Cell-free Hb Research Articles

Targeted Disruption of Nrf2 in Non-Hematopoietic Tissues Exacerbates Hemolysis and Accelerates Chronic Lung Disease in Transgenic Sickle Cell Anemia Mice

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a transcription factor that regulates the cellular defense mechanism by mediating a coordinate induction of cytoprotective antioxidant responsive element-driven genes. Nrf2 agonists augment fetal hemoglobin expression in hematopoietic progenitors and have emerged as a new class of drugs for therapeutic induction of fetal hemoglobin in sickle cell anemia (SCA). However, the cytoprotective effect of Nrf2 on the pathobiology of SCA has not been previously defined. To investigate the role and mechanism of Nrf2 in SCA independent of globin gene modulation, we generated chimeric mice with disruption of Nrf2 in non-hematopoietic tissues. A total of twenty-six Nrf2-/- mice were transplanted with bone marrow harvested from the Berkeley sickle and hemizygous (Hemi) mice and the Townes sickle (SS) and control (AA) mice. All sickle/Nrf2-null chimeras (SSNHNrf2-/-; n=13) developed classical hematological and intravascular hemolysis features of SCA after eight weeks of transplantation. Despite the presence of erythrocyte Nrf2, SSNHNrf2-/- chimeras developed more severe intravascular hemolysis than SS wild-type (SSWT) donor-litter-mates. The concentration of plasma cell-free Hb (p<0.01), plasma heme (p<0.05) and lactate dehydrogenase (p<0.05) were 1.5 to 2.0-fold higher in the chimeras than in the SSWT mice. The chimeras had markedly higher concentrations of soluble vascular cell adhesion molecule-1 (p<0.01) and soluble P-selectin in their plasma (p<0.05), and more oxidizing extracellular redox status (p<0.05). The latter finding was corroborated by markedly lower reduced-glutathione in the SSNHNrf2-/- mouse lungs (19.07±0.32 vs 26.5 ±1.5 nmol/mg protein, p<0.01). The worsening of hemolysis, vascular inflammation and oxidative stress in the SSNHNrf2-/- chimeras was unrelated to the transplantation procedure since none of these effects were seen in the AANHNrf2-/- and HemiNHNrf2-/- controls. Next, we found that the SSNHNrf2-/- chimeras had hypoxemia at baseline (oxygen saturation, 95.8±1.7%) while age-matched SSWT mice had normal oxygen saturation (99.2±0.2%, p<0.05). Consistent with these disparate pulmonary phenotypes, the SSNHNrf2-/- but not the SSWT mice developed pulmonary edema at a relatively young age, which was confirmed by histological evidence of alveolar edema. Remarkably, the severely damaged SSNHNrf2-/- mouse lungs contained a similar quantity of heme oxygenase-1 (HO-1) mRNA and protein as the relatively less inflamed SSWT lungs. In addition, the amount of HO-1 in the plasma was also similar in these animals. These results indicate that other transcription factors modulate HO-1 induction, and that HO-1 may play a lesser role in mitigating the chronic effects of SCA. To test the latter idea, we performed a longitudinal study in a cohort of thirty-two SS and AA mice of both sexes. At 8 weeks of age, plasma HO-1 was 3-fold higher in both male and female SS mice than in the AA mice (p<0.001). The HO-1 values did not change at 12, 24 and 40 weeks in any group, however intravascular hemolysis, vascular inflammation and endothelial barrier integrity deteriorated progressively only in the SSWT mice. Overall, our results demonstrate that the elevated HO-1 expression typical of SCA is insufficient to blunt many of the chronic effects of the disease in mice. However, other Nrf2 target genes in non-hematopoietic tissues can slow down this process. Such genes offer an attractive prophylactic therapeutic strategy to preserve organ function in adults with SCA. DisclosuresArcher:Global Blood Therapeutics: Consultancy.

Red blood cell, hemoglobin and heme in the progression of atherosclerosis.

For decades plaque neovascularization was considered as an innocent feature of advanced atherosclerotic lesions, but nowadays growing evidence suggest that this process triggers plaque progression and vulnerability. Neovascularization is induced mostly by hypoxia, but the involvement of oxidative stress is also established. Because of inappropriate angiogenesis, neovessels are leaky and prone to rupture, leading to the extravasation of red blood cells (RBCs) within the plaque. RBCs, in the highly oxidative environment of the atherosclerotic lesions, tend to lyse quickly. Both RBC membrane and the released hemoglobin (Hb) possess atherogenic activities. Cholesterol content of RBC membrane contributes to lipid deposition and lipid core expansion upon intraplaque hemorrhage. Cell-free Hb is prone to oxidation, and the oxidation products possess pro-oxidant and pro-inflammatory activities. Defense and adaptation mechanisms evolved to cope with the deleterious effects of cell free Hb and heme. These rely on plasma proteins haptoglobin (Hp) and hemopexin (Hx) with the ability to scavenge and eliminate free Hb and heme form the circulation. The protective strategy is completed with the cellular heme oxygenase-1/ferritin system that becomes activated when Hp and Hx fail to control free Hb and heme-mediated stress. These protective molecules have pharmacological potential in diverse pathologies including atherosclerosis.

Can gas replace protein function? CO abrogates the oxidative toxicity of myoglobin.

Outside their cellular environments, hemoglobin (Hb) and myoglobin (Mb) are known to wreak oxidative damage. Using haptoglobin (Hp) and hemopexin (Hx) the body defends itself against cell-free Hb, yet mechanisms of protection against oxidative harm from Mb are unclear. Mb may be implicated in oxidative damage both within the myocyte and in circulation following rhabdomyolysis. Data from the literature correlate rhabdomyolysis with the induction of Heme Oxygenase-1 (HO-1), suggesting that either the enzyme or its reaction products are involved in oxidative protection. We hypothesized that carbon monoxide (CO), a product, might attenuate Mb damage, especially since CO is a specific ligand for heme iron. Low density lipoprotein (LDL) was chosen as a substrate in circulation and myosin (My) as a myocyte component. Using oxidation targets, LDL and My, the study compared the antioxidant potential of CO in Mb-mediated oxidation with the antioxidant potential of Hp in Hb-mediated oxidation. The main cause of LDL oxidation by Hb was found to be hemin which readily transfers from Hb to LDL. Hp prevented heme transfer by sequestering hemin within the Hp-Hb complex. Hemin barely transferred from Mb to LDL, and oxidation appeared to stem from heme iron redox in the intact Mb. My underwent oxidative crosslinking by Mb both in air and under N2. These reactions were fully arrested by CO. The data are interpreted to suit several circumstances, some physiological, such as high muscle activity, and some pathological, such as rhabdomyolysis, ischemia/reperfusion and skeletal muscle disuse atrophy. It appear that CO from HO-1 attenuates damage by temporarily binding to deoxy-Mb, until free oxygen exchanges with CO to restore the equilibrium.

P99 Hydrogen-sulfide releasing molecules inhibit intraplaque hemorrhage-associated oxidative burst in the human atheroma and subsequent endothelial reactions

Background and aims Intraplaque hemorrhage (IH) contributes significantly to plaque progression and vulnerability. Following IH hemoglobin (Hb) is released form erythrocytes and can interact with plaque lipids. Interactions between cell-free Hb and plaque lipids provoke a vicious cycle promoting the oxidation of plaque lipids and Hb which in turn trigger endothelial activation and cytotoxicity. Hydrogen sulfide (H2S), the recently discovered endogenous gas, exhibits athero-protective properties. We aimed to test different H2S releasing molecules if they can inhibit interactions between Hb and lipids in human atheroma. Methods Specimens of human atherosclerotic lesions were obtained from aorta of beating-heart donors. LDL was isolated form human plasma by density-gradient ultracentrifugation. Hb was purified form human erythrocytes by anion-exchange chromatography. Conjugated dienes, lipid hydroperoxides (LOOHs) and thiobarbituric-acid reactive substances (TBARS) were measured to monitor LDL and plaque lipid oxidation. To assess Hb oxidation, covalently cross-linked Hb species were detected by western blotting. Human umbilical vein endothelial cells (HUVECs) were obtained from umbilical cords, and toxicity was determined by MTT assay. NaHS, GYY 4137, AP67 and AP72 were used as H2S releasing molecules throughout the study. Results H2S releasing molecules inhibited heme-mediated LDL oxidation, as well as Hb-mediated plaque lipid oxidation although there were differences in their potential, being AP67 the most powerful. H2S releasing molecules prevented accumulation of LOOHs resulting reduced cytotoxicity towards HUVECs. H2S donors inhibited both H2O2 and oxLDL-mediated formation of covalently cross-linked Hb species, those exhibit pro-oxidant and pro-inflammatory properties. Among the tested H2S releasing molecules AP67 was the most potent in preventing Hb oxidation. Conclusion H2S releasing molecules inhibit the vicious cycle initiated by IH. These data arises the potential therapeutic application of these compounds to prevent the progression of atherosclerosis.

Syncytiotrophoblast Vesicles Show Altered micro-RNA and Haemoglobin Content after Ex-vivo Perfusion of Placentas with Haemoglobin to Mimic Preeclampsia

BackgroundCell-free foetal haemoglobin (HbF) has been shown to play a role in the pathology of preeclampsia (PE). In the present study, we aimed to further characterize the harmful effects of extracellular free haemoglobin (Hb) on the placenta. In particular, we investigated whether cell-free Hb affects the release of placental syncytiotrophoblast vesicles (STBMs) and their micro-RNA content.MethodsThe dual ex-vivo perfusion system was used to perfuse isolated cotyledons from human placenta, with medium alone (control) or supplemented with cell-free Hb. Perfusion medium from the maternal side of the placenta was collected at the end of all perfusion phases. The STBMs were isolated using ultra-centrifugation, at 10,000×g and 150,000×g (referred to as 10K and 150K STBMs). The STBMs were characterized using the nanoparticle tracking analysis, identification of surface markers and transmission electron microscopy. RNA was extracted and nine different micro-RNAs, related to hypoxia, PE and Hb synthesis, were selected for analysis by quantitative PCR.ResultsAll micro-RNAs investigated were present in the STBMs. Mir-517a, mir-141 and mir-517b were down regulated after Hb perfusion in the 10K STBMs. Furthermore, Hb was shown to be carried by the STBMs.ConclusionThis study showed that Hb perfusion can alter the micro-RNA content of released STBMs. Of particular interest is the alteration of two placenta specific micro-RNAs; mir-517a and mir-517b. We have also seen that STBMs may function as carriers of Hb into the maternal circulation.

A1M/α1-Microglobulin Protects from Heme-Induced Placental and Renal Damage in a Pregnant Sheep Model of Preeclampsia

Preeclampsia (PE) is a serious pregnancy complication that manifests as hypertension and proteinuria after the 20th gestation week. Previously, fetal hemoglobin (HbF) has been identified as a plausible causative factor. Cell-free Hb and its degradation products are known to cause oxidative stress and tissue damage, typical of the PE placenta. A1M (α1-microglobulin) is an endogenous scavenger of radicals and heme. Here, the usefulness of A1M as a treatment for PE is investigated in the pregnant ewe PE model, in which starvation induces PE symptoms via hemolysis. Eleven ewes, in late pregnancy, were starved for 36 hours and then treated with A1M (n = 5) or placebo (n = 6) injections. After injections, the ewes were re-fed and observed for additional 72 hours. They were monitored for blood pressure, proteinuria, blood cell distribution and clinical and inflammation markers in plasma. Before termination, the utero-placental circulation was analyzed with Doppler velocimetry and the kidney glomerular function was analyzed by Ficoll sieving. At termination, blood, kidney and placenta samples were collected and analyzed for changes in gene expression and tissue structure. The starvation resulted in increased amounts of the hemolysis marker bilirubin in the blood, structural damages to the placenta and kidneys and an increased glomerular sieving coefficient indicating a defect filtration barrier. Treatment with A1M ameliorated these changes without signs of side-effects. In conclusion, A1M displayed positive therapeutic effects in the ewe starvation PE model, and was well tolerated. Therefore, we suggest A1M as a plausible treatment for PE in humans.

Prevalence and risk factors for pulmonary hypertension in patients with hemoglobin E/β‐thalassemia disease

To find the prevalence and risk factors of pulmonary hypertension (PHT) in adult patients with hemoglobin E/β-thalassemia disease (E/β-Thal). One hundred and ten clinically stable E/β-Thal outpatients, sixty-one of whom had undergone splenectomy, were prospectively studied using their clinical profiles, selected blood tests, chest roentgenogram, and transthoracic echocardiogram. Based on the pulmonary artery systolic pressure (PASP) values estimated by the echocardiogram of ≥36mmHg, they were dichotomized into those with (PHT+) and without (PHT-) PHT. PHT was found in 41 (37.3%) patients without gender preponderance. It was not due to the left heart and was not severe (PASP=46.3±10.4mmHg). PASP was higher in splenectomized patients (48.0±11 vs. 40.3±4.7mmHg (P=0.004)). PHT was found in 32 of 61 (52.5%) splenectomized patients, mostly (53%) in the second decade, and rarely (6.3%) during the first 5yr after splenectomy. PHT+ patients had more hemolysis (P=0.001-0.04 depending on the parameters), more asplenic cases (P<0.001), and higher serum soluble vascular cell adhesion molecule-1 (sVCAM-1) and high-sensitivity C-reactive protein levels (P=0.004 and 0.008, respectively). Strong risk factors by univariate analysis were serum sVCAM-1 levels ≥1600ng/mL, serum cell-free Hb≥3mg/dL, asplenia, and amount of NRBCs/100 WBCs >40. Prevalence of PHT in E/β-Thal patients was 37.3% without gender preponderance. Those with severe hemolysis and asplenia invariably had severer PHT. Strong risk factors were asplenia and associated markedly elevated values of sVCAM-1, cell-free Hb, and NRBCs in blood.

Mechanisms of hemolysis‐associated platelet activation

Intravascular hemolysis occurs after blood transfusion, in hemolytic anemias, and in other conditions, and is associated with hypercoagulable states. Hemolysis has been shown to potently activate platelets invitro and invivo, and several mechanisms have been suggested to account for this, including: (i) direct activation by hemoglobin (Hb); (ii) increase in reactive oxygen species (ROS); (iii) scavenging of nitric oxide (NO) by released Hb; and (iv) release of intraerythrocytic ADP. To elucidate the mechanism of hemolysis-mediated platelet activation. We used flow cytometry to detect PAC-1 binding to activated platelets for invitro experiments, and a Siemens' Advia120 hematology system to assess platelet aggregation by using platelet counts from invivo experiments in a rodent model. We found that Hb did not directly activate platelets. However, ADP bound to Hb could cause platelet activation. Furthermore, platelet activation caused by shearing of red blood cells (RBCs) was reduced in the presence of apyrase, which metabolizes ADP to AMP. The use of ROS scavengers did not affect platelet activation. We also found that cell-free Hb enhanced platelet activation by abrogating the inhibitory effect of NO on platelet activation. Invivo infusions of ADP and purified (ADP-free) Hb, as well as hemolysate, resulted in platelet aggregation, as shown by decreased platelet counts. Two primary mechanisms account for RBC hemolysis-associated platelet activation: ADP release, which activates platelets; and cell-free Hb release, which enhances platelet activation by lowering NO bioavailability.

Targeted Treatment Of Acute Chest Syndrome In Transgenic Sickle Mice

Acute chest syndrome (ACS) is a major cause of morbidity and mortality in sickle cell disease (SCD). The diagnosis, prevention and treatment of ACS pose major clinical concerns in SCD partly because the mechanism underlying the pathogenesis of this syndrome remains elusive. Our group first reported that excess intravascular hemin causes a lethal acute lung injury (ALI) in transgenic SCD mice reminiscent of ACS (Ghosh and Ofori-Acquah, Blood 116 Suppl 1:944, 2010). Subsequently, large-scale genomics studies by Bean et al., (Blood 120:3822-8, 2012) and Galarneau et al., (Blood, 122:434-42, 2013) have implicated hemin catabolism and inflammation in the pathogenesis of ACS. In addition, we have reported recently that raised plasma free hemin increases the odds of ACS in children with SCD (Adisa et al., Br J Haematol. 2013). Collectively, these studies support a new theme of ACS pathogenesis involving extracellular hemin. In the current study, we validated the respiratory dysfunction of this ACS model, tested the hypothesis that toll-like receptor 4 (TLR4) mediates the associated lung injury, and examined the efficacy of two strategies to treat the condition in mice. Arterial blood gas analysis of SS mice with the ACS-like disease confirmed severe hypoxemia (PaO2; 40.23 ±3.85 mmHg, SO2; 58.72±6.6%, p<0.001), and revealed worsening hypercapnia (PaCO2; 56.08±3.64 mmHg) and acidosis (mean pH; 7.21), which are all typical of severe ALI. Blood gas parameters remained normal in control AA mice exposed to the same concentration of extracellular hemin. Next, we generated two types of sickle bone marrow chimeric mice that lack expression of TLR4 in non-hematopoietic cells (SSNHTLR4-/-) or express TLR4 on all cell types (SSTLR4+/+). Induction of extracellular hemin crisis with 70 micromoles/kg of hemin resulted in rapid oxygen desaturation, reduced breath rate, pulmonary infiltration, high lung wet/dry weight ratio and sudden death in the SSTLR4+/+ chimeras (n=6). Although all the SSNHTLR4-/- mice acquired a similar phenotype of hemin crisis as the SSTLR4+/+ mice, defined by acute intravascular hemolysis (∼1g/dl drop in Hb, ∼1g/dl increase in cell-free Hb), exhaustion of plasma hemopexin, ∼3-fold increase in plasma free hemin, they did not develop any respiratory symptoms (n=7). Next, we tested two potential therapies, recombinant human hemopexin and TAK-242 a small molecule inhibitor of TLR4. Both agents protected SS mice from developing ACS when they were administered immediately after the induction of hemin crisis. When SS mice were treated after developing respiratory distress evident by significant reductions in oxygen saturation and breath rate, only the recombinant human hemopexin was effective in preventing respiratory failure. In conclusion, we show that TLR4 expressed by the vessel wall, most likely the endothelium, mediates the inflammatory response to excess free hemin associated with severe ACS in mice. Pharmacological inhibition of TLR4 signaling and hemin sequestration may be effective in preventing the development of ACS. In addition, hemin sequestration offers a targeted approach that may halt the progression of ACS in those with more advanced disease. Disclosures:No relevant conflicts of interest to declare.

Extracellular Hemin Auto-Amplification Intensifies Tissue Injury In Sickle Cell Disease

Acute organ failure is a major clinical concern in sickle cell disease (SCD). However, the mechanism responsible for this potentially lethal complication is poorly understood. We tested the hypothesis that extracellular hemin liberates an intracellular danger molecule that promotes acute organ failure in SCD. Transgenic homozygous SCD (SS), sickle-trait (AS) and normal human hemoglobin (Hb) AA mice were infused with purified hemin (35 µmoles/kg), which raised total plasma hemin by ∼0.45 mM (equivalent to 0.72 g/dl Hb) within 5 min in all three groups of mice. In agreement with our previous results, SS but not AA and AS mice (n= 6 for each genotype) developed cardiopulmonary depression at 30 min evident by reductions in oxygen saturation (99.88±0.23% to 92.1±1.3%, p<0.001), breath rate (175.4±20.6 to 77.36±2.25, p<0.001, breath per min), heart rate (574.5±22.7 to 361.9±23.25 beats per min, p<0.001) and pulse distension (512.8±18.7 to 238.8±17.6 µm, p<0.001), and ∼70% of these animals died within 2 hours. Markedly raised lung wet/dry weight ratio in SS mice that succumbed to hemin suggests that the cardiopulmonary depression was secondary to a severe pulmonary edema. To identify biological correlates for the acute adverse effects in the SS mice, cohorts of both sickle and control mice were challenged with the same dose of hemin, blood samples were drawn at baseline (i.e. time=0 min), and 5 and 30 min after the hemin infusion and analyzed for markers of oxidative stress, tissue damage, plasma scavengers and high mobility group box-1 (HMGB-1), a prototypical danger molecule. Plasma hemopexin decreased by ∼80% at 5 min compared to baseline values in all three groups of mice regardless of the Hb genotype. The catabolism of hemopexin was associated with clearance of ∼50% of the hemin infusion from the circulation of AS and AA mice at 30 min. Paradoxically, the plasma concentration of hemin in the SS mice during this same time interval increased by ∼0.2 mM (p<0.001, n=6). The magnitude of this increase was dependent on the dose of hemin administered exogenously. We discovered that the de novo hemin release in the SS mice was preceded by acute intravascular hemolysis (mean decrease in total Hb: ∼1.4 g/dl, p<0.001, n=9, mean increase in cell-free Hb: 1.0 g/dl, p=0.001, n=9), oxidation of oxyHbS to metHbS (mean increase: 12%, p<0.001, n=6) and persistence of metHbS. It is noteworthy that de novo hemin release did not occur in AS mice suggesting that this phenomenon is dominantly influenced by sickle erythrocytes and not by the presence of intracellular HbS per se. Auto-amplification of hemin may help to explain an observation made nearly fifty years ago that SCD patient plasma contains more hemin than the plasma of patients with more severe intravascular hemolysis involving normal adult Hb (e.g. paroxysmal nocturnal hemoglobinuria), who have higher plasma Hb. To determine whether this phenomenon is critical to the cardiopulmonary depression in the SS mice, recombinant human hemopexin was administered 5 min after the infusion to sequester the endogenous hemin release. In hemin challenged SS mice with respiratory distress, intravenous recombinant human hemopexin rapidly halted the decline in oxygen saturation and breath rate and averted inevitable respiratory failure. In conclusion, we have identified a phenomenon of extracellular hemin auto-amplification that appears to be unique to SCD, and may play a critical role in propagating tissue injury in this disorder. Factors that inhibit erythrocyte lysis and accelerate metHb reduction may help to limit extracellular hemin amplification and preserve organ function during episodes of acute exacerbations in SCD. Disclosures:No relevant conflicts of interest to declare.

Nitric Oxide Synthase 3 Deficiency Does Not Prevent Inflammatory Adverse Effects Of Transfusing Syngeneic Stored Blood In a Murine Model Of Hemorrhagic Shock

IntroductionBlood transfusion is a lifesaving treatment for hemorrhagic shock. During storage, red blood cells (RBC) undergo progressive deleterious functional, biochemical and structural alterations, which are collectively termed the “storage lesion”. The association between transfusion of blood stored for more than 14 days and adverse clinical outcomes (increased infection, multi-organ failure and mortality) is controversial. Studying mice with hemorrhagic shock, we recently found that resuscitation with blood stored for prolonged periods (SRBC) was associated with worse outcomes than was resuscitation with fresh blood (FRBC). The mechanisms responsible for the adverse effects associated with transfusion of SRBC are incompletely characterized. However, it is known that transfusion of SRBC increases plasma levels of hemoglobin (Hb), which can scavenge vascular nitric oxide (NO). Intravenous infusion of a solution containing cell-free Hb induces systemic hypertension in wild-type (WT) mice, but not in mice that are congenitally deficient in NO synthase 3 (NOS3-/-). In the present study, we sought to determine if NOS3-/- mice are protected from the adverse effects associated with resuscitation of hemorrhagic shock with SRBC. MethodsLeukoreduced, packed RBC from WT C57BL6 mice were stored with 14% CPDA-1 anticoagulant at 4°C for either ≤24 h (FRBC) or 2 weeks (SRBC). Mice, of each genotype that were not subjected to hemorrhagic shock or resuscitation, served as control groups. Anesthetized WT mice and NOS3-/- mice (on a C57BL6 background) were bled to a mean arterial pressure (MAP) of 40 mmHg over 10 min. After 90 min of hemorrhagic shock, mice were resuscitated with FRBC or SRBC. Survival rates for up to 7 days were determined. In addition, blood and tissue samples were collected at 4 h after resuscitation to measure plasma markers of liver and kidney injury, plasma Hb and interleukin 6 (IL-6) levels, tissue IL-6 mRNA levels, and pulmonary myeloperoxidase activity and mRNA levels. All data are expressed as mean±SD. ResultsBaseline MAP under anesthesia was higher in NOS3-/- mice than in WT mice (111±4 vs 83±5 mmHg; P<0.01). After hemorrhagic shock and resuscitation with either FRBC or SRBC, MAP returned to the respective baseline values in each genotype. Survival rates at one week did not differ between WT or NOS3-/- mice resuscitated with either FRBC or SRBC. In both genotypes, after hemorrhagic shock, resuscitation with FRBC elevated plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities similarly as compared to a control group (P<0.01). Plasma levels of ALT and AST were greater after resuscitation with SRBC than after resuscitation with FRBC in both genotypes at 4 h (P<0.01). Resuscitation with SRBC, but not FRBC, increased plasma blood urea nitrogen and creatinine levels similarly in WT and NOS3-/- mice. Plasma Hb levels were greater in mice resuscitated with SRBC than in those resuscitated with FRBC in both genotypes at 4 h after resuscitation (WT: 202±79 vs 5±2 µM, respectively; NOS3-/-: 240±51 vs 14±3 µM, respectively; P<0.001 for both). At 4 h after resuscitation, plasma IL-6 levels were greater in mice treated with SRBC than in mice treated with FRBC in both genotypes (WT: 0.8±0.1 vs 0.5±0.1 ng/ml, respectively; NOS3-/-: 0.8±0.1 vs 0.4±0.1 ng/ml; P<0.01 for both). In both WT and NOS3-/- mice, IL-6 mRNA levels were greater in the liver, kidney, and spleen after resuscitation with SRBC than after resuscitation with FRBC (P<0.01, all values differ SRBC vs FRBC for both genotypes). Pulmonary myeloperoxidase activity and mRNA levels were greater in both genotypes after resuscitation with SRBC than mice resuscitated with FRBC (P<0.01, all values differ SRBC vs FRBC for both genotypes). ConclusionsSurvival rate after hemorrhagic shock did not differ in WT and NOS3-/- mice resuscitated with either FRBC or SRBC. Resuscitation with SRBC induced greater tissue injury and a more marked inflammatory response than did resuscitation with FRBC, but there was no difference between the genotypes. Our data suggest that mice with NOS3 deficiency are not protected from the adverse effects associated with resuscitation of hemorrhagic shock with SRBC. These findings suggest that the adverse effects of transfusing blood stored for prolonged periods in mice with hemorrhagic shock are not exclusively attributable to scavenging of NOS3-generated NO by increased plasma Hb levels. Disclosures:No relevant conflicts of interest to declare.

Isoprostane and isofuran lipid mediators accumulate in stored red blood cells and influence platelet function in vitro

Stored red blood cells (RBCs) release hemoglobin (Hb) that leads to oxidative damage, which may contribute to thrombosis in susceptible transfusion recipients. Oxidative stress stimulates the generation of a new class of lipid mediators called F2 -isoprostanes (F2 -IsoPs) and isofurans (IsoFs) that influence cellular behavior. This study investigated RBC-derived F2 -IsoPs and IsoFs during storage and their influence on human platelets (PLTs). F2 -IsoP and IsoF levels in RBC supernatants were measured by mass spectrometry during storage and after washing. The effects of stored supernatants, cell-free Hb, or a key F2 -IsoP, 8-iso-prostaglandin F2α (PGF2α ), on PLT function were examined in vitro. F2 -IsoPs, IsoFs, and Hb accumulated in stored RBC supernatants. Prestorage leukoreduction reduced supernatant F2 -IsoPs and IsoFs levels, which increased again over storage time. Stored RBC supernatants and 8-iso-PGF2α induced PLT activation marker CD62P (P-selectin) expression and prothrombotic thromboxane A2 release. Cell-free Hb did not alter PLT mediator release, but did inhibit PLT spreading. Poststorage RBC washing reduced F2 -IsoP and IsoF levels up to 24 hours. F2 -IsoPs and IsoFs are produced by stored RBCs and induce adverse effects on PLT function in vitro, supporting a potential novel role for bioactive lipids in adverse transfusion outcomes. F2 -IsoP and IsoF levels could be useful biomarkers for determining the suitability of blood components for transfusion. A novel finding is that cell-free Hb inhibits PLT spreading and could adversely influence wound healing. Poststorage RBC washing minimizes harmful lipid mediators, and its use could potentially reduce transfusion complications.

Hemoglobin-Induced Endothelial Cell Permeability Is Controlled, in Part, via a Myeloid Differentiation Primary Response Gene–88–Dependent Signaling Mechanism

The release of hemoglobin (Hb) with hemolysis causes vascular dysfunction. New evidence implicates Hb-induced NF-κB and hypoxia inducible factor (HIF) activation, which may be under the control of a Toll-like receptor (TLR)-signaling pathway. Nearly all TLR-signaling pathways activate the myeloid differentiation primary response gene-88 (MyD88) that regulates NF-κB. We hypothesized that the differing transition states of Hb influence endothelial cell permeability via NF-κB activation and HIF regulation through a MyD88-dependent pathway. In cultured human dermal microvascular endothelial cells (HMECs-1), we examined the effects of Hb in the ferrous (HbFe(2+)), ferric (HbFe(3+)), and ferryl (HbFe(4+)) transition states on NF-κB and HIF activity, HIF-1α and HIF-2α mRNA up-regulation, and monolayer permeability, in the presence or absence of TLR4, MyD88, NF-κB, or HIF inhibition, as well as superoxide dismutase (SOD) and catalase. Our data showed that cell-free Hb, in each transition state, induced NF-κB and HIF activity, up-regulated HIF-1α and HIF-2α mRNA, and increased HMEC-1 permeability. The blockade of either MyD88 or NF-κB, but not TLR4, attenuated Hb-induced HIF activity, the up-regulation HIF-1 and HIF-2α mRNA, and HMEC-1 permeability. The inhibition of HIF activity exerted less of an effect on Hb-induced monolayer permeability. Moreover, SOD and catalase attenuated NF-κB, HIF activity, and monolayer permeability. Our results demonstrate that Hb-induced NF-κB and HIF are regulated by two mechanisms, either MyD88 activation or Hb transition state-induced ROS formation, that influence HMEC-1 permeability.

P58 Potential anti-atherogenic effects of hydrogen-sulfide releasing molecules

Introduction Oxidative modifications such as lipid peroxidation and hemoglobin (Hb) oxidation are important events in atherogenesis and can contribute to plaque progression. Hydrogen sulfide (H 2 S), the most recently discovered endogenous gaseous mediator is a reducing agent with the ability to prevent oxidative modification of low-density lipoprotein (LDL). Recently several H 2 S-releasing molecules have been introduced to deliver controlled and stable delivery of H 2 S to cells and tissues. In this work we compared the ability of different H 2 S releasing molecules to prevent lipid peroxidation and Hb oxidation; two key reactions contributing to atherosclerotic plaque progression. Methods NaHS, GYY, AP67 and AP72 were tested (2–200 μmol/L). Human LDL and Hb were isolated from human volunteers. Human Umbilical Vein Endothelial Cells (HUVEC) were isolated form umbilical cords. Results All the tested H 2 S releasing molecules inhibited heme-mediated LDL oxidation, although there were differences in their potential, being AP67 the most powerful. H 2 S releasing molecules were also able to prevent the lipid peroxidation of lipids derived from human soft plaques. During lipid peroxidation, lipid hydroperoxides are formed those are particularly toxic toward vascular endothelial cells. H 2 S releasing molecules prevented the accumulation of lipid hydroperoxides and reduced endothelial cytotoxicity. H 2 S donors also inhibited both H 2 O 2 and oxLDL-mediated Hb oxidation and thus the formation of pro-oxidant and pro-inflammatory Hb species. Among the tested H 2 S releasing molecules AP67 was the most potent in preventing Hb oxidation. Conclusion Interactions between cell-free Hb and plaque lipids provoke a vicious cycle promoting the oxidation of plaque lipids and Hb which in turn trigger endothelial activation and cytotoxicity. Both lipid peroxidation and Hb oxidation were potently inhibited by H 2 S releasing molecules. These data highlight the potential therapeutic application of these and related compounds in the prevention and/or treatment of atherosclerosis and endothelial dysfunction.

Hemoglobin induces inflammation after preterm intraventricular hemorrhage by methemoglobin formation

BackgroundCerebral intraventricular hemorrhage (IVH) is a major cause of severe neurodevelopmental impairment in preterm infants. To date, no therapy is available that prevents infants from developing serious neurological disability following IVH. Thus, to develop treatment strategies for IVH, it is essential to characterize the initial sequence of molecular events that leads to brain damage. In this study, we investigated extracellular hemoglobin (Hb) as a causal initiator of inflammation in preterm IVH.MethodsUsing a preterm rabbit pup model, we investigated the molecular mechanisms and events following IVH. We also characterized the concentrations of cell-free Hb metabolites and pro-inflammatory mediators in the cerebrospinal fluid (CSF) of preterm human infants and rabbit pups. Finally, Hb metabolites were evaluated as causal initiators of inflammation in primary rabbit astrocyte cell cultures.ResultsFollowing IVH in preterm rabbit pups, the intraventricular CSF concentration of cell-free methemoglobin (metHb) increased from 24 to 72 hours and was strongly correlated with the concentration of TNFα at 72 hours (r2 = 0.896, P <0.001). Also, the mRNA expression of TNFα, IL-1β, and Toll-like receptor-4 and TNFα protein levels were significantly increased in periventricular tissue at 72 hours, which was accompanied by extensive astrocyte activation (that is, glial fibrillary acidic protein (GFAP)staining). Furthermore, exposure of primary rabbit astrocyte cell cultures to metHb caused a dose-dependent increase in TNFα mRNA and protein levels, which was not observed following exposure to oxyhemoglobin (oxyHb) or hemin. Finally, a positive correlation (r2 = 0.237, P <0.03) between metHb and TNFα concentrations was observed in the CSF of preterm human infants following IVH.ConclusionsFollowing preterm IVH, increased metHb formation in the intraventricular space induces expression of pro-inflammatory cytokines. Thus, the formation of metHb might be a crucial initial event in the development of brain damage following preterm IVH. Accordingly, removal, scavenging, or neutralization of Hb could present a therapeutic opportunity and plausible approach to decreasing the damage in the immature brain following preterm IVH.

Can haptoglobin attenuate hemoglobin‐induced vascular damage?

Hemolytic diseases and infections can lead to intravascular hemolysis, which causes increased plasma hemoglobin (Hb) concentration. Over time, this “cell free Hb” is toxic to the blood vessels, exerting oxidative damage, nitric oxide (NO) scavenging, or inflammatory responses within the vasculature. The exposure of increased Hb on the lung tissue can lead to pulmonary hypertension (PH), which is high blood pressure in the pulmonary arteries due to changes in arterial wall. Haptaglobin is an acute phase protein responsible for binding and clearance of Hb. We hypothesized that haptoglobin (Hp) can attenuate PH secondarily to vascular hemoglobin exposure. To test our hypothesis, Hb was chronically administered to rats at physiologically relevant concentrations as observed in the sickle cell disease (SCD) (0.2 μmole/day) for 5 weeks. To further mimic the SCD process, rats were placed in hypobaric hypoxic chambers (~17,000 ft). Animals were divided into 4 groups; (1) Control (sham), (2) Hb infusion, (3) Hp injections (biweekly), and (4) Hb: Hp (chronically infused with Hb treated biweekly with Hp). At the end of the 5 weeks, we evaluated pulmonary blood pressure, cardiac output, and pulmonary vascular wall thickness. Additionally, lung analysis for indicators of endothelial nitric oxide synthase (eNOS) and intercellular adhesion molecule 1 (ICAM‐1) expression were performed. Preliminary data demonstrates that groups infused with haptoglobin had lower pulmonary blood pressure, decreased vessel wall thickness, and decreased ICAM‐1 expression. We observed no difference in eNOS expression between groups. This suggests that Hb induced inflammation may be attenuated by Hp.

P40: Platelet activation in the presence of nitrite, nitric oxide and heme

Background NO stimulates intercellular cGMP production leading to a reduction in platelet activation. Nitrite is reduced to nitric oxide (NO) by deoxygenated hemoglobin and therefore acts as a potential source of biological NO. Nitrite in the presence of red blood cells (RBCs) has been shown to reduce platelet aggregation and this reduction in aggregation is enhanced when RBCs are deoxygenated. In addition nitrite in the presences of deoxygenated RBCs has been shown to reduce platelet expression of p-selectin when platelets are activated by ADP. Methods Platelet rich plasma was separated from whole blood by centrifugation at 120 g for 10 min. Plasma was diluted 10-fold into phosphate buffered saline and exposed to nitrite, NO, and/or RBC, as indicated. Platelets were then activated by ADP After 10 min of incubation with ADP, platelets were labeled with fluorescent antibodies for platelet recognition (CD41 or CD61) and activation (Pac-1 or CD62P) and measured using flow cytometry. Results Here we confirm NO reduces platelet activation. However, in the presence of oxyhemoglobin (Hb) NO no longer reduces platelet activation. MetHb, which does not rapidly scavenge NO does not affect platelet activation reduction by NO. In our research we see similar trends in decreased platelet activation with increasing concentrations of nitrite in the presences on RBCs. However our results are not as sensitive to nitrite concentrations as previously published data. In addition we see trends of decreasing platelet activation with increasing nitrite concentration in the presence of Hb. Conclusions Our results verify the effect of NO on platelet activation. They also show a trend which agrees with previously published data showing a decrease in platelet activation in the presence of nitrite and red blood cells. Additionally we see the same trend in the presence of nitrite and cell free Hb. Disclosure Supported by NIH Grants HL058091 and HL098032.

Hemoglobin infusion does not alter murine pulmonary vascular tone

Plasma hemoglobin (Hb) scavenges endothelium-derived nitric oxide (NO), producing systemic and pulmonary vasoconstriction in many species. We hypothesized that i.v. administration of murine cell-free Hb would produce pulmonary vasoconstriction and enhance hypoxic pulmonary vasoconstriction (HPV) in mice.To assess the impact of plasma Hb on basal pulmonary vascular tone in anesthetized mice we measured left lung pulmonary vascular resistance (LPVRI) before and after infusion of Hb at thoracotomy. To confirm the findings obtained at thoracotomy, measurements of right ventricular systolic pressure (RVSP) and systemic arterial pressure (SAP) were obtained in closed-chest wild-type mice. To elucidate whether pretreatment with Hb augments HPV we assessed the increase in LPVRI before and during regional lung hypoxia produced by left mainstem bronchial occlusion (LMBO) in wild-type mice pretreated with Hb.Infusion of Hb increased SAP but did not change pulmonary arterial pressure (PAP), left lung pulmonary arterial flow (QLPA) or LPVRI in either wild-type or diabetic mice with endothelial dysfunction. Scavenging of NO by plasma Hb did not alter HPV in wild-type mice. Inhibition of NO synthase with l-NAME did not change the basal LPVRI, but augmented HPV during LMBO.Our data suggest that scavenging of NO by plasma Hb does not alter pulmonary vascular tone in mice. Therefore, generation of NO in the pulmonary circulation is unlikely to be responsible for the low basal pulmonary vascular tone of mice.

Atherogenesis May Involve the Prooxidant and Proinflammatory Effects of Ferryl Hemoglobin

Oxidized cell-free hemoglobin (Hb), including covalently cross-linked Hb multimers, is present in advanced atherosclerotic lesions. Oxidation of Hb produces methemoglobin (Fe3+) and ferryl hemoglobin (Fe4+ = O2−). Ferryl iron is unstable and can return to the Fe3+ state by reacting with specific amino acids of the globin chains. In these reactions globin radicals are produced followed by termination reactions yielding covalently cross-linked Hb multimers. Despite the evanescent nature of the ferryl state, herein we refer to this oxidized Hb as “ferryl Hb.” Our aim in this work was to study formation and biological effects of ferrylHb. We demonstrate that ferrylHb, like metHb, can release its heme group, leading to sensitization of endothelial cells (ECs) to oxidant-mediated killing and to oxidation of low-density lipoprotein (LDL). Furthermore, we observed that both oxidized LDL and lipids derived from human atherosclerotic lesions trigger Hb oxidation and subsequent production of covalently cross-linked ferrylHb multimers. Previously we showed that ferrylHb disrupts EC monolayer integrity and induces expression of inflammatory cell adhesion molecules. Here we show that when exposed to ferrylHb, EC monolayers exhibit increased permeability and enhanced monocyte adhesion. Taken together, interactions between cell-free Hb and atheroma lipids engage in a vicious cycle, amplifying oxidation of plaque lipids and Hb. These processes trigger EC activation and cytotoxicity.

Natural History of the Bruise: Formation, Elimination, and Biological Effects of Oxidized Hemoglobin

Numerous disease states are associated with hemolysis or hemorrhage. Because red cells in the extravascular space tend to lyse quickly, hemoglobin (Hb) is released and is prone to autoxidation producing MetHb. Inorganic and organic peroxides may convert Hb and MetHb to higher oxidation states such as ferrylHb. FerrylHb is not a single chemical entity but is a mixture of globin- and porphyrin-centered radicals and covalently cross-linked Hb multimers. Oxidized Hb species are potent prooxidants caused mainly by heme release from oxidized Hb. Moreover, ferrylHb is a strong proinflammatory agonist that targets vascular endothelial cells. This proinflammatory effect of ferrylHb requires actin polymerization, is characterized by the upregulation of proinflammatory adhesion molecules, and is independent of heme release. Deleterious effects of native Hb are controlled by haptoglobin (Hp) that binds cell-free Hb avidly and facilitates its removal from circulation through the CD163 macrophage scavenger receptor-mediated endocytosis. Under circumstances of Hb oxidation, Hp can prevent heme release from MetHb, but unfortunately the Hp-mediated removal of Hb is severely compromised when Hb is structurally altered such as in ferrylHb allowing deleterious downstream reactions to occur even in the presence of Hp.