Involvement of Phosphatases in Proliferation, Maturation, and Hemoglobinization of Developing Erythroid Cells

Production of RBCs is triggered by the action of erythropoietin (Epo) through its binding to surface receptors (Epo-R) on erythroid precursors in the bone marrow. The intensity and the duration of the Epo signal are regulated by several factors, including the balance between the activities of kinesase and phosphatases. The Epo signal determines the proliferation and maturation of the precursors into hemoglobin (Hb)-containing RBCs. The activity of various protein tyrosine phosphatases, including those involved in the Epo pathway, can be inhibited by sodium orthovanadate (Na3VO4, vanadate). Adding vanadate to cultured erythroid precursors of normal donors and patients with β-thalassemia enhanced cell proliferation and arrested maturation. This was associated with an increased production of fetal hemoglobin (HbF). Increased HbF in patients with β-hemoglobinopathies (β-thalassemia and sickle cell disease) ameliorates the clinical symptoms of the disease. These results raise the possibility that specific and nontoxic inhibitors of phosphatases may be considered as a therapeutic modality for elevating HbF in patients with β-hemoglobinopathies as well as for intensifying the Epo response in other forms of anemia.

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Hydroxyurea Treatment for Sickle Cell Disease

The Scientific World JOURNAL ◽

10.1100/tsw.2002.295 ◽

2002 ◽

Vol 2 ◽

pp. 1706-1728 ◽

Cited By ~ 13

Author(s):

Martin H. Steinberg

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Fetal Hemoglobin ◽

Hemoglobin Concentration ◽

Pharmacologic Therapy ◽

Acute Chest Syndrome ◽

Cell Disease ◽

Globin Chains ◽

Hydroxyurea Treatment ◽

Erythroid Precursors

High fetal hemoglobin (HbF) levels inhibit the polymerization of sickle hemoglobin (HbS) and reduce the complications of sickle cell disease. Pharmacologic agents that can reverse the switch from γ- to β-chain synthesis — γ-globin chains characterize HbF, and sickle β-globin chains are present in HbS — or selectively increase the proportion of adult erythroid precursors that maintain the ability to produce HbF are therapeutically useful. Hydroxyurea promotes HbF production by perturbing the maturation of erythroid precursors. This treatment increases the total hemoglobin concentration, reduces the vaso-occlusive complications of pain and acute chest syndrome, and attenuates mortality in adults. It is a promising beginning for pharmacologic therapy of sickle cell disease. Still, its effects are inconsistent, trials in infants and children are ongoing, and its ultimate value — and peril — when started early in life are still unknown.

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Morphologic Studies in a Recently Described Dyserythropoietic State

Blood ◽

10.1182/blood.v40.4.473.473 ◽

1972 ◽

Vol 40 (4) ◽

pp. 473-486 ◽

Cited By ~ 4

Author(s):

Arnold I. Freeman ◽

John A. Edwards ◽

Maimon Cohen ◽

Lucius F. Sinks

Keyword(s):

Bone Marrow ◽

Light Microscopy ◽

Blood Cells ◽

Marker Chromosome ◽

Fetal Hemoglobin ◽

Nuclear Bodies ◽

Hemoglobin F ◽

Cytoplasmic Inclusions ◽

Structural Abnormalities ◽

Erythroid Precursors

Abstract Studies of fetal hemoglobin and bone marrow ultrastructure were performed on a patient with a recently described syndrome consisting of lymphosarcoma, short stature, defective cellular immunity, hypogammaglobulinemia, and mosaicism for a marker chromosome. Abnormalities of the erythroid cells, including multinucleated erythroblasts, nuclear bodies, megaloblasts, and frayed cytoplasm, were seen by light microscopy. Ultrastructure confirmed the findings by light microscopy and showed in greater detail nuclear malformation, loss of nuclear membrane, and loss of cytoplasmic background, as well as peculiar cytoplasmic inclusions. An increased level of fetal hemoglobin (Hb F), ranging between 12%-15%, was detected. The Hb F was discreetly distributed, occurring in a small per cent of the red blood cells. It is speculated that a clone of chromosomally abnormal erythroid precursors might be responsible for producing the erythroblasts with the structural abnormalities described and the erythrocytes with the high hemoglobin F content.

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In Vivo Characterization of Ftx-6058, a Novel Small Molecular Fetal Hemoglobin Inducer for Sickle Cell Disease

Blood ◽

10.1182/blood-2020-139566 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 26-27

Author(s):

Keqiang Xie ◽

Mark Roth ◽

Ivan Efremov ◽

Serena Silver ◽

Lucienne Ronco ◽

...

Keyword(s):

Bone Marrow ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Fetal Hemoglobin ◽

Erythroid Cells ◽

Cell Disease ◽

Publicly Traded ◽

F Cells ◽

Biological Target

Sickle cell disease (SCD) results from genetic mutation in the β-globin gene encoding a subunit of the adult form of hemoglobin (HbA), leading to red blood cell (RBC) deformation and disease pathology. It has been demonstrated that reactivation of the fetal ortholog of the hemoglobin beta subunit, HBγ (also referred to as HBG proteins), can prevent or reduce disease-related pathophysiology. In SCD, the presence of HBG protein in hemoglobin tetramers prevents sickle hemoglobin polymerization under deoxygenated conditions and therefore may be of therapeutic benefit in SCD. FTX 6058, a novel orally bioavailable small molecule, is in development for the treatment of sickle cell disease (SCD) by Fulcrum Therapeutics. FTX-6058 was demonstrated to inhibit the novel biological target and elevate the expression of HBγ, resulting in induction of fetal hemoglobin (HbF) tetramer in differentiated human primary CD34+ cells. The in vivo target engagement (TE) and pharmacologic effects of FTX-6058 were characterized in wild-type CD-1 mice and humanized Townes SCD mice, with TE also confirmed in non-human primates. In CD-1 mice, once-daily (QD) FTX-6058 oral administration induced TE in a time- and dose-dependent manner and most markedly in erythroid lineage (Ter119+) cells derived from bone marrow, the putative therapeutic compartment, and increased transcript levels of Hbb-bh1, a murine embryonic hemoglobin surrogate for human HBG gene. Steady state TE in circulating monocytes, following repeated QD FTX-6058 administration, correlated well with that in bone marrow-derived erythroid cells, suggesting peripheral monocytes as a suitable surrogate for assessing erythroid TE activity in Fulcrum's Phase 1 study. In non-human primate cynomolgus monkeys, QD oral dosing of FTX-6058 as early as for 7 days induced robust and comparable TE in bone marrow derived CD34+ erythroid progenitors and circulating monocytes, further supporting the use of monocytes to assess TE in bone marrow. Mouse data also provided evidence of the reversibility of TE effects once dosing is stopped. In repeat-dose studies in the humanized Townes SCD mouse model, FTX-6058 was superior to standard of care hydroxyurea as measured by human HBG1 transgene induction and increased %F-cells and HBG1 protein levels. Furthermore, the induction of %F cells was sustainable for several days after dosing cessation. These in vivo studies have demonstrated that FTX-6058 engages its novel biological target in multiple preclinical models and induces HbF expression at plasma concentrations likely to b e readily achievable in clinic, and peripheral monocytes is a suitable surrogate for assessing TE in bone marrow erythroid cells, which could be beneficial to patients with SCD. Disclosures Xie: Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Roth:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Efremov:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Silver:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Ronco:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Thompson:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Stickland:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Moxham:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Wallace:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company.

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Sickled Erythrocyte Morphology in the Bone Marrow of Patients with Sickle Cell Disease: Previously Unrecognized Forms of Sickled Erythrocytes May Have a Pathogenetic Role in Bone Pain of Painful Crises.

Blood ◽

10.1182/blood.v106.11.3802.3802 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3802-3802

Author(s):

George T. Roberts

Keyword(s):

Bone Marrow ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Peripheral Blood ◽

Morphological Characteristics ◽

Fetal Hemoglobin ◽

Cell Disease ◽

Erythrocyte Morphology ◽

Pathogenetic Role ◽

Hbf Level

Abstract Background: The painful crises of sickle cell disease (SCD) are characteristically localized to the skeleton. Various lines of evidence suggest that bone and bone marrow (BM) infarction contribute to the pathophysiology of such pain in which sickled red blood cells (SRBC) are believed to play a part. However, there is little information about the morphological characteristics and role of SRBC in BM during the crises or indeed, in the “steady state”. Occasional light microscopic observations in the BM of SCD patients show numerous markedly distorted cells, presumed to be SRBC, types of which have not been previously reported from this site or the peripheral blood (PB). We therefore studied a series of BM biopsies from SCD patients to determine whether such changes are consistently present. Methods: We microscopically reviewed archival BM biopsies from SCD patients for abnormally shaped RBC and compared the findings with SRBC on Wright-stained smears of PB collected simultaneously with the biopsies. The material consisted of Wright-Giemsa stained smears of aspirated BM and hematoxylin-eosin stained histological sections of clotted aspirate and / or trephine biopsies. We also examined fresh BM RBC by scanning and transmission electron microscopy (EM). We determined the relationship between the percentage of SRBC in BM and PB on the one hand, and fetal hemoglobin (HbF) levels on the other. Results: We studied the BM of 43 SCD patients. By LM, we observed markedly distorted RBC in the BM of the majority of patients who also had BM SRBC that were more typical forms. The distorted forms included vastly elongated erythrocytes, measuring up to 40 microns in length and less than 1 micron in width, some fixed into rod like structures, but others as sinuous, snake-like forms that have not been previously described. Scanning EM appearances also confirmed the features observed by LM. By contrast, none of PB samples showed similarly distorted SRBC. Although the percentage of typical SRBC in BM was concordant with that in the PB (p<0.001) SRBC numbers in both PB and BM were inversely proportional to the level of HbF.(Table 1) Conclusion: We conclude that the proportions of markedly distorted SRBC present in the BM of SCD patients are inversely proportional to HbF level and may thus play a pathogenetic role in the localization of severe pain to the skeleton during painful crises. Table 1. Correlations between sickled RBC estimates and blood fetal hemoglobin levels MSRBC MARROW MBMSRBC MSRBCLOT+TREPHINE MSRBC PB MSRBC= Mean SRBC marrow and peripheral blood together. MBMSRBC = Mean SRBC in bone marrow aspirates only. MSRBCLOT+TREPHINE = Mean SRBC, clotted marrow aspirate and trephine together. MSRBCPB = Mean SRBC in peripheral blood. MHbF = Mean HbF level in blood. r = correlation coefficient. NS = Not significant. MHbF MHbF MHbF MHbF r −0.3806 −0.3293 −0.2685 −0.384 p < 0.05 < 0.05 NS < 0.05

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Leukocyte Immunoglobulin-Like Receptor B Regulates Erythroblastic Island Size in Human in-Vitro Erythroblastic Islands

Blood ◽

10.1182/blood.v120.21.3190.3190 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3190-3190

Author(s):

Ajay Perumbeti ◽

Alin Girnita ◽

Jose A. Cancelas

Keyword(s):

Bone Marrow ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Close Contact ◽

Surface Receptors ◽

Erythroblastic Island ◽

Erythroid Precursors ◽

Surface Receptor

Abstract Abstract 3190 Erythropoiesis occurs in a unique bone marrow (BM) microenvironment known as erythroblastic islands. Erythroblastic islands are composed of erythroid precursors in close contact with specialized stromal macrophages, also referred to as central or island macrophages. Human erythroblastic island macrophages have been difficult to discern from other macrophage subpopulations by cell surface markers, although they have reduced expression of C3b receptor (CD35) (Lee et al., 1988). The unique non-antigen presenting nature of erythroblastic island macrophages led us to hypothesize that it may share surface receptors of other tolerogenic macrophages subpopulations, including leukocyte immunoglobulin-like transcript B receptors (LILRB). We first identified a distinct subset of human bone marrow macrophages with flow cytometry, which express high amounts of LILRB1 and LILRB2 surface receptors, have low expression of C3b surface receptor (p<0.05), and have increased expression of VCAM-1 surface receptor. This macrophage subpopulation consisted of 1 in 500 (0.02%) of bone marrow mononuclear cells. Functionally, when we FACS sorted human BM enriched erythroid precursors (CD45−/CD71+) and combined them with FACS sorted LILRB1+/LILRB2+ (LILRB+) or LILRB1−/LILRB2- (LILRB−) BM macrophages, there were no significant differences in erythroblastic island reconstitution, suggesting that the adhesion of primary BM erythroblasts to island macrophages is not related to macrophage LILRB expression. Since our ex-vivo erythroid population was enriched for late precursors, we tested island reconstitution with CD34+ cell-derived cultured erythroblasts and FACS sorted LILRB+ or LILRB- BM macrophages, which resulted in a significantly higher number of large proliferative erythroblastic islands (>25 erythroblasts/island macrophage) in LILRB+ compared with LILRB- macrophages (p<0.05) (Figure 1). Incubation with neutralizing anti-LILRB1/LILRB2 antibodies did not prevent primary erythroblastic island reconstitution independent of expression of LILRB by macrophages. However, anti-LILRB1/LILRB2 did prevent the formation of large erythroblastic islands indicating a specific role of LILRB in optimal island reconstitution and erythropoiesis (p<0.05) (Figure 1). In conclusion, we demonstrate that LILRB+ BM macrophages increase the formation of large in-vitro erythroblastic islands and LILRB receptors are important candidate receptors for functional regulation of erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

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δ-Aminolevulinate induces fetal hemoglobin expression by enhancing cellular heme biosynthesis

Experimental Biology and Medicine ◽

10.1177/1535370219872995 ◽

2019 ◽

Vol 244 (14) ◽

pp. 1220-1232

Author(s):

Li Liu ◽

Xingguo Zhu ◽

Alexander Yu ◽

Christina M Ward ◽

Betty S Pace

Keyword(s):

Therapeutic Strategy ◽

Clinical Symptoms ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Heme Biosynthesis ◽

Erythroid Cell ◽

Nuclear Factor ◽

Cell Disease ◽

Erythroid Progenitors ◽

Novel Therapeutic

Sickle cell disease (SCD) and β-thalassemia are inherited blood disorders caused by genetic defects in the β-globin gene on chromosome 11, producing severe disease in people worldwide. Induction of fetal hemoglobin consisting of two α-globin and two γ-globin chains ameliorates the clinical symptoms of both disorders. In the present study, we investigated the ability of δ-aminolevulinate (ALA), the heme precursor, to activate γ-globin gene expression as well as its effects on cellular functions in erythroid cell systems. We demonstrated that ALA induced γ-globin expression at both the transcriptional and protein levels in the KU812 erythroid cell line. Using inhibitors targeting two enzymes in the heme biosynthesis pathway, we showed that cellular heme biosynthesis was involved in ALA-mediated γ-globin activation. Moreover, the transcription factor NRF2 (nuclear factor [erythroid-derived 2]-like 2), a critical regulator of the cellular antioxidant response, was activated by ALA and contributed to mechanisms of γ-globin activation; ALA did not affect cell proliferation and was not toxic to cells. Subsequent studies demonstrated ALA-induced γ-globin activation in erythroid progenitors generated from normal human CD34+ stem cells. These data support future study to explore the potential of stimulating intracellular heme biosynthesis by ALA or similar compounds as a novel therapeutic strategy for treating SCD and β-thalassemia. Impact statement Inherited mutations in the β-globin-like genes result in the most common forms of genetic blood disease including sickle cell disease (SCD) and β-thalassemia worldwide. Therefore, effective inexpensive therapies that can be distributed widely are highly desirable. Currently, drug-mediated fetal hemoglobin (HbF) induction can ameliorate clinical symptoms of SCD and β-thalassemia and is the most effective strategy for developing new therapeutic options. In the current study, we confirmed that δ-Aminolevulinate (ALA), the precursor of heme, induces γ-globin expression at both the transcriptional and translational levels in primary human erythroid progenitors. Moreover, the results indicate activation of the transcription factor NRF2 (nuclear factor (erythroid-derived 2)-like 2) by ALA to enhance HbF expression. These data support future study to explore the potential of stimulating intracellular heme biosynthesis by ALA or similar compounds as a novel therapeutic strategy for treating SCD and β-thalassemia.

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Adenosine Base Editing of γ-Globin Promoters Induces Fetal Hemoglobin and Inhibit Erythroid Sickling

Blood ◽

10.1182/blood-2020-141498 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 21-22

Author(s):

Thiyagaraj Mayuranathan ◽

Jonathan S. Yen ◽

Gregory A. Newby ◽

Yu Yao ◽

Shaina N. Porter ◽

...

Keyword(s):

Bone Marrow ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Fetal Hemoglobin ◽

Erythroid Cell ◽

Cell Disease ◽

Private Company ◽

Hematopoietic Stem ◽

Benign Condition ◽

Base Editing

Rare variants in the γ-globin (HBG2 and HBG1) promoters cause sustained postnatal expression of fetal hemoglobin (HbF, α2γ2) in red blood cells (RBCs). This benign condition is termed hereditary persistence of fetal hemoglobin (HPFH). Individuals with HPFH variants are protected from β-hemoglobinopathies including sickle cell disease and β-thalassemia. Our group and others have used CRIPSR/Cas9-mediated non-homologous end joining to generate HPFH-like insertion-deletion (indel) mutations in the γ-globin promoter. However, simultaneous double-stranded breaks (DSBs) in the tandem duplicated γ-globin genes can result in loss or inversion of the intervening genetic material and/or chromosomal rearrangements. More generally, Cas9-associated DSBs can elicit a cytotoxic DNA repair response leading to cell death or evoke p53 loss with malignant transformation. Base editor (BE) proteins represent a promising approach to install precise nucleotide substitutions without DSBs. Adenosine base editors (ABEs), consisting of catalytically impaired Cas9 fused to a modified adenosine deaminase, create targeted A:T-to-G:C mutations. Here we describe the use of ABEs to recapitulate naturally occurring HPFH variants in hematopoietic stem cells (HSCs). We electroporated ABE7.10-single guide (sg) RNA ribonucleoprotein (RNP) complex into mobilized peripheral blood CD34+ hematopoietic stem and progenitor cells (HSPCs) to recreate 3 different HPFH variants in the HBG1/2 promoters (-198 T>C, -175 T>C and -113 A>G). Measured editing frequency was maximal on day 10 after electroporation and transferred to erythroid differentiation media. 20% editing efficiency was observed for the -198 site, 58% for -175 and 50% for -113. Indel frequencies were <2% at each of the three sites, reflecting a low rate of DSBs. Fetal hemoglobin levels in erythroid cells generated in vitro from A base-edited CD34+ HSPCs were 26±4% (-198 T>C), 60±10% (-175 T>C), and 42±7% (-113 A>G) versus14±2% in unedited control cells. Base editing at the -175 site in sickle cell disease (SCD) donor CD34+ HSPCs resulted in the induction of HbF to 55% in erythroid progeny compared to 6% in controls. After exposure to hypoxia (2% oxygen), reticulocytes generated from -175 T>C-edited CD34+ HSPCs exhibited sickling rates of 24%, compared to 52% in controls. Thus, creation of this variant, which generates a de novo binding site for the transcriptional activator TAL1, reactivates erythroid cell HbF to levels that inhibit sickle hemoglobin polymerization and cell sickling. To assess base editing in HSCs, we used ABE RNP to modify the -175 site in SCD donor CD34+ HSPCs, followed by transplantation into NBSGW mice. The editing frequency in CD34+ HSPCs before transplantation was ~30% and declined to approximately 20% in bone marrow-repopulating donor cells at 16 weeks post-transplantation. Editing frequencies were similar in CD34+ donor cell-derived myeloid, erythroid, and B cells, indicating that hematopoietic differentiation was not altered. Bone marrow erythroblasts derived from base-edited and control CD34+ HSPCs exhibited similar maturation profiles and enucleation. Erythroblasts generated in vivo from SCD patient HSPCs exhibited 32±2% HbF compared to unedited controls (4±1%) (n=4, P>0.0001). Our studies provide proof of concept that adenosine base editors can be used therapeutically for β-hemoglobinopathies. Specifically, generation of the -175 T>C HPFH mutation in patient HSCs followed by autologous transplantation represents a new therapeutic approach for SCD and β-thalassemia. Disclosures Yen: Beam Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Sharma:Spotlight Therapeutics: Consultancy; Magenta Therapeutics: Other: Research Collaboration; CRISPR Therapeutics, Vertex Pharmaceuticals, Novartis: Other: Clinical Trial PI. Liu:Pairwise Plants: Consultancy, Patents & Royalties; Editas Medicine: Consultancy, Patents & Royalties; Beam Therapeutics: Consultancy, Patents & Royalties; Prime Medicine: Consultancy, Patents & Royalties. Weiss:Beam Therapeuticcs: Consultancy, Current equity holder in private company; Esperion Therapeutics: Consultancy, Current equity holder in private company; Novartis: Consultancy, Current equity holder in private company; Cellarity Inc.: Consultancy, Current equity holder in private company; Rubius Inc.: Consultancy, Current equity holder in private company.

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Experimental Type 2 Diabetes Differently Impacts on the Select Functions of Bone Marrow-Derived Multipotent Stromal Cells

Cells ◽

10.3390/cells10020268 ◽

2021 ◽

Vol 10 (2) ◽

pp. 268

Author(s):

Jonathan Ribot ◽

Cyprien Denoeud ◽

Guilhem Frescaline ◽

Rebecca Landon ◽

Hervé Petite ◽

...

Keyword(s):

Type 2 Diabetes ◽

Bone Marrow ◽

Stromal Cells ◽

Adipogenic Differentiation ◽

Therapeutic Modality ◽

Multipotent Stromal Cells ◽

Cell Therapies

Bone marrow-derived multipotent stromal cells (BMMSCs) represent an attractive therapeutic modality for cell therapy in type 2 diabetes mellitus (T2DM)-associated complications. T2DM changes the bone marrow environment; however, its effects on BMMSC properties remain unclear. The present study aimed at investigating select functions and differentiation of BMMSCs harvested from the T2DM microenvironment as potential candidates for regenerative medicine. BMMSCs were obtained from Zucker diabetic fatty (ZDF; an obese-T2DM model) rats and their lean littermates (ZL; controls), and cultured under normoglycemic conditions. The BMMSCs derived from ZDF animals were fewer in number, with limited clonogenicity (by 2-fold), adhesion (by 2.9-fold), proliferation (by 50%), migration capability (by 25%), and increased apoptosis rate (by 2.5-fold) compared to their ZL counterparts. Compared to the cultured ZL-BMMSCs, the ZDF-BMMSCs exhibited (i) enhanced adipogenic differentiation (increased number of lipid droplets by 2-fold; upregulation of the Pparg, AdipoQ, and Fabp genes), possibly due to having been primed to undergo such differentiation in vivo prior to cell isolation, and (ii) different angiogenesis-related gene expression in vitro and decreased proangiogenic potential after transplantation in nude mice. These results provided evidence that the T2DM environment impairs BMMSC expansion and select functions pertinent to their efficacy when used in autologous cell therapies.

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Fetal hemoglobin induction by acetate, a product of butyrate catabolism [see comments]

Blood ◽

10.1182/blood.v84.9.3198.3198 ◽

1994 ◽

Vol 84 (9) ◽

pp. 3198-3204 ◽

Cited By ~ 32

Author(s):

G Stamatoyannopoulos ◽

CA Blau ◽

B Nakamoto ◽

B Josephson ◽

Q Li ◽

...

Keyword(s):

Fatty Acids ◽

Sickle Cell Disease ◽

Umbilical Cord Blood ◽

Sodium Acetate ◽

Fetal Hemoglobin ◽

Cell Disease ◽

Gamma Globin ◽

Hbf Induction ◽

Globin Synthesis

Abstract Butyrate induces fetal hemoglobin (HbF) synthesis in cultures of erythroid progenitors, in primates, and in man. The mechanism by which this compound stimulates gamma-globin synthesis is unknown. In the course of butyrate catabolism, beta oxidation by mitochondrial enzymes results in the formation of two acetate molecules from each molecule of butyrate. Studies were performed to determine whether acetate itself induces HbF synthesis. In erythroid burst-forming unit (BFU-E) cultures from normal persons, and individuals with sickle cell disease and umbilical-cord blood, dose-dependent increases in gamma-globin protein and gamma mRNA were consistently observed in response to increasing acetate concentrations. In BFU-E cultures from normal adults and patients with sickle cell disease, the ratio of gamma/gamma + beta mRNA increased twofold to fivefold in response to acetate, whereas the percentage of BFU-E progeny staining with an anti-gamma monoclonal antibody (MoAb) increased approximately twofold. Acetate-induced increases in gamma-gene expression were also noted in the progeny of umbilical cord blood BFU-E, although the magnitude of change in response to acetate was less because of a higher baseline of gamma- chain production. The effect of acetate on HbF induction in vivo was evaluated using transgenic mouse and primate models. A transgenic mouse bearing a 2.5-kb mu locus control region (mu LCR) cassette linked to a 3.3-kb A gamma gene displayed a near twofold increase in gamma mRNA during a 10-day infusion of sodium acetate at a dose of 1.5 g/kg/d. Sodium acetate administration in baboons, in doses ranging from 1.5 to 6 g/kg/d by continuous intravenous infusion, also resulted in the stimulation of gamma-globin synthesis, with the percentage of HbF- containing reticulocytes (F reticulocytes) approaching 30%. Surprisingly, a dose-response effect of acetate on HbF induction was not observed in the baboons, and HbF induction was not sustained with prolonged acetate administration. These results suggest that both two- carbon fatty acids (acetate) and four-carbon fatty acids (butyrate) stimulate synthesis of HbF in vivo.

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