OBJECTIVE

Clinical indications for erythropoietin (EPO) in the vascular system reach far beyond the treatment of anemia, but the development of EPO as a non-toxic agent rests heavily upon the cellular pathways controlled by EPO that require elucidation.

METHODS

We modulated gene activity and examined cellular trafficking of critical pathways during oxidative stress that may work in concert with EPO to protect primary cerebral endothelial cells (ECs) during oxidative stress, namely protein kinase B (Akt1), 14-3-3 protein, the Forkhead transcription factor FOXO3a.

RESULTS

Here, we show that preservation of ECs by EPO during oxygen-glucose deprivation (OGD) required the initial activation of the phosphatidylinositol 3-kinase (PI-3K) pathway through Akt1, since specific pharmacological blockade of Akt1 activity or gene silencing of Akt1 prevented EC protection by EPO. EPO subsequently involved a series of anti-apoptotic pathways to activate STAT3, STAT5, and ERK 1/2. Furthermore, EPO maintained the inhibitory phosphorylation and integrity of the 'pro-apoptotic' transcription factor FOXO3a, promoted the binding of FOXO3a to 14-3-3 protein and regulated the intracellular trafficking of FOXO3a. Additionally, gene silencing of FOXO3a during OGD significantly increased EC survival, but did not synergistically improve cytoprotection by EPO, illustrating that EPO relied upon the blockade of the FOXO3a pathway.

CONCLUSIONS

Our work defines a novel cytoprotective pathway in ECs that involves PI-3 K, STAT3, STAT5, ERK 1/2, 14-3-3 protein and FOXO3a, which can be targeted for the development of EPO as a clinically effective and safe agent in the vascular system.

Radioiodinated erythropoietin (Epo) was bound specifically to the cells of two non-erythroid clonal lines, PC12 and SN6, which expressed neuronal characteristics. The binding was time-, cell number-, and dose-dependent and was reversible. Although the cloned Epo receptor from PC12 cells (derived from rat adrenal medulla) was identical to that from rat erythroid cells, significant differences in the ligand binding properties between two cell lineages were found; 1) PC12 cells had a single class of binding sites with very low affinity (Kd = 16 nM), whereas erythroid cells had two classes of binding sites with different affinities (Kd = 95 pM for high affinity sites and 1.9 nM for low affinity sites), and 2) cross-linking experiments revealed one cross-linked product of 105 kDa for PC12 cells and two products of 140 and 120 kDa for erythroid cells. Taken together with additional results, the presence of a putative accessory protein(s) that may alter the ligand binding affinity through interaction with Epo receptor is discussed. The binding of Epo to PC12 cells caused a rapid increase in the cytosolic concentration of free calcium. The presence of EGTA had no effect on the Epo binding but completely inhibited the calcium increase, indicating that Epo stimulated the calcium influx from outside of the cells. The addition of Epo to the culture media of PC12 cells elevated the intracellular concentrations of monoamines.

We have developed a strategy for the rapid enrichment and identification of cDNAs encoded by large genomic regions. The basis of this "direct selection" scheme is the hybridization of an entire library of cDNAs to an immobilized genomic clone. Nonspecific hybrids are eliminated and selected cDNAs are eluted. These molecules are then amplified and are either cloned or subjected to further selection/amplification cycles. This scheme was tested using a 550-kilobase yeast artificial chromosome clone that contains the EPO gene. Using this clone and a fetal kidney cDNA library, we have achieved a 1000-fold enrichment of EPO cDNAs in one cycle of enrichment. More significantly, we have further investigated one of the "anonymous" cDNAs that was selectively enriched. We confirmed that this cDNA was encoded by the yeast artificial chromosome. Its frequency in the starting library was 1 in 1 x 10(5) cDNAs and after selection comprised 2% of the selected library. DNA sequence analysis of this cDNA and of the yeast artificial chromosome clone revealed that this gene encodes the beta 2 subunit of the human guanine nucleotide-binding regulatory proteins. Restriction mapping and hybridization data position this gene (GNB2) to within 30-70 kilobases of the EPO gene. The selective isolation and mapping of GNB2 confirms the feasibility of this direct selection strategy and suggests that it will be useful for the rapid isolation of cDNAs, including disease-related genes, across extensive portions of the human genome.

We tested the effects of four eosinophil granule cationic proteins: major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin (EDN), on guinea pig tracheal epithelium in vitro. Examination by inverted microscopy revealed that MBP, both the form stabilized by alkylation of sulfhydryl groups as well as the native form of the molecule, ECP, EPO by itself, as well as EPO + H2O2 + halide, but not EDN, cause dose-related damage to the tracheal epithelium. The lowest concentrations of MBP and ECP causing damage were 10 and 100 micrograms/ml, respectively. In contrast, EDN, although biochemically similar to ECP, did not damage the tracheal epithelium in concentrations of up to 200 micrograms/ml. MBP caused exfoliation, as well as bleb formation and ciliostasis. EPO in the presence of the H2O2-producing enzyme glucose oxidase (GO), Cl-, 0.11 M, and iodide caused ciliostasis, bleb formation, and exfoliation of epithelial cells at concentrations as low as 1 U/ml (3.9 micrograms/ml). EPO + GO in the presence of Cl-, 0.11 M, alone or with Cl- and l-, 10(-4) M, or Cl- and Br-, 5 x 10(-5) M, were all toxic to epithelium. Surprisingly, EPO by itself caused partial ciliostasis, bleb formation, and exfoliation of epithelial cells in a dose-related manner at concentrations as low as 10 to 30 U/ml (39 to 121 micrograms/ml). These results confirm prior observations showing the toxicity of MBP to tracheal epithelium and indicate that ECP and EPO alone, as well as EPO + GO + halide, cause damage. Thus, several eosinophil granule proteins are able to damage respiratory epithelium.

The JAK2(V617F) mutation is present in almost all patients with polycythemia vera (PV), large proportions of patients with essential thrombocythemia and idiopathic myelofibrosis, and less frequently in atypical myeloproliferative disorders (MPD). We show that transplantation of JAK2(V617F)-transduced bone marrow into BALB/c mice induces MPD reminiscent of human PV, characterized by erythrocytosis, granulocytosis, extramedullary hematopoiesis, and bone marrow fibrosis, but not thrombocytosis. Fluorescence-activated cell sorting of bone marrow and spleen showed proportional expansion of common myeloid progenitors, granulocyte-monocyte and megakaryocyte-erythrocyte progenitors. Megakaryocyte and late erythroid progenitors were dramatically increased, with only modest expansion of early erythroid progenitors. Erythropoietin (Epo) receptor expression was reduced on early, but normal on late erythroblasts. Serum levels of Epo and granulocyte colony-stimulating factor, but not granulocyte macrophage colony-stimulating factor, were reduced, whereas tumor necrosis factor-alpha was increased, possibly exerting a negative effect on JAK2(V617F)-negative hematopoiesis. These data suggest that erythrocytosis and granulocytosis in JAK2(V617F) mice are the net result of a complex interplay between cell intrinsic and extrinsic factors. There were no thromboembolic events and no animals succumbed to their disease, implicating additional factors in the manifestation of human disease. The disease was not transplantable and prolonged observation showed normalization of blood counts in most JAK2(V617F) mice, suggesting that the mutation may not confer self-renewal capacity.

The cytokine-inducible SH2 protein-3 (CIS3/SOCS-3/SSI-3) has been shown to inhibit the JAK/STAT pathway and act as a negative regulator of fetal liver erythropoiesis. Here, we studied the molecular mechanisms by which CIS3 regulates the erythropoietin (EPO) receptor (EPOR) signaling in erythroid progenitors and Ba/F3 cells expressing the EPOR (BF-ER). CIS3 binds directly to the EPOR as well as JAK2 and inhibits EPO-dependent proliferation and STAT5 activation. We have identified the region containing Tyr(401) in the cytoplasmic domain of the EPOR as a direct binding site for CIS3. Deletion of the Tyr(401) region of the EPOR reduced the inhibitory effect of CIS3, suggesting that binding of CIS3 to the EPOR augmented the negative effect of CIS3. Both N- and C-terminal regions adjacent to the SH2 domain of CIS3 were necessary for binding to EPOR and JAK2. In the N-terminal region of CIS3, the amino acid Gly(45) was critical for binding to the EPOR but not to JAK2, while Leu(22) was critical for binding to JAK2. The mutation of G45A partially reduced ability of CIS3 to inhibit EPO-dependent proliferation and STAT5 activation, while L22D mutant CIS3 was completely unable to suppress EPOR signaling. Moreover, overexpression of STAT5, which also binds to Tyr(401), reduced the binding of CIS3 to the EPOR, and the inhibitory effect of CIS3 against EPO signaling, while it did not affect JAB/SOCS-1/SSI-1. These data demonstrate that binding of CIS3 to the EPOR augments the inhibitory effect of CIS3. CIS3 binding to both EPOR and JAK2 may explain a specific regulatory role of CIS3 in erythropoiesis.

The plasma level of erythropoietin (Epo) in anemic patients suffering from inflammation is often low in relation to the blood hemoglobin concentration. Various proinflammatory cytokines have been tested for their action on the synthesis of Epo. Interleukin 1 (IL-1) and tumor necrosis factor-alpha(TNF-alpha) suppress Epo gene expression in isolated perfused rat kidneys and in human hepatoma cell cultures. IL-6 inhibits in the kidney, and conflicting results have been reported for its effect on Epo synthesis in hepatic cells. Several other cytokines tested were without effect. Thus, mainly IL-1 and TNF-alpha seem to be responsible for the defect in Epo production in severe systemic and renal inflammatory diseases.

OBJECTIVE

Experiments on human erythropoietin (EPO) demonstrated that there is a direct relationship between the sialic acid-containing carbohydrate content of EPO, its circulating half-life, and in vivo bioactivity. This led to the hypothesis that an EPO analogue engineered to contain additional oligosaccharide chains would have enhanced biological activity. Darbepoetin alfa, a hyperglycosylated recombinant human EPO (rHuEPO) analogue with two extra carbohydrate chains, was designed and developed to test this hypothesis.

METHODS

Comparative pharmacokinetic and pharmacodynamic studies and biochemical analyses of darbepoetin alfa and rHuEPO were performed to define the consequences of the increased carbohydrate content.

RESULTS

Due to its increased sialic acid-containing carbohydrate content, darbepoetin alfa has a higher molecular weight, a greater negative charge, and a approximately fourfold lower EPO receptor binding activity than rHuEPO. It also has a threefold longer circulating half-life than rHuEPO in rats and dogs. In spite of its lower receptor binding, and perhaps counterintuitively, darbepoetin alfa is significantly more potent in vivo than rHuEPO. Due to the pharmacokinetic differences, the relative potency of the two molecules varies as a function of the dosing frequency. Darbepoetin alfa is 3.6-fold more potent than rHuEPO in increasing the hematocrit of normal mice when each is administered thrice weekly, but when the administration frequency is reduced to once weekly, darbepoetin alfa is approximately 13-fold to 14-fold more potent than rHuEPO.

CONCLUSIONS

Increasing the sialic acid-containing carbohydrate content beyond the maximum found in EPO leads to a molecule with a longer circulating half-life and thereby an increased in vivo potency that can be administered less frequently.

The effects of the inflammatory cytokines interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6, transforming growth factor-beta (TGF-beta), and tumor necrosis factor-alpha (TNF-alpha) on erythropoietin (Epo) production in Hep3B cells were examined. The addition of IL-1 alpha, IL-1 beta, or TNF-alpha resulted in a dose-dependent inhibition of hypoxia-induced Epo production by as much as 89%. IL-1 beta was the most effective cytokine tested, demonstrating half-maximal inhibition at 0.4 U/mL compared with 1.0 and 10.0 U/mL for IL-1 alpha and TNF-alpha, respectively. TGF-beta also inhibited hypoxia-induced Epo production, but only by as much as 56%. In contrast to IL-1 alpha, IL-1 beta, TNF-alpha, and TGF-beta, the addition of IL-6 to hypoxic Hep3B cells resulted in a dose-dependent stimulation of hypoxia-induced Epo production by as much as 81%. However, IL-6 did not stimulate Epo synthesis in the absence of hypoxia, and was thus synergistic with hypoxia in inducing Epo production. Combinations of IL-1 alpha, TNF-alpha, and IL-6 were found to be additive in their effects on hypoxia-induced Epo production. By Northern blot analysis, Epo messenger RNA levels in Hep3B cells grown in 1% O2 were decreased when concurrently exposed to either IL-1 alpha or TNF-alpha. The effects that IL-1 alpha, IL-1 beta, TGF-beta, TNF-alpha, and IL-6 have on hypoxia-induced Epo production may provide new insights into the signal transduction pathway by which hypoxia leads to changes in gene expression. In addition, the effects of these inflammatory cytokines on hypoxia-induced Epo production in vitro suggest that in various inflammatory disorders these cytokines may affect Epo production in vivo and may play a significant role in the pathogenesis of the anemia of chronic disease.

Normal tissue function in mammals depends on adequate supply of oxygen through blood vessels. A discrepancy between oxygen supply and consumption (hypoxia) induces a variety of specific adaptation mechanisms at the cellular, local and systemic level. These mechanisms are in part governed by the activation of hypoxia-inducible transcription factors (HIF-1, HIF-2), which in turn modulate expression of hypoxically regulated genes such as those encoding vascular endothelial growth factor (VEGF) and erythropoietin (EPO). EPO is a glycoprotein that is produced mainly by interstitial fibroblasts in the kidneys of the adult and in hepatocytes in the foetus. Released into the circulation, EPO makes its way to the bone marrow, where it regulates red cell production by preventing apoptosis of erythroid progenitor cells. Recently, EPO has emerged as a multifunctional growth factor that plays a significant role in the nervous system. Both EPO and its receptor are expressed throughout the brain in glial cells, neurones and endothelial cells. Hypoxia and ischaemia have been recognised as important driving forces of EPO expression in the brain. EPO has potent neuroprotective properties in vivo and in vitro and appears to act in a dual way by directly protecting neurones from ischaemic damage and by stimulating endothelial cells and thus supporting the angiogenic effect of VEGF in the nervous system. Thus, hypoxia-induced gene products such as VEGF and EPO might be part of a self-regulated physiological protection mechanism to prevent neuronal injury, especially under conditions of chronically reduced blood flow (chronic ischaemia). In this review, I will briefly summarize the recent findings on the molecular mechanisms of hypoxia-regulated EPO expression in general and give an overview of its expression in the central nervous system, its action as a growth factor with non-haematopoietic functions and its potential clinical relevance in various brain pathologies.

Neonatal stroke leads to mortality and severe morbidity, but there is no effective treatment currently available. Erythropoietin (EPO) has been shown to promote cytoprotection and neurogenesis and decrease subventricular zone morphologic changes following brain injury. The long-term cellular response to EPO has not been defined, and local changes in cell fate decision may play a role in functional improvement. We performed middle cerebral artery occlusion in P10 rats. EPO treatment (5 U/g i.p.) significantly preserved hemispheric brain volume 6 weeks after injury. Furthermore, EPO increased the percentage of newly generated neurons while decreasing newly generated astrocytes following brain injury, without demonstrating long-term differences in the subventricular zone. These results suggest that EPO may neuroprotect and direct cell fate toward neurogenesis and away from gliogenesis in neonatal stroke.

The erythropoietin receptor (EPO-R) is a member of the recently described cytokine receptor superfamily. A constitutively active (hormone independent) form of the EPO-R was isolated that has a single amino acid change in the exoplasmic domain, converting arginine-129 to cysteine (R129C). Since EPO-Rs containing R129S, R129E, and R129P mutations are functionally wild type, the presence of cysteine at residue 129, and not the loss of arginine, is required for constitutive activity. Several mutant forms of the EPO-R were analyzed; all constitutively active mutants form disulfide-linked homodimers, whereas EPO-responsive or inactive forms of the receptor do not. Monomers and disulfide-linked dimers of the constitutive receptor are present on the plasma membrane and bind EPO with a single affinity. Homodimerization of the EPO-R is likely to play a role in ligand-induced signal transduction, and disulfide-linked dimerization of the constitutive receptor may mimic this step.

Binding of erythropoietin (Epo) to the Epo receptor (EpoR) is crucial for production of mature red cells. Although it is well established that the Epo-bound EpoR is a dimer, it is not clear whether, in the absence of ligand, the intact EpoR is a monomer or oligomer. Using antibody-mediated immunofluorescence copatching (oligomerizing) of epitope-tagged receptors at the surface of live cells, we show herein that a major fraction of the full-length murine EpoR exists as preformed dimers/oligomers in BOSC cells, which are human embryo kidney 293T-derived cells. This observed oligomerization is specific because, under the same conditions, epitope-tagged EpoR did not oligomerize with several other tagged receptors (thrombopoietin receptor, transforming growth factor beta receptor type II, or prolactin receptor). Strikingly, the EpoR transmembrane (TM) domain but not the extracellular or intracellular domains enabled the prolactin receptor to copatch with EpoR. Preformed EpoR oligomers are not constitutively active and Epo binding was required to induce signaling. In contrast to tyrosine kinase receptors (e.g., insulin receptor), which cannot signal when their TM domain is replaced by the strongly dimerizing TM domain of glycophorin A, the EpoR could tolerate the replacement of its TM domain with that of glycophorin A and retained signaling. We propose a model in which TM domain-induced dimerization maintains unliganded EpoR in an inactive state that can readily be switched to an active state by physiologic levels of Epo.

Administration of therapeutic proteins by methods other than injection is limited, in part, by inefficient penetration of epithelial barriers. Therefore, unique approaches to breaching these barriers are needed. The neonatal constant region fragment (Fc) receptor (FcRn), which is responsible for IgG transport across the intestinal epithelium in newborn rodents, is expressed in epithelial cells in adult humans and non-human primates. Here we show that FcRn-mediated transport is functional in the lung of non-human primates and that this transport system can be used to deliver erythropoietin (Epo) when it is conjugated to the Fc domain of IgG1. FcRn-dependent absorption was more efficient when the EpoFc fusion protein was deposited predominantly in the upper and central airways of the lung, where epithelial expression of FcRn was most prominently detected. To optimize fusion protein absorption in the lung, we created a recombinant "monomeric-Epo" Fc fusion protein comprised of a single molecule of Epo conjugated to a dimeric Fc. This fusion protein exhibited enhanced pharmacokinetic and pharmacodynamic properties. The bioavailability of the EpoFc monomer when delivered through the lung was approximately equal to that reported for unconjugated Epo delivered s.c. in humans. These studies show that FcRn can be harnessed to noninvasively deliver bioactive proteins into the systemic circulation in therapeutic quantities.

The glycoprotein hormone erythropoietin (EPO) is an essential viability and growth factor for the erythrocytic progenitors. EPO is mainly produced in the kidneys. EPO gene expression is induced by hypoxia-inducible transcription factors (HIF). The principal representative of the HIF-family (HIF-1, -2 and -3) is HIF-1, which is composed of an O2-labile alpha-subunit and a constant nuclear beta-subunit. In normoxia, the alpha-subunit of HIF is inactivated following prolyl- and asparaginyl-hydroxylation by means of alpha-oxoglutarate and Fe(2+)-dependent HIF specific dioxygenases. While HIF-1 and HIF-2 activate the EPO gene, HIF-3, GATA-2 and NFkappaB are likely inhibitors of EPO gene transcription. EPO signalling involves tyrosine phosphorylation of the homodimeric EPO receptor and subsequent activation of intracellular antiapoptotic proteins, kinases and transcription factors. Lack of EPO leads to anemia. Treatment with recombinant human EPO (rHuEPO) is efficient and safe in improving the management of the anemia associated with chronic renal failure. RHuEPO analogues with prolonged survival in circulation have been developed. Whether the recent demonstration of EPO receptors in various non-hemopoietic tissues, including tumor cells, is welcome or ominous still needs to be clarified. Evidence suggests that rHuEPO may be a useful neuroprotective agent.

Hypoxia in the tubulointerstitium has been thought to play pivotal roles in the pathophysiology of acute renal failure and the progression of chronic kidney disease. Pre-induction of hypoxia-inducible and renoprotective gene expression may protect subsequent ischemic injury. This study evaluated the efficacy of cobalt, which inhibits HIF-1 degradation and increases the expression level of hypoxia-related genes, in an acute ischemic tubulointerstitial injury model of rats. Ischemic renal injury was induced by 45-min clamping of renal pedicles with contralateral nephrectomy. Elevation of serum creatinine and morphologic injury after the ischemic insult was observed. Administration of cobalt chloride afforded striking functional improvement (mean +/- SEM creatinine in mg/dl: Co treatment group, 2.14 +/- 1.21; control, 3.69 +/- 1.43; P < 0.05) associated with amelioration of tubulointerstitial damage. Cobalt treatment also reduced macrophage infiltration significantly. In the kidney of rats treated with cobalt, mRNA levels of several genes that serve for tissue protection, such as HO-1, EPO, Glut-1, and VEGF, were increased before ischemic injury. Upregulation of HO-1 by cobalt was confirmed at the protein level. Subcutaneous injection of cobalt also ameliorated ischemic injury, which was associated with upregulation of renal HIF-1alpha protein expression. These results suggest that protection against hypoxic tubulointerstitial injury by cobalt administration is mediated by induction of renoprotective gene expression. HIF induction is one possible and attractive explanation for the observed effects.

OBJECTIVE

This meta-analysis assessed the pooled effect of each genetic variant reproducibly associated with diabetic nephropathy.

METHODS

PubMed, EMBASE and Web of Science were searched for articles assessing the association between genes and diabetic nephropathy. All genetic variants statistically associated with diabetic nephropathy in an initial study, then independently reproduced in at least one additional study, were selected. Subsequently, all studies assessing these variants were included. The association between these variants and diabetic nephropathy (defined as macroalbuminuria/proteinuria or end-stage renal disease [ESRD]) was calculated at the allele level and the main measure of effect was a pooled odds ratio. Pre-specified subgroup analyses were performed, stratifying for type 1/type 2 diabetes mellitus, proteinuria/ESRD and ethnic group.

RESULTS

The literature search yielded 3,455 citations, of which 671 were genetic association studies investigating diabetic nephropathy. We identified 34 replicated genetic variants. Of these, 21 remained significantly associated with diabetic nephropathy in a random-effects meta-analysis. These variants were in or near the following genes: ACE, AKR1B1 (two variants), APOC1, APOE, EPO, NOS3 (two variants), HSPG2, VEGFA, FRMD3 (two variants), CARS (two variants), UNC13B, CPVL and CHN2, and GREM1, plus four variants not near genes. The odds ratios of associated genetic variants ranged from 0.48 to 1.70. Additional variants were detected in subgroup analyses: ELMO1 (Asians), CCR5 (Asians) and CNDP1 (type 2 diabetes).

CONCLUSIONS

This meta-analysis found 24 genetic variants associated with diabetic nephropathy. The relative contribution and relevance of the identified genes in the pathogenesis of diabetic nephropathy should be the focus of future studies.

Significant morbidity and mortality among patients with diabetes mellitus result largely from a greatly increased incidence of microvascular complications. Proliferative diabetic retinopathy (PDR) and end stage renal disease (ESRD) are two of the most common and severe microvascular complications of diabetes. A high concordance exists in the development of PDR and ESRD in diabetic patients, as well as strong familial aggregation of these complications, suggesting a common underlying genetic mechanism. However, the precise gene(s) and genetic variant(s) involved remain largely unknown. Erythropoietin (EPO) is a potent angiogenic factor observed in the diabetic human and mouse eye. By a combination of case-control association and functional studies, we demonstrate that the T allele of SNP rs1617640 in the promoter of the EPO gene is significantly associated with PDR and ESRD in three European-American cohorts [Utah: P = 1.91 x 10(-3); Genetics of Kidneys in Diabetes (GoKinD) Study: P = 2.66 x 10(-8); and Boston: P = 2.1 x 10(-2)]. The EPO concentration in human vitreous body was 7.5-fold higher in normal subjects with the TT risk genotype than in those with the GG genotype. Computational analysis suggests that the risk allele (T) of rs1617640 creates a matrix match with the EVI1/MEL1 or AP1 binding site, accounting for an observed 25-fold enhancement of luciferase reporter expression as compared with the G allele. These results suggest that rs1617640 in the EPO promoter is significantly associated with PDR and ESRD. This study identifies a disease risk-associated gene and potential pathway mediating severe diabetic microvascular complications.

Although erythropoietin (Epo) is essential for the production of mature red blood cells, the cooperation with other factors is required for a proper balance between progenitor proliferation and differentiation. In avian erythroid progenitors, steroid hormones cooperate with tyrosine kinase receptors to induce renewal of erythroid progenitors. We examined the role of corticosteroids in the in vitro expansion of primary human erythroid cells in liquid cultures and colony assays. Dexamethasone (Dex), a synthetic glucocorticoid hormone, cooperated with Epo and stem cell factor to induce erythroid progenitors to undergo 15 to 22 cell divisions, corresponding to a 10(5)- to 10(6)-fold amplification of erythroid cells. Dex acted directly on erythroid progenitors and maintained the colony-forming capacity of the progenitor cells expanded in liquid cultures. The hormone delayed terminal differentiation into erythrocytes, which was assayed by morphology, hemoglobin accumulation, and the expression of genes characteristic for immature cells. Sustained proliferation of erythroid progenitors could be induced equally well from purified erythroid burst-forming units (BFU-E), from CD34(+) blast cells, and from bone marrow depleted from CD34(+) cells.

Erythropoietin (EPO) has emerged as a versatile growth factor that has transcended its traditional role as a mediator of erythroid maturation to one that modulates stem cell development, cellular protection and angiogenesis in the brain. As a possible candidate for nervous system disorders, it becomes crucial to understand the cellular mechanisms that foster cytoprotection rather than cytotoxicity for EPO. EPO offers novel neuronal and vascular protection not only through the maintenance of cellular integrity, but also through the prevention of cellular inflammation. The protective and anti-inflammatory capacities of EPO originate with the Janus tyrosine kinase 2 protein and protein kinase B (Akt). Downstream cellular pathways include FOXO3a, GSK-3beta, Bad, Bcl-xL, NF-kappaB, mitochondrial membrane permeability, APAF-1 and caspases. Further understanding of the cellular pathways that are susceptible to modulation by EPO will be crucial to foster the development of this agent as a robust and efficacious therapy for the brain.

The effect of human eosinophil major basic protein (MBP) as well as other eosinophil proteins, on binding of [3H]N-methyl-scopolamine ([3H]NMS: 1 x 10(-10) M) to muscarinic M2 receptors in heart membranes and M3 receptors in submandibular gland membranes was studied. MBP inhibited specific binding of [3H]NMS to M2 receptors but not to M3 receptors. MBP also inhibited atropine-induced dissociation of [3H]NMS-receptor complexes in a dose-dependent fashion, demonstrating that the interaction of MBP with the M2 muscarinic receptor is allosteric. This effect of MBP suggests that it may function as an endogenous allosteric inhibitor of agonist binding to the M2 muscarinic receptor. Inhibition of [3H]NMS binding by MBP was reversible by treatment with heparin, which binds and neutralizes MBP. Eosinophil peroxidase (EPO) also inhibited specific binding of [3H]NMS to M2 receptors but not to M3 receptors and inhibited atropine-induced dissociation of [3H]NMS-receptor complexes. On a molar basis, EPO is less potent than MBP. Neither eosinophil cationic protein nor eosinophil-derived neurotoxin affected binding of [3H]NMS to M2 receptors. Thus both MBP and EPO are selective allosteric antagonists at M2 receptors. The effects of these proteins may be important causes of M2 receptor dysfunction and enhanced vagally mediated bronchoconstriction in asthma.

OBJECTIVE

Erythropoietin (EPO), an oxygen-regulated hormone stimulating erythrocyte production, was recently found to be critical for retinal angiogenesis. EPO mRNA expression levels in retina are highly elevated during the hypoxia-induced proliferation phase of retinopathy. The authors investigated the inhibition of retinal EPO mRNA expression with RNA interference as a potential strategy to suppress retinal neovascularization and to prevent proliferative retinopathy.

METHODS

The authors used a mouse model of oxygen-induced retinopathy. Retinal EPO and Epo receptor (EpoR) expression during retinopathy development were quantified with real-time RT-PCR in whole retina and on laser-captured retinal vessels and neuronal layers. Retinal hypoxia was assessed with an oxygen-sensitive hypoxyprobe. A small interference RNA (siRNA) targeting EPO or control negative siRNA was injected intravitreally at postnatal (P) day 12, P14, and P15 during the hypoxic phase, and the effect on neovascularization was evaluated in retinal flatmounts at P17.

RESULTS

Retinal EPO mRNA expression in total retina was suppressed during the initial phase of vessel loss in retinopathy and was significantly elevated during the hypoxia-induced proliferative phase in all three neuronal layers in the retina, corresponding to an increased level of retinal hypoxia. EpoR mRNA expression levels also increased during the second neovascular phase, specifically in hypoxia-induced neovascular vessels. Intravitreous injection of EPO siRNA effectively inhibited approximately 60% of retinal EPO mRNA expression and significantly suppressed retinal neovascularization by approximately 40%.

CONCLUSIONS

Inhibiting EPO mRNA expression with siRNA is effective in suppressing retinal neovascularization, suggesting EPO siRNA is a potentially useful pharmaceutical intervention for treating proliferative retinopathy.

The European Position Paper on Rhinosinusitis and Nasal Polyps 2020 is the update of similar evidence based position papers published in 2005 and 2007 and 2012. The core objective of the <em>EPOS</em>2020 guideline is to provide revised, up-to-date and clear evidence-based recommendations and integrated care pathways in ARS and CRS. <em>EPOS</em>2020 provides an update on the literature published and studies undertaken in the eight years since the <em>EPOS</em>2012 position paper was published and addresses areas not extensively covered in <em>EPOS</em>2012 such as paediatric CRS and sinus surgery. <em>EPOS</em>2020 also involves new stakeholders, including pharmacists and patients, and addresses new target users who have become more involved in the management and treatment of rhinosinusitis since the publication of the last <em>EPOS</em> document, including pharmacists, nurses, specialised care givers and indeed patients themselves, who employ increasing self-management of their condition using over the counter treatments. The document provides suggestions for future research in this area and offers updated guidance for definitions and outcome measurements in research in different settings. <em>EPOS</em>2020 contains chapters on definitions and classification where we have defined a large number of terms and indicated preferred terms. A new classification of CRS into primary and secondary CRS and further division into localized and diffuse disease, based on anatomic distribution is proposed. There are extensive chapters on epidemiology and predisposing factors, inflammatory mechanisms, (differential) diagnosis of facial pain, allergic rhinitis, genetics, cystic fibrosis, aspirin exacerbated respiratory disease, immunodeficiencies, allergic fungal rhinosinusitis and the relationship between upper and lower airways. The chapters on paediatric acute and chronic rhinosinusitis are totally rewritten. All available evidence for the management of acute rhinosinusitis and chronic rhinosinusitis with or without nasal polyps in adults and children is systematically reviewed and integrated care pathways based on the evidence are proposed. Despite considerable increases in the amount of quality publications in recent years, a large number of practical clinical questions remain. It was agreed that the best way to address these was to conduct a Delphi exercise . The results have been integrated into the respective sections. Last but not least, advice for patients and pharmacists and a new list of research needs are included. The full document can be downloaded for free on the website of this journal: http://www.rhinologyjournal.com.

Erythropoietin (EPO) possesses generalized neuroprotective and neurotrophic actions. We tested the efficacy of recombinant human EPO (rhEPO) in preventing and reversing nerve dysfunction in streptozotocin (STZ)-induced diabetes in rats. Two days after STZ [60 mg/kg of body weight (b.w.), i.p.], diabetic animals were administered rhEPO (40 microg/kg of b.w.) three times weekly for 5 weeks either immediately (preventive) before or after a 5-week delay (therapeutic) after induction of hyperglycemia or at a lower dose (8 microg/kg of b.w. once per week) for 8 weeks (prolonged). Tail-nerve conduction velocities (NCV) was assessed at 5 and 11 weeks for the preventive and therapeutic schedule, respectively. Compared to nondiabetic rats, NCV was 20% lower after 5 weeks in the STZ group, and this decrease was attenuated 50% by rhEPO. Furthermore, the reduction of Na(+),K(+)-ATPase activity of diabetic nerves (by 55%) was limited to 24% in the rhEPO-treated group. In the therapeutic schedule, NCV was reduced by 50% after 11 weeks but by only 23% in the rhEPO-treated group. rhEPO treatment attenuated the decrease in compound muscle action potential in diabetic rats. In addition, rhEPO treatment was associated with a preservation of footpad cutaneous innervation, as assessed by protein gene product 9.5 immunostaining. Diabetic rats developed alterations in mechanical and thermal nociception, which were partially reversed by rhEPO given either in a preventative or therapeutic manner. These observations suggest that administration of rhEPO or its analogues may be useful in the treatment of diabetic neuropathy.