Impairment of neutrophil oxidative burst in children with sickle cell disease is associated with heme oxygenase-1

Abstract Sickle cell disease is a common hemolytic disorder with a broad range of complications, including vaso-occlusive episodes, acute chest syndrome (ACS), pain, and stroke. Heme oxygenase-1 (gene HMOX1; protein HO-1) is the inducible, rate-limiting enzyme in the catabolism of heme and might attenuate the severity of outcomes from vaso-occlusive and hemolytic crises. A (GT)n dinucleotide repeat located in the promoter region of the HMOX1 gene is highly polymorphic, with long repeat lengths linked to decreased activity and inducibility. We examined this polymorphism to test the hypothesis that short alleles are associated with a decreased risk of adverse outcomes (hospitalization for pain or ACS) among a cohort of 942 children with sickle cell disease. Allele lengths varied from 13 to 45 repeats and showed a trimodal distribution. Compared with children with longer allele lengths, children with 2 shorter alleles (4%; ≤ 25 repeats) had lower rates of hospitalization for ACS (incidence rate ratio 0.28, 95% confidence interval, 0.10-0.81), after adjusting for sex, age, asthma, percentage of fetal hemoglobin, and α-globin gene deletion. No relationship was identified between allele lengths and pain rate. We provide evidence that genetic variation in HMOX1 is associated with decreased rates of hospitalization for ACS, but not pain. This study is registered at www.clinicaltrials.gov as #NCT00072761.

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Excess heme upregulates heme oxygenase 1 and promotes cardiac ferroptosis in mice with sickle cell disease

Blood ◽

10.1182/blood.2020008455 ◽

2021 ◽

Author(s):

Archita Venugopal Menon ◽

Jing Liu ◽

Hanting Phoebe Tsai ◽

Lingxue Zeng ◽

Seungjeong Yang ◽

...

Keyword(s):

Lipid Peroxidation ◽

Sickle Cell Disease ◽

Heme Oxygenase ◽

Sickle Cell ◽

Cardiovascular Complications ◽

Heme Oxygenase 1 ◽

Cell Disease ◽

Cardiac Damage ◽

Downstream Effects ◽

Free Heme

Sickle cell disease (SCD) is characterized by increased hemolysis which results in plasma heme overload and ultimately cardiovascular complications. Here, we hypothesized that increased heme in SCD causes upregulation of heme oxygenase 1 (Hmox1) which consequently drives cardiomyopathy through ferroptosis, an iron-dependent non-apoptotic form of cell death. First, we demonstrated that the Townes SCD mice had higher levels of hemopexin-free heme in the serum and increased cardiomyopathy, which was corrected by hemopexin supplementation. Cardiomyopathy in SCD mice was associated with upregulation of cardiac Hmox1, and inhibition or induction of Hmox1 improved or worsened cardiac damage, respectively. Since free iron, a product of heme degradation through Hmox1, has been implicated in toxicities including ferroptosis, we evaluated the downstream effects of elevated heme in SCD. Consistent with Hmox1 upregulation and iron overload, levels of lipid peroxidation and ferroptotic markers increased in SCD mice, which were corrected by hemopexin administration. Moreover, ferroptosis inhibitors decreased cardiomyopathy, whereas a ferroptosis inducer erastin exacerbated cardiac damage in SCD and induced cardiac ferroptosis in non-sickling mice. Finally, inhibition or induction of Hmox1 decreased or increased cardiac ferroptosis in SCD mice, respectively. Together, our results identify ferroptosis as a key mechanism of cardiomyopathy in SCD.

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Oxidative Stress and Induction of Heme Oxygenase-1 in the Kidney in Sickle Cell Disease

American Journal Of Pathology ◽

10.1016/s0002-9440(10)64037-0 ◽

2001 ◽

Vol 158 (3) ◽

pp. 893-903 ◽

Cited By ~ 130

Author(s):

Karl A. Nath ◽

Joseph P. Grande ◽

Jill J. Haggard ◽

Anthony J. Croatt ◽

Zvonimir S. Katusic ◽

...

Keyword(s):

Oxidative Stress ◽

Sickle Cell Disease ◽

Heme Oxygenase ◽

Sickle Cell ◽

Heme Oxygenase 1 ◽

Cell Disease

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Determinants Of Heme-Oxygenase-1 Upregulation In Patients With Sickle Cell Disease

Blood ◽

10.1182/blood.v122.21.2235.2235 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2235-2235 ◽

Cited By ~ 2

Author(s):

Olufolake Adisa ◽

Benjamin Yaw Owusu ◽

Yijuan Hu ◽

Samit Ghosh ◽

Fang Tan ◽

...

Keyword(s):

Steady State ◽

Sickle Cell Disease ◽

Heme Oxygenase ◽

Sickle Cell ◽

Conflicts Of Interest ◽

Dinucleotide Repeat ◽

Acute Chest Syndrome ◽

Heme Oxygenase 1 ◽

Cell Disease ◽

The Mean

Abstract Inflammation is a cardinal component of the pathogenesis of sickle cell disease (SCD). Increased plasma concentration of the inflammatory agonist hemin increases the odds of acute chest syndrome (ACS) in children with SCD (Adisa et al., Br. J Haematol, 2013). In addition, free hemin promotes the development of a lethal ACS-like disease in transgenic sickle mice (Ghosh et al., J Clin Invest, 2013). Hemin degradation is controlled by the rate-limiting enzyme heme oxygenase-1 (HO-1). Polymorphism of a (GT)n dinucleotide repeat in the HO-1 promoter, which enhances expression of the gene, is associated with lower rates of hospitalization for ACS in children. Over-expression of HO-1 reduces stasis in a mouse model of SCD vaso-occlusion. However, the role of plasma HO-1 in SCD patients is entirely unknown. In this study, we measured steady-state plasma HO-1 in two cohorts of patients. Cohort 1 in Atlanta (n=98) consisted of children with a mean age of 10.07±0.42 years (range 2-19 years) and cohort 2 from Accra (n=80) consisted of older patients (mean age 25.30±1.0 years, range 13-58 years). The mean plasma HO-1 of both cohorts was significantly higher compared to the mean value of age- and ethnic-matched individuals with normal adult Hb; Atlanta: 10.19±5.80 vs. 2.08± 1.16, p<0.0001 and Accra: 13.7±8.14 vs. 2.57± 0.82, p<0.0001. Plasma HO-1 varied by 25-fold in both cohorts and it correlated with the white blood cell count (Atlanta: r=0.3361, p<0.0001, Accra: r=0.25, p=0.02). Fifty-four percent (n=53) of subjects in the Atlanta cohort were on hydroxyurea. The mean plasma HO-1 of this subgroup was lower (8.1 ± 4.5) compared to the hydroxyurea naïve Accra cohort (p=<0.0001). Further studies of the Accra cohort revealed significant correlations between HO-1 and multiple markers of vascular inflammation; sICAM-1(r=0.2794, p=0.03, n=60), sE-selectin (r= 0.4209, p=0.0017, n=58) and sP-selectin (r=0.3855, p=0.0028, n=58). The number of the (GT)n dinucleotide in the HO-1 promoter ranged 17 to 45; the distribution was trimodal with peaks at 23, 30 and 41 repeats. The overwhelming majority of patients had medium and large size alleles that are generally hypo-response to induction. Plasma HO-1 level correlated with the length of the (GT)n dinucleotide repeat (p=0.003, n=80). In a multivariable regression model, WBC, sICAM-1, sE-selectin and sP-selectin accounted for 13.4% of the total variance of plasma HO-1 level, and the (GT)n polymorphism accounted for 9.8%. In conclusion, the concentration of plasma HO-1 is generally raised among SCD patients at steady-state. However, a large proportion of patients have a relatively modest level that is probably inadequate to counter the severity of inflammation typical of SCD, due in part to a hypo-responsive HO-1 promoter. Therapeutic strategies that complement induction of the endogenous HO-1 gene may be critical to ameliorate inflammation in SCD. Disclosures: No relevant conflicts of interest to declare.

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Haptoglobin attenuates hemoglobin-induced heme oxygenase-1 in renal proximal tubule cells and kidneys of a mouse model of sickle cell disease

Blood Cells Molecules and Diseases ◽

10.1016/j.bcmd.2014.12.001 ◽

2015 ◽

Vol 54 (3) ◽

pp. 302-306 ◽

Cited By ~ 28

Author(s):

Narendranath Reddy Chintagari ◽

Julia Nguyen ◽

John D. Belcher ◽

Gregory M. Vercellotti ◽

Abdu I. Alayash

Keyword(s):

Sickle Cell Disease ◽

Mouse Model ◽

Proximal Tubule ◽

Heme Oxygenase ◽

Sickle Cell ◽

Heme Oxygenase 1 ◽

Cell Disease ◽

Proximal Tubule Cells ◽

Tubule Cells ◽

Renal Proximal Tubule Cells

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Haptoglobin and hemopexin inhibit vaso-occlusion and inflammation in murine sickle cell disease: Role of heme oxygenase-1 induction

PLoS ONE ◽

10.1371/journal.pone.0196455 ◽

2018 ◽

Vol 13 (4) ◽

pp. e0196455 ◽

Cited By ~ 28

Author(s):

John D. Belcher ◽

Chunsheng Chen ◽

Julia Nguyen ◽

Fuad Abdulla ◽

Ping Zhang ◽

...

Keyword(s):

Sickle Cell Disease ◽

Heme Oxygenase ◽

Sickle Cell ◽

Heme Oxygenase 1 ◽

Cell Disease

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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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Heme Oxygenase-1 Gene Therapy in a Murine Model of Sickle Cell Disease.

Blood ◽

10.1182/blood.v114.22.1527.1527 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1527-1527

Author(s):

Belcher D. Belcher ◽

Julie V. Vineyard ◽

Carol M Bruzzone ◽

Chunsheng Chen ◽

Joan D Beckman ◽

...

Keyword(s):

Amino Acids ◽

Gene Therapy ◽

Sickle Cell Disease ◽

Transposable Element ◽

Heme Oxygenase ◽

Sickle Cell ◽

Conflicts Of Interest ◽

Sleeping Beauty ◽

Heme Oxygenase 1 ◽

Cell Disease

Abstract Abstract 1527 Poster Board I-550 We previously demonstrated that increases in heme oxygenase-1 (HO-1) and its products, carbon monoxide and biliverdin, inhibit inflammation and vasoocclusion in mouse models of sickle cell disease (SCD). In this study, an albumin promoter driven Sleeping Beauty transposase plasmid with a wild type rat hmox-1 gene (wt-HO-1) transposable element (289 amino acids) was delivered by hydrodynamic tail vein injections to SCD mice. Eight weeks after injection, SCD mice had 3-fold increases in HO-1 activity in their livers, similar to hemin-treated mice. mRNA transcription of the transposable element was confirmed by qRT-PCR RFLP analysis. HO-1 overexpressing mice had marked activation of the phospho-p38 MAPK and phospho-Akt proteins and decreased NF-kB p65 in liver nuclear extracts. Hypoxia-induced stasis, a characteristic of SCD but not normal mice, was inhibited in dorsal skin fold chambers in wt-HO-1 SCD mice. None of these effects were seen in SCD mice injected with Sleeping Beauty containing a nonsense rat hmox-1 gene (ns-HO-1) that encodes for amino acids 1-241 with little or no enzymatic activity. Immunohistochemistry of HO-1 in livers demonstrated perinuclear HO-1 staining in both hepatocytes and sinusoidal Kupffer cells in wt- and ns-HO-1-treated mice. We conclude HO-1 gene therapy targeted to the liver is beneficial in SCD by activating cytoprotective signaling pathways and inhibiting vascular stasis at sites distal to transgene expression. Disclosures No relevant conflicts of interest to declare.

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