The complexity of tissue- and day time-specific regulation of thousands of clock-controlled genes (CCGs) suggests that many regulatory mechanisms contribute to the transcriptional output of the circadian clock. We aim to predict these mechanisms using a large scale promoter analysis of CCGs.Our study is based on a meta-analysis of DNA-array data from rodent tissues. We searched in the promoter regions of 2065 CCGs for highly overrepresented transcription factor binding sites. In order to compensate the relatively high GC-content of CCG promoters, a novel background model to avoid a bias towards GC-rich motifs was employed. We found that many of the transcription factors with overrepresented binding sites in CCG promoters exhibit themselves circadian rhythms. Among the predicted factors are known regulators such as CLOCKratioBMAL1, DBP, HLF, E4BP4, CREB, RORalpha and the recently described regulators HSF1, STAT3, SP1 and HNF-4alpha. As additional promising candidates of circadian transcriptional regulators PAX-4, C/EBP, EVI-1, IRF, E2F, AP-1, HIF-1 and NF-Y were identified. Moreover, GC-rich motifs (SP1, EGR, ZF5, AP-2, WT1, NRF-1) and AT-rich motifs (MEF-2, HMGIY, HNF-1, OCT-1) are significantly overrepresented in promoter regions of CCGs. Putative tissue-specific binding sites such as HNF-3 for liver, NKX2.5 for heart or Myogenin for skeletal muscle were found. The regulation of the erythropoietin (Epo) gene was analysed, which exhibits many binding sites for circadian regulators. We provide experimental evidence for its circadian regulated expression in the adult murine kidney. Basing on a comprehensive literature search we integrate our predictions into a regulatory network of core clock and clock-controlled genes. Our large scale analysis of the CCG promoters reveals the complexity and extensiveness of the circadian regulation in mammals. Results of this study point to connections of the circadian clock to other functional systems including metabolism, endocrine regulation and pharmacokinetics.

In population-based studies of osteoporosis, ascertainment of fractures is typically based on self-report, with subsequent verification by medical records. The aim of this analysis was to assess the validity of self-report of incident nonspine fractures using a postal questionnaire. The degree of overreporting of fracture (false positives) was assessed by comparing self-reports of new fracture from respondents in the multicenter European Prospective Osteoporosis Study with data from other sources including radiographs and medical records. In the analysis, 563 subjects reported nonspine fractures. Verification of the presence of fracture was possible in 510 subjects. Of these, fractures were not confirmed in 11% (false positives). The percentage of false positives was greater in men than in women (15% vs 9%, p = 0.04), and less for fractures of the distal forearm and hip than for fractures at other sites. In a separate study, the degree of underreporting (false negatives) was assessed by follow-up of 251 individuals with confirmed fracture ascertained from the records of fracture clinics in three European centers (Lubeck, Oviedo, Warsaw). Questionnaire responses were received from 174 (69%) subjects. Of these, 12 (7%) did not recall sustaining a fracture (false negatives). The percentage of false negatives was lower for hip and distal forearm fractures with only 3 of 90 (3%) such fractures not recalled. Using the combined data from both studies, of those who reported a 'date' of fracture on the questionnaire, 91% of subjects were correct to within 1 month of the actual date of the fracture. A postal questionnaire is a relatively simple and accurate method for obtaining information about the occurrence of hip and distal forearm fractures, including their timing. Accuracy of ascertainment of fractures at other sites is less good and where possible self-reported fractures at such sites should be verified from other sources.

Erythropoietin (EPO) is neuroprotective in models of stroke and traumatic brain injury (TBI) when administered prior to or within the first few hours after injury. We seek to demonstrate that EPO also has neurorestorative effects when administered late (i.e., 1 day) after TBI in the rat. Twelve rats were subjected to TBI. Six rats were treated with EPO daily for 14 days starting 1 day after injury, and an additional six rats were treated with saline. Bromodeoxyuridine (BrdU) was administered daily for 14 days. Memory tests using a Morris Water Maze were performed prior to and after injury and treatment. Animals were sacrificed at 15 days after TBI, and their brains were prepared for histological analysis of damage to the dentate gyrus (DG) and for evaluation of newly formed neurons using double labeling of BrdU and MAP-2. The data revealed a significant improvement in spatial memory and significant increase in the number of newly formed neurons with EPO treatment compared with control animals. These data suggest that EPO treatment initiated 1 day after TBI is neurorestorative by enhancing neurogenesis, as well as neuroprotective.

Erythropoietin (EPO) is the hormonal regulator of red cell production and provided the paradigm for oxygen-regulated gene expression that led to the discovery of hypoxia-inducible factor (HIF). In this issue of the JCI, Rankin and colleagues show, using targeted gene inactivation, that induction of Epo expression in murine liver is dependent on the integrity of HIF-2alpha, and not HIF-1alpha (see the related article beginning on page 1068). These results demonstrate distinct functions for different HIF-alpha isoforms that could potentially be exploited in therapeutic approaches to anemia.

Caspase-3 is activated during both terminal differentiation and erythropoietin-starvation-induced apoptosis of human erythroid precursors. The transcription factor GATA-1, which performs an essential function in erythroid differentiation by positively regulating promoters of erythroid and anti-apoptotic genes, is cleaved by caspases in erythroid precursors undergoing cell death upon erythropoietin starvation or engagement of the death receptor Fas. In contrast, by an unknown mechanism, GATA-1 remains uncleaved when these cells undergo terminal differentiation upon stimulation with Epo. Here we show that during differentiation, but not during apoptosis, the chaperone protein Hsp70 protects GATA-1 from caspase-mediated proteolysis. At the onset of caspase activation, Hsp70 co-localizes and interacts with GATA-1 in the nucleus of erythroid precursors undergoing terminal differentiation. In contrast, erythropoietin starvation induces the nuclear export of Hsp70 and the cleavage of GATA-1. In an in vitro assay, Hsp70 protects GATA-1 from caspase-3-mediated proteolysis through its peptide-binding domain. The use of RNA-mediated interference to decrease the Hsp70 content of erythroid precursors cultured in the presence of erythropoietin leads to GATA-1 cleavage, a decrease in haemoglobin content, downregulation of the expression of the anti-apoptotic protein Bcl-X(L), and cell death by apoptosis. These effects are abrogated by the transduction of a caspase-resistant GATA-1 mutant. Thus, in erythroid precursors undergoing terminal differentiation, Hsp70 prevents active caspase-3 from cleaving GATA-1 and inducing apoptosis.

Recently we found that the level of anti-infarct tolerance afforded by ischemic preconditioning (IPC) and erythropoietin (EPO) infusion was closely correlated with the level of Ser9-phospho-GSK-3beta upon reperfusion in the heart. To get an insight into the mechanism by which phospho-GSK-3beta protects the myocardium from ischemia/reperfusion injury, we examined the effects of IPC and EPO on interactions between GSK-3beta and subunits of the mitochondrial permeability transition pore (mPTP) in this study. Rat hearts were subjected to 25-min global ischemia and 5-min reperfusion in vitro with or without IPC plus EPO infusion (5 units/ml) before ischemia. Ventricular tissues were sampled before or after ischemia/reperfusion to separate subcellular fractions for immunoblotting and immunoprecipitation. Reperfusion increased mitochondrial GSK-3beta by 2-fold and increased phospho-GSK-3beta level in all fractions examined. Major subunits of mPTP, adenine nucleotide translocase (ANT) and voltage-dependent anion channel (VDAC), were co-immunoprecipitated with GSK-3beta after reperfusion. Phospho-GSK-3beta was co-immunoprecipitated with ANT but not with VDAC. IPC+EPO significantly increased the levels of GSK-3beta and phospho-GSK-3beta that were co-immunoprecipitated with ANT to 145+/-8% and 143+/-16%, respectively, of baseline but did not induce phospho-GSK-3beta-VDAC binding. A PKC inhibitor and a PI3 kinase inhibitor suppressed the IPC+EPO-induced increase in the level of phospho-GSK-3beta-ANT complex. The level of cyclophilin D co-immunoprecipitated with ANT after reperfusion was significantly reduced to 39+/-10% of the control by IPC+EPO. These results suggest that reduction in affinity of ANT to cyclophilin D by increased phospho-GSK-3beta binding to ANT may be responsible for suppression of mPTP opening and myocardial protection afforded by IPC+EPO.

The methods for continuous assessment of cerebral autoregulation using correlation, phase shift, or transmission (either in time- or frequency-domain) were introduced a decade ago. They express dynamic relationships between slow waves of transcranial Doppler (TCD), blood flow velocity (FV) and cerebral perfusion pressure (CPP), or arterial pressure (ABP). We review a methodology and clinical application of indices useful for monitoring cerebral autoregulation and pressure-reactivity in various scenarios of neuro-critical care.

UNASSIGNED

Poor autoregulation and loss of pressure-reactivity are independent predictors of fatal outcome following head injury. Autoregulation is impaired by too low or too high CPP when compared to autoregulation with normal CPP (usually between 60 and 85 mmHg; and these limits are highly individual). Hemispheric asymmetry of the bi-laterally assessed autoregulation has been associated with asymmetry of CT scan findings: autoregulation was found to be worse ipsilateral to contusion or lateralized edema causing midline shift. The pressure-reactivity (PRx index) correlated with a state of low CBF and CMRO2 revealed using PET studies. The PRx is easier to monitor over prolonged periods of time than the TCD-based indices as it does not require fixation of external probes. Continuous monitoring with the PRx can be used to direct CPP-oriented therapy by determining the optimal CPP for pressure-reactivity. Autoregulation indices are able to reflect transient changes of autoregulation, as seen during plateau waves of ICP. However, minute-to-minute assessment of autoregulation has a poor signal-to-noise ratio. Averaging across time (30 min) or by combining with other relevant parameters improves the accuracy. MYTHS: It is debatable whether the TCD-based indices in head injured patients can be calculated using ABP instead of CPP. Thresholds for functional and disturbed autoregulation dramatically depends on arterial tension of CO2--therefore, comparison between patients cannot be performed without comparing their PaCO2. The TCD pulsatility index cannot accurately detect the lower limit of autoregulation. MISSING LINKS: We still do not know whether autoregulation-oriented therapy can be understood as a consensus between CPP-directed protocols and the Lund-concept. What are the links between endothelial function and autoregulation indices? Can autoregulation after head injury be improved with statins or EPO, as in subarachnoid hemorrhage? In conclusion, monitoring cerebral autoregulation can be used in a variety of clinical scenarios and may be helpful in delineating optimal therapeutic strategies.

Anaemia is frequently diagnosed in patients with cancer, and may have a detrimental effect on quality of life (QoL). We previously conducted a systematic literature review (1996-2003) to produce evidence-based guidelines on the use of erythropoietic proteins in anaemic patients with cancer.[Bokemeyer C, Aapro MS, Courdi A, et al. EORTC guidelines for the use of erythropoietic proteins in anaemic patients with cancer. Eur J Cancer 2004;40:2201-2216.] We report here an update to these guidelines, including literature published through to November 2005. The results of this updated systematic literature review have enabled us to refine our guidelines based on the full body of data currently available. Level I evidence exists for a positive impact of erythropoietic proteins on haemoglobin (Hb) levels when administered to patients with chemotherapy-induced anaemia or anaemia of chronic disease, when used to prevent cancer anaemia, and in patients undergoing cancer surgery. The addition of further Level I studies confirms our recommendation that in cancer patients receiving chemotherapy and/or radiotherapy, treatment with erythropoietic proteins should be initiated at a Hb level of 9-11 g/dL based on anaemia-related symptoms rather than a fixed Hb concentration. Early intervention with erythropoietic proteins may be considered in asymptomatic anaemic patients with Hb levels 11.9 g/dL provided that individual factors like intensity and expected duration of chemotherapy are considered. Patients whose Hb level is below 9 g/dL should primarily be evaluated for need of transfusions potentially followed by the application of erythropoietic proteins. We do not recommend the prophylactic use of erythropoietic proteins to prevent anaemia in patients undergoing chemotherapy or radiotherapy who have normal Hb levels at the start of treatment, as the literature has not shown a benefit with this approach. The addition of further supporting studies confirms our recommendation that the target Hb concentration following treatment with erythropoietic proteins should be 12-13 g/dL. Once this level is achieved, maintenance doses should be titrated individually. There is Level I evidence that dosing of erythropoietic proteins less frequently than three times per week is efficacious when used to treat chemotherapy-induced anaemia or prevent cancer anaemia, with studies supporting the use of epoetin alfa and epoetin beta weekly and darbepoetin alfa given every week or every 3 weeks. We do not recommend the use of higher than standard initial doses of erythropoietic proteins with the aim of producing higher haematological responses, due to the limited body of evidence available. There is Level I evidence that, within reasonable limits of body weight, fixed doses of erythropoietic proteins can be used to treat patients with chemotherapy-induced anaemia. This analysis confirms that there are no baseline predictive factors of response to erythropoietic proteins that can be routinely used in clinical practice if functional iron deficiency or vitamin deficiency is ruled out; a low serum erythropoietin (EPO) level (only in haematological malignancies) appears to be the only predictive factor to be verified in Level I studies. Further studies are needed to investigate the value of hepcidin, c-reactive protein, and other measures as predictive factors. In these updated guidelines, we explored a new question of whether oral or intravenous iron supplementation increases the response rate to erythropoietic proteins. We found no evidence of increased response with the addition of oral iron supplementation, but there is Level II evidence of improved response to erythropoietic proteins with the addition of intravenous iron. However, the doses and schedules for intravenous iron supplementation are not yet well defined, and further studies in this area are warranted. The two major goals of erythropoietic protein therapy are prevention or elimination of transfusions and improvement of QoL. The total body of evidence shows that red blood cell (RBC) transfusion requirements are reduced following treatment with erythropoietic proteins. This analysis also confirms that QoL is significantly improved in patients with chemotherapy-induced anaemia and in those with anaemia of chronic disease following erythropoietic protein therapy, with more robust evidence now available that QoL was improved in studies investigating early intervention in cases of chemotherapy- or radiotherapy-induced anaemia. There is only indirect evidence that patients with chemotherapy-induced anaemia or anaemia of chronic disease initially classified as non-responders to standard doses proceed to respond to treatment following a dose increase. None of the studies addressed the question in a prospective, randomised fashion, and so the Taskforce does not recommend dose escalation as a general approach in all patients who are not responding. There is still insufficient data to determine the effect on survival following treatment with erythropoietic proteins in conjunction with chemotherapy or radiotherapy. Our analysis of survival endpoints in studies involving patients receiving radio(chemo)therapy found that most studies were inconclusive, with no clear link between the use of erythropoietic proteins and survival. Likewise, we found no clear link between erythropoietic therapy and other endpoints such as local tumour control, time to progression, and progression-free survival. There is no evidence that pure red cell aplasia occurs in cancer patients following treatment with erythropoietic proteins, and the fear of this condition developing should not lead to erythropoietic proteins being withheld in patients with cancer. There is Level I evidence that the risk of thromboembolic events and hypertension are slightly elevated in patients with chemotherapy-induced anaemia receiving erythropoietic proteins. Additional trials are warranted, especially to define the optimal doses and schedules of intravenous iron supplementation during erythropoietic therapy. While our review did not address cost benefit evaluations in detail, the consensus is that studies taking into account all real determinants of cost and benefit need to be performed prospectively.

In the ischemic or hypoxic brain, astrocytes appear to be one of the main sources of erythropoietin (EPO). In this study, we investigated the differential contribution of hypoxia inducible factor (HIF) isoforms to the regulation of hypoxic EPO expression in cultured astrocytes. In addition, using an in vitro model of oxygen-glucose deprivation (OGD), we studied the role of HIF-1alpha and HIF-2alpha in the generation of paracrine protective signals by astrocytes that modulate the survival of neurons exposed to OGD. Expression of HIF-1alpha or HIF-2alpha was abrogated by infecting astrocytes with lentiviral particles encoding small interference RNA specific for HIF-1alpha or HIF-2alpha (siHIF-1alpha or siHIF-2alpha). Astrocytes infected with siHIF-1alpha showed abrogated hypoxic induction of vascular endothelial growth factor (VEGF) and lactate dehydrogenase (LDH) but normal EPO induction. In contrast, reduction of HIF-2alpha expression by siHIF-2alpha led to a drastic decrease of EPO hypoxic expression, but it did not affect LDH or VEGF upregulation. To further test whether HIF-2 is sufficient to drive EPO upregulation, we expressed oxygen-insensitive mutant forms of HIF-1alpha (mtHIF-1alpha) (P402A/P577A) and HIF-2alpha (mtHIF-2alpha) (P405A/P530A). Expression of mtHIF-2alpha but not mtHIF-1alpha in normoxic astrocytes resulted in a significant upregulation of EPO mRNA and protein. Accordingly, HIF-2alpha but not HIF-1alpha was found to be associated with the EPO hypoxia-response element by a chromatin immunoprecipitation assay. Interestingly, conditioned medium from astrocytes challenged by sublethal OGD improved neuronal survival to OGD; however, this effect was abolished during the downregulation of astrocytic HIF-2alpha using siHIF-2alpha. These results indicate that HIF-2alpha mediates the transcriptional activation of EPO expression in astrocytes, and this pathway may promote astrocytic paracrine-dependent neuronal survival during ischemia.

Erythropoietin (Epo), along with its receptor EpoR, is the principal regulator of red cell development. Upon Epo addition, the EpoR signaling through the Janus kinase 2 (JAK2) activates multiple pathways including Stat5, phosphoinositide-3 kinase (PI-3K)/Akt, and p42/44 mitogen-activated protein kinase (MAPK). The adaptor protein Lnk is implicated in cytokine receptor signaling. Here, we showed that Lnk-deficient mice have elevated numbers of erythroid progenitors, and that splenic erythroid colony-forming unit (CFU-e) progenitors are hypersensitive to Epo. Lnk(-/-) mice also exhibit superior recovery after erythropoietic stress. In addition, Lnk deficiency resulted in enhanced Epo-induced signaling pathways in splenic erythroid progenitors. Conversely, Lnk overexpression inhibits Epo-induced cell growth in 32D/EpoR cells. In primary culture of fetal liver cells, Lnk overexpression inhibits Epo-dependent erythroblast differentiation and induces apoptosis. Lnk blocks 3 major signaling pathways, Stat5, Akt, and MAPK, induced by Epo in primary erythroblasts. In addition, the Lnk Src homology 2 (SH2) domain is essential for its inhibitory function, whereas the conserved tyrosine near the C-terminus and the pleckstrin homology (PH) domain of Lnk are not critical. Furthermore, wild-type Lnk, but not the Lnk SH2 mutant, becomes tyrosine-phosphorylated following Epo administration and inhibits EpoR phosphorylation and JAK2 activation. Hence, Lnk, through its SH2 domain, negatively modulates EpoR signaling by attenuating JAK2 activation, and regulates Epo-mediated erythropoiesis.

Hepcidin is the principal iron regulatory hormone, controlling the systemic absorption and remobilization of iron from intracellular stores. Recent in vivo studies have shown that hepcidin is down-regulated by erythropoiesis, anemia, and hypoxia, which meets the need of iron input for erythrocyte production. Erythropoietin (EPO) is the primary signal that triggers erythropoiesis in anemic and hypoxic conditions. Therefore, a direct involvement of EPO in hepcidin regulation can be hypothesized. We report here the regulation of hepcidin expression by EPO, in a dose-dependent manner, in freshly isolated mouse hepatocytes and in the HepG2 human hepatocyte cell model. The effect is mediated through EPOR signaling, since hepcidin mRNA levels are restored by pretreatment with an EPOR-blocking antibody. The transcription factor C/EBPalpha showed a pattern of expression similar to hepcidin, at the mRNA and protein levels, following EPO and anti-EPOR treatments. Chromatin immunoprecipitation experiments showed a significant decrease of C/EBPalpha binding to the hepcidin promoter after EPO supplementation, suggesting the involvement of this transcription factor in the transcriptional response of hepcidin to EPO.

Erythropoietin (Epo) is a hormone regulating proliferation and differentiation of erythroid cells. The hypothesis that hematopoietic and endothelial cells share a common hemangioblast progenitor among others is based on the finding that both cell lineages express cell surface antigens like CD31 and CD34. In this study we investigated the angiogenic potential of recombinant human erythropoietin (rHuEpo) on endothelial cells derived from human adult myocardial tissue. In addition, we compared the angiogenic potential of rHuEpo to that of other cytokines (VEGF, aFGF) and combinations of growth factors. Samples of myocardial tissue (cardiac auricle) were obtained during coronary bypass surgery, embedded in a fibrin gel matrix, and cultured for 21 days. Capillary sprouting was measured with an eye-piece graticule under an inverted-phase contrast microscope. Tube-forming endothelial cells were characterized by immunohistochemistry and RT-PCR. Using a concentration of 2.5 U/ml, we found that rHuEpo stimulates capillary outgrowth up to 220%, compared to the nonstimulated physiological outgrowth. Epo therefore exhibits the same angiogenic potential on endothelial cells in our in vitro assay as VEGF(165) (230% increase). Erythropoietin stimulates capillary outgrowth in an in vitro angiogenesis assay using adult human myocardial tissue. This implies a role of erythropoietin in vasoproliferative processes. rHuEpo may serve as a direct angiogenic substance in patients with ischemic heart disease.

No longer considered exclusive for the function of the hematopoietic system, erythropoietin (EPO) is now considered as a viable agent to address central nervous system injury in a variety of cellular systems that involve neuronal, vascular, and inflammatory cells. Yet, it remains unclear whether the protective capacity of EPO may be effective for chronic neurodegenerative disorders such as Alzheimer's disease (AD) that involve beta-amyloid (Abeta) apoptotic injury to hippocampal neurons. We therefore investigated whether EPO could prevent both early and late apoptotic injury during Abeta exposure in primary hippocampal neurons and assessed potential cellular pathways responsible for this protection. Primary hippocampal neuronal injury was evaluated by trypan blue dye exclusion, DNA fragmentation, membrane phosphatidylserine (PS) exposure, and nuclear factor-kappaB (NF-kappaB) expression with subcellular translocation. We show that EPO, in a concentration specific manner, is able to prevent the loss of both apoptotic genomic DNA integrity and cellular membrane asymmetry during Abeta exposure. This blockade of Abeta generated neuronal apoptosis by EPO is both necessary and sufficient, since protection by EPO is completely abolished by co-treatment with an anti-EPO neutralizing antibody. Furthermore, neuroprotection by EPO is closely linked to the expression of NF-kappaB p65 by preventing the degradation of this protein by Abeta and fostering the subcellular translocation of NF-kappaB p65 from the cytoplasm to the nucleus to allow the initiation of an anti-apoptotic program. In addition, EPO intimately relies upon NF-kappaB p65 to promote neuronal survival, since gene silencing of NF-kappaB p65 by RNA interference removes the protective capacity of EPO during Abeta exposure. Our work illustrates that EPO is an effective entity at the neuronal cellular level against Abeta toxicity and requires the close modulation of the NF-kappaB p65 pathway, suggesting that either EPO or NF-kappaB may be used as future potential therapeutic strategies for the management of chronic neurodegenerative disorders, such as AD.

The erythropoietin (EPO) receptor (EPO-R), a member of a large cytokine receptor superfamily, has a 236-amino-acid cytoplasmic region which contains no obvious tyrosine kinase or other catalytic domain. In order to delineate the linear functional domains of the cytoplasmic tail, we generated truncated mutant cDNAs which were transfected into a murine interleukin-3-dependent cell line, Ba/F3, and the EPO-dependent growth characteristics of the stable transfectants were assayed. We identified two unique domains of the cytoplasmic tail. A membrane-proximal positive signal transduction domain of less than or equal to 103 amino acids, in a region highly similar to the interleukin-2 receptor beta chain, was sufficient for EPO-mediated signal transduction. A carboxy-terminal negative-control domain, a serine-rich region of approximately 40 amino acids, increased the EPO requirement for the Ba/F3 transfectants without altering EPO-R cell surface expression, affinity for EPO, receptor oligosaccharide processing, or receptor endocytosis. Truncation of this negative-control domain allowed the Ba/F3 transfectants to grow maximally in only 1 pM EPO, 1/10 the concentration required for growth of cells expressing the wild-type EPO-R. All truncated EPO-R mutants which retained the transmembrane region of the EPO-R polypeptide bound to the gp55 envelope protein of Friend spleen focus-forming virus. Only the functional EPO-R mutants were activated by the gp55, however, suggesting that gp55- and EPO-mediated signaling occur via a similar mechanism.

Erythropoietin (EPO) stimulates the growth of erythroblasts in the bone marrow (C. Lacombe and P. Mayeux, NEPHROL: DIAL: TRANSPLANT:, 14 (SUPPL: 2): 22-28, 1999). We report basal and hypoxia-stimulated expression of EPO and its receptor, EPOR, in human breast cancer cells, and we demonstrate EPO-stimulated tyrosine phosphorylation and the proliferation of these cells in vitro. In 50 clinical specimens of breast carcinoma, we report high levels of EPO and EPOR associated with malignant cells and tumor vasculature but not with normal breast, benign papilloma, or fibrocystic tissue. Hypoxic tumor regions display the highest levels of EPO and EPOR expression. Enhanced EPO signaling may contribute to the promotion of human cancer by tissue hypoxia.

The kidney is a highly sensitive oxygen sensor and plays a central role in mediating the hypoxic induction of red blood cell production. Efforts to understand the molecular basis of oxygen-regulated erythropoiesis have led to the identification of erythropoietin (EPO), which is essential for normal erythropoiesis and to the purification of hypoxia-inducible factor (HIF), the transcription factor that regulates EPO synthesis and mediates cellular adaptation to hypoxia. Recent insights into the molecular mechanisms that control and integrate cellular and systemic erythropoiesis-promoting hypoxia responses and their potential as a therapeutic target for the treatment of renal anemia are discussed in this review.

Red blood cells (RBCs), which constitute the most abundant cell type in the body, come in two distinct flavors- primitive and definitive. Definitive RBCs in mammals circulate as smaller, anucleate cells during fetal and postnatal life, while primitive RBCs circulate transiently in the early embryo as large, nucleated cells before ultimately enucleating. Both cell types are formed from lineage-committed progenitors that generate a series of morphologically identifiable precursors that enucleate to form mature RBCs. While definitive erythroid precursors mature extravascularly in the fetal liver and postnatal marrow in association with macrophage cells, primitive erythroid precursors mature as a semi-synchronous cohort in the embryonic bloodstream. While the cytoskeletal network is critical for the maintenance of cell shape and the deformability of definitive RBCs, little is known about the components and function of the cytoskeleton in primitive erythroblasts. Erythropoietin (EPO) is a critical regulator of late-stage definitive, but not primitive, erythroid progenitor survival. However, recent studies indicate that EPO regulates multiple aspects of terminal maturation of primitive murine and human erythroid precursors, including cell survival, proliferation, and the rate of terminal maturation. Primitive and definitive erythropoiesis share central transcriptional regulators, including Gata1 and Klf1, but are also characterized by the differential expression and function of other regulators, including myb, Sox6, and Bcl11A. Flow cytometry-based methodologies, developed to purify murine and human stage-specific erythroid precursors, have enabled comparative global gene expression studies and are providing new insights into the biology of erythroid maturation.

The Friend spleen focus-forming virus envelope glycoprotein, gp55, binds to the murine erythropoietin receptor (EPO-R) and triggers growth activation in the absence of EPO. Interleukin 3-dependent lymphoid cell lines that have been stably transfected with the EPO-R cDNA grow in the presence of EPO or interleukin 3. In these cells, the EPO-R is synthesized as a minor 62-kDa unglycosylated form and a major 64-kDa form carrying one high-mannose N-linked oligosaccharide. A fraction of the 64-kDa form is processed to a 66-kDa species with complex-type sugars. Very little of the EPO-R is expressed on the cell surface and all three forms of EPO-R are degraded rapidly. Cells transfected with both EPO-R and gp55 cDNAs grow in the absence of EPO. Most of the EPO-R associated with gp55 is endoglycosidase H-sensitive, suggesting that the interactions between these proteins occur in the endoplasmic reticulum. Furthermore, the endoglycosidase H-sensitive EPO-R is more stable than in the absence of gp55, a result suggesting that interaction of gp55 with the EPO-R causes it to remain within the rough endoplasmic reticulum. It is possible that gp55 EPO-R complexes within this compartment send a growth-promoting signal to the cell.

Erythropoietin (EPO) plays a prominent role in the regulation of the hematopoietic system, but the potential function of this trophic factor as a cytoprotectant in the cerebral vascular system is not known. The authors examined the ability of EPO to modulate a series of death-related cellular pathways during free radical-induced injury in cerebral microvascular endothelial cells (ECs). Endothelial cell injury was evaluated by trypan blue, DNA fragmentation, membrane phosphatidylserine exposure, apoptotic protease-activating factor-1 (Apaf-1), and Bcl-XL expression, mitochondrial membrane potential, cytochrome c release, and cysteine protease activity. They show that constitutive EPO is present in ECs but is insufficient to prevent cellular injury. Signaling through the EPO receptor, however, remains biologically responsive to exogenous EPO administration to offer significant protection against nitric oxide-induced injury. Exogenous EPO maintains both genomic DNA integrity and cellular membrane asymmetry through parallel pathways that prevent the induction of Apaf-1 and preserve mitochondrial membrane potential in conjunction with enhanced Bcl-XL expression. Consistent with the modulation of Apaf-1 and the release of cytochrome c, EPO also inhibits the activation of caspase-9 and caspase-3-like activities. Identification of novel cytoprotective pathways used by EPO may serve as therapeutic targets for cerebral vascular disease.

Leptin signals the status of body energy stores via the leptin receptor (LR), a member of the Type I cytokine receptor family. Type I cytokine receptors mediate intracellular signaling via the activation of associated Jak family tyrosine kinases. Although their COOH-terminal sequences vary, alternatively spliced LR isoforms (LRa-LRd) share common NH(2)-terminal sequences, including the first 29 intracellular amino acids. The so-called long form LR (LRb) activates Jak-dependent signaling and is required for the physiologic actions of leptin. In this study, we have analyzed Jak activation by intracellular LR sequences under the control of the extracellular erythropoeitin (Epo) (Epo receptor/LRb chimeras). We show that Jak2 is the requisite Jak kinase for signaling by the LRb intracellular domain and confirm the requirement for the Box 1 motif for Jak2 activation. A minimal LRb intracellular domain for Jak2 activation includes intracellular amino acids 31-48. Although the sequence requirements for intracellular amino acids 37-48 are flexible, intracellular amino acids 31-36 of LRb play a critical role in Jak2 activation and contain a loose homology motif found in other Jak2-activating cytokine receptors. The failure of short form sequences to function in Jak2 activation reflects the absence of this motif.

OBJECTIVE

Erythropoietin (EPO) improves cardiac function and induces neovascularization in chronic heart failure (CHF), although the exact mechanism has not been elucidated. We studied the effects of EPO on homing and incorporation of endothelial progenitor cells (EPC) into the myocardial microvasculature and myocardial expression of angiogenic factors.

RESULTS

CHF was induced in rats by coronary artery ligation resulting in myocardial infarction (MI) after bone marrow had been replaced by human placental alkaline phosphatase (hPAP) transgenic cells. We studied the effects of darbepoetin alfa treatment (EPO, 40 microg/kg, every 3 weeks, starting 3 weeks after MI) on longitudinal changes in left ventricular (LV) function, circulating EPC, myocardial histology, and expression of vascular endothelial growth factor (VEGF) determined 9 weeks after MI. EPO prevented LV-dilatation and improved cardiac function (all P < 0.05), which was associated with 42% increased capillary growth (P < 0.01). EPO-induced mobilization of EPC from the bone marrow (P < 0.01), which resulted in a three-fold increased homing of EPC into the cardiac microvasculature. The percentage of the endothelium that consisted of bone marrow derived cells was significantly increased (3.9 +/- 0.5 vs. 11.4 +/- 1%, P < 0.001) comprising 30% of the newly formed capillaries. In addition, EPO treatment resulted in a 4.5-fold increased myocardial expression of VEGF, which correlated strongly with neovascularization (r = 0.67; P < 0.001). VEGF was equally expressed by endothelial cells of myocardial and bone marrow origin.

CONCLUSIONS

EPO-induced neovascularization in post-MI heart failure is mediated through a combination of EPC recruitment from the bone marrow and increased myocardial expression of VEGF.

Erythropoietin (Epo) is required for the production of mature red blood cells. The requirement for Epo and its receptor (EpoR) for normal heart development and the response of vascular endothelium and cells of neural origin to Epo provide evidence that the function of Epo as a growth factor or cytokine to protect cells from apoptosis extends beyond the hematopoietic lineage. We now report that the EpoR is expressed on myoblasts and can mediate a biological response of these cells to treatment with Epo. Primary murine satellite cells and myoblast C2C12 cells, both of which express endogenous EpoR, exhibit a proliferative response to Epo and a marked decrease in terminal differentiation to form myotubes. We also observed that Epo stimulation activates Jak2/Stat5 signal transduction and increases cytoplasmic calcium, which is dependent on tyrosine phosphorylation. In erythroid progenitor cells, Epo stimulates induction of transcription factor GATA-1 and EpoR; in C2C12 cells, GATA-3 and EpoR expression are induced. The decrease in differentiation of C2C12 cells is concomitant with an increase in Myf-5 and MyoD expression and inhibition of myogenin induction during differentiation, altering the pattern of expression of the MyoD family of transcription factors during muscle differentiation. These data suggest that, rather than acting in an instructive or specific mode for differentiation, Epo can stimulate proliferation of myoblasts to expand the progenitor population during differentiation and may have a potential role in muscle development or repair.

We estimated whether single collections of cord blood contained sufficient cells for hematopoietic engraftment of adults by evaluating numbers of cord blood and adult bone marrow myeloid progenitor cells (MPCs) as detected in vitro with steel factor (SLF) and hematopoietic colony-stimulating factors (CSFs). SLF plus granulocyte-macrophage (GM)-CSF detected 8- to 11-fold more cord blood GM progenitors [colony-forming units (CFU)-GM] than cells stimulated with GM-CSF or 5637 conditioned medium (CM), growth factors previously used to estimate cord blood CFU-GM numbers. SLF plus erythropoietin (Epo) plus interleukin 3 (IL-3) enhanced detection of cord blood multipotential (CFU-GEMM) progenitors 15-fold compared to stimulation with Epo plus IL-3. Under the same conditions, bone marrow CFU-GM and CFU-GEMM were only enhanced in detection 2- to 4- and 6- to 8-fold. Increased detection of cord blood CFU-GEMM correlated directly with decreased detection of cord blood erythroid burst-forming units (BFU-E). In contrast, adult bone marrow CFU-GEMM and BFU-E numbers were both enhanced by SLF plus Epo plus IL-3. This suggests that most cord blood BFU-E may actually be CFU-GEMM. Cord blood collections (n = 17) contained numbers of MPCs (especially CFU-GM) similar to the number found in nine autologous bone marrow collections. To assess additional sources of MPCs, the peripheral blood of 1-day-old infants was assessed. However, average concentrations of MPCs circulating in these infants were only 30-46% that in their cord blood. Expansion of cord blood MPCs was also evaluated. Incubation of cord blood cells for 7 days with SLF resulted in 7.9-, 2.2-, and 2.7-fold increases in numbers of CFU-GM, BFU-E, and CFU-GEMM compared to starting numbers; addition of a CSF with SLF resulted in even greater expansion of MPCs. The results suggest that cord blood contains a larger number of early profile MPCs than previously recognized and that there are probably sufficient numbers of cells in a single cord blood collection to engraft an adult. Although the expansion data must be considered with caution, as human marrow repopulating cells cannot be assessed directly, in vitro expansion of cord blood stem and progenitor cells may be feasible for clinical transplantation.

Acute traumatic spinal cord injury (SCI) results in a devastating loss of neurological function below the level of injury and adversely affects multiple systems within the body. The pathobiology of SCI involves a primary mechanical insult to the spinal cord and activation of a delayed secondary cascade of events, which ultimately causes progressive degeneration of the spinal cord. Whereas cell death from the mechanical injury is predominated by necrosis, secondary injury events trigger a continuum of necrotic and apoptotic cell death mechanisms. These secondary events include vascular abnormalities, ischemia-reperfusion, glutamate excitotoxicity and disturbances in ionic homeostasis, oxidative cell injury, and a robust inflammatory response. No gold standard therapy for SCI has been established, although clinical trials with methylprednisolone (NASCIS II and III) and GM-1 ganglioside (Maryland and Sygen) have demonstrated modest, albeit potentially important therapeutic benefits. In light of the overwhelming impact of SCI on the individual, other therapeutic interventions are urgently needed. A number of promising pharmacological therapies are currently under investigation for neuroprotective abilities in animal models of SCI. These include the sodium (Na+) channel blocker riluzole, the tetracycline derivative minocycline, the fusogen copolymer polyethylene glycol (PEG), and the tissue-protective hormone erythropoietin (EPO). Moreover, clinical trials investigating the putative neuroprotective and neuroregenerative properties ascribed to the Rho pathway antagonist, Cethrin (BioAxone Therapeutic, Inc.), and implantation of activated autologous macrophages (ProCord; Proneuron Biotechnologies) in patients with thoracic and cervical SCI are now underway. We anticipate that these studies will harken an era of renewed interest in translational clinical trials. Ultimately, due to the multi-factorial pathophysiology of traumatic SCI, effective therapies will require combined approaches.