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1.  Nitrate and nitrite in biology, nutrition and therapeutics 
Nature chemical biology  2009;5(12):865-869.
Inorganic nitrate and nitrite from endogenous or dietary sources are metabolized in vivo to nitric oxide (NO) and other bioactive nitrogen oxides. The nitrate-nitrite-NO pathway is emerging as an important mediator of blood flow regulation, cell signaling, energetics and tissue responses to hypoxia. The latest advances in our understanding of the biochemistry, physiology and therapeutics of nitrate, nitrite and NO were discussed during a recent two-day meeting at the Nobel Forum, Karolinska Institutet in Stockholm.
PMCID: PMC4038383  PMID: 19915529
2.  Nitrite Signaling in Pulmonary Hypertension: Mechanisms of Bioactivation, Signaling, and Therapeutics 
Antioxidants & Redox Signaling  2013;18(14):1797-1809.
Significance: Pulmonary arterial hypertension (PAH) is a disorder characterized by increased pulmonary vascular resistance and mean pulmonary artery pressure leading to impaired function of the right ventricle, reduced cardiac output, and death. An imbalance between vasoconstrictors and vasodilators plays an important role in the pathobiology of PAH. Recent Advances: Nitric oxide (NO) is a potent vasodilator in the lung, whose bioavailability and signaling pathway are impaired in PAH. It is now appreciated that the oxidative product of NO metabolism, the inorganic anion nitrite (NO2−), functions as an intravascular endocrine reservoir of NO bioactivity that can be reduced back to NO under physiological and pathological hypoxia. Critical Issues: The conversion of nitrite to NO is controlled by coupled electron and proton transfer reactions between heme- and molybdenum-containing proteins, such as hemoglobin and xanthine oxidase, and by simple protonation and disproportionation, and possibly by catalyzed disproportionation. The two major sources of nitrite (and nitrate) are the endogenous l-arginine–NO pathway, by oxidation of NO, and the diet, with conversion of nitrate from diet into nitrite by oral commensal bacteria. In the current article, we review the enzymatic formation of nitrite and the available data regarding its use as a therapy for PAH and other cardiovascular diseases. Future Directions: The successful efficacy demonstrated in several animal models and safety in early clinical trials suggest that nitrite may represent a promising new therapy for PAH. Antioxid. Redox Signal. 18, 1797–1809.
PMCID: PMC3619206  PMID: 22871207
3.  Antioxidant Therapy for the Treatment of Pulmonary Hypertension 
Antioxidants & Redox Signaling  2013;18(14):1723-1726.
Substantial experimental evidence suggests the usefulness of antioxidants for the treatment of various forms of pulmonary hypertension. However, no recommendations have yet been made if patients with pulmonary hypertension should receive pharmacologic and/or dietary antioxidants. Our understanding of antioxidants has evolved greatly over the last two decades, from the primitive use of natural antioxidant vitamins to the modulation of vascular oxidases, such as NAD(P)H oxidases. These oxidases and their products not only regulate pulmonary vascular tone and intimal and smooth muscle thickening, but also modulate the adaptation of the right ventricle to increased afterload. It is important that well-designed randomized clinical trials be conducted to test the importance of oxidase-reactive oxygen species activation in the pathogenesis and treatment of pulmonary hypertension. The purpose of this Forum on Pulmonary Hypertension is to summarize the available preclinical information, which may aid in designing and conducting future randomized clinical trials for evaluating the efficacy of antioxidants for the treatment of pulmonary hypertension. The complexity of oxidative pathways contributed to the tremendous difficulties and challenges in selecting agents, doses, and designing clinical trials. Further studies using human, animal, and cell culture models may be needed to define optimal trials. This Forum on Pulmonary Hypertension should stimulate new thinking and provide essential background information to better define the challenges of developing successful randomized clinical trials in the near future. Antioxid. Redox Signal. 18, 1723–1726.
PMCID: PMC3941794  PMID: 23330936
4.  Establishment of a Transgenic Sickle-Cell Mouse Model to Study the Pathophysiology of Priapism 
The journal of sexual medicine  2009;6(9):2494-2504.
Priapism is a poorly understood disease process with little information on the etiology and pathophysiology of this erectile disorder. One group of patients with a high prevalence of priapism is men with sickle-cell disease.
Establish an in vivo transgenic sickle-cell mouse model to study the pathophysiology of sickle-cell disease-associated priapism.
Transgenic sickle-cell disease mice, expressing human sickle hemoglobin, were utilized. Three groups of mice were used: (i) wild type (WT), (ii) sickle-cell heterozygotes (Hemi), and (ii) sickle-cell homozygotes (Sickle). Two age groups of each cohort of mice were utilized: young adult (4–6 months) and aged (18–22 months).
Main Outcome Measures
Histological (trichrome stain to measure ratio of collagen to smooth muscle), penile hydroxyproline content (collagen content), and transmission electron microscopic analysis of WT, Hemi, and Sickle mice penes, as well as in vivo erectile responses [change in intracavernous pressure (ICP)] to cavernous nerve stimulation (CNS), were determined. The frequency of erectile responses (erections/hour) pre- and poststimulation was also measured in each of the experimental groups.
Sickle mice had increased (P < 0.05) collagen to smooth muscle ratio and hydroxyproline content in the penis when compared with WT and Hemi mice penes. Transmission electron microscopy demonstrated thickened smooth muscle cell bundles, disruption of the endothelial lining of the corporal sinusoids, and increased (P < 0.05) caveolae number. Sickle mice had significantly (P < 0.05) higher ICP to CNS and increased (P < 0.05) frequency of erections pre- and post-CNS when compared with WT and Hemi mice erectile responses. Sickle mice did develop ED (change in ICP in response to CNS) with increasing age.
The morphometric changes of the penis and exaggerated in vivo erectile responses support the use of this transgenic sickle-cell disease animal model to study the pathophysiological mechanisms involved in sickle-cell disease-associated priapism.
PMCID: PMC4011713  PMID: 19523035
Erectile Dysfunction; Ischemic Priapism; Endothelium; Fibrosis; Cavolae; Nitric Oxide
5.  Hemoglobin as a Nitrite Anhydrase: Modeling Methemoglobin-Mediated N2O3 Formation 
Nitrite has recently been recognized as a storage form of NO in blood and as playing a key role in hypoxic vasodilation. The ion is readily reduced to NO by hemoglobin in red blood cells, which, however, also presents a conundrum. Given NO’s enormous affinity of ferrous heme, a key question concerns how it escapes capture by hemoglobin as it diffuses out of the red cells and to the endothelium, where vasodilation takes place. Dinitrogen trioxide (N2O3) has been proposed as a vehicle that transports NO to the endothelium, where it dissociates to NO and NO2. Although N2O3 formation might be readily explained via the reaction
Hb-Fe3++NO2-+NO⇆Hb-Fe2++N2O3, the exact manner in which methemoglobin (Hb-Fe3+), nitrite and NO interact with one another is unclear. Both an ‘Hb-Fe3+-NO2− + NO’ pathway and an ‘Hb-Fe3+-NO + NO2−’ pathway have been proposed. Neither pathway has been established experimentally. Nor has there been any attempt until now to theoretically model N2O3 formation, the so-called nitrite anhydrase reaction. Both pathways have been examined here in a detailed density functional theory (DFT, B3LYP/TZP) study and both have been found to be feasible based on energetics criteria. Modeling the ‘Hb-Fe3+-NO2− + NO’ pathway proved complex. Not only are multiple linkage-isomeric (N- and O- coordinated) structures conceivable for methemoglobin-nitrite, multiple isomeric forms are also possible for N2O3 (the lowest-energy state has an N-N-bonded nitro-nitrosyl structure, O2N-NO). We considered multiple spin states of methemoglobin-nitrite as well as ferromagnetic and antiferromagnetic coupling of the Fe(III) and NO spins. Together, the isomerism and spin variables result in a diabolically complex combinatorial space of reaction pathways. Fortunately, transition states could be successfully calculated for the vast majority of these reaction channels, both MS = 0 and MS = 1. For a six-coordinate Fe3+-O-nitrito starting geometry, which is plausible for methemoglobin-nitrite, we found that N2O3 formation entails barriers of about 17–20 kcal/mol, which is reasonable for a physiologically relevant reaction. For the ‘Hb-Fe3+-NO + NO2−’ pathway, which was also found to be energetically reasonable, our calculations indicate a two-step mechanism. The first step involves transfer of an electron from NO2− to the Fe3+-heme-NO ({FeNO}6) center, resulting in formation of nitrogen dioxide and an Fe2+-heme-NO ({FeNO}7) center. Subsequent formation of N2O3 entails a barrier of only 8.1 kcal mol−1. From an energetics point of view, the nitrite anhydrase reaction thus is a reasonable proposition. Although it is tempting to interpret our results as favoring the ‘{FeNO}6 + NO2−’ pathway over the ‘FeIII-nitrite + NO’ pathway, both pathways should be considered energetically reasonable for a biological reaction and it seems inadvisable to favor a unique reaction channel based solely on quantum chemical modeling.
PMCID: PMC3954847  PMID: 21590821
6.  Physiologic Changes in a Nonhuman Primate Model of HIV-Associated Pulmonary Arterial Hypertension 
Pulmonary arterial hypertension (PAH) is increased in HIV, but its pathogenesis is not fully understood. Nonhuman primates infected with simian immunodeficiency virus (SIV) or SIV-HIV chimeric virus (SHIV) exhibit histologic changes characteristic of human PAH, but whether hemodynamic changes accompany this pathology is unknown. Repeated measurements of pulmonary artery pressures would permit longitudinal assessments of disease development and provide insights into pathogenesis. We tested the hypothesis that SIV-infected and SHIV-infected macaques develop physiologic manifestations of PAH. We performed right heart catheterizations, echocardiography, and computed tomography (CT) scans in macaques infected with either SIV (ΔB670) or SHIV (89.6P), and compared right heart and pulmonary artery pressures, as well as pulmonary vascular changes on CT scans, with those in uninfected control animals. Right atrial, right ventricular systolic, and pulmonary artery pressures (PAPs) were significantly elevated in 100% of macaques infected with either SIV or SHIV compared with control animals, with no difference in pulmonary capillary wedge pressure. PAPs increased as early as 3 months after SIV infection. Radiographic evidence of pulmonary vascular pruning was also found. Both SIV-infected and SHIV-infected macaques exhibited histologic changes in pulmonary arteries, predominantly consisting of intimal and medial hyperplasia. This report is the first to demonstrate SHIV-infected and SIV-infected macaques develop pulmonary hypertension at a high frequency, with physiologic changes occurring as early as 3 months after infection. These studies establish an important nonhuman primate model of HIV-associated PAH that will be useful in studies of disease pathogenesis and the efficacy of interventions.
PMCID: PMC3604086  PMID: 23239493
pulmonary hypertension; HIV; SIV; SHIV; macaque model
7.  Nitric Oxide Scavenging by Red Cell Microparticles and Cell Free Hemoglobin as a Mechanism for the Red Cell Storage Lesion 
Circulation  2011;124(4):465-476.
Intravascular red cell hemolysis impairs NO-redox homeostasis, producing endothelial dysfunction, platelet activation and vasculopathy. Red blood cell storage under standard conditions results in reduced integrity of the erythrocyte membrane, with formation of exocytic microvesicles or “microparticles” and hemolysis, which we hypothesized could impair vascular function and contribute to the putative “storage lesion” of banked blood.
Methods and Results
We now find that storage of human red blood cells under standard blood banking conditions results in the accumulation of cell free and microparticle-encapsulated hemoglobin which, despite 39 days of storage, remains in the reduced ferrous oxyhemoglobin redox state and stoichiometrically reacts with and scavenges the vasodilator nitric oxide (NO). Using stopped-flow spectroscopy and laser triggered NO release from a caged NO compound we found that both free hemoglobin and microparticles react with NO about 1000 times faster than with intact erythrocytes. In complementary in vivo studies we show that hemoglobin, even at concentrations below 10 μM (in heme), produces potent vasoconstriction when infused into the rat circulation, while controlled infusions of methemoglobin and cyanomethemoglobin, which do not consume NO, have substantially reduced vasoconstrictor effects. Infusion of the plasma from stored human red cell units into the rat circulation produces significant vasoconstriction related to the magnitude of storage related hemolysis.
The results of these studies suggest new mechanisms for endothelial injury and impaired vascular function associated with the most fundamental of storage lesions, hemolysis.
PMCID: PMC3891836  PMID: 21747051
Hemoglobin; microparticles; nitric oxide; blood transfusion; storage lesion; reactive oxygen species
8.  Mechanism of faster NO scavenging by older stored red blood cells☆ 
Redox Biology  2014;2:211-219.
The blood storage lesion involves morphological and biochemical changes of red blood cells (RBCs) that occur during storage. These include conversion of the biconcave disc morphology to a spherical one, decreased mean corpuscular hemoglobin concentration, varied mean corpuscular volume, reduced integrity of the erythrocyte membrane with formation of microparticles, and increased cell-free hemoglobin. We studied the extent that older stored red blood cells scavenge nitric oxide (NO) faster than fresher stored red blood cells. Using electron paramagnetic resonance spectroscopy and stopped-flow absorption spectroscopy to measure the rate of NO uptake and reaction with hemoglobin in red cells, we found that older stored red blood cells scavenge NO about 1.8 times faster than fresher ones. Based on these experimental data, we simulated NO scavenging by fresher or older stored red blood cells with a biconcave or spherical geometry, respectively, in order to explore the mechanism of NO scavenging related to changes that occur during blood storage. We found that red blood cells with a spherical geometry scavenges NO about 2 times slower than ones with a biconcave geometry, and a smaller RBC hemoglobin concentration or volume increases NO scavenging by red blood cells. Our simulations demonstrate that even the most extreme possible changes in mean corpuscular hemoglobin concentration and mean corpuscular volume that favor increased NO scavenging are insufficient to account for what is observed experimentally. Therefore, RBC membrane permeability must increase during storage and we find that the permeability is likely to increase between 5 and 70 fold. Simulations using a two-dimensional blood vessel show that even a 5-fold increase in membrane permeability to NO can reduce NO bioavailability at the smooth muscle.
Transfusion of older stored blood may be harmful.
Older stored red blood cells scavenge nitric oxide more than fresher cells.
As stored red blood cells age, structural and biochemical changes occur that lead to faster scavenging.
Increased nitric oxide scavenging by red blood cells as a function of storage age contributes to deleterious effects upon transfusion.
Graphical abstract
•Older stored red blood cells scavenge NO faster.•The biconcave geometry of fresh red cells favors faster NO scavenging.•A smaller RBC MCHC or MCV leads to increased NO scavenging.•RBC membrane permeability to NO needs to increase in order to explain our experimental results.•Even a 5-fold increase of RBC membrane permeability to NO can reduce NO bioavailability.
PMCID: PMC3909782  PMID: 24494195
NO, nitric oxide; RBC, red blood cell; Hb, hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular hemoglobin volume; PBS, phosphate buffered saline; MetHb, methemoglobin; Nitric oxide; Erythrocyte; Blood; Kinetics; Electron paramagnetic resonance (EPR); Blood storage lesion
9.  Cardiovascular Abnormalities in Sickle Cell Disease 
Journal of the American College of Cardiology  2012;59(13):10.1016/j.jacc.2011.10.900.
Sickle cell disease is characterized by recurrent episodes of ischemia-reperfusion injury to multiple vital organ systems and a chronic hemolytic anemia, both contributing to progressive organ dysfunction. The introduction of treatments that induce protective fetal hemoglobin and reduce infectious complications has greatly prolonged survival. However, with increased longevity, cardiovascular complications are increasingly evident, with the notable development of a progressive proliferative systemic vasculopathy, pulmonary hypertension (PH) and left ventricular diastolic dysfunction. Pulmonary hypertension is reported in autopsy studies and numerous clinical studies have shown that increased pulmonary pressures are an important risk marker for mortality in these patients. In epidemiological studies, the development of PH is associated with intravascular hemolysis, cutaneous leg ulceration, renal insufficiency, iron overload and liver dysfunction. Chronic anemia in sickle cell disease results in cardiac chamber dilation and a compensatory increase in left ventricular mass. This is often accompanied by left ventricular diastolic dysfunction which has also been a strong independent predictor of mortality patients with sickle cell disease. Both PH and diastolic dysfunction are associated with marked abnormalities in exercise capacity in these patients. Sudden death is an increasingly recognized problem and further cardiac investigations are necessary to recognize and treat high-risk patients.
PMCID: PMC3881188  PMID: 22440212
Sickle; Cell; Disease
10.  Depletion of circulating blood NOS3 increases severity of myocardial infarction and left ventricular dysfunction 
Nitric oxide (NO) derived from endothelial NO synthase (NOS3) plays a central role in myocardial ischemia/reperfusion (I/R)-injury. Subsets of circulating blood cells, including red blood cells (RBCs), carry a NOS3 and contribute to blood pressure regulation and RBC nitrite/nitrate formation. We hypothesized that the circulating blood born NOS3 also modulates the severity of myocardial infarction in disease models. We cross-transplanted bone marrow in wild-type and NOS3−/− mice with wild-type mice, producing chimeras expressing NOS3 only in vascular endothelium (BC−/EC+) or in both blood cells and vascular endothelium (BC+/EC+). After 60-min closed-chest coronary occlusion followed by 24 h reperfusion, cardiac function, infarct size (IS), NOx levels, RBCs NO formation, RBC deformability, and vascular reactivity were assessed. At baseline, BC−/EC+ chimera had lower nitrite levels in blood plasma (BC−/EC+: 2.13 ± 0.27 μM vs. BC+/EC+ 3.17 ± 0.29 μM; *p < 0.05), reduced DAF FM associated fluorescence within RBCs (BC−/EC+: 538.4 ± 12.8 mean fluorescence intensity (MFI) vs. BC+/EC+: 619.6 ± 6.9 MFI; ***p < 0.001) and impaired erythrocyte deformability (BC−/EC+: 0.33 ± 0.01 elongation index (EI) vs. BC+/EC+: 0.36 ± 0.06 EI; *p < 0.05), while vascular reactivity remained unaffected. Area at risk did not differ, but infarct size was higher in BC−/EC+ (BC−/EC+: 26 ± 3 %; BC+/EC+: 14 ± 2 %; **p < 0.01), resulting in decreased ejection fraction (BC−/EC+ 46 ± 2 % vs. BC+/EC+: 52 ± 2 %; *p < 0.05) and increased end-systolic volume. Application of the NOS inhibitor S-ethylisothiourea hydrobromide was associated with larger infarct size in BC+/EC+, whereas infarct size in BC−/EC+ mice remained unaffected. Reduced infarct size, preserved cardiac function, NO levels in RBC and RBC deformability suggest a modulating role of circulating NOS3 in an acute model of myocardial I/R in chimeric mice.
Electronic supplementary material
The online version of this article (doi:10.1007/s00395-013-0398-1) contains supplementary material, which is available to authorized users.
PMCID: PMC3898535  PMID: 24346018
Nitric oxide; Myocardial ischemia/reperfusion; Circulating NOS3
11.  Angeli’s Salt Counteracts the Vasoactive Effects of Elevated Plasma Hemoglobin 
Free radical biology & medicine  2012;53(12):10.1016/j.freeradbiomed.2012.10.548.
Plasma hemoglobin (Hb) released during intravascular hemolysis has been associated with numerous deleterious effects that may stem from increased nitric oxide (NO) scavenging, but has also been associated with reactive oxygen species generation and platelet activation. Therapies that convert plasma oxyHb to metHb, or metHb to iron-nitrosyl Hb, could be beneficial because these species do not scavenge NO. In this study, we investigated the effects of Angeli’s Salt (AS, sodium α-oxyhyponitrite, Na2N2O3), a nitroxyl (HNO) and nitrite (NO2−) donor, on plasma Hb oxidation and formation of iron-nitrosyl Hb from metHb, and on the vasoactivity of plasma Hb. We hypothesized that AS could ameliorate hemolysis-associated pathology via its preferential reactivity with plasma Hb, as opposed to red cell encapsulated Hb, and through its intrinsic vasodilatory activity. To test this hypothesis, we infused (n=3 per group) (1) cell-free Hb and AS, (2) cell-free Hb + 0.9% NaCl, (3) AS + 3% Albumin, and (4) 3% Albumin + 0.9% NaCl (colloid controls for Hb and AS, respectively) in a canine model. Co-infusion of AS and cell-free Hb led to preferential conversion of plasma Hb to metHb, but the extent of conversion was lower than anticipated based on the in vivo concentration of AS relative to plasma Hb. This lower metHb yield was likely due to reactions of nitroxyl-derived AS with plasma components such as thiol-containing compounds. From a physiological and therapeutic standpoint, the infusion of Hb alone led to significant increases in mean arterial pressure (p=0.03) and systemic vascular resistance index (p=0.01) compared to controls. Infusion of AS alone led to significant decreases in these parameters and co-infusion of AS along with Hb had an additive effect on reversing the effects of Hb alone on the systemic circulation. Interestingly, in the pulmonary system, the decrease in pressure when AS is added to Hb was significantly less than would have been expected compared to the effects of Hb and AS alone suggesting that inactivation of scavenging with AS reduced the direct vasodilatory effects of AS on the vasculature. We also found that, AS reduced platelet activation when administered to whole blood in vitro. These data suggest that AS-like compounds could serve as a therapeutic agent to counteract the negative vasoconstrictive consequences of hemolysis that occur in hemolytic anemia’s, transfusion of stored blood, and other diseases. Increases in metHb in the red blood cell, the potential of AS for neurotoxicity, and hypotension would need to be carefully monitored in a clinical trial.
PMCID: PMC3600400  PMID: 23099417
Angeli’s Salt; cell-free hemoglobin; methemoglobin; nitroxyl
12.  Nitrate- nitrite- nitric oxide pathway in pulmonary arterial hypertension therapeutics 
Circulation  2012;125(23):10.1161/CIRCULATIONAHA.112.107821.
PMCID: PMC3864803  PMID: 22572912
Free radical biology & medicine  2012;52(9):10.1016/j.freeradbiomed.2012.02.041.
Pulmonary vascular disease can be defined as either a disease affecting the pulmonary capillaries and pulmonary arterioles, termed pulmonary arterial hypertension, or as a disease affecting the left ventricle, called pulmonary venous hypertension. Pulmonary arterial hypertension (PAH) is a disorder of the pulmonary circulation characterized by endothelial dysfunction, as well as intimal and smooth muscle proliferation. Progressive increases in pulmonary vascular resistance and pressure impair the performance of the right ventricle, resulting in declining cardiac output, reduced exercise capacity, right heart failure, and ultimately death. While the primary and heritable forms of the disease are thought to affect over 5,000 patients in the U.S., the disease can occur secondary to congenital heart disease, most advanced lung diseases, and many systemic diseases. Multiple studies implicate oxidative stress in the development of PAH. Further, this oxidative stress has been shown to be associated with alterations in reactive oxygen species (ROS), reactive nitrogen species (RNS) and nitric oxide (NO) signaling pathways, whereby bioavailable NO is decreased and ROS and RNS production are increased. Many canonical ROS and NO signaling pathways are simultaneously disrupted in PAH, with increased expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and xanthine oxidoreductase, uncoupling of endothelial NO synthase (eNOS), and reduction in mitochondrial number, as well as impaired mitochondrial function. Upstream dysregulation of ROS/NO redox homeostasis impairs vascular tone and contributes to the pathological activation of anti-apoptotic and mitogenic pathways, leading to cell proliferation and obliteration of the vasculature. This manuscript will review the available data regarding the role of oxidative and nitrosative stress and endothelial dysfunction in the pathophysiology of pulmonary hypertension, and provide a description of targeted therapies for this disease.
PMCID: PMC3856647  PMID: 22401856
Biochemistry  2012;51(26):10.1021/bi300570v.
Plant non-symbiotic hemoglobins possess hexa-coordinate heme geometry similar to the heme protein neuroglobin. We recently discovered that deoxygenated neuroglobin converts nitrite to nitric oxide (NO), an important signaling molecule involved in many processes in plants. We sought to determine whether Arabidopsis thaliana non-symbiotic hemoglobins class 1 and 2 (AHb1 and AHb2) might function as nitrite reductases. We found that the reaction of nitrite with deoxygenated AHb1 and AHb2 generates NO gas and iron-nitrosyl-hemoglobin species. The bimolecular rate constants for nitrite reduction to NO are 19.8 ± 3.2 and 4.9 ± 0.2 M−1s−1, at pH = 7.4 and 25°C, respectively. We determined the pH dependence of these bimolecular rate constants and found a linear correlation with the concentration of protons, indicating the requirement for one proton in the reaction. Release of free NO gas during reaction in anoxic and hypoxic (2% oxygen) conditions was confirmed by chemiluminescence detection. These results demonstrate that deoxygenated AHb1 and AHb2 reduce nitrite to form NO via a mechanism analogous to that observed for hemoglobin, myoglobin and neuroglobin. Our findings suggest that during severe hypoxia and in the anaerobic plant roots, especially in water submerged species, non-symbiotic hemoglobins provide a viable pathway for NO generation via nitrite reduction.
PMCID: PMC3857030  PMID: 22620259
15.  Normoxic Cyclic GMP-independent Oxidative Signaling by Nitrite Enhances Airway Epithelial Cell Proliferation and Wound Healing 
The airway epithelium provides important barrier and host defense functions. Recent studies reveal that nitrite is an endocrine reservoir of nitric oxide (NO) bioactivity that is converted to NO by enzymatic reductases along the physiological oxygen gradient. Nitrite signaling has been described as NO dependent activation mediated by reactions with deoxygenated redox active hemoproteins, such as hemoglobin, myoglobin, neuroglobin, xanthine oxidoreductase (XO) and NO synthase at low pH and oxygen tension. However, nitrite can also be readily oxidized to nitrogen dioxide (NO2•) via heme peroxidase reactions, suggesting the existence of alternative oxidative signaling pathways for nitrite under normoxic conditions. In the present study we examined normoxic signaling effects of sodium nitrite on airway epithelial cell wound healing. In an in vitro scratch injury model under normoxia, we exposed cultured monolayers of human airway epithelial cells to various concentrations of sodium nitrite and compared responses to NO donor. We found sodium nitrite potently enhanced airway epithelium wound healing at physiological concentrations (from 1uM). The effect of nitrite was blocked by the NO and NO2• scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (c-PTIO). Interestingly, nitrite treatment did not increase cyclic guanosine monophosphate (cGMP) levels under these normoxic conditions, even in the presence of a phosphodiesterase 5 inhibitor, suggesting cGMP independent signaling. Consistent with an oxidative signaling pathway requiring hydrogen peroxide (H2O2)/heme peroxidase/NO2• signaling, the effects of nitrite were potentiated by superoxide dismutase (SOD) and low concentration H2O2, whereas inhibited completely by catalase, followed by downstream extracellular-signal-regulated kinase (ERK) 1/2 activation. Our data represent the first description of normoxic nitrite signaling on lung epithelial cell proliferation and wound healing and suggest novel oxidative signaling pathways involving nitrite-H2O2 reactions, possibly via the intermediary, NO2•.
PMCID: PMC3854970  PMID: 22425780
sodium nitrite; nitric oxide; cyclic GMP; cell proliferation; airway epithelium
16.  Nitric oxide, hemolysis, and the red blood cell storage lesion: Interactions between transfusion, donor, and recipient 
Transfusion  2012;52(7):10.1111/j.1537-2995.2012.03748.x.
PMCID: PMC3855012  PMID: 22780890
17.  Nitrite Regulates Hypoxic Vasodilation via Myoglobin–Dependent Nitric Oxide Generation 
Circulation  2012;126(3):325-334.
Hypoxic vasodilation is a physiological response to low oxygen (O2) tension that increases blood supply to match metabolic demands. While this response has been characterized for more than 100 years, the underlying hypoxic sensing and effector signaling mechanisms remain uncertain. We have shown that deoxygenated myoglobin (deoxyMb) in the heart can reduce nitrite to nitric oxide (NO˙) and thereby contribute to cardiomyocyte NO˙ signaling during ischemia. Based on recent observations that Mb is expressed in the vasculature of hypoxia-tolerant fish, we hypothesized that endogenous nitrite may contribute to physiological hypoxic vasodilation via reactions with vascular Mb to form NO˙.
Methods and Results
We here show that Mb is expressed in vascular smooth muscle and contributes significantly to nitrite-dependent hypoxic vasodilation in vivo and ex vivo. The generation of NO˙ from nitrite reduction by deoxyMb activates canonical soluble guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP) signaling pathways. In vivo and ex vivo vasodilation responses, the reduction of nitrite to NO˙ and the subsequent signal transduction mechanisms were all significantly impaired in mice without myoglobin (Mb−/−). Hypoxic vasodilation studies in Mb, endothelial and inducible NO synthase knockout models (eNOS−/−, iNOS−/−) suggest that only Mb contributes to systemic hypoxic vasodilatory responses in mice.
Endogenous nitrite is a physiological effector of hypoxic vasodilation. Its reduction to NO˙ via the heme globin Mb enhances blood flow and matches O2 supply to increased metabolic demands under hypoxic conditions.
PMCID: PMC3410747  PMID: 22685116
hypoxic vasodilation; myoglobin; nitrite
18.  Pulmonary Complications of Sickle Cell Disease 
Sickle cell disease (SCD) is a common monogenetic disorder with high associated morbidity and mortality. The pulmonary complications of SCD are of particular importance, as acute chest syndrome and pulmonary hypertension have the highest associated mortality rates within this population. This article reviews the pathophysiology, diagnosis, and treatment of clinically significant pulmonary manifestations of SCD, including acute chest syndrome, asthma, and pulmonary hypertension in adult and pediatric patients. Clinicians should be vigilant in screening and treating such comorbidities to improve patient outcomes.
PMCID: PMC3373067  PMID: 22447965
sickle cell disease; hemolytic anemia; pulmonary hypertension; sudden death; nitric oxide
20.  Pulmonary Embolism in Sickle Cell Disease: A Case-Control Study 
Pulmonary embolism (PE) is a leading cause of mortality in hospitalized patients, yet the prevalence of PE in sickle cell disease (SCD) and its relation to disease severity or intrinsic hypercoagulability are not established.
We estimated inpatient PE incidence and prevalence among SCD and non-SCD populations in Pennsylvania, and compared severity of illness and mortality, using Pennsylvania Health Care Cost Containment Council (PHC4) discharge data, 2001-2006. Risk factors for PE were assessed in a case-control study of discharges from the University of Pittsburgh Medical Archival Records System (MARS).
The incidence of inpatient PE was higher in the SCD PA population than in the non-SCD Pennsylvania population, 2001-2006. The PE prevalence among SCD discharges ≤50 years of age, 0.57%, was similar to that in non-SCD Pennsylvania discharges, 0.60%, and unchanged after adjustment for race. Among SCD discharges, those developing PE were significantly older, with longer length ofstay, greater severity of illness, and higher mortality, p<0.001, than SCD without PE. Among PE discharges, SCD had similar severity of illness, p=0.77, and mortality, p=0.39, but underwent fewer computerized tomographic scans, p=0.006, than non-SCD with PE. In the local case-control study, no clinical or laboratory feature was associated with PE.
The incidence of PE is higher and chest CT utilization is lower in SCD than non-SCD inpatients, suggesting that PE may be under-diagnosed.
PMCID: PMC3343190  PMID: 22417249
Pulmonary embolism; sickle cell disease; venous thromboembolism
21.  Expression of Regulatory Platelet MicroRNAs in Patients with Sickle Cell Disease 
PLoS ONE  2013;8(4):e60932.
Increased platelet activation in sickle cell disease (SCD) contributes to a state of hypercoagulability and confers a risk of thromboembolic complications. The role for post-transcriptional regulation of the platelet transcriptome by microRNAs (miRNAs) in SCD has not been previously explored. This is the first study to determine whether platelets from SCD exhibit an altered miRNA expression profile.
Methods and Findings
We analyzed the expression of miRNAs isolated from platelets from a primary cohort (SCD = 19, controls = 10) and a validation cohort (SCD = 7, controls = 7) by hybridizing to the Agilent miRNA microarrays. A dramatic difference in miRNA expression profiles between patients and controls was noted in both cohorts separately. A total of 40 differentially expressed platelet miRNAs were identified as common in both cohorts (p-value 0.05, fold change>2) with 24 miRNAs downregulated. Interestingly, 14 of the 24 downregulated miRNAs were members of three families - miR-329, miR-376 and miR-154 - which localized to the epigenetically regulated, maternally imprinted chromosome 14q32 region. We validated the downregulated miRNAs, miR-376a and miR-409-3p, and an upregulated miR-1225-3p using qRT-PCR. Over-expression of the miR-1225-3p in the Meg01 cells was followed by mRNA expression profiling to identify mRNA targets. This resulted in significant transcriptional repression of 1605 transcripts. A combinatorial approach using Meg01 mRNA expression profiles following miR-1225-3p overexpression, a computational prediction analysis of miRNA target sequences and a previously published set of differentially expressed platelet transcripts from SCD patients, identified three novel platelet mRNA targets: PBXIP1, PLAGL2 and PHF20L1.
We have identified significant differences in functionally active platelet miRNAs in patients with SCD as compared to controls. These data provide an important inventory of differentially expressed miRNAs in SCD patients and an experimental framework for future studies of miRNAs as regulators of biological pathways in platelets.
PMCID: PMC3625199  PMID: 23593351
22.  Plasma thrombospondin-1 is increased during acute sickle cell vaso-occlusive events and associated with acute chest syndrome, hydroxyurea therapy, and lower hemolytic rates 
American journal of hematology  2012;87(3):326-330.
Platelets are activated in sickle cell disease (SCD), and particularly during vaso-occlusive episodes (VOE). Thrombospondin-1 (TSP1), a major secretory product of activated platelets, is increased in the circulation in VOE and binds to sickle red blood cells (RBC) promoting vascular adhesion. Thus, we hypothesized that TSP1 may represent a plasma biomarker of disease severity in SCD. We tested the plasma collected from patients in steady state (n = 27) and VOE (n = 14), as well as healthy controls (n = 17) at the University of Pittsburgh Medical Center (UPMC), and from patients in steady state enrolled in the walk-PHaSST clinical trial (n = 483). We found that TSP1 levels were increased in VOE in the UPMC cohort. Among steady-state patients at UPMC, TSP1 values correlated positively with lifetime history of acute chest syndrome (r = 0.72, P < 0.0001) and hemoglobin concentration (r = 0.49, P = 0.01), and negatively with markers of hemolysis, such as LDH (r = −0.50, P = 0.009). Analysis of the walk-PHaSST cohort also showed a positive association between TSP1 levels and hydroxyurea use (r = 0.14, P = 0.003), and confirmed the negative associations with the severity of hemolysis. Our results suggest that TSP1 levels are associated with more VOE, hydroxyurea use and lower rates of hemolysis. High TSP1 concentrations may indicate higher risk of the viscosity/vaso-occlusion phenotype of SCD.
PMCID: PMC3619659  PMID: 22318901
23.  Cardiopulmonary function in individuals with HIV infection in the antiretroviral therapy era 
AIDS (London, England)  2012;26(6):731-740.
To determine relationship of echocardiographic measures of pulmonary hypertension to lung function and inflammatory biomarkers in HIV-infected individuals.
Cross-sectional study of 116 HIV-infected outpatients.
Doppler-echocardiography and pulmonary function testing were performed. Induced sputum and plasma cytokines, sputum cell counts and differentials, markers of peripheral T cell activation, and serum N-terminal pro-brain natriuretic peptide (NT-proBNP) were measured. Univariate and multivariate analyses determined relationship of echocardiographic variables to pulmonary function, inflammation, and NT-proBNP.
Mean estimated pulmonary artery systolic pressure (PASP) was 34.3 mmHg (SD 6.9) and mean tricuspid regurgitant jet velocity (TRV) was 2.5 m/sec (SD 0.32). Eighteen participants (15.5%) had PASP of at least 40 mmHg, and 9 (7.8%) had TRV of at least 3.0 m/sec. Elevated TRV was significantly associated with CD4 cell counts below 200 cells/μl and higher log HIV RNA levels. Forced expiratory volume in one second (FEV1) percent predicted, FEV1/forced vital capacity (FVC), and diffusing capacity for carbon monoxide (DLco) percent predicted were significantly lower in those with elevated PASP or TRV. Sputum interleukin-8, peripheral interleukin-8, peripheral interferon-γ levels, and CD8+ T-cell expression of CD69+ were associated increased with increasing PASP and TRV. Log NT-proBNP was significantly higher with increasing PASP and TRV. Left ventricular function was not associated with PASP or TRV.
Echocardiographic manifestations of pulmonary hypertension are common in HIV and are associated with respiratory symptoms, more advanced HIV disease, airway obstruction, abnormal DLco, and systemic and pulmonary inflammation. Pulmonary hypertension and COPD coexist in HIV and may arise secondary to common inflammatory mechanisms.
PMCID: PMC3606053  PMID: 22210636
HIV; pulmonary hypertension; emphysema; COPD; inflammation
24.  Markers of severe vaso-occlusive painful episode frequency in children and adolescents with sickle cell anemia 
The Journal of Pediatrics  2011;160(2):286-290.
To identify factors associated with frequent severe vaso-occlusive pain crises in a contemporary pediatric cohort of sickle cell anemia (SCA)enrolled in a prospective study of pulmonary hypertension and the hypoxic response in sickle cell disease (SCD).
Study design
Clinical and laboratory characteristics of children with SCA who had ≥3 severe pain crises requiring health care in the preceding year were compared with subjects with <3 such episodes.
Seventy-five children (20%) reported ≥3 severe pain episodes in the preceding year, and 232 (61%) had none. Frequent pain episodes were associated with older age (OR 1.2; 95% CI 1.1–1.3; P<0.0001), α-thalassemia trait (OR 3.5; 1.6–6.7; P=0.002), higher median hemoglobin (OR 1.7; 95% CI: 1.2–2.4; P<0.003) and lower lactate dehydrogenase (LDH) concentration (OR 1.82; 95% CI: 1.07–3.11; P = 0.027). Children with high pain frequency also had an increased iron burden (serum ferritin 480 vs. 198 μg/L; P=0.006) and higher median tricuspid regurgitation jet velocity (2.41 vs. 2.31 m/s; P=0.001). Neither hydroxy urea use nor fetal hemoglobin levels were significantly different according to severe pain history.
In our cohort of children with SCA increasing age was associated with higher frequency of severe pain episodes as were α-thalassemia, iron overload, higher hemoglobin and lower LDH concentration and higher tricuspid regurgitation velocity.
PMCID: PMC3258348  PMID: 21890147
Sickle cell anemia; vaso-occlusive crisis; pain
25.  Hemolysis and cell-free hemoglobin drive an intrinsic mechanism for human disease 
The Journal of Clinical Investigation  2012;122(4):1205-1208.
Blood transfusion represents the first and most prescribed cell-based therapy; however, clinical safety and efficacy trials are lacking. Clinical cohort studies have suggested that massive transfusion and/or transfusion of aged stored blood may contribute to multiorgan dysfunction in susceptible patients. In this issue of the JCI, Baek and colleagues report that aged stored blood hemolyzes after massive transfusion in a guinea pig model. Hemolysis led to vascular and kidney injury that was mediated by cell-free plasma hemoglobin and prevented by coinfusion of the specific hemoglobin scavenger protein, haptoglobin. These studies support an expanding body of research indicating that intravascular hemolysis is a pathological mechanism in several human diseases, including multiorgan dysfunction after either massive red blood cell transfusion or hemoglobin-based blood substitute therapy, the hemoglobinopathies, malaria, and other acquired and genetic hemolytic conditions.
PMCID: PMC3314481  PMID: 22446184

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