Targeting Pulmonary Endothelial Hemoglobin α Improves Nitric Oxide Signaling and Reverses Pulmonary Artery Endothelial Dysfunction.

Pulmonary hypertension is characterized by pulmonary endothelial dysfunction. Previous work showed that systemic artery endothelial cells (ECs) express hemoglobin (Hb) α to control nitric oxide (NO) diffusion, but the role of this system in pulmonary circulation has not been evaluated. We hypothesized that up-regulation of Hb α in pulmonary ECs contributes to NO depletion and pulmonary vascular dysfunction in pulmonary hypertension. Primary distal pulmonary arterial vascular smooth muscle cells, lung tissue sections from unused donor (control) and idiopathic pulmonary artery (PA) hypertension lungs, and rat and mouse models of SU5416/hypoxia-induced pulmonary hypertension (PH) were used. Immunohistochemical, immunocytochemical, and immunoblot analyses and transfection, infection, DNA synthesis, apoptosis, migration, cell count, and protein activity assays were performed in this study. Cocultures of human pulmonary microvascular ECs and distal pulmonary arterial vascular smooth muscle cells, lung tissue from control and pulmonary hypertensive lungs, and a mouse model of chronic hypoxia-induced PH were used. Immunohistochemical, immunoblot analyses, spectrophotometry, and blood vessel myography experiments were performed in this study. We find increased expression of Hb α in pulmonary endothelium from humans and mice with PH compared with controls. In addition, we show up-regulation of Hb α in human pulmonary ECs cocultured with PA smooth muscle cells in hypoxia. We treated pulmonary ECs with a Hb α mimetic peptide that disrupts the association of Hb α with endothelial NO synthase, and found that cells treated with the peptide exhibited increased NO signaling compared with a scrambled peptide. Myography experiments using pulmonary arteries from hypoxic mice show that the Hb α mimetic peptide enhanced vasodilation in response to acetylcholine. Our findings reveal that endothelial Hb α functions as an endogenous scavenger of NO in the pulmonary endothelium. Targeting this pathway may offer a novel therapeutic target to increase endogenous levels of NO in PH.

Stability of the individual globin genes during erythroid differentiation.

The genes of sheep betaA, betaC, and gamma globin were all present in DNA from erythroid cells which synthesized only betaC globin. Similarly, selective excision of non-expressed genes was shown not to occur during human erythroid differentiation. In contrast, evolutionary deletion of the betaC gene accounts for the inability of many sheep to make this globin.

Erythroid progenitors circulating in the blood of adult individuals produce fetal hemoglobin in culture.

Erythroid colonies, raised from erythroid stem cells circulating in the peripheral blood of normal adult individuals, synthesize considerable amounts of fetal hemoglobin. In cultures from persons with sickling disorders, amounts of hemoglobin F that are known to inhibit sickling in vivo are produced. The results provide evidence that primitive erythroid progenitors are able to express the hemoglobin F production program and that cultures of mononuclear cells of the adult blood can be used to investigate the mechanisms involved in regulation of gamma-globin gene switching.

[Ikaros and erythropoiesis]. / Ikaros, facteur de transcription impliqué, aussi, dans l'érythropoïèse.

Mostly studied in murine models, Ikaros--a factor that positively or negatively controls gene transcription--was first described as essential to lymphopoiesis until its involvement in numerous hematopoietic lineages was documented. Indeed, Ikaros is also active in murine erythropoiesis and more precisely during fetal to adult globin switching. Recently, these observations were confirmed by our team in a human context. We here review some of the most important characteristics of Ikaros, and detail more precisely how defects of Ikaros activity either by gene inactivation or mutation in mice, or by forced expression of dominant negative isoforms in human precursor cells, modify the erythroid differentiation. An increased cell death, together with decreased cell proliferation, decreased expression of erythroid-specific genes including GATA1, and a delay in fetal to adult globin switching were observed. At the same time, myeloid differentiation was slightly favoured thus suggesting that Ikaros could be involved in the control of the myeloid/erythroid commitment.

Investigations of the simian ontogenic switch from fetal to adult hemoglobin at the progenitor cell level.

The ontogenic switch from fetal to adult hemoglobin could result from discontinuous events, such as replacement of fetal erythroid progenitor cells by adult ones, or gradual modulation of the hemoglobin program of a single progenitor cell pool. The former would result in progenitors at midswitch with skewed fractional beta-globin synthesis programs, the latter in a Gaussian distribution. For these studies, we obtained bone marrow from rhesus monkey fetuses at 141-153 d (midswitch). Mononuclear cells were cultured in methyl cellulose with erythropoietin, and single BFU-E-derived colonies were removed and incubated with [3H]leucine. Globin synthesis was examined by gel electrophoresis and fluorography. The beta-globin synthesis pattern of single fetal colonies was skewed, and did not fit a normal distribution. The fetal pattern resembled the pattern of an artificial mixture of fetal and adult progenitors, suggesting that the fetal progenitor pool could contain populations with different beta-globin programs. This non-Gaussian distribution in the progenitors of midswitch fetuses is consistent with a discontinuous model for hemoglobin switching during ontogeny.

Fetal hemoglobin accumulation in vitro. Effect of adherent mononuclear cells.

In clonal cultures of erythroid burst-forming units (BFU-E) obtained from blood, the accumulation of fetal and adult hemoglobins (Hb F and Hb A) was measured by radioligand immunoassay. Inclusion of adherent mononuclear cells in the culture promoted a striking increase in the relative amount of Hb F in each of 44 experiments with 14 donors. In two-thirds of the instances, this was accounted for by a selective increase in the absolute amount of Hb F. The differential effect on Hb F and Hb A accumulation was achieved without altering the maturity of the erythroid cells, their mean hemoglobin content, or the asynchrony of the production of the two hemoglobins. Virtually all bursts produced Hb F, and the population of BFU-E as a whole, rather than a selected subset, appeared to be the target of adherent cell action. When the adherent cells were excluded from the culture input, the base-line value of Hb F was reproducible for each donor over a period of several months, and correlated with the number of in vivo circulating F cells.

Synthesis of hemoglobin Aic and related minor hemoglobin by erythrocytes. In vitro study of regulation.

Factors that influence hemoglobin (Hb)A(Ic) synthesis by intact erythrocytes were studied in vitro. After incubation cells were lysed, and hemoglobins were separated by isoelectric focusing on polyacrylamide slab gels and quantitated by microdensitometry. HbA(Ic) increased with time, glucose concentrations (5-500 mM), and incubation temperature (4 degrees -37 degrees C). Low temperatures allowed prolonged incubations with minimal hemolysis. At 4 degrees C HbA(Ic) increased linearly with time for 6 wk; after incubation at the highest glucose concentration, HbA(Ic) comprised 50% of total hemoglobin. Insulin (1 and 0.1 mU/ml) did not affect HbA(Ic) synthesis in vitro. In addition to glucose, galactose and mannose, but not fructose, served as precursors to HbA(Ic). A good substrate for hexokinase (2-deoxyglucose) and a poor hexokinase substrate (3-O-methylglucose), were better precursors for HbA(Ic) synthesis than glucose, suggesting that enzymatic phosphorylation of glucose is not required for HbA(Ic) synthesis. Autoradiography after erythrocyte incubation with (32)P-phosphate showed incorporation of radioactivity into HbA(Ia1) and A(Ia2), but not HbA(Ib), A(Ic), or A. Acetylated HbA, generated during incubation with acetylsalicylate, migrated anodal to HbA(Ic) and clearly separated from it. Erythrocytes from patients with insulinopenic diabetes mellitus synthesized HbA(Ic) at the same rate as controls when incubated with identical glucose concentrations. Likewise, the rate of HbA(Ic) synthesis by erythrocytes from patients with cystic fibrosis and congenital spherocytosis paralleled controls. When erythrocytes from cord blood and from HbC and sickle cell anemia patients were incubated with elevated concentrations of glucose, fetal Hb, HbC, and sickle Hb decreased, whereas hemoglobins focusing at isoelectric points near those expected for the corresponding glycosylated derivatives appeared in proportionately increased amounts.

Postnatal fetal and adult hemoglobin synthesis is preterm infants whose birth weight was less than 1,000 grams.

To determine if environmental factors could effect the switchover from fetal hemoglobin (HbF) to adult hemoglobin (HbA) synthesis, studies were carried out on blood samples from eight infants born at less than 1,000 g, when they had reached their postconceptional age corresponding to term. All of these infants required prolonged intensive care, multiple blood transfusions, and two required exchange transfusions. Several were ventilated mechanically for 60 d and two infants had bronchopulmonary dysplasia at the time of the study. The blood samples were incubated in an amino acid mixture containing [14C]leucine followed by column chromatography on DEAE Sephadex for separation of radioactive HbA and HbF. In spite of the extreme prematurity and poor growth of these sick infants, the proportional synthesis of HbF and HbA, as determined by the incorporation of [14C]leucine during the erythrocyte incubations, was characteristic of the period of human development from which the erythrocytes were obtained.

Effects of butyrate and glucocorticoids on gamma- to beta-globin gene switching in somatic cell hybrids.

Butyrate and its analogs have been shown to induce fetal hemoglobin in humans and primates and in erythroid cell cultures. To obtain insights concerning the cellular mechanisms of butyrate action, we analyzed the effects of butyrate on human globin gene expression in hybrids produced by fusing mouse erythroleukemia cells (MEL) with human fetal erythroid cells (HFE). These hybrids initially express human fetal hemoglobin but subsequently switch to adult globin expression after several weeks in culture. We found that alpha-aminobutyric acid, a butyrate analog which does not induce terminal maturation, strikingly delays the rate of the gamma- to beta-globin gene (gamma-to-beta) switch in the HFE x MEL hybrids. The effect of butyrate on globin expression is transient, with the result that the delay of globin gene switching requires the continuous presence of this compound in culture. Furthermore, butyrate fails to induce fetal hemoglobin expression in hybrids which have switched, suggesting that the effect of this compound on gamma-globin expression is due to inhibition of gamma gene silencing rather than to induction of gamma gene transcription. Since in other cellular systems, glucocorticoids antagonize the action of butyrate, the effect of dexamethasone on the gamma-to-beta switch in HFE x MEL hybrids was examined. Dexamethasone strikingly accelerated the gamma-to-beta switch, and its effect was irreversible. The effects of dexamethasone and butyrate on the gamma-to-beta switch of the HFE x MEL hybrids appear to be codominant. These results indicate that steroids can have a direct effect on globin gene switching in erythroid cells.

Erythropoietin contributes to implantation: ectopic hemoglobin synthesis in decidual cells of mice.

Erythropoietin, by binding to its receptor, stimulates definitive erythroblasts to accumulate hemoglobin (Hb) by up-regulating erythroid-specific genes and causes differentiation of erythroblasts into erythrocytes. In mouse decidua we have found the expression of transcripts for the erythropoietin receptor, the function of which has not yet been elucidated. Erythropoietin signaling was inhibited by the injection of a soluble form of the erythropoietin receptor capable of binding with erythropoietin into the mouse uterine cavity on day 4 of gestation, and pale and defective decidual bodies appeared three days later. These pale decidual bodies contained defective embryos without extension to the ectoplacental region, while normal reddish decidual bodies contained normal developing embryos and expressed embryonic and adult Hb with characteristic location of the respective hemoglobins in which an epsilon- or beta-globin signal was confirmed. Furthermore, blocking of erythropoietin signaling destroyed Hb-containing cells and resulted in apoptosis that caused embryonic death. Thus, erythropoietin-mediated Hb synthesis is essential for the survival of decidual cells. In addition, although no transcripts for GATA-1 and erythroid heme enzymes could be detected, genes for beta-globin, as well as non-specific delta-aminolevulinate synthase, were expressed and regulated in an erythropoietin-dependent manner. This is the first evidence that ectopic Hb synthesis exists and that erythropoietin coregulates erythroid (globin) and nonerythroid (delta-aminolevulinate synthase) genes.

The effect of progesterone on hemoglobin synthesis in suspension cultures of fetal erythroid cells from calf liver.

When fetal calf liver erythroid cells were incubated in the presence of small amounts of progesterone (10(-7)-10(-8) M), the hemoglobin synthesis in these cells was significantly increased. The increase in the amount of radioactivity in de novo synthesized hemoglobins could be demonstrated when techniques such as isoelectric focusing, chromatography on DEAE-cellulose and gel chromatography on Sephadex G-100 were used to isolate the hemoglobin fraction. Using the latter technique, it was shown that the synthesis of cytoplasmic non-hemoglobin proteins in erythroid-cell lysates was also stimulated by progesterone. The presence of hepatocytes in culture nullified the hormone action. It was necessary that progesterone was present during the first hours of culture. Delayed addition of the steroid to the cells had no effect on hemoglobin synthesis. Erythropoietin was necessary to obtain stimulation by progesterone. These results suggest that the target cell of the hormone is an erythropoietin-sensitive cell. High concentrations of progesterone (10(-4) M) strongly inhibited hemoglobin synthesis in fetal calf erythroid cells. Culture of cells under this condition, however, gives rise to a cell population that preferentially synthesizes adult hemoglobin. Our results suggest that in the erythropoietic calf liver, high concentrations of progesterone may preferentially stimulate adult hemoglobin synthesis, or that those cells which have a high capacity to synthesize adult hemoglobins are less sensitive to toxic concentrations of the hormone. The effects of stimulation of hemoglobin synthesis in fetal calf erythroid cells occur at hormone concentrations that suggest a possible physiological role of progesterone in fetal, and eventually also in maternal, erythropoiesis.

Nonenzymatic glycosylation of protein: relevance to diabetes.

Glucose can react nonenzymatically with proteins to form stable covalent linkages. The most abundant minor hemoglobin component in human red cells is hemoglobin AIc: glucose is attached to the N-terminal amino group of the beta chain by a ketoamine linkage. Hemoglobin AIc is increased two to three-fold in the red cells of diabetic patients. It is formed slowly and continuously throughout the 120-day lifespan of the red cell. Measurement of hemoglobin AIc provides an index of average blood glucose levels over the preceding two or three months. Thus, hemoglobin AIc has proved to be useful in assessing diabetic control and, perhaps, in screening people for diabetes. Many other proteins, such as lens crystallins, collagen and proteins in serum and in red cell membrane, are modified by nonenzymatic glycosylation. This structural alteration may lead to impaired protein function and, perhaps, contribute to the long-term complications of diabetes.

Elevated levels of fetal hemoglobin synthesis in infants with bronchopulmonary dysplasia.

A study was devised to determine whether levels of fetal hemoglobin (HbF) synthesis are elevated in infants with bronchopulmonary dysplasia (BPD) when compared with the levels of HbF synthesis found in normal control infants. Twelve infants with BPD, whose postconceptional ages ranged from 40 to 62 weeks, were studied. The mean (+/- SD) gestational age and birth weight was 29 +/- 1.9 weeks and 1289 +/- 376 g, respectively. Elevation infants matched for birth weight, gestational age, and postnatal age served as the control subjects. Blood samples were incubated in an amino acid mixture containing [14C]leucine. The adult hemoglobin and HbF were then separated by column chromatography on diethylaminoethyl-Sephadex. The results demonstrated that the mean (+/- SD) level of HbF synthesis in infants with BPD was significantly higher than that in the control infants (42.6 +/- 22.9% vs 18.8 +/- 12.8%; P less than .01). When levels of HbF synthesis in the infants with BPD and the control infants were compared with data previously reported in normal infants, 7 of the 12 infants with BPD, but none of the control infants, were synthesizing amounts of HbF greater than would be expected for their postconceptional age. The results suggest that cardiopulmonary insufficiency could stimulate HbF synthesis during the first year of life as a result of an erythropoietic response to hypoxemia.

Red cell oxygen affinity, hemoglobin type, 2,3-diphosphoglycerate, and pH as a function of fetal development.

Studies were carried out on fresh cord blood obtained at delivery from nonstressed normal fetuses ranging from 24 to 42 weeks of gestation, to determine the relationship of 2,3-diphosphoglycerate (DPG), the intracellular red cell and extracellular pH, and the proportions of adult and fetal hemoglobin in regulating the position of fetal red cell oxygen affinity in utero. There was a significant positive correlation between P50 and gestational age (r = .62, P less than .001), the linear regression increased from 17.8 to 22.5 mm Hg. There was also a significant positive correlation between P50 and the percentage of adult type hemoglobin (HbA) (r = .67, P less than .001). In contrast gestational age had no effect of 2,3-DPG levels, the mean and SD was 14.86 +/- 2.04 mol/gm of Hb or delta pH between plasma and red cell, the mean was 0.187 +/- SD 0.032. However, there was a significant negative correlation between the intraerythrocyte hydrogen ion concentration and DPG level (r = .5, P less than .025). It is concluded therefore that the decrease in fetal oxygen affinity as gestation progresses is related mainly to the increase in the amount of HbA and the levels of DPG or delta pH between plasma and red cells are not a function of gestational age.

Effect on hemoglobin A1 of rapid normalization of glycemia with an artificial endocrine pancreas.

Six insulin-requiring chronically hyperglycemic diabetic patients underwent glucose control for 72 hours with an artificial endocrine pancreas, the Biostator Glucose Controller. Euglycemia was within 4 hours and was maintained (110 leads to 2 mg/dl) thereafter. The concentration of hemoglobin A (HbA) was elevated, at 12.5 leads to 0.6%, at the initiation of Biostator treatment. Decreases in HbA, of 0.5 leads to 0.1% (P less than 0.01) were observed within 4 hours of Biostator therapy. Over the first 16 hours, the slope was less steep, -0.018 leads to 0.004% per hour (P less than 0.01). After 72 hours, decreases in HbA of 1.8 leads to 0.4% (P less than 0.01) were observed. The current concept of HbA as an indication of integrated glycemia over weeks and months requires modification to account for a small, rapidly reversible component. Interpretation of the value of HbA when measured by a kit method must take into consideration that small changes in HbA may occur over a few hours as a result of a large and persistent change in glycemia.

Hemoglobin synthesis of both adult and fetal types in a human CML cell line.

We present evidence for the continuous erythroid differentiation of a cell line, KU-812-F, without the addition of an inducer. Erythroid differentiation of these cells was confirmed according to the following findings: 1) erythroid morphology; 2) existence of glycophorin A and carbonic anhydrase I as a membrane marker protein and an enzyme characteristic of erythroid cells, respectively; 3) synthesis of adult type (HbA and HbA2) and fetal type (HbF) hemoglobins, as detected on isoelectric focusing; and 4) transcription of the alpha-, beta-, and gamma-globin genes, as detected on Northern blot analysis.

Overview: mechanisms of the regulation of hemoglobin synthesis at the cellular level.

The concept that has emerged from our experiments and those of others is that erythroid stem cells are committed to undergo a program of erythroid differentiation with respect to the ultimate hemoglobin phenotype of their progeny erythrocytes. A clear distinction can be drawn between the switch from Hb A (or Hb F) to Hb C in sheep and the switch from Hb F to adult hemoglobin in humans. The former appears to be regulated in a relatively late erythroid stem cell with characteristics of CFU-E. In contrast, the CFU-E found in adult sheep bone marrow from animals that lack the beta C gene appear to be preprogrammed to produce only adult hemoglobin. Fetal stem cells may be induced to synthesize Hb C within a time frame that is similar to that seen in cultures of adult bone marrow. Thus, a common mechanism modulating the potential for expression of this gene and commitment of erythroid stem cells with respect to Hb C production in progeny erythroblasts seems quite likely. Again fetal CFU-E and BFU-E in animals lacking the beta C gene appear to be, for the most part, committed toward producing erythroblasts making Hb F. Further analysis will be required to determine at exactly which stage of stem cell differentiation this programming occurs and also the factors that are important in modulating the potential for fetal and adult hemoglobin synthesis.

Cellular regulation of fetal hemoglobin production.

In this paper, we put together several observations from studies in erythroid cultures that suggest that the major regulatory events determining Hb F and Hb A formation in the red cells are taken at the level of erythroid stem cells. We outlined the evidence that suggests that the program of Hb F expression changes during the differentiation of the primitive progenitors, known as burst-forming units, and we raised several possibilities on how this differentiation-dependent change in developmental programs could be accomplished. Although many questions remain still unanswered, the work done so far suggests that the mechanisms of regulation of Hb F at the cellular level can be successfully probed with existing methodologies. Delineation of the cellular mechanism of Hb F regulation is required in order to find out whether the therapeutic manipulation of Hb F in the patient with Cooley's anemia is possible or not.

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)