MP4CO, a Pegylated Hemoglobin Saturated with Carbon Monoxide, Inhibits Microvascular Stasis in Transgenic Sickle Mice Through Heme Oxygenase-1.

Abstract Abstract 2112 Our laboratory has shown in murine models of sickle cell disease (SCD) that intravascular heme promotes oxidative stress, inflammation and microvascular stasis through toll-like receptor-4 (TLR4) signaling. Furthermore, the heme degrading enzyme, heme-oxygenase-1 (HO-1) and its by-products biliverdin and carbon monoxide (CO), inhibit these effects. CO may induce salutary effects in SCD to decrease vaso-occlusion by inhibiting hemoglobin S polymerization, vasodilation and anti-inflammatory actions, including induction of HO-1. MP4CO is a 4.3 g/dL solution of human hemoglobin conjugated with polyethylene glycol and saturated with CO. In the current studies, we tested the hypothesis that MP4CO would induce HO-1 in transgenic sickle mice and inhibit microvascular stasis in response to hypoxia/reoxygenation (H/R). Microvascular stasis (% non-flowing venules) was examined by intravital microscopy following 1hr of hypoxia (7% O2) and 1hr of reoxygenation (room air) in NY1DD transgenic sickle mice implanted 3 days earlier with a dorsal skin fold chamber window (DSFC). Five treatment groups of 3–6 mice were studied initially: 1) lactated Ringer's solution (LRS); 2) MP4OX (oxygen saturated MP4); 3) MP4CO; 4) oxygen-saturated stroma-free hemoglobin (SFH); 5) hemin chloride, 40 nmols/g i.p. × 3 days was administered as a positive control based on the previously-demonstrated induction of HO-1. Other than hemin chloride, all solutions (LRS, MP4CO, MP4OX, SFH) were administered i.v., 0.008 mL/g. In the first study, LRS, MP4OX, MP4CO or SFH were infused 24hr prior to H/R and in the second study the same solutions were infused 30min after hypoxia, during the reoxygenation phase of the experiment. In sickle mice treated with LRS or MP4OX 24hr prior to H/R, 25% and 22% of the venules, respectively, became static in response to H/R. However, in sickle mice treated with MP4CO 24hr prior to H/R, only 9% of the venules became static (p<0.05 MP4CO vs. LRS and MP4OX). In contrast, sickle mice treated with SFH 24hr prior to H/R developed significantly more stasis (37% stasis) than sickle mice in the other treatment groups (p<0.05). As we have previously shown, pretreatment with hemin abrogated vascular stasis in sickle mice (3% stasis, p<0.05 vs. all other groups). In additional groups of sickle mice, LRS, MP4OX, MP4CO and SFH were administered 30min after hypoxia during the reoxygenation phase. After H/R, LRS-treated animals had 26% stasis, MP4OX-treated mice had 18% stasis (p<0.05 vs. LRS) and MP4CO-treated mice had11% stasis (p<0.05 vs. LRS). Infusion of SFH 30min post-hypoxia markedly worsened stasis compared to the other treatments (44% stasis, p<0.05 vs. MP4CO, LRS and MP4OX). Infusion of MP4CO, but not LRS, MP4OX or SFH, markedly induced expression of microsomal HO-1 activity and protein, suggesting HO-1 was responsible for inhibition of stasis by MP4CO. Indeed, the HO-1 inhibitor SnPP reversed the effect of MP4CO on H/R-induced stasis in sickle mice (27% stasis with SnPP + MP4CO vs. 10% with LRS + MP4CO, p<0.05). The mechanism of HO-1 induction by MP4CO was likely due to an increased expression of nuclear factor erythroid 2-related factor 2 (Nrf2), an important transcriptional regulator of HO-1. MP4CO induced strikingly more Nrf2 in liver nuclei than LRS, MP4OX or SFH. Induction of HO-1 by MP4CO decreased the inflammatory response in sickle mice as evidenced by a decrease in activated nuclear factor-kappa B (NF-kB) phospho-p65 in liver nuclei following H/R. We conclude that MP4CO enhances cytoprotective Nrf2-regulated proteins including HO-1 resulting in decreased NF-kB activation, inflammation and microvascular stasis in transgenic SCD mice. CO delivery via MP4CO may be beneficial in patients with sickle cell anemia. Disclosures: Belcher: Sangart Inc: Research Funding. Chen:Sangart Inc: Research Funding. Young:Sangart Inc: Employment. Burhop:Sangart Inc: Employment. Vercellotti:Sangart Inc: Consultancy, Research Funding.

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Tu1858 Role of Heme Oxygenase-1 and Nuclear Factor (Erythroid-Derived 2)-Like 2 Factor (NRF2) in Pathogenesis of Aspirin-Induced Gastric Damage and Protection by Carbon Monoxide

Gastroenterology ◽

10.1016/s0016-5085(16)33252-8 ◽

2016 ◽

Vol 150 (4) ◽

pp. S961-S962

Author(s):

Marcin Magierowski ◽

Katarzyna Magierowska ◽

Slawomir Kwiecien ◽

Juliusz Adamski ◽

Zbigniew Sliwowski ◽

...

Keyword(s):

Carbon Monoxide ◽

Heme Oxygenase ◽

Gastric Damage ◽

Heme Oxygenase 1 ◽

Nuclear Factor

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Carbon Monoxide Therapy Modulates Hematopoietic Stem Cell Development in Heme-Oxygenase-1 Knockout Mice

Blood ◽

10.1182/blood.v118.21.1318.1318 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1318-1318

Author(s):

Joan Denise Beckman ◽

Paul H Marker ◽

Julia Nguyen ◽

John D Belcher ◽

Anthony J. Croatt ◽

...

Keyword(s):

Bone Marrow ◽

Carbon Monoxide ◽

Stem Cell ◽

Heme Oxygenase ◽

Blood Cells ◽

Research Funding ◽

S Phase ◽

Cell Populations ◽

Heme Oxygenase 1 ◽

Cell Preservation

Abstract Abstract 1318 Previous studies have demonstrated that heme-oxygenase-1 (HO-1), the rate limiting enzyme in the catabolism of heme, is a regulator of the balance between hematopoiesis and stem cell preservation under stress. HO-1−/− mice display oxidative stress, anemia, and leukocytosis with chronic inflammation. We hypothesize that therapy with inhaled carbon monoxide (CO), a by-product of HO-1 activity with antioxidant and anti-apoptotic properties; will reduce stress hematopoiesis in HO-1−/− mice, reducing inflammation and redistributing hematopoietic potential. In order to test this hypothesis we treated 25 week old HO-1−/− mice and HO-1+/+ mice (n=7/strain) with 250 ppm inhaled CO for 1 h/day, 3 days/week for eight weeks and compared them to an equal number of untreated HO-1−/− and HO-1+/+ mice. After 8-weeks of treatment the mice were sacrificed and flow cytometry was performed on bone marrow to assess hematopoietic potential. Cell cycle analysis of the bone marrow demonstrates that untreated HO-1−/− mice have a significantly decreased percent of cells in S-phase compared to untreated HO-1+/+ mice. Treatment with CO significantly (p<0.05) increases the percent of cells in S-phase in HO-1−/− mice but not HO-1+/+ mice. Reactive oxygen species (ROS) production in lineage−, c-kit+, Sca-1+ (KLS) cell population was assessed using 5-(and 6-)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate dye. HO-1−/− mice have a significantly (p<0.001) increased proportion of ROS positive KLS cells compared with HO-1+/+ mice. Analysis of long-term (LT), short-term, and multipotent (MPP) progenitor cell populations was conducted. Treatment with CO significantly (p<0.05) increases the percent of LT-HSC and MPP progenitor cells in HO-1−/− mice but not HO-1+/+ mice. Concordantly, the total white blood cell count of the CO-treated mice increased significantly. The differential of the mature blood cells demonstrates significant shift in cell maturation, with significant increase in red blood cells, platelets, and lymphocytes and a significant decrease in monocytes. Combined this data indicates that CO therapy is able to modify the hematopoietic potential of HO-1−/− mice leading to a change in mature cell populations. We propose a model in which CO-mediated signaling initiates a homeostatic conditioning program in stem cells to balance hematopoiesis and stem cell preservation, ultimately leading to a change in the inflammatory milieu of the mice. Disclosures: Belcher: Sangart, Inc: Research Funding. Vercellotti:Sangart, Inc: Research Funding.

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Heme and the Vasculature: How the Endothelium Protects Itself Against Toxic Iron

Blood ◽

10.1182/blood.v116.21.sci-25.sci-25 ◽

2010 ◽

Vol 116 (21) ◽

pp. SCI-25-SCI-25

Author(s):

Gregory M. Vercellotti ◽

John D Belcher

Keyword(s):

Oxidative Stress ◽

Carbon Monoxide ◽

Endothelial Cells ◽

Endothelial Cell ◽

Mouse Models ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Research Funding ◽

Sleeping Beauty ◽

Heme Oxygenase 1

Abstract Abstract SCI-25 Iron-derived reactive oxygen species (ROS) are involved in the pathogenesis of numerous vascular disorders. Heme-derived iron plays an instrumental role in the pathology of intravascular hemolytic diseases including malaria, sickle cell anemia, transfusion reactions, DIC and PNH. Heme catalyzed oxidative stress promotes a pro-inflammatory/prothrombogenic endothelium, diminution of bio-available nitric oxide (NO) and attraction of leukocytes and platelets. The vasculature is protected against heme-catalyzed injury by plasma proteins including haptoglobin, hemopexin, albumin, alpha-1-microglobulin and by scavenger receptors for heme complexes including CD163 and CD91. Heme and its concomitant oxidative stress induces the cytoprotective and rate-limiting enzyme in heme catabolism, heme oxygenase-1 (HO-1). In the process, HO-1 releases three enzymatic byproducts: carbon monoxide (CO), biliverdin/bilirubin, and iron, which stimulates ferritin synthesis. These HO-1 by-products have established anti-oxidant and anti-inflammatory properties. Human patients and mouse models elevate HO-1 in response to chronic hemolysis. Of all sites in the body, the endothelium may be at greatest risk of exposure to heme. Heme greatly potentiates endothelial cell killing mediated by leukocytes and other sources of ROS. As a defense against heme, endothelial cells upregulate HO-1 and ferritin. If cultured endothelial cells are briefly pulsed with heme and are then incubated for a prolonged period (16 h), the cells become highly resistant to oxidant-mediated injury and to the accumulation of endothelial lipid peroxidation products. This protection is associated with induction of both HO-1 and ferritin. H-ferritin with its ferroxidase activity is especially cytoprotective. In animal models, increased expression of HO-1 has been shown to protect tissues against ischemia-reperfusion injury, oxidative stress, inflammation, transplant rejection, apoptosis, and cell proliferation. Conversely, HO-1 null mice (hmox-1−/−) and human patients deficient in HO-1 are especially prone to oxidative stress and inflammation. Sickle cell anemia is an archetypal example of heme-induced oxidative stress and cytoprotective adaptation. The sickle patient and sickle mouse models defend and adapt to hemolysis by increasing their defenses against heme. HO-1 plays an essential role in the inhibition and resolution of vaso-occlusion in sickle cell anemia. HO-1 and its products, carbon monoxide and biliverdin, modulate vaso-occlusion through multiple mechanisms including reducing oxidative stress, inhibiting NF-kB, down-regulating endothelial cell adhesion molecules, decreasing red blood cell hemolysis and altering vascular tone. However, sickle cell patients often have adaptive increases in HO-1 activity which are insufficient to completely handle the excessive heme burden, particularly during acute bouts of hemolysis. HO-1 gene therapy in sickle mice using Sleeping Beauty-mediated transposition of an HO-1 transgene provides a promising non-viral approach to significantly enhance HO-1 expression in sickle cell anemia. Strategies to minimize heme-iron activation of the vasculature including increasing HO-1 and its products, anti-oxidants, iron chelators, increasing haptoglobin, hemopexin and/or their receptors CD163/CD91 should be explored in hemolytic disease states. Disclosures: Vercellotti: Sangart: Consultancy, Research Funding. Belcher:Sangart: Research Funding.

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Prevention of Venular Stasis, Circulatory Collapse and Mortality in Transgenic Sickle Mice Administered MP4CO, a Pegylated Hemoglobin Saturated with Carbon Monoxide

Blood ◽

10.1182/blood.v118.21.850.850 ◽

2011 ◽

Vol 118 (21) ◽

pp. 850-850

Author(s):

John D Belcher ◽

Chunsheng Chen ◽

Mark Young ◽

Phuc Tran ◽

Gregory M Vercellotti

Keyword(s):

Carbon Monoxide ◽

Single Dose ◽

Arterial Pressure ◽

Sickle Cell ◽

Research Funding ◽

Heme Oxygenase 1 ◽

Wild Type ◽

Cell Disease ◽

Sickle Cell Crisis ◽

Hypoxia Reoxygenation

Abstract Abstract 850FN2 Patients with sickle cell disease experience recurrent episodes of painful vaso-occlusion with organ damage due to red cell deformation, hemolysis, activation of endothelium, adhesion of blood cells to each other and to the vessel wall, and ischemia/reperfusion injury. There are few therapeutic options available for patients experiencing a sickle cell crisis. Previous studies in our lab have shown that induction of heme oxygenase-1 or administration of its products, carbon monoxide (CO) and biliverdin can inhibit hypoxia-induced vaso-occlusion in transgenic sickle mice. MP4CO is a 4.3 g/dL solution of human hemoglobin conjugated with polyethylene glycol and saturated with CO. In the current studies, we tested the hypothesis that MP4CO would protect transgenic sickle mice from adverse responses to hypoxia/reoxygenation and intravenous administration of hemin. Vascular stasis was examined by intravital microscopy following hypoxia/reoxygenation in NY1DD transgenic sickle mice implanted 3–4 days earlier with a dorsal skin fold chamber window (DSFC). Four groups of 6 mice were studied: 1) Control untreated mice; 2) Hemin chloride, 40 μmols/kg i.p. × 3 days; 3) single dose MP4CO, 0.012 mL/g i.v. treated 30min after hypoxia; 4) two doses of MP4CO treated 24h prior to and 30 min after hypoxia. Flowing venules in the DSFC window were randomly selected at baseline. A total of 15–32 subcutaneous venules were selected in each mouse. After baseline selection of venules, the mice were exposed to hypoxia (7% oxygen, 93% nitrogen) for one hour then returned to room air and the same venules were re-examined for stasis at 1h and 4h after hypoxia. Venules without any visible blood flow were counted as static. Percent stasis was calculated by dividing the number of static venules at each time point by the total number of venules selected at baseline. In addition, bioavailability of CO was determined in four NY1DD sickle mice by gas chromatographic measurement of CO in exhaled air 0–3h and 24–27h after treatment with MP4CO. Control, untreated NY1DD mice exhibited 27% and 4% static venules at 1h and 4h, respectively, following hypoxia. This was significantly reduced by the positive control hemin (3%* and 0%*), single dose MP4CO (15%* and 3%), and two doses of MP4CO (0%* and 0%*) at 1h and 4h, respectively (*p<0.05). Exhaled CO increased approximately 6-fold from baseline of 0.8 to 4.63* nmol/hr/g body weight over the 3h following dosing, representing approximately 50% of administered CO. Based on these data, we evaluated MP4CO in a different transgenic mouse model of sickle cell disease. Intravenous injection of hemin is reported to induce vaso-occlusion more robustly than hypoxia/reoxygenation and to cause mortality in transgenic sickle mice. In this potentially more severe model, we studied the effects of hemin injection (50 μmol/kg, i.v.) in heterozygote Townes-AS mice. These mice were mildly anemic compared to wild type C57 (tHb 9.9 vs 14.0 g/dl) with elevated spleen weights measured at necropsy (269 vs. 87 g). Mice were anesthetized, mechanically ventilated, and catheterized to measure and record arterial pressure continuously. Hemin administration was followed 10 minutes later by 30 minute i.v. infusion of normal saline or MP4CO (0.012 mL/g). In saline treated transgenic sickle mice, hemin induced 100% lethality within 90 minutes, compared with 20% lethality following MP4CO (p<0.05, log rank test). In saline treated mice, hemin administration was followed by a progressive decline in mean arterial pressure, which was absent in MP4CO treated mice (p<0.01). Hemin had no effect on arterial pressure or survival in wild-type C57 mice. These data, from two different models of transgenic sickle mice, demonstrate that MP4CO reduces the vascular complications resulting from oxidative injury. The data suggest that MP4CO is an effective way to administer therapeutic levels of CO. The dose of MP4CO administered in these mice is known to induce a transient increase in total HbCO saturation to approximately 10% with return to baseline levels within 120 minutes. Taken together, these experiments suggest that MP4CO may provide a novel approach to treating patients with sickle cell crisis. Further experiments will evaluate the dose-response, timing of administration, and relative roles of CO administration vs. induction of heme oxygenase-1 by MP4CO. Disclosures: Belcher: Sangart, Inc: Research Funding. Chen:Sangart, Inc: Research Funding. Young:Sangart, Inc: Employment. Tran:Sangart, Inc: Employment. Vercellotti:Sangart, Inc: Research Funding.

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ML-0207/ASP8731: A Novel BACH1 Inhibitor That Induces Fetal Hemoglobin in Treatment of Sickle Cell Disease

Blood ◽

10.1182/blood-2021-151606 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 854-854

Author(s):

Selva Nataraja ◽

Maneet Singh ◽

Shilpa Demes ◽

Lyndsay Olson ◽

Jeff Stanwix ◽

...

Keyword(s):

Sickle Cell Disease ◽

Red Blood Cells ◽

Heme Oxygenase ◽

Sickle Cell ◽

Blood Cells ◽

Research Funding ◽

Heme Oxygenase 1 ◽

Cell Disease ◽

Nrf2 Pathway ◽

F Cells

Abstract Sickle cell disease (SCD) is a genetic disorder caused by a point mutation in the β-globin subunit resulting in hemoglobin S (HbS). Following deoxygenation of red blood cells, HbS forms polymers that can promote hemolysis and the release of free heme that cause pro-oxidative and pro-inflammatory stress, vaso-occlusive pain crises, and ischemia-reperfusion pathophysiology. Heme also functions as an intracellular activator of antioxidant and globin gene expression. Heme binds to the transcriptional repressor BTB and CNC homolog 1 (BACH1), which relieves BACH1's repression of gene transcription. The release of BACH1 repression increases the binding of nuclear factor erythroid 2-related factor 2 (NRF2) to antioxidant response elements (ARE) and the cell-specific transcription of antioxidant genes such as heme oxygenase-1 (HMOX1), glutathione reductase (GR), solute carrier family 7 member 11 (SLC7A11), and NAD(P)H dehydrogenase [quinone] 1 (NQO1). We have previously shown that pharmacologic activation of the NRF2 pathway in SCD mice provides protection against heme-induced vascular occlusion, is anti-inflammatory, and decreases hepatic necrosis. NRF2 activation also promotes erythroid expression of the A-gamma (HBG1) and G-gamma (HBG2) globins, which are subunits of hemoglobin F (HbF) that replace β S-globins and thus increase HbF and decrease HbS in red blood cells. Thus, BACH1 inhibitors have the potential to increase expression of antioxidant and HbF genes and prevent or reduce SCD-related pathophysiology, resulting in reduction of hemolysis, inflammation, and vaso-occlusive pain crises. Mitobridge is currently developing ML-0207/ASP8731, a highly potent, selective small molecule inhibitor of BACH1 capable of activating the Nrf2 pathway in human and murine models and investigated the ability of ML-0207 to modulate antioxidant and anti-inflammatory genes and induce HbF in human translational cellular models and a preclinical murine model of SCD. ML-0207 induced mRNA expression of Nrf2 target genes HGB1, HBG2, HMOX1, SLC7A11, GCLM, and NQO1 in human bone marrow-derived CD34+ cells differentiated to erythrocytes. We observed 2-fold increases in both the percentage and number of CD71+/HbF+ erythrocytes by FACS using 1 µM ML-0207 and 10 μM HU compared to DMSO control (Figure 1A). The combination of ML-0207 and HU induced significantly more HbF+ erythrocytes compared to each drug alone (Figure 1B). In a single healthy CD34+ donor non-responsive to 10 µM HU, we observed ML-0207 was able to significantly induce CD71+/HbF+ cells at 1 & 3 µM (Figure 1C). In Townes SCD mice, there were significant increases in heme oxygenase 1 and decreases in VCAM-1, ICAM-1, and decreases in phospho-p65 NF-ĸB protein. Furthermore, we observed a significant reduction in hemin-induced vaso-occlusion and an increase in the percentage of F-cells. The increases in F-cells were accompanied by increases in blood A-gamma globin and erythrocytes and decreases in leukocytes. Taken together, these data support BACH1 inhibitors as potential novel and effective treatments for SCD patients. Figure 1 Figure 1. Disclosures Nataraja: Mitobridge: Current Employment. Singh: Mitobridge: Current Employment. Demes: Astellas: Current Employment. Olson: Mitobridge: Current Employment. Stanwix: Mitobridge: Current Employment. Biddle: Rheos Medicine: Current Employment. Vercellotti: Mitobridge, an Astellas Company: Consultancy, Research Funding; CSL Behring: Research Funding. Belcher: Mitobridge/Astellas: Consultancy, Research Funding; CSL Behring: Research Funding.

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Faculty Opinions recommendation of Galantamine and carbon monoxide protect brain microvascular endothelial cells by heme oxygenase-1 induction.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1122912.580012 ◽

2008 ◽

Author(s):

George Perry

Keyword(s):

Carbon Monoxide ◽

Endothelial Cells ◽

Heme Oxygenase ◽

Microvascular Endothelial Cells ◽

Heme Oxygenase 1 ◽

Brain Microvascular Endothelial Cells ◽

Microvascular Endothelial

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Trilobatin Protects Cardiomyocytes from Hypoxia/ Reperfusion Injury by Regulating the Nuclear Factor Erythroid 2-Related Factor 2/Heme Oxygenase-1 Pathway

Current Topics in Nutraceutical Research ◽

10.37290/ctnr2641-452x.19:133-138 ◽

2020 ◽

Vol 19 (2) ◽

pp. 133-138

Author(s):

Wenyu Chen ◽

Hui He

Keyword(s):

Heme Oxygenase ◽

Molecular Mechanisms ◽

Heme Oxygenase 1 ◽

Related Factor ◽

Cellular Injury ◽

Nuclear Factor ◽

Oxygen Species ◽

H9c2 Cells ◽

Natural Plant ◽

Induced Changes

Trilobatin is a natural plant-derived glycosylated flavonoid that has been shown to exhibit multiple beneficial pharmacologic activities including protection of heart against H/R-induced cardiomyocyte injury. However, the molecular mechanisms underlying protection from H/R-induced cardiomyocyte injury remain unknown. Using H9C2 cells as a model, we examined the effect of trilobatin on H/R-induced cellular injury, apoptosis, and generation of reactive oxygen species. The results showed that trilobatin protected H9C2 cells not only from cell death and apoptosis, but also counteracted H/R-induced changes in malondialdehyde, superoxide dismutase, glutathione, and glutathione peroxidase. The evaluation of the mechanism underlying the effect of trilobatin on protection from H/R-induced cellular injury suggested changes in the regulation of nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathway.

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