Sickle erythrocytes, after sickling, regulate the expression of the endothelin-1 gene and protein in human endothelial cells in culture.

The molecular defect in sickle cell disease resides in the beta globin gene, with consequent defects in erythrocytes only, suggesting that the vascular occlusion and vasomotor instability which characterize this disease are the result of interactions between abnormal sickle erythrocytes and cells of the blood vessel wall. We explored whether sickle erythrocytes may have effects on vascular tone, exclusive of adhesion events. Exposure of human endothelial cells in culture to previously sickled sickle erythrocytes resulted in a four to eight-fold transcriptional induction of the gene encoding the potent vasoconstrictor endothelin-1 (ET-1). Unsickled sickle erythrocytes or normal erythrocytes exposed to "sickling" conditions had no effect on ET-1 gene induction. Contact of the sickled erythrocytes with the endothelium was not required. Elevations in the ET-1 transcript peaked at 3 h after exposure and persisted for up to 24 h. Four to sixfold increases in the amount of ET-1 peptide was released into the medium surrounding the endothelial cells after exposure to sickled sickle erythrocytes. This is the first demonstration of the regulation of gene expression in endothelial cells as a result of interaction with sickle cells, with induction of genes encoding vasoconstrictors. Furthermore, these findings suggest that sickle erythrocytes may have the capacity to affect local vasomotor tone directly.

Sleep-disordered breathing and nocturnal hypoxemia in young adults with sickle cell disease.

Sleep-disordered breathing (SDB) is reported in up to 69% of adolescents and children with sickle cell disease (SCD) [1], but data regarding the prevalence of SDB in adults with SCD are limited. In order to obtain a preliminary assessment of the frequency and degree of sleep-related hypoxemia and potential associations with cardiovascular function in adults with SCD, we conducted overnight sleep studies, 6-min walk tests, echocardiograms, and hematologic and chemistry panels, calculated the Pittsburgh sleep quality index (PSQI), and conducted fatigue- and health-related quality-of-life measurement in 20 young adults with SCD visiting a sickle cell clinic for routine care. Sleep apnea, defined as an apnea-hypopnea index (AHI) > 5 events/h, was found in 50% of patients. Traditional clinical indicators, such as obesity, the presence of snoring, and reported sleep complaints, did not reliably differentiate them. The patients with AHI > 5 had higher mean systolic blood pressure (p = 0.03), evidence of impaired left ventricular diastolic function (i.e., increased mitral valve E/A ratio, p = 0.05), a trend toward higher reduction in 6-min walk distances (p = 0.06), and lower health-related quality-of-life scores (p ≤ 0.01). Three of nine patients with more severe anemia (total Hb < 9.0) showed nocturnal hypoxemia in the absence of sleep apnea. As prolonged and frequent hypoxemic episodes likely increase risks for vaso-occlusive, cardiovascular, and neurologic complications of SCD, these results suggest that the prevalence and severity of SDB should be investigated further in studies of larger patient populations. If confirmed, these findings could identify opportunities to prevent or reduce nocturnal hypoxia and improve outcomes.

The biochemistry of erythropoietin: an approach to its mode of action.

The elucidation of the mechanism of action of erythropoietin depends upon a detailed assessment of its effects at the molecular level. We have now begun to examine the effects of human erythropoietin and other inducers on clonal lines of Rauscher murine erythroleukemia cells. Over 100 clonal lines have been examined by assessing the hemglobinization of colonies grown in plasma clot culture in response to erythropoietin and dimethylsulfoxide. Many of the clones respond to both inducers. However, some clones respond only to erythropoietin. The cells also differentiate in suspension culture, exhibiting striking morphological changes characteristic of erythroid development. This system should serve as an excellent model for the study of control mechanisms in erythropoiesis.

Chediak-Higashi syndrome: studies in long-term bone marrow culture.

The granulocyte defects of Chediak-Higashi syndrome (CHS) include neutropenia, characteristic giant lysosomal granule morphology, and functionally abnormal cell motility, degranulation, and bacterial killing. Findings of elevated levels of adenosine 3', 5' cyclic monophosphate nucleotide (cAMP) and of concanavalin A (Con A) capping have suggested a pathogenic role of a cyclic-nucleotide-related defect in microtubule polymerization, but not all patients exhibit these abnormalities. In order to test which defects derive from the cells' genetic program and which from the host environment, we examined granulocytes produced by CHS bone marrow progenitors in long-term in vitro bone marrow cultures. These cells exhibited the characteristic giant-granule morphology and defective cell motility of CHS. However, culture-derived CHS granulocytes had normal cAMP contents and normal spontaneous capping of Con A. Granulopoiesis diminished dramatically after five weeks in culture, with accompanying autophagocytosis by mononuclear phagocytes. In mixing experiments, the phenotype of the mature granulocytes corresponded to the genotype of the hematopoietic component of the culture rather than the stroma. These results indicate that the hallmark giant-granule morphology and cell motility defect of CHS are expressions of the genetic program of the hematopoietic cells. However the abnormalities in resting cyclic nucleotide levels and in Con-A capping may be secondary manifestations of the disease and are not essential to the pathogenesis of the chemotactic defect.

A physiological delay in human fetal hemoglobin switching is associated with specific globin DNA hypomethylation.

The human fetal-to-adult globin switch normally occurs on a fixed schedule, beginning at 32-34 weeks gestation, and recent studies have suggested an association between this developmental inactivation of the fetal (gamma) globin genes and the appearance of methylation within and around these genes. We have studied a population of infants in whom this switch does not occur before birth (infants of diabetic mothers, IDM) and examined the patterns of methylation surrounding their active gamma-globin genes, in comparison to the gamma-globin genes of age-matched controls who have switched their pattern of globin gene expression on schedule. All genomic DNA samples from infants with delays in the globin switch demonstrated extensive hypomethylation in the region of the gamma-globin genes, comparable to that found in the genomes of fetuses of less than 21 weeks gestation. DNA from the erythroid cells of infants of 32-40 weeks gestation had no detectable hypomethylation in the gamma-globin region. These findings support the concept that hypomethylation is an accurate developmental marker of globin gene switching, and suggest that globin gene expression in IDM may be arrested at an early preswitch stage.

Cellular and molecular effects of a pulse butyrate regimen and new inducers of globin gene expression and hematopoiesis.

Cooley's anemia is characterized by a deficiency of beta-globin chains, a relative excess of alpha-globin chains, and consequent accelerated programmed death of developing erythroid cells in the bone marrow. Increasing expression of the gamma-globin genes to adequately balance excess alpha-globin chains can ameliorate this disorder. Butyrates induce gamma-globin experimentally, but can also cause cell growth arrest with prolonged exposure or high concentrations, which in turn can accelerate apoptosis. To determine if these potentially opposing effects can be balanced to enhance therapeutic efficacy, an intermittent "pulsed" regimen of butyrate was evaluated. Following induction of gamma-globin mRNA and protein synthesis, total hemoglobin increased in beta-thalassemia patients by more than 2 g/dl above baseline, and Hb F increased above 20% in 5/8 sickle cell patients from baseline levels of 2% Hb F. Specific regulatory regions were identified in the gamma- and beta-globin gene promoters to which new binding of transcription factors, including alpha CP2 (an activator of gamma globin) occur during therapy solely in the butyrate-responsive patients. Other compounds which induce gamma globin, derivatives of acetic, phenoxyacetic, propionic, and cinnamic acids, and dimethylbutyrate, are under investigation. Some of these newer gamma-globin inducers (designed hemokines) provide better potential as therapeutics by also acting to increase hematopoietic cell viability and proliferation. Pharmacologic induction of expression of the endogenous gamma-globin genes is a realistic approach to therapy of the beta-globin disorders for many patients, with some effective agents available now and new therapeutics, with enhanced activities, under development.

Stopping the biologic clock for globin gene switching.

The developmental switch from production of fetal (gamma) to adult (beta) globin occurs on a normally set biologic clock which proceeds even if expression of the adult (beta) globin genes is defective and produces little or no protein, as in the beta-thalassemias. Preventing or reversing the globin gene switch could provide a way of keeping the abnormal globin genes "silent" and maintaining expression of the fetal globin gene. We have identified a class of agents which, when present in elevated plasma concentrations during gestation, inhibits the gamma----beta-globin gene switch in developing humans. Further investigation has shown that butyric acid and related compounds can increase gamma-globin and decrease beta-globin expression in cultured erythroid cells of patients with beta-thalassemia. Butyrate compounds were therefore infused in an in vivo fetal animal model, and the globin switch was inhibited and even reversed in some fetal lambs. Histone hyperacetylation, which maintains active chromatin structure, and an effect on the gamma-globin promoter appear to be mechanisms of action involved. These data suggest that inhibiting expression of abnormal beta-globin genes by pharmacologic means may in the future be possible for treatment of individuals with beta-globin disorders.

Butyric acid modulates developmental globin gene switching in man and sheep.

The developmental switch from production of fetal (gamma) to adult (beta) globin occurs on a normally set biologic clock which proceeds even if the adult (beta) globin genes are defective. Preventing or reversing the globin gene switch would be beneficial for subjects with abnormal beta globin genes. We have now identified a class of agents which, when present in elevated plasma concentrations during gestation, appears to inhibit the gamma beta globin gene switch in developing humans. Further investigation has shown that butyric acid and related compounds can increase gamma globin and decrease beta globin expression in erythroid cells cultured from subjects with diseases of abnormal beta globin. Butyrate compounds were therefore infused in an in vivo fetal animal model, and the globin switch was inhibited in most and reversed in some fetal lambs. These data suggest that inhibiting expression of abnormal beta globin genes may be possible in future generations. Histone modification may be a mechanism of action involved. The developmental switch from production of gamma globin to beta globin results in significant morbidity when the beta globin genes are defective. The globin switch has therefore been extensively studied, appearing to be set on a biologic clock and proceeding despite the site of blood production and solely on the basis of gestational age. We previously found that this developmental gene switch is delayed in human fetuses developing in the presence of maternal diabetes. A number of metabolites present in abnormal concentrations in these infants were therefore tested for effects on globin expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Butyrate in the treatment of sickle cell disease and beta-thalassemia.

The search for, and discovery of, a physiologic model in which the developmentally regulated switch from fetal to adult globin gene expression could be prevented has resulted in the development of a new class of therapeutic agents, consisting of simple fatty acids, such as butyric acid, for the treatment of the beta-hemoglobinopathies. Butyrate and related drugs stimulate fetal (gamma-) globin gene expression in erythroid cells cultured from patients, and in chicken, ovine, and primate animal models. The butyrates are perhaps the first class of drugs designed to transcriptionally activate specific genes--in this particular case, to reactivate the developmentally silenced fetal globin genes. Phase I-II clinical trials resulting from this basic research have been initiated on a small scale during the past 3 years. Analysis of two butyrate-derived therapeutic agents, one delivered intravenously and one orally, has shown initial efficacy in stimulating fetal hemoglobin expression in 50% to 85% of patients. Correction of the anemia from the beta-hemoglobinopathy has followed induction of fetal globin, and has been adequate to eliminate the need for erythrocyte transfusions in some patients with beta-thalassemia. These compounds have been relatively safe and without generalized cytotoxicity in patients, but drug tolerance develops in some patients after prolonged therapy. Third-generation, small two- to five-carbon butyrate derivatives are in development. The molecular basis for butyrate action is being defined. Binding of putative regulatory proteins to a specific region of the gamma-globin promoter is altered in vivo in patients receiving butyrate therapy. Further analysis of the mode of action may contribute to development of other therapeutic agents designed to regulate gene transcription.

Butyrate-induced reactivation of the fetal globin genes: a molecular treatment for the beta-hemoglobinopathies.

The inherited beta-hemoglobinopathies (sickle cell disease and beta thalassemia) are the result of a mutation in the adult (beta) globin gene. The fetal globin chain, encoded by the gamma globin genes, can substitute for the mutated or defective beta globin chain, but expression of the gamma globin gene is developmentally inactivated prior to birth. Re-inducing expression of the normal fetal globin genes is a preferred method of ameliorating sickle cell disease and the beta thalassemias. Stimulation of as little as 4-8% fetal globin synthesis in the bone marrow can produce > 20% fetal hemoglobin in the peripheral circulation, due to enhanced survival of red blood cells containing both sickle and fetal hemoglobin, compared to those containing sickle hemoglobin alone. Butyric acid and butyrate derivatives are generally safe compounds which induce fetal hemoglobin production by stimulating the promoter of the fetal globin genes. An initial trial with the parent compound, delivered as Arginine Butyrate, has demonstrated rapid stimulation of fetal globin expression to levels that have been shown to ameliorate these conditions. Phase 1 trials of an oral butyrate derivative with a long plasma half-life have just begun. These agents now provide a specific new approach for ameliorating these classic molecular disorders and merit further investigation in larger patient populations.

Prednisone stimulation of erythropoiesis in leukemic children during remission.

Children with acute leukemia in remission manifest reticulocytosis and a significant increase in hemoglobin concentration (mean increment of 2.3 +/- 1.1 g/dl) following prednisone pulse therapy. This hemoglobin increment is not associated with changes in serum erythropoietin activity. It is speculated that this stimulation of red cell production may be a direct effect of steroids on erythroid progenitor cells.

Epstein--Barr virus post-transplant lymphoproliferative disease and virus-specific therapy: pharmacological re-activation of viral target genes with arginine butyrate.

Lymphoproliferative disorders associated with the Epstein-Barr virus (EBV) include non-Hodgkin's lymphoma, Hodgkin's lymphoma, and "post-transplant lymphoproliferative disorders" (PTLD), which occur with immunosuppression after marrow and organ transplantation. PTLD is characterized by actively proliferating, latently infected EBV(+) B-lymphocytes, and often manifests a rapidly progressive fatal clinical course if the immunosuppression cannot be reversed. Lung transplant recipients are a subset of patients at special risk for developing PTLD. The incidence of PTLD development in these patients has been estimated at 5--10%. Whereas immunologic and antiviral therapy have been moderately effective for treating EBV-associated infections in the lytic phase, they have been less useful in the more common latent phase of the disease. One common treatment for herpesvirus infections has targeted the virus-specific enzyme thymidine kinase (TK). The lack of viral TK expression in EBV(+) tumor cells, due to viral latency, makes anti-viral therapy alone ineffective as an anti-neoplastic therapy, however. We have developed a strategy for the treatment of EBV-associated lymphomas/PTLD using pharmacologic induction of the latent viral TK gene and enzyme in the tumor cells, followed by treatment with ganciclovir. Arginine butyrate selectively activates the EBV TK gene in latently EBV-infected human lymphoid cells and tumor cells. A Phase I/II trial has been initiated, employing an intra-patient dose escalation of arginine butyrate combined with ganciclovir. In six patients with EBV-associated lymphomas or PTLD, all of which were resistant to conventional radiation and/or chemotherapy, this combination produced complete clinical responses in four of six patients, with a partial response occurring in a fifth patient. Pathologic examination in two of three patients demonstrated complete necrosis of the EBV lymphoma, with no residual disease, following a single three-week course of the combination therapy. Possible side-effects of the therapy included nausea and reversible lethargy at the highest doses. One patient suffered acute liver failure, thought to be secondary to release of FasL from the necrotic tumor. Analysis of patient-derived tumor cells in culture demonstrated that arginine butyrate produced selective induction of the EBV TK gene, which then conferred sensitivity to ganciclovir, resulting in tumor apoptosis. Additional patient accrual is sought for further evaluation of this therapy.

Induction of the Epstein-Barr virus thymidine kinase gene with concomitant nucleoside antivirals as a therapeutic strategy for Epstein-Barr virus-associated malignancies.

Lymphoproliferative diseases (LPDs) associated with the Epstein-Barr virus (EBV) include non-Hodgkin lymphomas, which occur in the setting of immunosuppression, including that induced by human immunodeficiency virus, and posttransplant lymphoproliferative disorders. These LPDs are characterized by actively proliferating, latently infected EBV-positive B lymphocytes and often follow a rapidly progressive fatal clinical course. Pharmacologic treatment for herpesvirus infections has targeted the virus-specific enzyme, thymidine kinase (TK), with nucleoside analogs. The lack of viral TK expression in EBV-positive tumors, caused by viral latency, however, makes antiviral therapy alone ineffective as an antineoplastic therapy. Arginine butyrate selectively activates the EBV TK gene in latently infected EBV-positive tumor cells. We have developed a strategy for treatment of EBV-associated lymphomas using pharmacologic induction of the latent viral TK gene and enzyme in tumor cells using arginine butyrate, followed by treatment with ganciclovir. A phase I/II trial, using an intrapatient dose escalation of arginine butyrate combined with ganciclovir, is underway. This combination therapy has produced complete clinical responses in 5 of 10 previously refractory patients, with partial responses occurring in 2 additional patients. This virus-targeted antitumor strategy may provide a new therapeutic approach to EBV-associated neoplasms.

Delay in the fetal globin switch in infants of diabetic mothers.

In the normal fetus, a switch from production of hemoglobin F (alpha 2 gamma 2) to hemoglobin A (alpha 2 beta 2) occurs at 28 to 34 weeks of gestation. In the fetus with beta-hemoglobinopathy or beta-thalassemia, this switch proceeds despite the morbidity that results when production of beta-globin is abnormal or reduced. Since insulin has recently been shown to induce renewed expression of some inactive genes, we studied globin biosynthesis during the natural evolution of the fetal globin switch under conditions of hyperinsulinemia, which occurs in infants of diabetic mothers. Such infants develop in a hyperglycemic environment, which produces reactive hyperinsulinemia. The normal increase in beta-globin production from pre-switch levels did not occur in 9 of 10 such infants at term, as compared with 11 normal infants, in whom the switch occurred by 36 to 39 weeks of gestation (P less than 0.0001). The delay in the switch from gamma-globin to beta-globin in this unique clinical setting may allow identification of physiologic factors that can modulate developmental gene suppression.

Hematopoietic hormones: from cloning to clinic.

In order to maintain adequate circulating numbers of blood cells, the bone marrow must produce billions of cells each day and must be able to rapidly increase production by 10-20-fold in response to infection and hemorrhage. The existence of circulating factors that regulate this process has been suspected for over 100 years. Recently, the genes encoding these growth factors were cloned and their functions are now identified. Interleukin-3 (IL-3) acts on the most primitive hematopoietic stem cell, driving this self-renewing cell to produce progeny of all hematopoietic lineages. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the granulocyte-macrophage progenitor cell, as well as cells committed to the erythroid lineage, to differentiate. G-CSF and M-CSF stimulate the most differentiated myeloid progenitors to produce granulocytes and monocytes/macrophages, respectively. Erythropoietin stimulates the differentiation of late erythroid progenitors. In the lymphoid progenitor lineage, IL-2 stimulates T cell differentiation; IL-4 and IL-6 stimulate differentiation of B cells. The colony-stimulating factors also enhance function and cause activation of the mature cells whose production they induce. In clinical trials, these hormones have successfully ameliorated anemia in renal failure, chronic disease, and in prematurity. They have improved pancytopenias in aplastic anemia, myelodysplastic syndromes, and congenital cytopenias, and they have hastened recovery from chemotherapy and bone marrow transplantation.

MB-02 cells undergo fetal to adult globin gene switching in response to erythropoietin.

The recently described MB-02 human cell line requires Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) for continuous growth and terminally differentiates into enucleate, hemoglobinized cells in response to erythropoietin. Here, analysis of globin production now demonstrates that uninduced MB-02 cells produce alpha globin and the fetal globin chains G gamma and A gamma in a ratio of 1:3. Addition of erythropoietin results in de novo synthesis of beta globin chains and a marked increase in total Hb/cell. Thus, the MB-02 cell line partially recapitulates the fetal to adult globin switch that occurs during erythroid and human fetal development and provides a new clonal human erythroid progenitor system for investigating the biochemical and molecular events involved in globin gene switching.

Hypoxia increases thrombospondin-1 transcript and protein in cultured endothelial cells.

The exposure of endothelial cells to hypoxic environments regulates the expression of a number of genes with products that are vasoactive or mitogenic for vascular tissue, including platelet-derived growth factor, endothelin-1, and endothelial nitric oxide synthase. Hypoxia is also known to alter the adhesive properties of endothelium toward a variety of blood cell types. Thrombospondin-1 (TSP-1) is a glycoprotein with major roles in cellular adhesion and vascular smooth muscle proliferation and migration. We report here that hypoxia induces TSP-1 gene and protein expression. Oxygen tensions of < or =30 torr resulted in TSP-1 transcript induction initially apparent at 1 to 6 hours, with maximal induction (6.5-fold+/-1.2-fold) within 24 to 48 hours in both human and bovine endothelial cells. TSP-1 protein levels remain elevated after 72 hours of continuous hypoxic exposure. The induction of TSP-1 steady-state transcript levels is caused in large part, if not entirely, by post-transcriptional stabilization of the TSP-1 mRNA. The TSP-1 induction by hypoxia is a graded and reversible physiologic response and can be mimicked by the use of cobalt chloride or the inhibition of nitric oxide production, suggesting both the involvement of a heme-containing oxygen sensor and a role for the endogenous production of nitric oxide in TSP-1 regulation. The effects of hypoxia both on the stabilization of the TSP-1 transcript and the stimulation of TSP-1 protein production are completely inhibited by arginine butyrate.

Erythroid progenitor proliferation is stimulated by phenoxyacetic and phenylalkyl acids.

Short-chain fatty acids, such as butyrate and propionate, are under investigation as therapeutic stimulants of fetal hemoglobin production in the beta-hemoglobin disorders. Significant limitations to these fatty acids and derivatives as optimal therapeutics are their rapid metabolism in vivo and their induction of cell growth arrest in the G1 phase of the cell cycle. This antiproliferative activity is related to their inhibition of metabolic transport pumps which are essential for cell proliferation. Other small carbon compounds, the phenylalkyl acids, phenoxyacetic acids, and phenylacetic acids, which are structurally resistant to oxidative metabolism, are shown here to induce fetal globin production in human erythroid cultures at concentrations of 0.2 mM, lower than those required for most other fatty acids. Certain of these compounds were found not to inhibit cellular neutral amino acid transport function in erythroid cells, nor to inhibit erythroid colony (Bfu-e) growth. Certain of these compounds even stimulated human Bfu-e proliferation in vitro beyond that induced by optimal concentrations of hematopoietic growth factors. The combination of increased fetal globin chain production by these compounds and their stimulatory effects on erythropoiesis result in an increase in Hb F-expressing erythroid cells in culture several-fold greater than that achieved by the butyrates. These new compounds thus have the potential to provide superior therapy for the beta-hemoglobinopathies and other anemias.

Hypoxia-activated ligand HAL-1/13 is lupus autoantigen Ku80 and mediates lymphoid cell adhesion in vitro.

Hypoxia is known to induce extravasation of lymphocytes and leukocytes during ischemic injury and increase the metastatic potential of malignant lymphoid cells. We have recently identified a new adhesion molecule, hypoxia-activated ligand-1/13 (HAL-1/13), that mediates the hypoxia-induced increases in lymphocyte and neutrophil adhesion to endothelium and hypoxia-mediated invasion of endothelial cell monolayers by tumor cells. In this report, we used expression cloning to identify this molecule as the lupus antigen and DNA-dependent protein kinase-associated nuclear protein, Ku80. The HAL-1/13-Ku80 antigen is present on the surface of leukemic and solid tumor cell lines, including T and B lymphomas, myeloid leukemias, neuroblastoma, rhabdomyosarcoma, and breast carcinoma cells. Transfection and ectopic expression of HAL-1/13-Ku80 on (murine) NIH/3T3 fibroblasts confers the ability of these normally nonadhesive cells to bind to a variety of human lymphoid cell lines. This adhesion can be specifically blocked by HAL-1/13 or Ku80-neutralizing antibodies. Loss of expression variants of these transfectants simultaneously lost their adhesive properties toward human lymphoid cells. Hypoxic exposure of tumor cell lines resulted in upregulation of HAL-1/13-Ku80 expression at the cell surface, mediated by redistribution of the antigen from the nucleus. These studies indicate that the HAL-1/13-Ku80 molecule may mediate, in part, the hypoxia-induced adhesion of lymphocytes, leukocytes, and tumor cells.