Therapeutic potential of erythropoietin and its structural or functional variants in the nervous system

SummaryThe growth factor erythropoietin (EPO) and erythropoietin receptors (EPOR) are expressed in the nervous system. Neuronal expression of EPO and EPOR peaks during brain development and is upregulated in the adult brain after injury. Peripherally administered EPO, and at least some of its variants, cross the blood-brain barrier, stimulate neurogenesis, neuronal differentiation, and activate brain neurotrophic, anti-apoptotic, anti-oxidant and anti-inflammatory signaling. These mechanisms underlie their tissue protective effects in nervous system disorders. As the tissue protective functions of EPO can be separated from its stimulatory action on hematopoiesis, novel EPO derivatives and mimetics, such as asialo-EPO and carbamoylated EPO have been developed. While the therapeutic potential of the novel EPO derivatives continues to be characterized in preclinical studies, the experimental findings in support for the use of recombinant human (rh)EPO in human brain disease have already been translated to clinical studies in acute ischemic stroke, chronic schizophrenia, and chronic progressive multiple sclerosis. In this review article, we assess the studies on EPO and, in particular, on its structural or functional variants in experimental models of nervous system disorders, and we provide a short overview of the completed and ongoing clinical studies testing EPO as neuroprotective/neuroregenerative treatment option in neuropsychiatric disease.

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title: Developments in the therapeutic use of erythropoiesis stimulating agents
abstract: Recombinant human erythropoietin (rHuEPO) has become the standard therapy for treatment of the anaemia of chronic kidney disease (CKD) during the past two decades. In addition, rHuEPO can be indicated for the treatment of cancer patients on chemotherapy and surgical patients to avoid allogeneic red blood cell transfusion. This review first describes recent attempts in developing rHuEPO congeners (mutated and pegylated rHuEPOs) and mimetics with prolonged half‐lives and improved application requirements. Secondly, the pathophysiological background of the regulatory guideline, that blood haemoglobin levels in anaemic CKD or cancer patients on chemotherapy should not be raised above the target value of 120 g/l, is discussed. Finally, potential novel indications are considered for the use of rHuEPO and its analogues as pleiotropic cytoprotectant agents for cardio‐, nephro‐, hepato‐ and neuroprotection.

title: A Potential Role for Erythropoietin in Focal Permanent Cerebral Ischemia in Mice
abstract: The present study describes, for the first time, a temporal and spatial cellular expression of erythropoietin (Epo) and Epo receptor (Epo-R) with the evolution of a cerebral infarct after focal permanent ischemia in mice. In addition to a basal expression of Epo in neurons and astrocytes, a postischemic Epo expression has been localized specifically to endothelial cells (1 day), microglia/macrophage-like cells (3 days), and reactive astrocytes (7 days after occlusion). Under these conditions, the Epo-R expression always precedes that of Epo for each cell type. These results support the hypothesis that there is a continuous formation of Epo, with its corresponding receptor, during the active evolution of a focal cerebral infarct and that the Epo/Epo-R system might be implicated in the processes of neuroprotection and restructuring (such as angiogenesis and gliosis) after ischemia. To support this hypothesis, a significant reduction in infarct volume (47%; P < 0.0002) was found in mice treated with recombinant Epo 24 hours before induction of cerebral ischemia. Based on the above, we propose that the Epo/Epo-R system is an endogenous mechanism that protects the brain against damages consequent to a reduction in blood flow, a mechanism that can be amplified by the intracerebroventricular application of exogenous recombinant Epo.

title: In vivo evidence that erythropoietin protects neurons from ischemic damage.
abstract: Erythropoietin (EPO) produced by the kidney and the liver (in fetuses) stimulates erythropoiesis. In the central nervous system, neurons express EPO receptor (EPOR) and astrocytes produce EPO. EPO has been shown to protect primary cultured neurons from N-methyl-D-aspartate (NMDA) receptor-mediated glutamate toxicity. Here we report in vivo evidence that EPO protects neurons against ischemia-induced cell death. Infusion of EPO into the lateral ventricles of gerbils prevented ischemia-induced learning disability and rescued hippocampal CA1 neurons from lethal ischemic damage. The neuroprotective action of exogenous EPO was also confirmed by counting synapses in the hippocampal CA1 region. Infusion of soluble EPOR (an extracellular domain capable of binding with the ligand) into animals given a mild ischemic treatment that did not produce neuronal damage, caused neuronal degeneration and impaired learning ability, whereas infusion of the heat-denatured soluble EPOR was not detrimental, demonstrating that the endogenous brain EPO is crucial for neuronal survival. The presence of EPO in neuron cultures did not repress a NMDA receptor-mediated increase in intracellular Ca2+, but rescued the neurons from NO-induced death. Taken together EPO may exert its neuroprotective effect by reducing the NO-mediated formation of free radicals or antagonizing their toxicity.

title: Survival of hippocampal neurons in culture upon hypoxia: effect of erythropoietin
abstract: The potential of erythropoietin (EPO) to reduce hypoxia-induced cell death has been investigated in 5-day-old primary cultures of rat postnatal hippocampal neurons. Application of EPO (100 pM) at the start of hypoxia resulted in a significant reduction of neuronal death (33.0 ± 7.5% in cells incubated with EPO vs 56.75 ± 7.3% in non-treated cells; n = 4, p < 0.021). Similar results were obtained upon application of cycloheximide (CHX; 1 μM) simultaneously with hypoxia (34.75 ± 5.6% vs 56.75 ± 7.3% with and without CHX, respectively, n = 4, p < 0.035), indicating that hypoxia-induced neuronal death is an active, protein synthesis-dependent process. Both, EPO and EPO receptor (EPOR) were found to be expressed after hypoxia in hippocampal neurons in vitro and in vivo. These results demonstrate for the first time that EPO can reverse hypoxia-induced neuronal death when applied simultaneously with the hypoxic stimulus.

title: Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress
abstract: Erythropoietin (EPO) promotes neuronal survival after hypoxia and other metabolic insults by largely unknown mechanisms. Apoptosis and necrosis have been proposed as mechanisms of cellular demise, and either could be the target of actions of EPO. This study evaluates whether antiapoptotic mechanisms can account for the neuroprotective actions of EPO. Systemic administration of EPO (5,000 units/kg of body weight, i.p.) after middle-cerebral artery occlusion in rats dramatically reduces the volume of infarction 24 h later, in concert with an almost complete reduction in the number of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling of neurons within the ischemic penumbra. In both pure and mixed neuronal cultures, EPO (0.1–10 units/ml) also inhibits apoptosis induced by serum deprivation or kainic acid exposure. Protection requires pretreatment, consistent with the induction of a gene expression program, and is sustained for 3 days without the continued presence of EPO. EPO (0.3 units/ml) also protects hippocampal neurons against hypoxia-induced neuronal death through activation of extracellular signal-regulated kinases and protein kinase Akt-1/protein kinase B. The action of EPO is not limited to directly promoting cell survival, as EPO is trophic but not mitogenic in cultured neuronal cells. These data suggest that inhibition of neuronal apoptosis underlies short latency protective effects of EPO after cerebral ischemia and other brain injuries. The neurotrophic actions suggest there may be longer-latency effects as well. Evaluation of EPO, a compound established as clinically safe, as neuroprotective therapy in acute brain injury is further supported.

title: Emerging biological roles for erythropoietin in the nervous system
abstract: (2005) In the third sentence of the first paragraph on page 490, the description of the human stroke study should have cited the following reference: Ehrenreich, H. et al. Erythropoietin therapy for acute stroke is both safe and beneficial.  (2002).

title: The non‐haematopoietic biological effects of erythropoietin
abstract: In the haematopoietic system, the principal function of erythropoietin (Epo) is the regulation of red blood cell production, mediated by its specific cell surface receptor (EpoR). Following the cloning of the Epo gene (EPO) and characterization of the selective haematopoietic action of Epo in erythroid lineage cells, recombinant Epo forms (epoetin‐alfa, epoetin‐beta and the long‐acting analogue darbepoetin‐alfa) have been widely used for treatment of anaemia in chronic kidney disease and chemotherapy‐induced anaemia in cancer patients. Ubiquitous EpoR expression in non‐erythroid cells has been associated with the discovery of diverse biological functions for Epo in non‐haematopoietic tissues. During development, Epo–EpoR signalling is required not only for fetal liver erythropoiesis, but also for embryonic angiogenesis and brain development. A series of recent studies suggest that endogenous Epo–EpoR signalling contributes to wound healing responses, physiological and pathological angiogenesis, and the body’s innate response to injury in the brain and heart. Epo and its novel derivatives have emerged as major tissue‐protective cytokines that are being investigated in the first human studies involving neurological and cardiovascular diseases. This review focuses on the scientific evidence documenting the biological effects of Epo in non‐haematopoietic tissues and discusses potential future applications of Epo and its derivatives in the clinic.

title: Erythropoietin and Erythropoietin Receptor in the Developing Human Central Nervous System
abstract: We have previously shown the presence of erythropoietin (Epo) within the spinal fluid of normal preterm and term infants, and the presence of Epo receptor (Epo-R) in the spinal cords of human fetuses. It is not known, however: 1) whether cells within the fetal central nervous system(CNS) express Epo; 2) if so, whether this expression changes with development; 3) which cells within the CNS express Epo-R;4) whether Epo-R expression within the CNS changes with development; and 5) whether Epo-R within the fetal CNS are functional. Expression of mRNA for Epo and Epo-R was sought by reverse transcription-PCR in mixed primary cultures of fetal spinal cords as well as NT2 and hNT cells, human cell lines of neuronal precursors and mature neurons, respectively. Epo was measured by ELISA in spent media from primary cell culture, and immunohistochemistry was used to identify Epo-R on neurons and glia in cell culture, and in brain sections. Developmental changes in Epo and Epo-R expression were sought in spinal cords and brains from fetuses of 7-24 wk postconception by semiquantitative PCR. To assess Epo-R function, NT2 cells were exposed to conditions which stimulate programmed cell death, and rescue from apoptosis by the addition of recombinant Epo was evaluated by nuclear matrix protein ELISA, cell counts, and by Klenow labeling of DNA fragments. Epo and Epo-R mRNA were expressed in mixed primary cultures of neural tissues and NT2 and hNT cells. Epo was detected by ELISA in media removed from mixed cell cultures, and immunohistochemical staining confirmed the presence of Epo-R on neurons and their supporting cells. Semiquantitative PCR revealed no significant change in expression of either Epo or Epo-R in spinal cords between 7 and 16 wk of gestation, with increased expression of Epo and Epo-R in brains from 8 to 24 wk of gestation. Epo mRNA expression from neurons doubled under conditions of hypoxia. Recombinant Epo decreased apoptotic cell death of neurons under conditions of hypoxia. Protein and mRNA for Epo and its receptor are expressed by human neurons and glial cells in spinal cord and brain during fetal development. These receptors appear to have a neuroprotective effect in conditions of hypoxia.

title: Erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons, and erythropoietin prevents in vitro glutamate-induced neuronal death
abstract: Recently, erythropoietin has been shown to be produced by astrocytes and its production is hypoxia-inducible. In the present study, we demonstrated, using a reverse transcription-polymerase chain reaction assay and immunostaining of the cells, that the erythropoietin receptor was expressed in cultured hippocampal and cerebral cortical neurons of day 19 rat embryo. Erythropoietin protected the cultured neurons from glutamate neurotoxicity. Neurons cultured for seven to 10 days were exposed to glutamate for 15 min and after culture for a further 24 h in the absence of glutamate the neuron survival was assayed. Significant protection was observed with erythropoietin from 3 pM (c. 100 pg/ml) in a dose-dependent manner. The protection was completely reversed by co-application of a soluble erythropoietin receptor, an extracellular domain capable of binding with erythropoietin. For exhibition of the neuroprotective effect, exposure of neurons to erythropoietin approximately 8 h prior to exposure to glutamate was required. Experiments with the inhibitors indicated that RNA and protein syntheses were necessary for the protection. However, exposure to erythropoietin for a short period (5 min or less) was sufficient to elicit the protective effect. The protective effect of erythropoietin was blocked by the simultaneous addition of EGTA. These findings and the previous finding that erythropoietin induces a rapid and transient increase in intracellular Ca2+ concentration in neuronal cells suggest that erythropoietin plays a neuroprotective role in brain injury caused by hypoxia or ischemia and that erythropoietin-induced Ca2+ influx from outside of the cells is a critical initial event yielding an enhanced resistance of the neurons to glutamate toxicity.

title: Role of erythropoietin in the brain.
abstract: Multi-tissue erythropoietin receptor (EPO-R) expression provides for erythropoietin (EPO) activity beyond its known regulation of red blood cell production. This review highlights the role of EPO and EPO-R in brain development and neuroprotection. EPO-R brain expression includes neural progenitor cells (NPC), neurons, glial cells and endothelial cells. EPO is produced in brain in a hypoxia sensitive manner, stimulates NPC proliferation and differentiation, and neuron survival, and contributes to ischemic preconditioning. Mice lacking EPO or EPO-R exhibit increased neural cell apoptosis during development before embryonic death due to severe anemia. EPO administration provides neural protection in animal models of brain ischemia and trauma, reducing the extent of injury and damage. Intrinsic EPO production in brain and EPO stimulation of endothelial cells contribute to neuroprotection and these are of particular importance since only low levels of EPO appear to cross the blood-brain barrier when administered at high dose intravenously. The therapeutic potential of EPO for brain ischemia/trauma and neurodegenerative diseases has shown promise in early clinical trial and awaits further validation.

title: Apoptosis dominant in the periinfarct area of human ischaemic stroke--a possible target of antiapoptotic treatments.
abstract: Animal experiments have suggested that apoptotic programmed cell death is responsible for an important portion of the delayed ischaemic brain damage. Antiapoptotic signalling through erythropoietin (EPO) binding to its receptor (EPOR) is triggered by systemic or local hypoxia and may exist in the post-ischaemic brain, and a neuroprotective effect by EPO was described recently and proposed for clinical stroke treatment. The objective of the study was to determine whether apoptosis occurs in human ischaemic stroke and to describe its topographical distribution. An autopsy cohort consisting of 13 cases of fatal ischaemic stroke (symptom duration from 15 h to 18 days) treated at the Department of Neurology, Helsinki University Central Hospital and 3 controls were studied. DNA damage was investigated by immunofluorescent TUNEL-labelling in combination with apoptotic cell morphology and by visualization of a major signalling system of apoptosis, Fas-FasL (Fas-ligand), by the immunoperoxidase technique. The relationship of EPO and EPOR in the face of TUNEL-labelled and necrotic cell death was co-registered in human cerebral neurons undergoing different stages of ischaemic change. TUNEL-labelled cells with apoptotic morphology were disproportionately more frequent, 148% (30) [mean (SE)] in the periinfarct versus 97% (22) in the core, as percentage of the cells in the contralateral hemisphere (P = 0.027). The apoptotic cell percentage reached up to 26% (2) of all cells in periinfarct area. A linear correlation was found for Fas and its counterpart FasL expression (r(S) = 0.774, P < 0.001). Ischaemia induced widespread neuronal expression of EPOR, which was inversely related to the severity of ischaemic neuronal necrosis (P < 0.05). To conclude, these data verify the predominance of apoptosis in the periphery of human ischaemic infarctions. Fas and FasL were linearly overexpressed supporting that this 'death-receptor' complex may promote the completion of cell death. Increased EPO signalling may be a cellular response for survival in less severely damaged areas. These results support antiapoptotic therapies against delayed neuronal cell death in human ischaemic stroke.

title: Erythropoietin and erythropoietin receptor in human ischemic/hypoxic brain
abstract: Using immunohistochemistry, expression of erythropoietin (EPO), a hypoxia-inducible neuroprotective factor, and its receptor (EPOR) were investigated in human brain tissue after ischemia/hypoxia. Autopsy brains of neuropathologically normal subjects were compared to those with ischemic infarcts or hypoxic damage. In normal brain, weak EPO/EPOR immunoreactivity was mainly neuronal. In fresh infarcts, EPO immunoreactivity appeared in vascular endothelium, EPOR in microvessels and neuronal fibers. In older infarcts reactive astrocytes exhibited EPO/EPOR immunoreactivity. Acute hypoxic brain damage was associated with vascular EPO expression, older hypoxic damage with EPO/EPOR immunoreactivity in reactive astrocytes. The pronounced up-regulation of EPO/EPOR in human ischemic/hypoxic brains underlines their role as an endogenous neuroprotective system and suggests a novel therapeutic potential in cerebrovascular disease for EPO, a clinically well-characterized and safe compound.

title: Pleiotropic functions and tissue-specific expression of erythropoietin.
abstract: Erythropoietin (EPO) is produced in the brain, uterus, and oviduct. Brain EPO plays a neuroprotective role, and uterine EPO is likely involved in estrogen-dependent angiogenesis. Hypoxic induction of brain EPO markedly differs from that in the kidney. EPO in the uterus and oviduct is estrogen inducible.

title: HIF1 and oxygen sensing in the brain
abstract: Of all the chemical elements, oxygen is the most vital to the human body. The brain is the most sensitive organ to oxygen deprivation (hypoxia), which, over an extended period, can cause coma, seizures, cognitive impairment and other neurological disabilities, and even brain death. However, during mild hypoxia of short duration, the brain develops adaptative mechanisms that allow it to maintain normal physiological conditions. In this review, we discuss some of the molecular mechanisms of oxygen sensing in the brain. Particular emphasis is placed on the oxygen-dependant regulation of the transcription factor HIF1 (hypoxia-inducible factor 1) — one of the main cellular responses to hypoxia that operates in numerous cell types.

title: The Brain Erythropoietin System and its Potential for Therapeutic Exploitation in Brain Disease
abstract: The discovery of the broad neuroprotective potential of erythropoietin (EPO), an endogenous hematopoietic growth factor, has opened new therapeutic avenues in the treatment of brain diseases. EPO expression in the brain is induced by hypoxia. Practically all brain cells are capable of production and release of EPO and expression of its receptor. EPO exerts multifaceted protective effects on brain cells. It protects neuronal cells from noxious stimuli such as hypoxia, excess glutamate, serum deprivation or kainic acid exposure in vitro by targeting a variety of mechanisms and involves neuronal, glial and endothelial cell functions. In rodent models of ischemic stroke, EPO reduces infarct volume and improves functional outcome, but beneficial effects have also been observed in animal models of subarachnoid hemorrhage, intracerebral hemorrhage, traumatic brain injury, and spinal cord injury. EPO has a convenient therapeutic window upon ischemic stroke and favorable pharmacokinetics. Results from first therapeutic trials in humans are promising, but will need to be validated in larger trials. The safety profile and effectiveness of EPO in a wide variety of neurologic disease models make EPO a candidate compound for a potential first-line therapeutic for neurologic emergencies.

title: Erythropoietin in the Cerebrospinal Fluid of Neonates Who Sustained CNS Injury
abstract: We previously reported that erythropoietin (Epo) is present in human cerebrospinal fluid (CSF). It is not known whether CSF Epo concentrations change under conditions of CNS injury or, if so, whether this change reflects loss of blood-brain barrier integrity or increased CNS Epo synthesis. We hypothesized that CSF Epo increases in conditions of neural injury including hypoxia, meningitis, and intraventricular hemorrhage (IVH) and that CSF Epo concentrations are independent of plasma Epo concentrations. To test these hypotheses, Epo concentrations were measured in 122 paired CSF and blood samples obtained from neonates and children categorized as follows: 16, asphyxia; 31, meningitis; 11, IVH; 41, controls. Twelve infants treated with recombinant Epo (rEpo) and 11 additional samples from children with miscellaneous neurologic problems were also evaluated. CSF and plasma Epo concentrations were significantly higher in asphyxiated infants than in controls (225.0 ± 155.0 versus 4.5 ± 0.5 mU/mL; mean ± SEM, p< 0.05, respectively, in CSF; 1806.7 ± 1254 versus 5.2 ± 0.5, p< 0.05 in plasma). Neonates with IVH had higher CSF Epo concentrations than controls (p< 0.01) but did not have higher plasma Epo concentrations than controls. Patients with meningitis did not have elevated CSF or plasma Epo concentrations. There was no correlation between CSF and plasma Epo concentrations in infants treated with rEpo. We conclude that Epo is selectively increased in the CSF by hypoxia, less so by IVH, and not at all by meningitis. rEpo treatment does not elevate CSF Epo. These findings suggest that rEpo does not cross the blood-brain barrier and that hypoxia induces increased CNS synthesis of Epo.

title: Vascular endothelial growth factor and erythropoietin concentrations in cerebrospinal fluid of children with hydrocephalus
abstract: Hypoxia induces transcription of vascular endothelial growth factor (VEGF) and erythropoietin (EPO) genes. We set up the hypothesis that elevated intracranial pressure in patients with hydrocephalus triggers release of VEGF and EPO into cerebrospinal fluid (CSF).  VEGF and EPO concentrations, measured in 57 CSF aliquots obtained from infants and children with hydrocephalus undergoing surgery or therapeutic taps, were significantly elevated compared with those in 41 CSF aliquots of sex- and age-matched children undergoing routine diagnostic lumbar puncture for unrelated reasons (P<0.001 and P=0.015, respectively). In hydrocephalus samples, median (interquartile range) VEGF concentrations were 135 (35–410) pg/ml, and 4 of 57 hydrocephalus samples had a VEGF concentration below the detection limit (1 pg/ml), compared with 38 of 41 control samples. Erythropoietin was undetectable (<0.1 pg/ml) in 34 of 57 hydrocephalus samples and in 34 of 41 controls.  We conclude that conditions necessitating surgical intervention in hydrocephalus patients result in increased CSF concentrations of VEGF and EPO.

title: Detection of erythropoietin in human liquor: intrinsic erythropoietin production in the brain.
abstract: Until now, erythropoietin (EPO) was thought to be produced exclusively in fetal liver and adult kidney and to regulate mammalian erythropoiesis. However, we recently showed that steady state levels of EPO mRNA could be induced up to 100-fold in primary mouse astrocytes cultured under hypoxic conditions, and also reported the presence of mRNA for EPO and its receptor in the brain of mouse, monkey and human. In extending these studies on humans we now show that immunoreactive EPO is present in ventricular cerebrospinal fluid (CSF) of 5 patients with traumatic brain injuries: EPO was found in 15 out of 15 CSF samples. There was no correlation between the serum EPO concentration and the concentration in the CSF. However, EPO concentrations in CSF correlated with the degree of blood-brain-barrier dysfunction. This suggests that EPO does not cross the intact blood-brain-barrier, implying that EPO is produced in the brain itself, most probably by astrocytes in an oxygen-dependent manner. In view that neuronal cells carry the EPO receptor, we propose that EPO acts in a paracrine fashion in the central nervous system and might function as a protective factor against hypoxia-induced damage of neurons.

title: Erythropoietin in the cerebrospinal fluid of patients with aneurysmal subarachnoid haemorrhage originates from the brain
abstract: Recent years' research has revealed a specific, neuroprotective erythropoietin (EPO) system in the central nervous system (CNS) that is upregulated by hypoxia. The presence and dynamics of EPO in the cerebrospinal fluid (CSF) of patients with subarachnoid haemorrhage (SAH) has not been investigated. We collected a total of 83 corresponding serum and CSF samples from 18 patients with aneurysmal SAH and compared the concentrations of EPO with those of blood-derived markers of blood-brain barrier function (albumin, transferrin, alpha(2)-macroglobulin) and with those of proteins with well-known CNS synthesis (prealbumin, apolipoprotein E). The EPO concentration in CSF was 0.93 (0.82) mU/ml (median and inter-quartile range). Nine patients presented CSF-EPO values above 1 mU/ml. CSF levels did not correlate with serum concentrations and were independent of blood-brain barrier integrity suggesting a synthesis in CNS rather than a blood-derived origin. Furthermore, the median CSF:serum ratio (Q(protein)) of EPO was similar to those of prealbumin and apolipoprotein E, and much higher than those of albumin, transferrin and alpha(2)-macroglobulin. When the Q(protein) of all proteins were plotted against Q(albumin), EPO showed dynamics similar to CNS-derived proteins. Our data indicate that EPO in the CSF of patients with aneurysmal SAH originates mainly from the CNS.

title: Ischemic tolerance and endogenous neuroprotection
abstract: Practically any stimulus capable of causing injury to a tissue or organ can, when applied close to (but below) the threshold of damage, activate endogenous protective mechanisms--thus potentially lessening the impact of subsequent, more severe stimuli. A sub-threshold ischemic insult applied to the brain, for example, activates certain cellular pathways that can help to reduce damage caused by subsequent ischemic episodes--a phenomenon known as 'ischemic preconditioning' (IP) or 'ischemic tolerance' (IT). Although investigated for some time in model organisms, IP/IT has recently been shown in human brain. This opens a window into endogenous neuroprotection and, potentially, a window of opportunity to utilize these mechanisms in the clinic to treat patients with stroke and other CNS disorders.

title: Erythropoietin receptor signalling is required for normal brain development.
abstract: Erythropoietin, known for its role in erythroid differentiation, has been shown to be neuroprotective during brain ischaemia in adult animal models. Although high levels of erythropoietin receptor are produced in embryonic brain, the role of erythropoietin during brain development is uncertain. We now provide evidence that erythropoietin acts to stimulate neural progenitor cells and to prevent apoptosis in the embryonic brain. Mice lacking the erythropoietin receptor exhibit severe anaemia and defective cardiac development, and die at embryonic day 13.5 (E13.5). By E12.5, in addition to apoptosis in foetal liver, endocardium and myocardium, the erythropoietin receptor null mouse shows extensive apoptosis in foetal brain. Lack of erythropoietin receptor affects brain development as early as E10.5, resulting in a reduction in the number of neural progenitor cells and increased apoptosis. Corresponding in vitro cultures of cortical cells from Epor(-/-) mice also exhibited decreases in neuron generation compared with normal controls and increased sensitivity to low oxygen tension with no surviving neurons in Epor(-/-) cortical cultures after 24 hour exposure to hypoxia. The viability of primary Epor(+/+) rodent embryonic cortical neurons was further increased by erythropoietin stimulation. Exposure of these cultures to hypoxia induced erythropoietin expression and a tenfold increase in erythropoietin receptor expression, increased cell survival and decreased apoptosis. Cultures of neuronal progenitor cells also exhibited a proliferative response to erythropoietin stimulation. These data demonstrate that the neuroprotective activity of erythropoietin is observed as early as E10.5 in the developing brain, and that induction of erythropoietin and its receptor by hypoxia may contribute to selective cell survival in the brain.

title: Erythropoietin: a candidate compound for neuroprotection in schizophrenia
abstract: Erythropoietin (EPO) is a candidate compound for neuroprotection in human brain disease capable of combating a spectrum of pathophysiological processes operational during the progression of schizophrenic psychosis. The purpose of the present study was to prepare the ground for its application in a first neuroprotective add-on strategy in schizophrenia, aiming at improvement of cognitive brain function as well as prevention/slowing of degenerative processes. Using rodent studies, primary hippocampal neurons in culture, immunohistochemical analysis of human post-mortem brain tissue and nuclear imaging technology in man, we demonstrate that: (1) peripherally applied recombinant human (rh) EPO penetrates into the brain efficiently both in rat and humans, (2) rhEPO is enriched intracranially in healthy men and more distinctly in schizophrenic patients, (3) EPO receptors are densely expressed in hippocampus and cortex of schizophrenic subjects but distinctly less in controls, (4) rhEPO attenuates the haloperidol-induced neuronal death in vitro, and (4) peripherally administered rhEPO enhances cognitive functioning in mice in the context of an aversion task involving cortical and subcortical pathways presumably affected in schizophrenia. These observations, together with the known safety of rhEPO, render it an interesting compound for neuroprotective add-on strategies in schizophrenia and other human diseases characterized by a progressive decline in cognitive performance.

title: Brain Erythropoietin Receptor Expression in Alzheimer Disease and Mild Cognitive Impairment
abstract: Cellular mechanisms conferring neuroprotection in the brains of patients with Alzheimer disease (AD) remain incompletely understood. Erythropoietin (Epo) and the erythropoietin receptor (EpoR) are expressed in neural tissues and protect against oxidative and other stressors in various models of brain injury and disease. Our objective in this study was to determine whether EpoR is upregulated in the brains of persons with sporadic AD and mild cognitive impairment (MCI). Postmortem hippocampus and temporal cortex from subjects with AD, MCI, and no cognitive impairment (NCI) were procured from the Religious Orders Study. Total immunoreactive EpoR protein was determined by Western blotting. Astrocytes expressing immunoreactive EpoR were quantified in 4 temporal and 6 hippocampal regions, and correlated with clinical, neuropsychologic, and neuropathologic indices. Total immunoreactive EpoR protein was markedly increased in AD and MCI temporal cortex versus NCI tissues. Composite measures of glial EpoR expression in temporal cortex layers I to IV were significantly greater in the MCI group compared with the NCI and AD groups. Hippocampal EpoR scores were increased in persons with MCI and AD relative to those with NCI. There was substantial subregional heterogeneity in disease-related EpoR expression patterns in AD and MCI temporal cortex and hippocampus. There was no association of EpoR-positive astrocytes with summary measures of global cognition or AD pathology. We conclude that upregulation of EpoR in temporal cortical and hippocampal astrocytes is an early, potentially neuroprotective, event in the pathogenesis of sporadic AD.

title: Structure, function, and activation of the erythropoietin receptor.
abstract: RYTHROPOIETIN (EPO) is the principal growth factor E that promotes the viability, proliferation, and differentiation of mammalian erythroid progenitor cells, functions that are transduced by the specific cell surface EPO receptor (EPO-R). In contrast to many other hematopoietic growth factors, EPO primarily affects relatively mature erythroid cells, although it may also influence the growth and behavior of other cells. The recent cloning of the EPO-R gene has greatly facilitated the molecular analysis of the structure and function of this receptor, and provided a fundamental link to earlier biochemical and cellular studies of erythropoiesis. Here we describe recent studies on the structure, function, and activation of this receptor. One focus will be on mechanisms by which the activated receptor can induce cellular proliferation in the absence of EPO. This can be achieved by binding of a retrovirus envelope glycoprotein to the EPO-R or by a specific point mutation in the exoplasmic domain of the EPOR. EPO-independent activation of the EPO-R can contribute to the development of erythroleukemia in animal model systems. A second focus will be on the structure and control of expression of the EPO-R gene.

title: Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-κB signalling cascades
abstract: Erythropoietin, a kidney cytokine regulating haematopoiesis (the production of blood cells), is also produced in the brain after oxidative or nitrosative stress. The transcription factor hypoxia-inducible factor-1 (HIF-1) upregulates EPO following hypoxic stimuli. Here we show that preconditioning with EPO protects neurons in models of ischaemic and degenerative damage due to excitotoxins and consequent generation of free radicals, including nitric oxide (NO). Activation of neuronal EPO receptors (EPORs) prevents apoptosis induced by NMDA (N-methyl-d-aspartate) or NO by triggering cross-talk between the signalling pathways of Janus kinase-2 (Jak2) and nuclear factor-κB (NF-κB). We show that EPOR-mediated activation of Jak2 leads to phosphorylation of the inhibitor of NF-κB (IκB), subsequent nuclear translocation of the transcription factor NF-κB, and NF-κB-dependent transcription of neuroprotective genes. Transfection of cerebrocortical neurons with a dominant interfering form of Jak2 or an IκBα super-repressor blocks EPO-mediated prevention of neuronal apoptosis. Thus neuronal EPORs activate a neuroprotective pathway that is distinct from previously well characterized Jak and NF-κB functions. Moreover, this EPO effect may underlie neuroprotection mediated by hypoxic–ischaemic preconditioning.

title: Erythropoietin mediates tissue protection through an erythropoietin and common beta-subunit heteroreceptor.
abstract: The cytokine erythropoietin (Epo) is tissue-protective in preclinical models of ischemic, traumatic, toxic, and inflammatory injuries. We have recently characterized Epo derivatives that do not bind to the Epo receptor (EpoR) yet are tissue-protective. For example, carbamylated Epo (CEpo) does not stimulate erythropoiesis, yet it prevents tissue injury in a wide variety of in vivo and in vitro models. These observations suggest that another receptor is responsible for the tissue-protective actions of Epo. Notably, prior investigation suggests that EpoR physically interacts with the common beta receptor (betacR), the signal-transducing subunit shared by the granulocyte-macrophage colony stimulating factor, and the IL-3 and IL-5 receptors. However, because betacR knockout mice exhibit normal erythrocyte maturation, betacR is not required for erythropoiesis. We hypothesized that betacR in combination with the EpoR expressed by nonhematopoietic cells constitutes a tissue-protective receptor. In support of this hypothesis, membrane proteins prepared from rat brain, heart, liver, or kidney were greatly enriched in EpoR after passage over either Epo or CEpo columns but covalently bound in a complex with betacR. Further, antibodies against EpoR coimmunoprecipitated betacR from membranes prepared from neuronal-like P-19 cells that respond to Epo-induced tissue protection. Immunocytochemical studies of spinal cord neurons and cardiomyocytes protected by Epo demonstrated cellular colocalization of Epo betacR and EpoR. Finally, as predicted by the hypothesis, neither Epo nor CEpo was active in cardiomyocyte or spinal cord injury models performed in the betacR knockout mouse. These data support the concept that EpoR and betacR comprise a tissue-protective heteroreceptor.

title: Derivatives of Erythropoietin That Are Tissue Protective But Not Erythropoietic
abstract: Erythropoietin (EPO) is both hematopoietic and tissue protective, putatively through interaction with different receptors. We generated receptor subtype–selective ligands allowing the separation of EPO's bioactivities at the cellular level and in animals. Carbamylated EPO (CEPO) or certain EPO mutants did not bind to the classical EPO receptor (EPOR) and did not show any hematopoietic activity in human cell signaling assays or upon chronic dosing in different animal species. Nevertheless, CEPO and various nonhematopoietic mutants were cytoprotective in vitro and conferred neuroprotection against stroke, spinal cord compression, diabetic neuropathy, and experimental autoimmune encephalomyelitis at a potency and efficacy comparable to EPO.

title: Erythropoietin and carbamylated erythropoietin are neuroprotective following spinal cord hemisection in the rat
abstract: The cytokine erythropoietin (EPO) has been shown to be neuroprotective in a variety of models of central and peripheral nervous system injury. Derivatives of EPO that lack its erythropoietic effects have recently been developed, and the initial reports suggest that they have a neuroprotective potential comparable to that of EPO. One such derivative is carbamylated EPO (CEPO). In the current study we compared the effects of treatment with EPO and CEPO on some of the early neurodegenerative events that occur following spinal cord injury (SCI) induced by hemisection. Adult male Wistar rats received a unilateral hemisection of the spinal cord. Thirty minutes and 24 h following injury, animals received an intraperitoneal injection of saline, EPO (40 µg/kg) or CEPO (40 µg/kg). Results indicated that 3 days post‐injury, both CEPO and EPO decreased to a similar extent the size of the lesion compared with control animals. Both compounds also decreased the number of terminal transferase‐mediated dUTP nick‐end labelling (TUNEL)‐labelled apopotic nuclei around the lesion site, as well as the number of axons expressing the injury marker β‐amyloid precursor protein. EPO and CEPO also increased Schwann cell infiltration into the lesion site, although neither compound had any effect on macrophage infiltration either within the lesion site itself or in the surrounding intact tissue. In addition, immunohistochemistry showed an increased expression of both the EPO receptor and the β common receptor subunit, the components of the receptor complex proposed to mediate the neuroprotective effects of EPO and CEPO in neurons near the site of the injury. The results show that not only does CEPO have an efficacy comparable to that of EPO in its neuroprotective potential following injury, but also that changes in the receptors for these compounds following SCI may underlie their neuroprotective efficacy.

title: A "classical" homodimeric erythropoietin receptor is essential for the antiapoptotic effects of erythropoietin on differentiated neuroblastoma SH-SY5Y and pheochromocytoma PC-12 cells.
abstract: The hematopoietic cytokine erythropoietin (Epo) exerts cytoprotective effects on several types of neuronal cells both in vivo and in culture. Detailed molecular mechanisms underlying this phenomenon have not been elucidated and even the identity of the cytoprotective Epo receptors in neuronal cells is controversial. Here we show that Epo prevents staurosporine-induced apoptosis of differentiated human neuroblastoma SH-SY5Y cells, and activates the STAT5, AKT and MAPK signaling pathways. Differentiated SH-SY5Y cells have fewer than 50 high affinity Epo surface binding sites per cell, which could not be detected by standard assays measuring binding of 125I-labeled Epo. However, by measuring endocytosis of 125I-Epo, we could reliably quantify very small numbers of high-affinity Epo surface binding sites. Using SH-SY5Y cells stably expressing an Epo receptor (EpoR) shRNA and thus lacking detectable EpoR expression, we show that high affinity binding of Epo to these neuronal cells is mediated by the hematopoietic EpoR, and that this EpoR is also essential for the antiapoptotic activity of Epo. In contrast, a mutant Epo that has an intact binding site 1 but a non-functional binding site 2 and hence binds only to one cell surface EpoR molecule ("site 2" Epo mutant) displays significantly lower antiapoptotic activity than wild-type Epo. Furthermore, expression of the GM-CSF/IL-3/IL-5 receptor common beta chain, which was proposed to be responsible for the cytoprotective activity of Epo on certain types of neuronal cells, was undetectable in differentiated SH-SY5Y cells. Epo also alleviated staurosporine-induced apoptosis of rat PC-12 pheochromocytoma cells while the R103A "site 2" Epo mutant did not, and we could not detect expression of the common beta chain in PC-12 cells. Together our results indicate that Epo exerts its antiapoptotic effects on differentiated SH-SY5Y and PC-12 cells through the standard stoichiometry of one molecule of Epo binding to two EpoR subunits, comprising the "classical" Epo receptor signaling complex.

title: The Sonic Hedgehog Pathway Mediates Carbamylated Erythropoietin-enhanced Proliferation and Differentiation of Adult Neural Progenitor Cells*
abstract: Carbamylated erythropoietin (CEPO), a well characterized erythropoietin (EPO) derivative, does not bind to the classical EPO receptor and does not stimulate erythropoiesis. Using neural progenitor cells derived from the subventricular zone of the adult mouse, we investigated the effect of CEPO on neurogenesis and the associated signaling pathways in vitro. We found that CEPO significantly increased neural progenitor cell proliferation and promoted neural progenitor cell differentiation into neurons, which was associated with up-regulation of Sonic hedgehog (Shh), its receptor ptc, and mammalian achaete-scute homolog 1 (Mash1), a pro-neuron basic helix-loop-helix protein transcription factor. Blockage of the Shh signaling pathway with a pharmacological inhibitor, cyclopamine, abolished the CEPO-induced neurogenesis. Attenuation of endogenous Mash1 expression by short-interfering RNA blocked CEPO-promoted neuronal differentiation. In addition, recombinant mouse Shh up-regulated Mash1 expression in neural progenitor cells. These results demonstrate that the Shh signaling pathway mediates CEPO-enhanced neurogenesis and Mash1 is a downstream target of the Shh signaling pathway that regulates CEPO-enhanced neuronal differentiation.

title: Functional erythropoietin receptor of the cells with neural characteristics. Comparison with receptor properties of erythroid cells.
abstract: 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.

title: Nonerythropoietic, tissue-protective peptides derived from the tertiary structure of erythropoietin
abstract: Erythropoietin (EPO), a member of the type 1 cytokine superfamily, plays a critical hormonal role regulating erythrocyte production as well as a paracrine/autocrine role in which locally produced EPO protects a wide variety of tissues from diverse injuries. Significantly, these functions are mediated by distinct receptors: hematopoiesis via the EPO receptor homodimer and tissue protection via a heterocomplex composed of the EPO receptor and CD131, the β common receptor. In the present work, we have delimited tissue-protective domains within EPO to short peptide sequences. We demonstrate that helix B (amino acid residues 58–82) of EPO, which faces the aqueous medium when EPO is bound to the receptor homodimer, is both neuroprotective in vitro and tissue protective in vivo in a variety of models, including ischemic stroke, diabetes-induced retinal edema, and peripheral nerve trauma. Remarkably, an 11-aa peptide composed of adjacent amino acids forming the aqueous face of helix B is also tissue protective, as confirmed by its therapeutic benefit in models of ischemic stroke and renal ischemia–reperfusion. Further, this peptide simulating the aqueous surface of helix B also exhibits EPO's trophic effects by accelerating wound healing and augmenting cognitive function in rodents. As anticipated, neither helix B nor the 11-aa peptide is erythropoietic in vitro or in vivo. Thus, the tissue-protective activities of EPO are mimicked by small, nonerythropoietic peptides that simulate a portion of EPO's three-dimensional structure.

title: Asialoerythropoietin is a nonerythropoietic cytokine with broad neuroprotective activity in vivo
abstract: Erythropoietin (EPO) is a tissue-protective cytokine preventing vascular spasm, apoptosis, and inflammatory responses. Although best known for its role in hematopoietic lineages, EPO also affects other tissues, including those of the nervous system. Enthusiasm for recombinant human erythropoietin (rhEPO) as a potential neuroprotective therapeutic must be tempered, however, by the knowledge it also enlarges circulating red cell mass and increases platelet aggregability. Here we examined whether erythropoietic and tissue-protective activities of rhEPO might be dissociated by a variation of the molecule. We demonstrate that asialoerythropoietin (asialoEPO), generated by total enzymatic desialylation of rhEPO, possesses a very short plasma half-life and is fully neuroprotective. In marked contrast with rhEPO, this molecule at doses and frequencies at which rhEPO exhibited erythropoiesis, did not increase the hematocrit of mice or rats. AsialoEPO appeared promptly within the cerebrospinal fluid after i.v. administration; intravenously administered radioiodine-labeled asialoEPO bound to neurons within the hippocampus and cortex in a pattern corresponding to the distribution of the EPO receptor. Most importantly, asialoEPO exhibits a broad spectrum of neuroprotective activities, as demonstrated in models of cerebral ischemia, spinal cord compression, and sciatic nerve crush. These data suggest that nonerythropoietic variants of rhEPO can cross the blood–brain barrier and provide neuroprotection.

title: Reduced Functional Deficits, Neuroinflammation, and Secondary Tissue Damage after Treatment of Stroke by Nonerythropoietic Erythropoietin Derivatives
abstract: Carbamylerythropoietin (CEPO) does not bind to the classical erythropoietin (EPO) receptor. Nevertheless, similarly to EPO, CEPO promotes neuroprotection on the histologic level in short-term stroke models. In the present study, we investigated whether CEPO and other nonerythropoietic EPO analogs could enhance functional recovery and promote long-term histologic protection after experimental focal cerebral ischemia. Rats were treated with the compounds after focal cerebral ischemia. Animals survived 1, 7, or 60 days and underwent behavioral testing (sensorimotor and foot-fault tests). Brain sections were stained and analyzed for Iba-1, myeloperoxidase, Tau-1, CD68 (ED1), glial fibrillary acidic protein (GFAP), Jade B staining, and overall infarct volumes. Treatment with CEPO reduced perifocal microglial activation (P<0.05), polymorphomonuclear cell infiltration (P<0.05), and white matter damage (P<0.01) at 1 day after occlusion. Carbamylerythropoietin-treated rats showed better functional recovery relative to vehicle-treated animals as assessed 1, 7, 14, 28, and 50 days after stroke. Both GFAP and CD68 were decreased within the ipsilateral thalamus of CEPO-treated animals 60 days postoperatively (P<0.01 and P<0.05, respectively). Furthermore, behavioral analysis showed efficacy of CEPO treatment even if administered 24 h after the stroke. Other nonerythropoietic derivatives such as carbamylated darbepoetin alfa and the mutant EPO-S100E were also found to protect against ischemic damage and to improve postischemic neurologic function. In conclusion, these results show that postischemic intravenous treatment with nonerythropoietic EPO derivatives leads to improved functional recovery, which may be linked to their long-term effects against neuroinflammation and secondary tissue damage.

title: High-dose erythropoietin alters platelet reactivity and bleeding time in rodents in contrast to the neuroprotective variant carbamyl-erythropoietin (CEPO).
abstract: The haematopoietic hormone erythropoietin (EPO) has neuroprotective properties and is currently being explored for treatment of stroke and other neurological disorders. Short-term, high-dose treatment with EPO seems to improve neurological function of stroke patients but may be associated with increased thrombotic risk, whereas alternative non-erythropoietic neuroprotective derivatives of EPO, such as carbamylated EPO (CEPO), may be devoid of such side-effects. We investigated the effects of short-term, high-dose treatment with EPO and CEPO on platelet function and haemostasis in healthy mice and rats. Animals received three daily doses of EPO or CEPO (50 microg/kg), and blood was compared with respect to alterations in haematology and platelet reactivity. In rats, treatment with EPO increased the haematocrit to >50% and the mean platelet volume by 37%, while CEPO had no effect on these parameters. Platelets from EPO-treated rats showed an increased sensitivity to thrombin receptor agonist peptides and elevated plasma levels of soluble P-selectin (sP-selectin) were found in treated mice. Further indicators of platelet hyperreactivity in EPO, but not CEPO-treated animals, were significantly increased aggregatory responses to collagen in whole blood and platelet-rich plasma (PRP). The increased platelet reactivity was paralleled by a decreased bleeding time after tail transection in rats. Samples from EPO-treated rats showed an attenuated response to ADP in whole blood aggregometry and thrombelastography (TEG) platelet mapping but not in apyrase-treated PRP, suggesting involvement of ADP receptor desensitization. These findings suggest that while EPO affects various aspects of platelet function, CEPO is devoid of such effects.

title: Stimulating Erythropoiesis: Future Perspectives
abstract: The introduction of recombinant human erythropoietin (rHuEpo) nearly 20 years ago has revolutionised the management of patients with CKD, providing the opportunity for safe long-term anaemia correction without the attendant risks identified with blood products. Based on our expanding knowledge in this area, there are many exciting and innovative new approaches to anaemia correction that stand on, or are close to, the threshold of yielding products ready for clinical use. Recently, an Epo-related molecule called continuous Epo receptor activator has been licensed in Europe, and other molecules are in various processes of development, including Epo mimetic peptide. The search goes on for orally active antianaemic therapies, and several strategies are being investigated. Furthermore, it is now clear that in addition to the anaemia-correction properties of erythropoiesis-stimulating agents, there is the potential for cytoprotection by prevention of cellular apoptosis. This effect could be used in the prevention of ischaemia-reperfusion injury as well as other conditions associated with acute kidney injury and other disease processes. The aim of this article is to discuss these possible future strategies, focusing in particular on those with a reasonable likelihood of a pharmaceutical product that is likely to be used clinically.

title: Darbepoetin alfa has a longer circulating half-life and greater in vivo potency than recombinant human erythropoietin.
abstract: 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.



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



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.



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.

title: Preclinical evaluation of Hematide, a novel erythropoiesis stimulating agent, for the treatment of anemia.
abstract: To evaluate the preclinical erythropoiesis stimulating properties of Hematide, a novel, PEGylated, synthetic peptide for the treatment of anemia associated with chronic kidney disease and cancer.



The in vitro activity of Hematide was assessed in competitive binding, proliferation, signal transduction, and apoptosis assays, and in erythroid colony-forming assays with CD34(+) cells purified from human bone marrow. Erythropoiesis and pharmacokinetics were evaluated in rat, monkey, and a rat chronic renal insufficiency (CRI) model following single administration. Erythropoiesis and immunogenicity were also evaluated following repeat administration in rats.



Hematide binds and activates the erythropoietin receptor and causes proliferation and differentiation of erythroid progenitor cells. Sustained circulatory persistence of Hematide is observed in rats and monkeys. In a rat CRI model, Hematide exhibited twofold lower clearance than in the normal rat, with hypothesis consistent with Hematide being cleared, at least partially, via the kidney. A dose-dependent rise in hemoglobin (Hgb) and duration of response was observed following single administration in rats and monkeys. Hematide was able to alleviate anemia in an experimental CRI rodent model. Repeat intravenous (IV) and subcutaneous (SC) administration in rats yielded similar erythrogenic responses, with no anti-Hematide antibodies being detected.



Hematide is a potent erythropoiesis stimulating agent with a prolonged half-life and slow clearance times. It is anticipated that similar prolonged clearance and activity will be observed in the clinic, potentially enabling dosing intervals of 3 to 4 weeks that may translate into improved patient convenience for the treatment of anemia.

title: Erythropoietin inhibits calcium-induced neurotransmitter release from clonal neuronal cells.
abstract: Erythropoietin (EPO) and EPO receptor (EPO-R) are expressed in the brain but their neuronal functions are not yet clarified. The effects of EPO on neurosecretion were studied using clonal rat pheochromocytoma PC12 cells. EPO suppressed Ca(2+)-induced dopamine release from PC12 cells in a concentration- and time-dependent manner. Inhibition was also produced by an EPO mimetic peptide 1 (EMP1), a small synthetic peptide agonist of EPO-R, but not by its inactive analogue in which Cys residues were substituted with Ser. Inhibition was abolished by genistein but not by genistin. EPO and EMP1 induced autophosphorylation of Janus kinase 2 (JAK 2), a tyrosine kinase that associates with EPO-R, and dephosphorylation of GAP-43 in a tyrosine kinase-dependent fashion. These results suggest that EPO suppresses neurotransmitter release through activation of EPO-R linked to JAK2.

title: Erythropoietin Receptor-mediated Inhibition of Exocytotic Glutamate Release Confers Neuroprotection during Chemical Ischemia*
abstract: Erythropoietin (EPO) reduced Ca2+-induced glutamate (Glu) release from cultured cerebellar granule neurons. Inhibition was also produced by EPO mimetic peptide 1 (EMP1), a small synthetic peptide agonist of EPO receptor (EPO-R), but not by iEMP1, an inactive analogue of EMP1. EPO and EMP1 induced autophosphorylation of Janus kinase 2 (JAK2), a tyrosine kinase that associates with EPO-R. Furthermore, genistein, but not genistin, antagonized both the phosphorylation of JAK2 and the suppression of Glu release induced by EPO and EMP1. During chemical ischemia, substantial amounts of Glu were released from cultured cerebellar and hippocampal neurons by at least two distinct mechanisms. In the early phase, Glu release occurred by exocytosis of synaptic vesicle contents, because it was abolished by botulinum type B neurotoxin (BoNT/B). In contrast, the later phase of Glu release mainly involved a BoNT/B-insensitive non-exocytotic pathway. EMP1 inhibited Glu release only during the early exocytotic phase. A 20-min exposure of hippocampal slices to chemical ischemia induced neuronal cell death, especially in the CA1 region and the dentate gyrus, which was suppressed by EMP1 but not iEMP1. However, EMP1 did not attenuate neuronal cell death induced by exogenously applied Glu. These results suggest that activation of EPO-R suppresses ischemic cell death by inhibiting the exocytosis of Glu.

title: Small Peptides as Potent Mimetics of the Protein Hormone Erythropoietin
abstract: Random phage display peptide libraries and affinity selective methods were used to isolate small peptides that bind to and activate the receptor for the cytokine erythropoietin (EPO). In a panel of in vitro biological assays, the peptides act as full agonists and they can also stimulate erythropoiesis in mice. These agonists are represented by a 14- amino acid disulfide-bonded, cyclic peptide with the minimum consensus sequence YXCXXGPXTWXCXP, where X represents positions allowing occupation by several amino acids. The amino acid sequences of these peptides are not found in the primary sequence of EPO. The signaling pathways activated by these peptides appear to be identical to those induced by the natural ligand. This discovery may form the basis for the design of small molecule mimetics of EPO.

title: Neuroprotection in Diet-Induced Ketotic Rat Brain after Focal Ischemia
abstract: Neuroprotective properties of ketosis may be related to the upregulation of hypoxia inducible factor (HIF)-1α, a primary constituent associated with hypoxic angiogenesis and a regulator of neuroprotective responses. The rationale that the utilization of ketones by the brain results in elevation of intracellular succinate, a known inhibitor of prolyl hydroxylase (the enzyme responsible for the degradation of HIF-1α) was deemed as a potential mechanism of ketosis on the upregulation of HIF-1α. The neuroprotective effect of diet-induced ketosis (3 weeks of feeding a ketogenic diet), as pretreatment, on infarct volume, after reversible middle cerebral artery occlusion (MCAO), and the upregulation of HIF-1α were investigated. The effect of β-hydroxybutyrate (BHB), as a pretreatment, via intraventricular infusion (4 days of infusion before stroke) was also investigated following MCAO. Levels of HIF-1α and Bcl-2 (anti-apoptotic protein) proteins and succinate content were measured. A 55% or 70% reduction in infarct volume was observed with BHB infusion or diet-induced ketosis, respectively. The levels of HIF-1α and Bcl-2 proteins increased threefold with diet-induced ketosis; BHB infusions also resulted in increases in these proteins. As hypothesized, succinate content increased by 55% with diet-induced ketosis and fourfold with BHB infusion. In conclusion, the biochemical link between ketosis and the stabilization of HIF-1α is through the elevation of succinate, and both HIF-1α stabilization and Bcl-2 upregulation play a role in ketone-induced neuroprotection in the brain.

title: Hypoxia-inducible Factor Prolyl 4-Hydroxylase Inhibition
abstract: Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases are a family of iron- and 2-oxoglutarate-dependent dioxygenases that negatively regulate the stability of several proteins that have established roles in adaptation to hypoxic or oxidative stress. These proteins include the transcriptional activators HIF-1α and HIF-2α. The ability of the inhibitors of HIF prolyl 4-hydroxylases to stabilize proteins involved in adaptation in neurons and to prevent neuronal injury remains unclear. We reported that structurally diverse low molecular weight or peptide inhibitors of the HIF prolyl 4-hydroxylases stabilize HIF-1α and up-regulate HIF-dependent target genes (e.g. enolase, p21waf1/cip1, vascular endothelial growth factor, or erythropoietin) in embryonic cortical neurons in vitro or in adult rat brains in vivo. We also showed that structurally diverse HIF prolyl 4-hydroxylase inhibitors prevent oxidative death in vitro and ischemic injury in vivo. Taken together these findings identified low molecular weight and peptide HIF prolyl 4-hydroxylase inhibitors as novel neurological therapeutics for stroke as well as other diseases associated with oxidative stress.

title: Beta-hydroxybutyrate, a cerebral function improving agent, protects rat brain against ischemic damage caused by permanent and transient focal cerebral ischemia.
abstract: In our previous study, beta-hydroxybutyrate (BHB) was found to prolong survival time and to inhibit cerebral edema by improving energy metabolism in the hypoxia, anoxia and global cerebral ischemia models. In this study, the cerebroprotective effect of BHB was examined in rats with permanent (p)-occlusion and transient (t)-occlusion of middle cerebral artery (MCA). BHB (30 mg x kg(-1) x h(-1) was continuously administered through the femoral vein. In rats with p-MCA occlusion, BHB significantly reduced infarct area at 24 h after the occlusion, but not at 72 h after the occlusion. In rats with 2-h t-MCA occlusion followed by 22-h reperfusion, BHB significantly reduced cerebral infarct area, edema formation, lipid peroxidation and neurological deficits. Moreover, in the t-MCA occlusion model, delayed administration of BHB started at 1 h after the initiation of the MCA occlusion also significantly reduced cerebral infarct area. Taking together the results obtained in our previous study into account, these results indicate that BHB decreased cerebral edema formation and infarct area by improving of the cerebral energy metabolism during ischemia and by inhibition of lipid peroxidation after reperfusion.

title: Neuroprotective Effect of Darbepoetin Alfa, a Novel Recombinant Erythropoietic Protein, in Focal Cerebral Ischemia in Rats
abstract: Darbepoetin alfa is a novel erythropoiesis-stimulating protein developed for treating anemia. In animal models, exogenous recombinant human erythropoietin has been reported to be beneficial in treating experimental cerebral ischemia. In this study, we determined whether darbepoetin alfa would protect in a rat model of transient focal cerebral ischemia.  Rats received 2-hour middle cerebral artery suture-occlusion. The drug (darbepoetin alfa, 10 &mgr;g/kg) or vehicle was administered intraperitoneally 2 hours after onset of middle cerebral artery occlusion. Animals were allowed to survive for 3 or 14 days. Behavioral tests were performed sequentially. Infarct volumes and brain swelling were determined.  Darbepoetin alfa-treated rats showed improved neuroscores relative to vehicle-treated animals beginning within 1 hour of treatment and persisting throughout the 14-day survival period. Darbepoetin alfa significantly reduced corrected total (cortical + subcortical) infarct volume (56.3±20.6 and 110.8±6.8 mm3, respectively) and total infarct areas at multiple levels compared with vehicle in the 14-day survival group. Brain swelling was not affected by treatment.  Darbepoetin alfa confers behavioral and histological neuroprotection after focal ischemia in rats.

title: Erythropoietin Selectively Attenuates Cytokine Production and Inflammation in Cerebral Ischemia by Targeting Neuronal Apoptosis
abstract: Ischemic brain injury resulting from stroke arises from primary neuronal losses and by inflammatory responses. Previous studies suggest that erythropoietin (EPO) attenuates both processes. Although EPO is clearly antiapoptotic for neurons after experimental stroke, it is unknown whether EPO also directly modulates EPO receptor (EPO-R)–expressing glia, microglia, and other inflammatory cells. In these experiments, we show that recombinant human EPO (rhEPO; 5,000 U/kg body weight, i.p.) markedly reduces astrocyte activation and the recruitment of leukocytes and microglia into an infarction produced by middle cerebral artery occlusion in rats. In addition, ischemia-induced production of the proinflammatory cytokines tumor necrosis factor, interleukin 6, and monocyte chemoattractant protein 1 concentration is reduced by >50% after rhEPO administration. Similar results were also observed in mixed neuronal-glial cocultures exposed to the neuronal-selective toxin trimethyl tin. In contrast, rhEPO did not inhibit cytokine production by astrocyte cultures exposed to neuronal homogenates or modulate the response of human peripheral blood mononuclear cells, rat glial cells, or the brain to lipopolysaccharide. These findings suggest that rhEPO attenuates ischemia-induced inflammation by reducing neuronal death rather than by direct effects upon EPO-R–expressing inflammatory cells.

title: Treatment of Stroke With Erythropoietin Enhances Neurogenesis and Angiogenesis and Improves Neurological Function in Rats
abstract: Erythropoietin (EPO) promotes proliferation and differentiation of erythroid progenitors and the survival of maturing erythroid cells. Here, we investigated the role of EPO in brain repair after stroke.  Rats were treated with recombinant human EPO (rhEPO) at 24 hours after the onset of embolic stroke. An array of behavior tests was performed. Rats were euthanized 28 days after stroke for measurements of infarct volume, angiogenesis, and neurogenesis. In vitro, neurospheres derived from the subventricular zone (SVZ) of the rat and cerebral endothelial cells derived from the mouse were treated with rhEPO. Capillary-like tube formation and neuronal differentiation were measured.  Treatment with rhEPO significantly improved functional recovery, along with increases in density of cerebral microvessels at the stroke boundary and numbers of BrdU, doublecortin, and nestin immunoreactive cells in the SVZ. rhEPO treatment significantly increased brain levels of vascular endo...

title: Post‐ischemic treatment with erythropoietin or carbamylated erythropoietin reduces infarction and improves neurological outcome in a rat model of focal cerebral ischemia
abstract: Recombinant human erythropoietin (rhEPO; Epoetin‐α; PROCRIT™) has been shown to exert neuroprotective and restorative effects in a variety of CNS injury models. However, limited information is available regarding the dose levels required for these beneficial effects or the neuronal responses that may underlie them. Here we have investigated the dose‐response to rhEPO and compared the effects of rhEPO with those of carbamylated rhEPO (CEPO) in a model of cerebral stroke in rats.

title: Intranasal recombinant human erythropoietin protects rats against focal cerebral ischemia
abstract: Erythropoietin (EPO) is a hematopoietic growth factor with tissue-protective properties, and can protect animals from cerebral ischemic injury. However, the central nervous effects of EPO as a glycoprotein is limited by the potential complication resulted from its erythropoietic activity and the problem of the penetration through blood-brain barrier (BBB). To avoid these limitations, in this study we administered recombinant human EPO (rhEPO) intranasally (i.n.) to evaluate its neuroprotective effect in the rats with focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO). We found that rhEPO i.n. at doses of 4.8, 12 and 24 U (administered 10 min after MCAO and 1h after reperfusion) reduced infarct volume, brain swelling and cell damage in the ischemic hemispheres, and improved behavioral dysfunction 24 h after cerebral ischemia. Intraperitoneal rhEPO (5000 U/kg) also showed the protective effect, but the heat-inactivated rhEPO did not show any effect. Thus, intranasal administration of relatively small doses of rhEPO protects rats from acute injury after focal cerebral ischemia, suggesting that intranasal rhEPO may be a more effective and safer administration route for treatments of ischemic or other brain diseases.

title: Erythropoietin Improves Functional and Histological Outcome in Neonatal Stroke
abstract: Neonatal stroke is a condition that leads to disability in later life, and as yet there is no effective treatment. Recently, erythropoietin (EPO) has been shown to be cytoprotective following brain injury and may promote neurogenesis. However, the effect of EPO on functional outcome and on morphologic changes in neonatal subventricular zone (SVZ) following experimental neonatal stroke has not been described. We used a transient focal model of neonatal stroke in P10 rat. Injury was documented by diffusion weighted MRI during occlusion. Immediately upon reperfusion, either EPO (5U/gm) or vehicle was administered intraperitoneally and animals were allowed to grow for 2 wk. Sensorimotor function was assessed using the cylinder rearing test and then brains were processed for volumetric analysis of the SVZ. Stroke induced SVZ expansion proportional to hemispheric volume loss. EPO treatment markedly preserved hemispheric volume and decreased the expansion of SVZ unilaterally. Furthermore, EPO treatment significantly improved the asymmetry of forelimb use following neonatal stroke. This functional improvement directly correlated with the amount of preserved hemispheric volume. These results suggest EPO may be a candidate in the treatment of neonatal stroke.

title: Erythropoietin: Novel Approaches to Neuroprotection in Human Brain Disease
abstract: With the increased life expectancy in western industrialized countries, the incidence and prevalence of brain diseases dramatically increased. Stroke and a wide spectrum of neuropsychiatric illnesses such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, traumatic head injury, and schizophrenia all lead to severe disability. However, targeted effective therapies for treatment of these diseases are lacking. Even more frustrating is the fact that we do not yet clearly understand the basic mechanisms underlying the disease processes in these conditions. We propose a hypothesis of loss of neuronal function via a final common deleterious pathway in this clinically very heterogeneous disease group. This review presents a novel neuroprotective concept for treatment of brain disease: Erythropoietin (EPO). EPO is a natural body-own-protein hormone that has been used for treatment of anemia for more than a decade. The neuroprotective approach using EPO in brain disease represents a totally new frontier. The “Göttingen EPO-stroke trial” represents the first effective use in man of a neuroprotective therapy in an acute brain disease while the experimental EPO therapy to combat cognitive decline in patients with schizophrenia will be introduced as an example of a neuroprotective strategy for a chronic brain disease.

title: Erythropoietin Enhances Long-Term Neuroprotection and Neurogenesis in Neonatal Stroke
abstract: 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 IP) 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.

title: Erythropoietin improves long-term spatial memory deficits and brain injury following neonatal hypoxia–ischemia in rats
abstract: It is well known that neonatal hypoxic-ischemic brain injury leads to mental retardation and deficits in cognitive abilities such as learning and memory in human beings. The ameliorative effect of erythropoietin (Epo) on experimental hypoxic-ischemic brain injury in neonatal rats has been recently reported. However, the effect of Epo on cognitive abilities in the hypoxic-ischemic brain injury model is unknown. The aim of this study is to investigate the effects of Epo on learning-memory, behavior and neurodegeneration induced by hypoxia-ischemia. Seven days old Wistar Albino rat pups have been used in the study (n = 28). Experimental groups in the study were: (1) saline-treated hypoxia-ischemia group, (2) Epo-treated (i.p., 1000 U/kg) hypoxia-ischemia group, (3) sham-operated group, (4) control group. In hypoxia-ischemia groups, left common carotid artery was ligated permanently on the seventh postnatal day. Two hours after the procedure, hypoxia (92% nitrogen and 8% oxygen) was induced for 2.5 h. Epo was administered as a single dose immediately after the hypoxia period. When pups were 22 days old, learning experiments were performed using Morris water maze. On the 20th week, when brain development is accepted to be complete, learning experiments were repeated. Rats were then perfused and brains removed for macroscopic and microscopic evaluation. Epo treatment immediately after hypoxic-ischemic insult significantly improved long-term neurobehavioral achievements when tested during the subsequent phase of brain maturation and even into adulthood. Histopathological evaluation demonstrated that Epo also significantly diminished brain injury and spared hippocampal CA1 neurons. In conclusion, Epo administrated as a single dose immediately after neonatal hypoxic-ischemic insult provides benefit over a prolonged period in the still developing rat brain. Since the wide use of Epo in premature newborns, this agent may be potentially beneficial in treating asphyxial brain damage in the perinatal period.

title: Erythropoietin prevents long-term sensorimotor deficits and brain injury following neonatal hypoxia–ischemia in rats
abstract: Perinatal asphyxia accounts for behavioral dysfunctions that often manifest as sensorimotor, learning or memory disabilities throughout development and into maturity. Erythropoietin (Epo) has been shown to exert neuroprotective effects in different models of brain injury including experimental models of perinatal asphyxia. However, the effect of Epo on functional abilities following cerebral hypoxia-ischemia (HI) in neonatal rats is not known. The aim of the present study is to investigate the effect of Epo on sensorimotor deficits and brain injury induced by hypoxia-ischemia. Seven-day-old rats underwent unilateral, permanent carotid artery ligation followed by 1 h of hypoxia. Epo was administered as a single dose immediately after the hypoxic insult (2000 U/kg). The neuroprotective effect of Epo was evaluated at postnatal day 42 by using a battery of behavioral tests and histological analysis. The results of the present study suggest that Epo treatment immediately after HI insult significantly facilitated recovery of sensorimotor function. Consistently, histopathological evaluation demonstrated that Epo significantly attenuated brain injury and preserved the integrity of cerebral cortex. These findings indicate that long-term neuroprotective effect of Epo on neonatal HI-induced brain injury might be associated with the preservation of sensorimotor functions.

title: Beyond erythropoiesis: novel applications for recombinant human erythropoietin.
abstract: Erythropoietin (EPO) primarily is produced in the kidney and acts as a principal mediator of the physiologic response to hypoxia by increasing red blood cell production. Astrocytes and neurons in the central nervous system (CNS) also are known to produce EPO in response to hypoxia/ischemia. EPO appears to play a neuroprotective role based on preclinical data demonstrating the ability of recombinant human erythropoietin (r-HuEPO) to shield neurons from hypoxic/ischemic stress when administered intracerebraventricularly. In CNS models, systemically administered r-HuEPO has not been intensely investigated because large glycosylated molecules generally were deemed incapable of crossing the blood-brain barrier (BBB). A collaborative research effort identified expression of EPO receptors on human brain capillaries and a specific receptor-mediated transport of r-HuEPO across the BBB after a single intraperitoneal (IP) injection in rodents, with subsequent protection against various types of neuronal damage. For example, administration of r-HuEPO 24 hours before or up to 6 hours after focal ischemic stroke significantly reduced the extent of infarction. r-HuEPO also attenuated concussive brain injury, kainate-induced seizure activity, and autoimmune encephalomyelitis. These preclinical findings suggest that r-HuEPO may have therapeutic potential for stroke, head trauma, and epilepsy; additional studies are needed to confirm and extend these encouraging observations in animal models.

title: Erythropoietin: Multiple Physiological Functions and Regulation of Biosynthesis
abstract: Erythropoietin (Epo), which is produced by the kidney in the adult and by the liver in the fetus, increases red blood cells by supporting the survival of erythroid progenitor cells and stimulating their differentiation and proliferation via binding to Epo receptor (EpoR). The main signal in the control of Epo production is oxygen; hypoxia stimulates Epo production through activation of Epo gene transcription. Tremendous progress in our understanding of molecular mechanisms of Epo action on erythroid cells and regulation of the Epo production has been made by manipulation of cDNAs and genes of Epo and EpoR. Studies on hypoxic induction of Epo gene transcription led to the identification of hypoxia-inducible factor (HIF-1), a transcriptional factor, that functions as a global regulator of hypoxic gene expression. Paracrine Epo/EpoR systems that are independent of the endocrine erythropoietic system (kidney/bone marrow) have been found in the central nervous system and uterus. Novel functions of Epo at these local sites and tissue-specific regulation of Epo production including a newly found potent regulator (estrogen) have been proposed. The tissue-specific regulation rationalizes the specific functions of Epo produced by individual tissues.

title: Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury.
abstract: Erythropoietin (EPO), recognized for its central role in erythropoiesis, also mediates neuroprotection when the recombinant form (r-Hu-EPO) is directly injected into ischemic rodent brain. We observed abundant expression of the EPO receptor at brain capillaries, which could provide a route for circulating EPO to enter the brain. In confirmation of this hypothesis, systemic administration of r-Hu-EPO before or up to 6 h after focal brain ischemia reduced injury by approximately 50-75%. R-Hu-EPO also ameliorates the extent of concussive brain injury, the immune damage in experimental autoimmune encephalomyelitis, and the toxicity of kainate. Given r-Hu-EPO's excellent safety profile, clinical trials evaluating systemically administered r-Hu-EPO as a general neuroprotective treatment are warranted.

title: Therapeutic window for nonerythropoietic carbamylated-erythropoietin to improve motor function following multiple infarct ischemic strokes in New Zealand white rabbits
abstract: Carbamylated erythropoietin (CEPO) is a novel neuroprotective agent that does not bind to the classical erythropoietin receptor or affect hematocrit. Since CEPO has not been systematically studied in a fully blinded and randomized manner in an embolic stroke model, we determined if CEPO would be useful to attenuate clinical deficits associated with multiple infarct ischemia using the rabbit small clot embolic stroke model (RSCEM). Rabbits were embolized and treated with vehicle or CEPO within 6 h of embolization and behavioral analysis was conducted 48 h after embolization. Using quantal analysis, we determined the quantity of blood clot (mg) in brain that produce neurologic dysfunction in 50% of the rabbits (P(50)), with intervention considered beneficial if it increased the P(50) compared to controls. CEPO administered between 5 min and 3 h after embolization significantly (p<0.05) improved behavioral function and increased the P(50) value by 55-216%. However, CEPO administration did not improve behavior when administered 6 h following embolization. In conclusion, in the RSCEM, CEPO had a therapeutic window of at least 3 h, where it effectively improved clinical rating scores and motor function. Our results suggest that CEPO may be useful to treat acute ischemic stroke and supports the study of CEPO in stroke patients.

title: Inhibitory effect on cerebral inflammatory agents that accompany traumatic brain injury in a rat model: A potential neuroprotective mechanism of recombinant human erythropoietin (rhEPO)
abstract: Erythropoietin (EPO) has recently been shown to have a neuroprotective effect in animal models of traumatic brain injury (TBI). However, the precise mechanisms remain unclear. Cerebral inflammation plays an important role in the pathogenesis of secondary brain injury after TBI. We, therefore, tried to analyze how recombinant human erythropoietin (rhEPO) might effect the inflammation-related factors common to TBI: nuclear factor kappa B (NF-kappaB), interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1) in a rat TBI model. Male rats were given 0 or 5000 units/kg injections of rhEPO 1h post-injury and on days 1, 2 and 3 after surgery. Brain samples were extracted at 3 days after trauma. We measured NF-kappaB by electrophoretic mobility shift assay (EMSA); IL-1beta, TNF-alpha and IL-6 by enzyme-linked immunosorbent assay (ELISA); ICAM-1 by immunohistochemistry; brain edema by wet/dry method; blood-brain barrier (BBB) permeability by Evans blue extravasation and cortical apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. We found that NF-kappaB, pro-inflammatory cytokines and ICAM-1 were increased in all injured animals. In animals given rhEPO post-TBI, NF-kappaB, IL-1beta, TNF-alpha and ICAM-1 were decreased in comparison to vehicle-treated animals. Measures of IL-6 showed no change after rhEPO treatment. Administration of rhEPO reduced brain edema, BBB permeability and apoptotic cells in the injured brain. In conclusion, post-TBI rhEPO administration may attenuate inflammatory response in the injured rat brain, and this may be one mechanism by which rhEPO improves outcome following TBI.

title: Neuroprotection by erythropoietin administration after experimental traumatic brain injury
abstract: A large body of evidence indicates that the hormone erythropoietin (EPO) exerts beneficial effects in the central nervous system (CNS). To date, EPO's effect has been assessed in several experimental models of brain and spinal cord injury. This study was conducted to validate whether treatment with recombinant human EPO (rHuEPO) would limit the extent of injury following experimental TBI. Experimental TBI was induced in rats by a cryogenic injury model. rHuEPO or placebo was injected intraperitoneally immediately after the injury and then every 8 h until 2 or 14 days. Forty-eight hours after injury brain water content, an indicator of brain edema, was measured with the wet-dry method and blood-brain barrier (BBB) breakdown was evaluated by assay of Evans blue extravasation. Furthermore, extent of cerebral damage was assessed. Administration of rHuEPO markedly improved recovery from motor dysfunction compared with placebo group (P<0.05). Brain edema was significantly reduced in the cortex of the EPO-treated group relative to that in the placebo-treated group (80.6+/-0.3% versus 91.8%+/-0.8% respectively, P<0.05). BBB breakdown was significantly lower in EPO-treated group than in the placebo-treated group (66.2+/-18.7 mug/g versus 181.3+/-21 mug/g, respectively, P<0.05). EPO treatment reduced injury volume significantly compared with placebo group (17.4+/-5.4 mm3 versus 37.1+/-5.3 mm3, P<0.05). EPO, administered in its recombinant form, affords significant neuroprotection in experimental TBI model and may hold promise for future clinical applications.

title: Erythropoietin Protects from Post-Traumatic Edema in the Rat Brain
abstract: Erythropoietin (Epo) is gaining interest in various neurological insults as a possible neuroprotective agent. We determined the effects of recombinant human Epo (rhEpo, 5000 IU per kg bw) on brain edema induced in rats by traumatic brain injury (TBI; impact-acceleration model; rhEpo administration 30 mins after injury). Magnetic resonance imaging (MRI) and a gravimetric technique were applied. In the MRI experiments, the apparent diffusion coefficient (ADC) and the tissue T1 relaxation time were measured hourly in the neocortex and caudoputamen, during a 6 h time span after TBI. In the gravimetric experiments, brain water content (BWC) was determined in these two regions, 6 h after TBI. Apparent diffusion coefficient measurements showed that rhEpo decreased brain edema early and durably. Gravimetric measurements showed that rhEpo decreased BWC at H6 in the neocortex as well as in the caudoputamen. No significant differences in ADC, in T1, or in BWC were found between rhEpo treated-TBI rats and sham-operated rats. Our findings show that post-traumatic administration of rhEpo can significantly reduce the development of brain edema in a model of diffuse TBI. Further studies should be conducted to identify the biochemical mechanisms involved in these immediate effects and to assess the use of rhEpo as a possible therapy for post-traumatic brain edema.

title: Global brain atrophy after unilateral parietal lesion and its prevention by erythropoietin.
abstract: In humans, neurotrauma is suspected to cause brain atrophy and accelerate slowly progressive neurodegenerative disorders, such as Alzheimer's disease or schizophrenia. However, a direct link between brain injury and subsequent delayed global neurodegeneration has remained elusive. Here we show that juvenile (4-week-old) mice that are given a discrete unilateral lesion of the parietal cortex, develop to adulthood without obvious clinical symptoms. However, when monitored 3 and 9 months after lesioning, using high-resolution three-dimensional MRI and behavioural testing, the same mice display global neurodegenerative changes. Surprisingly, erythropoietin, a haematopoietic growth factor with potent neuroprotective activity, prevents behavioural abnormalities, cognitive dysfunction and brain atrophy when given for 2 weeks after acute brain injury. This demonstrates that a localized brain lesion is a primary cause of delayed global neurodegeneration that can be efficiently counteracted by neuroprotection.

title: Erythropoietin exerts an anti-inflammatory effect on the CNS in a model of experimental autoimmune encephalomyelitis
abstract: In recent work we reported that systemically administered erythropoietin (EPO) crosses the blood-brain barrier and has protective effects in animal models of cerebral ischemia, brain trauma and in a rat model of experimental autoimmune encephalomyelitis (EAE). Here we characterize the effect of systemic EPO on the inflammatory component of actively induced, acute EAE in Lewis rats. Administration of EPO at doses of 500-5000 U/kg bw i.p., daily from day 3 after immunization with myelin basic protein (MBP), delayed the onset of EAE and decreased its clinical score at peak time (days 12-13). Immunohistochemical analysis of the spinal cord using anti-glial fibrillary acidic protein (GFAP) and anti-CD11b antibodies showed that EPO markedly diminished inflammation and glial activation/proliferation. EAE induced significant levels of TNF and IL-6 in the spinal cord, where IL-6 was maximum at the onset of the disease (day 10) and TNF at its peak (day 12). EPO delayed the increase of TNF levels, without altering their peak levels, and markedly reduced those of IL-6 suggesting that the decreased inflammation and clinical score may be in part upon attenuation of IL-6. On the other hand, EPO was without effect in a model of adjuvant-induced arthritis in Lewis rats, suggesting a specificity towards autoimmune demyelinating diseases. These data suggest that EPO might act as a protective cytokine in inflammatory pathologies of the CNS.

title: Beneficial effect of erythropoietin on experimental allergic encephalomyelitis
abstract: We have known for a long time that erythropoietin signaling plays a key role in bone marrow erythrocyte proliferation. However, recent studies have indicated that erythropoietin also may have protective effects on the nervous system. This unexpected role remains incompletely characterized. To investigate the potential neuroprotective role of erythropoietin in the central nervous system, we assessed its effects on a well‐characterized autoimmune demyelinating model of multiple sclerosis–myelin oligodendrocyte glycoprotein‐induced experimental autoimmune encephalomyelitis (EAE) in the mouse. We found that erythropoietin administered intravenously for 14 days after the onset of symptoms reduced both disease severity and duration of maximum impairment at dose levels as low as 50U/kg (p < 0.001). We assessed the neuropathology of diseased spinal cords and found that erythropoietin‐treated EAE animals had reduced axonal damage, inflammatory cell infiltration and demyelination, and diminished blood–brain barrier leakage when compared with saline‐treated EAE controls. Moreover, the pronounced upregulation of spinal cord major histocompatibility complex (MHC) class II expression found in saline‐treated EAE was significantly reduced in erythropoietin‐treated animals, a finding we replicated in vitro, using microglial cultures. The notion that short‐term erythropoietin therapy might be of clinical benefit in human autoimmune demyelinating diseases needs investigation.

title: Neuroprotective effects and intracellular signaling pathways of erythropoietin in a rat model of multiple sclerosis
abstract: AbstractIn 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.

title: Delayed administration of erythropoietin and its non-erythropoietic derivatives ameliorates chronic murine autoimmune encephalomyelitis
abstract: Erythropoietin (EPO) mediates a wide range of neuroprotective activities, including amelioration of disease and neuroinflammation in rat models of EAE. However, optimum dosing parameters are currently unknown. In the present study, we used a chronic EAE model induced in mice by immunization with the myelin oligodendrocyte glycoprotein peptide (MOG35-55) to compare the effect of EPO given with different treatment schedules. EPO was administered intraperitoneally at 0.5, 5.0 or 50 microg/kg three times weekly starting from day 3 after immunization (preventive schedule), at the onset of clinical disease (therapeutic schedule) or 15 days after the onset of symptoms (late therapeutic schedule). The results show that EPO is effective even when given after the appearance of clinical signs of EAE, but with a reduced efficacy compared to the preventative schedule. To determine whether this effect requires the homodimeric EPO receptor (EPOR2)-mediated hematopoietic effect of EPO, we studied the effect of carbamylated EPO (CEPO) that does not bind EPOR2. CEPO, ameliorated EAE without changing the hemoglobin concentration. Another non-erythropoietic derivative, asialoEPO was also effective. Both EPO and CEPO equivalently decreased the EAE-associated production of TNF-alpha, IL-1beta and IL-1Ra in the spinal cord, and IFN-gamma by peripheral lymphocytes, indicating that their action involves targeting neuroinflammation. The lowest dosage tested appeared fully effective. The possibility to dissociate the anti-neuroinflammatory action of EPO from its hematopoietic action, which may cause undesired side effects in non-anemic patients, present new avenues to the therapy of multiple sclerosis.

title: Erythropoietin treatment improves neurological functional recovery in EAE mice
abstract: Erythropoietin (EPO), originally recognized for its central role in erythropoiesis, has been shown to improve neurological outcome after stroke. Here, we investigated the treatment of experimental autoimmune encephalomyelitis (EAE) in mice with EPO. Mice were treated with recombinant human EPO (rhEPO) upon onset of paresis. Neurological functional tests were scored daily by grading of clinical signs (score 0-5). Hematoxylin and eosin (HE) staining of cerebral tissue was performed to detect inflammatory infiltrates. Double staining for Luxol fast blue and Bielshowsky was used to demonstrate myelin and axons, respectively. Immunohistochemistry was performed to measure the expression of bromodeoxyuridine (BrdU, a marker for cell proliferation), NG2 (a marker for oligodendrocyte progenitor cells) and brain-derived neurotrophic factor (BDNF). Treatment with rhEPO significantly improved neurological functional recovery, reduced inflammatory infiltrates and demyelination, and increased oligodendrocyte progenitor cell proliferation and BDNF+ cells compared to the EAE controls. These data indicate that rhEPO treatment improved functional recovery after EAE in mice, possibly, via stimulating oligodendrogenesis, downregulating proinflammatory infiltrates and by elevating BDNF expression.

title: Recombinant human erythropoietin prevents the death of mice during cerebral malaria.
abstract: Cerebral involvement during malaria is a complication that leads to seizure, coma, and death. The effect of new neuroprotective therapies has not yet been investigated, although cerebral malaria shares some features with neurological stroke. Erythropoietin (EPO) is one of the more promising drugs in this area. We measured the effect of EPO on the survival of mice infected with Plasmodium berghei ANKA and demonstrated that inoculations of recombinant human EPO at the beginning of the clinical manifestations of cerebral malaria protect >90% of mice from death. This drug has no effect on the course of parasitemia. The effect of EPO was not related to either the inhibition of apoptosis in the brain or the regulation of the increase and decrease of nitric oxide production in the brain and blood, respectively. Tumor necrosis factor-alpha and interferon-gamma mRNA overexpression was inhibited by EPO, and treated mice had fewer brain hemorrhages. EPO has been used in patients with chronic diseases for years, and more recently it has been used to treat acute ischemic stroke. The data presented here provide the first evidence indicating that this cytokine could be useful for the symptomatic prevention of mortality during the acute stage of cerebral malaria.

title: Recombinant human erythropoietin increases survival and reduces neuronal apoptosis in a murine model of cerebral malaria
abstract: BackgroundCerebral malaria (CM) is an acute encephalopathy with increased pro-inflammatory cytokines, sequestration of parasitized erythrocytes and localized ischaemia. In children CM induces cognitive impairment in about 10% of the survivors. Erythropoietin (Epo) has – besides of its well known haematopoietic properties – significant anti-inflammatory, antioxidant and anti-apoptotic effects in various brain disorders. The neurobiological responses to exogenously injected Epo during murine CM were examined.MethodsFemale C57BL/6j mice (4–6 weeks), infected with Plasmodium berghei ANKA, were treated with recombinant human Epo (rhEpo; 50–5000 U/kg/OD, i.p.) at different time points. The effect on survival was measured. Brain pathology was investigated by TUNEL (Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-digoxigenin nick end labelling), as a marker of apoptosis. Gene expression in brain tissue was measured by real time PCR.ResultsTreatment with rhEpo increased survival in mice with CM in a dose- and time-dependent manner and reduced apoptotic cell death of neurons as well as the expression of pro-inflammatory cytokines in the brain. This neuroprotective effect appeared to be independent of the haematopoietic effect.ConclusionThese results and its excellent safety profile in humans makes rhEpo a potential candidate for adjunct treatment of CM.

title: Carbamylated Erythropoietin Reduces Radiosurgically-Induced Brain Injury
abstract: Gamma knife radiosurgery is an attractive noninvasive treatment of brain tumors and vascular malformations that minimizes collateral tissue damage. However, exposure of normal tissue to even low-dose radiation triggers a cascade of acute and chronic injury and potentially significant morbidity and mortality. Because many irradiated patients now survive for years, identifying methods to prevent radiotherapy-induced collateral tissue damage is a major focus of current research. Erythropoietin (EPO), a cytokine produced locally by many tissues in response to injury, antagonizes apoptosis, reduces inflammation, and promotes healing. Systemic administration of recombinant EPO, widely used for treatment of anemia, provides robust protection from numerous insults in a variety of tissues, including the brain. Although irradiation injury is likely sensitive to EPO, the hematopoietic activity of EPO is undesirable in this setting, increasing erythrocyte number and predisposing to thrombosis. To avoid these potential adverse effects, we developed carbamylated EPO (CEPO) which does not stimulate the bone marrow. In this study, we show that CEPO (50 µg kg−1 intraperitoneally) improves functional outcome when administered to adult rats just before, and then once daily for 10 d after, a necrotizing dose of radiation (100 Gy) to the right striatum. Immediately following irradiation, use and reflex movements of the contralateral forelimb to vibrissae stimulation were abnormal but rapidly improved in animals receiving CEPO. Moreover, histological examination revealed that the extent of brain necrosis after 90 days was reduced by ~ 50%. These findings further extend the kinds of injury for which administration of a tissue-protective cytokine provides benefit.

title: Neuroprotective potential of erythropoietin and darbepoetin alfa in an experimental model of sciatic nerve injury. Laboratory investigation.
abstract: OBJECT
The objectives of this study were to examine whether the systemic administration of recombinant human erythropoietin (rHuEPO) and its long-lasting derivative darbepoetin alfa expedited functional recovery in a rat model of sciatic nerve injury, and to compare the effects of these agents in the model.



Thirty male Sprague-Dawley rats received a crush injury to the left sciatic nerve and subsequently underwent either placebo treatment, daily injections of rHuEPO, or weekly injections of darbepoetin alfa.



Both rHuEPO and darbepoetin alfa were effective in reducing neurological impairment and improving compound muscle action potentials following nerve injury. Darbepoetin alfa, however, shortened the duration of peripheral nerve recovery'and facilitated recovery from the neurological and electrophysiological impairment following crush injury significantly better than rHuEPO. Examination of the footprint length factor data revealed that darbepoetin alfa-treated animals recovered preinjury function by postoperative Day 10, 4 days earlier than animals treated with rHuEPO and 11 days earlier than animals treated with placebo.



These results suggest that recovery of neurological function in a model of peripheral nerve injury is more rapid with weekly administration of darbepoetin alfa than with daily rHuEPO treatment. Agents that facilitate nerve regeneration have the potential to limit the extent of motor endplate loss and muscle atrophy. The administration of EPO in its long-lasting recombinant forms affords significant neuroprotection in peripheral nerve injury models and may hold promise for future clinical applications.

title: Erythropoietin reduces Schwann cell TNF‐α, Wallerian degeneration and pain‐related behaviors after peripheral nerve injury
abstract: Chronic sciatic nerve constriction injury (CCI) induces Wallerian degeneration and exaggerated pain‐like behaviors. These effects are mediated in large part by pro‐inflammatory cytokines, such as tumor necrosis factor alpha (TNF‐α). In this study, we demonstrate that systemically administered recombinant human erythropoietin (rhEpo) facilitates recovery from chronic neuropathic pain associated with CCI in rats. Because TNF‐α has been implicated in the development of pain‐related behaviors, we measured TNF‐α mRNA at the nerve injury site. Systemically or locally administered rhEpo decreased TNF‐α mRNA, compared with that observed in untreated animals. RhEpo also significantly (P < 0.05) decreased axonal degeneration. Immunohistochemistry of CCI nerve showed abundant TNF‐α in Schwann cells, axoplasm and macrophages. In rhEpo‐treated animals, TNF‐α immunopositivity was decreased selectively in Schwann cells. These results suggest a model in which rhEpo counteracts the effects of TNF‐α in CCI by blocking expression of TNF‐α in Schwann cells. To further test this model, we studied primary Schwann cell cultures. RhEpo inhibited TNF‐α expression in response to lipopolysaccharide, supporting the conclusions of our in vivo CCI experiments. In addition, rhEpo directly counteracted Schwann cell death induced by exogenously added TNF‐αin vitro. These results indicated that rhEpo regulates TNF‐α by multiple mechanisms; rhEpo regulates TNF‐α mRNA expression by Schwann cells but also may directly counteract TNF‐α signaling pathways that lead to injury, chronic pain and/or death.

title: A novel endogenous erythropoietin mediated pathway prevents axonal degeneration
abstract: Clinically relevant peripheral neuropathies (such as diabetic and human immunodeficiency virus sensory neuropathies) are characterized by distal axonal degeneration, rather than neuronal death. Here, we describe a novel, endogenous pathway that prevents axonal degeneration. We show that in response to axonal injury, periaxonal Schwann cells release erythropoietin (EPO), which via EPO receptor binding on neurons, prevents axonal degeneration. We demonstrate that the relevant axonal injury signal that stimulates EPO production from surrounding glial cells is nitric oxide. In addition, we show that this endogenous pathway can be therapeutically exploited by administering exogenous EPO. In an animal model of distal axonopathy, systemic EPO administration prevents axonal degeneration, and this is associated with a reduction in limb weakness and neuropathic pain behavior. Our in vivo and in vitro data suggest that EPO prevents axonal degeneration and therefore may be therapeutically useful in a wide variety of human neurological diseases characterized by axonopathy.

title: Erythropoietin is neuroprotective in models of HIV sensory neuropathy
abstract: HIV-associated sensory neuropathy (HIV-SN) is the most common neurological complication of HIV infection. Presently, there are no effective therapies for this painful neuropathy. The pathology of HIV-SN is characterized by 'dying back' sensory axonal degeneration and a more modest loss of dorsal root ganglion (DRG) sensory neurons. It has been hypothesized that HIV-SN results from neurotoxicity by secreted viral proteins, such as the HIV envelope glycoprotein gp120. Furthermore, neurotoxicity by dideoxynucleoside (DDX) agents, results in the observed higher incidence of HIV-SN in HIV-infected patients taking these antiretroviral drugs. In this study we show that administration of picomolar amounts of the hormone erythropoietin (EPO) prevents sensory axonal degeneration and in vitro DRG neuronal death by both gp120 and ddC (a neurotoxic DDX drug). Our results suggest that EPO may be useful in the treatment of HIV-SN.

title: Erythropoietin and its carbamylated derivative prevent the development of experimental diabetic autonomic neuropathy in STZ-induced diabetic NOD-SCID mice
abstract: Autonomic neuropathy is a significant diabetic complication resulting in increased morbidity and mortality. Studies of autopsied diabetic patients and several rodent models demonstrate that the neuropathologic hallmark of diabetic sympathetic autonomic neuropathy in prevertebral ganglia is the occurrence of synaptic pathology resulting in distinctive dystrophic neurites ("neuritic dystrophy"). Our prior studies show that neuritic dystrophy is reversed by exogenous IGF-I administration without altering the metabolic severity of diabetes, i.e. functioning as a neurotrophic substance. The description of erythropoietin (EPO) synergy with IGF-I function and the recent discovery of EPO's multifaceted neuroprotective role suggested it might substitute for IGF-I in treatment of diabetic autonomic neuropathy. Our current studies demonstrate EPO receptor (EPO-R) mRNA in a cDNA set prepared from NGF-maintained rat sympathetic neuron cultures which decreased with NGF deprivation, a result which demonstrates clearly that sympathetic neurons express EPO-R, a result confirmed by immunohistochemistry. Treatment of STZ-diabetic NOD-SCID mice have demonstrated a dramatic preventative effect of EPO and carbamylated EPO (CEPO, which is neuroprotective but not hematopoietic) on the development of neuritic dystrophy. Neither EPO nor CEPO had a demonstrable effect on the metabolic severity of diabetes. Our results coupled with reported salutary effects of EPO on postural hypotension in a few clinical studies of EPO-treated anemic diabetic and non-diabetic patients may reflect a primary neurotrophic effect of EPO on the sympathetic autonomic nervous system, rather than a primary hematopoietic effect. These findings may represent a major clinical advance since EPO has been widely and safely used in anemic patients due to a variety of clinical conditions.

title: HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration
abstract: Erythropoietin (Epo) is upregulated by hypoxia and provides protection against apoptosis of erythroid progenitors in bone marrow and brain neurons. Here we show in the adult mouse retina that acute hypoxia dose-dependently stimulates expression of Epo, fibroblast growth factor 2 and vascular endothelial growth factor via hypoxia-inducible factor-1α (HIF-1α) stabilization. Hypoxic preconditioning protects retinal morphology and function against light-induced apoptosis by interfering with caspase-1 activation, a downstream event in the intracellular death cascade. In contrast, induction of activator protein-1, an early event in the light-stressed retina, is not affected by hypoxia. The Epo receptor required for Epo signaling localizes to photoreceptor cells. The protective effect of hypoxic preconditioning is mimicked by systemically applied Epo that crosses the blood–retina barrier and prevents apoptosis even when given therapeutically after light insult. Application of Epo may, through the inhibition of apoptosis, be beneficial for the treatment of different forms of retinal disease.

title: Intravitreal injection of erythropoietin protects both retinal vascular and neuronal cells in early diabetes.
abstract: To explore and evaluate the protective effect of erythropoietin (EPO) on retinal cells of chemically induced diabetic rats after EPO was injected intravitreally at the onset of diabetes.



Diabetes was induced in Sprague-Dawley rats by intraperitoneal injection of streptozotocin (STZ). At the onset of diabetes, a single intravitreal injection of EPO (0.05-200 ng/eye) was performed. In the following 6 weeks, the blood retinal barrier (BRB) was evaluated by Evans blue permeation (EBP). Retinal cell death in different layers was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The retinal thickness and cell counts were examined at the light microscopic level. Electron microscopy (EM) was used to scrutinize retinal vascular and neuronal injury. Neurosensory retinas of normal and diabetic rats were used as the sources of reverse transcription-polymerase chain reaction (RT-PCR) and Western blot for the detection of EPO, EPO receptor (EpoR), and products of the extracellular signal-regulated kinase (ERK) and the signal transducers and activators of transcription 5 (STAT5) pathways. The distribution of EpoR in retinal layers was demonstrated by immunohistochemistry (IHC).



In the diabetic rats, BRB breakdown was detected soon after the onset of diabetes, peaked at 2 weeks, and reached a plateau at 2 to 4 weeks. The number of TUNEL-positive cells increased in the neurosensory retina, especially, the outer nuclear layer (ONL) at 1 week after diabetes onset and reached a peak at 4 to 6 weeks. The retinal thickness and the number of cells in the ONL were reduced significantly. EM observations demonstrated vascular and photoreceptor cell death starting soon after the onset of diabetes. All these changes were largely prevented by EPO treatment. Upregulation of EpoR in the neurosensory retina was detected at both the transcriptional and protein levels 4 to 8 weeks after the onset of diabetes, whereas, the endogenous EPO levels of neurosensory retinas were essentially unchanged during the same period observed. In EPO-treated diabetic groups, EpoR expression remained at upregulated levels. Within 2 weeks of the onset of diabetes, activation of the ERK but not the STAT5 pathway was detected in the diabetic retina treated with EPO.



These data demonstrate that apoptosis is an major contributor to neuronal cell death in the early course of diabetic retinopathy (DR). The upregulation of EpoR may be a compensatory response of retinal cells and tissue to diabetic stresses. The EPO/EpoR system as a maintenance-survival mechanism of retinal neurons responds to the insults of early diabetes other than ischemia. The protective function of EPO/EpoR at the least acts through the EpoR-mediated ERK pathway. Exogenous EPO administration by intravitreal injection in early diabetes may prevent retinal cell death and protect the BRB function. Therefore, this is a novel approach for treatment of early DR.

title: [Erythropoietin as a retinal angiogenic factor in proliferative diabetic retinopathy].
abstract: Although retinal neovascularization in proliferative diabetic retinopathy(PDR) is a major cause of legal blindness, its mechanism is not fully understood. In this study, we focused on the angiogenic activity of erythropoietin (Epo) and evaluated its potential role in treating retinal angiogenesis in PDR.



We measured Epo and vascular endothelial growth factor (VEGF) levels in the vitreous fluids of 73 PDR patients and 71 nondiabetic (NDM) patients. We evaluated Epo expression and regulation in retinal neovascularization with soluble Epo receptor.



The vitreous Epo level was significantly higher in patients with PDR than in NDM patients. Multivariate logistic-regression analyses indicated that Epo and VEGF were independently associated with PDR and that Epo was more strongly associated with PDR than VEGF was. The blockade of Epo inhibited retinal neovascularization in vitro and in vivo.



Our data suggest that Epo is a potent angiogenic factor that acts independently of VEGF during retinal angiogenesis in PDR.

title: Erythropoietin requires NF-kappaB and its nuclear translocation to prevent early and late apoptotic neuronal injury during beta-amyloid toxicity.
abstract: 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.

title: Erythropoietin restores glutathione peroxidase activity in 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine-induced neurotoxicity in C57BL mice and stimulates murine astroglial glutathione peroxidase production in vitro
abstract: Recently, we have reported that erythropoietin (Epo) provides neuroprotection in 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP)-induced neurotoxicity in vivo. In the present study, we investigated the effects of single Epo administration on brain antioxidant enzyme (superoxide dismutase (SOD) and glutathion peroxidase (GSHPx)) activities in this model in C57BL/6 mice. We found that MPTP treatment decreased GSHPx activity in both substantia nigra and striatum, and Epo restores nigral GSHPx activity decreased by MPTP. SOD enzyme activity was not significantly changed by MPTP and Epo treatment. Further, Epo stimulated astroglial GSHPx production in neonatal murine astroglial cell culture suggesting that the possible cell source for the stimulation of GSHPx activity by Epo in the MPTP-induced neurotoxicity model are astroglia. In conclusion, modulation of the astroglial antioxidant defense system might be one of the mechanisms by which Epo exerts a beneficial effect in MPTP-induced Parkinsonism.

title: Erythropoietin exerts neuroprotection in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated C57/BL mice via increasing nitric oxide production
abstract: Erythropoietin (EPO), produced by the kidney and fetal liver, is a cytokine-hormone that stimulates erythropoiesis under hypoxic conditions. It has been shown that EPO is produced in the central nervous system and its receptor is expressed on neurons. Since EPO has neuroprotective effects in vitro and in vivo against brain injury, we investigated the effect of EPO treatment on locomotor activities of animals, survival of nigral dopaminergic neurons and nitrate levels in substantia nigra and striatum in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced animal model of Parkinsonism in C57/BL mice. Our findings suggest that EPO has protective and treating effect in MPTP-induced neurotoxicity in this mouse model of Parkinson's Disease via increasing nitric oxide production.

title: Erythropoietin delays disease onset in an amyotrophic lateral sclerosis model
abstract: Erythropoietin (Epo) has been shown in the recent years to have neuroprotective activity in a variety of settings. In this study, we investigated its impact on the progression of paralysis in a mouse model simulating the human disorder amyotrophic lateral sclerosis (ALS). We found that Epo can delay the onset of motor deterioration in transgenic SOD G93A mice without prolonging their survival. Notably this effect was selective for the females only. These initial findings encourage further investigation of this biological avenue in the search for improved remedies for this fatal disease.

title: Nonhematopoietic Erythropoietin Derivatives Prevent Motoneuron Degeneration In Vitro and In Vivo
abstract: Chronic treatment with asialo erythropoietin (ASIALO-EPO) or carbamylated erythropoietin (CEPO) improved motor behavior and reduced motoneuron loss and astrocyte and microglia activation in the cervical spinal cord of wobbler mice, an animal model of amyotrophic lateral sclerosis, but had no effect on hematocrit values. ASIALO-EPO and CEPO, like the parent compound EPO, protected primary motoneuron cultures from kainate-induced death in vitro. Both EPO receptor and the common CD131 β chain were expressed in cultured motoneurons and in the anterior horn of wobbler mice spinal cord. Our results strongly support a role for the common β chain CD131 in the protective effect of EPO derivatives on motoneuron degeneration. Thus CEPO, which does not bind to the classical homodimeric EPO receptor and is devoid of hematopoietic activity, could be effective in chronic treatment aimed at reducing motoneuron degeneration.

title: Exogenous erythropoietin provides neuroprotection of grafted dopamine neurons in a rodent model of Parkinson's disease
abstract: Parkinson's disease (PD) is a neurodegenerative disease marked by severe loss of dopamine (DA) neurons in the nigrostriatal system, which results in depletion of striatal DA. Transplantation of embryonic ventral mesencephalic (VM) DA neurons into the striatum is a currently explored experimental treatment aimed at replacing lost DA in the nigrostriatal system, but is plagued with poor survival (5-20%) of implanted neurons. Here, we tested the ability of erythropoietin (Epo) to provide neuroprotection for embryonic day 14 (E14) VM DA neurons. Epo was tested in vitro for the ability to augment tyrosine hydroxylase-immunoreactive (TH-ir) neuron survival under normal cell culture conditions. In vitro, Epo did not increase the number of TH-ir neurons when administered at the time of plating the E14 VM cells in culture. We also tested the efficacy of Epo to enhance E14 VM transplants in vivo. Rats unilaterally lesioned with 6-hydroxydopamine received transplants that were incubated in Epo. Treatment with Epo produced significant increases in TH-ir neuron number, soma size, and staining intensity. Animals receiving Epo-treated grafts exhibited significantly accelerated functional improvements and significantly greater overall improvements from rotational asymmetry compared to control grafted rats. These data indicate that the survival of embryonic mesencephalic TH-ir neurons is increased when Epo is administered with grafted cells in a rodent model of PD. As direct neurotrophic effects of Epo were not observed in vitro, the mechanism of Epo neuroprotection remains to be elucidated.

title: Asialoerythropoetin is not effective in the R6/2 line of Huntington's disease mice
abstract: BackgroundHuntington's disease (HD) is a hereditary neurodegenerative disorder caused by an expanded CAG repeat in the HD gene. Both excitotoxicity and oxidative stress have been proposed to play important roles in the pathogenesis of HD. Since no effective treatment is available, this study was designed to explore the therapeutic potential of erythropoietin (EPO), a cytokine that has been found to prevent excitotoxicity, and to promote neurogenesis. To avoid the side effects of a raised hematocrit, we used asialoerythropoietin (asialoEPO), a neuroprotective variant of EPO that lacks erythropoietic effects in mice. R6/2 transgenic HD mice were treated with this cytokine from five to twelve weeks of age.ResultsWe provide new evidence that cell proliferation in the dentate gyrus of the R6/2 hippocampus is reduced by 50% compared to wild-type littermate controls. However, we found that the asialoEPO treatment did not affect the progression of motor symptoms, weight loss or the neuropathological changes. Furthermore, cell proliferation was not enhanced.ConclusionsWe conclude that the chosen protocol of asialoEPO treatment is ineffective in the R6/2 model of HD. We suggest that reduced hippocampal cell proliferation may be an important and novel neuropathological feature in R6 HD mice that could be assessed when evaluating potential therapies.

title: Recommendations for standards regarding preclinical neuroprotective and restorative drug development.
abstract: The plethora of failed clinical trials with neuroprotective drugs for acute ischemic stroke have raised justifiable concerns about how best to proceed for the future development of such interventions. Preclinical testing of neuroprotective drugs is an important aspect of assessing their therapeutic potential, but guidelines concerning how to perform preclinical development of purported neuroprotective drugs for acute ischemic stroke are lacking. This conference of academicians and industry representatives was convened to suggest such guidelines for the preclinical evaluation of neuroprotective drugs and to recommend to potential clinical investigators the data they should review to reassure themselves that a particular neuroprotective drug has a reasonable chance to succeed in an appropriately designed clinical trial. Without rigorous, robust, and detailed preclinical evaluation, it is unlikely that novel neuroprotective drugs will prove to be effective when tested in large, time-consuming, and expensive clinical trials. Additionally, similar recommendations are provided for drugs with the potential to enhance recovery after acute ischemic stroke, a burgeoning new field with great potential but little currently available data. The suggestions contained in this document are meant to serve as overall guidelines that must be adapted to the individual characteristics related to particular drugs and their preclinical and clinical development needs.

title: Exploring recombinant human erythropoietin in chronic progressive multiple sclerosis.
abstract: The neurodegenerative aspects of chronic progressive multiple sclerosis (MS) have received increasing attention in recent years, since anti-inflammatory and immunosuppressive treatment strategies have largely failed. However, successful neuroprotection and/or neuroregeneration in MS have not been demonstrated yet. Encouraged by the multifaceted neuroprotective effects of recombinant human erythropoietin (rhEPO) in experimental models, we performed an investigator-driven, exploratory open label study (phase I/IIa) in patients with chronic progressive MS. Main study objectives were (i) evaluating safety of long-term high-dose intravenous rhEPO treatment in MS, and (ii) collecting first evidence of potential efficacy on clinical outcome parameters. Eight MS patients, five randomly assigned to high-dose (48,000 IU), three to low-dose (8000 IU) rhEPO treatment, and, as disease controls, two drug-naïve Parkinson patients (receiving 48,000 IU) were followed over up to 48 weeks: A 6-week lead-in phase, a 12-week treatment phase with weekly EPO, another 12-week treatment phase with bi-weekly EPO, and a 24-week post-treatment phase. Clinical and electrophysiological improvement of motor function, reflected by a reduction in expanded disability status scale (EDSS), and of cognitive performance was found upon high-dose EPO treatment in MS patients, persisting for three to six months after cessation of EPO application. In contrast, low-dose EPO MS patients and drug-naïve Parkinson patients did not improve in any of the parameters tested. There were no adverse events, no safety concerns and a surprisingly low need of blood-lettings. This first pilot study demonstrates the necessity and feasibility of controlled trials using high-dose rhEPO in chronic progressive MS.

title: An Approach to Using Recombinant Erythropoietin for Neuroprotection in Very Preterm Infants
abstract: Erythropoietin has been shown to be protective against hypoxic-ischemic and inflammatory injuries in cell culture, animal models of brain injury, and clinical trials of adult humans. The rationale for our study was that early administration of high-dose recombinant human erythropoietin may reduce perinatal brain injury (intraventricular hemorrhage and periventricular leukomalacia) in very preterm infants and improve neurodevelopmental outcome. We investigated whether administration of high-dose recombinant human erythropoietin to very preterm infants shortly after birth and subsequently during the first 2 days is safe in terms of short-term outcome.  This was a randomized, double-masked, single-center trial with a 2:1 allocation in favor of recombinant human erythropoietin. Preterm infants (gestational age: 24 to 31 weeks) were given recombinant human erythropoietin or NaCl 0.9% intravenously 3, 12 to 18, and 36 to 42 hours after birth.  The percentage of infants who survived without brain injury or retinopathy was 53% in the recombinant human erythropoietin group and 60% in the placebo group. There were no relevant differences regarding short-term outcomes such as intraventricular hemorrhage, retinopathy, sepsis, necrotizing enterocolitis, and bronchopulmonary dysplasia. For 5 infants who were in the recombinant human erythropoietin group and had a gestational age of <26 weeks, withdrawal of intensive care was decided (3 of 5 with severe bilateral intraventricular hemorrhage, 2 of 5 with pulmonary insufficiency); no infant of the control group died. Recombinant human erythropoietin treatment did not result in significant differences in blood pressure, cerebral oxygenation, hemoglobin, leukocyte, and platelet count.  No significant adverse effects of early high-dose recombinant human erythropoietin treatment in very preterm infants were identified. These results enable us to embark on a large multicenter trial with the aim of determining whether early high-dose administration of recombinant human erythropoietin to very preterm infants improves neurodevelopmental outcome at 24 months' and 5 years' corrected age.

title: A Phase I/II Trial of High-Dose Erythropoietin in Extremely Low Birth Weight Infants: Pharmacokinetics and Safety
abstract: High-dose recombinant erythropoietin is neuroprotective in animal models of neonatal brain injury. Extremely low birth weight infants are at high risk for brain injury and neurodevelopmental problems and might benefit from recombinant erythropoietin. We designed a phase I/II trial to test the safety and determine the pharmacokinetics of high-dose recombinant erythropoietin in extremely low birth weight infants.  In a prospective, dose-escalation, open-label trial, we compared 30 infants who were treated with high-dose recombinant erythropoietin with 30 concurrent control subjects. Eligible infants were <24 hours old, ≤1000 g birth weight, and ≤28 weeks of gestation and had an umbilical artery catheter in place. Each infant received 3 intravenous doses of 500, 1000, or 2500 U/kg at 24-hour intervals beginning on day 1 of age. Blood samples were collected at scheduled intervals to determine recombinant erythropoietin pharmacokinetics. Safety parameters were also evaluated. In the concurrent control group, only clinical data were collected.  Mean erythropoietin concentrations 30 minutes after recombinant erythropoietin infusion were 5973 ± 266, 12291 ± 403, and 34197 ± 1641 mU/mL after 500, 1000, or 2500 U/kg, respectively. High-dose recombinant erythropoietin followed nonlinear pharmacokinetics as a result of decreasing clearance from the lowest dosage (17.3 mL/hour per kg for 500 U/kg) to the highest dosage (8.2 mL/hour per kg for 2500 U/kg). Steady state was achieved within 24 to 48 hours. Both 1000 and 2500 U/kg recombinant erythropoietin produced peak serum erythropoietin concentrations that were comparable to neuroprotective concentrations that previously were seen in experimental animals. No excess adverse events occurred in the recombinant erythropoietin–treated infants compared with control infants.  Early high-dose recombinant erythropoietin is well tolerated by extremely low birth weight infants, causing no excess morbidity or mortality. Recombinant erythropoietin dosages of 1000 and 2500 U/kg achieved neuroprotective serum levels.

title: Erythropoietin in patients with aneurysmal subarachnoid haemorrhage: a double blind randomised clinical trial
abstract: Erythropoietin (EPO) is neuroprotective in experimental models of stroke and subarachnoid haemorrhage (SAH) and possibly in patients with thromboembolic stroke. We studied the efficacy and safety of EPO in patients with SAH.
 Methods. A larger scale clinical trial was planned but preliminarily terminated because of a lower than expected inclusion rate. However, 73 patients were randomised to treatment with EPO (500 IU/kg/day for three days) or placebo. The primary endpoint was Glasgow Outcome Score at six months. We further studied surrogate measures of secondary ischaemia, i.e. transcranial Doppler (TCD) flow velocity, symptomatic vasospasm, cerebral metabolism (microdialysis) and jugular venous oximetry, biochemical markers of brain damage (S-100β and neuron specific enolase) and blood–brain barrier integrity.
 Findings. The limited sample size precluded our primary hypotheses being verified and refuted. However, data from this study are important for any other study of SAH and as much raw data as possible are presented and can be included in future meta analyses. On admission the proportion of patients in a poor condition was higher in the EPO group compared with the placebo group but the difference was statistically insignificant. In the EPO-treated patients the CSF concentration of EPO increased 600-fold. Except for a higher extracelullar concentration of glycerol in the EPO group probably caused by the poorer clinical condition of these patients, there were no statistically significant group differences in the primary or secondary outcome measures. EPO was well tolerated.
 Conclusions. Beneficial effects of EPO in patients with SAH cannot be excluded or concluded on the basis of this study and larger scale trials are warranted.

title: Friedreich's ataxia: clinical pilot trial with recombinant human erythropoietin
abstract: To determine the role of recombinant human erythropoietin as a possible treatment option in Friedreich's ataxia, we performed an open‐label clinical pilot study. Primary outcome measure was the change of frataxin levels at week 8 versus baseline. Twelve Friedreich's ataxia patients received 5,000 units recombinant human erythropoietin three times weekly subcutaneously. Frataxin levels were measured in isolated lymphocytes by enzyme‐linked immunosorbent assay. In addition, urinary 8‐hydroxydeoxyguanosine and serum peroxides, were measured. Treatment with recombinant human erythropoietin showed a persistent and significant increase in frataxin levels after 8 weeks (p < 0.01). All patients showed a reduction of oxidative stress markers.

title: Improvement of cognitive functions in chronic schizophrenic patients by recombinant human erythropoietin
abstract: Schizophrenia is increasingly recognized as a neurodevelopmental disease with an additional degenerative component, comprising cognitive decline and loss of cortical gray matter. We hypothesized that a neuroprotective/neurotrophic add-on strategy, recombinant human erythropoietin (rhEPO) in addition to stable antipsychotic medication, may be able to improve cognitive function even in chronic schizophrenic patients. Therefore, we designed a double-blind, placebo-controlled, randomized, multicenter, proof-of-principle (phase II) study. This study had a total duration of 2 years and an individual duration of 12 weeks with an additional safety visit at 16 weeks. Chronic schizophrenic men (N=39) with defined cognitive deficit (⩾1 s.d. below normal in the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS)), stable medication and disease state, were treated for 3 months with a weekly short (15 min) intravenous infusion of 40 000 IU rhEPO (N=20) or placebo (N=19). Main outcome measure was schizophrenia-relevant cognitive function at week 12. The neuropsychological test set (RBANS subtests delayed memory, language–semantic fluency, attention and Wisconsin Card Sorting Test (WCST-64) – perseverative errors) was applied over 2 days at baseline, 2 weeks, 4 weeks and 12 weeks of study participation. Both placebo and rhEPO patients improved in all evaluated categories. Patients receiving rhEPO showed a significant improvement over placebo patients in schizophrenia-related cognitive performance (RBANS subtests, WCST-64), but no effects on psychopathology or social functioning. Also, a significant decline in serum levels of S100B, a glial damage marker, occurred upon rhEPO. The fact that rhEPO is the first compound to exert a selective and lasting beneficial effect on cognition should encourage new treatment strategies for schizophrenia.

title: Erythropoietin enhances hippocampal long-term potentiation and memory
abstract: BackgroundErythropoietin (EPO) improves cognition of human subjects in the clinical setting by as yet unknown mechanisms. We developed a mouse model of robust cognitive improvement by EPO to obtain the first clues of how EPO influences cognition, and how it may act on hippocampal neurons to modulate plasticity.ResultsWe show here that a 3-week treatment of young mice with EPO enhances long-term potentiation (LTP), a cellular correlate of learning processes in the CA1 region of the hippocampus. This treatment concomitantly alters short-term synaptic plasticity and synaptic transmission, shifting the balance of excitatory and inhibitory activity. These effects are accompanied by an improvement of hippocampus dependent memory, persisting for 3 weeks after termination of EPO injections, and are independent of changes in hematocrit. Networks of EPO-treated primary hippocampal neurons develop lower overall spiking activity but enhanced bursting in discrete neuronal assemblies. At the level of developing single neurons, EPO treatment reduces the typical increase in excitatory synaptic transmission without changing the number of synaptic boutons, consistent with prolonged functional silencing of synapses.ConclusionWe conclude that EPO improves hippocampus dependent memory by modulating plasticity, synaptic connectivity and activity of memory-related neuronal networks. These mechanisms of action of EPO have to be further exploited for treating neuropsychiatric diseases.

title: UNRESOLVED ISSUES IN DIALYSIS: Managing Erythropoietin Hyporesponsiveness
abstract: The anemia of chronic kidney disease is associated with cardiovascular disease, decreased quality of life, and mortality. The introduction of recombinant human erythropoietin (rHuEPO) has transformed the management of this condition. However, a significant proportion of patients fail to respond to even high doses of rHuEPO. Several factors have been implicated in the hyporesponsiveness to rHuEPO. Iron deficiency, whether absolute or functional, is considered the most important, and maintenance of adequate iron stores reduces rHuEPO requirements among patients on hemodialysis. However, traditional indices of iron that are currently utilized may not reflect iron stores accurately, and there is also increasing concern regarding the potential long‐term toxicity of parenteral iron therapy. Infection and inflammation also influence the response to rHuEPO, both by disruption of iron metabolism and by eliciting the release of cytokines that inhibit erythropoiesis. Oxidative stress may contribute to rHuEPO hyporesponsiveness directly by promoting lipid peroxidation in cell membranes, leading to increased erythrocyte fragility and reduced life span and also through its strong association with inflammation. Severe hyperparathyroidism can lead to a reduced number of erythroid progenitor cells. Inadequate dialysis dose, aluminum overload, nutritional factors such as deficiencies of carnitine, vitamin B12, folic acid, and vitamin C can also reduce the efficacy of rHuEPO therapy. Hyporesponsiveness to rHuEPO presents a challenge to both diagnosis and management in an era where optimizing response to rHuEPO is critical both in limiting the burgeoning costs of anemia management and improving clinical outcomes in the dialysis population.

title: Intravenous erythropoietin (rHuEPO) administration increases plasma endothelin and blood pressure in hemodialysis patients.
abstract: The correction of anemia in end stage renal disease with recombinant human erythropoietin (rHuEPO) is associated with hypertension in about a third of hemodialysis patients. In the present study, we investigated the role of endothelin (ET-1) on rHuEPO associated hypertension and the effect of the rHuEPO administration route on plasma ET-1 levels. We studied 50 stable chronic hemodialysis patients who were divided into three groups: 26 patients received rHuEPO intravenously (IV) and 21 subcutaneously (SC). The control group was nine patients who were treated with nandrolone decanoate (ND). Prehemodialysis ET-1 plasma levels were correlated with mean arterial pressure (MAP), hematocrit (Hct), time on dialysis, and rHuEPO doses. The antihypertensive therapeutic index (ATI) was used to determine the changes in blood pressure medication intake. We observed that the ET-1 levels were significantly higher in the IV group (19.3 +/- 2) than the SC (5.0 +/- 0.6) or ND groups (3.6 +/- 0.4 pg/mL) (P < 0.001, IV v SC or ND). After IV rHuEPO treatment, there were increases in both MAP (pre- v post-rHuEPO, P < .001) and in ATI (pre- v post-rHuEPO, P < .001). In the SC group, the increases in MAP and ATI were not significant. Only the IV group showed a significant correlation between MAP and ET-1 levels (r = .05, P = .02). To accomplish the same Hct, the IV group received higher rHuEPO doses than those of the SC (180 +/- 15 v 87 +/- 12 U/kg/week) (P < .001). No significant correlations were found between the plasma ET-1 levels and Hct, time on dialysis and rHuEPO doses.(ABSTRACT TRUNCATED AT 250 WORDS)

title: Blood pressure response to erythropoietin injection in hemodialysis and predialysis patients.
abstract: Recombinant human erythropoietin (rHuEPO) has been reported to induce hypertension. We investigated the effect of a single injection of rHuEPO on blood pressure in patients receiving hemodialysis (HD) and in patients with predialysis chronic renal failure (CRF). Forty-one patients receiving HD and 36 patients with predialysis CRF received an intravenous injection of rHuEPO, and blood pressure and plasma endothelin-1 were measured before and 30 min after the injection. Mean blood pressure was increased significantly in HD patients, but not in CRF patients (HD: 103+/-5 to 105+/-6 mmHg, p<0.05; CRF: 103+/-4 to 103+/-6, NS). The percentage of patients with increased mean blood pressure of more than 10 mmHg after rHuEPO injection was significantly larger in the HD than in the CRF group (27.0% vs. 5.5%, p<0.01). A positive correlation was found between changes in endothelin-1 level and mean blood pressure in the HD (r=0.43, p<0.01) but not in predialysis chronic renal failure. In conclusion, a single injection of rHuEPO increased blood pressure with a positive correlation with endothelin-1 release in hemodialysis patients, but not in predialysis chronic renal failure patients.

title: Pure red cell aplasia and anti-erythropoietin antibodies in patients treated with epoetin.
abstract: Recombinant human erythropoietin (epoetin) was first used for the treatment of renal anaemia in 1986. During the first 10 years of its use, epoetin-induced antibodies were a rare complication and only three cases of patients with epoetin-induced antibodies associated with pure red cell aplasia (PRCA) were published. Since 1998, however, there has been a significant increase in the number of patients developing severe anaemia during the course of epoetin treatment due to neutralizing antibodies. Patients with PRCA present with an absolute resistance to epoetin therapy and then rapidly develop severe anaemia with a very low reticulocyte count (<10 000/mm(3)). Consequently, patients become dependent on blood transfusions to maintain an acceptable level of haemoglobin. By December 2002, approximately 142 patients worldwide had been diagnosed with antibody-positive PRCA after receiving epoetin. The vast majority of these patients had been treated with the Eprex/Erypo brand of epoetin alfa, but there were also some cases in which patients had been receiving epoetin beta (NeoRecormon). To date, there have been no cases of antibody-mediated PRCA reported with the sole use of darbepoetin alfa (Aranesp). All patients with epoetin-induced anti-erythropoietin antibodies had received the drug subcutaneously (s.c.), and almost all had chronic kidney disease-related anaemia. To our knowledge, no patient treated exclusively by intravenous (i.v.) administration has developed anti-erythropoietin antibodies. The increase in reported cases coincides with the removal of human serum albumin from the ex-US formulation of epoetin alfa, in order to comply with new regulations from the European regulatory authorities. It has been proposed that the new formulation is less stable, allowing aggregates of erythropoietin molecules to form, which increases the probability of antibody formation. Treatment with epoetin must be discontinued if PRCA is suspected. Patients do not respond to an increase in dose. Furthermore, patients must not be switched to another form of erythropoietic therapy as the antibodies cross-react with all erythropoietic therapies available. In around 70% of cases, immunosuppressive regimens are effective in eliminating the antibodies; cessation of epoetin therapy without concomitant immunosuppression is rarely effective. Kidney transplantation seems to provide an immediate and effective cure.

title: Pure red cell aplasia induced by erythropoiesis-stimulating agents.
abstract: Pure red cell aplasia in patients who are treated for anemia of chronic kidney disease with erythropoiesis-stimulating agents such as epoetin was first reported in 1998. Although the incidence of pure red cell aplasia peaked in 2002, it remains important for nephrologists to know how to investigate a suspected case of pure red cell aplasia and how to identify other causes of hyporesponsiveness to erythropoiesis-stimulating agents, which account for the vast majority of such cases. The authors reviewed the current status of information in the literature and drew on their personal experiences with patients regarding the diagnosis and management of epoetin-induced pure red cell aplasia. The mechanism for development of epoetin-induced pure red cell aplasia remains unconfirmed. It generally occurs after the production of neutralizing anti-erythropoietin antibodies. Elucidation of a suspected pure red cell aplasia case requires a systematic approach, beginning with simple measurements such as blood cell counts, because most cases of erythropoiesis-stimulating agent hyporesponsiveness are attributable to other causes. If these criteria indicate that the patient's response to erythropoiesis-stimulating agent therapy is very poor, then bone marrow examination and measurement of anti-erythropoietin antibodies is justified. If pure red cell aplasia is confirmed, then cessation of erythropoiesis-stimulating agent therapy and initiation of immunosuppressive therapy are recommended. Continued study of epoetin-induced pure red cell aplasia is needed to help nephrologists prevent or manage future cases and will have implications for the use of other protein-based therapeutic agents.