Control of red blood cell mass during spaceflight

Data are reviewed from twenty-two astronauts from seven space missions in a study of red blood cell mass. The data show that decreased red cell mass in all astronauts exposed to space for more than nine days, although the actual dynamics of mass changes varies with flight duration. Possible mechanisms for these changes, including alterations in erythropoietin levels, are discussed.     

EFFECT OF ERYTHROPOIETIN ON PROLIFERATION OF ERYTHROPOIETIN-RESPONSIVE CELLS

A single dose of Myleran suppressed CFU in polycythemic mice to around 1% of normal for a period of 2 weeks and permitted the study of effects of erythropoietin on unipotential, erythroid stem cells (erythropoietin-responsive cells, ERC) in the absence of cell inflow from the CFU compartment. Without erythropoietin no ERC were detectable for 12 days after Myleran. Injections of erythropoietin had no effect on CFU but restored ERC populations in proportion to the dose of erythropoietin. Hydroxyurea given after erythropoietin markedly inhibited ERC repopulation and the latter is attributed to a stimulation of ERC proliferation by erythropoietin. Evidence in support of an age structure in the ERC population is presented. Daily erythropoietin injection resulted in stable ERC populations, indicating that ERC loss through differentiation and ERC self-replication were in balance.     

The effect of erythropoietin on the growth and development of spleen colony-forming cells

The progressive growth and development of spleen colonies was studied in heavily irradiated host mice in which erythropoiesis was modified by various procedures. Erythropoietic activity in non-polycythemic hosts bearing spleen colonies was not increased by injections of exogenous erythropoietin. Detectable levels of erythropoietin were found in the heavily irradiated host mice suggesting that the failure of exogenous erythropoietin to modify erythropoiesis was because the host mice were already maximally stimulated by the high endogenous erythropoietin levels. Spleen colonies do not become erythroid in polycythemic mice. The injection of exogenous erythropoietin into heavily irradiated polycythemic hosts did not decrease the total number of spleen colonies produced by a given bone marrow transplant, as would be expected if erythropoietin acted directly on the colony-forming cells. Comparison of growth curves for colony-forming cells in the spleens of polycythemic hosts either receiving or not receiving erythropoietin indicated that the overall doubling time of colony-forming cells during the first ten days after transplantation was not changed by the daily injection of erythropoietin. These experiments are consistent with the concept that erythropoietin is necessary for the development of erythroid colonies. Erythropoietin acts upon some progeny of the colony-forming cell rather than the colony-forming cell itself.     

Multimeric structure of the membrane erythropoietin receptor of murine erythroleukemia cells (Friend cells). Cross-linking of erythropoietin with the spleen focus-forming virus envelope protein.

In erythroleukemia cells infected with the polycythemia strain of the Friend virus complex, erythropoietin could be cross-linked mainly to a protein of 63 kDa when using disuccinimidyl suberate. In contrast, erythropoietin in other erythroleukemia cells cross-linked to two proteins of 85 and 100 kDa. When native erythropoietin receptor complexes were immunoprecipitated, the 63-kDa erythropoietin-cross-linked protein could be precipitated both by antibodies directed against the intracellular part of the cloned chain of the erythropoietin receptor and by antibodies directed against the envelope proteins of the Friend virus. However, after denaturation of the complexes, the 63-kDa protein was only precipitated by antibodies directed against the envelope proteins of the Friend virus. Enzymatic deglycosylation confirmed that erythropoietin was cross-linked with the envelope protein of the defective virus and bidimensional diagonal gel electrophoresis analyses showed that some of the erythropoietin cross-linked envelope proteins were dimerized by disulfide bonds. Thus, the main erythropoietin-receptor complex in the plasma membrane of these cells consisted of a molecule of the cloned chain of the erythropoietin receptor noncovalently associated with one or two disulfide-bonded molecule(s) of the envelope protein of the defective virus. Moreover, our results also showed that the viral envelope protein associated with the cloned chain of the erythropoietin receptor at a site distinct from the erythropoietin binding site.     

Effect of Iron on Erythropoietin Production in Anaemic Piglets

In the present study the response of plasma erythropoietin to iron injection in anaemic piglets was examined. At the age of 16 days, 4 piglets from the same litter were given 180 mg iron subcutane-ously. After iron injection, blood samples for estimation of erythropoietin activity in plasma, haemoglobin concentration, and reticulocyte counts were taken every 6 or 12 h for 3 Vi days. Plasma erythropoietin activity was estimated by a monoclonal enzyme-im-munoassay (ELISA), developed for human erythropoietin. On the day of iron injection, haemoglobin concentration ranged between 41 and 48 g/1, reticulocyte counts from 9 to 17 percent, and plasma erythropoietin between 22 and 144 mU/ml. In 3 of the 4 piglets, iron injection resulted in a 2-6 fold increase in erythropoietin activity. Maximal eryth-ropoietin activities were observed 24-42 h after injection, and after 66 h, the activities were close to the pretreatment values. It is concluded that in our experiment, iron, per se, has stimulated erythropoietin production.     

Comparative analysis of the effects of the unpurified preparations of murine erythropoietin and human recombinant erythropoietin on erythroid and granulocyte-macrophage progenitor cells in semi-solid mouse bone marrow cultures]

Effects of unpurified murine erythropoietin and unpurified human recombinant erythropoietin on the growth of erythroid--BFU-E and granulocyte--macrophage progenitor cells--CFU--GM from the mouse bone marrow were compared using a methylcellulose culture system. Average erythropoietin titers for murine serum and supernatant human recombinant erythropoietin were 16 U/ml and 33 U/ml, respectively. The maximal stimulation was observed at 1-2 U/ml culture recombinant erythropoietin and 0.5 U/ml culture murine erythropoietin. Murine erythropoietin was more effective then human one. Murine and human recombinant erythropoietin had no significant effect on the number of CFU-GM colonies. But human recombinant erythropoietin could be preferentially used when studying the mechanism of erythropoiesis in man and animals because there were erythropoiesis inhibitors in mouse serum.     

Lack of maternal to fetal transfer of 125I-labelled erythropoietin in sheep.

We administered tracer quantities of biologically active 125I-labelled recombinant human erythropoietin by intravenous bolus injection to seven late gestation pregnant ewes. Maternal and fetal blood was sampled over the subsequent six hours and assayed for erythropoietin-specific radioactivity. Despite the expected increase in maternal plasma immunoprecipitable 125I-labelled erythropoietin radioactivity, fetal plasma levels remained unchanged throughout the study. In addition, erythropoietin receptors were not detected in ovine and human placental tissue. We conclude that biologically active 125I-labelled erythropoietin does not cross the placenta from mother to fetus in measurable quantities in sheep, and likely in humans. Thus, these data indicate the levels of erythropoietin measured in fetal plasma are reflective of fetal, and not maternal, erythropoietin production and elimination.     

Elevated cyclic GMP concentrations in rabbit bone marrow culture and mouse spleen following erythropoietic stimulation.

The effects of erythropoietin and hypoxia on cyclic nucleotide concentrations in erythroid tissue were evaluated. A rabbit bone marrow culture system and a mouse spleen model provided evidence that erythropoietin and an hypoxic stimulus which increases erythropoietin production may enhance erythropoiesis by initiating reciprocal changes in erythroid cell cyclic nucleotide levels. Cyclic GMP appears to be the active signal in mediating the response to erythropoietin, whereas cyclic AMP may be a passive signal allowing full expression of the cyclic GMP response. Whether the type of response mediated by cyclic nucleotides is proliferative, differentiative or both is not clear, but our data and that of other investigators suggest that cyclic GMP mediates the proliferative actions of erythropoietin.     

Dietary Protein-induced Changes of the Erythropoietin Level in Rat Serum

Erythropoietin, a glycoprotein, is the primary regulator of erythropoiesis. The most convenient and sensitive assay for active erythropoietin is to measure its stimulatory effect on in vitro ³H-thymidine incorporation into DNA of erythropoietin-responsive cells. An attempt with this method to estimate the erythropoietin level in rat serum, however, was unsuccesful because of the presence of inhibitory substance(s) and non-erythropoietic factor(s) stimulating ³H-thymidine incorporation. Pretreatment of the serum by heating, extraction of erythropoietin from denatured-protein aggregates, and subsequent concentration of erythropoietin in the extract with alcohol precipitation made it possible to measure the serum erythropoietin levels. Rabbit anti-erythropoietin antibody was used for a quantitative estimation of erythropoietin in the concentrated extracts. Erythropoietin levels in sera of rats fed on varied amounts of casein for 7 days were measured with these procedures to find if the impairment of erythropoiesis upon protein deprivation was due to changes in the erythropoietin level. We found that the level in protein-deprived rats was less than 1/8 that of 20% casein-fed rats, a level undetectable by the present assay, and that the serum erythropoietin increased as the protein content in the diet was increased up to 20%, then leveled off. The erythropoietin in serum decreased rapidly after protein deprivation; the level at 12hr after deprivation began was about 1/5 that in 20% casein-fed rats. Thus, the depression of erythropoiesis upon protein deprivation is primarily caused by the lowered level of erythropoietin.     

Inactivation of erythropoietin leads to defects in cardiac morphogenesis.

Erythropoietin is an essential growth factor that promotes survival, proliferation, and differentiation of mammalian erythroid progenitor cells. Erythropoietin(-/-) and erythropoietin receptor(-/-) mouse embryos die around embryonic day 13.5 due, in part, to failure of erythropoiesis in the fetal liver. In this study, we demonstrated a novel role of erythropoietin and erythropoietin receptor in cardiac development in vivo. We found that erythropoietin receptor is expressed in the developing murine heart in a temporal and cell type-specific manner: it is initially detected by embryonic day 10.5 and persists until day 14.5. Both erythropoietin(-/-) and erythropoietin receptor(-/-) embryos suffered from ventricular hypoplasia at day 12-13 of gestation. This defect appears to be independent from the general state of hypoxia and is likely due to a reduction in the number of proliferating cardiac myocytes in the ventricular myocardium. Cell proliferation assays revealed that erythropoietin acts as a mitogen in cells isolated from erythropoietin(-/-) mice, while it has no effect in hearts from erythropoietin receptor(-/-) animals. Erythropoietin(-/-) and erythropoietin receptor(-/-) embryos also suffered from epicardium detachment and abnormalities in the vascular network. Finally, through a series of chimeric analysis, we provided evidence that erythropoietin acts in a manner which is non-cell-autonomous. Our results elucidate a novel role of erythropoietin in cardiac morphogenesis and suggest a combination of anemia and cardiac failure as the cause of embryonic lethality in the erythropoietin(-/-) and erythropoietin receptor(-/-) animals.     

An extra high dose of erythropoietin fails to support the proliferation of erythropoietin dependent cell lines

Erythropoietin is responsible for the red blood cell formation by stimulating the proliferation and the differentiation of erythroid precursor cells. Erythropoietin triggers the conformational change in its receptor thereby induces the phosphorylation of JAK2. In this study, we show that an extra high dose of erythropoietin, however, fails to activate the erythropoietin receptor, to stimulate the phosphorylation of JAK2 and to support the cell proliferation of Ep-FDC-P2 cell. Moreover, high dose of EPO also inhibited the proliferation of various erythropoietin-dependent cell lines, suggesting that excess amount of EPO could not trigger the conformational change of the receptor. In the presence of an extra high dose of erythropoietin as well as in the absence of erythropoietin, the cells caused the DNA fragmentation, a typical symptom of apoptosis. The impairment of cell growth and the DNA fragmentation at the extremely high concentration of EPO was rescued by the addition of erythropoietin antibody or soluble form of erythropoietin receptor by titrating the excess erythropoietin. These results suggest that two erythropoietin binding sites on erythropoietin receptor dimer should be occupied by a single erythropoietin molecule for the proper conformational change of the receptor and the signal transduction of erythropoietin, instead, when two erythropoietin binding sites on the receptor are shared by two erythropoietin molecules, it fails to evoke the conformational change of erythropoietin receptor adequate for signal transduction.     

The frequency of bone marrow cells that bind erythropoietin

The frequencies of rat and mouse bone marrow cells capable of binding erythropoietin were studied by both direct fluorescence and indirect immunofluorescence. We found that between 1-2% of the cells bound erythropoietin, that the binding was specific, and that the number of cells that bound erythropoietin was, in part, a function of the erythropoietic state of the donor animal. A statistical method for evaluating the data obtained is included.     

Hemoglobin switching in sheep and goats. V. Effect of erythropoietin concentration on in vitro erythroid colony growth and globin synthesis

Erythroid colonies were generated in response to erythropoietin in plasma clot cultures of sheep and goat bone marrow cells. At low concentration erythropoietin only hemoglobin A (betaA globin) was synthesized in goat cultures, but at high concentrations 50% of the hemoglobin synthesized was hemoglobin C (betaC globin). This effect of erythropoietin on the expression of a specific beta globin gene was manifested only after 72 h in vitro and followed the development of erythroid colonies. Sheep colonies behaved differently from those of goat in that little or no betaC globin synthesis occurred even at high erythropoietin concentration. To investigate this difference, sheep marrow cells were fractionated by unit gravity sedimentation. The erythroid colony-forming cells sedimented more rapidly (3.5-6mm/h) than the hemoglobinized eththroid precursors (1-3.5 mm/h), suggesting that the colonies were formed from an early erythroid precursor, However, the colonies formed from the sheep marrow fractions synthesized only betaA globin even at concentrations of erythropoietin sufficient to stimulate betaC globin synthesis in goat colonies. Morphologically, the goat colonies were larger and more mature than those of the sheep. By 96 h in vitro three-fourths of the goat colonies contained enucleated red cells compared to only 3% of the sheep colonies. Thus, erythropoietin had an equivalent effect in stimulating erythroid colony growth from the marrow of both species although there were both biochemical and morphological differences between the colonies. Hemoglobin switching appeared to require exposure of an early precursor to high erythropoietin concentration, but the results with sheep marrow suggested that the rate of colony growth and cellular maturation might also be important.     

促红细胞生成素治疗慢性心力衰竭156例临床观察

目的:观察EPO对慢性充血性心力衰竭(CHF)治疗效果。方法:选择CHF患者212例,随机分为非EPO治疗组(56例)和EPO治疗组(156例)。其中非EPO治疗组采用常规抗心衰治疗,EPO治疗组接受常规抗心衰治疗和EPO治疗,直至临床观察结束。治疗后观察心功能指标改变。结果:156例心衰患者经过EPO治疗后,左室射血分数,每搏输出量,每分输出量,心指数均较非EPO治疗对照组患者明显改善(P<0.05),其中尤以左室射血分数改善最为明显(P<0.01)。156例EPO治疗组患者中116例心衰伴贫血患者心功能各项指标的改善较40例心衰不伴贫血患者更为明显,具有显著统计学意义(P<0.01)。结论:EPO对CHF患者心功能的改善主要是因为EPO的促红细胞生成作用。EPO对于慢性心功能不全有独立于促红细胞生成以外的治疗作用。     

Impaired erythropoietin synthesis in chronic kidney disease is caused by alterations in extracellular matrix composition

Renal fibrosis and anaemia are two of the most relevant events in chronic kidney disease. Fibrosis is characterized by the accumulation of extracellular matrix proteins in the glomeruli and tubular interstitium. Anaemia is the consequence of a decrease in erythropoietin production in fibrotic kidneys. This work analyses the possibility that the accumulation of abnormal collagens in kidney interstitium could be one of the mechanisms responsible for erythropoietin decreased synthesis. In renal interstitial fibroblast grown on collagen I, erythropoietin mRNA expression and HIF‐2α protein decreased, whereas focal adhesion kinase protein (FAK) phosphorylation and proteasome activity increased, compared to cells grown on collagen IV. Proteasome inhibition or FAK inactivation in cells plated on collagen I restored erythropoietin and HIF‐2α expression. FAK inhibition also decreased the collagen I‐dependent proteasome activation. In a model of tubulointerstitial fibrosis induced by unilateral ureteral obstruction in mice, increased collagen I protein content and an almost complete disappearance of erythropoietin mRNA expression were observed in the ureteral ligated kidney with respect to the contralateral control. Interestingly, erythropoietin synthesis was recovered in obstructed mice treated with proteasome inhibitor. These data suggest that reduced kidney erythropoietin synthesis could be caused by the accumulation of abnormal extracellular matrix proteins.     

Comparative study of the asparagine-linked sugar chains of human erythropoietins purified from urine and the culture medium of recombinant Chinese hamster ovary cells

The asparagine-linked sugar chains of human erythropoietin produced by recombinant Chinese hamster ovary cells and naturally occurring human urinary erythropoietin were liberated by hydrazinolysis and fractionated by paper electrophoresis, lectin affinity chromatography, and Bio-Gel P-4 column chromatography. Both erythropoietins had three asparagine-linked sugar chains in one molecule, all of which were acidic complex type. Structural analysis of them revealed that the sugar chains from both erythropoietins are quite similar except for sialyl linkage. All sugar chains of erythropoietin produced by recombinant Chinese hamster ovary cells contain only the NeuAc alpha 2----3Gal linkage, while those of human urinary erythropoietin contain the NeuAc alpha 2----6Gal linkage together with the NeuAc alpha 2----3Gal linkage. The major sugar chains were of fucosylated tetraantennary complex type with and without N-acetyllactosamine repeating units in their outer chain moieties in common, and small amounts of 2,4- and 2,6-branched triantennary and biantennary sugar chains were detected. This paper proved, for the first time, that recombinant technique can produce glycoprotein hormone whose carbohydrate structures are common to the major sugar chains of the native one.     

Erythropoietin receptor signalling is required for normal brain development.

Erythropoietin, known for its role in erythroid differentiation, has been shown to be neuroprotective during brain ischaemia in adult animal models. Although high levels of erythropoietin receptor are produced in embryonic brain, the role of erythropoietin during brain development is uncertain. We now provide evidence that erythropoietin acts to stimulate neural progenitor cells and to prevent apoptosis in the embryonic brain. Mice lacking the erythropoietin receptor exhibit severe anaemia and defective cardiac development, and die at embryonic day 13.5 (E13.5). By E12.5, in addition to apoptosis in foetal liver, endocardium and myocardium, the erythropoietin receptor null mouse shows extensive apoptosis in foetal brain. Lack of erythropoietin receptor affects brain development as early as E10.5, resulting in a reduction in the number of neural progenitor cells and increased apoptosis. Corresponding in vitro cultures of cortical cells from Epor(-/-) mice also exhibited decreases in neuron generation compared with normal controls and increased sensitivity to low oxygen tension with no surviving neurons in Epor(-/-) cortical cultures after 24 hour exposure to hypoxia. The viability of primary Epor(+/+) rodent embryonic cortical neurons was further increased by erythropoietin stimulation. Exposure of these cultures to hypoxia induced erythropoietin expression and a tenfold increase in erythropoietin receptor expression, increased cell survival and decreased apoptosis. Cultures of neuronal progenitor cells also exhibited a proliferative response to erythropoietin stimulation. These data demonstrate that the neuroprotective activity of erythropoietin is observed as early as E10.5 in the developing brain, and that induction of erythropoietin and its receptor by hypoxia may contribute to selective cell survival in the brain.     

脑源性促红细胞生成素在短暂性前脑缺血沙土鼠海马的表达变化

目的 探讨短暂性前脑缺血鼠海马脑源性促红细胞生成素蛋白的表达变化,揭示脑缺血时中枢神经系统发生内源性脑保护的机制。方法 阻断沙土鼠双侧颈总动脉3．5min造成前脑缺血模型,再灌注1h,6h,12h,1d,3d,7d,应用免疫组织化学和免疫印迹法观察海马脑源性促红细胞生成素蛋白的表达变化。结果 脑缺血再灌注6h,可检测到脑源性促红细胞生成素的表达,再灌注12h,脑源性促红细胞生成素表达达到较高水平,以后随时间延长逐渐下调。结论 脑源性促红细胞生成素在脑缺血再灌注后的表达,可能是机体发生内源性脑保护的机制之一。     

Lowered plasma erythropoietin in hypoxic rats with kidney tubule lesions

The role of the kidney tubules in the renal formation of erythropoietin is incompletely understood. Therefore, the capability to produce erythropoietin in response to hypoxia was studied in rats with tubular lesions. Nephron damage was induced in two different ways. First, rats were treated with the nephrotoxic aminoglycoside gentamicin (67.5 mg/kg and day) for 14 days. The animals were then subjected to simulated altitude (6,800 m) for 6 h. The resulting plasma erythropoietin concentration was significantly lower (0.5 IU/ml) than in saline treated control rats exposed to hypoxia (1.0 IU/ml). Second, unilateral hydronephrosis was induced by ureteral ligation. The contralateral kidney was removed immediately before the animals were exposed to simulated altitude for 6 h. The plasma erythropoietin concentration in the ureter-ligated rats did not increase above the value (0.3 IU/ml) in hypoxia exposed anephric rats. These results indicate that the production of erythropoietin is reduced following tubular injury. Tubule cells may directly produce the hormone or interfere with the O2-sensing mechanisms controlling its synthesis. The latter hypothesis would seem to be supported by our failure to demonstrate in vitro erythropoietin production by the two established kidney tubule cell lines, LLC-PK1 and PK-15.