Differentiation Of Erythroid Progenitor Cells Research Articles

Epobis is a Nonerythropoietic and Neuroprotective Agonist of the Erythropoietin Receptor with Anti-Inflammatory and Memory Enhancing Effects.

The cytokine erythropoietin (EPO) stimulates proliferation and differentiation of erythroid progenitor cells. Moreover, EPO has neuroprotective, anti-inflammatory, and antioxidative effects, but the use of EPO as a neuroprotective agent is hampered by its erythropoietic activity. We have recently designed the synthetic, dendrimeric peptide, Epobis, derived from the sequence of human EPO. This peptide binds the EPO receptor and promotes neuritogenesis and neuronal cell survival. Here we demonstrate that Epobis in vitro promotes neuritogenesis in primary motoneurons and has anti-inflammatory effects as demonstrated by its ability to decrease TNF release from activated AMJ2-C8 macrophages and rat primary microglia. When administered systemically Epobis is detectable in both plasma and cerebrospinal fluid, demonstrating that the peptide crosses the blood-brain barrier. Importantly, Epobis is not erythropoietic, but systemic administration of Epobis in rats delays the clinical signs of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, and the peptide has long-term, but not short-term, effects on working memory, detected as an improved social memory 3 days after administration. These data reveal Epobis to be a nonerythropoietic and neuroprotective EPO receptor agonist with anti-inflammatory and memory enhancing properties.

ОСОБЛИВОСТІ ПРОЛІФЕРАЦІЇ ТА ДИФЕРЕНЦІЮВАННЯ КЛІТИН-ПОПЕРЕДНИКІВ ЕРИТРОПОЕЗУ ПРИ ХРОНІЧНІЙ МІЄЛОЇДНІЙ ЛЕЙКЕМІЇ В КУЛЬТУРІ КЛІТИН IN VIVO

The article presents a study of proliferation and differentiation features of erythroid progenitor cells of patients with chronic myeloid leukemia during the treatment with tyrosine kinase inhibitors – imatinib and nilotinib. The cultivation results showed an increase in proliferative activity of erythropoiesis progenitor cells both in the case of patients with leukemia, diagnosed for the first time, and in the case of bone marrow samples of patients with a resistance of leukemic cells clone to treatment with tyrosine kinase inhibitors. Moreover, the results showed an inhibition of erythroid progenitor cell differentiation and acquisition of resistance to tyrosine kinase inhibitors by leukemic cells clone.

Erythropoietin Receptor Antagonist Suppressed Ectopic Hemoglobin Synthesis in Xenografts of HeLa Cells to Promote Their Destruction.

The aim of this study is to explore a cause-oriented therapy for patients with uterine cervical cancer that expresses erythropoietin (Epo) and its receptor (EpoR). Epo, by binding to EpoR, stimulates the proliferation and differentiation of erythroid progenitor cells into hemoglobin-containing red blood cells. In this study, we report that the HeLa cells in the xenografts expressed ε, γ, and α globins as well as myoglobin (Mb) to produce tetrameric α2ε2 and α2γ2 and monomeric Mb, most of which were significantly suppressed with an EpoR antagonist EMP9. Western blotting revealed that the EMP9 treatment inhibited the AKT-pAKT, MAPKs-pMAPKs, and STAT5-pSTAT5 signaling pathways. Moreover, the treatment induced apoptosis and suppression of the growth and inhibited the survival through disruption of the harmonized hemoprotein syntheses in the tumor cells concomitant with destruction of vascular nets in the xenografts. Furthermore, macrophages and natural killer (NK) cells with intense HIF-1α expression recruited significantly more in the degenerating foci of the xenografts. These findings were associated with the enhanced expressions of nNOS in the tumor cells and iNOS in macrophages and NK cells in the tumor sites. The treated tumor cells exhibited a substantial number of perforations on the cell surface, which indicates that the tumors were damaged by both the nNOS-induced nitric oxide (NO) production in the tumor cells as well as the iNOS-induced NO production in the innate immune cells. Taken together, these data suggest that HeLa cells constitutively acquire ε, γ and Mb synthetic capacity for their survival. Therefore, EMP9 treatment might be a cause-oriented and effective therapy for patients with squamous cell carcinoma of the uterine cervix.

NR2F6 (EAR-2) Is a Novel Leukemia Oncogene Whose Cellular Function Is to Regulate Terminal Differentiation of Erythrocytes at the Proerythroblast Stage

The leukemia stem cell model suggests that elucidation of the genes that regulate growth ability within the leukemia cell hierarchy will have important clinical relevance. We showed that the expression of NR2F6 (EAR-2), is greater in clonogenic leukemia single cells than in leukemia cells that do not divide, and that this gene is over-expressed in patients with acute myeloid leukemia and myelodysplastic syndrome. In vivo, overexpression of EAR-2 using a retroviral vector in a chimeric mouse model leads to a condition that resembles myelodysplastic syndrome with hypercellular bone marrow, increased blasts, abnormal localization of immature progenitors, morphological dysplasia of the erythroid lineage and a competitive advantage over wild-type cells, that eventually leads to AML in a subset of the mice, or after secondary-transplantation. Interestingly, animals transplanted with bone marrow that over-expresses EAR-2 develop leukemia that is preceded by expansion of the stem cell compartment in the transplanted mice—suggesting that EAR-2 is an important regulator of hematopoietic stem cell differentiation. Here we report that over-expression of EAR-2 also has a profound effect on the differentiation of erythroid progenitor cells both in vitro and in vivo.Studies of the roles of EAR-2 in normal primary bone marrow cells in vitro showed that overexpression of EAR-2 profoundly impaired differentiation along the erythroid lineage. EAR-2 over-expressing bone marrow cells formed 40% fewer BFU-E colonies, but had greatly extended replating capacity in colony assays. While knockdown of EAR-2 increased the number of cells produced per BFU-E colony 300%.Normal mice transplanted with grafts of purified bone marrow cells that over-expressed EAR-2 developed a rapidly fatal leukemia characterized by pancytopenia, enlargement of the spleen, and infiltration of blasts into the spleen, liver and peripheral blood. Sick animals had profound reduction of peripheral blood cell counts, particularly anemia with a 55% reduction in hemoglobin levels. Anemia was evident even on gross inspection of the blood and the liver in EAR-2 overexpressing animals.Analysis of the leukemic cells revealed an erythroblastic morphology, with the immunophenotype lineageneg, CD71high, TER119med. Hence, we wondered weather EAR-2 caused leukemia by arresting erythroid progenitor cell differentiation. Examination of the bone marrow of pre-leukemic animals showed a four-fold increase in cells with a pro-erythroblastic immunophenotype (CD71highTER119med , region I), and a four-fold decrease in orthochromatophilic erythroblasts (CD71lowTER119high , region IV). We observed no change in the numbers of basophilic erythroblasts (CD71highTER119high , region II) or late basophilic and polychromatophilic erythroblasts (CD71medTER119high, region III). These data suggests that over-expression of EAR-2 blocks erythroid cell differentiation at the pro-erythroblastic stage.Since EAR-2 over-expressing recipients died within 4 week, we wanted to definitively test whether animals had compromised radioprotection. We showed that decreasing the size of the bone marrow graft, reduced survival of the EAR-2 over-expressing cohort by a week, but had no effect on control animals proving that EAR-2 over-expression has a profound effect on erythropoietic reconstitution in vivo.Mechanistically, we show that DNA binding is necessary for EAR-2 function, and that EAR-2 functions in an HDAC-dependent manner, regulating expression of several genes. Pre-leukemic pro-erythroblastic cells (CD71highTER119med) that over-expressed EAR-2 had lower expression of genes involved in erythroid differentiation such as GATA1, EBF1, inhibitor of NFKB (NFKBia), ETV6, CEBP/a, LMO2, and Nfe2, and increased expression of GATA2, GLI1, ID1 and PU.1 than GFP control pro-erythroblasts.These data establish that EAR-2 is a novel oncogene whose cellular function is to regulate terminal differentiation of erythroid cells at the proerythroblastic (CD71highTER119med) stage by deregulating gene expression necessary for erythroid differentiation. DisclosuresIchim:Entest BioMedical: Employment, Equity Ownership, Patents & Royalties, Research Funding. Koos:Entest BioMedical: Employment, Equity Ownership, Patents & Royalties, Research Funding.

Neutrophil Gelatinase-Associated Lipocalin Levels in Patients with Thalassemia and Sickle Cell Disease: Correlation with Renal Injury

Background and Aims: Neutrophil gelatinase-associated lipocalin (NGAL) is a protein belonging to the lipocalin superfamily initially found in activated neutrophils, in accordance with its role as an innate antibacterial factor. However, it subsequently was shown that many other types of cells, including in the kidney tubule, may produce NGAL in response to various injuries. The increase in NGAL production and release from tubular cells after harmful stimuli of various kinds may have self-defensive intent based on the activation of specific iron-dependent pathways, which in all probability also represent the mechanism through which NGAL promotes kidney growth and differentiation. NGAL levels clearly correlate with severity of renal impairment, probably expressing the degree of active damage underlying the chronic condition. For all these reasons, NGAL may become one of the most promising next-generation biomarkers in clinical nephrology and beyond. We aimed to investigate the clinical significance of NGAL levels and its correlation with renal function in patients with hemoglobinopathies.Patients and Methods: 117 adult patients with hemoglobinopathies were included in the study divided in 3 groups. Group A: 30 patients with transfusion-dependent thalassemia major (TM); Group B: 29 patients with Non-Transfusion-Dependent Thalassemia (TI) and Group C: 58 patients with HbS/βthal disease, while 20 apparently healthy individuals served as controls (Group D). In patients and controls along with standard blood and urine chemistry. Measurements of serum Cystatin C and NGAL were performed by means of immunonephelometric and immunoenzymatic techniques, (Siemens Healthcare Diagnostics, Liederbach, Germany and R&D Systems, Minneapolis, MN, USA), respectively. Estimated Glomerular Filtration Rate (eGFR) values were calculated with an unadjusted for body surface Cystatin C based equation: eGFR (mL/min)=77.24(Cys C)-1.2623.Results: The main results of the study showed that: a) NGAL levels were significantly higher in all the groups of patients compared to controls: Group A 95.0±45.0µg/L, Group B 139.1±86.1µg/L, Group C 117.8±37.3µg/L vs Group D 50.3±11.3µg/L (p<0.001), b) Cystatin C levels were significantly higher in patients of Group A 0.94±0.34mg/L and Group C 1.04±0.50mg/L compared to controls 0.75±0.09mg/L (p<0.01), while no significant difference was found between Group B 0.72±0.13mg/L and controls (p>0.300), c) NGAL levels and eGFR values (Group A: 96.9±39.8, Group B: 117.0±26.0, Group C: 86.2±27.8 and group D: 109.6±15.0mL/min, respectively) correlated significantly in patients of Group A and Group C (r=-0.739, p<0.001 and r=-0.735, p<0.001, respectively), while NGAL values are independent from eGFR values in patients of Group B.Conclusions: These findings illustrate the tubular-glomerular activation feedback mirrored by NGAL in patients with transfusion-dependent thalassemia major and HbS/βthal disease, who suffer from renal injuries, indicating that tubular damage precedes GFR reduction. Upregulation of NGAL in patients with non-transfusion-dependent thalassemia independently of renal injuries may reflect the compensatory, protective role of NGAL in response to diverse cellular stresses, including inflammation and oxidative stress. However, recent reports have implicated NGAL upregulation as a mechanism that contributes to anemia in the setting of chronic low grade inflammation. In experimental models, systemic and medullary NGAL has been demonstrated to induce inhibition of erythropoiesis through induction of apoptosis and arrest of differentiation of erythroid progenitor cells. DisclosuresNo relevant conflicts of interest to declare.

Disturbed Tryptophan Metabolism in Cardiovascular Disease

Atherosclerosis (AS), a major pathologic consequence of obesity, is the main etiological factor of cardiovascular disease (CVD), which is the most common cause of death in the western world. A systemic chronic low grade immune- mediated inflammation (scLGI) is substantially implicated in AS and its consequences. In particular, proinflammatory cytokines play a major role, with Th1-type cytokine interferon-γ (IFN-γ) being a key mediator. Among various other molecular and cellular effects, IFN-γ activates the enzyme indoleamine 2,3-dioxygenase (IDO) in monocyte-derived macrophages, dendritic, and other cells, which, in turn, decreases serum levels of the essential amino acid tryptophan (TRP). Thus, people with CVD often have increased serum kynurenine to tryptophan ratios (KYN/TRP), a result of an increased TRP breakdown. Importantly, increased KYN/TRP is associated with a higher likelihood of fatal cardiovascular events. A scLGI with increased production of the proinflammatory adipokine leptin, in combination with IFN-γ and interleukin-6 (IL-6), represents another central link between obesity, AS, and CVD. Leptin has also been shown to contribute to Th1-type immunity shifting, with abdominal fat being thus a direct contributor to KYN/TRP ratio. However, TRP is not only an important source for protein production but also for the generation of one of the most important neurotransmitters, 5-hydroxytryptamine (serotonin), by the tetrahydrobiopterin-dependent TRP 5-hydroxylase. In prolonged states of scLGI, availability of free serum TRP is strongly diminished, affecting serotonin synthesis, particularly in the brain. Additionally, accumulation of neurotoxic KYN metabolites such as quinolinic acid produced by microglia, can contribute to the development of depression via NMDA glutamatergic stimulation. Depression had been reported to be associated with CVD endpoints, but it most likely represents only a secondary loop connecting excess adipose tissue, scLGI and cardiovascular morbidity and mortality. Accelerated catabolism of TRP is further involved in the pathogenesis of the anemia of scLGI. The pro-inflammatory cytokine IFN-γ suppresses growth and differentiation of erythroid progenitor cells, and the depletion of TRP limits protein synthesis and thus hemoglobin production, and, through reduction in oxygen supply, may contribute to ischemic vascular disease. In this review we discuss the impact of TRP breakdown and the related complex mechanisms on the prognosis and individual course of CVD. Measurement of TRP, KYN concentrations, and calculation of the KYN/TRYP ratio will contribute to a better understanding of the interplay between inflammation, metabolic syndrome, mood disturbance, and anemia, all previously described as significant predictors of an unfavorable outcome in patients with CVD. The review leads to a novel framework for successful therapeutic modification of several cardinal pathophysiological processes leading to adverse cardiovascular outcome.

Soluble expression and partial purification of recombinant human erythropoietin from E. coli

Human erythropoietin (hEpo) is an essential regulator of erythrocyte production that induces the division and differentiation of erythroid progenitor cells in the bone marrow into mature erythrocytes. It is widely used for the treatment of anemia resulting from chronic kidney disease, chemotherapy, and cancer-related therapies. Active hEpo, and hEpo analogs, have been purified primarily from mammalian cells, which has several disadvantages, including low yields and high production costs. Although an Escherichia coli (E. coli) expression system may provide economic production of therapeutic proteins, it has not been used for the production of recombinant hEpo (rhEpo) because it aggregates in inclusion bodies in the E. coli cytoplasm and is not modified post-translationally. We investigated the soluble overexpression of active rhEpo with various protein tags in E. coli, and found out that several tags increased the solubility of rhEpo. Among them maltose binding protein (MBP)-tagged rhEpo was purified using affinity and gel filtration columns. Non-denaturing electrophoresis and MALDI-TOF MS analysis demonstrated that the purified rhEpo had two intra-disulfide bonds identical to those of the native hEpo. An in vitro proliferation assay showed that rhEpo purified from E. coli had similar biological activity as rhEpo derived from CHO cells. Therefore, we report for the first time that active rhEpo was overexpressed as a soluble form in the cytoplasm of E. coli and purified it in simple purification steps. We hope that our results offer opportunities for progress in rhEpo therapeutics.

A protective role of heme-regulated eIF2α kinase in cadmium-induced toxicity in erythroid cells

Although a number of studies have demonstrated that cadmium (Cd) can incur damage to mature red cells, the potential injuries of Cd to erythroid progenitor cells have not been investigated thus far. Heme-regulated eIF2α kinase (Hri) is essential for translational regulation and survival of erythroid precursors in the setting of iron deficiency. Hri has been demonstrated to activate Atf4 signaling in reducing oxidative stress and in promoting erythroid differentiation during stress erythropoiesis. Here, we demonstrated that Cd significantly provoked cell death and suppressed erythroid differentiation of erythroid progenitor cells. Importantly, our results established a crucial role of Hri in ameliorating Cd-induced impairment to erythropoiesis. Upon Cd treatment, Hri-eIF2αP-Atf4 signaling was activated to protect cells from cell death and differentiation attenuation in Wt fetal liver erythroblasts; in contrast, Hri−/− erythroblasts suffered from enhanced oxidative stress, as evidenced by increased levels of reactive oxygen species (ROS) and consequentially elevated apoptosis. As for Cd administration in vivo, impaired erythropoiesis in bone marrow and dramatic extramedullary erythropoiesis in spleen were observed in Hri−/− mice. Taken together, our combined data highlighted a crucial role of Hri in protecting survival and differentiation of erythroid progenitor cells upon Cd treatment.

Molecular and functional characterization of erythropoietin of the goldfish (Carassius auratus L.)

Erythropoietin is the principal regulator of erythropoiesis and promotes the survival, proliferation and differentiation of erythroid progenitor cells in mammals. In this study we report on the molecular and functional characterization of erythropoietin from the goldfish. Quantitative expression analysis of goldfish epo revealed the highest mRNA levels in heart, followed by brain, liver, spleen and kidney tissues. There was no marked change of epo expression in goldfish primary kidney macrophage cultures, as progenitor cell to macrophage development progressed, indicating that erythropoietin is not involved in monopoiesis. Recombinant goldfish erythropoietin induced proliferation of progenitor cells in a dose-dependent manner, and up-regulated the expression of erythroid transcription factors gata1 and lmo2 in progenitor cells. Furthermore, recombinant goldfish erythropoietin stimulated erythroid colony formation in a dose-dependent manner and promoted survival of erythroid progenitor cells as colony-forming cells. Our results demonstrate that the function of erythropoietin in the goldfish is similar to that of mammals and suggest a highly conserved mechanism of early erythrocyte development in lower and higher vertebrates.

Critical Role of the Matricellular Protein SPARC in Mediating Erythroid Progenitor Cell Development in Zebrafish

Sparc (osteonectin) is a multifunctional matricellular glycoprotein expressed by many differentiated cells. Members of this family mediate cell-matrix interactions rather than acting as structural components of the extracellular matrix (ECM); therefore, they can influence many remodelling events, including haematopoiesis. We have investigated the role of sparc in embryonic haematopoiesis using a morpholino antisense oligonucleotide-based knockdown approach. Knockdown of sparc function resulted in specific erythroid progenitor cell differentiation defects that were highlighted by changes in gene expression and morphology, which could be rescued by injection of sparc mRNA. Furthermore, a comparison of blood phenotypes of sparc and fgfs knockdowns with similar defects and the sparc rescue of the fgf21 blood phenotype places sparc downstream of fgf21 in the genetic network regulating haematopoiesis in zebrafish. These results establish a role for an ECM protein (Sparc) as an important regulator of embryonic haematopoiesis during early development in zebrafish.

Measurement of generation‐dependent proliferation rates and death rates during mouse erythroid progenitor cell differentiation

Herein, we describe an experimental and computational approach to perform quantitative carboxyfluorescein diacetate succinimidyl ester (CFSE) cell-division tracking in cultures of primary colony-forming unit-erythroid (CFU-E) cells, a hematopoietic progenitor cell type, which is an important target for the treatment of blood disorders and for the manufacture of red blood cells. CFSE labeling of CFU-Es isolated from mouse fetal livers was performed to examine the effects of stem cell factor (SCF) and erythropoietin (EPO) in culture. We used a dynamic model of proliferation based on the Smith-Martin representation of the cell cycle to extract proliferation rates and death rates from CFSE time-series. However, we found that to accurately represent the cell population dynamics in differentiation cultures of CFU-Es, it was necessary to develop a model with generation-specific rate parameters. The generation-specific rates of proliferation and death were extracted for six generations (G(0) -G(5) ) and they revealed that, although SCF alone or EPO alone supported similar total cell outputs in culture, stimulation with EPO resulted in significantly higher proliferation rates from G(2) to G(5) and higher death rates in G(2) , G(3) , and G(5) compared with SCF. In addition, proliferation rates tended to increase from G(1) to G(5) in cultures supplemented with EPO and EPO + SCF, while they remained lower and more constant across generations with SCF. The results are consistent with the notion that SCF promotes CFU-E self-renewal while EPO promotes CFU-E differentiation in culture.

Current Perspectives on Protective Roles of Erythropoietin in Cardiovascular System: Erythropoietin Receptor as a Novel Therapeutic Target

Erythropoietin (EPO) is a principal regulator that promotes proliferation and terminal differentiation of erythroid progenitor cells. EPO receptors are expressed not only in hematopoietic lineage cells but also in the cardiovascular system. We performed animal experiments using transgene-rescued EPO receptor null mutant mice (EpoR-/- rescued) that express the EPO receptor exclusively in the hematopoietic cells. The results of these experiments suggest that endogenous EPO/EPO receptor system in the heart exerts cardioprotective effects against myocardial injury induced by ischemia followed by reperfusion and pressure-overload induced left ventricular dysfunction. Many animal experiments have shown that the administration of recombinant human EPO also elicits cardioprotective effects against myocardial injury induced by ischemia and reperfusion. In contrast to the promising results of these animal experiments, recent clinical trials failed to demonstrate the reduction in infarct size or improvement of cardiac function by the administration of recombinant human EPO in patients with acute myocardial infarction who underwent primary percutaneous coronary intervention. It should be tested in future clinical studies whether a relatively low dose of recombinant human EPO or its derivatives that have no erythropoietic action reduces infarct size and ameliorates cardiac dysfunction in patients with acute myocardial infarction. In this article, we review implications of anemia associated with chronic heart failure, roles of the endogenous EPO/EPO receptor system, and the effects of the administration of erythropoiesis-stimulating agents in pathologic conditions of the heart by focusing on the EPO receptor as a potential candidate of novel therapeutic targets in cardiovascular diseases.

The protective effects of erythropoietin/erythropoietin receptor system on optic nerve and retinal diseases

Erythropoietin(EPO) has an anti-apoptotic effect,and promotes the proliferation and differentiation of erythroid progenitor cells. Several studies have indicated that EPO can protect photoreceptor cells from the lightinduced retinal degeneration ;protect retinal neurons from ischemia-reperfusion injury and retinal ganglion cells after acute and chronic ocular hypertension; promote ganglion cell survival and axonal regeneration after optic nerve transaction; attenuate inflammation in multiple sclerosis optic neuritis; reduce the permeability of the retinal barrier and protect retinal neurons in diabetic retinopathy; enhance the stability of hypoxic retinal vessels in retinopathy of prematurity. Herein,we review the distribution of EPO and its receptor in retina,their expression in animal model of retinal diseases,and their effects and mechanisms in protection of retinal neurons and optic nerve. Key words: Erythropoietin; Erythropoietin receptor; Optic nerve; Retina; Neuroprotection

Erythropoietin levels in endocrinopathies

Erythropoietin (EPO) is an oxygenregulated hormone promoting the differentiation of erythroid progenitor cells. Apart fromhypoxia, few data is available about release by secretagogues including hormones. To investigate EPO serum concentration in subjects with endocrine diseases. A retrospective study evaluating serumEPO concentrations in serumleftovers fromsubjects with various endocrine disorders. EPO is not noticeably influenced by thyroid hormone or cortisol concentrations and the relationship with hemoglobin concentration is preserved. In acromegalic patients, the latter is lost but EPO is neither statistically influenced by GH/IGF-I. This may reflect a dual action of GH and/or IGF-I on erythroid progenitors proliferation as well as on EPO synthesis. EPO is not noticeably modified by endocrine disorders although GH and or IGF-I may alter EPO relationship with blood hemoglobin concentration.

Cytosolic lysine residues enhance anterograde transport and activation of the erythropoietin receptor

Lysine residues are key residues in many cellular processes, in part due to their ability to accept a wide variety of post-translational modifications. In the present study, we identify the EPO-R [EPO (erythropoietin) receptor] cytosolic lysine residues as enhancers of receptor function. EPO-R drives survival, proliferation and differentiation of erythroid progenitor cells via binding of its ligand EPO. We mutated the five EPO-R cytosolic lysine residues to arginine residues (5KR EPO-R), eliminating putative lysine-dependent modifications. Overexpressed 5KR EPO-R displayed impaired ubiquitination and improved stability compared with wt (wild-type) EPO-R. Unexpectedly, fusion proteins consisting of VSVGtsO45 (vesicular stomatitis virus glycoprotein temperature-sensitive folding mutant) with wt or 5KR EPO-R cytosolic domains demonstrated delayed glycan maturation kinetics upon substitution of the lysine residues. Moreover, VSVG-wt EPO-R, but not VSVG-5KR EPO-R, displayed endoplasmic reticulum-associated ubiquitination. Despite similar cell-surface EPO-binding levels of both receptors and the lack of EPO-induced ubiquitination by 5KR EPO-R, the lysine-less mutant produced weaker receptor activation and signalling than the wt receptor. We thus propose that EPO-R cytosolic lysine residues enhance receptor function, most probably through ubiquitination and/or other post-translational modifications.

Neuroprotective properties of a novel, non-haematopoietic agonist of the erythropoietin receptor

Erythropoietin, a member of the type 1 cytokine superfamily, controls proliferation and differentiation of erythroid progenitor cells through binding to and dimerization of the erythropoietin receptor. Both erythropoietin and its receptor are also expressed in the central nervous system, where they are involved in tissue protection. However, the use of erythropoietin as a neuroprotective agent may be hampered by its erythropoietic activity. Therefore, developing non-haematopoietic erythropoietin mimetics is important. Based on the crystal structure of the complex of erythropoietin and its receptor, we designed a peptide, termed Epotris, corresponding to the C α-helix region (amino-acid residues 92-111) of human erythropoietin. The peptide specifically bound to the erythropoietin receptor and promoted neurite outgrowth and survival of primary neurons with the same efficiency as erythropoietin, but with 10(3)-fold lower potency. Knockdown of the erythropoietin receptor or interference with its downstream signalling inhibited the Epotris-induced neuritogenic and pro-survival effect. Similarly to erythropoietin, Epotris penetrated the blood-brain barrier. Moreover, treatment with the peptide attenuated seizures, decreased mortality and reduced neurodegeneration in an in vivo model of kainic acid-induced neurotoxicity. In contrast to erythropoietin, Epotris did not stimulate erythropoiesis upon chronic administration. Thus, Epotris is a novel neuroprotective non-haematopoietic erythropoietin mimetic that may offer new opportunities for the treatment of neurological disorders.

Erythropoietin levels in endocrinopathies.

Background: Erythropoietin (EPO) is an oxygen-regulated hormone promoting the differentiation of erythroid progenitor cells. Apart from hypoxia, few data is available about release by secretagogues including hormones. Aim: To investigate EPO serum concentration in subjects with endocrine diseases. Material and methods: A retrospective study evaluating serum EPO concentrations in serum leftovers from subjects with various endocrine disorders. Results: EPO is not noticeably influenced by thyroid hormone or cortisol concentrations and the relationship with hemoglobin concentration is preserved. In acromegalic patients, the latter is lost but EPO is neither statistically influenced by GH/IGF-I. This may reflect a dual action of GH and/or IGF-I on erythroid progenitors proliferation as well as on EPO synthesis. Conclusion: EPO is not noticeably modified by endocrine disorders although GH and or IGF-I may alter EPO relationship with blood hemoglobin concentration.

Erythroid Progenitor Cells Expanded from Peripheral Blood without Mobilization or Preselection: Molecular Characteristics and Functional Competence

BackgroundContinued development of in-vitro procedures for expansion and differentiation of erythroid progenitor cells (EPC) is essential not only in hematology and stem cell research but also virology, in light of the strict erythrotropism of the clinically important human parvovirus B19.Methodology/Principal FindingsWe cultured EPC directly from ordinary blood samples, without ex vivo stem cell mobilization or CD34+ cell in vitro preselection. Profound increase in the absolute cell number and clustering activity were observed during culture. The cells obtained expressed the EPC marker combination CD36, CD71 and glycophorin, but none of the lymphocyte, monocyte or NK markers. The functionality of the generated EPC was examined by an in vitro infection assay with human parvovirus B19, tropic for BFU-E and CFU-E cells. Following infection (i) viral DNA replication and mRNA production were confirmed by quantitative PCR, and (ii) structural and nonstructural proteins were expressed in >50% of the cells. As the overall cell number increased 100–200 fold, and the proportion of competent EPC (CD34+ to CD36+) rose from <0.5% to >50%, the in vitro culture procedure generated the EPC at an efficiency of >10 000-fold. Comparative culturing of unselected PBMC and ex vivo-preselected CD34+ cells produced qualitatively and quantitatively similar yields of EPC.Conclusions/SignificanceThis approach yielding EPC directly from unmanipulated peripheral blood is gratifyingly robust and will facilitate the study of myeloid infectious agents such as the B19 virus, as well as the examination of erythropoiesis and its cellular and molecular mechanisms.

The erythropoietin and regenerative medicine: a lesson from fish

Erythropoietin (EPO), the main haematopoietic growth factor for the proliferation and differentiation of erythroid progenitor cells, is also known for its angiogenic and regenerative properties. In this study, we aimed to test the regenerative effects of EPO administration in an experimental model of Sea bass (Dicentrarchus labrax) subjected to amputation of the caudal fin. Erythropoietin-treated fishes (3000 UI of human recombinant EPO-alpha immediately after cutting and after 15 days) showed an increased growth rate of their fins compared with those untreated (anova variance: P: 0.01 vs. P: 0.04). By analysing fin length at established times (15 and 30 days after cut), EPO-treated fishes always showed an increased length compared with untreated ones (T-15: 1.1 +/- 0.2 vs. 0.7 +/- 0.2 cm, P: 0.03; T-30: 1.9 +/- 0.3 vs. 1.2 +/- 0.2 cm, P: 0.01). Moreover, exogenous EPO administration induced an enormous increase in EPO-blood levels at each observation time (T-15: 2240 +/- 210 vs. 16.7 +/- 1.8 mU mL(-1), P < 0.001; T-30: 2340 +/- 190 vs. 17.1 +/- 1.9 mU mL(-1), P < 0.001), whereas these levels remained quite unmodified in untreated fishes. Immunochemical analyses performed by confocal laser scanning microscopic observations showed an increased expression of EPO-receptors and PECAM-1 (an endothelial surface marker of vessels sprout) in the regenerating tissue, whereas no signs of inflammation or fibrosis were recognisable. All these findings confirm EPO as a new factor involved in regenerative processes, also suggesting a potential, future utility for new therapeutical applications in the field of human regenerative medicine.

Chronische Entzündungsanämie – Epiphänomen oder Adaptationsmechanismus der Entzündung?

Die chronische Entzündungsanämie (anaemia of chronic disease, ACD) ist die häufigste Anämie bei hospitalisierten Patienten und findet sich vor allem bei Personen, die an chronischen Infektionen, Autoimmunerkrankungen und malignen Tumoren leiden. Alle der ACD zugrunde liegenden pathophysiologischen Mechanismen sind durch die mit jener der Grundkrankheit assoziierten Immunaktivierung bedingt. Zytokine und Akutphaseproteine führen zu einer Retention von Eisen im retikuloendothelialen System und damit zu einer eisenlimitierten Erythropoese, da Eisen nicht für die Hämbiosynthese im Knochenmark zur Verfügung steht. Ferner hemmen vor allem pro-inflammatorische Zytokine die Proliferation und Differenzierung von erythroiden Vorläuferzellen, die Bildung und biologische Wirkung von Erythropoetin und führen wohl auch zu einer direkten Schädigung von Erythrozyten durch die Bildung von Radikalen und dadurch zu einer verminderten Lebensdauer der Erythrozyten. Die Entwicklung der ACD hat auch potentielle Vorteile, da durch die Limitierung der Eisenverfügbarkeit das Wachstum von eingedrungenen Pathogenen blockiert wird. Weiters führt der Entzug von Eisen zu einer Stärkung der durch IFN-γ induzierten Immunabwehrmechanismen von Makrophagen, da Eisen einen negativen Effekt auf pro-inflammatorische, zellvermittelte Immuneffektorwege ausübt. Während diese Strategie bei Infektion und Tumorerkrankungen wichtig ist, erweißt sich diese bei Autoimmunerkrankung wohl als kontraproduktiv. Während die alleinige therapeutische Gabe von Eisen zur Anämiebehandlung bei ACD Patienten mit Infektionen und Tumorerkrankungen aufgrund der Wachstumsförderung von Pathogenen sowie der durch das Metall vermittelten negativen immunologischen Effekte im Regelfall kontraindiziert ist, kann diese umgekehrt bei Patienten mit Erkrankungen des rheumatischen Formenkreises aufgrund der immunsupprimierenden Eigenschaften des Metalls positive Effekte auf die Krankheitsaktivität ausüben, was klinisch zu verifizieren sein wird.