPa, YC: 2120 ± 362 kPa; P = 0.03). Endothelial function. ABT-treated mice had greater peak EDD relative to [V]-treated animals and comparable to YC (ABT: 94 ± 3% vs. V: 78 ± 5%, P = 0.01; YC: 91 ±2). Group differences in peak EDD were abolished in the presence of L-NAME, suggesting that ABT rescued EDD by restoring NO bioavailability. TEMPOL restored peak EDD in [V]-treated old mice to YC levels (92 ±5%, P = 0.003 vs. ACh alone), while having no effect in ABT or YC animals, suggesting that ABT selectively ameliorated the tonic ROS-related suppression of EDD with aging. Conclusion Cellular senescence is mechanistically implicated in age-related arterial dysfunction, and treatment with the senolytic compound ABT-263 may be a therapeutic strategy for improving arterial function, with the potential for reducing CVD risk with aging.

Edible Bird's Nest Protects Against Hyperglycemia-Induced Oxidative Stress and Endothelial Dysfunction.

Increased oxidative stress by hyperglycemia is a major cause of vascular complications in diabetes. Bird’s nest, which is made from the saliva of swiftlets has both medicinal and nutritional values dated back to ancient China. However, its role in improving endothelial dysfunction due to diabetes is yet to be elucidated. The present study examined the protective effect and mechanism of action of the aqueous extract of hydrolyzed edible bird nest (HBN) on endothelium in models of diabetes, in vitro and in vivo. Male db/m+ and db/db mice were orally administered with or without HBN and glibenclamide for 28 days, followed by vascular reactivity studies in mouse aortas. Human umbilical vein endothelial cells (HUVECs) and isolated mouse aorta from C57BL/6J were treated with high glucose (HG), HBN, sialic acid (SA), glibenclamide, and apocynin, respectively. The effects of HBN on reactive oxygen species (ROS) production and nitric oxide (NO) bioavailability were assessed by Western blot, 2′,7′-dichlorofluorescin-diacetate (DCF-DA), and 4-amino-5-methylamino-2′,7′ difluorofluorescein (DAF-FM DA) in HUVECs, isolated mouse aorta, and db/db diabetic mice. HBN significantly reversed the endothelial dysfunction in diabetic mice and isolated mouse aorta. HBN normalized ROS over-production of NOX2 and nitrotyrosine, reversed the reduction of anti-oxidant marker, SOD-1 as well as restored NO bioavailability in both HUVECs challenged with HG and in db/db diabetic mice. Similarly, HG-induced elevation of oxidative stress in HUVECs were reversed by SA, glibenclamide, and apocynin. This attests that HBN restores endothelial function and protects endothelial cells against oxidative damage induced by HG in HUVECs, isolated mouse aorta, and db/db diabetic mice via modulating ROS mechanism, which subsequently increases NO bioavailability. This result demonstrates the potential role of HBN in preserving endothelial function and management of micro- or macrovascular complications in diabetes.

A Phase II Trial of Topical Sodium Nitrite in Patients with Sickle Cell Disease and Leg Ulcers

Background Leg ulcerations are a serious and debilitating complication of sickle cell disease (SCD). Patients with SCD and leg ulcers have biomarkers of severe hemolytic anemia, a state associated with low bioavailability of nitric oxide (NO). Therapies directed at restoring NO bioavailability might prove beneficial. We selected topical sodium nitrite for clinical development due to its safety profile and vascular, anti-microbial, antiplatelet, and pro-keratinocyte functions. The nitrite anion is a vasodilator in vivo by generating NO in tissues with low oxygen tension and pH, conditions that are likely present in chronic wounds. Data from our successful phase 1 study showed improved regional blood flow, decreased ulcer pain, and appeared to improve ulcer healing. The dosing data informed the concentration of active ingredient to be used and suggested sodium nitrite efficacy in SCD patients with leg ulcers (Minniti, CP et al, Lancet Haematol 1, e95-e103). Study Design and Methods This is a multicenter, phase 2, prospective, randomized, placebo-controlled study of topical sodium nitrite in patients with SCD and leg ulcers. Primary aim is to determine the safety, tolerability, and effectiveness of twice a week topical application of study ointment for 10 weeks. We hypothesize that sodium nitrite will 1) accelerate wound healing (>25% improvement over placebo arm); and/or 2) decrease pain at the wound site (>20% over placebo). Ulcers measured by ImageJ planimetry software to increase accuracy. The secondary aims are to: a) evaluate the effect of hydroxyurea (HU) on leg ulcer healing in combination with topical sodium nitrite or placebo; b) assess changes in ulcer microbiome after application of sodium nitrite or placebo and how these changes may relate to healing; c) evaluate the dermal composition and microvascular structure in the ulcer beds. We plan to enroll 50 adults with all SCD genotypes and leg ulcers not individually >100 cm2, such that, after an expected 20% dropout, 40 subjects will complete > 8 weeks of treatment. Exclusion criteria: use of PDE5 inhibitors, NO, L-arginine, nitroprusside, nitroglycerine; acute bacterial infection; pre-existing methemoglobinemia (>3.5% on two different occasions); G6PD deficiency. Randomization is stratified by HU use to minimize potential confounding effects on study outcomes. Funded by the Food and Drug Administration (FDA) Division of Orphan Drug Development (# FD-R-0005729); active at two sites (ClinicalTrials.gov NCT02863068). Results We have screened 68 subjects with known history of leg ulcer: 46% were not eligible as the ulcer was closed at the time of screening. Of the 30 eligible, 13 subjects enrolled (12 at Montefiore Medical Center, 1 at the University of Pittsburgh), with 9 subjects randomized. Reasons for screen failure, other than ulcer closed, are depicted in fig 1. Three subjects experienced ulcer decrease >25% in the run-in period and did not have ointment application as per protocol. One subject withdrew due to lack of adherence. Therefore the overall dropout rate at this time is 33%, higher than the 20% anticipated. No study ointment SAEs have been noted, AEs have been minor and non-ointment related. Most SAEs have been VOCs, expected in this patient population and wound infections. Discussion As expected, enrollment of subjects with a rare complication of a rare disease is challenging. The recurrent pattern of ulcers in SCD was the reason why the majority of patients were not able to enroll, as the ulcer was closed at the time of screening. We monitor them closely for possible re-opening. Simplification of protocol-related procedures, such as decrease in number of required visits from twice to once a week by packaging dose-specific blister packs that the patient takes home for self-administration of ointment, has facilitated enrollment. Travel to the center is being addressed. A close relationship between the subjects and study team is essential. Co-location of the wound specialist in the sickle cell clinic and training the research nurse for wound care has helped with recruitment. Disclosures Ogu: Vertex Pharmaceuticals: Consultancy. Kato:Novartis, Global Blood Therapeutics: Consultancy, Research Funding; Bayer: Research Funding. Minniti:Doris Duke Foundation: Research Funding.

S-Nitrosothiols as potential therapeutics to induce a mobilizable vascular store of nitric oxide to counteract endothelial dysfunction

Endothelial dysfunction predisposing to cardiovascular diseases is defined as an imbalance in the production of vasodilating factors, such as nitric oxide (NO), and vasoconstrictive factors. To insure its physiological role, NO, a radical with very short half-life, requires to be stored and transported to its action site. S-nitrosothiols (RSNOs) like S-nitrosoglutathione (GSNO) represent the main form of NO storage within the vasculature. The NO store formed by RSNOs is still bioavailable to trigger vasorelaxation. In this way, RSNOs are an emerging class of NO donors with a potential to restore NO bioavailability within cardiovascular disorders.The aim of this study was to compare S-nitrosothiols ability, formed of peptide (glutathione) like the physiologic GSNO or derived from amino acids (cysteine, valine) like the synthetics S-nitroso-N-acetylcysteine (NACNO) and S-nitroso-N-acetylpenicillamine (SNAP), respectively, to produce a vascular store of NO either in endothelium-intact or endothelium-removed aortae in order to evaluate whether RSNOs can be used as therapeutics to compensate endothelial dysfunction. Sodium nitroprusside (SNP), a marketed drug already in clinics, was used as a non-RSNO NO-donor. Endothelium-intact or endothelium-removed aortae, isolated from normotensive Wistar rats, were exposed to RSNOs or SNP. Then, NO-derived (NOx) species, representing the NO store inside the vascular wall, were quantified using the diaminonaphthalene probe coupled to mercuric ions. The bioavailability of the NO store and its ability to induce vasodilation was tested using N-acetylcysteine, then its ability to counteract vasoconstriction was challenged using phenylephrine (PHE).All the studied RSNOs were able to generate a NO store materialized by a three to five times increase in NOx species inside aortae. NACNO was the most potent RSNO to produce a vascular NO store bioavailable for vasorelaxation and the most efficient to induce vascular hyporeactivity to PHE in endothelium-removed aortae. GSNO and SNAP were equivalent and more efficient than SNP. In endothelium-intact aortae, the NO store was also formed whereas it seemed less available for vasorelaxation and did not influence PHE-induced vasoconstriction. In conclusion, RSNOs – NACNO in a better extent – are able to restore NO bioavailability as a functional NO store within the vessel wall, especially when the endothelium is removed. This was associated with a hyporeactivity to the vasoconstrictive agent phenylephrine. Treatment with RSNOs could present a benefit to restore NO-dependent functions in pathological states associated with injured endothelium.

Maternal Exposure to Bisphenol A Combined with High-Fat Diet-Induced Programmed Hypertension in Adult Male Rat Offspring: Effects of Resveratrol.

Maternal exposure to endocrine disrupting chemicals (EDCs) and a high-fat intake may induce the developmental programming of hypertension in adult offspring. Bisphenol A (BPA) is one of the most commonly environmental EDCs. As the nitric oxide (NO) and aryl hydrocarbon receptor (AHR) signaling pathways both contribute to the pathogenesis of hypertension, we evaluated whether resveratrol, an antioxidant and an AHR antagonist, can prevent hypertension programmed by a maternal BPA and HF diet. Sixteen-week-old male rat offspring were assigned to six groups (n = 8 per group): Control, HF (D12331, Research Diets), BPA (50 μg/kg/day), HF + BPA, BPA + R (resveratrol 50mg/L in drinking water throughout pregnancy and lactation), and HF + BPA + R. Maternal BPA exposure exacerbated hypertension programmed by HF consumption in adult male offspring, which was protected by maternal resveratrol therapy. The BPA and HF diet synergistically induced oxidative stress in offspring kidneys, which resveratrol treatment prevented. We observed that HF + BPA-induced programmed hypertension was associated with a decreased NO bioavailability, increased oxidative stress, and an activated AHR signaling pathway. The beneficial effects of resveratrol are relevant to restoring NO bioavailability, reducing oxidative stress, and antagonizing the AHR signaling pathway. Our results cast a new light on resveratrol as a reprogramming strategy to protect against hypertension programmed by combined BPA and HF exposure, but this strategy has yet to be translated into clinical applications.

Nitric oxide pathology and therapeutics in sickle cell disease.

Sickle cell disease is caused by a mutant form of hemoglobin that polymerizes under hypoxic conditions which leads to red blood cell (RBC) distortion, calcium-influx mediated RBC dehydration, increased RBC adhesivity, reduced RBC deformability, increased RBC fragility, and hemolysis. These impairments in RBC structure and function result in multifaceted downstream pathology including inflammation, endothelial cell activation, platelet and leukocyte activation and adhesion, and thrombosis, all of which contribute vascular occlusion and substantial morbidity and mortality. Hemoglobin released upon RBC hemolysis scavenges nitric oxide (NO) and generates reactive oxygen species (ROS) and thereby decreases bioavailability of this important signaling molecule. As the endothelium-derived relaxing factor, NO acts as a vasodilator and also decreases platelet, leukocyte, and endothelial cell activation. Thus, low NO bioavailability contributes to pathology in sickle cell disease and its restoration could serve as an effective treatment. Despite its promise, clinical trials based on restoring NO bioavailability have so far been mainly disappointing. However, particular "NO donating" agents such as nitrite, which unlike some other NO donors can improve sickle RBC properties, may yet prove effective.

The novel organic mononitrate NDHP attenuates hypertension and endothelial dysfunction in hypertensive rats

RationaleDevelopment and progression of cardiovascular diseases, including hypertension, are often associated with impaired nitric oxide synthase (NOS) function and nitric oxide (NO) deficiency. Current treatment strategies to restore NO bioavailability with organic nitrates are hampered by undesirable side effects and development of tolerance. In this study, we evaluated NO release capability and cardiovascular effects of the newly synthesized organic nitrate 1, 3-bis (hexyloxy) propan-2-yl nitrate (NDHP). MethodsA combination of in vitro and in vivo approaches was utilized to assess acute effects of NDHP on NO release, vascular reactivity and blood pressure. The therapeutic value of chronic NDHP treatment was assessed in an experimental model of angiotensin II-induced hypertension in combination with NOS inhibition. ResultsNDHP mediates NO formation in both cell-free system and small resistance arteries, a process which is catalyzed by xanthine oxidoreductase. NDHP-induced vasorelaxation is endothelium independent and mediated by NO release and modulation of potassium channels. Reduction of blood pressure following acute intravenous infusion of NDHP was more pronounced in hypertensive rats (two-kidney-one-clip model) than in normotensive sham-operated rats. Toxicological tests did not reveal any harmful effects following treatment with high doses of NDHP. Finally, chronic treatment with NDHP significantly attenuated the development of hypertension and endothelial dysfunction in rats with chronic NOS inhibition and angiotensin II infusion. ConclusionAcute treatment with the novel organic nitrate NDHP increases NO formation, which is associated with vasorelaxation and a significant reduction of blood pressure in hypertensive animals. Chronic NDHP treatment attenuates the progression of hypertension and endothelial dysfunction, suggesting a potential for therapeutic applications in cardiovascular disease.

Sodium nitrite attenuates hypertension-in-pregnancy and blunts increases in soluble fms-like tyrosine kinase-1 and in vascular endothelial growth factor

Preeclampsia is a pregnancy-associated disorder characterized by hypertension with uncertain pathogenesis. Increases in antiangiogenic soluble fms-like tyrosine kinase-1 (sFlt-1) and reductions in nitric oxide (NO) bioavailability have been observed in preeclamptic women. However, the specific mechanisms linking these detrimental changes to the hypertension-in-pregnancy are not clearly understood. In this regard, while recent findings have suggested that nitrite-derived NO formation exerts antihypertensive and antioxidant effects, no previous study has examined these responses to orally administered nitrite in hypertension-in-pregnancy. We then hypothesized restoring NO bioavailability with sodium nitrite in pregnant rats upon NO synthesis inhibition with N(omega)-nitro-l-arginine methyl ester (L-NAME) attenuates hypertension and high circulating levels of sFlt-1. Number and weight of pups and placentae were recorded to assess maternal-fetal interface. Plasma sFlt-1, vascular endothelial growth factor (VEGF) and biochemical determinants of NO formation and of antioxidant function were measured. We found that sodium nitrite blunts the hypertension-in-pregnancy and restores the NO bioavailability, and concomitantly prevents the L-NAME-induced high circulating sFlt-1 and VEGF levels. Also, our results suggest that nitrite-derived NO protected against reductions in litter size and placental weight caused by L-NAME, improving number of viable and resorbed fetuses and antioxidant function. Therefore, the present findings are consistent with the hypothesis that nitrite-derived NO may possibly be the driving force behind the maternal and fetal beneficial effects observed with sodium nitrite during hypertension-in-pregnancy. Certainly further investigations are required in preeclampsia, since counteracting the damages to the mother and fetal sides resulting from hypertension and elevated sFlt-1 levels may provide a great benefit in this gestational hypertensive disease.

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 (3μM, 1 hour) and chronic (10mg/L, 8 weeks) treatments with BM-573 on vasodilatory function, nitric oxide (NO) bioavailability, oxidative stress and systolic blood pressure in 15 weeks old apolipoprotein E-deficient (ApoE-KO) mice. ApoE-KO mice showed a reduced endothelium-derived relaxation. In addition, NO bioavailability was reduced and oxidative stress and blood pressure were increased in ApoE-KO mice versus wild-type mice. BM-573 treatments were able to improve the relaxation profile in ApoE-KO mice. Short-term effects of BM-573 were mainly mediated by an increased phosphorylation of both eNOS and Akt, whereas BM-573 in vivo treatment also reduced oxidative stress and restored NO bioavailability. In addition, chronic administration of BM-573 reduced systolic blood pressure in ApoE-KO mice. In conclusion, pharmacological modulation of TxA2 biosynthesis and biological activities by dual TP antagonism/TxAS inhibition with BM-573, already known to prevent plaque formation, has the potential to correct vasodilatory dysfunction at the early stages of atherosclerosis.

Comparison of the effects of levocetirizine and losartan on diabetic nephropathy and vascular dysfunction in streptozotocin-induced diabetic rats

This work was designed to investigate the effects of levocetirizine, a histamine H1 receptor antagonist, on diabetes-induced nephropathy and vascular disorder, in comparison to an angiotensin II receptor antagonist, losartan. Diabetes was induced in male Sprague Dawley rats by a single intraperitoneal injection of streptozotocin (50mg/kg). Diabetic rats were divided into three groups; diabetic, diabetic-levocetirizine (0.5mg/kg/day) and diabetic-losartan (25mg/kg/day). Treatments were started two weeks following diabetes induction and continued for additional eight weeks. At the end of the experiment, urine was collected and serum was separated for biochemical measurements. Tissue homogenates of kidney and aorta were prepared for measuring oxidative stress, nitric oxide (NO), transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α). Moreover, histological analyses were conducted and aortic vascular reactivity was investigated.Levocetirizine improved renal function in diabetic rats (evidenced by mitigation of diabetes-induced changes in kidney to body weight ratio, serum albumin, urinary proteins and creatinine clearance). Moreover, levocetirizine attenuated the elevated renal levels of TNF-α and TGF-β1, ameliorated renal oxidative stress and restored NO bioavailability in diabetic kidney. These effects were comparable to or surpassed those produced by losartan. Moreover, levocetirizine, similar to losartan, reduced the enhanced responsiveness of diabetic aorta to phenylephrine. Histological evaluation of renal and aortic tissues further confirmed the beneficial effects of levocetirizine on diabetic nephropathy and revealed a greater attenuation of diabetes-induced vascular hypertrophy by levocetirizine than by losartan. In conclusion, levocetirizine may offer comparable renoprotective effect to, and possibly superior vasculoprotective effects than, losartan in streptozotocin-diabetic rats.

Rapid and body weight-independent improvement of endothelial and high-density lipoprotein function after Roux-en-Y gastric bypass: role of glucagon-like peptide-1.

Roux-en-Y gastric bypass (RYGB) reduces body weight and cardiovascular mortality in morbidly obese patients. Glucagon-like peptide-1 (GLP-1) seems to mediate the metabolic benefits of RYGB partly in a weight loss-independent manner. The present study investigated in rats and patients whether obesity-induced endothelial and high-density lipoprotein (HDL) dysfunction is rapidly improved after RYGB via a GLP-1-dependent mechanism. Eight days after RYGB in diet-induced obese rats, higher plasma levels of bile acids and GLP-1 were associated with improved endothelium-dependent relaxation compared with sham-operated controls fed ad libitum and sham-operated rats that were weight matched to those undergoing RYGB. Compared with the sham-operated rats, RYGB improved nitric oxide (NO) bioavailability resulting from higher endothelial Akt/NO synthase activation, reduced c-Jun amino terminal kinase phosphorylation, and decreased oxidative stress. The protective effects of RYGB were prevented by the GLP-1 receptor antagonist exendin9-39 (10 μg·kg(-1)·h(-1)). Furthermore, in patients and rats, RYGB rapidly reversed HDL dysfunction and restored the endothelium-protective properties of the lipoprotein, including endothelial NO synthase activation, NO production, and anti-inflammatory, antiapoptotic, and antioxidant effects. Finally, RYGB restored HDL-mediated cholesterol efflux capacity. To demonstrate the role of increased GLP-1 signaling, sham-operated control rats were treated for 8 days with the GLP-1 analog liraglutide (0.2 mg/kg twice daily), which restored NO bioavailability and improved endothelium-dependent relaxations and HDL endothelium-protective properties, mimicking the effects of RYGB. RYGB rapidly reverses obesity-induced endothelial dysfunction and restores the endothelium-protective properties of HDL via a GLP-1-mediated mechanism. The present translational findings in rats and patients unmask novel, weight-independent mechanisms of cardiovascular protection in morbid obesity.

Mitigation of lead-induced neurotoxicity by the naringin: erythrocytes as neurons substitute markers.

This study aimed to investigate the effect of lead (Pb) on neuronal nitric oxide synthase (nNOS) activity using erythrocytes as neurons surrogate markers. Moreover, the protective effect of naringin (NAR) against lead acetate (PbAc)-induced neurotoxicity was investigated. Human erythrocytes were incubated with L-arginine (L-Arg), Nω-nitro-L-Arginine methyl ester ( L-NAME), NAR, PbAc, PbAc+L-Arg, PbAc+NAR, or PbAc+L-Arg+NAR. The present results revealed that incubation of erythrocytes with PbAc inhibited NOS activity and decreased nitrite levels as an index for nitric oxide (NO) production to values similar that of L-NAME as known NOS inhibitor. Likewise, PbAc induced a significant decrease in activities of ATPases and acetylcholinesterase compared to control cells. Furthermore, PbAc exposure significantly increased protein carbonyl content (PCC) and malondialdehyde (MDA) levels while significantly decrease the levels of reduced glutathione (GSH). On the contrary, incubation of erythrocytes with PbAc in the presence of L-Arg+NAR synergistically ameliorated the investigated parameters compared to erythrocytes incubated with PbAc alone. These data suggest that NAR can restore NO bioavailability in a situation of Pb-induced cellular damage. This attributed to antioxidant activity and restoration NOS activity.

Two different approaches to restore renal nitric oxide and prevent hypertension in young spontaneously hypertensive rats: l-citrulline and nitrate

Nitric oxide (NO) deficiency mediates oxidative stress in the kidney and is involved in the development of hypertension. NO synthesis occurs via 2 pathways: nitric oxide synthase (NOS) dependent and NOS-independent. We tested whether the development of hypertension is prevented by restoration of NO by dietary l-citrulline or nitrate supplementation in young spontaneously hypertensive rats (SHRs). Male SHRs and normotensive Wistar Kyoto control rats (WKYs)s age 4weeks were assigned to 4groups: untreated SHRs and WKYs, and SHRs and WKYs that received 0.25% l-citrulline for 8weeks. In our second series of studies, we replaced l-citrulline with 1mmol/kg/d sodium nitrate. All rats were sacrificed at age 12weeks. We found an increase in the blood pressure of SHRs was prevented by dietary supplementation of l-citrulline or nitrate. Both treatments restored NO bioavailability and reduced oxidative stress in SHR kidneys. l-Citrulline therapy reduced levels of l-arginine and asymmetric dimethylarginine (ADMA)-an endogenous inhibitor of NOS-and increased the l-arginine-to-ADMA ratio in SHR kidneys. Nitrate treatment reduced plasma levels of l-arginine and ADMA concurrently in SHRs. Our findings suggest that both NOS-dependent and -independent approaches in the prehypertensive stage toward augmentation of NO can prevent the development of hypertension in young SHRs.

Erythrocyte nitric oxide synthase as a surrogate marker for mercury-induced vascular damage: The modulatory effects of naringin

In this study, endothelial nitric oxide synthase activity and nitric oxide (NO) production by human erythrocytes in the presence and absence of mercuric chloride (HgCl2 ), L-arginine (L-ARG), N ω- nitro-L-arginine methyl ester (L-NAME), and naringin (NAR) were investigated. In addition, the levels of reduced glutathione (GSH) and related enzymes were estimated in erythrocytes hemolysate. The protein carbonyl content (PCC) and thiobarbituric acid-reactive substances (TBARS) levels were also determined. The results of this study revealed that the treatment of erythrocytes with either HgCl2 or L-NAME induced a significant decrease in NOS activity and nitrite levels compared with control cells. Furthermore, mercury exposure significantly increased the levels of PCC and TBARS but reduced the GSH level. The activities of glucose-6-phosphate dehydrogenase, glutathione reductase, glutathione peroxidase, and glutathione-S-transferase (GST) were inhibited. The exposure of erythrocytes to HgCl2 in combination with L-ARG, NAR, or both ameliorated the investigated parameters compared with erythrocytes incubated with HgCl2 alone. These results indicate that mercury exposure decreased both erythrocyte NOS activity and nitrite production, and that these parameters might be indicative of mercury exposure. The data also suggest that concomitant treatment with NAR can restore NO bioavailability through either its metal-chelating properties or its antioxidant activity.

Role of cellular L-arginine uptake and nitric oxide production on renal blood flow and arterial pressure regulation

L-Arginine (L-Arg) is the substrate for nitric oxide (NO) formation. Reduced NO bioavailability, particularly within the renal circulation, has been identified as a key factor in the pathogenesis of hypertension. This review focuses on the pathogenic role of abnormal L-Arg transport, particularly within the kidney, in hypertension. Most recent studies have attempted to restore NO bioavailability in cardiovascular diseases with the use of antioxidants to reduce NO inactivation, but this approach has failed to provide beneficial effects in the clinical setting. We argue that this may be due to reduced NO formation in hypertension, which has largely been overlooked as a means of restoring NO bioavailability in cardiovascular diseases. Recent data indicate that renal L-Arg transport plays an important role in regulating both renal perfusion and function and the long-term set point of arterial pressure in health. Perturbations in the renal L-Arg transport system can give rise to abnormal renal perfusion and function, initiating hypertension and related renal damage. Accordingly, we propose that L-Arg transporters are a new treatment target in hypertension and in disease states where renal NO bioavailability is disturbed.

Acute Exposure to Low Glucose Rapidly Induces Endothelial Dysfunction and Mitochondrial Oxidative Stress

Hypoglycemia is associated with increased mortality. The reasons for this remain unclear, and the effects of low glucose exposure on vascular endothelial function remain largely unknown. We endeavored to determine the effects of low glucose on endothelial cells and intact human arterioles. We exposed human umbilical vein endothelial cells to low glucose conditions in a clinically relevant range (40-70 mg/dL) and found rapid and marked reductions in nitric oxide (NO) bioavailability (P<0.001). This was associated with concomitantly increased mitochondrial superoxide production (P<0.001) and NO-dependent mitochondrial hyperpolarization (P<0.001). Reduced NO bioavailability was rapid and attributable to reduced endothelial nitric oxide synthase activity and destruction of NO. Low glucose rapidly activated AMP kinase, but physiological activation failed to restore NO bioavailability. Pharmacological AMP kinase activation led to phosphorylation of endothelial nitric oxide synthase's Ser633 activation site, reversing the adverse effects of low glucose. This protective effect was prevented by L-NG-Nitroarginine methyl ester. Intact human arterioles exposed to low glucose demonstrated marked endothelial dysfunction, which was prevented by either metformin or TEMPOL. Our data suggest that moderate low glucose exposure rapidly impairs NO bioavailability and endothelial function in the human endothelium and that pharmacological AMP kinase activation inhibit this effect in an NO-dependent manner.

Reversal of hemoglobin-induced vasoconstriction with sustained release of nitric oxide

Erythrocyte free hemoglobin (Hb) induces vasoconstriction due to nitric oxide (NO) scavenging, limiting the NO available for vascular smooth muscle. The central objective of this study was to restore NO bioavailability using long-lived circulating NO-releasing nanoparticles (NO-np) to reverse the vasoconstriction and hypertension induced by polymerized bovine Hb (PBH) NO scavenging. PBH (13 g/dl) was infused in a volume equal to 10% of the animal blood volume. Intravascular NO supplementation was provided with an infusion of NO-np (10 and 20 mg/kg body wt). This study was performed using the hamster window chamber model to concurrently access systemic and microvascular hemodynamics. Infusion of PBH increased blood pressure and induced vasoconstriction. Treatment with 10 and 20 mg/kg NO-np reduced the blood pressure and vasoconstriction induced by PBH. Moreover, the higher dose of NO-np decreased blood pressure and induced vasodilation compared with baseline, respectively. Treatment with NO-np to decrease PBH-induced vasoconstriction increased methemoglobin levels and plasma nitrite and nitrate. In conclusion, NO-np counteracted both systemic hypertension and decreased the vasoconstrictor effects of PBH infusion, improving systemic and microvascular function. Based on the observed physiological properties, NO-np has clear potential as a therapeutic agent to replenish NO in situations where NO production is impaired, insufficient, or consumed, thereby preventing vascular complications.

Inorganic nitrite and chronic tissue ischaemia: a novel therapeutic modality for peripheral vascular diseases

Ischaemic tissue damage represents the ultimate form of tissue pathophysiology due to cardiovascular disease, which is the leading cause of morbidity and mortality across the globe. A significant amount of basic research and clinical investigation has been focused on identifying cellular and molecular pathways to alleviate tissue damage and dysfunction due to ischaemia and subsequent reperfusion. Over many years, the gaseous molecule nitric oxide (NO) has emerged as an important regulator of cardiovascular health as well as protector against tissue ischaemia and reperfusion injury. However, clinical translation of NO therapy for these pathophysiological conditions has not been realized for various reasons. Work from our laboratory and several others suggests that a new form of NO-associated therapy may be possible through the use of nitrite anion (sodium nitrite), a prodrug which can be reduced to NO in ischaemic tissues. In this manner, nitrite anion serves as a highly selective NO donor in ischaemic tissues without substantially altering otherwise normal tissue. This surprising and novel discovery has reinvigorated hopes for effectively restoring NO bioavailability in vulnerable tissues while continuing to reveal the complexity of NO biology and metabolism within the cardiovascular system. However, some concerns may exist regarding the effect of nitrite on carcinogenesis. This review highlights the emergence of nitrite anion as a selective NO prodrug for ischaemic tissue disorders and discusses the potential therapeutic utility of this agent for peripheral vascular disease.