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1.  Erythrocyte eNOS does not modulate red blood cell storage hemolysis 
Transfusion  2012;53(5):981-989.
Background
The red blood cell (RBC) endothelial nitric oxide synthase (eNOS) has been shown to regulate intrinsic erythrocyte rheological properties, such as membrane deformability, suggesting that a functional eNOS could be important in RBC viability and function during storage. This study examines the correlation between RBC eNOS deficiency and the propensity of RBCs to hemolyze under selected stress conditions including prolonged hypothermic storage.
Experimental design
Fresh or stored RBCs from normal and eNOS knock out (KO) mice or from healthy human volunteers were subjected to selected hemolytic stress conditions including mechanical stress hemolysis, osmotic stress hemolysis, oxidation stress hemolysis, and evaluated during standard storage in CPDA-1 solutions.
Results
Fresh RBCs from normal and eNOS KO mice demonstrated comparable susceptibility to hemolysis triggered by mechanical stress (mechanical fragility index = 6.5±0.5 in eNOS KO versus 6.4±0.4 for controls; n=8–9), osmotic stress, and oxidative stress. Additionally, RBCs from both mouse groups exhibited similar hemolytic profile at the end of 14-day hypothermic storage, analogous to 42 days of human RBC storage. Storage of human RBCs (28 days in CPDA-1) in the presence of NOS cofactors (L-arginine and tetrahydro-L-biopterin) or inhibitor (L-NMMA) did not affect cell recovery or hemolytic response to the selected stressors.
Conclusion
These studies suggest that RBC eNOS does not modulate susceptibility to hemolysis in response to selected stress conditions or prolonged hypothermic storage. Other strategies to increase NO bioactivity following prolonged storage utilizing NOS-independent pathways such as the nitrate-nitrite-NO pathway may prove a more promising approach.
doi:10.1111/j.1537-2995.2012.03850.x
PMCID: PMC4313879  PMID: 22897637
Hemolysis; red blood cell storage lesion; RBC endothelial nitric oxide synthase (eNOS)
2.  Direct sGC Activation Bypasses NO Scavenging Reactions of Intravascular Free Oxy-Hemoglobin and Limits Vasoconstriction 
Antioxidants & Redox Signaling  2013;19(18):2232-2243.
Abstract
Aims: Hemoglobin-based oxygen carriers (HBOC) provide a potential alternative to red blood cell (RBC) transfusion. Their clinical application has been limited by adverse effects, in large part thought to be mediated by the intravascular scavenging of the vasodilator nitric oxide (NO) by cell-free plasma oxy-hemoglobin. Free hemoglobin may also cause endothelial dysfunction and platelet activation in hemolytic diseases and after transfusion of aged stored RBCs. The new soluble guanylate cyclase (sGC) stimulator Bay 41-8543 and sGC activator Bay 60-2770 directly modulate sGC, independent of NO bioavailability, providing a potential therapeutic mechanism to bypass hemoglobin-mediated NO inactivation. Results: Infusions of human hemoglobin solutions and the HBOC Oxyglobin into rats produced a severe hypertensive response, even at low plasma heme concentrations approaching 10 μM. These reactions were only observed for ferrous oxy-hemoglobin and not analogs that do not rapidly scavenge NO. Infusions of L-NG-Nitroarginine methyl ester (L-NAME), a competitive NO synthase inhibitor, after hemoglobin infusion did not produce additive vasoconstriction, suggesting that vasoconstriction is related to scavenging of vascular NO. Open-chest hemodynamic studies confirmed that hypertension occurred secondary to direct effects on increasing vascular resistance, with limited negative cardiac inotropic effects. Intravascular hemoglobin reduced the vasodilatory potency of sodium nitroprusside (SNP) and sildenafil, but had no effect on vasodilatation by direct NO-independent activation of sGC by BAY 41-8543 and BAY 60-2770. Innovation and Conclusion: These data suggest that both sGC stimulators and sGC activators could be used to restore cyclic guanosine monophosphate-dependent vasodilation in conditions where cell-free plasma hemoglobin is sufficient to inhibit endogenous NO signaling. Antioxid. Redox Signal. 19, 2232–2243.
doi:10.1089/ars.2013.5181
PMCID: PMC3869448  PMID: 23697678
4.  Elevated Pulse Pressure is Associated with Hemolysis, Proteinuria and Chronic Kidney Disease in Sickle Cell Disease 
PLoS ONE  2014;9(12):e114309.
A seeming paradox of sickle cell disease is that patients do not suffer from a high prevalence of systemic hypertension in spite of endothelial dysfunction, chronic inflammation and vasculopathy. However, some patients do develop systolic hypertension and increased pulse pressure, an increasingly recognized major cardiovascular risk factor in other populations. Hence, we hypothesized that pulse pressure, unlike other blood pressure parameters, is independently associated with markers of hemolytic anemia and cardiovascular risk in sickle cell disease. We analyzed the correlates of pulse pressure in patients (n  =  661) enrolled in a multicenter international sickle cell trial. Markers of hemolysis were analyzed as independent variables and as a previously validated hemolytic index that includes multiple variables. We found that pulse pressure, not systolic, diastolic or mean arterial pressure, independently correlated with high reticulocyte count (beta  =  2.37, p  =  0.02) and high hemolytic index (beta  =  1.53, p = 0.002) in patients with homozygous sickle cell disease in two multiple linear regression models which include the markers of hemolysis as independent variables or the hemolytic index, respectively. Pulse pressure was also independently associated with elevated serum creatinine (beta  =  3.21, p  =  0.02), and with proteinuria (beta  =  2.52, p  =  0.04). These results from the largest sickle cell disease cohort to date since the Cooperative Study of Sickle Cell Disease show that pulse pressure is independently associated with hemolysis, proteinuria and chronic kidney disease. We propose that high pulse pressure may be a risk factor for clinical complications of vascular dysfunction in sickle cell disease. Longitudinal and mechanistic studies should be conducted to confirm these hypotheses.
doi:10.1371/journal.pone.0114309
PMCID: PMC4257593  PMID: 25478953
5.  In vivo reduction of cell-free methemoglobin to oxyhemoglobin results in vasoconstriction in canines 
Transfusion  2013;53(12):10.1111/trf.12162.
BACKGROUND
Cell-free hemoglobin (Hb) in the vasculature leads to vasoconstriction and injury. Proposed mechanisms have been based on nitric oxide (NO) scavenging by oxyhemoglobin (oxyHb) or processes mediated by oxidative reactions of methemoglobin (metHb). To clarify this, we tested the vascular effect and fate of oxyHb or metHb infusions.
STUDY DESIGN AND METHODS
Twenty beagles were challenged with 1 h similar infusions of (200uM) metHb (n=5), oxyHb (n=5), albumin (n=5), or saline (n=5). Measurements were taken over 3 h.
RESULTS
Infusions of the two pure Hb species resulted in increases in mean arterial blood pressure (MAP), systemic vascular resistance index, and NO consumption capacity of plasma (all p<0.05) with the effects of oxyHb being greater than that from metHb (MAP; increase 0 to 3h; 27±6 % vs.7±2 %, respectively) (all p<0.05). The significant vasoconstrictive response of metHb (vs. albumin and saline controls) was related to in vivo auto-reduction of metHb to oxyHb, and the vasoactive Hb species that significantly correlated with MAP was always oxyHb, either from direct infusion or after in vivo reduction from metHb. Clearance of total Hb from plasma was faster after metHb than oxyHb infusion (p<0.0001).
CONCLUSION
These findings indicate that greater NO consumption capacity makes oxyHb more vasoactive than metHb. Additionally, metHb is reduced to oxyHb post-infusion and cleared faster or is less stable than oxyHb. Although we found no direct evidence that metHb itself is involved in acute vascular effects, in aggregate, these studies suggest that metHb is not inert and its mechanism of vasoconstriction is due to its delayed conversion to oxyHb by plasma-reducing agents.
doi:10.1111/trf.12162
PMCID: PMC3686899  PMID: 23488474
methemoglobin; cell-free hemoglobin; nitric oxide; vasoconstriction; hemoglobin
6.  Nitric Oxide Scavenging by Red Cell Microparticles 
Free radical biology & medicine  2013;65:10.1016/j.freeradbiomed.2013.09.002.
Red cell microparticles form during the storage of red blood cells and in diseases associated with red cell breakdown and asplenia, including hemolytic anemias such as sickle cell disease. These small phospholipid vesicles that are derived from red blood cells have been implicated in the pathogenesis of transfusion of aged stored blood and hemolytic diseases, via activation of the hemostatic system and effects on nitric oxide (NO) bioavailability. Red cell microparticles react with the important signaling molecule NO almost as fast as cell-free hemoglobin, about one-thousand times faster than red cell encapsulated hemoglobin. The degree to which this fast reaction with NO by red cell microparticles impacts NO bioavailability depends on several factors that are explored here. In the context of stored blood preserved in ADSOL, we find that both cell-free hemoglobin and red cell microparticles increase as a function of duration of storage and the proportion of extra erythrocytic hemoglobin in the red cell microparticles fraction is about 20% throughout storage. Normalized by hemoglobin concentration, the NO scavenging ability of cell-free hemoglobin is slightly higher than red cell microparticles as determined by a chemiluminescent NO scavenging assay. Computational simulations show that the degree to which red cell microparticles scavenge NO will depend substantially on whether they enter the cell-free zone next to the endothelial cells. Single microvessel myography experiments performed under laminar flow conditions demonstrate that microparticles significantly enter the cell-free zone and inhibit acetylcholine, endothelial-dependent and NO-dependent vasodilation. Taken together, these data suggest that as little as five micromolar hemoglobin in red cell microparticles, an amount formed after the infusion of one unit of aged stored packed red blood cells, has the potential to reduce NO bioavailability and impair endothelial-dependent vasodilation.
doi:10.1016/j.freeradbiomed.2013.09.002
PMCID: PMC3859830  PMID: 24051181
Nitric oxide; Blood storage; Red cell microparticles; Cell-free hemoglobin
7.  Mechanisms for Cellular NO Oxidation and Nitrite Formation in Lung Epithelial Cells 
Airway lining fluid contains relatively high concentrations of nitrite and arterial blood levels of nitrite are higher than venous levels, suggesting the lung epithelium may represent an important source of nitrite in vivo. To investigate whether lung epithelial cells possess the ability to convert NO to nitrite by oxidation, and the effect of oxygen reactions on nitrite formation, the NO donor DETA NONOate was incubated with or without A549 cells or primary human bronchial epithelial (HBE) cells for 24 hrs under normoxic (21% O2) and hypoxic (1% O2) conditions. Nitrite production was significantly increased under all conditions in the presence of A549 or HBE cells, suggesting that both A549 and HBE cells have the capacity to oxidize NO to nitrite even under low oxygen conditions. The addition of oxy-hemoglobin (oxy-Hb) to the A549 cell media decreased the production of nitrite, consistent with NO scavenging limiting nitrite formation. Heat-denatured A549 cells produced much lower nitrite and bitrate, suggesting an enzymatic activity is required. This NO oxidation activity was found to be highest in membrane bound proteins with molecular sizes < 100 kDa. In addition, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one] (ODQ) and cyanide inhibited formation of nitrite in A549 cells. It has been shown that ceruloplasmin (Cp) possesses an NO oxidase and nitrite synthase activity in plasma based on NO oxidation to nitrosonium cation (NO+). We observed that Cp is expressed intracellularly in lung epithelial A549 cells and secreted into medium under basal conditions and during cytokine stimulation. However, an analysis of Cp expression level and activity measured via ρ-phenylenediamine oxidase activity assay revealed very low activity compared with plasma, suggesting that there is insufficient Cp to contribute to detectable NO oxidation to nitrite in A549 cells. Additionally, Cp levels were knocked down using siRNA by more than 75% in A549 cells, with no significant change in either nitrite or cellular S-nitrosothiol (SNO) formation compared to scrambled siRNA control under basal conditions or cytokine stimulation. These data suggest that lung epithelial cells possess NO oxidase activity, which is enhanced in cell membrane associated proteins and not regulated by intracellular or secreted Cp, indicating that alternative NO oxidases determine hypoxic and normoxic nitrite formation from NO in human lung epithelial cells.
doi:10.1016/j.freeradbiomed.2013.04.031
PMCID: PMC3883890  PMID: 23639566
ceruloplasmin; NO oxidase; nitrite; nitric oxide; human lung epithelial cells; hypoxia
8.  Genetic determinants of haemolysis in sickle cell anaemia 
British journal of haematology  2013;161(2):270-278.
Summary
Haemolytic anaemia is variable among patients with sickle cell anaemia and can be estimated by reticulocyte count, lactate dehydrogenase, aspartate aminotransferase and bilirubin levels. Using principal component analysis of these measurements we computed a haemolytic score that we used as a subphenotype in a genome-wide association study. We identified in one cohort and replicated in two additional cohorts the association of a single nucleotide polymorphism in NPRL3 (rs7203560; chr16p13·3) (P = 6·04 × 10−07). This association was validated by targeted genotyping in a fourth independent cohort. The HBA1/HBA2 regulatory elements, hypersensitive sites (HS)-33, HS-40 and HS-48 are located in introns of NPRL3. Rs7203560 was in perfect linkage disequilibrium (LD) with rs9926112 (r2 = 1) and in strong LD with rs7197554 (r2 = 0·75) and rs13336641 (r2 = 0·77); the latter is located between HS-33 and HS-40 sites and next to a CTCF binding site. The minor allele for rs7203560 was associated with the −∝3·7thalassaemia gene deletion. When adjusting for HbF and ∝ thalassaemia, the association of NPRL3 with the haemolytic score was significant (P = 0·00375) and remained significant when examining only cases without gene deletion∝ thalassaemia (P = 0·02463). Perhaps by independently down-regulating expression of the HBA1/HBA2 genes, variants of the HBA1/HBA2 gene regulatory loci, tagged by rs7203560, reduce haemolysis in sickle cell anaemia.
doi:10.1111/bjh.12245
PMCID: PMC4129543  PMID: 23406172
haemolysis; sickle cell anaemia; haemolytic anaemia; genetic analysis; thalassaemia
9.  Circulating Blood eNOS Contributes to the Regulation of Systemic Blood Pressure and Nitrite Homeostasis 
Arteriosclerosis, thrombosis, and vascular biology  2013;33(8):10.1161/ATVBAHA.112.301068.
Objective
Mice genetically deficient in endothelial nitric oxide synthase (eNOS−/−) are hypertensive with lower circulating nitrite levels, indicating the importance of constitutively produced nitric oxide (NO•) to blood pressure regulation and vascular homeostasis. While the current paradigm holds that this bioactivity derives specifically from expression of eNOS in endothelium, circulating blood cells also express eNOS protein. A functional red cell eNOS that modulates vascular NO• signaling has been proposed.
Approach and Results
To test the hypothesis that blood cells contribute to mammalian blood pressure regulation via eNOS-dependent NO• generation, we cross-transplanted WT and eNOS−/− mice, producing chimeras competent or deficient for eNOS expression in circulating blood cells. Surprisingly, we observed a significant contribution of both endothelial and circulating blood cell eNOS to blood pressure and systemic nitrite levels, the latter being a major component of the circulating NO• reservoir. These effects were abolished by the NOS inhibitor L-NAME and repristinated by the NOS substrate L-Arginine, and were independent of platelet or leukocyte depletion. Mouse erythrocytes were also found to carry an eNOS protein and convert 14C-Arginine into 14C-Citrulline in a NOS-dependent fashion.
Conclusions
These are the first studies to definitively establish a role for a blood borne eNOS, using cross transplant chimera models, that contributes to the regulation of blood pressure and nitrite homeostasis. This work provides evidence suggesting that erythrocyte eNOS may mediate this effect.
doi:10.1161/ATVBAHA.112.301068
PMCID: PMC3864011  PMID: 23702660
10.  Risk Factors for Death in 632 Patients with Sickle Cell Disease in the United States and United Kingdom 
PLoS ONE  2014;9(7):e99489.
Background
The role of pulmonary hypertension as a cause of mortality in sickle cell disease (SCD) is controversial.
Methods and Results
We evaluated the relationship between an elevated estimated pulmonary artery systolic pressure and mortality in patients with SCD. We followed patients from the walk-PHaSST screening cohort for a median of 29 months. A tricuspid regurgitation velocity (TRV)≥3.0 m/s cuttof, which has a 67–75% positive predictive value for mean pulmonary artery pressure ≥25 mm Hg was used. Among 572 subjects, 11.2% had TRV≥3.0 m/sec. Among 582 with a measured NT-proBNP, 24.1% had values ≥160 pg/mL. Of 22 deaths during follow-up, 50% had a TRV≥3.0 m/sec. At 24 months the cumulative survival was 83% with TRV≥3.0 m/sec and 98% with TRV<3.0 m/sec (p<0.0001). The hazard ratios for death were 11.1 (95% CI 4.1–30.1; p<0.0001) for TRV≥3.0 m/sec, 4.6 (1.8–11.3; p = 0.001) for NT-proBNP≥160 pg/mL, and 14.9 (5.5–39.9; p<0.0001) for both TRV≥3.0 m/sec and NT-proBNP≥160 pg/mL. Age >47 years, male gender, chronic transfusions, WHO class III–IV, increased hemolytic markers, ferritin and creatinine were also associated with increased risk of death.
Conclusions
A TRV≥3.0 m/sec occurs in approximately 10% of individuals and has the highest risk for death of any measured variable.
The study is registered in ClinicalTrials.gov with identifier
NCT00492531
doi:10.1371/journal.pone.0099489
PMCID: PMC4079316  PMID: 24988120
11.  Clinical correlates of acute pulmonary events in children and adolescents with sickle cell disease* 
Objectives
We aimed to identify risk factors for acute pulmonary events in children and adolescents in the Pulmonary Hypertension and the Hypoxic Response in SCD (PUSH) study.
Methods
Patients with hemoglobin SS (n=376) and other sickle cell genotypes (n=127) aged 3-20 years were studied at four centers in a cross-sectional manner. A sub-group (n=293) was followed for a median of 21 months (range 9-35).
Results
A patient-reported history of one or more acute pulmonary events, either acute chest syndrome (ACS) or pneumonia, was obtained in 195 hemoglobin SS patients (52%) and 51 patients with other genotypes (40%). By logistic regression, history of acute pulmonary events was independently associated with patient-reported history of asthma (p<0.0001), older age (p=0.001), >3 severe pain episodes in the preceding 12 months (p=0.002), higher tricuspid regurgitation velocity (TRV) (p=0.028), and higher white blood cell (WBC) count (p=0.043) among hemoglobin SS patients. History of acute pulmonary events was associated with >3 severe pain episodes (p=0.009) among patients with other genotypes. During follow-up, 43 patients (15%) had at least one new ACS episode including 11 without a baseline history of acute pulmonary events. History of acute pulmonary events (odds ratio 5.4; p<0.0001) and younger age (odds ratio 0.9; p=0.010) were independently associated with developing a new episode during follow-up.
Conclusions
Asthma history, frequent pain and higher values for TRV and WBC count were independently associated with history of acute pulmonary events in hemoglobin SS patients and frequent pain was associated in those with other genotypes. Measures to reduce pain episodes and control asthma may help to decrease the incidence of acute pulmonary events in SCD.
doi:10.1111/ejh.12118
PMCID: PMC3689858  PMID: 23560516
sickle cell disease; acute chest syndrome; vaso-occlusive crisis; asthma; pain
13.  Computed tomography correlates with cardiopulmonary hemodynamics in pulmonary hypertension in adults with sickle cell disease 
Pulmonary Circulation  2014;4(2):319-329.
Our objective was to determine whether computed tomography angiography (CTA) measurements of pulmonary artery size can noninvasively assess hemodynamics and diagnose pulmonary hypertension (PH) secondary to sickle cell disease (SCD). Twenty SCD patients with confirmed PH were compared with 20 matched controls. Diameters of the pulmonary artery trunk and branches were measured with CTA and a semiautomatic segmentation algorithm. Measurements were normalized by body size and correlated (Spearman rank) with hemodynamic markers from right-heart catheterization. Receiver operating characteristic (ROC) curves were used to investigate the role of pulmonary artery sizes in diagnosing PH. Analysis of pulmonary artery sizes adjusted for body surface area (BSA) resulted in the most significant discrimination between subjects with PH secondary to SCD and controls (P < 0.001); PH was diagnosed accurately with an area under the ROC curve of 0.99. There was significant correlation between pulmonary artery sizes and body mass index (BMI) and BSA only in controls (r = 0.46–0.68, P < 0.04 for all). The most significant correlations with hemodynamic markers were found between BMI-adjusted pulmonary artery sizes and high systolic pulmonary arterial pressure, high pulmonary vascular resistance, high systemic vascular resistance, and low cardiac output (r = 0.47, 0.62, 0.61, and 0.66, respectively; P < 0.04 for all). BMI-adjusted CTA measures of the pulmonary artery relate to high pulmonary vascular resistance and reduced cardiac output in patients with SCD and PH. CTA with quantitative image analysis is a powerful noninvasive diagnostic tool for PH in SCD and shows promise as estimator of hemodynamic markers.
doi:10.1086/675997
PMCID: PMC4070777  PMID: 25006451
CT angiography; cardiopulmonary hemodynamics; pulmonary hypertension; sickle cell disease; arterial size; quantitative imaging
14.  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.
doi:10.1038/nchembio.260
PMCID: PMC4038383  PMID: 19915529
15.  Nitrite Signaling in Pulmonary Hypertension: Mechanisms of Bioactivation, Signaling, and Therapeutics 
Antioxidants & Redox Signaling  2013;18(14):1797-1809.
Abstract
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.
doi:10.1089/ars.2012.4833
PMCID: PMC3619206  PMID: 22871207
16.  Antioxidant Therapy for the Treatment of Pulmonary Hypertension 
Antioxidants & Redox Signaling  2013;18(14):1723-1726.
Abstract
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.
doi:10.1089/ars.2013.5193
PMCID: PMC3941794  PMID: 23330936
17.  Establishment of a Transgenic Sickle-Cell Mouse Model to Study the Pathophysiology of Priapism 
The journal of sexual medicine  2009;6(9):2494-2504.
Introduction
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.
Aim
Establish an in vivo transgenic sickle-cell mouse model to study the pathophysiology of sickle-cell disease-associated priapism.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1111/j.1743-6109.2009.01359.x
PMCID: PMC4011713  PMID: 19523035
Erectile Dysfunction; Ischemic Priapism; Endothelium; Fibrosis; Cavolae; Nitric Oxide
18.  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.
doi:10.1002/chem.201003578
PMCID: PMC3954847  PMID: 21590821
19.  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.
doi:10.1165/rcmb.2011-0434OC
PMCID: PMC3604086  PMID: 23239493
pulmonary hypertension; HIV; SIV; SHIV; macaque model
20.  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.
Background
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.
Conclusions
The results of these studies suggest new mechanisms for endothelial injury and impaired vascular function associated with the most fundamental of storage lesions, hemolysis.
doi:10.1161/CIRCULATIONAHA.110.008698
PMCID: PMC3891836  PMID: 21747051
Hemoglobin; microparticles; nitric oxide; blood transfusion; storage lesion; reactive oxygen species
21.  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.
Background
Transfusion of older stored blood may be harmful.
Results
Older stored red blood cells scavenge nitric oxide more than fresher cells.
Conclusion
As stored red blood cells age, structural and biochemical changes occur that lead to faster scavenging.
Significance
Increased nitric oxide scavenging by red blood cells as a function of storage age contributes to deleterious effects upon transfusion.
Graphical abstract
Highlights
•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.
doi:10.1016/j.redox.2013.12.014
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
22.  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.
doi:10.1016/j.jacc.2011.10.900
PMCID: PMC3881188  PMID: 22440212
Sickle; Cell; Disease
23.  Depletion of circulating blood NOS3 increases severity of myocardial infarction and left ventricular dysfunction 
Basic Research in Cardiology  2013;109(1):398.
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.
doi:10.1007/s00395-013-0398-1
PMCID: PMC3898535  PMID: 24346018
Nitric oxide; Myocardial ischemia/reperfusion; Circulating NOS3
24.  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.
doi:10.1016/j.freeradbiomed.2012.10.548
PMCID: PMC3600400  PMID: 23099417
Angeli’s Salt; cell-free hemoglobin; methemoglobin; nitroxyl
25.  Nitrate- nitrite- nitric oxide pathway in pulmonary arterial hypertension therapeutics 
Circulation  2012;125(23):10.1161/CIRCULATIONAHA.112.107821.
doi:10.1161/CIRCULATIONAHA.112.107821
PMCID: PMC3864803  PMID: 22572912

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