Unbalanced Globin Synthesis Research Articles

Treatment of 100 chronic thalassemic leg wounds by plasma-rich platelets.

Thalassemia is a heterogeneous group of congenital hemoglobinopathies caused by mutations in the globin gene complex that result in an unbalanced globin synthesis. Unmatched globin chains bind to the cytosolic surface of red blood cell membrane where they cause oxidative damage that might in part be responsible for membrane weakness. The deformability of red blood cells and hypercoagulable state in thalassemic patients have been incriminated in leg ulcer formation, as this might cause ischemia to the skin and consequently friability and ulceration. Platelet-rich plasma (PRP) gel is considered an advanced wound therapy for chronic and acute wounds. PRP gel consists of cytokines, growth factors, chemokines, and a fibrin scaffold derived from a patient's blood. In this study, we treated 100 thalassemic leg wounds using PRP. There was wound size reduction in patients after 4 weeks of treatment. In wounds with 2-3.5cm2 surface area, complete closure happened after 12.5weeks, 5-12cm2 wounds completely closed after 13.2weeks, and finally, 4.5-6cm2 wounds healed completely after 14weeks. None of the wounds reopened after 8months of monitoring. This study will help extrapolation of the use of PRP for at least thalassemic leg wound treatment.

Heme Oxygenase 1 Plays An Unexpected Role During Erythroid Differentiation

Abstract 344Red blood cells (RBC) are produced at a rate of 2.3 × 106 cells per second by a dynamic and exquisitely regulated process known as erythropoiesis. During this development, RBC precursors synthesize the highest amounts of total organismal heme (75–80%), which is a complex of iron with protoporphyrin IX. Heme is essential for the function of all aerobic cells, but if left unbound to protein, it can promote free radical formation and peroxidation reactions leading to cell damage and tissue injury. Therefore, in order to prevent the accumulation of ‘free’ heme, it is imperative that cells maintain a balance of heme biosynthesis and catabolism. Physiologically, the only enzyme capable of degrading heme are heme oxyganase 1 & 2 (HO). Red blood cells contain the majority of heme destined for catabolism; this process takes place in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Heme oxygenase, in particular its heme-inducible isoform HO1, has been extensively studied in hepatocytes and many other non-erythroid cells. In contrast, virtually nothing is known about the expression of HO1 in developing RBC. Likewise, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using primary erythroid cells isolated from mouse fetal livers (FL), we have shown that HO1 mRNA and protein are expressed in undifferenetiated FL cells and that its levels, somewhat surprisingly, increase during erythropoietin-induced erythroid differentiation. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase, the second enzyme in the heme biosynthesis pathway. Moreover, we have found that down-regulation of HO1 via siRNA increases globin protein levels in DMSO-induced murine erythroleukemic (MEL) cells. Similarly, compared to wild type mice, FL cells isolated from HO1 knockout mice (FL/HO1−/−) exhibited increased globin and transferrin receptor levels and a decrease in ferritin levels when induced for differentiation with erythropoietin. Following induction, compared to wild type cells, FL/HO1−/− cells showed increased iron uptake and its incorporation into heme. We therefore conclude that the normal hemoglobinization rate appears to require HO1. On the other hand, MEL cells engineered to overexpress HO1 displayed reduced globin mRNA and protein levels when induced to differentiate. This finding suggests that HO1 could play a role in some pathophysiological conditions such as unbalanced globin synthesis in thalassemias. Disclosures:No relevant conflicts of interest to declare.

α-Thalassemia-Like Globin Gene Expression by Primitive Erythrocytes Derived from Human Embryonic Stem Cells

Under culture conditions that promote hematopoietic differentiation, human embryonic stem cells (huESC) give rise to primitive erythroid cells that closely resemble the nucleated erythrocytes of early-stage human embryos. The globin chain distribution of these cells is similar to that seen during the embryonic and fetal stages of development. Here we show that huESC-derived erythroid cells produce substantial quantities of homotetrameric hemoglobin (Hb) composed exclusively of γ-globin-containing subunits. The globin synthesis of these erythroid cells was also significantly unbalanced, with a substantial decrease of α-like globin chain synthesis in relation to that of their β-like globins, a pattern characteristically associated with α-thalassemia (α-thal). This pattern of unbalanced globin synthesis appears to be an inherent feature of human erythroid cells that synthesize predominantly embryonic-stage globins.

The Role of Heme Oxygenase 1 in Erythroid Differentiation.

Heme is a complex of iron with protoporphyrin IX that is essential for the function of all aerobic cells. However, if left unguarded, non-protein-bound heme promotes free radical formation, resulting in cell damage and tissue injury. The highest amounts of organismal heme (75–80%) are present in circulating red blood cells (RBC) whose precursors synthesize heme with rates that are at least 1 order of magnitude higher than those in the liver (on the per cell basis), which is the second most active heme producer in the body. The only physiological mechanism of heme degradation is performed by heme oxygenases (HO1 and HO2), which catalyze the rate-limiting step in the oxidative degradation of heme and are, therefore, involved in the control of cellular heme levels. Red blood cells contain the majority of heme destined for catabolism; this process takes place in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Although the heme-inducible HO isoform, HO1, has been extensively studied in hepatocytes and many other non-erythroid cells, virtually nothing is known about the expression of HO1 in developing RBC. Similarly, it is unknown whether HO-1 plays any role in erythroid cell development under physiological or pathophysiological conditions. In this study we have shown that HO1 protein is expressed in uninduced murine erythroleukemic (MEL) cells and that its levels, somewhat surprisingly, do not decrease during DMSO-induced erythroid differentiation. Moreover, we demonstrated that heme significantly induces HO1 in both uninduced and induced MEL cells. Additionally, we investigated the effect of overexpressed HO1 on heme and iron metabolism in stably transfected MEL cells (MEL-HO1) and their non-transfected counterparts. Compared to wild type cells, DMSO-treated MEL-HO1 cells displayed a reduction in heme stability (measured by the incorporation of 59Fe into heme) in addition to impairment of erythroid differentiation. Moreover, although wild type and transfected cells expressed similar levels of transferrin receptors in the uninduced state, MEL-HO1 cells, as compared to wild type MEL cells, showed only a small increase in transferrin receptors upon treatment with DMSO. Finally, we measured apoptosis using annexin-V and observed an increase in the number of apoptotic cells in HO1 transfectants, but not in wild type MEL cells. These results suggest that an as yet unknown mechanism exists to protect heme against endogenous HO1 action during physiological erythroid differentiation. In addition, our results showing that high levels of HO1 in erythroid cells cause heme catabolism and a defect in erythroid differentiation raise the possibility that HO1 could play a role in some pathophysiological conditions such as unbalanced globin synthesis in thalassemias.

Unbalanced Globin Chain Synthesis in Erythroid Precursor Cells of Heterozygous αThalassaemia

Globin biosynthesis was studied in both erythroid precursors and reticulocytes of three individuals with heterozygous alpha-thalassaemia. In contrast to the finding of equal or nearly equal alpha and beta chain synthesis in the marrow of patients with heterozygous beta-thalassaemia previously examined, our studies showed equal degrees of unbalanced globin synthesis in both reticulocytes and nucleated-erythroid cells of alpha-thalassaemia heterozygotes. Greater stability and less susceptibility to proteolysis of the excess beta-chain formed in alpha-thalassaemia may explain our findings.

Isolation and in vitro differentiation of human erythroid precursor cells

There is decreased beta-globin production in beta-thalassemic reticulocytes and nucleated erythroid cells. In this study, we have examined whether unbalanced globin synthesis is expressed at all stages of human erythroid cell maturation. In order to determine the pattern of globin synthesis in early erythroid cells during erythroid cell maturation, an in vitro culture system using human bone marrow erythroid precursor cells has been developed. Early erythroid precursor cells (proerythroblasts and basophilic erythroblasts) have been isolated from nonthalassemic and thalassemic human bone marrows by lysing more mature erythroid cells, using complement and a rabbit antiserum prepared against normal human red cells. In the presence of erythropoietin, differentiation and proliferation of erythroid cells in demonstrable in liquid suspension culture for 24–48 hr, as determined by morphological criteria and by an increase in globin synthesis. The ratio of alpha- to beta-globin chain synthesis in nonthalassemic cells in approximately 1 at all stages of erythroid cell differentiation during culture. In cells from four patients with homozygous beta- thalassemia there is decreased beta-globin synthesis compared to alpha- globin synthesis, both in early erythroid precursor cells and during their maturation in culture. These findings indicate that unbalanced globin chain synthesis is expressed at all stages of red cell maturation in homozygous beta-thalassemia.

Isolation and In Vitro Differentiation of Human Erythroid Precursor Cells

There is decreased beta-globin production in beta-thalassemic reticulocytes and nucleated erythroid cells. In this study, we have examined whether unbalanced globin synthesis is expressed at all stages of human erythroid cell maturation. In order to determine the pattern of globin synthesis in early erythroid cells during erythroid cell maturation, an in vitro culture system using human bone marrow erythroid precursor cells has been developed. Early erythroid precursor cells (proerythroblasts and basophilic erythroblasts) have been isolated from nonthalassemic and thalassemic human bone marrows by lysing more mature erythroid cells, using complement and a rabbit antiserum prepared against normal human red cells. In the presence of erythropoietin, differentiation and proliferation of erythroid cells in demonstrable in liquid suspension culture for 24-48 hr, as determined by morphological criteria and by an increase in globin synthesis. The ratio of alpha- to beta-globin chain synthesis in nonthalassemic cells in approximately 1 at all stages of erythroid cell differentiation during culture. In cells from four patients with homozygous beta- thalassemia there is decreased beta-globin synthesis compared to alpha-globin synthesis, both in early erythroid precursor cells and during their maturation in culture. These findings indicate that unbalanced globin chain synthesis is expressed at all stages of red cell maturation in homozygous beta-thalassemia.

Heterozygous beta thalassaemia of unusual severity.

The proband of each of three families of Northern European or Italian extraction had an unusual form of heterozygous beta-thalassaemia, confirmed by haematological, genetic and peripheral blood globin synthesis studies. The unusual severity of this disorder was indicated by chronic haemolysis leading to splenectomy and cholecystectomy, by numerous nucleated red cells and reticulocytes in the peripheral blood, and by leg ulcers in one family. The diversity of clinical expression in many family members with heterozygous beta-thalassaemia was striking. Bone marrow examination in the probands showed numerous large inclusion bodies of the type usually found only in thalassaemia major. In addition, there was unbalanced globin synthesis in the bone marrow, in contrast to the more balanced synthesis found in asymptomatic beta-thalassaemia trait. The amount of newly synthesized alpha-chain found in the free alpha-chain pool was markedly elevated. The unbalanced globin synthesis and alpha-chain inclusions in the bone marrow cells suggest that the severity of the disorder in these patients may be due to the inability of their red cell precursors to fully compensate for the thalassaemic defect or to remove excess alpha-chains. The diversity of clinical expression suggests the influence of undefined acquired or genetic factors on the expression of beta-thalassaemia in these families.

Synthesis of globin chains in sickle -thalassemia.

In five patients with sickle beta-thalassemia there was balanced alpha- and beta-globin synthesis in the bone marrow and decreased total beta-chain synthesis relative to that of alpha-chain in the peripheral blood. These findings are similar to those in patients with simple beta-thalassemia trait. Despite a range of hemoglobin concentrations from 6.8 to 12.5 g/100 ml in the patients with sickle thalassemia, there was no evidence of a significant excess of alpha-chains in the red cells of the bone marrow which could contribute to the hemolysis and anemia. In patients heterozygous for beta-thalassemia the capacity to synthesize beta-chain decreases more rapidly than that for alpha-chain. In nonthalassemic subjects the rates of beta- and alpha-chain synthesis decrease equally as the red cell matures. The beta(S)- and beta(A)-chains serve as convenient markers for globin synthesis due to the nonthalassemic and thalassemic alleles in patients with sickle beta-thalassemia. The unbalanced globin synthesis in the peripheral blood of these patients is explained by the decrease in relative synthesis of beta(S)-chain, in comparison with that of alpha-chain. This instability is not present in sickle cell trait. The beta(A)-chain synthesis was only unstable in the two patients who had the most marked anemia. The major mechanism for achieving balanced globin production in the bone marrow in the presence of one thalassemic gene appears to be increased synthesis of beta-chain due to the nonthalassemic allele. In addition, there may be a decrease of total alpha-chain synthesis in some patients.