Metabolic syndrome (MetS) is associated with reduced nitric oxide (NO) bioavailability and impaired cerebrovascular function, potentially increasing cognitive decline risk. Dynamic cerebral autoregulation (dCA) maintains cerebral blood flow and may involve NO-related vascular mechanisms. Whether dietary nitrate supplementation influences dCA in MetS remains unclear. This pilot trial evaluated feasibility and explored effects of nitrate supplementation on dCA. Twenty-two older adults with MetS (55-75 years) were randomized in a double-blind, placebo-controlled design to receive nitrate-rich beetroot juice (BRJ; 6.4 mmol NO3 -·day-1) or nitrate-depleted placebo for 4 week. dCA was assessed using transfer function analysis (TFA) of beat-to-beat blood pressure and functional near-infrared spectroscopy-derived cerebral oxygenation. Primary analyses examined frontal dCA holistically, with exploratory regional and cognitive outcomes. dCA was indexed by TFA phase and gain. The intervention was well tolerated, adherence was high, and BRJ increased plasma nitrate concentrations versus placebo (p < 0.001). No significant effects were observed for frontal dCA or cognition. Exploratory analyses indicated region-specific differences in dCA in select frontal areas, characterized by improvements in TFA phase and gain. This pilot trial demonstrates the feasibility of dietary nitrate supplementation in MetS. While no effects were observed in primary outcomes, exploratory analyses suggest potential region-specific improvements in dCA.

Nitric oxide (NO) is formed via the oxidation of L-arginine in a reaction catalyzed by the NO synthase enzymes or via reduction of inorganic nitrite (NO2–) by deoxygenated hemoproteins and molybdopterin enzymes. We have recently demonstrated that NO can form a stable, labile ferrous heme-nitrosyl complex (NO-ferroheme) that protects NO from scavenging reactions in blood and has potent vasodilatory and platelet signaling activity. To enable future in vivo and in vitro studies establishing physiological NO-ferroheme formation, transport, and signaling, accurate and sensitive detection methods specifically for NO-ferroheme in biological milieu must be developed and validated. NO-heme complexes can be oxidized to release NO into the gas-phase for ozone-based chemiluminescence detection, which has been used for detection of iron-nitrosylated hemoglobin. In the current studies, we extend classical assays such as acidic potassium triiodide – with and without acidified sulfanilamide (AS) pre-treatment to eliminate NO2– and mercury(II) chloride (HgCl2) pretreatment to eliminate S-nitrosothiols – to detect and quantify NO-ferroheme, S-nitrosothiols, and nitrite. We also developed a new potassium ferricyanide/cyanide-based assay for sensitive and specific NO-ferroheme detection. All assays are sensitive and specific for NO-ferroheme to concentrations as low as 5 nM, with validated detection in buffer, plasma, whole blood and in vivo studies in mice. Additionally, we detect and quantify in vivo plasma NO-ferroheme formation and levels in mice after treatment with lipopolysaccharide (LPS), modeling pathological sepsis. These studies validate highly sensitive and specific assays for NO-ferroheme quantification and for the first time demonstrate plasma NO-ferroheme formation in vivo in a sepsis model.

Myoglobin plays a key role in oxygen storage and delivery through oxygen binding and release from the ferrous (Fe+2) heme. However, when the heme is oxidized to its ferric (Fe+3) form it can no longer bind oxygen and further oxidation leads to lipid peroxidation which can lead to a variety of pathological consequences. Ferric heme can be reduced to the ferrous form by nitric oxide (NO•) through classical reductive nitrosylation but this reaction, where a second NO• molecule binds to the reduced heme to form iron nitrosyl myoglobin, is very slow and inefficient. We have recently demonstrated that glutathione (and other thiols) can catalyze reductive nitrosylation of free ferric heme (hemin) when solubilized in albumin or red blood cell ghosts and that the resultant NO-ferroheme has potent NO• signaling properties. In this work we show this catalysis is also viable in myoglobin and that ascorbate is a better catalyst than glutathione with reductive nitrosylation occurring 1000 times faster in the presence of ascorbate compared to its absence under some conditions. Our data support a mechanism involving ascorbate-mediated heme reduction forming an ascorbyl radical. Only one NO• molecule and 0.5 ascorbate molecules are needed to convert one ferric myoglobin to one nitrosyl myoglobin molecule. Addition of NO• and ascorbate are shown to be more protective against lipid peroxidation due to myoglobin oxidation by hydrogen peroxide than either ascorbate or NO• alone. This reaction has potential application in meat preservation and treatment of ischemic reperfusion injury, various myopathies, and rhabdomyolysis.

Anthracycline‐induced cardiotoxicity remains a major limitation in cancer therapy, affecting long‐term cardiovascular health in survivors. Dietary nitrate supplementation has shown cardioprotective effects in preclinical models of doxorubicin (Dox)‐induced and ischemia–reperfusion injury, but it is unclear whether nitrate and/or nitrite (NOx) would have adverse effects on the anticancer efficacy of the drug. To evaluate Dox efficacy against triple‐negative breast cancer (TNBC) in the presence of dietary nitrate and nitrite, tumor‐bearing BALB/c mice ( N = 5 mice per group, 10 mice total) were treated with four weekly intravenous doses of Dox with or without NOx supplementation of their drinking water. Cardiac tissue from the NOx‐treated mice exhibited less fibrosis and lower levels of 4‐hydroxynonenal‐modified proteins, a marker of lipid oxidation and oxidative stress. Tumor sizes varied, but most regressed by the final Dox dose. Importantly, NOx supplementation did not compromise the antitumor efficacy of Dox nor did it promote pulmonary metastasis; instead, a trend toward fewer metastatic lesions was observed. These findings support the potential clinical use of dietary nitrate and nitrite as adjuncts to Dox treatment to mitigate cardiotoxicity without impairing anticancer outcomes.

Carbon monoxide (CO) poisoning causes 50,000 to 100,000 emergency department visits and ~1,500 deaths in the United States annually. Current treatments are limited to supplemental and/or hyperbaric oxygen to accelerate CO elimination. Even with oxygen therapy, nearly half of CO poisoning survivors suffer long-term cardiac and neurocognitive deficits related to slow CO clearance, highlighting a need for point of care antidotal therapies. Given the natural interaction between CO and ferrous heme, we hypothesized that the hemoprotein RcoM, a transcriptional regulator of microbial CO metabolism, would make an ideal platform for CO-selective scavenging from endogenous hemoproteins. We engineered an RcoM truncate (RcoM-HBD-CCC) that exhibits high CO affinity ( K a,CO = 2.8 × 10 10 M −1 ), remarkable selectivity for CO over oxygen ( K a,O2 = 1.4 × 10 5 M −1 ; K a,CO / K a,O2 = 1.9 × 10 5 ), thermal stability (T m = 72 °C), and slow autoxidation rate ( k ox = 1.1 h −1 ). In a murine model of acute CO poisoning, infused RcoM-HBD-CCC accelerated CO clearance from hemoglobin in red blood cells (RBCs) and was rapidly excreted in urine. Moreover, infused RcoM-HBD-CCC elicited minimal hypertension in mice compared to infused globins (hemoglobin, myoglobin, and neuroglobin), attributed to a comparatively limited reactivity toward nitric oxide (NO) via dioxygenation [ k NOD (RcoM) = 6 to 8 × 10 6 M −1 s −1 vs k NOD (Hb) = 6 to 8 × 10 7 M −1 s −1 ]. These data suggest that RcoM-HBD-CCC is a safe, selective, and efficacious CO scavenger. By limiting hypertension through minimal NO scavenging, RcoM-HBD-CCC improves end-organ adverse effects compared with other hemoprotein-based therapeutics.

We recently demonstrated a rapid reaction between labile ferric heme and nitric oxide (NO) in the presence of reduced glutathione (GSH) or other small thiols in a process called thiol-catalyzed reductive nitrosylation, yielding a novel signaling molecule, labile nitrosyl ferrous heme (NO-ferroheme), which we and others have shown can regulate vasodilation and platelet homeostasis. Red blood cells (RBCs) contain high concentrations of GSH, and NO can be generated in the RBC via nitrite reduction and/or RBC endothelial nitric oxide synthase (eNOS) so that NO-ferroheme could, in principle, be formed in the RBC. NO-ferroheme may also form in other cells and compartments, including in plasma, where another small and reactive thiol species, hydrogen sulfide (H2S/HS-), is also present and may catalyze NOferroheme formation akin to GSH. Here, we compare the reactivity of GSH and hydrogen sulfide with hemin in physiologically relevant media, including human serum albumin (HSA) and RBC membranes. Strikingly, hydrogen sulfide demonstrated a second-order rate constant over 10 times higher than GSH. We propose that the increased solubility of H2S vs GSH in lipophilic environments-where labile heme is most readily found-and the increased steric hindrance of the bulkier GSH account for the faster reaction kinetics observed with hydrogen sulfide. Our findings suggest that the hydrogen sulfide-catalyzed reductive nitrosylation reaction produces thionitrous acid (HSNO), which readily undergoes further reactions with excess hydrogen sulfide to form nitrosopersulfide (SSNO-) and polysulfides. These results suggest a common theme in thiol-catalyzed reductive nitrosylation of labile ferric heme that could play an important role in NO signaling.

IntroductionCardiovascular disease (CVD) is the leading cause of death in women, with increased risk following menopause. Dietary intake of beetroot juice and other plant-based nitrate-rich foods is a promising non-pharmacological strategy for increasing systemic nitric oxide and improving endothelial function in elderly populations. The purpose of this randomized, placebo-controlled, double-blind, crossover clinical trial was to determine the effects of short-term dietary nitrate (NO3−) supplementation, in the form of beetroot juice, on resting macrovascular endothelial function and endothelial resistance to whole-arm ischemia–reperfusion (IR) injury in postmenopausal women at two distinct stages of menopause.MethodsEarly-postmenopausal [1–6 years following their final menstrual period (FMP), n = 12] and late-postmenopausal (6+ years FMP, n = 12) women consumed nitrate-rich (400 mg NO3−/70 mL) and nitrate-depleted beetroot juice (approximately 40 mg NO3−/70 mL, placebo) daily for 7 days. Brachial artery flow-mediated dilation (FMD) was measured pre-supplementation (Day 0), and approximately 24 h after the last beetroot juice (BR) dose (Day 8, post-7-day BR). Consequently, FMD was measured immediately post-IR injury and 15 min later (recovery).ResultsResults of the linear mixed-effects model revealed a significantly greater increase in resting FMD with 7 days of BRnitrate compared to BRplacebo (mean difference of 2.21, 95% CI [0.082, 4.34], p = 0.042); however, neither treatment blunted the decline in post-IR injury FMD in either postmenopausal group. Our results suggest that 7-day BRnitrate-mediated endothelial protection is lost within the 24-h period following the final dose of BRnitrate.ConclusionOur findings demonstrate that nitrate-mediated postmenopausal endothelial protection is dependent on the timing of supplementation in relation to IR injury and chronobiological variations in dietary nitrate metabolism.Clinical trial registrationhttps://classic.clinicaltrials.gov/ct2/show/NCT03644472

BackgroundPeripheral arterial disease (PAD) is a prevalent vascular disorder characterized by atherosclerotic occlusion of peripheral arteries, resulting in reduced blood flow to the lower extremities and poor walking ability. Older patients with PAD are also at a markedly increased risk of cardiovascular events, including myocardial infarction. Recent evidence indicates that inorganic nitrate supplementation, which is abundant in certain vegetables, augments nitric oxide (NO) bioavailability and may have beneficial effects on walking, blood pressure, and vascular function in patients with PAD.ObjectiveWe sought to determine if short-term nitrate supplementation (via beetroot juice) improves peak treadmill time and coronary hyperemic responses to plantar flexion exercise relative to placebo (nitrate-depleted juice) in older patients with PAD. The primary endpoints were peak treadmill time and the peak coronary hyperemic response to plantar flexion exercise.MethodsEleven PAD patients (52–80 yr.; 9 men/2 women; Fontaine stage II) were randomized (double-blind) to either nitrate-rich (Beet-IT, 0.3 g inorganic nitrate twice/day; BRnitrate) or nitrate-depleted (Beet-IT, 0.04 g inorganic nitrate twice/day, BRplacebo) beetroot juice for 4 to 6 days, followed by a washout of 7 to 14 days before crossing over to the other treatment. Patients completed graded plantar flexion exercise with their most symptomatic leg to fatigue, followed by isometric handgrip until volitional fatigue at 40% of maximum on day 4 of supplementation, and a treadmill test to peak exertion 1–2 days later while continuing supplementation. Hemodynamics and exercise tolerance, and coronary blood flow velocity (CBV) responses were measured.ResultsAlthough peak walking time and claudication onset time during treadmill exercise did not differ significantly between BRplacebo and BRnitrate, the diastolic blood pressure response at the peak treadmill walking stage was significantly lower in the BRnitrate condition. Increases in CBV from baseline to peak plantar flexion exercise after BRplacebo and BRnitrate showed a trend for a greater increase in CBV at the peak workload of plantar flexion with BRnitrate (p = 0.06; Cohen’s d = 0.56).ConclusionOverall, these preliminary findings suggest that inorganic nitrate supplementation in PAD patients is safe, well-tolerated, and may improve the coronary hyperemic and blood pressure responses when their calf muscles are most predisposed to ischemia.Clinical trial registration:https://clinicaltrials.gov/, identifier NCT02553733.

Men and women have different cardiovascular responses to spaceflight; however, few studies have focused on direct comparisons between sexes. We investigated the mechanisms of aortic stiffening in socially and sexually mature 20-week-old male and female Sprague Dawley (SD) rats exposed to hindlimb unloading (HLU) for 14 days. Pulse wave velocity (PWV) was greater in the aortic arch of females after HLU versus control females (n = 6-8). HLU had no effect on aortic PWV in males (n = 5-6). Aortic alpha smooth muscle actin, myosin, collagen, elastin, and collagen-to-elastin ratio were not different in rats of either sex following HLU. The levels of G protein-coupled estrogen receptor (GPER) were lower in the aorta of SD females exposed to HLU compared with female controls but were not altered in males. HLU females also had lower aortic PPAR gamma, increased oxidative stress markers, and diastolic dysfunction compared with control females. GPER agonist G1 prevented the increase in PWV and 8-hydroxy-2'-deoxyguanosine without altering PPAR gamma or p47phox in HLU females (n = 4 in each group) suggesting that lower GPER may contribute to arterial stiffening in the setting of simulated microgravity. This study highlights sex-specific vascular adaptations to the state of simulated microgravity.

Malaria is a highly oxidative parasitic disease in which anemia is the most common clinical symptom. A major contributor to malarial anemia pathogenesis is the destruction of bystander, uninfected red blood cells (RBCs). Metabolic fluctuations are known to occur in the plasma of individuals with acute malaria, emphasizing the role of metabolic changes in disease progression and severity. Here, we report that conditioned media from Plasmodium falciparum culture induces oxidative stress in uninfected, catalase-depleted RBCs. As cell permeable precursors to glutathione, we show a benefit of pre-exposure to exogenous glutamine, cysteine, and glycine (QCG) amino acids for RBCs and that this pre-treatment intrinsically prepares RBCs to mitigate oxidative stress.

Postmenopausal cardiovascular health is a critical determinant of longevity. Consumption of beetroot juice (BR) and other nitrate-rich foods is a safe, effective non-pharmaceutical intervention to increase systemic bioavailability of the vasoprotective molecule, nitric oxide, through the exogenous nitrate (NO3 (-))-nitrite (NO2 (-))-nitric oxide (NO) pathway. We hypothesized that a single dose of nitrate-rich beetroot juice (BRnitrate 600 mg NO3 (-)/140 mL, BRplacebo similar to 0 mg/140 mL) would improve resting endothelial function and resistance to ischemia-reperfusion (IR) injury to a greater extent in early-postmenopausal (1-6 years following their final menstrual period (FMP), n = 12) compared to late-postmenopausal (6+ years after FMP, n = 12) women. Analyses with general linear models revealed a significant (p < 0.05) time*treatment interaction effect for brachial artery adjusted flow-mediated dilation (FMD). Pairwise comparisons revealed that adjusted FMD was significantly lower following IR-injury in comparison to all other time points with BRplacebo (early FMD 2.51 +/- 1.18%, late FMD 1.30 +/- 1.10, p < 0.001) and was lower than post-IR with BRnitrate (early FMD 3.84 +/- 1.21%, late FMD 3.21 +/- 1.13%, p = 0.014). A single dose of BRnitrate significantly increased resting macrovascular function in the late postmenopausal group only (p = 0.005). Considering the postmenopausal stage-dependent variations in endothelial responsiveness to dietary nitrate, we predict differing mechanisms underpin macrovascular protection against IR injury.

BACKGROUND:Donor genetic variation is associated with red blood cell (RBC) storage integrity and post-transfusion recovery. Our previous large-scale genome-wide association study demonstrated that the African G6PD deficient A- variant (rs1050828, Val68Met) is associated with higher oxidative hemolysis after cold storage. Despite a high prevalence of X-linked G6PD mutation in African American population (>10%), blood donors are not routinely screened for G6PD status and its importance in transfusion medicine is relatively understudied. STUDY DESIGN AND METHODS:To further evaluate the functional effects of the G6PD A- mutation, we created a novel mouse model carrying this genetic variant using CRISPR-Cas9. We hypothesize that this humanized G6PD A- variant is associated with reduced G6PD activity with a consequent effect on RBC hemolytic propensity and post-transfusion recovery. RESULTS:G6PD A- RBCs had reduced G6PD protein with ~5% residual enzymatic activity. Significantly increased in vitro hemolysis induced by oxidative stressors was observed in fresh and stored G6PD A- RBCs, along with a lower GSH:GSSG ratio. However, no differences were observed in storage hemolysis, osmotic fragility, mechanical fragility, reticulocytes, and post-transfusion recovery. Interestingly, a 14% reduction of 24-h survival following irradiation was observed in G6PD A- RBCs compared to WT RBCs. Metabolomic assessment of stored G6PD A- RBCs revealed an impaired pentose phosphate pathway (PPP) with increased glycolytic flux, decreasing cellular antioxidant capacity. DISCUSSION:This novel mouse model of the common G6PD A- variant has impaired antioxidant capacity like humans and low G6PD activity may reduce survival of transfused RBCs when irradiation is performed.

Heme is a ubiquitous cofactor used in a variety of proteins including hemoglobin, myoglobin, nitric oxide synthase, soluble guanylyl cyclase, cytochrome c, and cytochrome c oxidase. On the other hand, free heme can facilitate various deleterious redox reactions via activation of danger-associated molecular pattern signaling pathways and the inflammasome. Given the multitude of functions of heme proteins and the potential toxicity of free heme, it is no wonder that the use of heme-containing proteins as well as heme scavenger molecules has been developed for therapeutic use. In this chapter, we review these developments, including hemoglobin-based oxygen carriers for use as "blood substitutes," heme-globins for use as antidotes for carbon monoxide poisoning, and S-nitroso hemoglobin for use in a variety of conditions. We also describe nitrite-based therapies for cardiovascular diseases that rely on nitrite reactions with hemoglobin. Finally, we review work on NO ferroheme (nitric oxide bound to a ferrous heme), related to recent discoveries on its chemistry of formation and vascular effects.

Sickle cell anemia is caused by a single mutation in the gene encoding the beta subunit of hemoglobin. Due to this mutation, sickle cell hemoglobin (HbS) polymerizes under hypoxic conditions, decreasing red blood cell deformability and leading to multiple pathological effects that cause substantial morbidity and mortality. Several pre-clinical and human studies have demonstrated that the anion nitrite has potential therapeutic benefits for patients with sickle cell disease. Nitrite is reduced to nitric oxide (NO) by deoxygenated hemoglobin contributing to vasodilation, decreasing platelet activation, decreasing cellular adhesion to activated endothelium, and decreasing red cell hemolysis; all of which could ameliorate patient morbidities. Previous work on extracellular hemoglobin has shown that solution phase HbS reduces nitrite to NO faster than normal adult hemoglobin (HbA), while polymerized HbS reduces nitrite slower than HbA. In this work, we compared the rate of nitrite reduction to NO measured by the formation of nitrosyl hemoglobin in sickle and normal red blood cells at varying hemoglobin oxygen saturations. We found the overall rate of nitrite reduction between normal and sickle red blood cells was similar and confirmed this result under partially oxygenated conditions, but normal red blood cells reduced nitrite faster than sickle red blood cells under anoxia where HbS polymerization is maximal. These results are consistent with previous work using extracellular hemoglobin where the rate of reduction by solution phase HbS makes up for the slower reduction by polymer phase HbS under partially oxygenated conditions, but the polymer phase kinetics dominates in the complete absence of oxygen.

Nitric oxide (NO) is an endogenously produced physiological signaling molecule that regulates blood flow and platelet activation. However, both the intracellular and intravascular diffusion of NO is severely limited by scavenging reactions with hemoglobin, myoglobin, and other hemoproteins, raising unanswered questions as to how free NO can signal in hemoprotein-rich environments, like blood and cardiomyocytes. We explored the hypothesis that NO could be stabilized as a ferrous heme-nitrosyl complex (Fe 2+ -NO, NO-ferroheme) either in solution within membranes or bound to albumin. Unexpectedly, we observed a rapid reaction of NO with free ferric heme (Fe 3+ ) and a reduced thiol under physiological conditions to yield NO-ferroheme and a thiyl radical. This thiol-catalyzed reductive nitrosylation reaction occurs readily when the hemin is solubilized in lipophilic environments, such as red blood cell membranes, or bound to serum albumin. NO-ferroheme albumin is stable, even in the presence of excess oxyhemoglobin, and potently inhibits platelet activation. NO-ferroheme-albumin administered intravenously to mice dose-dependently vasodilates at low- to mid-nanomolar concentrations. In conclusion, we report the fastest rate of reductive nitrosylation observed to date to generate a NO-ferroheme molecule that resists oxidative inactivation, is soluble in cell membranes, and is transported intravascularly by albumin to promote potent vasodilation.

Device thrombosis occurs in otherwise life-saving procedures involving blood-contacting medical devices. Despite the use of systemic blood thinners, anticoagulants, and antiplatelet agents, device thrombosis can lead to substantial neurological damage, limb loss, death, and prolonged illness. Systemic treatments can also lead to bleeding. New methods to locally reduce thrombosis are urgently needed. Earlier work has shown that nitrite is a unique nitric oxide (NO) donor that is well-suited to use in blood and that its ability to inhibit platelet activation is potentiated by far-red light. In this study, we have applied our combined nitrite/light treatment in a prototypical technique used to prevent device thrombosis in extracorporeal circulation. We show that circuit pressure and survival are improved by an average of 213 percent with our treatment compared to the control. In addition, the dual therapy preserved platelet numbers at the end of the circulation time (%17 difference in platelet loss), and it reduced circuit hemolysis 2.3 fold. Thus, the combination of nitrite and red-light illumination has potential to prevent device thrombosis and to lead new clinical applications and practices.