Emerging biological roles for erythropoietin in the nervous system

Erythropoietin mediates an evolutionarily conserved, ancient immune response that limits damage to the heart, the nervous system and other tissues following injury. New evidence indicates that erythropoietin specifically prevents the destruction of viable tissue surrounding the site of an injury by signalling through a non-haematopoietic receptor. Engineered derivatives of erythropoietin that have a high affinity for this receptor have been developed, and these show robust tissue-protective effects in diverse preclinical models without stimulating erythropoiesis. A recent successful proof-of-concept clinical trial that used erythropoietin to treat human patients who had suffered a stroke encourages the evaluation of both this cytokine and non-erythropoietic derivatives as therapeutic agents to limit tissue injury.     

Delayed administration of pyroglutamate helix B surface peptide (pHBSP), a novel nonerythropoietic analog of erythropoietin, attenuates acute kidney injury

In preclinical studies, erythropoietin (EPO) reduces ischemia-reperfusion-associated tissue injury (for example, stroke, myocardial infarction, acute kidney injury, hemorrhagic shock and liver ischemia). It has been proposed that the erythropoietic effects of EPO are mediated by the classic EPO receptor homodimer, whereas the tissue-protective effects are mediated by a hetero-complex between the EPO receptor monomer and the β-common receptor (termed "tissue-protective receptor"). Here, we investigate the effects of a novel, selective-ligand of the tissue-protective receptor (pyroglutamate helix B surface peptide [pHBSP]) in a rodent model of acute kidney injury/dysfunction. Administration of pHBSP (10 μg/kg intraperitoneally [i.p.] 6 h into reperfusion) or EPO (1,000 IU/kg i.p. 4 h into reperfusion) to rats subjected to 30 min ischemia and 48 h reperfusion resulted in significant attenuation of renal and tubular dysfunction. Both pHBSP and EPO enhanced the phosphorylation of Akt (activation) and glycogen synthase kinase 3β (inhibition) in the rat kidney after ischemia-reperfusion, resulting in prevention of the activation of nuclear factor-κB (reduction in nuclear translocation of p65). Interestingly, the phosphorylation of endothelial nitric oxide synthase was enhanced by EPO and, to a much lesser extent, by pHBSP, suggesting that the signaling pathways activated by EPO and pHBSP may not be identical.     

Localization of erythropoietin in and around growing cartilage

Erythropoietin (EPO) is known to be a hematopoietic growth factor and a regulator of red blood cell production. Recently, EPO has also been reported to function as a tissue-protective cytokine and as an angiogenesis promoting factor. EPO is mainly regulated by hypoxia through the action of hypoxia inducible factors (HIF-1α and HIF-2α). The localization of the EPO protein and the HIF-2α protein were immunohistochemically analyzed in developing porcine embryos. Both proteins were localized in developing cartilage tissue. HIF-2α and EPO protein were expressed in the peripheral chondrocytes of cartilage anlagen, in the perichondrium and in the cell condensations that will eventually differentiate into cartilage tissue. The results of this study reveal that EPO might play a role as a survival factor or as a mitogen in developing cartilage tissue. Moreover, the presence of both proteins at the same locations supports the hypothesis that EPO expression is regulated by HIF-2α.     

Carbamylated erythropoietin reduces radiosurgically-induced brain injury

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 microg 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 approximately 50%. These findings further extend the kinds of injury for which administration of a tissue-protective cytokine provides benefit.     

Erythropoietin in cancer: an update

Erythropoietin (EPO) has long been recognized as the major hematopoietic cytokine regulating normal erythropoiesis. Moreover, there is a growing interest in the non-erythropoietic, tissue-protective effects of EPO. Because of its potential to correct anemia, EPO has been increasingly prescribed to cancer patients. However, although recombinant human Epo (rHuEPO) significantly reduces the risk for red blood cell transfusions in cancer patients, recent clinical studies have reported decreased survival and disease control following rHuEPO treatment in patients with different cancer types. The issue of EPOR expression in tumor cells is critical in this respect. The expression of EPOR in tumor cells raises the possibility that exogenous rHuEPO may directly influence tumor growth or sensitivity to chemo-radiation therapy. In addition, EPOR expression in endothelial cells suggests what potential effects EPO may have on tumor capillaries, such as the stimulation of angiogenesis. However, as experimental studies reveal, the overall direct effect of EPO-EPOR signaling on cancer progression and therapy is not a straightforward one. The current paper provides an update on the biology of EPO, and discusses its utility in the treatment of cancer patients.     

Selective modulation of the erythropoietic and tissue-protective effects of erythropoietin: time to reach the full therapeutic potential of erythropoietin

Erythropoietin (EPO) has been used clinically both as an erythropoietic stimulating agent in the treatment of anemia and as a tissue-protective agent in diverse clinical settings including stroke, multiple sclerosis, acute myocardial infarction and others. However, use of EPO or EPO-analogues leads to simultaneous targeting of both the erythropoietic and tissue-protective properties of EPO, and this strategy has been associated with several problems. Specifically, the benefit of correction of cancer-related anemia can be offset by the tissue-protective effects of EPO, which may lead to stimulation of cancer cell proliferation. Conversely, the benefit of tissue-protection in patients with stroke or myocardial infarction can be offset by adverse effects associated with the erythropoietic effects of EPO such as elevation of red blood cell mass, hypertension and prothrombotic phenomena. The finding that the erythropoietic and tissue-protective properties of EPO are conferred via two distinct receptor systems raises the interesting possibility of discovering novel drugs that selectively stimulate either the erythropoietic or the tissue-protective activities of EPO. This article reviews the current status of the clinical use of EPO and EPO-analogues in the treatment of cancer-related anemia and for tissue protection, outlines the distinct molecular biology of the tissue-protective and erythropoietic effects of EPO and discusses strategies of selective targeting of these activities with the goal of exploiting the full therapeutic potential of EPO.     

A new agonist of the erythropoietin receptor, Epobis, induces neurite outgrowth and promotes neuronal survival

Apart from its hematopoietic activity, erythropoietin (EPO) is also known as a tissue-protective cytokine. In the brain, EPO and its receptor are up-regulated in response to insult and exert pro-survival effects. EPO binds to its receptor (EPOR) via high- and low-affinity binding sites (Sites 1 and 2, respectively), inducing conformational changes in the receptor, followed by the activation of downstream signaling cascades. Based on the crystal structure of the EPO:EPOR(2) complex, we designed a peptide, termed Epobis, whose sequence encompassed amino acids from binding Site 1. The present study shows that the Epobis peptide specifically binds to EPOR and induces neurite outgrowth from primary neurons in an EPOR-expression dependent manner. Furthermore, Epobis promoted the survival of hippocampal and cerebellar neuronal cultures after kainate treatment and KCl deprivation, respectively. Thus, we identified a new functional agonist of EPOR with the potential to promote neuroregeneration and neuroprotection.     

N-Glycosylation engineering of tobacco plants to produce asialoerythropoietin

Erythropoietin (EPO) is a glycoprotein hormone that displays both hematopoietic and tissue-protective functions by binding to two distinct receptors. Recombinant human EPO (rhuEPO) is widely used for the treatment of anemia, but its use for tissue protection is limited because of potentially harmful increases in red blood cell mass when higher doses of rhuEPO are used. Recent studies have shown that asialoerythropoietin (asialo-rhuEPO), a desialylated form of rhuEPO, lacks hematopoietic activity, but retains cytoprotective activity. Currently, a small amount of asialo-rhuEPO is produced by enzymatic desialylation of rhuEPO. The prohibitive cost of rhuEPO, however, is a major limitation of this method. Plants have the ability to synthesize complex N-glycans, but lack enzymatic activities to add sialic acid and β1,4-galactose to N-glycan chains. Plants could be genetically engineered to produce asialo-rhuEPO by introducing human β1,4-galactosyltransferase. The penultimate β1,4-linked galactose residues are important for in vivo biological activity. In this proof of concept study, we show that tobacco plants co-expressing human β1,4-galactosyltransferase and EPO genes accumulated asialo-rhuEPO. Purified asialo-rhuEPO binds to an Erythrina cristagalli lectin column, indicating that its N-glycan chains bear terminal β1,4-galactose residues and that the co-expressed GalT is functionally active. Asialo-rhuEPO interacted with the EPO receptor (EPOR) with similar affinity as rhuEPO, implying that it was properly folded. The strategy described here provides a straightforward way to produce asialo-rhuEPO for research and therapeutic purposes. KEY MESSAGE: N-glycosylation pathway in tobacco plants could be genetically engineered to produce a tissue-protective cytokine, asialoerythropoietin (a desialylated form of human hormone erythropoietin).     

Erythropoietin mimetic compound AGEM400(HES) binds to the same receptor as erythropoietin but displays a different spectrum of activities

EPO mimetic peptides (EMPs) have a completely different structure than erythropoietin (EPO) or new generation recombinant erythropoiesis stimulating agents (ESAs) like Darbepoietin alfa (Aranesp) and continuous erythropoiesis stimulating agent (CERA). This study intended to compare the effects of a novel compound called AGEM400(HES), consisting of a dimeric EMP conjugated to hydroxyethyl starch (HES), to those of recombinant EPO. AGEM400(HES) efficiently stimulated erythropoiesis in vitro and efficiently stimulated survival of EPO-dependent cell line UT7/EPO. It also efficiently induced phosphorylation of signaling proteins in these models. However, AGEM400(HES) was shown to have weak or absent effects on survival of, and signaling in, three different EPO-responsive hematopoietic cell lines. In the latter models, when added in excess to moderate concentrations of EPO, AGEM400(HES) inhibited the activity of EPO in a fashion indicating receptor binding competition between EPO and AGEM400(HES). It was furthermore shown, using stably transfected BA/F3 cells, that the degree of responsiveness of a cell to AGEM400(HES) relative to its responsiveness to EPO, correlated with the level of EPO receptor surface expression. The findings presented raise intriguing possibilities because they imply that not all side-effects said to be associated with EPO must necessarily be elicited by AGEM400(HES) too.     

Up-regulated Endogenous Erythropoietin/Erythropoietin Receptor System and Exogenous Erythropoietin Rescue Retinal Ganglion Cells after Chronic Ocular Hypertension

Aims Recent studies have showed that erythropoietin (EPO) is a neuroprotectant for central nerve system neurons in addition to being a hematopoietic cytokine in response to hypoxia. In this study, we investigate the role of the EPO/EPO receptor (EPOR) system in the rat retina after ocular hypertension injury that mimics glaucoma. Methods Elevated intraocular pressure was induced by laser coagulation of the episcleral and limbal veins. Expression of EPO and EPOR in the normal and glaucomous retinas was investigated by immunohistochemistry and Western blot. To examine the effects of endogenous EPO on the survival of retinal ganglion cells (RGCs) subjected to hypertensive injury, soluble EPOR was directly injected into the vitreous body. Recombinant human EPO was both intravitreally and systemically administrated to study the effect of exogenous EPO on the survival of RGCs after ocular hypertension injury. Results Immunohistochemistry studies identified Müller cells as the main source of EPO in the normal retina. Expression of EPO and EPOR proteins was increased significantly 2 weeks after ocular hypertension. RGCs, amacrine and bipolar cells all demonstrated an increased expression of EPOR after ocular hypertension. Neutralization of endogenous EPO with soluble EPOR exacerbated ocular hypertensive injury, suggesting a role of the EPO/EPOR system in the survival of RGCs after injury. Similarly, topical and systemic administration of recombinant human EPO rescues RGCs after chronic ocular hypertension. Conclusions These results indicate that an endogenous EPO/EPOR system participates in intrinsic recovery mechanisms after retina injury and RGCs might be rescued by exogenous administration of EPO.     

Low Oxygen Tension Primes Aortic Endothelial Cells to the Reparative Effect of Tissue-Protective Cytokines

Erythropoietin (EPO) has both erythropoietic and tissue-protective properties. The EPO analogues carbamylated EPO (CEPO) and pyroglutamate helix B surface peptide (pHBSP) lack the erythropoietic activity of EPO but retain the tissue-protective properties that are mediated by a heterocomplex of EPO receptor (EPOR) and the β common receptor (βCR). We studied the action of EPO and its analogues in a model of wound healing where a bovine aortic endothelial cells (BAECs) monolayer was scratched and the scratch closure was assessed over 24 h under different oxygen concentrations. We related the effects of EPO and its analogues on repair to their effect on BAECs proliferation and migration (evaluated using a micro-Boyden chamber). EPO, CEPO and pHBSP enhanced scratch closure only at lower oxygen (5%), while their effect at atmospheric oxygen (21%) was not significant. The mRNA expression of EPOR was doubled in 5% compared with 21% oxygen, and this was associated with increased EPOR assessed by immunofluorescence and Western blot. By contrast, βCR mRNA levels were similar in 5% and 21% oxygen. EPO and its analogues increased both BAECs proliferation and migration, suggesting that both may be involved in the reparative process. The priming effect of low oxygen tension on the action of tissue-protective cytokines may be of relevance to vascular disease, including atherogenesis and restenosis.     

Changes in kidney tissue and effects of erythropoietin after acute heart failure

Impairment of cardiac function causes renal damage. Renal failure after heart failure is attributed to hemodynamic derangement including reduced renal perfusion and increased venous pressure. One mechanism involves apoptosis and is defined as cardiorenal syndrome type 1. Erythropoietin (EPO) is a cytokine that induces erythropoiesis under hypoxic conditions. Hypoxia inducible factor 1 alpha (HIF-1α) plays a regulatory role in cellular response to hypoxia. Protective effects of EPO on heart, kidney and nervous system are unrelated to red blood cell production. We investigated early changes in and effects of EPO on renal tissues of rats with myocardial infarction by morphology and immunohistochemistry. Coronary artery ligation was used to induce myocardial infarction in Wistar rats. Group 1 comprised sham operated rats; groups 2, 3 and 4 included rats after coronary artery ligation that were sacrificed 6 h after ligation and that were treated with saline, 5,000 U/kg EPO or 10,000 U/kg EPO, respectively; group 5 included rats sacrificed 1 h after ligation. Group 2 showed increased renal tubule damage. Significantly less tubule damage was observed in EPO treated groups. EPO and EPO receptor (EPO-R) immunostaining intensities increased slightly for group 5 and became more intense for group 2. EPO and EPO-R immunostaining was observed in the interstitial area, glomerular cells and tubule epithelial cells of EPO treated groups. HIF-1α immunostaining was observed in collecting tubules in the medulla only in group 2. Caspase-3 immunostaining is an indicator of apoptosis. Caspase-3 staining intensity decreased in renal medulla of EPO treated groups. EPO treatment may exert a protective effect on the renal tissues of patients with cardiorenal syndrome.     

Effect of Trypanosoma brucei brucei on erythropoiesis in infected rats

Anemia generated from African trypanosome infection is considered an important symptom in humans and in domestic animals. In order to recover from anemia, the process of erythropoiesis is essential. Erythropoiesis is affected by erythropoietin (EPO), an erythropoietic hormone, supplying iron and inflammatory and proinflammatory cytokines. However, the role of these factors in erythropoiesis during African trypanosome infection remains unclear. In the present study, we analyze how erythropoiesis is altered in anemic Trypanosoma brucei brucei (interleukin-tat 1.4 strain [ILS])-infected rats. We report that the packed cell volume (PCV) of blood from ILS-infected rats was significantly lower 4 days after infection, whereas the number of reticulocytes, as an index of erythropoiesis, did not increase. The level of EPO mRNA in ILS-infected rats did not increase from the third day to the sixth day after infection, the same time that the PCV decreased. Kidney cells of uninfected rats cultured with ILS trypanosome strain for 8 hr in vitro decreased EPO mRNA levels. Treatment of both ILS and cobalt chloride mimicked hypoxia, which restrained the EPO-production-promoting effect of the cobalt. Messenger RNA levels of β-globin and transferrin receptor, as markers of erythropoiesis in the bone marrow, also decreased in ILS-infected rats. Levels of hepcidin mRNA, which controls the supply of iron to the marrow in liver, were increased in ILS-infected rats; however, the concentration of serum iron did not change. Furthermore, mRNA levels of interleukin-12, interferon-γ, tumor necrosis factor-α, and macrophage migration inhibitory factor in the spleen, factors that have the potential to restrain erythropoiesis in bone marrow, were elevated in the ILS-infected rats. These results suggest that ILS infection in rats affect erythropoiesis, which responds by decreasing EPO production and restraining its function in the bone marrow.     

Plant recombinant erythropoietin attenuates inflammatory kidney cell injury

Human erythropoietin (EPO) is a pleiotropic cytokine with remarkable tissue-protective activities in addition to its well-established role in red blood cell production. Unfortunately, conventional mammalian cell cultures are unlikely to meet the anticipated market demands for recombinant EPO because of limited capacity and high production costs. Plant expression systems may address these limitations to enable practical, cost-effective delivery of EPO in tissue injury prevention therapeutics. In this study, we produced human EPO in tobacco and demonstrated that plant-derived EPO had tissue-protective activity. Our results indicated that targeting to the endoplasmic reticulum (ER) provided the highest accumulation levels of EPO, with a yield approaching 0.05% of total soluble protein in tobacco leaves. The codon optimization of the human EPO gene for plant expression had no clear advantage; furthermore, the human EPO signal peptide performed better than a tobacco signal peptide. In addition, we found that glycosylation was essential for the stability of plant recombinant EPO, whereas the presence of an elastin-like polypeptide fusion had a limited positive impact on the level of EPO accumulation. Confocal microscopy showed that apoplast and ER-targeted EPO were correctly localized, and N -glycan analysis demonstrated that complex plant glycans existed on apoplast-targeted EPO, but not on ER-targeted EPO. Importantly, plant-derived EPO had enhanced receptor-binding affinity and was able to protect kidney epithelial cells from cytokine-induced death in vitro . These findings demonstrate that tobacco plants may be an attractive alternative for the production of large amounts of biologically active EPO.     

Erythropoietin protects cardiac myocytes from hypoxia-induced apoptosis through an Akt-dependent pathway

Apoptosis is a contributing cause of myocyte loss in ischemic heart disease. Recent work has shown that erythropoietin (EPO) offers protection against apoptosis in a wide variety of tissues. We demonstrate that the erythropoietin receptor (EPOR) is expressed in the neonatal rat ventricular myocyte (NRVM). Exposure of NRVMs to hypoxia, with recombinant human EPO, significantly decreased apoptosis as measured by TUNEL, flow cytometry, and caspase 3/7 like activity when compared to hypoxia treatment alone. EPO administered at the initiation of coronary artery occlusion in the rat significantly decreased apoptosis in the myocardial ischemic region. In the NRVM, EPO increased the activity of Akt. The anti-apoptotic effect of EPO was abrogated by co-treatment with LY294002, a specific blocker of phosphatidylinositol 3-kinase (PI3-K). Our study demonstrates that EPO inhibits apoptosis in the NRVM exposed to hypoxia, through an Akt-dependent pathway. EPO also inhibits apoptosis in the in vivo rat model of myocardial ischemia.     

Erythropoietin and the skin: a role for epidermal oxygen sensing?

Erythropoietin (EPO), long appreciated as the chief endocrine regulator of red blood cell formation, is now recognized to exert many additional functions outside the bone marrow. Thus, the quest is on to define the full range of EPO functions in the physiology and pathology of non‐hematopoietic tissues. Two recent studies in man and mice have highlighted the importance of the mammalian skin as one peripheral tissue with a previously unsuspected role in EPO biology; both, as a target and as a source of EPO. In addition, the skin has been proposed to function as an oxygen sensor. The present hypothesis essay critically reviews the currently available evidence for this and provides a unifying theoretical scenario for intracutaneous EPO functions and for a potential role of the skin in the control of EPO production. Mainly, we propose that the skin itself directly contributes to the up‐regulation of EPO plasma levels in response to hypoxia.