Ferryl Hemoglobin and Heme Induce A1-Microglobulin in Hemorrhaged Atherosclerotic Lesions with Inhibitory Function against Hemoglobin and Lipid Oxidation.

Infiltration of red blood cells into atheromatous plaques and oxidation of hemoglobin (Hb) and lipoproteins are implicated in the pathogenesis of atherosclerosis. α1-microglobulin (A1M) is a radical-scavenging and heme-binding protein. In this work, we examined the origin and role of A1M in human atherosclerotic lesions. Using immunohistochemistry, we observed a significant A1M immunoreactivity in atheromas and hemorrhaged plaques of carotid arteries in smooth muscle cells (SMCs) and macrophages. The most prominent expression was detected in macrophages of organized hemorrhage. To reveal a possible inducer of A1M expression in ruptured lesions, we exposed aortic endothelial cells (ECs), SMCs and macrophages to heme, Oxy- and FerrylHb. Both heme and FerrylHb, but not OxyHb, upregulated A1M mRNA expression in all cell types. Importantly, only FerrylHb induced A1M protein secretion in aortic ECs, SMCs and macrophages. To assess the possible function of A1M in ruptured lesions, we analyzed Hb oxidation and heme-catalyzed lipid peroxidation in the presence of A1M. We showed that recombinant A1M markedly inhibited Hb oxidation and heme-driven oxidative modification of low-density lipoproteins as well plaque lipids derived from atheromas. These results demonstrate the presence of A1M in atherosclerotic plaques and suggest its induction by heme and FerrylHb in the resident cells.

Oxidative pathways in the sickle cell and beyond.

Polymerization of deoxy sickle cell hemoglobin (HbS) is well recognized as the primary event that triggers the classic cycles of sickling/unsickling of patients red blood cells (RBCs). RBCs are also subjected to continuous endogenous and exogenous oxidative onslaughts resulting in hemolytic rate increases which contribute to the evolution of vasculopathies associated with this disease. Compared to steady-state conditions, the occurrences of vaso-occlusive crises increase the levels of both RBC-derived microparticles as well as extracellular Hb in circulation. Common byproduct resulting from free Hb oxidation and from Hb-laden microparticles is heme (now recognized as damage associated molecular pattern (DAMP) molecule) which has been shown to initiate inflammatory responses. This review provides new insights into the interplay between microparticles, free Hb and heme focusing on Hb's pseudoperoxidative activity that drives RBC's cytosolic, membrane changes as well as oxidative toxicity towards the vascular system. Emerging antioxidative strategies that include the use of protein and heme scavengers in controlling Hb oxidative pathways are discussed.

Mechanisms involved in hemoglobin-mediated oxidation of lipids in washed fish muscle and inhibitory effects of phospholipase A2.

BACKGROUND: Hemoglobin (Hb) is a lipid oxidation promoter in fish muscle. Phospholipase A2 (PLA2; EC 3.1.1.4) is linked to an increased resistance to lipid oxidation of frozen-thawed cod fillets via an unknown mechanism. The present study aimed to investigate the mechanism of Hb-mediated lipid oxidation with a focus on ferryl Hb and methemoglobin (metHb), the pro-oxidative Hb species, and to examine how porcine pancreatic PLA2 inhibits Hb-mediated lipid oxidation in washed cod muscle (WCM). Lipid hydroperoxides (LOOHs) and thiobarbituric acid reactive substances (TBARS) were measured as primary and secondary lipid oxidation products, respectively. The formation of metHb and ferryl Hb was also monitored. RESULTS: Ferryl Hb and metHb formed during the Hb-mediated lipid oxidation. PLA2 inhibited the formation of LOOHs and TBARS and suppressed the formation of metHb and ferryl Hb. WCM was pre-oxidized by hemin to increase the amount of LOOHs. PLA2 promoted the depletion of LOOHs in the pre-oxidized WCM with limited TBARS formation at the expense of the heme moiety of Hb. CONCLUSION: The results of the present study suggest that ferryl Hb may play a role in Hb-mediated lipid oxidation and that PLA2 from pig pancreas may work together with Hb as a novel antioxidant with an ability to remove pre-formed LOOHs from a lipid substrate. © 2017 Society of Chemical Industry.

Interaction of the chlorite-based drug WF10 and chlorite with hemoglobin, methemoglobin and ferryl hemoglobin.

The interaction of the chlorite-based drug solution WF10 with human oxyhemoglobin and oxidized hemoglobin forms was investigated monitoring the corresponding spectral changes in heme states. The chlorite component of WF10 converts oxyhemoglobin into methemoglobin with a rate of 35.4 M(-1)s(-1). Methemoglobin is also formed upon the interaction of ferryl hemoglobin and WF10/chlorite. The rate of this interconversion depends on the oxidation state of ferryl hemoglobin. This rate is 114 M(-1)s(-1), when ferryl hemoglobin was generated upon reaction of oxyhemoglobin and hydrogen peroxide. A considerable higher rate (6600 M(-1)s(-1)) is measured between the chlorite components of WF10 and ferryl hemoglobin after formation of the latter species from methemoglobin. WF10/chlorite inactivates also methemoglobin as evidenced by the continuous decrease of the Soret band and all other absorbances with a rate of 8.3 M(-1)s(-1). In all interconversions, the chlorite component of WF10 was the active principle as shown in experiments applying pure chlorite at the same concentration as in WF10. Thus, WF10 is able to diminish efficiently the yield of cytotoxic hemoglobin species that might appear after excessive hemolysis of red blood cells under pathologic situations.

Caffeic acid: an antioxidant with novel antisickling properties.

It is well documented that caffeic acid (3,4-dihydroxycinnamic acid) (CA) interacts with and inhibits the oxidative reactions of myoglobin (Mb) and hemoglobin (Hb), and this interaction underlies its antioxidative action in meat. Sickle cell hemoglobin (HbS) is known for its tendency to oxidize more readily than normal HbA in the presence of hydrogen peroxide (H2 O2 ), which leads to a more persistent and highly oxidizing ferryl Hb (HbFe4+ ). We have investigated the effects of CA on HbS oxidation intermediates, specifically on the ferric/ferryl forms. At a low concentration of H2 O2 (0.5-fold over heme), we observed a fivefold reduction in the amount of HbFe4+ accumulated in a mixture of ferric and H2 O2 solution. Higher levels of H2 O2 (onefold and twofold over heme) led to a lesser threefold and twofold reduction in the content of HbFe4+ , respectively, possibly due to the saturation of the binding sites on the Hb molecule. The most intriguing finding was that when 5-molar excess CA over heme was used, and a considerable increase in the delay time of HbS polymerization to approximately 200 s was observed. This delay in polymerization of HbS is theoretically sufficient to avoid microcapillary blockage and prevent vasoconstrictions in vivo. Mass spectrometry analysis indicated that CA was more extensively covalently bonded to ßCys93 than to ßCys112 and αCys104 . The dual antioxidant and antisickling properties of CA may be explored further to maximize its therapeutic potential in SCD.

Oxidized hemoglobin forms contribute to NLRP3 inflammasome-driven IL-1ß production upon intravascular hemolysis.

Damage associated molecular patterns (DAMPs) are released form red blood cells (RBCs) during intravascular hemolysis (IVH). Extracellular heme, with its pro-oxidant, pro-inflammatory and cytotoxic effects, is sensed by innate immune cells through pattern recognition receptors such as toll-like receptor 4 and nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3), while free availability of heme is strictly controlled. Here we investigated the involvement of different hemoglobin (Hb) forms in hemolysis-associated inflammatory responses. We found that after IVH most of the extracellular heme molecules are localized in oxidized Hb forms. IVH was associated with caspase-1 activation and formation of mature IL-1ß in plasma and in the liver of C57BL/6 mice. We showed that ferrylHb (FHb) induces active IL-1ß production in LPS-primed macrophages in vitro and triggered intraperitoneal recruitment of neutrophils and monocytes, caspase-1 activation and active IL-1ß formation in the liver of C57BL/6 mice. NLRP3 deficiency provided a survival advantage upon IVH, without influencing the extent of RBC lysis or the accumulation of oxidized Hb forms. However, both hemolysis-induced and FHb-induced pro-inflammatory responses were largely attenuated in Nlrp3-/- mice. Taken together, FHb is a potent trigger of NLRP3 activation and production of IL-1ß in vitro and in vivo, suggesting that FHb may contribute to hemolysis-induced inflammation. Identification of RBC-derived DAMPs might allow us to develop new therapeutic approaches for hemolytic diseases.

Oxygen Self-Production Red Blood Cell Carrier System for MRI Mediated Cancer Therapy: Ferryl-Hb, Sonodynamic, and Chemical Therapy.

Hypoxia in tumors can lead to insufficient oxygen supply during sonodynamic therapy (SDT), which in turn strengthens tumor resistance to sonodynamic efficacy. To conquer hypoxia in tumors and improve the treatment effectiveness, we developed oxygen self-production red blood cell (RBC) carrier system to decompose tumor endogenic H2O2 into O2 and combine triplex cancer therapy: ferryl-hemoglobin (ferryl-Hb), sonodynamic, and chemical therapy. Both hydrophilic sonosensitizer and doxorubicin (DOX) were encapsulated inside RBCs (DOX/Mn-TPPS@RBCs). The drug release can be improved by combining the effects of H2O2 and ultrasonic irradiation. Here, we introduced a contrast agent, meso-tetra (4-sulfonatephenyl) porphyrinate manganese(III) complex (Mn-TPPS), which could be used to enhance the signal intensity of magnetic resonance imaging (MRI) of the tumor site. The feasibility of Mn-TPPS as a sonosensitizer was investigated during SDT. Importantly, DOX/Mn-TPPS@RBCs overcame hypoxia in the tumor and improved the efficacy of SDT owing to the O2 generation by the catalase-catalyzed decomposition of tumor endogenic H2O2. Hemoglobin was simultaneously oxidized into highly oxidative ferryl-Hb species by H2O2 and reactive oxygen species, resulting in cytotoxicity. Overall, this drug delivery system is a promising therapeutic agent involving in situ production of oxygen inside the tumor, triplex therapy, and MRI.

Oxidized Mutant Human Hemoglobins S and E Induce Oxidative Stress and Bioenergetic Dysfunction in Human Pulmonary Endothelial Cells.

Cell free hemoglobin (Hb), becomes oxidized in the circulation during hemolytic episodes in sickle cell disease (SCD) or thalassemia and may potentially cause major complications that are damaging to the vascular system. Hemolytic anemias are commonly associated with pulmonary hypertension (PH) and often result from dysfunction of lung endothelial cells. The aim of this study was to determine the effect of different Hbs on cultured human lung endothelial function. Toward this goal, endothelial permeability, oxidative stress response parameters, glycolytic and mitochondrial bioenergetic functions were monitored in cultured human pulmonary arterial endothelial cells (HPAEC) following incubation with human adult Hb (HbA), and Hb isolated from patients with sickle cell Hb (HbS, ßV6E) and HbE (ßE26K) that commonly co-exist with ß-thalassemia. These mutant Hbs are known for their distinct oxidative profiles. HPAEC treated with the ferrous forms of HbE, HbS for 24 h showed higher loss of endothelial monolayer integrity with concomitant rise in reactive oxygen radical production, lipid hydroperoxide formation and higher expressions of oxidative stress response proteins including heme oxygenase-1 (HO-1) accompanied by a rise in uncoupled mitochondrial respiration. Loss of membrane permeability was diminished in part by haptoglobin (Hp, protein scavenger), hemopexin (Hpx, heme scavenger) or ascorbate (reducing agent). To understand the role of Hb oxidation, HPAEC were exposed to ferric or ferryl states of the mutant Hbs. Ferryl forms of all proteins caused a significant damage to the endothelial monolayer integrity at a higher degree than their respective ferric Hbs. Ferryl forms of HbS and HbE also caused a loss of respiratory chain complex activities in isolated endothelial mitochondria and basal oxygen consumption in HPAEC. However, longer incubation with ferryl Hbs produced bioenergetic reprogramming including higher degree of uncoupled respiration and glycolytic rate. The data in this report collectively indicate that higher oxidation forms of HbS and HbE cause endothelial dysfunction through distinct damaging mechanisms involving mitochondrial bioenergetic function.

Differential heme release from various hemoglobin redox states and the upregulation of cellular heme oxygenase-1.

Despite advances in our understanding of the oxidative pathways mediated by free hemoglobin (Hb), the precise contribution of its highly reactive redox forms to tissue and organ toxicities remains ambiguous. Heme, a key degradation byproduct of Hb oxidation, has recently been recognized as a damage-associated molecular pattern (DAMP) molecule, able to trigger inflammatory responses. Equally damaging is the interaction of the highly redox active forms of Hb with other biological molecules. We determined the kinetics of heme loss from individual Hb redox states-ferrous (Fe(2+)), ferric (Fe(3+)), and ferryl (Fe(4+))-using two different heme receptor proteins: hemopexin (Hxp), a naturally occurring heme scavenger in plasma, and a double mutant (H64Y/V86F), apomyoglobin (ApoMb), which avidly binds heme released from Hb. We show for the first time that ferric Hb (Fe(3+)) loses heme at rates substantially higher than that of ferryl Hb (Fe(4+)). This was also supported by a higher expression of heme oxygenase-1 (HO-1) when ferric Hb was added to cultured lung alveolar epithelial cells (E10). The reported cytotoxicity of Hb may therefore be attributed to a combination of accelerated heme loss from the ferric form and protein radical formation associated with ferryl Hb. Targeted therapeutic interventions can therefore be designed to curb specific oxidative pathways of Hb in hemolytic anemias and when Hb is used as an oxygen-carrying therapeutic.

Tyrosine can protect against oxidative stress through ferryl hemoglobin reduction.

The toxic mechanism of hemoglobin (Hb) under oxidative stress is linked to the formations of highly cytotoxic ferryl species and subsequently heme-to-protein cross-linked derivative of Hb (Hb-X). In this study, we have examined the effects of free tyrosine and its analogues (3-chlorotyrosine, phenylalanine) on the stability of ferryl hemoglobin and the formation of Hb-X. The results showed that free tyrosine (not phenylalanine, 10-500 µM) was an efficient reducing agent of ferryl species and also effective at preventing the formation of cytotoxic Hb-X. Meanwhile, the dimeric tyrosine was formed as the oxidation product of tyrosine during Hb redox reaction. Compared with free tyrosine, 3-chlorotyrosine, an oxidation product of tyrosine and a proposed biomarker for hypochlorous acid (HOCl) in vivo, exhibited stronger antioxidant properties in Hb-induced oxidative stress, which was consistent with its more efficient ability in the reduction of ferryl species. These results showed that the presence of tyrosine and its derivative in vivo and vitro could ameliorate oxidative damage through ferryl heme reduction. The antioxidant ability, therefore, may provide new insights into the nutritional and physiological significance of free tyrosine with redox active heme proteins-related oxidative stress.