Erythropoietin and megakaryocytopoiesis

To determine if erythropoietin influences megakaryocytopoiesis, the purified recombinant human hormone (rEpo) was added to serum-free liquid cultures of murine marrow. A dose-related increment in acetylcholinesterase (AchE) production was observed. To assess if increments in this relatively megakaryocyte-specific enzyme marker were mediated by a direct hormone-megakaryocyte interaction rather than via an accessory cell population, rEpo was added to cultures of isolated single megakaryocytes. A significant, dose-related increase in cell size was noted in the presence of the hormone, accompanied by a high probability of an increase in cellular DNA content. The data show that rEpo does directly influence some aspects of megakaryocytic maturation, although the physiologic significance of this effect remains unknown.     

红细胞生成刺激素(EPO)的纯化

 本文用中空纤维柱超滤浓缩尿,再经离子交换层析、聚焦层析、凝胶过滤和制备型聚丙烯酰胺凝胶电泳(PAGE)-层析等四步从15L再生障碍性贫血患者尿中获得约2mg EPO制品。比活性达10 300U/mg蛋白。该制品在分析型PAGE中呈一条区带。     

Up-regulated Endogenous Erythropoietin/Erythropoietin Receptor System and Exogenous Erythropoietin Rescue Retinal Ganglion Cells after Chronic Ocular Hypertension

Aims Recent studies have showed that erythropoietin (EPO) is a neuroprotectant for central nerve system neurons in addition to being a hematopoietic cytokine in response to hypoxia. In this study, we investigate the role of the EPO/EPO receptor (EPOR) system in the rat retina after ocular hypertension injury that mimics glaucoma. Methods Elevated intraocular pressure was induced by laser coagulation of the episcleral and limbal veins. Expression of EPO and EPOR in the normal and glaucomous retinas was investigated by immunohistochemistry and Western blot. To examine the effects of endogenous EPO on the survival of retinal ganglion cells (RGCs) subjected to hypertensive injury, soluble EPOR was directly injected into the vitreous body. Recombinant human EPO was both intravitreally and systemically administrated to study the effect of exogenous EPO on the survival of RGCs after ocular hypertension injury. Results Immunohistochemistry studies identified Müller cells as the main source of EPO in the normal retina. Expression of EPO and EPOR proteins was increased significantly 2 weeks after ocular hypertension. RGCs, amacrine and bipolar cells all demonstrated an increased expression of EPOR after ocular hypertension. Neutralization of endogenous EPO with soluble EPOR exacerbated ocular hypertensive injury, suggesting a role of the EPO/EPOR system in the survival of RGCs after injury. Similarly, topical and systemic administration of recombinant human EPO rescues RGCs after chronic ocular hypertension. Conclusions These results indicate that an endogenous EPO/EPOR system participates in intrinsic recovery mechanisms after retina injury and RGCs might be rescued by exogenous administration of EPO.     

Erythropoietin: current status

Understanding the regulation of red blood cell production has been greatly enhanced by the cloning and expression of the gene for human erythropoietin (Epo) and its receptor. The availability of recombinant human erythropoietin (rhEpo) for administration to patients has ushered in a new era in molecular medicine. Intravenous or subcutaneous administration of rhEpo can reliably cure the anemia of chronic renal failure and may be effective in the treatment of anemias secondary to chronic inflammation, malignancy, and marrow suppression from chemotherapy. In addition, rhEpo therapy will probably play a prominent role in transfusion medicine, both in preparing patients for auto-transfusions as well as in minimizing red cell transfusion requirements in the post-operative period.     

Recombinant Erythropoietin in Humans Has a Prolonged Effect on Circulating Erythropoietin Isoform Distribution

The membrane-assisted isoform immunoassay (MAIIA) quantitates erythropoietin (EPO) isoforms as percentages of migrated isoforms (PMI). We evaluated the effect of recombinant human EPO (rhEPO) on the distribution of EPO isoforms in plasma in a randomized, placebo-controlled, double-blinded, cross-over study. 16 healthy subjects received either low-dose Epoetin beta (5000 IU on days 1, 3, 5, 7, 9, 11 and 13); high-dose Epoetin beta (30.000 IU on days 1, 2 and 3 and placebo on days 5, 7, 9, 11 and 13); or placebo on all days. PMI on days 4, 11 and 25 was determined by interaction of N-acetyl glucosamine with the glycosylation dependent desorption of EPO isoforms. At day 25, plasma-EPO in both rhEPO groups had returned to values not different from the placebo group. PMI with placebo, reflecting the endogenous EPO isoforms, averaged 82.5 (10.3) % (mean (SD)). High-dose Epoetin beta decreased PMI on days 4 and 11 to 31.0 (4.2)% (p<0.00001) and 45.2 (7.3)% (p<0.00001). Low-dose Epoetin beta decreased PMI on days 4 and 11 to 46.0 (12.8)% (p<0.00001) and 46.1 (10.4)% (p<0.00001). In both rhEPO groups, PMI on day 25 was still decreased (high-dose Epoetin beta: 72.9 (19.4)% (p = 0.029); low-dose Epoetin beta: 73.1 (17.8)% (p = 0.039)). In conclusion, Epoetin beta leaves a footprint in the plasma-EPO isoform pattern. MAIIA can detect changes in EPO isoform distribution up til at least three weeks after administration of Epoetin beta even though the total EPO concentration has returned to normal.     

Kinetic Folding Mechanism of Erythropoietin

This report describes what to our knowledge is the first kinetic folding studies of erythropoietin, a glycosylated four-helical bundle cytokine responsible for the regulation of red blood cell production. Kinetic responses for folding and unfolding reactions initiated by manual mixing were monitored by far-ultraviolet circular dichroism and fluorescence spectroscopy, and folding reactions initiated by stopped-flow mixing were monitored by fluorescence. The urea concentration dependence of the observed kinetics were best described by a three-state model with a transiently populated intermediate species that is on-pathway and obligatory. This folding scheme was further supported by the excellent agreement between the free energy of unfolding and m-value calculated from the microscopic rate constants derived from this model and these parameters determined from separate equilibrium unfolding experiments. Compared to the kinetics of other members of the four-helical bundle cytokine family, erythropoietin folding and unfolding reactions were slower and less susceptible to aggregation. We tentatively attribute these slower rates and protection from association events to the large amount of carbohydrate attached to erythropoietin at four sites.