Peroxiredoxin-2 recycling is slower in denser and pediatric sickle cell red cells.

Peroxiredoxin-2 (Prx-2) is a critical antioxidant protein in red blood cells (RBC). Prx-2 is oxidized to a disulfide covalently-bound dimer by H2 O2 , and then reduced back by the NADPH-dependent thioredoxin-thioredoxin reductase system. The reduction of oxidized Prx-2 is relatively slow in RBCs. Since Prx-2 is highly abundant, Prx-2s' peroxidase catalytic cycle is not considered to be limiting under normal conditions. However, whether Prx-2 recycling becomes limiting when RBCs are exposed to stress is not known. Using three different model systems characterized by increased oxidative damage to RBCs spanning the physiologic (endogenous RBCs of different ages), therapeutic (cold-stored RBCs in blood banks) and pathologic (RBCs from sickle cell disease (SCD) patients and humanized SCD mice) spectrum, basal levels of Prx-2 oxidation and Prx-2 recycling kinetics after addition of H2 O2 were determined. The reduction of oxidized Prx-2 was significantly slower in older versuin older versus younger RBCs, in RBCs stored for 4-5 weeks compared to 1 week, and in RBC from pediatric SCD patients compared to RBCs from control non-SCD patients. Similarly, the rate of Prx-2 recycling was slower in humanized SCD mice compared to WT mice. Treatment of RBC with carbon monoxide (CO) to limit heme-peroxidase activity had no effect on Prx-2 recycling kinetics. Treatment with glucose attenuated slowed Prx-2 recycling in older RBCs and SCD RBCs, but not stored RBCs. In conclusion, the reduction of oxidized Prx-2 can be further slowed in RBCs, which may limit the protection afforded by this antioxidant protein in settings associated with erythrocyte stress.

Different-sized extracellular vesicles derived from stored red blood cells package diverse cargoes and cause distinct cellular effects.

BACKGROUND: The formation of extracellular vesicles (EVs) occurs during cold storage of RBCs. Transfusion of EVs may contribute to adverse responses in recipients receiving RBCs. However, EVs are poorly characterized with limited data on whether distinct vesicles are formed, their composition, and potential biological effects. STUDY DESIGN AND METHODS: Stored RBC-derived EVs were purified using protocols that separate larger microvesicle-like EVs (LEVs) from smaller exosome-like vesicles (SEVs). Vesicles were analyzed by electron microscopy, content of hemoglobin, heme, and proteins (by mass spectrometry), and the potential to mediate lipid peroxidation and endothelial cell permeability in vitro. RESULTS: SEVs were characterized by having an electron-dense double membrane whereas LEVs had more uniform electron density across the particles. No differences in hemoglobin nor heme levels per particle were observed, however, due to smaller volumes, SEVs had higher concentrations of oxyHb and heme. Both particles contained antioxidant proteins peroxiredoxin-2 and copper/zinc superoxide dismutase, these were present in higher molecular weight fractions in SEVs suggesting either oxidized proteins are preferentially packaged into smaller vesicles and/or that the environment associated with SEVs is more pro-oxidative. Furthermore, total glutathione (GSH + GSSG) levels were lower in SEVs. Both EVs mediated oxidation of liposomes that were prevented by hemopexin, identifying heme as the pro-oxidant effector. Addition of SEVs, but not LEVs, induced endothelial permeability in a process also prevented by hemopexin. CONCLUSION: These data show that distinct EVs are formed during cold storage of RBCs with smaller particles being more likely to mediate pro-oxidant and inflammatory effects associated with heme.

Xanthine Oxidase Drives Hemolysis and Vascular Malfunction in Sickle Cell Disease.

OBJECTIVE: Chronic hemolysis is a hallmark of sickle cell disease (SCD) and a driver of vasculopathy; however, the mechanisms contributing to hemolysis remain incompletely understood. Although XO (xanthine oxidase) activity has been shown to be elevated in SCD, its role remains unknown. XO binds endothelium and generates oxidants as a byproduct of hypoxanthine and xanthine catabolism. We hypothesized that XO inhibition decreases oxidant production leading to less hemolysis. Approach and Results: Wild-type mice were bone marrow transplanted with control (AA) or sickle (SS) Townes bone marrow. After 12 weeks, mice were treated with 10 mg/kg per day of febuxostat (Uloric), Food and Drug Administration-approved XO inhibitor, for 10 weeks. Hematologic analysis demonstrated increased hematocrit, cellular hemoglobin, and red blood cells, with no change in reticulocyte percentage. Significant decreases in cell-free hemoglobin and increases in haptoglobin suggest XO inhibition decreased hemolysis. Myographic studies demonstrated improved pulmonary vascular dilation and blunted constriction, indicating improved pulmonary vasoreactivity, whereas pulmonary pressure and cardiac function were unaffected. The role of hepatic XO in SCD was evaluated by bone marrow transplanting hepatocyte-specific XO knockout mice with SS Townes bone marrow. However, hepatocyte-specific XO knockout, which results in >50% diminution in circulating XO, did not affect hemolysis levels or vascular function, suggesting hepatocyte-derived elevation of circulating XO is not the driver of hemolysis in SCD. CONCLUSIONS: Ten weeks of febuxostat treatment significantly decreased hemolysis and improved pulmonary vasoreactivity in a mouse model of SCD. Although hepatic XO accounts for >50% of circulating XO, it is not the source of XO driving hemolysis in SCD.

Safety and toxicology assessment of sodium nitrite administered by intramuscular injection.

Intramuscular (IM) injection of nitrite (1-10 mg/kg) confers survival benefit and protects against lung injury after exposure to chlorine gas in preclinical models. Herein, we evaluated safety/toxicity parameters after single, and repeated (once daily for 7 days) IM injection of nitrite in male and female Sprague Dawley rats and Beagle dogs. The repeat dose studies were performed in compliance with the Federal Drug Administration's (FDA) Good Laboratory Practices Code of Federal Regulations (21 CFR Part 58). Parameters evaluated consisted of survival, clinical observations, body weights, clinical pathology, plasma drug levels, methemoglobin and macroscopic and microscopic pathology. In rats and dogs, single doses of ≥100 mg/kg and 60 mg/kg resulted in death and moribundity, while repeated administration of ≤30 or ≤ 10 mg/kg/day, respectively, was well tolerated. Therefore, the maximum tolerated dose following repeated administration in rats and dogs were determined to be 30 mg/kg/day and 10 mg/kg/day, respectively. Effects at doses below the maximum tolerated dose (MTD) were limited to emesis (in dogs only) and methemoglobinemia (in both species) with clinical signs (e.g. blue discoloration of lips) being dose-dependent, transient and reversible. These signs were not considered adverse, therefore the No Observed Adverse Effect Level (NOAEL) for both rats and dogs was 10 mg/kg/day in males (highest dose tested for dogs), and 3 mg/kg/day in females. Toxicokinetic assessment of plasma nitrite showed no difference between male and females, with Cmax occurring between 5 mins and 0.5 h (rats) or 0.25 h (dogs). In summary, IM nitrite was well tolerated in rats and dogs at doses previously shown to confer protection against chlorine gas toxicity.

Hemoglobin ß93 Cysteine Is Not Required for Export of Nitric Oxide Bioactivity From the Red Blood Cell.

BACKGROUND: Nitrosation of a conserved cysteine residue at position 93 in the hemoglobin ß chain (ß93C) to form S-nitroso (SNO) hemoglobin (Hb) is claimed to be essential for export of nitric oxide (NO) bioactivity by the red blood cell (RBC) to mediate hypoxic vasodilation and cardioprotection. METHODS: To test this hypothesis, we used RBCs from mice in which the ß93 cysteine had been replaced with alanine (ß93A) in a number of ex vivo and in vivo models suitable for studying export of NO bioactivity. RESULTS: In an ex vivo model of cardiac ischemia/reperfusion injury, perfusion of a mouse heart with control RBCs (ß93C) pretreated with an arginase inhibitor to facilitate export of RBC NO bioactivity improved cardiac recovery after ischemia/reperfusion injury, and the response was similar with ß93A RBCs. Next, when human platelets were coincubated with RBCs and then deoxygenated in the presence of nitrite, export of NO bioactivity was detected as inhibition of ADP-induced platelet activation. This effect was the same in ß93C and ß93A RBCs. Moreover, vascular reactivity was tested in rodent aortas in the presence of RBCs pretreated with S-nitrosocysteine or with hemolysates or purified Hb treated with authentic NO to form nitrosyl(FeII)-Hb, the proposed precursor of SNO-Hb. SNO-RBCs or NO-treated Hb induced vasorelaxation, with no differences between ß93C and ß93A RBCs. Finally, hypoxic microvascular vasodilation was studied in vivo with a murine dorsal skin-fold window model. Exposure to acute systemic hypoxia caused vasodilatation, and the response was similar in ß93C and ß93A mice. CONCLUSIONS: RBCs clearly have the fascinating ability to export NO bioactivity, but this occurs independently of SNO formation at the ß93 cysteine of Hb.

Correction: Role of heme in lung bacterial infection after trauma hemorrhage and stored red blood cell transfusion: A preclinical experimental study.

[This corrects the article DOI: 10.1371/journal.pmed.1002522.].

The role of redox-dependent mechanisms in heme release from hemoglobin and erythrocyte hemolysates.

Toxicity mediated by free heme has emerged as an important element of end organ injuries and adverse outcomes in critically ill disease states. Free heme is thought to be derived from oxidative denaturation of free hemoglobin, secondary to red cell hemolysis. In this study, we evaluated the ability of oxidants (H2O2, nitrite, peroxynitrite and hypochlorous acid) formed during inflammation to cause heme release from purified hemoglobin and hemolysates, at pH 7.4 and 6.8. Supraphysiological concentrations of nitrite, peroxynitrite or hypochlorous acid were required to cause appreciable heme release from either free hemoglobin or hemolysates. However, H2O2 administered as a bolus or generated in situ, was more potent at promoting free heme release with free hemoglobin. With hemolysates, only in situ H2O2 formation resulted in significant free heme release. In all cases, free heme release was higher at lower pH and required oxidation of ferrous heme, but was not dependent on ferrylHb formation. Moreover, ligating ferric heme with cyanide or blocking the ß93Cys did not prevent, but in fact increased free heme release. The salient observations from this study are that free heme release is likely mediated by continuous generation of H2O2 versus other heme oxidants, and facilitated at low pH.

Role of heme in lung bacterial infection after trauma hemorrhage and stored red blood cell transfusion: A preclinical experimental study.

BACKGROUND: Trauma is the leading cause of death and disability in patients aged 1-46 y. Severely injured patients experience considerable blood loss and hemorrhagic shock requiring treatment with massive transfusion of red blood cells (RBCs). Preclinical and retrospective human studies in trauma patients have suggested that poorer therapeutic efficacy, increased severity of organ injury, and increased bacterial infection are associated with transfusion of large volumes of stored RBCs, although the mechanisms are not fully understood. METHODS AND FINDINGS: We developed a murine model of trauma hemorrhage (TH) followed by resuscitation with plasma and leukoreduced RBCs (in a 1:1 ratio) that were banked for 0 (fresh) or 14 (stored) days. Two days later, lungs were infected with Pseudomonas aeruginosa K-strain (PAK). Resuscitation with stored RBCs significantly increased the severity of lung injury caused by P. aeruginosa, as demonstrated by higher mortality (median survival 35 h for fresh RBC group and 8 h for stored RBC group; p < 0.001), increased pulmonary edema (mean [95% CI] 106.4 µl [88.5-124.3] for fresh RBCs and 192.5 µl [140.9-244.0] for stored RBCs; p = 0.003), and higher bacterial numbers in the lung (mean [95% CI] 1.2 × 10(7) [-1.0 × 10(7) to 2.5 × 10(7)] for fresh RBCs and 3.6 × 10(7) [2.5 × 10(7) to 4.7 × 10(7)] for stored RBCs; p = 0.014). The mechanism underlying this increased infection susceptibility and severity was free-heme-dependent, as recombinant hemopexin or pharmacological inhibition or genetic deletion of toll-like receptor 4 (TLR4) during TH and resuscitation completely prevented P. aeruginosa-induced mortality after stored RBC transfusion (p < 0.001 for all groups relative to stored RBC group). Evidence from studies transfusing fresh and stored RBCs mixed with stored and fresh RBC supernatants, respectively, indicated that heme arising both during storage and from RBC hemolysis post-resuscitation plays a role in increased mortality after PAK (p < 0.001). Heme also increased endothelial permeability and inhibited macrophage-dependent phagocytosis in cultured cells. Stored RBCs also increased circulating high mobility group box 1 (HMGB1; mean [95% CI] 15.4 ng/ml [6.7-24.0] for fresh RBCs and 50.3 ng/ml [12.3-88.2] for stored RBCs), and anti-HMGB1 blocking antibody protected against PAK-induced mortality in vivo (p = 0.001) and restored macrophage-dependent phagocytosis of P. aeruginosa in vitro. Finally, we showed that TH patients, admitted to the University of Alabama at Birmingham ER between 1 January 2015 and 30 April 2016 (n = 50), received high micromolar-millimolar levels of heme proportional to the number of units transfused, sufficient to overwhelm endogenous hemopexin levels early after TH and resuscitation. Limitations of the study include lack of assessment of temporal changes in different products of hemolysis after resuscitation and the small sample size precluding testing of associations between heme levels and adverse outcomes in resuscitated TH patients. CONCLUSIONS: We provide evidence that large volume resuscitation with stored blood, compared to fresh blood, in mice increases mortality from subsequent pneumonia, which occurs via mechanisms sensitive to hemopexin and TLR4 and HMGB1 inhibition.

Non-invasive analysis of stored red blood cells using diffuse resonance Raman spectroscopy.

A method to acquire the Raman spectra of sub-surface components using diffusely focused radiation in a microscope sampling configuration is described. This procedure generates Raman scattering at various sample depths by producing a converging beam at the back aperture of the objective lens. This method requires illumination of the sample with a defocused laser, while simultaneously increasing the number of CCD pixels that are binned along the spatial axis of the detector. We applied this diffuse sampling method to the analysis of stored red blood cells (RBCs). During storage, biochemical changes to RBCs occur (the "storage lesion"). However, there are no existing non-invasive methods to assess this. We evaluated the instrumental parameters needed to maximize the diffusely scattered signal, including pixel binning, slit width, and bandwidth. We demonstrated the effectiveness of this diffuse resonance Raman spectroscopy (DRRS) method by detecting RBCs through a blood bag segment (1 mm wall thickness). We directly compared the DRRS method to the more common stand-off Raman spectroscopy (SORS) method using both 633 nm and 785 nm excitation. Time-dependent DRRS spectra were used in a multivariate model for classification of RBCs in polymer segments by storage age. Young (6-8 day) RBCs were differentiated from old (35-40) RBCs with 100% sensitivity and 98.5% selectivity. These data indicated that DRRS is a promising, non-invasive technique for acquiring the spectra of sub-surface components, and is particularly applicable when the underlying sample can be resonantly enhanced.

Formation of chlorinated lipids post-chlorine gas exposure.

Exposure to chlorine (Cl2) gas can occur during accidents and intentional release scenarios. However, biomarkers that specifically indicate Cl2 exposure and Cl2-derived products that mediate postexposure toxicity remain unclear. We hypothesized that chlorinated lipids (Cl-lipids) formed by direct reactions between Cl2 gas and plasmalogens serve as both biomarkers and mediators of post-Cl2 gas exposure toxicities. The 2-chloropalmitaldehyde (2-Cl-Pald), 2-chlorostearaldehyde (2-Cl-Sald), and their oxidized products, free- and esterified 2-chloropalmitic acid (2-Cl-PA) and 2-chlorostearic acid were detected in the lungs and plasma of mouse and rat models of Cl2 gas exposure. Levels of Cl-lipids were highest immediately post-Cl2 gas exposure, and then declined over 72 h with levels remaining 20- to 30-fold higher at 24 h compared with baseline. Glutathione adducts of 2-Cl-Pald and 2-Cl-Sald also increased with levels peaking at 4 h in plasma. Notably, 3-chlorotyrosine also increased after Cl2 gas exposure, but returned to baseline within 24 h. Intranasal administration of 2-Cl-PA or 2-Cl-Pald at doses similar to those formed in the lung after Cl2 gas exposure led to increased distal lung permeability and inflammation and systemic endothelial dysfunction characterized by loss of eNOS-dependent vasodilation. These data suggest that Cl-lipids could serve as biomarkers and mediators for Cl2 gas exposure and toxicity.

Detection of electrophile-sensitive proteins.

BACKGROUND: Redox signaling is an important emerging mechanism of cellular function. Dysfunctional redox signaling is increasingly implicated in numerous pathologies, including atherosclerosis, diabetes, and cancer. The molecular messengers in this type of signaling are reactive species which can mediate the post-translational modification of specific groups of proteins, thereby effecting functional changes in the modified proteins. Electrophilic compounds comprise one class of reactive species which can participate in redox signaling. Electrophiles modulate cell function via formation of covalent adducts with proteins, particularly cysteine residues. SCOPE OF REVIEW: This review will discuss the commonly used methods of detection for electrophile-sensitive proteins, and will highlight the importance of identifying these proteins for studying redox signaling and developing novel therapeutics. MAJOR CONCLUSIONS: There are several methods which can be used to detect electrophile-sensitive proteins. These include the use of tagged model electrophiles, as well as derivatization of endogenous electrophile-protein adducts. GENERAL SIGNIFICANCE: In order to understand the mechanisms by which electrophiles mediate redox signaling, it is necessary to identify electrophile-sensitive proteins and quantitatively assess adduct formation. Strengths and limitations of these methods will be discussed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Predicting storage-dependent damage to red blood cells using nitrite oxidation kinetics, peroxiredoxin-2 oxidation, and hemoglobin and free heme measurements.

BACKGROUND: Storage-dependent damage to red blood cells (RBCs) varies significantly. Identifying RBC units that will undergo higher levels of hemolysis during storage may allow for more efficient inventory management decision-making. Oxidative-stress mediates storage-dependent damage to RBCs and will depend on the oxidant:antioxidant balance. We reasoned that this balance or redox tone will serve as a determinant of how a given RBC unit stores and that its assessment in "young" RBCs will predict storage-dependent hemolysis. STUDY DESIGN AND METHODS: RBCs were sampled from bags and segments stored for 7 to 42 days. Redox tone was assessed by nitrite oxidation kinetics and peroxiredoxin-2 (Prx-2) oxidation. In parallel, hemolysis was assessed by measuring cell-free hemoglobin (Hb) and free heme (hemin). Correlation analyses were performed to determine if Day 7 measurements predicted either the level of hemolysis at Day 35 or the increase in hemolysis during storage. RESULTS: Higher Day 7 Prx-2 oxidation was associated with higher Day 35 Prx-2 oxidation, suggesting that early assessment of this variable may identify RBCs that will incur the most oxidative damage during storage. RBCs that oxidized nitrite faster on Day 7 were associated with the greatest levels of storage-dependent hemolysis and increases in Prx-2 oxidation. An inverse relationship between storage-dependent changes in oxyhemoglobin and free heme was observed underscoring an unappreciated reciprocity between these molecular species. Moreover, free heme was higher in the bag compared to paired segments, with opposite trends observed for free Hb. CONCLUSION: Measurement of Prx-2 oxidation and nitrite oxidation kinetics early during RBC storage may predict storage-dependent damage to RBC including hemolysis-dependent formation of free Hb and heme.

Nitrite therapy improves survival postexposure to chlorine gas.

Exposure to relatively high levels of chlorine (Cl2) gas can occur in mass-casualty scenarios associated with accidental or intentional release. Recent studies have shown a significant postexposure injury phase to the airways, pulmonary, and systemic vasculatures mediated in part by oxidative stress, inflammation, and dysfunction in endogenous nitric oxide homeostasis pathways. However, there is a need for therapeutics that are amenable to rapid and easy administration in the field and that display efficacy toward toxicity after chlorine exposure. In this study, we tested whether nitric oxide repletion using nitrite, by intramuscular injection after Cl2 exposure, could prevent Cl2 gas toxicity. C57bl/6 male mice were exposed to 600 parts per million Cl2 gas for 45 min, and 24-h survival was determined with or without postexposure intramuscular nitrite injection. A single injection of nitrite (10 mg/kg) administered either 30 or 60 min postexposure significantly improved 24-h survival (from ∼20% to 50%). Survival was associated with decreased neutrophil accumulation in the airways. Rendering mice neutropenic before Cl2 exposure improved survival and resulted in loss of nitrite-dependent survival protection. Interestingly, female mice were more sensitive to Cl2-induced toxicity compared with males and were also less responsive to postexposure nitrite therapy. These data provide evidence for efficacy and define therapeutic parameters for a single intramuscular injection of nitrite as a therapeutic after Cl2 gas exposure that is amenable to administration in mass-casualty scenarios.

Gene expression and ultra-structural evidence for metabolic derangement in the primary mitral regurgitation heart.

Aims: Chronic neurohormonal activation and haemodynamic load cause derangement in the utilization of the myocardial substrate. In this study, we test the hypothesis that the primary mitral regurgitation (PMR) heart shows an altered metabolic gene profile and cardiac ultra-structure consistent with decreased fatty acid and glucose metabolism despite a left ventricular ejection fraction (LVEF) > 60%. Methods and results: Metabolic gene expression in right atrial (RA), left atrial (LA), and left ventricular (LV) biopsies from donor hearts (n = 10) and from patients with moderate-to-severe PMR (n = 11) at surgery showed decreased mRNA glucose transporter type 4 (GLUT4), GLUT1, and insulin receptor substrate 2 and increased mRNA hexokinase 2, O-linked N-acetylglucosamine transferase, and O-linked N-acetylglucosaminyl transferase, rate-limiting steps in the hexosamine biosynthetic pathway. Pericardial fluid levels of neuropeptide Y were four-fold higher than simultaneous plasma, indicative of increased sympathetic drive. Quantitative transmission electron microscopy showed glycogen accumulation, glycophagy, increased lipid droplets (LDs), and mitochondrial cristae lysis. These findings are associated with increased mRNA for glycogen synthase kinase 3ß, decreased carnitine palmitoyl transferase 2, and fatty acid synthase in PMR vs. normals. Cardiac magnetic resonance and positron emission tomography for 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) uptake showed decreased LV [18F]FDG uptake and increased plasma haemoglobin A1C, free fatty acids, and mitochondrial damage-associated molecular patterns in a separate cohort of patients with stable moderate PMR with an LVEF > 60% (n = 8) vs. normal controls (n = 8). Conclusion: The PMR heart has a global ultra-structural and metabolic gene expression pattern of decreased glucose uptake along with increased glycogen and LDs. Further studies must determine whether this presentation is an adaptation or maladaptation in the PMR heart in the clinical evaluation of PMR.

Cell signalling by reactive lipid species: new concepts and molecular mechanisms.

The process of lipid peroxidation is widespread in biology and is mediated through both enzymatic and non-enzymatic pathways. A significant proportion of the oxidized lipid products are electrophilic in nature, the RLS (reactive lipid species), and react with cellular nucleophiles such as the amino acids cysteine, lysine and histidine. Cell signalling by electrophiles appears to be limited to the modification of cysteine residues in proteins, whereas non-specific toxic effects involve modification of other nucleophiles. RLS have been found to participate in several physiological pathways including resolution of inflammation, cell death and induction of cellular antioxidants through the modification of specific signalling proteins. The covalent modification of proteins endows some unique features to this signalling mechanism which we have termed the 'covalent advantage'. For example, covalent modification of signalling proteins allows for the accumulation of a signal over time. The activation of cell signalling pathways by electrophiles is hierarchical and depends on a complex interaction of factors such as the intrinsic chemical reactivity of the electrophile, the intracellular domain to which it is exposed and steric factors. This introduces the concept of electrophilic signalling domains in which the production of the lipid electrophile is in close proximity to the thiol-containing signalling protein. In addition, we propose that the role of glutathione and associated enzymes is to insulate the signalling domain from uncontrolled electrophilic stress. The persistence of the signal is in turn regulated by the proteasomal pathway which may itself be subject to redox regulation by RLS. Cell death mediated by RLS is associated with bioenergetic dysfunction, and the damaged proteins are probably removed by the lysosome-autophagy pathway.

Plasma Exosome Hemoglobin Released During Surgery Is Associated With Cardiac Injury in Animal Model.

BACKGROUND: Patients with valvular heart disease require cardiopulmonary bypass and cardiac arrest. Here, we test the hypothesis that exosomal hemoglobin formed during cardiopulmonary bypass mediates acute cardiac injury in humans and in an animal model system. METHODS: Plasma exosomes were collected from arterial blood at baseline and 30 minutes after aortic cross-clamp release in 20 patients with primary mitral regurgitation and 7 with aortic stenosis. These exosomes were injected into Sprague-Dawley rats and studied at multiple times up to 30 days. Tissue was examined by hematoxylin and eosin stain, immunohistochemistry, transmission electron microscopy, and brain natriuretic peptide. RESULTS: Troponin I levels increased from 36 ± 88 ng/L to 3622 ± 3054 ng/L and correlated with exosome hemoglobin content (Spearman r = 0.7136, < .0001, n = 24). Injection of exosomes isolated 30 minutes after cross-clamp release into Sprague-Dawley rats resulted in cardiomyocyte myofibrillar loss at 3 days. Transmission electron microscopy demonstrated accumulation of electron dense particles of ferritin within cardiomyocytes, in the interstitial space, and within exosomes. At 21 days after injection, there was myofibrillar and myosin breakdown, interstitial fibrosis, elevated brain natriuretic peptide, and left ventricle diastolic dysfunction measured by echocardiography/Doppler. Pericardial fluid exosomal hemoglobin content is fourfold higher than simultaneous plasma exosome hemoglobin, suggesting a cardiac source of exosomal hemoglobin. CONCLUSIONS: Red blood cell and cardiac-derived exosomal hemoglobin may be involved in myocardial injury during cardiopulmonary bypass in patients with valvular heart disease.

Elevated cardiac hemoglobin expression is associated with a pro-oxidative and inflammatory environment in primary mitral regurgitation.

BACKGROUND: Primary mitral regurgitation (PMR) is associated with oxidative and inflammatory myocardial damage. We reported greater exosome hemoglobin (Hb) in pericardial fluid (PCF) versus plasma, suggesting a cardiac source of Hb. OBJECTIVE: Test the hypothesis that Hb is produced in the PMR heart and is associated with increased inflammation. METHODS AND RESULTS: Hb gene expression for subunits alpha (HBA) and beta (HBB) was assessed in right atria (RA), left atria (LA) and left ventricular (LV) tissue from donor hearts (n = 10) and PMR patient biopsies at surgery (n = 11). PMR patients (n = 22) had PCF and blood collected for macrophage markers, pro-inflammatory cytokines, and matrix metalloproteinases (MMPs). In-situ hybridization for HBA mRNA and immunohistochemistry for Hb-alpha (Hbα) and Hb-beta (Hbß) protein was performed on PMR tissue. RESULTS: HBA and HBB genes are significantly increased (>4-fold) in RA, LA, and LV in PMR vs. normal hearts. In PMR tissue, HBA mRNA is expressed in both LV cardiomyocytes and interstitial cells by in-situ hybridization; however, Hbα and Hbß protein is only expressed in interstitial cells by immunohistochemistry. PCF oxyHb is significantly increased over plasma along with low ratios (<1.0) of haptoglobin:oxyHb and hemopexin:heme supporting a highly oxidative environment. Macrophage chemotactic protein-1, tumor necrosis factor-α, interleukin-6, and MMPs are significantly higher in PCF vs. plasma. CONCLUSION: There is increased Hb production in the PMR heart coupled with the inflammatory state of the heart, suggests a myocardial vulnerability of further Hb delivery and/or production during cardiac surgery that could adversely affect LV functional recovery.

Red blood cell exosome hemoglobin content increases after cardiopulmonary bypass and mediates acute kidney injury in an animal model.

OBJECTIVE: Hemolysis, characterized by formation of free hemoglobin (Hb), occurs in patients undergoing cardiopulmonary bypass (CPB). However, there is no study of the dynamic changes in red blood cell (RBC)-derived exosomes (Exos) released during CPB, nor whether these particles mediate acute kidney injury (AKI). METHODS: This study is a comprehensive time-course analysis, at baseline, 30 minutes, to 24 hours post-crossclamp release (XCR) to determine (1) Exos Hb content; (2) free Hb/heme, haptoglobin, hemopexin; and (3) urinary markers of AKI over the same time period. In addition, we developed a model system in Sprague-Dawley rats to test for AKI after intravenous injection of Exos Hb released during CPB. RESULTS: In 30 patients undergoing CPB, there is a significant increase in plasma Hb-positive Exos but not microvesicles 30 minutes post-XCR versus other time points, with a simultaneous decrease in the haptoglobin/Hb ratio. These changes presage a significant increase in urine neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 at 24 hours. Intravenous injection of plasma Exos (109-10 particles obtained 30 minutes post-XCR) into rats causes AKI at 72 hours, manifested by multifocal degeneration of proximal tubular epithelium. At 21 days, there is persistent tubular injury and interstitial fibrosis. Intravenous injection of Exos from 35-day-old stored RBCs into rats results in glomerular-tubular injury, increased kidney ferritin and hemoxygenase-1 expression, and significant elevation of kidney injury molecule-1 and proteinuria at 72 hours. CONCLUSIONS: These combined studies raise the potential for RBC-derived Exos, released during CPB, to target the kidney and mediate AKI.

Modulation of mammary cancer cell migration by 15-deoxy-delta(12,14)-prostaglandin J(2): implications for anti-metastatic therapy.

Recently, a number of steps in the progression of metastatic disease have been shown to be regulated by redox signalling. Electrophilic lipids affect redox signalling through the post-translational modification of critical cysteine residues in proteins. However, the therapeutic potential as well as the precise mechanisms of action of electrophilic lipids in cancer cells is poorly understood. In the present study, we investigate the effect of the electrophilic prostaglandin 15d-PGJ2 (15-deoxy-Delta12,14-prostaglandin J2) on metastatic properties of breast cancer cells. 15d-PGJ2 was shown to decrease migration, stimulate focal-adhesion disassembly and cause extensive F-actin (filamentous actin) reorganization at low concentrations (0.03-0.3 microM). Importantly, these effects seem to be independent of PPARgamma (peroxisome-proliferator-activated receptor gamma) and modification of actin or Keap1 (Kelch-like ECH-associated protein 1), which are known protein targets of 15d-PGJ2 at higher concentrations. Interestingly, the p38 inhibitor SB203580 was able to prevent both 15d-PGJ2-induced F-actin reorganization and focal-adhesion disassembly. Taken together, the results of the present study suggest that electrophiles such as 15d-PGJ2 are potential anti-metastatic agents which exhibit specificity for migration and adhesion pathways at low concentrations where there are no observed effects on Keap1 or cytotoxicity.

Human and rodent red blood cells do not demonstrate xanthine oxidase activity or XO-catalyzed nitrite reduction to NO.

A number of molybdopterin enzymes, including xanthine oxidoreductase (XOR), aldehyde oxidase (AO), sulfite oxidase (SO), and mitochondrial amidoxime reducing component (mARC), have been identified as nitrate and nitrite reductases. Of these enzymes, XOR has been the most extensively studied and reported to be a substantive source of nitric oxide (NO) under inflammatory/hypoxic conditions that limit the catalytic activity of the canonical NOS pathway. It has also been postulated that XOR nitrite reductase activity extends to red blood cell (RBCs) membranes where it has been immunohistochemically identified. These findings, when combined with countervailing reports of XOR activity in RBCs, incentivized our current study to critically evaluate XOR protein abundance/enzymatic activity in/on RBCs from human, mouse, and rat sources. Using various protein concentrations of RBC homogenates for both human and rodent samples, neither XOR protein nor enzymatic activity (xanthine → uric acid) was detectable. In addition, potential loading of RBC-associated glycosaminoglycans (GAGs) by exposing RBC preparations to purified XO before washing did not solicit detectable enzymatic activity (xanthine → uric acid) or alter NO generation profiles. To ensure these observations extended to absence of XOR-mediated contributions to overall RBC-associated nitrite reduction, we examined the nitrite reductase activity of washed and lysed RBC preparations via enhanced chemiluminescence in the presence or absence of the XOR-specific inhibitor febuxostat (Uloric®). Neither addition of inhibitor nor the presence of the XOR substrate xanthine significantly altered the rates of nitrite reduction to NO by RBC preparations from either human or rodent sources confirming the absence of XO enzymatic activity. Furthermore, examination of the influence of the age (young cells vs. old cells) of human RBCs on XO activity also failed to demonstrate detectable XO protein. Combined, these data suggest: 1) that XO does not contribute to nitrite reduction in/on human and rodent erythrocytes, 2) care should be taken to validate immuno-detectable XO by demonstrating enzymatic activity, and 3) XO does not associate with human erythrocytic glycosaminoglycans or participate in nonspecific binding.