Human eosinophil peroxidase (EPO) was purified from eosinophil granules derived from the peripheral blood of patients with eosinophilia. The molecular mass of the H and L subunits was determined by gel filtration to be 57,000 and 11,000 daltons, respectively. The partial amino acid sequences of both subunits were used to construct oligonucleotides for the screening of several cDNA libraries, including one derived from human-induced umbilical cord mononuclear cells. A cDNA clone was isolated corresponding to EPO. The nucleotide sequence revealed an open reading frame of 2,106 bp, corresponding to a prosequence, L chain, and H chain, in this order. Comparison of the EPO nucleotide sequence with other peroxidases, such as myeloperoxidase, suggests the existence of a multigene family.

The aim of this analysis was to determine the influence of lifestyle, anthropometric and reproductive factors on the subsequent risk of incident vertebral fracture in men and women aged 50-79 years. Subjects were recruited from population registers from 28 centers across Europe. At baseline, they completed an interviewer-administered questionnaire and had lateral thoraco-lumbar spine radiographs performed. Repeat spinal radiographs were performed a mean of 3.8 years later. Incident vertebral fractures were defined morphometrically and also qualitatively by an experienced radiologist. Poisson regression was used to determine the influence of the baseline risk factor variables on the occurrence of incident vertebral fracture. A total of 3173 men (mean age 63.1 years) and 3402 women (mean age 62.2 years) contributed data to the analysis. In total there were 193 incident morphometric and 224 qualitative fractures. In women, an age at menarche 16 years or older was associated with an increased risk of vertebral fracture (RR = 1.80; 95%CI 1.24, 2.63), whilst use of hormonal replacement was protective (RR = 0.58; 95%CI 0.34, 0.99). None of the lifestyle factors studied including smoking, alcohol intake, physical activity or milk consumption showed any consistent associations with incident vertebral fracture. In men and women, increasing body weight and body mass index were associated with a reduced risk of vertebral fracture though, apart from body mass index in men, the confidence intervals embraced unity. For most variables the strengths of the associations observed were similar using the qualitative and morphometric approaches to fracture definition. In conclusion our data suggest that modification of other lifestyle risk factors is unlikely to have a major impact on the population occurrence of vertebral fractures. The important biological mechanisms underlying vertebral fracture risk need to be explored using new investigational strategies.

In multiple sclerosis (MS), long-term disability is primarily caused by axonal and neuronal damage. We demonstrated in a previous study that neuronal apoptosis occurs early during experimental autoimmune encephalomyelitis, a common animal model of MS. In the present study, we show that, in rats suffering from myelin oligodendrocyte glycoprotein (MOG)-induced optic neuritis, systemic application of erythropoietin (Epo) significantly increased survival and function of retinal ganglion cells (RGCs), the neurons that form the axons of the optic nerve. We identified three independent intracellular signaling pathways involved in Epo-induced neuroprotection in vivo: Protein levels of phospho-Akt, phospho-MAPK 1 and 2, and Bcl-2 were increased under Epo application. Using a combined treatment of Epo together with a selective inhibitor of phosphatidylinositol 3-kinase (PI3-K) prevented upregulation of phospho-Akt and consecutive RGC rescue. We conclude that in MOG-EAE the PI3-K/Akt pathway has an important influence on RGC survival under systemic treatment with Epo.

Stem cell factor (SCF), erythropoietin (Epo), and GATA-1 play an essential role(s) in erythroid development. We examined how these proteins interact functionally in G1E cells, a GATA-1(-) erythroblast line that proliferates in an SCF-dependent fashion and, upon restoration of GATA-1 function, undergoes GATA-1 proliferation arrest and Epo-dependent terminal maturation. We show that SCF-induced cell cycle progression is mediated via activation of the Src kinase/c-Myc pathway. Restoration of GATA-1 activity induced G1 cell cycle arrest coincident with repression of c-Kit and its downstream effectors Vav1, Rac1, and Akt. Sustained expression of each of these individual signaling components inhibited GATA-1-induced cell cycle arrest to various degrees but had no effects on the expression of GATA-1-regulated erythroid maturation markers. Chromatin immunoprecipitation analysis revealed that GATA-1 occupies a defined Kit gene regulatory element in vivo, suggesting a direct mechanism for gene repression. Hence, in addition to its well-established function as an activator of erythroid genes, GATA-1 also participates in a distinct genetic program that inhibits cell proliferation by repressing the expression of multiple components of the c-Kit signaling axis. Our findings reveal a novel aspect of molecular cross talk between essential transcriptional and cytokine signaling components of hematopoietic development.

Erythropoietin is a hypoxia-induced hormone that is essential for normal erythropoiesis. The production of recombinant human erythropoietin (rHuEpo) has revolutionized the treatment of anemia associated with chronic renal failure and chemotherapy, and has been used as prophylaxis to prevent anemia after surgery. The erythropoietin receptor is widely distributed in the cardiovascular system, including endothelial cells, smooth muscle cells and cardiomyocytes. Epo has potentially beneficial effects on the endothelium including anti-apoptotic, mitogenic and angiogenic activities. On the other hand, some reports suggest that rHuEpo may have pro-thrombotic or platelet-activating effects. Hypertension develops in 20-30% of renal patients treated with rHuEpo. Many patients with heart failure have anemia. Despite some potential adverse effects, early studies in heart failure patients with anemia suggest that rHuEpo therapy is safe and effective in reducing left ventricular hypertrophy, enhancing exercise performance and increasing ejection fraction. Further studies are warranted to define the role of rHuEpo in chronic heart failure and other cardiovascular settings.

BACKGROUND

We reported recently that normal human, rat, and mouse tubular cells express authentic erythropoietin-receptors (EPO-R) through which EPO stimulates mitogenesis. The present study examines whether EPO could elicit such a proliferative and thereby potentially detrimental response in cells of human renal-cell carcinoma (RCC).

METHODS

Nephrectomy samples were screened from patients with RCC (one chromophilic, two clear cell) as well as cell lines of human (Caki-2, 786-0) and mouse (RAG) renal adenocarcinomas for expression of EPO-R transcripts and protein. Cells were further tested for specific 125I-EPO binding and mitogenic response to EPO.

RESULTS

Authentic EPO-R transcripts and protein (approximately 72 kD) were detected in renal tumors and cell lines. Tumors showed low-level EPO expression, while cell lines did not. In cells, specific 125I-EPO binding to a single class of EPO-R (apparent Kd 1. 3 to 1.4 nmol/L, Bmax 2.2 to 2.6 fmol/mg protein) was observed. EPO stimulated cell proliferation dose dependently, and the individual mitogenic effects of either EPO or 10% newborn calf serum were markedly amplified when both were coadministered.

CONCLUSIONS

These data are the first to demonstrate, to our knowledge, that human RCCs express EPO-R message and protein and that receptor activation stimulates their proliferation in vitro. If these mitogenic effects of EPO are also operative in patients with RCC, endogenous EPO or its administration for the treatment of anemia could potentially hasten proliferation of renocellular malignancies.

OBJECTIVE

The normochromic normocytic anemia of erythropoietin (EPO) deficiency is recognized in advanced renal failure but not in early renal disease. The aim of this study was to determine whether anemia with EPO deficiency is found in type 1 diabetic patients with diabetic nephropathy in the absence of advanced renal failure and to compare them with patients with nondiabetic renal disease of similar severity.

METHODS

A total of 27 type 1 diabetic patients with diabetic nephropathy (DN), defined as having persistent proteinuria (mean 1,086 mg/day [CI 120-5,1901), a serum creatinine < or = 180 micromol/l, and retinopathy, were compared with 26 nondiabetic patients with glomerulonephritis (GN) and persistent proteinuria (1,874 mg/day [349-5,005]). The Hb concentration, red cell indexes, and serum EPO levels were measured, and other causes for the anemia were excluded. The EPO values were compared with a normal reference range obtained from nondiabetic patients with a microcytic anemia. The DN patients were tested for signs of diabetic peripheral and autonomic neuropathy.

RESULTS

We found that 13 of the 27 DN patients were anemic (Hb 10.6 +/- 0.9 g/dl) in marked contrast to none of the GN patients (Hb 13.7 +/- 1.4 g/dl, P < 0.005). In the DN group, serum EPO concentrations failed to increase in response to anemia compared with the response seen in patients with microcytic anemia. Thus, the anemia of the DN group was associated with EPO deficiency. The anemic DN patients showed evidence of more severe proteinuria and diabetic neuropathy than the nonanemic DN patients.

CONCLUSIONS

Anemia associated with EPO deficiency can occur early in DN before the onset of advanced renal failure, but does not normally occur in nondiabetic renal disease of similar severity. The pathogenesis requires elucidation.

Global use of erythropoietin (EPO) continues to increase as a proven agent for the treatment of anemia. Yet, EPO is no longer believed to have exclusive biological activity in the hematopoietic system and is now considered applicable for a variety of disorders such as diabetes, Alzheimer's disease, and cardiovascular disease. Treatment with EPO is considered to be robust and can prevent metabolic compromise, neuronal and vascular degeneration, and inflammatory cell activation. On the converse side, observations that EPO administration is not without risk have fueled controversy. Here we present recent advances that have elucidated a number of novel cellular pathways governed by EPO to open new therapeutic avenues for this agent and avert its potential deleterious effects.

To study the existence of the erythropoietin receptor (Epo-R) mRNA in brain capillary endothelial cells, the reverse transcription (RT) PCR was performed using total RNAs from rat brain capillary endothelial cells (RBECs) and MBEC4, which is one of the established mouse brain capillary endothelial cell lines. Southern analysis of the RT-PCR products indicated that both RBECs and MBEC4 expressed an authentic form of Epo-R mRNA as a minor form and an intron-5-inserted form of Epo-R mRNA, thus a soluble form of Epo-R mRNA, as a major form. Furthermore, the effect of recombinant human erythropoietin (rHuEpo) on the DNA synthesis in RBECs was analyzed. rHuEpo showed a dose-dependent mitogenic action on RBECs as a competence factor. Radioiodinated rHuEpo was bound specifically to RBECs with time, cell number and dose dependencies. Binding studies with 125I-rHuEpo showed that RBECs had a single class of receptors with low-affinity (Kd = 860 pM) and that the number of sites/cell (10300) was abundant. These results suggest that brain capillary endothelial cells express not only an authentic form of Epo-R but also a soluble form of Epo-R and that erythropoietin acts directly on brain capillary endothelial cells as a competence factor.

Tissue injury triggers reparative processes that often involve endothelial progenitor cells (EPCs) recruitment. We hypothesized that atherosclerotic renal artery stenosis (ARAS) activates homing signals that would be detectable in both the kidney and EPCs, and attenuated on renal repair using selective cell-based therapy. Pigs were treated with intrarenal autologous EPC after 6 weeks of ARAS. Four weeks later, expression of homing-related signals in EPC and kidney, single kidney function, microvascular (MV) density, and morphology were compared with untreated ARAS and normal control pigs (n = 7 each). Compared with normal EPC, EPC from ARAS pigs showed increased stromal cell-derived factor (SDF)-1, angiopoietin-1, Tie-2, and c-kit expression, but downregulation of erythropoietin (EPO) and its receptor. The ARAS kidney released the c-kit-ligand stem cell factor, uric acid, and EPO, and upregulated integrin beta2, suggesting activation of corresponding homing signaling. However, angiopoietin-1 and SDF-1/CXCR4 were not elevated. Administration of EPC into the stenotic kidney restored angiogenic activity, improved MV density, renal hemodynamics and function, decreased fibrosis and oxidative stress, and attenuated endogenous injury signals. The ARAS kidney releases specific homing signals corresponding to cognate receptors expressed by EPC. EPC show plasticity for organ-specific recruitment strategies, which are upregulated in early atherosclerosis. EPC are renoprotective as they attenuated renal dysfunction and damage in chronic ARAS, and consequently decreased the injury signals. Importantly, manipulation of homing signals may potentially allow therapeutic opportunities to increase endogenous EPC recruitment.

BACKGROUND

Since 1988, millions of patients have received epoetin products intravenously (IV) and subcutaneously. In 1998, epoetin-associated pure red cell aplasia (PRCA) was first reported and causation was attributed to formulations without human serum albumin (HSA), subcutaneous administration, and uncoated rubber stoppers.

METHODS

Data on erythropoietin (EPO)-associated PRCA were obtained from the Food and Drug Administration (FDA), regulatory authorities in other countries, and the manufacturers of epoetin alfa, epoetin beta, and darbepoetin. The data included information on numbers of PRCA cases and estimated exposure-adjusted incidence rates by EPO product, anemia etiology, administration route, country of PRCA identification, and date reported.

RESULTS

In 1999, academicians in Paris identified 12 EPO-treated patients with antibody-mediated PRCA; 11 of these patients were on hemodialysis and had received subcutaneous Eprex (Johnson & Johnson). In 2002, authorities in Europe, Australia, Singapore, and Canada mandated Eprex by IV route to hemodialysis patients, and the relevant manufacturers added Teflon coating to prefilled syringes of Eprex; PRCA cases subsequently decreased by 90 percent. By 2003, 180 Eprex-associated PRCA cases were identified in Europe, Canada, Australia, and Asia, despite improvements in handling. Since 2002, FDA safety databases include information on 59 new cases of antibody-associated PRCA, primarily associated with subcutaneous epoetin alfa and darbepoetin that does not contain HSA.

CONCLUSIONS

Independent actions by regulatory authorities, manufacturers, and academic researchers identified significant numbers of PRCA cases between 1998 and 2003 and characterized the probable etiology. Today, antibody-mediated PRCA is an infrequent class toxicity occurring among some hemodialysis patients on EPOs.

Erythrocytosis can arise from deregulation of the erythropoietin (Epo) axis resulting from defects in the oxygen-sensing pathway. Epo synthesis is controlled by the hypoxia inducible factor (HIF) complex, composed of an alpha and a beta subunit. There are 2 main alpha subunits, HIF-1 alpha and HIF-2 alpha. Recently, a HIF-2 alpha Gly537Trp mutation was identified in a family with erythrocytosis. This raises the possibility of HIF2A mutations being associated with other cases of erythrocytosis. We now report a subsequent analysis of HIF2A in a cohort of 75 erythrocytosis patients and identify 4 additional patients with novel heterozygous Met535Val and Gly537Arg mutations. All patients presented at a young age with elevated serum Epo. Mutations at Gly-537 account for 4 of 5 HIF2A mutations associated with erythrocytosis. These findings support the importance of HIF-2 alpha in human Epo regulation and warrant investigation of HIF2A in patients with unexplained erythrocytosis.

Erythroid cells from mice infected with the polycythemia-inducing strain of Friend spleen focus-forming virus (SFFVP), unlike normal erythroid cells, can proliferate and differentiate in apparent absence of the erythroid hormone erythropoietin (Epo). The unique envelope glycoprotein encoded by SFFV has been shown to be responsible for this biological effect. The recent isolation of an Epo-dependent erythroleukemia cell line, HCD-57, derived from a mouse infected at birth with Friend murine leukemia virus, afforded us the opportunity to study the direct effect of SFFVP on a homogeneous population of factor-dependent cells. The introduction of SFFVP in complex with various helper viruses into these Epo-dependent cells efficiently and reproducibly gave rise to lines which expressed high levels of SFFV and were factor independent. SFFV appears to be unique in its ability to abrogate the factor dependence of Epo-dependent HCD-57 cells, since infection of these cells with retroviruses carrying a variety of different oncogenes had no effect. The induction of Epo independence by SFFV does not appear to involve a classical autocrine mechanism, since there is no evidence that the factor-independent cells synthesize or secrete Epo or depend on it for their growth. However, the SFFV-infected, factor-independent cells had significantly fewer receptors available for binding Epo than their factor-dependent counterparts had, raising the possibility that the induction of factor independence by the virus may be due to the interaction of an SFFV-encoded protein with the Epo receptor.

Chronic administration of protein therapeutics may elicit unacceptable immune responses to the specific protein. Our hypothesis is that the immunogenicity of protein drugs can be ascribed to a few immunodominant helper T lymphocyte (HTL) epitopes, and that reducing the MHC binding affinity of these HTL epitopes contained within these proteins can generate drugs with lower immunogenicity. To test this hypothesis, we studied the protein therapeutic erythropoietin (Epo). Two regions within Epo, designated Epo 91-120 and Epo 126-155, contained HTL epitopes that were recognized by individuals with numerous HLA-DR types, a property common to immunodominant HTL epitopes. We then engineered analog epitopes with reduced HLA binding affinity. These analog epitopes were associated with reduced in vitro immunogenicity. Two modified forms of Epo containing these substitutions were shown to be bioactive and nonimmunogenic in vitro. These findings support our hypothesis and demonstrate that immunogenicity of protein drugs can be reduced in a systematic and predictable manner.

Stable delivery of a therapeutic protein under pharmacologic control was achieved through in vivo somatic gene transfer. This system was based on the expression of two chimeric, human-derived proteins that were reconstituted by rapamycin into a transcription factor complex. A mixture of two adeno-associated virus vectors, one expressing the transcription factor chimeras and one containing erythropoietin (Epo) under the control of a promoter responsive to the transcription factor, was injected into skeletal muscle of immune-competent mice. Administration of rapamycin resulted in 200-fold induction of plasma Epo. Stable engraftment of this humanized system in immune-competent mice was achieved for 6 months with similar results for at least 3 months in a rhesus monkey.

Therapy with human recombinant erythropoietin (EPO) has been accepted as effective for renal anemia in dialysis patients. However, studies in rats have shown that correcting anemia with EPO may affect the progression of renal dysfunction. In humans, however, the effect of EPO on residual renal function is a matter of controversy. We, therefore, investigated whether the long-term administration of EPO to predialysis patients influences residual renal function. Anemic patients at the predialysis stage with a serum creatinine (Cr) concentration ranging from 2 to 4 (average 2.9) mg/dl and a hematocrit (Ht) of less than 30% were randomly assigned to two groups which consisted of anemic patients not treated with EPO (group I, untreated anemic controls, n = 31) and anemic patients treated with EPO (group II, treated anemics, n = 42). Patients with nonsevere or moderate anemia (Ht>> 30%) with a Cr ranging from 2 to 4 (average 2.6) mg/dl were also recruited as nonanemic controls (group III, untreated nonanemic controls, n = 35). Blood pressure was controlled to the same degree among the three groups by combined treatment with calcium antagonists and angiotensin-converting enzyme inhibitors. All patients were kept strictly on a low-protein (0.6 g/kg/day) and a low-salt (7 g/day) diet. The degree of control of dietary protein and blood pressure and the frequency of angiotensin-converting enzyme inhibitor administration were comparable among the three groups. The primary end point for each patient was a doubling of the baseline Cr which yielded cumulative renal survival rates which were plotted against time. Ht rose significantly from 27.0+/-2.3 to 32.1+/-3.2% in group II (n = 42, p < 0.001) with a rate of increase of 0.4+/-0.06%/week. However, it declined from 27.9+/-1.8 to 25.3+/-1.9% in group I (n = 31, p < 0.001) and from 35.9+/-3.5 to 32.2+/-3.9% in group III (n = 35, p < 0.001). Cr doubled in 26 patients (84%) in group I as compared with 22 (52%) in group II and 21 (60%) in group III. The cumulative renal survival rates in groups II and III were significantly better than that in group I: p = 0.0003 (group I vs. group II) and p = 0.0024 (group I vs. group III). However, there was no difference in the renal survival rate between groups II and III (p = 0.3111). The better survival rate obtained in group II was attributable to the better survival rate for the nondiabetic patients in this group. The present study suggests that anemia, per se, is a factor in the progression of end-stage renal failure and that reversal of anemia by EPO can retard the progression of renal failure, especially in nondiabetic patients, provided that blood pressure control, rate of increase in Ht, and dietary protein restriction are appropriate.

Erythropoietin (EPO) is a hypoxia-induced hormone produced in the kidneys that stimulates hematopoiesis in the bone marrow. However, recent studies have also shown important nonhematopoietic effects of EPO. A functional EPO receptor is found in the cardiovascular system, including endothelial cells and cardiomyocytes. In animal studies, treatment with EPO during ischemia/reperfusion in the heart has been shown to limit the infarct size and the extent of apoptosis. In the longer term, EPO may promote ischemia-induced neovascularization, either by stimulating endothelial cells in situ or by mobilizing endothelial progenitor cells from bone marrow. The effects of EPO in the ischemic heart support the concept of EPO as a pleiotropic, tissue-protective agent for other organs expressing the EPO receptor. We recently performed a first randomized clinical study showing the safety and feasibility of EPO administration in patients with acute myocardial infarction. Future clinical studies are warranted to translate the beneficial effects of EPO from basic experiments to cardiac patients.

BACKGROUND

Anemia is now recognized as being a common finding in CHF and is associated with increased mortality and morbidity. However, it is uncertain whether the anemia is actually causing the worse prognosis or is merely a marker of more severe cardiac disease. Previous intervention studies with subcutaneous (s.c.) beta-EPO in combination with iron have either been uncontrolled or case-controlled studies. We report a randomized, double-blind, placebo-controlled study of the combination of s.c. EPO and oral iron versus oral iron alone in patients with anemia and resistant CHF.

OBJECTIVE

The present study examines, in patients with advanced congestive heart failure (CHF) and anemia, the effects of beta-erythropoietin (EPO) and oral iron on the anemia and on cardiac and renal functional parameters.

METHODS

Forty consecutive subjects with moderate to severe CHF and anemia (hemoglobin [Hb] <11 g/dL) were studied. They were randomized to receive, in a double-blind fashion, either (a) (group A, the treatment group, 20 patients) s.c. beta-EPO for 3 months twice weekly, in addition to daily oral iron, or (b) (group B, the placebo group, 20 patients) normal saline in s.c. injections and daily oral iron. Two patients in group B were eventually excluded because of a fall of Hb <8 g/dL requiring transfusion, leaving 18 patients in group B. After the 3-months study, the group A patients were maintained on the same treatment for an additional 9 months, whereas in Group B, the placebo and oral iron were stopped.

RESULTS

In group A, after a mean of 3.5 +/- 0.8 months of treatment, there was a significant increase in Hb from 10.4 +/- 0.6 to 12.4 +/- 0.8 g/dL (P < .01); a significant improvement in New York Heart Association functional class from 3.5 +/- 0.6 to 2.8 +/- 0.5 (P < .05); a longer endurance time on exercise testing, from 5.8 +/- 2.2 to 7.8 +/- 2.5 minutes (P < .01); a greater distance walked on exercise testing, from 278 +/- 55 to 356 +/- 88 meters (P < .01); a significant increase in the peak oxygen consumption (VO2) from 12.8 +/- 2.8 to 15.1 +/- 2.8 mL/kg per minute (<.05); and the VO2 at the anaerobic threshold, from 9.2 +/- 2.0 to 13.2 +/- 3.6 mL/kg minute (P < .01). There was also a significant fall in plasma B-type natriuretic peptide levels from 568 +/- 320 to 271 +/- 120 pg/mL (P < .01), a significant reduction in serum creatinine (P < .01), and an increase in estimated creatinine clearance (P < .05). In group B, there were no significant changes in any of the above parameters over the study period. At the end of the 1-year study, the Hb was still higher in group A than group B, and the rate of hospital admissions/patients over the year averaged 0.8 +/- 0.2 in group A and 1.7 +/- 0.8 in group B (P < .01).

CONCLUSIONS

In anemic CHF patients, correction of anemia with EPO and oral iron leads to improvement in New York Heart Association status, measured exercise endurance, oxygen use during exercise, renal function and plasma B-type natriuretic peptide levels and reduces the need for hospitalization.

The JAK2(V617F) mutation is frequently observed in classical myeloproliferative disorders, and disease progression is associated with a biallelic acquisition of the mutation occurring by mitotic recombination. In this study, we examined whether JAK2 activation could lead to increased homologous recombination (HR) and genetic instability. In a Ba/F3 cell line expressing the erythropoietin (EPO) receptor, mutant JAK2(V617F) and, to a lesser extent, wild-type (wt) JAK2 induced an increase in HR activity in the presence of EPO without modifying nonhomologous end-joining efficiency. Moreover, a marked augmentation in HR activity was found in CD34(+)-derived cells isolated from patients with polycythemia vera or primitive myelofibrosis compared with control samples. This increase was associated with a spontaneous RAD51 foci formation. As a result, sister chromatid exchange was 50% augmented in JAK2(V617F) Ba/F3 cells compared with JAK2wt cells. Moreover, JAK2 activation increased centrosome and ploidy abnormalities. Finally, in JAK2(V617F) Ba/F3 cells, we found a 100-fold and 10-fold increase in mutagenesis at the HPRT and Na/K ATPase loci, respectively. Together, this work highlights a new molecular mechanism for HR regulation mediated by JAK2 and more efficiently by JAK2(V617F). Our study might provide some keys to understand how a single mutation can give rise to different pathologies.

p38 MAP kinase (p38) and JNK have been described as playing a critical role in the response to a variety of environmental stresses and proinflammatory cytokines. It was recently reported that hematopoietic cytokines activate not only classical MAP kinases (ERK), but also p38 and JNK. However, the physiological function of these kinases in hematopoiesis remains obscure. We found that all MAP kinases examined, ERK1, ERK2, p38, JNK1, and JNK2, were rapidly and transiently activated by erythropoietin (Epo) stimulation in SKT6 cells, which can be induced to differentiate into hemoglobinized cells in response to Epo. Furthermore, p38-specific inhibitor SB203580 but not MEK-specific inhibitor PD98059 significantly suppressed Epo-induced differentiation and antisense oligonucleotides of p38, JNK1, and JNK2, but neither ERK1 nor ERK2 clearly inhibited Epo-induced hemoglobinization. However, in Epo-dependent FD-EPO cells, inhibition of either ERKs, p38, or JNKs suppressed cell growth. Furthermore, forced expression of a gain-of-function MKK6 mutant, which specifically activated p38, induced hemoglobinization of SKT6 cells without Epo. These results indicate that activation of p38 and JNKs but not of ERKs is required for Epo-induced erythroid differentiation of SKT6 cells, whereas all of these kinases are involved in Epo-induced mitogenesis of FD-EPO cells.

Myocardial infarction leads to scar formation and subsequent reduced cardiac performance. The ultimate therapy after myocardial infarction would pursue stem cell-based regeneration. The aim of stem cell-mediated cardiac repair embodies restoration of cardiac function by regeneration of healthy myocardial tissue, which is accomplished by neo-angiogenesis and cardiogenesis. A major reservoir of adult autologous stem cells distal from the heart is the bone marrow. Adequate regulation of signaling between the bone marrow, the peripheral circulation and the infarcted myocardium is important in orchestrating the process of mobilization, homing, incorporation, survival, proliferation and differentiation of stem cells, that leads to myocardial regeneration. In this review, we discuss key signaling factors, including cytokines, chemokines and growth factors, which are involved in orchestrating the stem cell driven repair process. We focus on signaling factors known for their mobilizing and chemotactic abilities (SDF-1, G-CSF, SCF, IL-8, VEGF), signaling factors that are expressed after myocardial infarction involved in the patho-physiological healing process (TNF-alpha, IL-8, IL-10, HIF-1alpha, VEGF, G-CSF) and signaling factors that are involved in cardiogenesis and neo-angiogenesis (VEGF, EPO, TGF-beta, HGF, HIF-1alpha, IL-8). The future therapeutic application and capacity of secreted factors to modulate tissue repair after myocardial infarction relies on the intrinsic potency of factors and on the optimal localization and timing of a combination of signaling factors to stimulate stem cells in their niche to regenerate the infarcted heart.

Oxidative stress significantly impacts multiple cellular pathways that can lead to the initiation and progression of varied disorders throughout the body. It therefore becomes imperative to elucidate the components and function of novel therapeutic strategies against oxidative stress to further clinical diagnosis and care. In particular, both the growth factor and cytokine erythropoietin (EPO) and members of the mammalian forkhead transcription factors of the O class (FoxOs) may offer the greatest promise for new treatment regimens since these agents and the cellular pathways they oversee cover a range of critical functions that directly influence progenitor cell development, cell survival and degeneration, metabolism, immune function, and cancer cell invasion. Furthermore, both EPO and FoxOs function not only as therapeutic targets, but also as biomarkers of disease onset and progression, since their cellular pathways are closely linked and overlap with several unique signal transduction pathways. However, biological outcome with EPO and FoxOs may sometimes be both unexpected and undesirable that can raise caution for these agents and warrant further investigations. Here we present the exciting as well as complicated role EPO and FoxOs possess to uncover the benefits as well as the risks of these agents for cell biology and clinical care in processes that range from stem cell development to uncontrolled cellular proliferation.

Cerebral malaria (CM) in children is associated with a high mortality and long-term neurocognitive sequelae. Both erythropoietin (Epo) and vascular endothelial growth factor (VEGF) have been shown to be neuroprotective. We hypothesized that high plasma and cerebrospinal fluid (CSF) levels of these cytokines would prevent neurological sequelae in children with CM. We measured Epo, VEGF, and tumor necrosis factor in paired samples of plasma and CSF of Kenyan children admitted with CM. Logistic regression models were used to identify risk and protective factors associated with the development of neurological sequelae. Children with CM (n = 124) were categorized into three groups: 76 without sequelae, 32 with sequelae, and 16 who died. Conditional logistic regression analysis matching the 32 patients with CM and neurological sequelae to 64 patients with CM without sequelae stratified for hemoglobin level estimated that plasma Epo (>200 units/liter) was associated with >80% reduction in the risk of developing neurological sequelae [adjusted odds ratio (OR) 0.18; 95% C.I. 0.05-0.93; P = 0.041]. Admission with profound coma (adjusted OR 5.47; 95% C.I. 1.45-20.67; P = 0.012) and convulsions after admission (adjusted OR 16.35; 95% C.I. 2.94-90.79; P = 0.001) were also independently associated with neurological sequelae. High levels of Epo were associated with reduced risk of neurological sequelae in children with CM. The age-dependent Epo response to anemia and the age-dependent protective effect may influence the clinical epidemiology of CM. These data support further study of Epo as an adjuvant therapy in CM.

This study was designed to determine the effect of erythropoietin (Epo) on cerebral blood flow (CBF), nitric oxide (NO) concentration, and neurological outcome after traumatic brain injury. In one experiment, the hemodynamic effects of Epo were determined after controlled cortical impact injury (CCII) by measuring mean arterial pressure, intracranial pressure, CBF using laser Doppler flowmetry, and brain tissue NO concentrations using an NO electrode. In total, 41 rats were given either Epo (5000 U/kg) or saline s.c. 3 days before injury. In animals pretreated with saline, L-arginine but not D-arginine administration resulted in a significant increase in tissue NO concentrations and an improvement in CBF at the impact site. Likewise, in animals pretreated with Epo, L-arginine but not D-arginine given postinjury increased brain tissue NO concentrations and increased CBF. In another experiment, 74 rats underwent CCII (3-mm deformation, velocity 5 m/s), and they were given saline or Epo 5000 U/kg s.c. at 5 min, 1 h, 3 h, 6 h, 9 h, or 12 h postinjury. The contusion volume and cell counts of viable neurons in the CA1 and CA3 regions of the hippocampus were assessed at 2 weeks postinjury. The contusion volume was significantly reduced at 5 min, 1 h, 3 h, and 6 h postinjury Epo administration. The neuron density in the CA1 and CA3 region of the hippocampus was increased at 1, 3, and 6 h after injury. These data demonstrate the neuroprotective effects of Epo in traumatic injury, and the effects are optimal when Epo is given within 6 h of injury.