Enhancing Nitric Oxide Bioavailability Research Articles

S-Nitroso Human Serum Albumin Enhances Left Ventricle Hemodynamic Performance and Reduces Myocardial Damage after Local Ischemia-Reperfusion Injury.

Endothelial nitric oxide (NO) production is crucial in maintaining vascular homeostasis. However, in the context of ischemia-reperfusion (I/R) injury, uncoupled endothelial nitric oxide synthase (eNOS) can exacerbate reactive oxygen species (ROS) generation. Supplementation with S-nitroso human serum albumin (S-NO-HSA) offers a potential solution by mitigating eNOS uncoupling, thereby enhancing NO bioavailability. In a study conducted at the University of Verona, male rats underwent thoracotomy followed by 30 min left anterior descendant coronary (LAD) occlusion and subsequent reperfusion. Hemodynamic parameters were meticulously assessed using a conductance catheter inserted via the carotid artery. The rats were stratified into two main groups based on reperfusion duration and the timing of drug infusion, with the effects of S-NO-HSA evaluated after 2 or 24 h. Remarkably, intravenous administration of S-NO-HSA, initiated before or during ischemia, exhibited notable benefits. It significantly improved left ventricular function, safeguarded energetic substrates such as phosphocreatine and ATP, and sustained glutathione levels akin to basal conditions, indicative of diminished oxidative stress. The data from this study strongly suggest a protective role for S-NO-HSA in mitigating I/R injury induced by LAD artery occlusion, a phenomenon observed at both 2 and 24 h post-reperfusion. These findings underscore the promising therapeutic potential of NO supplementation in alleviating myocardial damage subsequent to ischemic insult.

Tsantan Sumtang, a traditional Tibetan medicine, protects pulmonary vascular endothelial function of hypoxia-induced pulmonary hypertension rats through AKT/eNOS signaling pathway

Ethnopharmacological relevanceTsantan Sumtang (TS), originated from the Four Tantras, is an empirical Tibetan medicine prescription, which has been widely used for treating cardiovascular diseases in the clinic in Qinghai Province of China. Our previous studies found that TS alleviated hypoxia-induced pulmonary hypertension (HPH) in rats. However, the effect and bioactive fractions of TS on hypoxia-injured pulmonary vascular endothelium are unknown. Aim of the studyTo investigate the effect, bioactive fractions and pharmacological mechanism of TS on hypoxia-injured pulmonary vascular endothelium in vivo and in vitro. Materials and methodsIn vivo studies, HPH animal model was established, and TS was administrated for four weeks. Then, hemodynamic indexes, ex vivo pulmonary artery perfusion experiment, morphological characteristics, nitric oxide (NO) production, and the protein expression of protein kinase B (AKT)/endothelial nitric oxide synthase (eNOS) and AMP-activated protein kinase (AMPK)/eNOS signaling were determined. In vitro studies, 1% O2-induced pulmonary artery endothelial cells (PAECs) injury model was applied for screening bioactive fractions of TS by cell proliferation assay and NO production measurement. The associated proteins of AKT/eNOS signaling were further measured to elucidate underlying mechanism of bioactive fraction of TS via using phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002. Ultra-high performance liquid chromatography with hybrid quadrupole-orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS) was used to reveal the chemical profile of bioactive fraction of TS. ResultsTS showed protective effect on the integrity of distal pulmonary arterial endothelium in HPH rats. Tsantan Sumtang dilated pulmonary arterial rings in HPH rats. TS enhanced NO bioavailability in lung tissue via regulating AKT/eNOS signaling. Furthermore, in the cellular level, cell viability as well as NO content of hypoxia-injured PAECs were elevated by fraction 17 of water extract of TS (WTS), through activating the AKT/eNOS signaling. Ellagic acid could be one of compositions in fraction 17 of WTS to produce NO in hypoxia-injured PAECs. ConclusionTS restored pulmonary arterial endothelial function in HPH rats. The bioactive fraction 17 was screened, which protected hypoxia-injured PAECs via upregulating AKT/eNOS signaling.

Analysis of dietary nitrate incorporation in porcine ocular tissues after supplementing 15N-labeled sodium nitrate

The mechanisms for the generation of nitric oxide (NO) include reduction of nitrate (NO 3 - ) and nitrite (NO 2 - ) as well as the classic nitric oxide synthase (NOS) pathways. In the reductive pathways, ingested nitrate is reduced to nitrite, mainly by oral bacteria and to some extent by tissue enzymes, then these nitrite ions can be reduced to NO by several endogenous proteins or, in stomach, by acid disproportionation. Dietary nitrate, rich in green leafy vegetables and beets, has beneficial effects in lowering blood pressure and improving vascular health by enhancing NO bioavailability. Previously, we showed that porcine eye components contain substantial amounts of nitrate and nitrite ions that are similar to those in blood. More importantly, cornea and sclera exhibited a nitrate reduction capability. To extend our knowledge on nitrate metabolism in the eye, in the present study, we fed pigs (Yorkshire domestic cross swine weighing 35-40kg, housed in an AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care)-accredited facility with environmental enrichment) with either NaCl or Na 15 NO 3 and assessed the amount of 15 NO 3 - and 15 NO 2 - in various parts of the eye by LC-MS/MS at 3 or 24h after supplementation. We also measured the total concentrations of nitrate and nitrite ions using a standard chemiluminescence nitric oxide analyzer. At 3h, there was a marked increase in both 15 NO 3 - and 15 NO 2 - in all parts of the eye; in particular, aqueous and vitreous humor showed very high 15 NO 3 - contents (77.5 and 74.5% respectively), similar to plasma (77.1%) and even higher 15 NO 2 - contents (39.9% and 35.3% respectively) than plasma (19.8%). Cornea (64.4%) and sclera (63.4%) also showed notable increases in 15 NO 3 - content at 3h, then retina (48.8%) and lens (44.8%) followed. In 15 NO 2 - percent analysis at 3h, except aqueous and vitreous humor, other eye parts showed similar levels to plasma. At 24h, 15 NO 3 - contents decreased significantly compared to the 3h point, but it was still higher than the baseline in all ocular components. The 15 NO 2 - contents at 24h were almost back to the baseline. Absolute values of nitrate and nitrite ions in these tissues show similar patterns as shown in 15 N analysis. These results indicate that dietary nitrate supplementation has a vast impact on the levels of nitrate and nitrite and likely NO as well in the eye. NO generation via the nitrate-nitrite reductive pathway may contribute to various physiological and pathophysiological processes in the eye and may be useful therapeutically. This work was supported by intramural NIDDK/NIH grant ZIA DK 025104-15 to Alan N. Schechter. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dipeptidyl peptidase 4 inhibition rescues PKA-eNOS signaling and suppresses aortic hypercontractility in male rats with heart failure

AimsVascular dysfunction and elevated circulating dipeptidyl peptidase 4 (DPP4) activity are both reported to be involved in the progression of heart failure (HF). While the cardiac benefits of DPP4 inhibitors (DPP4i) have been extensively studied, little is known about the effects of DPP4i on vascular dysfunction in nondiabetic HF. This study tested the hypothesis that vildagliptin (DPP4i) mitigates aortic hyperreactivity in male HF rats. Materials and methodsMale Wistar rats were subjected to left ventricle (LV) radiofrequency ablation to HF induction or sham operation (SO). Six weeks after surgery, radiofrequency-ablated rats who developed HF were treated with vildagliptin (120 mg⸱kg−1⸱day−1) or vehicle for 4 weeks. Thoracic aorta reactivity, dihydroethidium fluorescence, immunoblotting experiments, and enzyme-linked immunosorbent assays were performed. Key findingsDPP4i ameliorated the hypercontractility of HF aortas to the α-adrenoceptor agonist phenylephrine towards SO levels. In HF, the reduced endothelium and nitric oxide (NO) anticontractile effect on phenylephrine response was restored by DPP4i. At the molecular level, this vasoprotective effect of DPP4i was accompanied by (i) reduced oxidative stress and NADPH oxidase 2 (Nox2) expression, (ii) enhanced total endothelial nitric oxide synthase (eNOS) expression and phosphorylation at Ser1177, and (iii) increased PKA activation, which acts upstream of eNOS. Additionally, DPP4i restored the higher serum angiotensin II concentration towards SO. SignificanceOur data demonstrate that DPP4i ameliorates aortic hypercontractility, most likely by enhancing NO bioavailability, showing that the DPP4i-induced cardioprotection in male HF may arise from effects not only in the heart but also in conductance arteries.

Effect of Nitrates on Endurance Athletes- A Case Control Study

Beetroot itself is a precursor of Nitric Oxide (NO), it is used as a supplement. Increased NO availability will enhance oxygen and nutrient delivery to the working muscle by lowering the ATP cost of muscle contractile force production and the oxygen cost of aerobic exercise. The mechanisms contributed to these effects are linked to mitochondrial respiration and biogenesis which shows that enhancing NO bioavailability positively influences exercise performance in endurance athletes.VO2 max (maximal oxygen intake) is a familiar measurement related to aerobic endurance that numerous athletes use to determine their overall fitness as it measures the maximum amount of oxygen that a person can utilize throughout the intense exercise.

HIGH-INTENSITY STATINS COMBINED WITH EICOSAPENTAENOIC ACID (EPA) IMPROVES ENDOTHELIAL FUNCTION DURING EXPOSURE TO OXIDIZED LDL

BackgroundDuring atherosclerosis, endothelial cell (EC) dysfunction results in reduced nitric oxide (NO) bioavailability and increased cytotoxic peroxynitrite (ONOO−). This loss of NO bioavailability results in abnormal vasodilation and inflammatory changes. Eicosapentaenoic acid (EPA) administered as icosapent ethyl (IPE) reduced cardiovascular (CV) events in high-risk patients treated with statins (REDUCE-IT). We tested the effects of high-intensity statins and EPA in ECs exposed to oxidized LDL (oxLDL).Methods and ResultsHuman umbilical vein ECs (HUVECs) were pretreated with 20 mg/dL oxLDL for 20 min, then treated with atorvastatin (active metabolite, ATM) and rosuvastatin (rosuva) at 1.0 μM ± EPA (10μM) for 1 hr. Cells were stimulated with calcium and assayed for the NO/ONOO− release ratio using nanosensors. ECs exposed to oxLDL showed a 60% reduction in NO release compared with vehicle (386±29 to 156±18 nM, p< 0.001) concomitant with a pronounced increase in ONOO− release (205±31 to 283±16 nM, p< 0.001), resulting in a >70% decrease in the NO/ONOO− release ratio (p < 0.001). ECs treated with ATM had an improved NO/ONOO− release ratio (53%) that increased in combination with EPA by 216% (p < 0.01). Similar results were observed for EPA in combination with rosuvastatin. When either statin was combined with EPA, there was also decreased ONOO− release compared to statin alone (p < 0.01).ConclusionIn combination with high intensity statins, EPA enhanced NO bioavailability in dysfunctional human ECs. The ability of EPA to reverse vascular EC dysfunction may lead to reduced ischemic events in statin-treated patients, as evidenced in outcome trials. BackgroundDuring atherosclerosis, endothelial cell (EC) dysfunction results in reduced nitric oxide (NO) bioavailability and increased cytotoxic peroxynitrite (ONOO−). This loss of NO bioavailability results in abnormal vasodilation and inflammatory changes. Eicosapentaenoic acid (EPA) administered as icosapent ethyl (IPE) reduced cardiovascular (CV) events in high-risk patients treated with statins (REDUCE-IT). We tested the effects of high-intensity statins and EPA in ECs exposed to oxidized LDL (oxLDL). During atherosclerosis, endothelial cell (EC) dysfunction results in reduced nitric oxide (NO) bioavailability and increased cytotoxic peroxynitrite (ONOO−). This loss of NO bioavailability results in abnormal vasodilation and inflammatory changes. Eicosapentaenoic acid (EPA) administered as icosapent ethyl (IPE) reduced cardiovascular (CV) events in high-risk patients treated with statins (REDUCE-IT). We tested the effects of high-intensity statins and EPA in ECs exposed to oxidized LDL (oxLDL). Methods and ResultsHuman umbilical vein ECs (HUVECs) were pretreated with 20 mg/dL oxLDL for 20 min, then treated with atorvastatin (active metabolite, ATM) and rosuvastatin (rosuva) at 1.0 μM ± EPA (10μM) for 1 hr. Cells were stimulated with calcium and assayed for the NO/ONOO− release ratio using nanosensors. ECs exposed to oxLDL showed a 60% reduction in NO release compared with vehicle (386±29 to 156±18 nM, p< 0.001) concomitant with a pronounced increase in ONOO− release (205±31 to 283±16 nM, p< 0.001), resulting in a >70% decrease in the NO/ONOO− release ratio (p < 0.001). ECs treated with ATM had an improved NO/ONOO− release ratio (53%) that increased in combination with EPA by 216% (p < 0.01). Similar results were observed for EPA in combination with rosuvastatin. When either statin was combined with EPA, there was also decreased ONOO− release compared to statin alone (p < 0.01). Human umbilical vein ECs (HUVECs) were pretreated with 20 mg/dL oxLDL for 20 min, then treated with atorvastatin (active metabolite, ATM) and rosuvastatin (rosuva) at 1.0 μM ± EPA (10μM) for 1 hr. Cells were stimulated with calcium and assayed for the NO/ONOO− release ratio using nanosensors. ECs exposed to oxLDL showed a 60% reduction in NO release compared with vehicle (386±29 to 156±18 nM, p< 0.001) concomitant with a pronounced increase in ONOO− release (205±31 to 283±16 nM, p< 0.001), resulting in a >70% decrease in the NO/ONOO− release ratio (p < 0.001). ECs treated with ATM had an improved NO/ONOO− release ratio (53%) that increased in combination with EPA by 216% (p < 0.01). Similar results were observed for EPA in combination with rosuvastatin. When either statin was combined with EPA, there was also decreased ONOO− release compared to statin alone (p < 0.01). ConclusionIn combination with high intensity statins, EPA enhanced NO bioavailability in dysfunctional human ECs. The ability of EPA to reverse vascular EC dysfunction may lead to reduced ischemic events in statin-treated patients, as evidenced in outcome trials. In combination with high intensity statins, EPA enhanced NO bioavailability in dysfunctional human ECs. The ability of EPA to reverse vascular EC dysfunction may lead to reduced ischemic events in statin-treated patients, as evidenced in outcome trials.

Nitric oxide, aging and aerobic exercise: Sedentary individuals to Master's athletes

Aging is associated with a decline in physiological function and exercise performance. These effects are mediated, at least in part, by an age-related decrease in the bioavailability of nitric oxide (NO), a ubiquitous gasotransmitter and regulator of myriad physiological processes. The decrease in NO bioavailability with aging is especially apparent in sedentary individuals, whereas older, physically active individuals maintain higher levels of NO with advancing age. Strategies which enhance NO bioavailability (including nutritional supplementation) have been proposed as a potential means of reducing the age-related decrease in physiological function and enhancing exercise performance and may be of interest to a range of older individuals including those taking part in competitive sport. In this brief review we discuss the effects of aging on physiological function and endurance exercise performance, and the potential role of changes in NO bioavailability in these processes. We also provide a summary of current evidence for dietary supplementation with substrates for NO production — including inorganic nitrate and nitrite, l-arginine and l-citrulline — for improving exercise capacity/performance in older adults. Additionally, we discuss the (limited) evidence on the effects of (poly)phenols and other dietary antioxidants on NO bioavailability in older individuals. Finally, we provide suggestions for future research.

Exercise training improves cerebral endothelial function in juvenile and mature Ossabaw miniature‐pigs

BackgroundChronic hyperglycemia promotes excessive production of endothelial reactive oxygen species (ROS), which may impair nitric oxide (NO) signaling, a hallmark of vascular disfunction. How maturation and exercise training influence this process in the cerebrovasculature has yet to be explored. The purpose of this study was to examine the role of ROS on endothelial‐dependent cerebral dilation at different stages of maturation in sedentary and exercise trained hyperglycemic pigs. It was hypothesized that mature pigs would display reduced endothelial NO‐dependent dilation coinciding with augmented NAD(P)H Oxidase (NOX; involved in ROS production) activity and reduced superoxide dismutase (SOD; involved in ROS clearance) activity compared with juvenile pigs. Furthermore, compared with sedentary (SED) pigs, pigs that completed high‐intensity interval training (HIIT) would exhibit enhanced endothelial NO‐dependent dilation coupled with decreased NOX and increased SOD signaling.MethodsTwenty juvenile (n=10F/10M; 3±1 mo.) and 17 mature (n=9F/7M; 14±1 mo.) Ossabaw miniature‐pigs were divided into SED or HIIT groups. The HIIT consisted of 3 sessions per week (5X3 min intervals interspersed with 1 min recovery periods performed on a motorized treadmill) for 8 weeks. Animals were group housed with access to 1 kg of feed/pig/day and ad libitum access to a 10% sugar water solution. At the end of the intervention pigs were euthanized, and cerebral arteries were harvested for arterial myography experiments. To examine endothelial NO‐dependent relaxation, vasomotor responses to bradykinin (BK; 3e‐15M to 1e‐6M) were studied in the presence or absence of NO synthase inhibition (L‐NAME; 3e‐4 M). Further, to examine the role of ROS signaling, vasodilatory responses to BK were studied under the following conditions: 1) NOX inhibition (Apocynin; 1e‐4 M) and 2) SOD mimetic (TEMPOL; 1e‐4 M).ResultsAt the end of the trial all groups were considered hyperglycemic (mean plasma glucose&gt; 8.5 mmol/L), and plasma glucose values were lower in mature (8.8±1.6 mmol//L) vs. juvenile pigs (10.3±2.2 mmol/L; main effect; P&lt;0.05). Regarding cerebral vasomotor control, BK‐induced dilation was greater in HIIT vs. SED groups (main effect; P&lt;0.01), owing to enhanced NO signaling (main effect; P=0.05). Treatment with a NOX inhibitor increased BK‐induced dilation in juvenile (P=0.05) and mature SED pigs (P=0.01). Treatment with a SOD mimetic did not influence dilation in juvenile SED pigs (P=0.87), and increases in BK‐induced dilation in mature SED pigs approached significance (P=0.07).ConclusionThese results show that despite being hyperglycemic, HIIT improved endothelial‐dependent cerebral dilation in juvenile and mature pigs likely owing to enhanced NO bioavailability, secondary to decreased NOX and increased SOD signaling. Maturity was associated with lower levels of hyperglycemia and did not impair cerebral endothelial‐dependent dilation.

The root of the matter: nitrate-rich beetroot juice reduces skeletal muscle O2 uptake during exercise.

The root of the matter: nitrate-rich beetroot juice reduces skeletal muscle O2 uptake during exercise.

Anthocyanins attenuate endothelial dysfunction through regulation of uncoupling of nitric oxide synthase in aged rats.

Endothelial dysfunction is one of the main age‐related arterial phenotypes responsible for cardiovascular disease (CVD) in older adults. This endothelial dysfunction results from decreased bioavailability of nitric oxide (NO) arising downstream of endothelial oxidative stress. In this study, we investigated the protective effect of anthocyanins and the underlying mechanism in rat thoracic aorta and human vascular endothelial cells in aging models. In vitro, cyanidin‐3‐rutinoside (C‐3‐R) and cyanidin‐3‐glucoside (C‐3‐G) inhibited the d‐galactose (d‐gal)‐induced senescence in human endothelial cells, as indicated by reduced senescence‐associated‐β‐galactosidase activity, p21, and p16INK4a. Anthocyanins blocked d‐gal‐induced reactive oxygen species (ROS) formation and NADPH oxidase activity. Anthocyanins reversed d‐gal‐mediated inhibition of endothelial nitric oxide synthase (eNOS) serine phosphorylation and SIRT1 expression, recovering NO level in endothelial cells. Also, SIRT1‐mediated eNOS deacetylation was shown to be involved in anthocyanin‐enhanced eNOS activity. In vivo, anthocyanin‐rich mulberry extract was administered to aging rats for 8 weeks. In vivo, mulberry extract alleviated endothelial senescence and oxidative stress in the aorta of aging rats. Consistently, mulberry extract also raised serum NO levels, increased phosphorylation of eNOS, increased SIRT1 expression, and reduced nitrotyrosine in aortas. The eNOS acetylation was higher in the aging group and was restored by mulberry extract treatment. Similarly, SIRT1 level associated with eNOS decreased in the aging group and was restored in aging plus mulberry group. These findings indicate that anthocyanins protect against endothelial senescence through enhanced NO bioavailability by regulating ROS formation and reducing eNOS uncoupling.

Intermittent Hypoxic Exposure Reduces Endothelial Dysfunction.

Intermittent exposure to hypoxia (IHE) increases the production of reactive oxygen and nitrogen species as well as erythropoietin (EPO), which stimulates the adaptation to intense physical activity. However, several studies suggest a protective effect of moderate hypoxia in cardiovascular disease (CVD) events. The effects of intense physical activity with IHE on oxi-inflammatory mediators and their interaction with conventional CVD risk factors were investigated. Blood samples were collected from elite athletes (control n = 6, IHE n = 6) during a 6-day IHE cycle using hypoxicator GO2 altitude. IHE was held once a day, at least 2 hours after training. In serum, hydrogen peroxide (H2O2), nitric oxide (NO), 3-nitrotyrosine (3-Nitro), proinflammatory cytokines (IL-1β and TNFα), high sensitivity C-reactive protein (hsCRP), and heat shock protein 27 (HSP27) were determined by the commercial immunoenzyme (ELISA kits) or colorimetric methods. Serum erythropoietin (EPO) level was measured by ELISA kit every day of hypoxia. IHE was found to significantly increase H2O2, NO, and HSP27 but to decrease 3NT concentrations. The changes in 3NT and HSP27 following hypoxia proved to enhance NO bioavailability and endothelial function. In the present study, the oxi-inflammatory mediators IL-1β and hsCRP increased in IHE group but they did not exceed the reference values. The serum EPO level increased on the 3rd day of IHE, then decreased on 5th day of IHE, and correlated with NO/H2O2 ratio (rs = 0.640, P < 0.05). There were no changes in haematological markers contrary to lipoproteins such as low-density lipoprotein (LDL) and non-high-density lipoprotein (non-HDL) which showed a decreasing trend in response to hypoxic exposure. The study demonstrated that IHE combined with sports activity reduced a risk of endothelial dysfunction and atherogenesis in athletes even though the oxi-inflammatory processes were enhanced. Therefore, 6-day IHE seems to be a potential therapeutic and nonpharmacological method to reduce CVD risk, especially in elite athletes participating in strenuous training.

Effects of short‐term therapy with high dose nutritional inorganic nitrite and nitrate on vascular dysfunction, oxidative damage and epigenetic regulation in experimental arterial hypertension

BackgroundArterial hypertension is one of the major risk factors leading to highly relevant diseases like coronary artery disease, stroke or peripheral artery disease. Even short‐term application of angiotensin‐II (AT‐II) in animal models of arterial hypertension is leading to vascular dysfunction, enhanced production of reactive oxygen species (ROS) and modification of epigenetic processes. Nitric oxide (NO) represents an important opponent to AT‐II induced vasoconstriction and high blood pressure. Dietary uptake (e.g. vegetables) of inorganic nitrite (NO2−) and nitrate (NO3−) leads to enhanced NO bioavailability and provides antihypertensive effects. The present study aims to understand the underlying vasoprotective effects of nutritional NO2− and NO3− co‐therapy in hypertensive mice.Methods and resultsHigh‐dose AT‐II (1mg/kg/d for 1 week) was used to induce arterial hypertension in male C57BL/6 mice. In parallel high dose inorganic nitrite (7.5mg/kg/d) and inorganic nitrate (150mg/kg/d) was administered via drinking water. Hemodynamic effects were evaluated by blood pressure measurements and isometric tension studies. To elucidate underlying epigenetic regulation processes gene expression analyses and quantification of ROS formation were performed.Administration of AT‐II induced hypertension, cardiac hypertrophy and impaired endothelial function. Co‐therapy with high dose inorganic nitrite, but not with nitrate, normalised vascular function and arterial hypertension. In addition, oxidative stress markers and inflammatory pathways were enhanced in hypertensive mice and mostly normalized by nitrite therapy. We observed hypertension induced changes, at least by trend, of epigenetic proteins like histone deacetylase 2 or lysine demethylase 3A in cardiac tissue, which were mostly normalized by nitrite therapy.ConclusionNutritional nitric oxide precursors represent complementary therapy to classical antihypertensive drugs, representing a dietary treatment applicable to the general population (e.g. by eating certain vegetables). Short‐term therapy with inorganic nitrite showed beneficial effects on vascular function, oxidative stress and epigenetic regulation in experimental hypertension, which were less pronounced with inorganic nitrate. The observed differences may rely on superior bioconversion of nitrite to NO or intoxication through methaemoglobinaemia because of high dose inorganic nitrate therapy. Therefore, in particular inorganic nitrite seems to represent a cost‐efficient, drug‐free, alternative in antihypertensive therapy. Further clinical research is necessary to further support these preclinical findings and to demonstrate whether the incidence and severity of hypertension in particular and cardiovascular outcome in general can be influenced by inorganic nitrite therapy.Support or Funding InformationWe gratefully acknowledge the financial support by Stiftung Mainzer Herz (to M.O. and A.D.).

Pharmaceutical Preconditioning With Nitric Oxide Synthase and L-Arginine in Ischemic Tissues.

Nitric oxide (NO) is a multifunctional signaling molecule involved in regulating vascular tone and tissue oxygenation. It is also an important cytoprotective agent against ischemia-reperfusion injury (IRI). Enhancing NO bioavailability via exogenous NO synthases (NOSs) and L-arginine promotes conversation to NO, circumventing the problem of nonfunctioning NOSs under hypoxic and acidic conditions. In this study, the authors evaluated the therapeutic efficacy of neuronal, inducible, and endothelial NOS and L-arginine on reperfusion-induced skin flap alterations. The vascular pedicle isolated rat skin flap model was used and underwent 3 hours of ischemia. At 30 minutes before ischemia, normal saline, endothelial-, inducible-, and neuronal NOSs (1/2 IU) and L-arginine (100 mg/kg body weight) were administered by means of intravenous infusion. The IRI-induced alterations were measured 5 days after the operation. The 3 isoforms of NOS increased the flap vitality rate (VR) from 10% to 23% compared with the control group. L-Arginine treatment also increased the VR by approximately 15%. The combination of L-arginine with NOS resulted in even higher flap VRs. The best results could be achieved with the combination of endothelial NOS (2 IU) and L-arginine. Modulation of NO bioavailability via exogenous application of NOSs and L-arginine significantly improved VRs in a skin flap rat model. This pharmacologic preconditioning has the potential to attenuate IRI-induced alterations in skin flaps.

Inducible Nitric Oxide Synthase and L-Arginine Optimizes Nitric Oxide Bioavailability in Ischemic Tissues Under Diabetes Mellitus Type 1.

The mechanisms influencing the balance of nitric oxide (NO) bioavailability in tissues are negatively affected under diabetic and also under ischemic conditions. Free tissue transplantation for diabetic patients has to deal with both ischemic and diabetic circumstances, which lead to a significantly decrease in providing NO, thus increasing ischemia-reperfusion injury. In previous studies, we could prove that enhancing NO bioavailability leads to attenuated ischemia-reperfusion injury macrocirculatory and microcirculatory alterations in healthy and also in diabetes type 2 rats. This study is evaluating the role of inducible nitric oxide synthase in different dosages and L-arginine under diabetes type 1 conditions. Diabetic type 1 conditions were established via streptozotocin over a period of 4 weeks and verified via blood sugar, insulin, and C-peptide levels. Vascular pedicle isolated rat skin flap model that underwent 3 hours of ischemia was used. At 30 minutes before ischemia, normal saline, inducible nitric oxide synthase (NOS) (1/2 IE), and L-arginine (50 mg/kg body weight) were administered systemically. Ischemia/reperfusion (I/R)-induced alterations were measured 5 days after the operation. The inducible NOS (iNOS) attenuated I/R-induced alterations under diabetic type 1 conditions significantly with vitality rates of 16.1% compared with control group (5.5%). Best results could be achieved with the combination of iNOS (1 IE) and L-arginine displaying vitality rates of 43%. Increased dosage of inducible nitric oxide (2 IE) led to decreased vitality rates (22.2%/27.4% without/with L-arginine). Supporting the mechanisms of NO bioavailability via exogenous application of iNOS and L-arginine significantly attenuated I/R-induced alterations in a skin flap rat model. This pharmacologic preconditioning could be an easy and effective interventional strategy to uphold conversation of L-arginine to NO even on ischemic and type 1 diabetic conditions.

Ginkgolide A protects adverse cardiac remodeling through enhancing antioxidation and nitric oxide utilization in mice with pressure overload.

In this study, we aimed to explore whether ginkgolide A (GA) would exhibit cardio-protective effects in mice with pressure overload through enhancing antioxidation and nitric oxide (NO) bioavailability. 21 male mice were randomly assigned into three groups as follows: sham group (10 ml/kg/day PBS, n=7), transverse aortic constriction (TAC) group (TAC and 10 ml/kg/day PBS, n=7) and GA group (TAC and 20 mg/kg/day GA, n=7). All groups received an intraperitoneal injection for four weeks. Heart and body mass were measured. Cardiac function was assessed by echocardiography. The collagen deposition, area of cardiomyocytes, number of capillaries and cell apoptosis were evaluated using Masson's staining, WGA staining, CD31 staining and TUNEL assay, respectively. Cadiac oxidative and antioxidative indexes were measured by colorimetry. Nitrotyrosine (NT) and transforming growth factor-β (TGF-β) were determined by ELISA. Phospho-endothelial NO synthases (eNOS) (Ser1177), phospho-eNOS (Thr 495), eNOS, neuronal NOS (nNOS), inducible NOS (iNOS) and GAPDH were analyzed by western blot. GA treatment greatly improved cardiac dysfunction, suppressed cardiac hypertrophy and increased capillary number at 4 weeks after TAC (P<0.05). Fibrotic area, cardiomyocyte area, and cell apoptosis of GA group were declined notably as compared to those of TAC group (P<0.05). GA administration substantially attenuated cardiac oxidative stress, and reduced NT and TGF-β levels (P<0.05). Besides, GA medication can enhance eNOS signaling, resulting in increased cardiac NO production (P<0.05). GA had a cardioprotective effect in mice with pressure overload, which was closely related with reducing oxidative stress and enhancing NO bioavailability in hearts.

Protective effect of Xin-Ji-Er-Kang on cardiovascular remodeling in high-salt induced hypertensive mice: Role ofoxidative stress and endothelial dysfunction

BackgroundXin-Ji-Er-Kang (XJEK) is a Chinese herbal formula that has beenreported to exert effective protection against cardiovascular diseases, such as hypertension and myocarditis. ObjectiveThe aim of the present study was to investigate the effect of XJEK on high-salt-induced hypertensive mice and its possible mechanism. MethodsThe model of hypertension was established through a high-salt diet. Sixty male Kunming mice were randomized into six groups, namely the Control, Model, Low-dose XJEK, Middle-dose XJEK, High-dose XJEK and Fosinopril groups (n=10 per group). Different steady interventions were given to each group: 0.9% Sodium chloride was added to the diet of the Control group and 8% sodium chloride to the diet of the other five groups from the very beginning. An additional 4, 8 and 12 g/kg/day XJEK were intragastrically administered to the Low-dose, Middle-dose and High-dose XJEK groups, respectively, and 2 mg/kg/day fosinopril to the fosinopril group, from the start of week 5. Systolic blood pressure (SBP) was measured weekly from weeks 1 to 8 using the tail-cuff method. At the end of week 8, left ventricular (LV) systolic pressure, LV end-diastolic pressure and rate of rise of LV pressure were examined using a TransonicScisense catheter (Transonic Systems Inc,Ithaca, NY,USA). Endothelium-dependent relaxations induced by acetylcholine were observed in an isolated thoracic aorta ring. Serum and heartsweresampled for the measurement of the following indexes:Serum nitric oxide (NO), superoxide dismutase (SOD) activity and malondialdehyde (MDA) content (determined by colorimetricanalysis); serum angiotensin II(Ang II), endothelin-1, endothelial NO synthase (eNOS), asymmetric dimethylarginine (ADMA), tetrahydrobiopterin (BH4) concentration and l-arginine (determined by enzyme-linked immunosorbent assay); heart to body weight (HW/BW) ratio; myocardial morphological change (determined by HE and VG staining); myocardial eNOS expression (determined by immunofluorescence), and myocardial endothelin receptor A (ETA) expression (determined by western blotting).Results:Statistical data showed that the HW/BW ratio was significantly decreased in the drug treatment group. XJEK treatment could improve the heart systolic and diastolic function and ameliorate hemodynamic parameters and vascular remodeling indexes. Colorimetric results showed that, compared with the model group, XJEK increased serum SOD, NOlevels, and decreased those of serum MDA and Ang II. XJEK reverted changes in cardiac pathology, decreased the myocardial cross-sectional area, collagen volume fraction and perivascular collagen area and improved endothelial dysfunction (ED) by promoting eNOS activity, enhancing NO bioavailability, increasing the expression of BH4 and decreasing ETA content. In addition, treatment with XJEK decreased ADMA content in the myocardium.Conclusion:In conclusion, XJEK mitigates cardiac remodeling in high-salt-induced hypertensive mice. The potential mechanism involves improved oxidative stress and endothelial dysfunction, independently of ameliorating BP.

Mulberry Extract Attenuates Endothelial Dysfunction through the Regulation of Uncoupling Endothelial Nitric Oxide Synthase in High Fat Diet Rats.

Dyslipidemia is associated with endothelial dysfunction, which is linked to nitric oxide (NO) biology. The coupling of endothelial NO synthase with cofactors is a major step for NO release. This study is aimed to investigate the vascular pharmacology effects of mulberry in rat thoracic aorta and human vascular endothelial cells. In vitro, we investigated the protective effects of the mulberry extract and its main component cyanidin-3-rutinoside (C-3-R), against oxidized low-density lipoprotein (ox-LDL)-induced endothelial nitric oxide synthase (eNOS) uncoupling. Whereas ox-LDL significantly decreased NO levels in endothelial cells, mulberry extract, and C-3-R significantly recovered NO levels and phospho-eNOS Thr495 and Ser1177 expression. In vivo, mulberry was administered to 60% of high-fat diet (w/w)-fed Sprague Dawley (SD) rats for six weeks, in which endothelium-dependent relaxations were significantly improved in organ bath studies and isometric tension recordings. Consistently, aortic expressions of phospho-eNOS and nitrotyrosine were increased. Mulberry also raised serum NO levels, increased phosphorylation of eNOS, and reduced nitrotyrosine and intracellular reactive oxygen species (ROS) in aortas, showing that mulberry preserves endothelium-dependent relaxation in aortas from high-fat diet rats. We suggest that this effect is mediated through enhanced NO bioavailability, in which the regulation of ROS and its reduced eNOS uncoupling are involved.

Chewing Well During Meals May Benefit Health Via the Enterosalivary Nitrate–Nitrite–Nitric Oxide Pathway

Saliva contains nitrates recycled from ingested foods (e.g., vegetables) via the enterosalivary pathway. During meals, thorough chewing enhances salivary flow and the reduction of salivary nitrate to nitrite by oral commensal bacteria and thus leads to a nitrite-rich gastric environment. Subsequently, nitric oxide (NO) and S-nitrosothiols, rather than N-nitroso compounds, are generated in the acidic stomach. These molecules may confer gastric and systemic health benefits by transducing NO-mediated signals, particularly in patients with vascular endothelial dysfunction and metabolic syndrome, which is associated with reduced endogenous NO generation consequent to impaired NO synthase activity. This article reviews literature suggesting that the thorough chewing of food produces a nitrite-rich gastric environment and promotes health benefits by enhancing NO bioavailability via the enterosalivary nitrate-nitrite-NO pathway.

Oxymatrine ameliorates diabetes-induced aortic endothelial dysfunction via the regulation of eNOS and NOX4.

Oxymatrine (OMT) is the major quinolizidine alkaloid extracted from the root of Sophora flavescens Ait (the Chinese herb Kushen) and exhibits diverse pharmacological actions. In this study, we investigated the effects of OMT on diabetes-associated aortic endothelial dysfunction in a rat model of diabetes and its mechanisms. Male Sprague-Dawley rats were randomly divided into five groups: control, diabetic rats, diabetic rats treated with OMT (60, 120 mg/kg per day, by gavage), and diabetic rats treated with metformin (20 mg/kg per day, by gavage). The serum fasting blood glucose, insulin, total cholesterol, triglyceride, and nitric oxide (NO) levels were determined with commercial kits. Biochemical indices reflecting oxidative stress, such as malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were analyzed with commercial kits. Mitochondrial reactive oxygen species 2',7'-dichlorofluorescein diacetate (DCFH-DA) was measured by fluorescence microscopy. Histological analyses were conducted to observe morphological changes. Western blot analysis was applied to detect the expression levels of eNOS and NOX4. Reverse transcription polymerase chain reaction was used to detect the expressions of eNOS and NOX4 messenger RNA (mRNA). The diabetic rats exhibited markedly reduced body weight and increased plasma glucose levels. Moreover, the diabetic rats showed oxidative stress (significantly increased MDA and decreased SOD, CAT, GSH-Px, and serum NO levels). Hyperglycemia caused significant endothelial injury and dysfunction, including vasodilative and histologic changes in the diabetic rats. The expressions of phospho-eNOS protein and mRNA were significantly decreased, while the NOX4 protein expression was increased in the aortas of the diabetic rats. All of these diabetes-induced effects were reversed by OMT in the diabetic rats. The OMT treatment ameliorates diabetic endothelial dysfunction through enhanced NO bioavailability by upregulating eNOS expression and downregulating expression of NOX4.

Abstract P242: Dietary Nitrate Does not Reduce Resting Metabolic Rate or Oxidative Stress in Healthy Males

Introduction: Nitric oxide (NO) is a vasodilator that increases blood flow by promoting relaxation of endothelium. Dietary nitrate supplementation increases plasma nitrite, a marker of overall NO bioavailability. Previously, acute dietary nitrate supplementation has been shown to reduce oxygen consumption and improve tolerance during submaximal exercise in healthy populations. Less is known about the effect of dietary nitrate on oxygen consumption at rest. Hypothesis: We hypothesized that dietary nitrate supplementation would reduce resting metabolic rate (RMR) and oxidative stress (8-isoprostane) at rest, via enhanced NO bioavailability via the oxygen independent Nitrate-Nitrite-Nitric Oxide pathway in healthy, young males. Methods: In a randomized, double-blind, cross-over study, ten healthy, young males (21 ± 2 years) visited the laboratory on 5 separate occasions. Participants completed informed consent paperwork and underwent protocol familiarization during visit 1. Prior to visits 2 and 4, participants fasted for 12 hours and adhered to an NIH-approved low-nitrate diet for 48 hours. During visits 2 and 4, an initial blood draw was performed to analyze baseline plasma nitrite and 8-isoprostane. Participants then completed a 30-minute resting metabolic rate (RMR) test. Two hours prior to visits 3 and 5, participants consumed either a placebo or dietary nitrate supplement (negligible and 6.2 mmol nitrate, respectively). During visits 3 and 5, participants also had blood drawn for analysis of the previously stated measurements, and completed an RMR test. Visits 2 and 3 were on consecutive days, followed by a week-long washout period between visit 3 and visit 4, while visit 4 and 5 also occurred on consecutive days. Results: Plasma nitrite significantly increased following dietary nitrate consumption compared to baseline values (27.56 ± 7.58 and 1.25 ± 1.51 arbitrary units, respectively). No difference was present between nitrate and baseline measurements for 8-isoprostane (155.75 ± 57.01 and 198.42 ± 66.44 pg/mL, respectively; p=0.55) and RMR (2086.60 ± 202.23 and 2050.00 ± 209.23 kcal/day, respectively; p=0.13). Conclusion: Dietary nitrate supplementation increases plasma nitrite, but does not change resting metabolic rate following an acute dose of dietary nitrate in healthy males. Individuals consuming dietary nitrate as an ergogenic aid during exercise may not, however, experience similar changes in their resting metabolism. The lack of change in oxidative stress may have been associated with the overall health of the cohort examined. Future research should investigate the clinical implications of dietary nitrate in populations with decreased NO bioavailability and associated endothelial disfunction (and elevated oxidative stress). In such populations, dietary nitrate may provide benefit. However, in a healthy cohort, dietary nitrate exerts minimal effects.