Constance Tom Noguchi

Hematologic Responses of Patients with Sickle Cell Disease to Treatment with Hydroxyurea

Because fetal hemoglobin contains gammaglobin chains instead of beta chains, it is not affected by the genetic defect that causes sickle cell disease. Increased levels of fetal hemoglobin decrease the tendency toward intracellular polymerization of sickle hemoglobin that characterizes this disease. Hydroxyurea is one of several cytostatic agents that have been shown to increase the production of fetal hemoglobin in some patients with sickle cell disease. We studied the effects of hydroxyurea administration in 10 hospitalized patients with sickle cell disease, each of whom was treated for three months. Seven patients responded with a 2- to 10-fold increase in fetal hemoglobin, from a mean (+/- SD) of 1.6 +/- 1.6 percent of total hemoglobin to 6.8 +/- 4.7 percent; three patients had fetal-hemoglobin levels of 10 to 15 percent of total hemoglobin. Three did not respond to treatment. Four of the patients who responded were retreated with hydroxyurea after one to four months without treatment and were found to have larger increases in fetal-hemoglobin levels. In most patients, levels were still rising at the end of the study, even after 90 days of therapy. Fetal-hemoglobin levels tended to peak at dosages of hydroxyurea that were myelosuppressive. In the patients who responded to treatment, there were significant increases in the percentage of reticulocytes and erythrocytes containing fetal hemoglobin and in the amount of fetal hemoglobin within these cells. The percentage of dense red cells decreased in the patients who responded to treatment. The tendency toward intracellular polymerization at physiologic oxygen saturation was reduced by about 33 percent in the cells containing fetal hemoglobin, whereas there was no change in the other cells. We conclude that hydroxyurea is effective in increasing the production of fetal hemoglobin, which in this study was found to be associated with a small decrease in hemolysis and an increase in hemoglobin levels despite myelosuppression. Controlled, prospective trials are necessary to establish whether these effects will lead to clinical benefit.

Erythropoietin Stimulates Proliferation and Interferes with Differentiation of Myoblasts

Erythropoietin (Epo) is required for the production of mature red blood cells. The requirement for Epo and its receptor (EpoR) for normal heart development and the response of vascular endothelium and cells of neural origin to Epo provide evidence that the function of Epo as a growth factor or cytokine to protect cells from apoptosis extends beyond the hematopoietic lineage. We now report that the EpoR is expressed on myoblasts and can mediate a biological response of these cells to treatment with Epo. Primary murine satellite cells and myoblast C2C12 cells, both of which express endogenous EpoR, exhibit a proliferative response to Epo and a marked decrease in terminal differentiation to form myotubes. We also observed that Epo stimulation activates Jak2/Stat5 signal transduction and increases cytoplasmic calcium, which is dependent on tyrosine phosphorylation. In erythroid progenitor cells, Epo stimulates induction of transcription factor GATA-1 and EpoR; in C2C12 cells, GATA-3 and EpoR expression are induced. The decrease in differentiation of C2C12 cells is concomitant with an increase in Myf-5 and MyoD expression and inhibition of myogenin induction during differentiation, altering the pattern of expression of the MyoD family of transcription factors during muscle differentiation. These data suggest that, rather than acting in an instructive or specific mode for differentiation, Epo can stimulate proliferation of myoblasts to expand the progenitor population during differentiation and may have a potential role in muscle development or repair.

Augmentation by erythropoietin of the fetal-hemoglobin response to hydroxyurea in sickle cell disease.

BACKGROUND Hydroxyurea increases the production of fetal hemoglobin in patients with sickle cell anemia, inhibiting the polymerization of hemoglobin S and potentially improving vaso-occlusive manifestations and hemolysis. Recombinant erythropoietin increases the number of reticulocytes containing fetal hemoglobin in laboratory animals and in humans. We studied whether hydroxyurea and erythropoietin might have a potentiating effect on the production of fetal hemoglobin in patients with sickle cell disease. METHODS We treated four patients who were receiving hydroxyurea for sickle cell disease (three who were homozygous for sickle cell anemia and one with sickle beta zero-thalassemia) with escalating doses of intravenous erythropoietin for seven weeks, along with oral iron sulfate. Doses of hydroxyurea on four consecutive days were alternated with doses of erythropoietin on three consecutive days. RESULTS There was a 28 percent increase in the number of reticulocytes containing fetal hemoglobin and a 48 percent increase in the percentage of fetal hemoglobin, as compared with the maximal values obtained with hydroxyurea alone. The percentage of erythrocytes containing fetal hemoglobin (F cells) increased from 64 to 78 percent. As compared with hydroxyurea alone, treatment with hydroxyurea and erythropoietin decreased the mean (+/- SD) serum indirect bilirubin level from 0.8 +/- 0.2 to 0.5 +/- 0.1 mg per deciliter (13.3 +/- 2.9 to 8.9 +/- 2.2 mumol per liter) (P = 0.02), suggesting a further decrease in hemolysis. Red-cell filterability improved. CONCLUSIONS Intravenous recombinant erythropoietin with iron supplementation alternating with hydroxyurea elevates fetal-hemoglobin and F-cell levels more than hydroxyurea alone. Such increases decrease intracellular polymerization of hemoglobin S and improve the overall rheologic characteristics of erythrocytes. A reduced dosage of hydroxyurea alternating with erythropoietin may prove less myelotoxic than hydroxyurea given daily or in pulsed-dose regimens. It may also increase levels of fetal hemoglobin in patients with sickle cell disease who have not been helped by hydroxyurea alone.

Levels of fetal hemoglobin necessary for treatment of sickle cell disease.

THE advent of pharmacologic strategies for increasing γ-chain synthesis in patients with sickle cell disease and other hemoglobinopathies1 2 3 4 5 6 7 has made it important to ascertain the benefit...

Regulated Human Erythropoietin Receptor Expression in Mouse Brain

Erythropoietin (Epo) is known for its role in erythropoiesis and acts by binding to its receptor (EpoR) on the surface of erythroid progenitors. EpoR activity follows the site of hematopoiesis from the embryonic yolk sac to the fetal liver and then the adult spleen and bone marrow. Expression of EpoR has also been observed in selected cells of non-hematopoietic origin, such as the embryonic mouse brain during mid-gestation, at levels comparable to adult bone marrow. EpoR transcripts in brain decrease during development falling by birth to less than 1–3% of the level in hematopoietic tissue. We have now recapitulated this pattern of expression using a human EpoR transgene consisting of an 80-kb human EpoR genomic fragment. The highest level of expression was observed in the embryonic yolk sac and fetal liver, analogous to the endogenous gene, in addition to expression in adult spleen and bone marrow. Although activity of this transgene in brain is initially lower than the endogenous gene, it does exhibit the down-regulation observed for the endogenous gene in adult brain. The expression pattern of hybrid transgenes of an hEpoR promoter fused to β-galactosidase in 9.5-day embryos suggested that the hEpoR promoter region between −1778 and −150 bp 5′ of the transcription start site is necessary to direct EpoR expression in the neural tube. EpoR expression in the neural tube may be the origin of the EpoR transcripts detected in brain during development. These data demonstrate that both the mouse and human EpoR genes contain regulatory elements to direct significant levels of expression in a developmentally controlled manner in brain and suggest that in addition to its function during erythropoiesis, EpoR may play a role in the development of selected non-hematopoietic tissue.

Periodic microcirculatory flow in patients with sickle-cell disease

We have applied the technique of laser-Doppler velocimetry to compare patterns of cutaneous blood flow in the forearms of patients with stable sickle-cell disease, with the patterns in normal subjects matched for age, race, and sex, and in patients with anemia due to beta+-thalassemia. The mean resting blood flow in the patients with sickle-cell disease was comparable to that of the control groups but was associated with large, local oscillations in flow with periods of 7 to 10 seconds and peak-to-trough magnitudes about half the mean flow. Oscillations occurred simultaneously at sites separated by 1 cm but were independent in phase and frequency. Since these laser-Doppler measurements represent the average flow pattern in about 1 mm3 of skin (i.e., in approximately 50 to 70 capillary loops), these results suggest that microcirculatory flow in patients with sickle-cell disease proceeds by synchronization of rhythmic flow in large domains of microvessels. These findings indicate that periodic flow may be a compensatory mechanism to offset the deleterious altered rheology of erythrocytes containing polymerized hemoglobin S, and suggest that laser-Doppler velocimetry may be a useful method to investigate microvascular physiology in patients with sickle-cell disease.

Endogenous erythropoietin signaling is required for normal neural progenitor cell proliferation.

Erythropoietin (Epo) and its receptor (EpoR), critical for erythropoiesis, are expressed in the nervous system. Prior to death in utero because of severe anemia EpoR-null mice have fewer neural progenitor cells, and differentiated neurons are markedly sensitive to hypoxia, suggesting that during development Epo stimulates neural cell proliferation and prevents neuron apoptosis by promoting oxygen delivery to brain or by direct interaction with neural cells. Here we present evidence that neural progenitor cells express EpoR at higher levels compared with mature neurons; that Epo stimulates proliferation of embryonic neural progenitor cells; and that endogenous Epo contributes to neural progenitor cell proliferation and maintenance. EpoR-null mice were rescued with selective EpoR expression driven by the endogenous EpoR promoter in hematopoietic tissue but not in brain. Although these mice exhibited normal hematopoiesis and erythrocyte production and survived to adulthood, neural cell proliferation and viability were affected. Embryonic brain exhibited increased neural cell apoptosis, and neural cell proliferation was reduced in the adult hippocampus and subventricular zone. Neural cells from these animals were more sensitive to hypoxia/glutamate neurotoxicity than normal neurons in culture and in vivo. These observations demonstrate that endogenous Epo/EpoR signaling promotes cell survival in embryonic brain and contributes to neural cell proliferation in adult brain in regions associated with neurogenesis. Therefore, Epo exerts extra-hematopoietic function and contributes directly to brain development, maintenance, and repair by promoting cell survival and proliferation independent of insult, injury, or ischemia.

Inhaled nitric oxide augments nitric oxide transport on sickle cell hemoglobin without affecting oxygen affinity

Nitric oxide (NO) inhalation has been reported to increase the oxygen affinity of sickle cell erythrocytes. Also, proposed allosteric mechanisms for hemoglobin, based on S-nitrosation of beta-chain cysteine 93, raise the possibility of altering the pathophysiology of sickle cell disease by inhibiting polymerization or by increasing NO delivery to the tissue. We studied the effects of a 2-hour treatment, using varying concentrations of inhaled NO. Oxygen affinity, as measured by P(50), did not respond to inhaled NO, either in controls or in individuals with sickle cell disease. At baseline, the arterial and venous levels of nitrosylated hemoglobin were not significantly different, but NO inhalation led to a dose-dependent increase in mean nitrosylated hemoglobin, and at the highest dosage, a significant arterial-venous difference emerged. The levels of nitrosylated hemoglobin are too low to affect overall hemoglobin oxygen affinity, but augmented NO transport to the microvasculature seems a promising strategy for improving microvascular perfusion.

The Sonic Hedgehog Pathway Mediates Carbamylated Erythropoietin-enhanced Proliferation and Differentiation of Adult Neural Progenitor Cells

Carbamylated erythropoietin (CEPO), a well characterized erythropoietin (EPO) derivative, does not bind to the classical EPO receptor and does not stimulate erythropoiesis. Using neural progenitor cells derived from the subventricular zone of the adult mouse, we investigated the effect of CEPO on neurogenesis and the associated signaling pathways in vitro. We found that CEPO significantly increased neural progenitor cell proliferation and promoted neural progenitor cell differentiation into neurons, which was associated with up-regulation of Sonic hedgehog (Shh), its receptor ptc, and mammalian achaete-scute homolog 1 (Mash1), a pro-neuron basic helix-loop-helix protein transcription factor. Blockage of the Shh signaling pathway with a pharmacological inhibitor, cyclopamine, abolished the CEPO-induced neurogenesis. Attenuation of endogenous Mash1 expression by short-interfering RNA blocked CEPO-promoted neuronal differentiation. In addition, recombinant mouse Shh up-regulated Mash1 expression in neural progenitor cells. These results demonstrate that the Shh signaling pathway mediates CEPO-enhanced neurogenesis and Mash1 is a downstream target of the Shh signaling pathway that regulates CEPO-enhanced neuronal differentiation.

LSD1-mediated epigenetic modification is required for TAL1 function and hematopoiesis

TAL1 is a critical transcription factor required for hematopoiesis. However, perturbation of its activity often leads to T cell leukemia. Whether and how its transcriptional activities are regulated during hematopoiesis remains to be addressed. Here, we show that TAL1 is associated with histone demethylase complexes containing lysine-specific demethylase 1 (LSD1), RE1 silencing transcription factor corepressor (CoREST), histone deacetylase 1 (HDAC1), and histone deacetylase 2 in erythroleukemia and T cell leukemia cells. The enzymatic domain of LSD1 plays an important role in repressing the TAL1-directed transcription of GAL4 reporter linked to a thymidine kniase minimal promoter. Furthermore, we demonstrate that the TAL1-associated LSD1, HDAC1, and their enzymatic activities are coordinately down-regulated during the early phases of erythroid differentiation. Consistent with the rapid changes of TAL1–corepressor complex during differentiation, TAL1 recruits LSD1 to the silenced p4.2 promoter in undifferentiated, but not in differentiated, murine erythroleukemia (MEL) cells. Finally, shRNA-mediated knockdown of LSD1 in MEL cells resulted in derepression of the TAL1 target gene accompanied by increasing dimeH3K4 at the promoter region. Thus, our data revealed that histone lysine demethylase LSD1 may negatively regulate TAL1-mediated transcription and suggest that the dynamic regulation of TAL1-associated LSD1/HDAC1 complex may determine the onset of erythroid differentiation programs.