Hemichrome Formation Research Articles

Reactive Oxygen Species and Their Involvement in Red Blood Cell Damage in Chronic Kidney Disease.

Reactive oxygen species (ROS) released in cells are signaling molecules but can also modify signaling proteins. Red blood cells perform a major role in maintaining the balance of the redox in the blood. The main cytosolic protein of RBC is hemoglobin (Hb), which accounts for 95-97%. Most other proteins are involved in protecting the blood cell from oxidative stress. Hemoglobin is a major factor in initiating oxidative stress within the erythrocyte. RBCs can also be damaged by exogenous oxidants. Hb autoxidation leads to the generation of a superoxide radical, of which the catalyzed or spontaneous dismutation produces hydrogen peroxide. Both oxidants induce hemichrome formation, heme degradation, and release of free iron which is a catalyst for free radical reactions. To maintain the redox balance, appropriate antioxidants are present in the cytosol, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and peroxiredoxin 2 (PRDX2), as well as low molecular weight antioxidants: glutathione, ascorbic acid, lipoic acid, α-tocopherol, β-carotene, and others. Redox imbalance leads to oxidative stress and may be associated with overproduction of ROS and/or insufficient capacity of the antioxidant system. Oxidative stress performs a key role in CKD as evidenced by the high level of markers associated with oxidative damage to proteins, lipids, and DNA in vivo. In addition to the overproduction of ROS, a reduced antioxidant capacity is observed, associated with a decrease in the activity of SOD, GPx, PRDX2, and low molecular weight antioxidants. In addition, hemodialysis is accompanied by oxidative stress in which low-biocompatibility dialysis membranes activate phagocytic cells, especially neutrophils and monocytes, leading to a respiratory burst. This review shows the production of ROS under normal conditions and CKD and its impact on disease progression. Oxidative damage to red blood cells (RBCs) in CKD and their contribution to cardiovascular disease are also discussed.

Mechanisms involved in the inhibitory effects of free fatty acids on lipid peroxidation in turkey muscle

The pro-oxidant and anti-oxidant effects of free fatty acids (FFA) in meat remain ambiguous. To clarify the role of FFA in lipid oxidation of muscle food, the FFA was added into two systems (turkey mince and washed turkey muscle (WTM)) and lipid oxidation was investigated. A mixture of FFA inhibited lipid oxidation in both systems. The absorbance spectrum of oxyHb and metHb was determined in the presence of C18:1 and C18:2. Conversion to hemichrome spectra was observed and was particularly rapid when metHb was mixed with C18:2. C18:2 was also reacted with metHb followed by a chromatography step to remove unbound C18:2, and termed ‘modified Hb’. Electron spin resonance (ESR) spectra of the modified Hb was indicative of hemichrome formation. The modified Hb did not promote lipid oxidation in washed turkey muscle during storage. This suggested that hemichrome formation due to added FFA diminished the lipid oxidation capacity of Hb.

Methemoglobin determination by multi-component analysis in coho salmon (Oncorhynchus kisutch) possessing unstable hemoglobin

Hemoglobin derivatives are often quantified in blood to establish cardio-respiratory status and possible causes of impaired oxygen transport. The derivative known as methemoglobin results from oxidation of hemoglobin and is pathologically relevant because it cannot transport oxygen. In species and individuals possessing unstable methemoglobin, methemoglobin formation leads to rapid hemichrome formation and precipitation. Oxidizing reagents in standard methemoglobin analysis techniques therefore prevent accurate quantification of hemoglobin oxidative degradation products in species possessing unstable hemoglobin. In this study, we demonstrated that individual coho salmon (Oncorhynchus kisutch) possess unstable methemoglobin. Because molar absorptivities of coho methemoglobin, hemichrome and carboxyhemoglobin were significantly different from humans, the use of previous standard methods leads to an overestimation of methemoglobin in coho. Spontaneous conversion of methemoglobin to hemichrome was also demonstrated in Chinook (O. tshawytscha), pink (O. gorbuscha) and chum salmon (O. keta), but not steelhead (O. mykiss), indicating there may be a frequent need to account for unstable hemoglobin when quantifying methemoglobin in salmonids.•Our method builds upon multi-component analysis (MCA) by using a multivariate modeling technique to derive the coho-specific molar absorptivities of major hemoglobin derivatives•This approach fills a current need for the accurate quantification of methemoglobin in fishes possessing unstable hemoglobin

Ligand accessibility to heme cytochrome b5 coordinating sphere and enzymatic activity enhancement upon tyrosine ionization.

Recently, we observed that at extreme alkaline pH, cytochrome b5 (Cb5) acquires a peroxidase-like activity upon formation of a low spin hemichrome associated with a non-native state. A functional characterization of Cb5, in a wide pH range, shows that oxygenase/peroxidase activities are stimulated in alkaline media, and a correlation between tyrosine ionization and the attained enzymatic activities was noticed, associated with an altered heme spin state, when compared to acidic pH values at which the heme group is released. In these conditions, a competitive assay between imidazole binding and Cb5 endogenous heme ligands revealed the appearance of a binding site for this exogenous ligand that promotes a heme group exposure to the solvent upon ligation. Our results shed light on the mechanism behind Cb5 oxygenase/peroxidase activity stimulation in alkaline media and reveal a role of tyrosinate anion enhancing Cb5 enzymatic activities on the distorted protein before maximum protein unfolding.

Correcting Non-Transfusion Dependent β-Thalassemia by Utilizing a Combined Therapy that Modulates EPO Activity by Limiting Erythroid Cellular Iron Intake

Β-thalassemia intermedia is a disorder characterized by ineffective erythropoiesis (IE), anemia, splenomegaly and systemic iron overload. Administration of agents that increase erythropoiesis (such as erythropoietin-Epo) can potentially improve anemia by enhancing the production of red blood cells (RBC). However, this improvement will likely result in exacerbation of splenomegaly and worsening of iron overload due to further suppression of hepcidin, the hormone that controls dietary iron absorption. However, we postulated that, following Epo administration, some level of iron restriction will limit the splenomegaly and iron overload without preventing the improvement of anemia.To increase circulating Epo levels and increase erythropoiesis, we infused animals with primary dermal fibroblasts transduced with a helper dependent adenoviral vector carrying the Epo gene (TARGTEpo). Pharmacokinetic studies indicated that 1e6 cells were sufficient to significantly increase Epo synthesis (up to 47.6%in Hbbth3/+ and 90% in WT). This led to an increase in hemoglobin levels in both mouse models (average of 3-4 g/dL) through the end of the treatment (6 weeks). In Hbbth3/+ mice, Hb levels rose to 10.7±1.0 g/dL in the first week and remained elevated through the end of the 6-weeks of treatment (compared to 7.7±0.7 g/dL in controls). As expected, stimulation of erythropoiesis led to worsening of splenomegaly and suppression of hepcidin.To test our hypothesis, we then combined TARGTEpo with low iron diet or antisense oligonucleotides targeting Tmprss6 mRNA (Tmprss6-ASO; the last treatment only for Hbbth3/+ animals). It has already been shown that inhibition of Tmprss6 increases hepcidin expression leading to decreased iron absorption and limited erythroid iron intake, improving anemia, IE, splenomegaly and iron overload in Hbbth3/+ mice (Guo et al, JCI, 2013).In WT animals, after three weeks, the combination of TARGTEpo with low iron diet significantly reduced Hb levels (-40%), RBC number (-38%) and reticulocytes (-80%), when compared to animals overexpressing Epo and receiving a normal iron diet. In contrast, Hbbth3/+ animals on an iron-deficient diet or treated with Tmprss6-ASO in the presence of TARGTEpo showed decreased hemichrome formation and improved anemia through the end of treatment. In particular, Hb levels increased (on average 4 g/dL), reaching levels of 11-12g/dl, corresponding to +36% more than baseline levels in Hbbth3/+ (7.7g/dL) and +25% more compared to Hbbth3/+ mice treated with Tmprss6-ASO alone (9.0g/dL). Animals receiving TARGTEpo with Tmprss6-ASO showed a significant reduction in splenomegaly when compared to baseline levels (-35%) and to animals treated only with TARGTEpo (-52.4%). Preliminary data indicate that organ iron concentration improved or normalized compared to control Hbbth3/+ mice when iron restriction was applied.We then inquired how these two treatments combined improved erythropoiesis. We quantified the number of nucleated erythroid cells positive for the phosphorylated form of pAkt and pJak2, proteins activated by Epo but whose activity is potentially limited by iron restriction. For pAkt+ cells in the spleen, the number of proerythroblasts were increased in Hbbth3/+ treated with TARGTEpo (+35% compared to control Hbbth3/+ mice), while they were reduced in Hbbth3/+ treated with Tmprss6-ASO or TARGTEpo+Tmprss6-ASO (-72% and -43% respectively, compared to control Hbbth3/+ mice). Other erythroid populations showed a comparable profile for both pAkt+ and pJak2+ nucleated erythroid cells. The total number of progenitor erythroid cells (i.e. BFUe) also followed a similar trend. Cell cycle analysis of splenic erythroid progenitor cells showed a decrease in the percentage of nucleated erythroid cells in mice treated with Tmprss6-ASO or TARGTEpo+Tmprss6-ASO compared to control or TARGTEpo-treated Hbbth3/+ mice. Given that the number of RBC in circulation was highest in mice treated with TARGTEpo+Tmprss6-ASO, this data suggests that co-administration of these two drugs maximize the ability of each erythroid cell to develop into a functional RBC.In conclusion, we propose that in β-thalassemia, combination of erythropoietic stimulating agents with iron restriction could maximize correction of anemia while improving splenomegaly and iron overload. DisclosuresCasu:Aevi Genomic Medicine, Inc: Research Funding; Ionis Pharmaceuticals, Inc.: Research Funding. Aghajan:Ionis Pharmaceuticals, Inc: Employment. Neil:Aevi Genomic Medicine, Inc: Employment, Other: Officer and share holder. Guo:Ionis Pharmaceuticals, Inc: Employment. Rivella:Ionis Pharmaceuticals, Inc: Consultancy; MeiraGTx: Other: SAB; Disc Medicine: Consultancy; Protagonist: Consultancy.

Peroxidase-like activity of cytochrome b5 is triggered upon hemichrome formation in alkaline pH

In alkaline media (pH12) a catalytic peroxidase activity of cytochrome b5 was found associated to a different conformational state. Upon incubation at this pH, cytochrome b5 electronic absorption spectrum was altered, with disappearance of characteristic bands of cytochrome b5 at pH7.0. The appearance of new electronic absorption bands and EPR measurements support the formation of a cytochrome b5 class B hemichrome with an acquired ability to bind polar ligands. This hemichrome is characterized by a negative formal redox potential and the same folding properties than cytochrome b5 at pH7. The acquired peroxidase-like activity of cytochrome b5 found at pH12, driven by a hemichrome formation, suggests a role of this protein in peroxidation products propagation.

Electrochemical behavior of immobilized hemoglobin in alkaline solution

Glassy carbon electrode was modified with different synthesized hybrid clay-based materials and tested in alkaline solution with and without H2O2. The hybrid materials were obtained by immobilizing hemoglobin (Hb) on acid activated (AA) clay, or on AA clay modified with different sodium dodecyl sulfate (SDS) loadings. The obtained materials were characterized using DR UV–vis and ESR spectroscopy, elemental analysis, and SEM. The characterization confirmed higher degree of hemoglobin incorporation in the presence of SDS. The presence of SDS on the surface of clay particles resulted in the partial oxidation/denaturation of hemoglobin and formation of hemichrome. Cyclic voltammetry was used for the investigation of the electrochemical behavior of immobilized hemoglobin in alkaline solution. Two cathodic peaks at −0.45V and −0.70V were recorded and ascribed to the reduction of heme Fe(III)/Fe(II), and formation of HbFe(I) – highly reduced form of hemoglobin − respectively. The latter peak reflects hemoglobin denaturation. The presence of H2O2 in the alkaline solution increased current intensities corresponding to both peaks (−0.45V and −0.7V). Linear response of peak current intensity vs. H2O2 concentration was monitored for all investigated samples within different H2O2 concentration ranges. The AA-SDS1.0-Hb electrode exhibited the highest current response with linear regression equation in the following form: I(μA)=7.99+1.056×[H2O2] (mM) (R=0.996). The limit of detection of 28μM was estimated using the 3 sigma method.Different modified electrodes exhibited different degrees of denaturation resistance. The obtained values of Michaelis-Menten constant indicated that prolonged cycling in the presence of SDS increases protein denaturation.

Administration of Minihepcidins to Animals Affected By β-Thalassemia Major Reduces Anemia and Splenomegaly

Both β-thalassemia intermedia and major are characterized by formation of hemichromes in erythroid cells, impairing their survival and the lifespan of red blood cells (RBC). Minihepcidins (MH) are novel compounds that function as hepcidin agonists and reduce iron absorption and transferrin saturation. Hbbth3/+ mice show features of β-thalassemia intermedia, such as ineffective erythropoiesis (IE), anemia and reduced hepcidin synthesis, but do not require blood transfusion for survival (non-transfusion dependent thalassemia or NTDT). As we have previously shown, the administration of MH in these animals decreased transferrin saturation, erythroid iron intake, heme synthesis and hemichrome formation, with a significant beneficial effect on RBC quality, lifespan and anemia (Casu et al, Blood 2016). In order to test if this approach could also benefit animals affected by β-thalassemia major we focused on generating a model that exhibited a low production of RBCs, severe anemia and a blood transfusion requirement for survival, as in patients affected by transfusion dependent thalassemia or TDT. We have previously shown that engraftment of Hbbth3/th3fetal liver cells (FLCs) into normal mice leads to a very severe anemia that requires blood transfusion for survival (Gardenghi et al, Blood 2007). However, Hbbth3/th3FLCs do not contain any adult or fetal-globin genes and are unable to make hemoglobin in the transplanted animals, in contrast to human β-thalassemia. Therefore, animals cannot benefit from therapies that decrease hemichrome formation and target IE such as MH. To overcome this limitation, we crossed Hbbth3/+ mice with additional models of NTDT, indicated as Hbbth1/th1 and Hbbth2/+. These mice harbor alternative mutations so that the synthesis of the mouse b-globin genes is different in each model. Hbbth1/th2 and Hbbth1/th3 pups were alive at birth, but unable to survive more than a couple of days even with the support of blood transfusion. However, recipient transgenic animals expressing GFP and engrafted with Hbbth1/th2andHbbth1/th3FLCs showed the desired phenotype 3 months post-transplant including production of GFP- RBCs (with less than 2% of host GFP+ RBC) and a different degree of anemia, respectively 5.6±0.5 g/dL and 3.1±1.5 g/dL. In the long term these animals require blood transfusion for survival. Therefore these models are useful to test drugs that have the potential to modify erythropoiesis and RBC production. Ten weeks following engraftment with Hbbth1/th2 FLCs, mice were treated for six weeks with two different doses of MH (5.25 mg/kg and 2.625 mg/kg administered every other day) in absence of blood transfusion. Animals treated with vehicleshowed severe ineffective erythropoiesis and worsening anemia over 6 weeks (from 5.6±0.5 g/dL on D0 to 5.0±0.7 g/dL on D42 of treatment). In contrast, animals treated with MH showed reversal of anemia at 3 weeks (6.6±0.3 g/dL and 6.1±0.6 g/dL in the 5.25 mg/kg and 2.625 mg/kg group, respectively, compared to 5.3±0.9 g/dL in controls), while at 6 weeks the differences were reduced compared to vehicle treated mice (6.0±0.4 g/dL and 5.7±0.5 g/dL in the 5.25 mg/kg and 2.625 mg/kg group, respectively, compared to 4.9±0.7 g/dL in controls). The RBC number followed the same trend. Furthermore, the RBC morphology of animals treated with MH was improved compared to control animals. At 6 weeks, splenomegaly was also improved in the treatment groups (13.8±2.7 mg and 16.9±2.7 mg respectively in the 5.25 mg/Kg and 2.625 mg/Kg group compared to 26.9±3.5 mg in controls). Comparing the data at 3 versus 6 weeks, we speculate that, while the MH has a positive effect on RBC quality and production, this is insufficient, in the long term, to prevent the severe splenomegaly and the consequent entrapment of the RBC, which exacerbates the anemia over time. However, we hypothesized that administration of MH could have longer lasting beneficial effects in presence of blood transfusion, which would limit the splenomegaly. Presently, we are testing this hypothesis using both the Hbbth1/th2 and Hbbth1/th3 models. Complete characterization of these models and their parameters (CBC, erythropoiesis, iron metabolism and organ iron content) is in progress. In conclusion, these models can be utilized to characterize severe thalassemia phenotypes and new drugs that have the potential to ameliorate IE and improve RBCs generation. DisclosuresMacDonald:Merganser: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Ex Vivo Gene Therapy Approach By Targt Technology for the Treatment of β-Thalassemia Intermedia

β-Thalassemia intermedia or non-transfusion dependent thalassemia (NTDT) is a genetic blood disorder characterized by ineffective erythropoiesis (IE), anemia, splenomegaly and systemic iron overload mediated by down-regulation of hepcidin. Using Hbbth3/+animals affected by NTDT, we have shown that iron restriction improves anemia and reduces splenomegaly by reducing the formation of hemichromes and improving the lifespan of red blood cells (RBC; Gardenghi et al, JCI, 2010; Guo et al, JCI, 2013; Casu et all Blood 2016). These findings emphasize the strong correlation between erythropoiesis, RBC synthesis and iron metabolism. In theory, agents that can stimulate erythropoiesis (such as erythropoietin or EPO) could improve anemia by increasing the production of RBC and, under conditions of reduced iron absorption, correct the iron overload by consuming the excess of iron stored in organs. Here we used a new ex vivo technology called TARGT (Transduced Autologous Restorative Gene Therapy) for prolonged production and secretion of therapeutic agents. Primary dermal fibroblasts transduced with helper dependent adenovirus expressing mouse Epo were embedded in Matrigel and implanted in the dorsal area of 2-3 months old Hbbth3/+and wild-type (WT) C57BL/6 mice. Three groups were generated for each genotype using different amounts of genetically modified fibroblasts (10e5, 5x10e5 and 1x10e6 transduced cells), while one group received no transduced fibroblasts as control. Complete blood count and serum harvest for Epo levels measurement were performed one week after implantation and then every ten days until the end of the experiment, at six weeks. In WT mice, already one week after fibroblasts implantation, hemoglobin (Hb) levels raised 2/3 g/dL, showing erythrocytosis. At the end of the treatment animals showed significantly increased Hb levels (18.5±1.7 g/dL, 19.4±0.5 g/dL and 19.8±0.8 g/dL in the groups implanted with 10e5, 5x10e5 and 1x10e6 fibroblasts, respectively, compared to 13.5±0.8 g/dL in controls). RBC numbers and HTC follow the same trend, showing increases in the range of 27% to 39%.In Hbbth3/+mice Hb levels rose about 3 g/dl already one week after fibroblasts implantation and remained elevated until the end experiment (8.7±0.3 g/dL, 10.7±1.0 g/dL and 10.6±1.8 g/dL in the groups implanted with 10e5, 5x10e5 and 1x10e6 fibroblasts, respectively, compared to 7.7±0.7 g/dL in controls). RBC numbers and HTC follow the same trend, showing increases in the range of 7% to 27%. At the end of the treatment, serum Epo levels were markedly increased at all doses both in Hbbth3/+and WT animals (more than 50% and up to 90%) when compared with their respective controls.However, as expected, stimulation of erythropoiesis led to worsening of splenomegaly and suppression of hepcidin, likely preventing the beneficial effect of erythroid-mediated consumption of stored iron in Hbbth3/+animals. Here we postulated that, in presence of agents that increase erythropoiesis such as Epo, some level of iron restriction is required to improve the anemia, prevent exacerbation of the splenomegaly and, concurrently, decreased iron overload. As a proof of principle of the ability of this technology to improve anemia and decrease iron overload, TARGTEPO was combined with low iron diet or iron chelation. We expect that decreased iron intake will improve the quality of the RBC, while the larger number of RBC produced should utilize more stored iron, reversing the iron overload in Hbbth3/+animals. Preliminary results at three weeks from the beginning of the experiment indicated that in WT implanted-animals the combination of TARGTEPO with low iron diet significantly reduced Hb levels (-40%), HTC (-42%), RBC number (-38%) and reticulocytes (-80%) when compared to animals overexpressing Epo and receiving normal iron diet or iron chelation. This indicates that in WT animals, after 3 weeks, the stored iron is insufficient to support the increased erythropoiesis. In contrast, in Hbbth3/+animals this drop in Hb was not seen, suggesting that this effect is delayed due to the highest level of stored iron. Complete characterization of these models and their parameters is in progress. These preliminary results suggest that the TARGTplatformcould be utilized, in combination with drugs that limit iron intake, to improve anemia and decrease iron overload in NTDT. Additional experiments are in progress to test this hypothesis. DisclosuresCasu:Ionis Pharmaceuticals: Research Funding; Medgenics LLC: Research Funding; Merganser Biotech: Research Funding. Neil:Medgenics: Employment. Miari:Medgenics: Employment. Shapir:Medgenics: Employment.

Interactions between peroxiredoxin 2, hemichrome and the erythrocyte membrane

Peroxiredoxin 2 (Prx2) is an abundant antioxidant protein in erythrocytes that protects against hemolytic anemia resulting from hemoglobin oxidation and Heinz body formation. A small fraction of Prx2 is bound to the cell membrane, but the mechanism and relevance of binding are not clear. We have investigated Prx2 interactions with the erythrocyte membrane and oxidized hemoglobin and whether these interactions are dependent on Prx2 redox state. Membrane binding of Prx2 in erythrocytes decreased when the cells were treated with H2O2, but studies with purified Prx2 and isolated ghosts showed that the interaction was independent of Prx2 redox state. Hemoglobin oxidation leads to the formation of hemichrome, a denatured form of the protein that binds to Band3 protein in the cell membrane as part of the senescence process and is a precursor of Heinz bodies. Hemichrome competed with Prx2 and decreased Prx2 binding to the membrane, potentially explaining the decreased binding in oxidant-exposed cells. The increased membrane binding of Prx2 seen with increasing intracellular calcium was less sensitive to H2O2 or hemichrome, suggesting an alternative mode of binding. Prx2 was also shown to exhibit chaperone-like activity by retarding the precipitation of pre-formed hemichrome. Our results suggest that Prx2, by restricting membrane binding of hemichrome, could impede Band3 clustering and exposure of senescence antigens. This mechanism, plus the observed chaperone activity for oxidized hemoglobin, may help protect against hemolytic anemia.

Combination of Tmprss6-ASO and the Iron Chelator Deferiprone Improves Erythropoiesis and Reduces Iron Overload in a Mouse Model of Beta-Thalassemia

Patients affected by non-transfusion dependent thalassemia (NTDT) do not require chronic blood transfusion for survival. However, transfusion-independence in such patients is not without side effects. Ineffective erythropoiesis (IE), the hallmark of disease, leads to a variety of serious clinical morbidities. In NTDT the master regulator of iron homeostasis, hepcidin, is chronically repressed. Consequently, patients absorb abnormally high levels of iron, which eventually requires iron chelation to prevent the clinical sequelaes associated with iron overload. It has been shown that in mice affected by NTDT (Hbbth3/+), a second-generation antisense oligonucleotide (Tmprss6-ASO) can reduce expression of transmembrane serine protease Tmprss6, the major suppressor of hepcidin expression. This leads to reduction of hemichrome formation in erythroid cells, amelioration of IE and splenomegaly, and increased hemoglobin levels (Guo et al, JCI, 2013). Now we propose the use of Tmprss6-ASO in combination with iron chelators for the treatment of NTDT using Hbbth3/+ mice as a preclinical model. Our hypothesis is that use of chelators will benefit from the positive effect of Tmprss6-ASO on erythropoiesis and iron absorption, further ameliorating organ iron content. To this end, Hbbth3/+ animals were treated with Tmprss6-ASO at 100 mg/kg/week for 6 weeks with or without the iron chelator deferiprone (DFP) at a dose of 1.25 mg/ml. Additional animals were treated with DFP alone. We fed the animals with a commercial or physiological diet, containing 200 or 35 ppm of iron, respectively.We did not observe major differences in the treated animals fed the commercial or physiological iron diet and, for this reason, the data were combined for simplicity. Administration of DFP alone was successful in decreasing organ iron content. Compared to untreated Hbbth3/+ animals, we observed a reduction of 30% and 33% in the liver and spleen, respectively, and no change in the kidney. However, erythropoiesis was not improved (looking at IE, splenomegaly, RBC production and total Hb levels). This was associated with increased serum iron levels (+25%). In Tmprss6-ASO treated Hbbth3/+ animals, we observed an improvement in liver iron content (36% reduction), amelioration of IE, and increased RBC and Hb synthesis (~2 g/dL). Compared to treatment with Tmprss6-ASO alone, combination of DFP with Tmprss6-ASO achieved the same level of suppression of Tmprss6 in the liver (~90%) and reduction of serum iron parameters. This was associated with improvement of IE, decreased reticulocyte counts and splenomegaly, and increased RBC and Hb synthesis (~2 g/dL). While we observed that both Tmprss6-ASO and DFP separately reduced liver iron content to the same extent (~30-36%), combination treatment further reduced iron concentrations in the liver and kidney (69% and 19%, respectively), with no changes in the spleen. Additional analyses are in progress to evaluate the amount of hepcidin in serum as well as expression of erythroferrone, the erythroid regulator of hepcidin.Our first conclusion is that administration of an iron chelator alone is not sufficient to improve erythropoiesis despite that organ iron content is reduced. We speculate that when iron is removed from the liver, hepcidin expression becomes more susceptible to the suppressive effect of IE rather than the enhancing effect of reduced liver organ iron concentration. In addition, the combined effect of iron mobilized from organs and unchanged (or even augmented) iron absorption leads to increased serum iron concentration. As we have shown previously, amelioration of IE in this model requires decreased erythroid iron intake and hemichrome formation. Therefore, iron chelation alone is likely insufficient to improve erythropoiesis. Additional experiments are in progress to further elucidate this mechanism.Our second conclusion is that use of Tmprss6-ASO together with DFP combines the best effects of these two drugs, in particular on erythropoiesis and organ iron content. In animals that received the combined treatment, kidney and liver iron concentrations were further decreased compared to the single treatments. This indicates that Tmprss6-ASO might be extremely helpful in the treatment of NTDT and it could further improve iron related-chelation therapies. DisclosuresCasu:Merganser Biotech LLC: Employment; Isis Pharmaceuticals, Inc.: Employment. Aghajan:Isis Pharmaceuticals, Inc.: Employment. Guo:Isis Pharmaceuticals, Inc.: Employment. Monia:Isis Pharmaceuticals, Inc.: Employment. Rivella:bayer: Consultancy, Research Funding; isis Pharmaceuticals, Inc.: Consultancy, Research Funding; merganser Biotech LLC: Consultancy, Research Funding, Stock options , Stock options Other.

High‐resolution Raman imaging reveals spatial location of heme oxidation sites in single red blood cells of dried smears

Raman confocal microscopy with 488 nm excitation wavelength supported with atomic force microscopy (AFM), scanning near‐field optical microscopy (SNOM) and UV–Vis spectrometry was used to investigate air‐dried erythrocytes (red blood cells, RBCs) in whole human blood smears. The central internal part of the cell was dominated by the laser‐induced O2 dissociated oxyHb form as evidenced by the Fe2+ marker band appearing at 1356 cm−1. The existence of a thin outer layer of hemoglobin in the periphery of RBCs was assigned to hemichrome. Evidence for hemichrome includes the oxidation state marker band appearing at 1376 cm−1, the absence of FeO2 band at 570 cm−1 and a UV–Vis spectrum consistent with hemichrome. This is the first time that distributions of Fe2+/Fe3+ hemes inside the single RBC have been reported. The outer layer formation of hemichrome was additionally studied when RBCs were in contact with leucocytes and carotenoids of blood plasma. Copyright © 2014 John Wiley & Sons, Ltd.

Treatment With Minihepcidin Peptide Improves Anemia and Iron Overload In a Mouse Model Of Thalassemia Intermedia

IntroductionBeta-thalassemia is a inherited disorder characterized by mutations in the beta-globin gene that lead to reduced or absent beta-globin synthesis. As a consequence, in erythroid cells there is a relative excess of alpha globin chains and heme forming toxic hemichromes, which lead to oxidative damage, impaired erythroid cell maturation, premature cell death and anemia. Under these conditions, the anemia and the elevated erythropoietin levels increase the proliferation of the erythroid progenitors, suppressing the expression of the iron master regulator hepcidin. Hepcidin targets the iron exporter ferroportin present on enterocytes and macrophages. High levels of hepcidin limit iron absorption and iron recycling but low levels of hepcidin increase iron absorption. Chronic low levels of hepcidin, as in β-thalassemia, eventually lead to iron overload. The th3/+ mouse model of thalassemia intermedia (Non-Transfusion Dependent Thalassemia or NTDT), reproduces the main features of the disease including anemia, increased erythropoiesis, splenomegaly and iron overload due to low levels of hepcidin. It has been previously demonstrated that modestly increasing hepcidin levels in these mice ameliorates both anemia and iron overload (Gardenghi et al, JCI, 2010). MethodsWe used minihepcidin M004, a modified peptide analog of the 9 N-terminal amino acids of hepcidin, to reproduce the biological effects of hepcidin. In a dose-ranging study, we treated th3/+ animals with daily intraperitoneal (ip) injections of M004 at doses of 6.25-100 µg/day for 2-6 weeks or with subcutaneous (sc) dosing of 52.5 μg twice a week for 6 weeks. ResultsIn animals treated with the lower ip doses of M004 (6.25-25 μg/day for 2-4 weeks), mild iron restriction was observed (Tf sat ≥15%). Flow cytometry studies using the markers CD44 and Ter119 demonstrated an increase in the proportion of mature erythroid cells. Consistent with the flow cytometry findings, a 30% increase in peripheral red cells (rbc) was observed associated with a reduction in reticulocyte count (>62%) and a reduction in spleen size (>40%), reflecting improved erythropoietic efficiency. At the 12.5 and 25 µg doses, Hb level increased by 1.8-2.5 g/dL. Longer treatment (6 weeks) with 12.5-25 µg/day led to greater iron restriction and the loss of therapeutic effect at 6 weeks. In the same animals, measurement of total organ iron concentrations demonstrated no changes in the liver and kidney, but a 4-fold iron increase in the spleen. Prussian blue staining confirmed increased sequestration of iron in splenic macrophages and Kupffer cells.At high ip doses of M004 (100 μg/day for 2 weeks) the mice developed severe iron restriction (Tf sat = 6%) and exacerbation of anemia (Hb reduction of 4.5 g/dL). Accumulation of immature erythroid precursors was observed by flow cytometry studies, reflecting the erythroid maturation block caused by lack of iron for hemoglobin generation.Subsequent pharmacokinetic studies in rats indicated that plasma levels of M004 increased gradually with sc dosing and were still increasing 24h after dosing, indicating accumulation of drug levels and iron restriction with daily administration. We therefore explored whether less frequent dosing may be preferable to achieve sustained improvement in anemia and normalization of tissue iron. Sc dosing of M004 at 52.5 µg twice weekly was associated with an increase in rbc at 6 weeks to levels observed in WT animals, and an Hb increase of almost 3 g/dL. Reduced spleen weight (-51%) and reticulocyte count (-61%) reflected improved erythropoietic efficiency, further corroborated by flow cytometry studies. Reduction in hemichrome and ROS formation coupled with improvement in rbc morphology and reduced RDW indicated that hematological improvement was likely caused by improved erythroid cell survival as a result of reduced oxidative damage secondary to decreased erythroid iron uptake. Evaluation of total organ iron concentrations showed a reduction of 77% and 54% in the liver and kidney, respectively. Remarkably, the liver iron values were normalized compared to WT animals. Splenic iron increased only ∼1.5 times or 148%. However, when the total weight of the spleen was taken in account, the total splenic iron was reduced (-39%). ConclusionMinihepcidin peptides may be beneficial for the treatment of beta thalassemia intermedia by improving anemia and reducing tissue iron burden. Disclosures:Casu:Merganser: Research Funding. Goldberg:Merganser: Research Funding. Nemeth:Merganser: Equity Ownership. Ganz:Merganser: Equity Ownership. MacDonald:Merganser : Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Rivella:Alexion: Consultancy; Biomarin: Consultancy; Merganser: Equity Ownership, Research Funding; Isis: Consultancy, Research Funding; Bayer: Consultancy; Novartis: Consultancy; Imago: Consultancy.

The role of T56 in controlling the flexibility of the distal histidine in dehaloperoxidase-hemoglobin from Amphitrite ornata

The activation of dehaloperoxidase-hemoglobin (DHP) to form a ferryl intermediate requires the distal histidine, H55, to act as an acid base catalyst. The lack of ancillary amino acids in the distal pocket to assist in this process makes H55 even more important to the formation of active intermediates than in conventional peroxidases. Therefore, one can infer that the precise conformation H55 may greatly affect the enzymatic activity. Using site-direct mutagenesis at position T56, immediately adjacent to H55, we have confirmed that subtle changes in the conformation of H55 affect the catalytic efficiency of DHP. Mutating T56 to a smaller amino acid appears to permit H55 to rotate with relatively low barriers between conformations in the distal pocket, which may lead to an increase in catalytic activity. On the other hand, larger amino acids in the neighboring site appear to restrict the rotation of H55 due to the steric hindrance. In the case of T56V, which is an isosteric mutation, H55 appears less mobile, but forced to be closer to the heme iron than in wild type. Both proximity to the heme iron and flexibility of motion in some of the mutants can result in an increased catalytic rate, but can also lead to protein inactivation due to ligation of H55 to the heme iron, which is known as hemichrome formation. A balance of enzymatic rate and protein stability with respect to hemichrome formation appears to be optimum in wild type DHP (WT-DHP).

Removal of Macrophages From the Erythroid Niche Impairs Stress Erythropoiesis but Improves Pathophysiology of Polycythemia Vera and Beta-Thalassemia

AbstractAbstract 81We investigated the contribution of macrophages to physiological and pathological conditions in which erythropoietic activity is enhanced. We utilized mouse models of a) anemia by phlebotomy-induced stress erythropoiesis (SE); b) increased erythropoiesis by erythropoietin (Epo) administration; c) Polycythemia Vera (Jak2V617F/+ or PV) and d) beta-thalassemia intermedia (Hbbth3/+ or BTI) in which macrophages were chemically depleted by injection of liposome-clodronate (LC).While chronic injection (up to 3 months) of LC in normal mice had little effect on steady state erythropoiesis, depletion of macrophages severely impaired recovery from anemia following phlebotomy and significantly limited the increase in hematocrit (Htc) in animals treated with Epo. To exclude that this effect was mediated by decreased serum iron parameters, we used mice iron overloaded by dietary means or affected by hemochromatosis (Hfe-KO and Hamp-KO). In these mice, recovery from anemia was still impaired following macrophage depletion, even though serum iron and transferrin saturation levels were elevated and unaffected by LC administration.In vitro studies using both mouse and human primary erythroblasts (EBs) indicated that EBs in S-phase were twice as many compared to EBs cultured in absence of macrophages. The numbers of terminally mature erythroid cells were up to six fold higher in co-culture conditions. Experiments using transwells indicate that direct contact between EBs and macrophages was required to generate this effect.Since our data highlighted an important role of macrophages in enhancing erythropoiesis, we investigated two disorders in which the pool of erythroid progenitor cells is expanded, such as PV and BTI. Chronic administration of LC in PV mice completely reversed splenomegaly and the Htc (P<0.001). BTI mice exhibited normal spleen, amelioration of ineffective erythropoiesis (by accelerating the differentiation of EBs to erythrocytes), improvement of red blood cell (RBC) morphology, red cell distribution width (RDW, P<0.001) and increased hemoglobin levels (∼2g/dL, P<0.01). This effect was due to an increased RBC lifespan following LC administration (P<0.001), which was associated with a decrease in hemichrome formation, but not with a reduction in erythophagocytosis.Our observations indicate that macrophages directly modulate stress- and pathological erythropoiesis. Several adhesion molecules participate in the formation of interactions within the erythroblastic islands, including integrins. Interestingly, βeta1integrin and its associated protein, focal adhesion kinase-1 (Fak1), were reported to be necessary for the compensatory response to anemia, suggesting that this pathway might be involved in the macrophage-EB cross-talk. More EBs co-cultured with macrophages retained cell surface expression of βeta1integrin molecule during the last stage of cell differentiation compared to EBs cultured alone, even though other differentiation markers did not shown any variation. Fak1 phosphorylation in EBs was induced by co-culturing them with splenic macrophages, suggesting that Fak1 signaling is one of the pathways activated in EBs through contact with macrophages. Administration of a FAK1 inhibitor (FAK1i) decreased proliferation of EB co-cultured with macrophages, while delayed recovery from anemia and decreased the spleen size in phlebotomized animals (40% decrease compared to phlebotomized control animals at day 4; P=0.032). Finally, short-term administration of FAK1i to BTI animals rapidly reverted splenomegaly with a concurrent reduction of erythroid expansion in both BM and spleen and led to amelioration of anemia, supported by increased RBCs count.Our data indicate that, while macrophages allow proper erythroid response under conditions of induced anemia or increased erythropoiesis in wt mice, they contribute to the pathological progression of PV and BTI. Activation of Fak1 promotes erythroid proliferation and pathological development, while its inhibition limits ineffective erythropoiesis and splenomegaly in BTI. In conclusion, we identified a new mechanism contributing to the pathophysiology of these disorders, which we believe will have critical scientific and therapeutic implications in the near future. Disclosures:Levine:Agios Pharmaceuticals: Research Funding. Rivella:Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding.

Membrane association of peroxiredoxin-2 in red cells is mediated by the N-terminal cytoplasmic domain of band 3

Band 3 (B3), the anion transporter, is an integral membrane protein that plays a key structural role by anchoring the plasma membrane to the spectrin-based membrane skeleton in the red cell. In addition, it also plays a critical role in the assembly of glycolytic enzymes to regulate red cell metabolism. However, its ability to recruit proteins that can prevent membrane oxidation has not been previously explored. In this study, using a variety of experimental approaches including cross-linking studies, fluorescence and dichroic measurements, surface plasmon resonance analysis, and proteolytic digestion assays, we document that the antioxidant protein peroxiredoxin-2 (PRDX2), the third most abundant cytoplasmic protein in RBCs, interacts with the cytoplasmic domain of B3. The surface electrostatic potential analysis and stoichiometry measurements revealed that the N-terminal peptide of B3 is involved in the interaction. PRDX2 underwent a conformational change upon its binding to B3 without losing its peroxidase activity. Hemichrome formation induced by phenylhydrazine of RBCs prevented membrane association of PRDX2, implying overlapping binding sites. Documentation of the absence of binding of PRDX2 to B3 Neapolis red cell membranes, in which the initial N-terminal 11 amino acids are deleted, enabled us to conclude that PRDX2 binds to the N-terminal cytoplasmic domain of B3 and that the first 11 amino acids of this domain are crucial for PRDX2 membrane association in intact RBCs. These findings imply yet another important role for B3 in regulating red cell membrane function.

On the stability of the extracellular hemoglobin of Glossoscolex paulistus, in two iron oxidation states, in the presence of urea

The stability of the Glossoscolex paulistus hemoglobin (HbGp), in two iron oxidation states (and three forms), as monitored by optical absorption, fluorescence emission and circular dichroism (CD) spectroscopies, in the presence of the chaotropic agent urea, is studied. HbGp oligomeric dissociation, denaturation and iron oxidation are observed. CD data show that the cyanomet-HbGp is more stable than the oxy-form. Oxy- and cyanomet-HbGp show good fits on the basis of a two state model with critical urea concentrations at 220–222nm of 5.1±0.2 and 6.1±0.1mol/L, respectively. The three-state model was able to reveal a subtle second transition at lower urea concentration (1.0–2.0mol/L) associated to partial oligomeric dissociation. The intermediate state for oxy- and cyanomet-HbGp is very similar to the native state. For met-HbGp, a different equilibrium, in the presence of urea, is observed. A sharp transition at 1.95±0.05mol/L of denaturant is observed, associated to oligomeric dissociation and hemichrome formation. In this case, analysis by a three-state model reveals the great similarity between the intermediate and the unfolded states. Analysis of spectroscopic data, by two-state and three-state models, reveals consistency of obtained thermodynamic parameters for HbGp urea denaturation.

Role of the tertiary and quaternary structure in the formation of bis-histidyl adducts in cold-adapted hemoglobins

All tetrameric hemoglobins from Antarctic fish, including that from Trematomus bernacchii, HbTb form in the ferric state, promptly and distinctively from all the other tetrameric hemoglobins, a mixture of aquo-met at the α subunits and bis-histidyl adduct (hemichrome) at the β subunits. The role of the tertiary and quaternary structure in the hemichrome formation is unknown. Here we report the cloning, expression, purification, spectroscopic and computational characterization of the β-chain of HbTb (β-HbTb). Similarly to the human β-chains, β-HbTb self-assembles to form the homotetramer β4-HbTb; however, the latter quantitatively forms reversible ferric and ferrous bis-histidyl adducts, which are only partially present in the human tetramer (β4-HbA). A molecular dynamics study of the isolated β subunit of the two Hbs indicates that the ability to form hemichrome is an intrinsic feature of the chain; moreover, the greater propensity of β-HbTb to form the bis-histidyl adduct is probably linked to the higher flexibility of the CD loop region. On the bases of these experimental and computational results on the isolated chain, the influence of the quaternary structure on the stability of the endogenous ferrous and ferric hexa-coordination is also discussed.

Perfluorodecanoic Acid Binding to Hemoproteins: New Insights from Spectroscopic Studies

Perfluorodecanoic acid (PFDA), a representative of the perfluoroalkyl acids, poses a great threat to humans and animals via food and other potential sources. In this work, we determined the effects of PFDA binding to two hemoproteins, bovine hemoglobin (BHb) and myoglobin (Mb). Using fluorescence spectroscopy, we found that PFDA greatly enhanced the fluorescence intensity of both hemoproteins, while perfluorooctanoic acid (PFOA) and perfluoropentanoic acid (PFPA) have minimal effects on the fluorescence. UV-vis absorption (UV) spectroscopy showed that PFDA induced the unfolding of the hemoproteins accompanied by exposure of the heme pocket and facilitating the formation of hemichrome. Additionally, as shown by the circular dichroism (CD) data, PFDA altered the secondary structure of both BHb and Mb. This work elucidates the interaction mechanism of PFDA with two hemoproteins.

Molecular Biosensing Mechanisms in the Spleen for the Removal of Aged and Damaged Red Cells from the Blood Circulation

Heinz bodies are intraerythrocytic inclusions of hemichrome formed as a result of hemoglobin (Hb) oxidation. They typically develop in aged red cells. Based on the hypothesis that hemichrome formation is an innate characteristic of physiologically normal Hb molecules, we present an overview of our previous findings regarding the molecular instability of Hb and the formation of hemichrome, as well as recent findings on Heinz body formation within normal human erythrocytes. Human adult Hb (HbO2 A) prepared from healthy donors showed a tendency to produce hemichrome, even at close to physiological temperature and pH. Recent studies found that the number of Heinz bodies formed in red cells increased with increasing temperature when freshly drawn venous blood from healthy donors was subjected to mild heating above 37 °C. These findings suggest that Hb molecules control the removal of non-functional erythrocytes from the circulation via hemichrome formation and subsequent Heinz body clustering. In this review, we discuss the molecular biosensing mechanisms in the spleen, where hemichrome formation and subsequent Heinz body clustering within erythrocytes play a key role in the removal of aged and damaged red cells from the blood circulation.