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

Blood ◽  
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.

Carbon Monoxide Therapy Reduces Reactive Oxygen Species Production and the Short-Term Hematopoietic Stem Cell Population In Heme-Oxygenase-1 Knockout Mice

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4767-4767
Author(s):  
Joan D. Beckman ◽  
Paul H Marker ◽  
Julia Nguyen ◽  
John D Belcher ◽  
Robert P Hebbel ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Bone Marrow ◽  
Carbon Monoxide ◽  
Stem Cell ◽  
Research Funding ◽  
S Phase ◽  
Heme Oxygenase 1 ◽  
Oxygen Species ◽  
Short Term ◽  
Cell Preservation

Abstract Abstract 4767 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 produces three by-products, carbon monoxide, biliverdin/bilirubin, and iron/ferritin, all of which have demonstrated antioxidant and anti-apoptotic properties. HO-1-/- mice display oxidative stress, anemia, and leukocytosis with chronic inflammation. We hypothesize that therapy with inhaled carbon monoxide (CO) may reduce stress hematopoiesis in HO-1-/- mice, restoring them to normal hematopoiesis. We have previously reported that treatment with 250 ppm CO for 1 h raises mouse carboxyhemoglobin to 14–20% which returns to baseline within 24 hours. Therefore, 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. We demonstrate that after four weeks, CO-treated HO-1-/- mice have a total white blood cell count of 11.9 ± 3.2 K/μ L compared to 22.6 ± 3.2 K/μ L in untreated HO-1-/- mice (p<0.05), with HO-1+/+ mice having no treatment effect. After 8-weeks of treatment the mice were sacrificed and methylcellulose colony-forming unit assays and flow cytometry were performed on bone marrow to assess their hematopoietic potential. Total methylcellulose colony-forming units were similar in both HO-1-/- and HO-1+/+ mice with CO-treatments significantly (p<0.05) increasing the total number of colonies per animal. Of note, in both the CO-treated HO-1-/- and HO-1+/+ mice the CFU-GM were not significantly affected. Cell cycle analysis of the bone marrow using propidium iodide 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. Similarly, untreated HO-1-/- mice have a lower frequency of cells in sub-G1 phase compared to untreated HO-1+/+ mice, however CO-treatments significantly (p<0.01) increase this frequency in HO-1-/- mice to levels comparable with untreated and CO-treated 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. Interestingly, the CO-treated HO-1-/- mice had a significantly higher proportion of KLS cells staining positive for ROS, but the mean fluorescent intensity of this population was significantly (p<0.05) decreased compared to untreated HO-1-/- mice. This indicates that there is an overall decrease in ROS in the KLS cells of CO-treated HO-1-/- mice compared to untreated HO-1-/- mice. Analysis of long-term, short-term, and multipotent progenitor cell populations was conducted and reveals significant changes at the level of the short-term hematopoietic progenitor population (ST-HSC). Specifically, untreated HO-1-/- mice have a significantly increased percent of ST-HSC cells compared to untreated HO-1+/+ mice. Treatment with CO significantly (p<0.05) decreases the percent of ST-HSC cells in HO-1-/- mice but not HO-1+/+ mice. Combined this data indicates that CO therapy is able to modify the hematopoietic potential of HO-1-/- mice. We propose a model in which CO-mediated signaling initiates a homeostatic conditioning program in stem cells to balance hematopoiesis and stem cell preservation. Disclosures: Belcher: Sangart: Research Funding. Vercellotti:Sangart: Consultancy, Research Funding.

scholarly journals Donor-Derived Myeloid Heme Oxygenase-1 Controls the Development of Graft-Versus-Host Disease

2021 ◽  
Vol 11 ◽  
Author(s):  
Chloé Spilleboudt ◽  
Virginie De Wilde ◽  
Philippe Lewalle ◽  
Ludovic Cabanne ◽  
Mathieu Leclerc ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Heme Oxygenase ◽  
Graft Versus Host Disease ◽  
Acute Gvhd ◽  
Heme Oxygenase 1 ◽  
Hematopoietic Stem ◽  
Graft Versus Host

Graft-versus-host disease (GVHD) remains a major clinical drawback of allogeneic hematopoietic stem cell transplantation (HSCT). Here, we investigated how the stress responsive heme catabolizing enzyme heme oxygenase-1 (HO-1, encoded by HMOX1) regulates GVHD in response to allogeneic hematopoietic stem cell transplantation in mice and humans. We found that deletion of the Hmox1 allele, specifically in the myeloid compartment of mouse donor bone marrow, promotes the development of aggressive GVHD after allogeneic transplantation. The mechanism driving GVHD in mice transplanted with allogeneic bone marrow lacking HO-1 expression in the myeloid compartment involves enhanced T cell alloreactivity. The clinical relevance of these observations was validated in two independent cohorts of HSCT patients. Individuals transplanted with hematopoietic stem cells from donors carrying a long homozygous (GT)n repeat polymorphism (L/L) in the HMOX1 promoter, which is associated with lower HO-1 expression, were at higher risk of developing severe acute GVHD as compared to donors carrying a short (GT)n repeat (S/L or S/S) polymorphism associated with higher HO-1 expression. In this study, we showed the unique importance of donor-derived myeloid HO-1 in the prevention of lethal experimental GVHD and we corroborated this observation by demonstrating the association between human HMOX1 (GT)n microsatellite polymorphisms and the incidence of severe acute GVHD in two independent HSCT patient cohorts. Donor-derived myeloid HO-1 constitutes a potential therapeutic target for HSCT patients and large-scale prospective studies in HSCT patients are necessary to validate the HO-1 L/L genotype as an independent risk factor for developing severe acute GVHD.

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

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2112-2112
Author(s):  
John D Belcher ◽  
Chunsheng Chen ◽  
Mark Young ◽  
Kenneth Burhop ◽  
Gregory M Vercellotti
Keyword(s):  
Carbon Monoxide ◽  
Heme Oxygenase ◽  
Sickle Cell ◽  
Research Funding ◽  
The Other ◽  
Heme Oxygenase 1 ◽  
Nuclear Factor ◽  
Treatment Groups ◽  
Liver Nuclei ◽  
Hemin Chloride

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.

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

Blood ◽  
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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