Date Published: February 26, 2012
Publisher: Hindawi Publishing Corporation
Author(s): Mawadda Alnaeeli, Li Wang, Barbora Piknova, Heather Rogers, Xiaoxia Li, Constance Tom Noguchi.
Erythropoietin is known as the requisite cytokine for red blood cell production. Its receptor, expressed at a high level on erythroid progenitor/precursor cells, is also found on endothelial, neural, and other cell types. Erythropoietin and erythropoietin receptor expression in the developing and adult brain suggest their possible involvement in neurodevelopment and neuroprotection. During ischemic stress, erythropoietin, which is hypoxia inducible, can contribute to brain homeostasis by increasing red blood cell production to increase the blood oxygen carrying capacity, stimulate nitric oxide production to modulate blood flow and contribute to the neurovascular response, or act directly on neural cells to provide neuroprotection as demonstrated in culture and animal models. Clinical studies of erythropoietin treatment in stroke and other diseases provide insight on safety and potential adverse effects and underscore the potential pleiotropic activity of erythropoietin. Herein, we summarize the roles of EPO and its receptor in the developing and adult brain during health and disease, providing first a brief overview of the well-established EPO biology and signaling, its hypoxic regulation, and role in erythropoiesis.
Erythropoietin (EPO) is produced primarily in the adult kidney and secreted into the circulation to regulate red blood cell production in the bone marrow. EPO stimulates erythroid progenitor cell survival, proliferation, and differentiation to satisfy the daily requirement of about 200 billion new red blood cells due in part to the limited red blood cell lifespan of 120 days. Human recombinant EPO has been used clinically for more than 2 decades to treat anemia associated with conditions such as chronic kidney disease, antiviral HIV therapy, and cancer patients on chemotherapy. EPO production is hypoxia inducible and thus increases during anemia and hypoxic stress. Interestingly, EPO production has also been detected in brain in response to hypoxic stress. The finding that EPO receptor (EpoR) expression extends beyond hematopoietic tissue to include neural and endothelial cells and the accumulating evidence for EPO antiapoptotic properties such as its neuroprotective activity have collectively led to investigations of EPO as a pleiotropic cytokine. In this paper, we review the nonhematopoietic activity of EPO in the developing as well as adult brain, and summarize its roles during health and disease.
EPO is a glycoprotein hormone consisting of a single polypeptide of 166 amino acids folded into four α-helices with two disulphide bridges between cysteines 6 and 161 and between cysteines 29 and 33 [1–3]. EPO is composed of 40% to 60% carbohydrate, with a molecular mass of 30 to 34 kDa, depending on carbohydrate content. Three N-glycosylation sites at asparagines 24, 38, and 83 can each accommodate up to four sialic residues and one O-glycosylation site at serine 126 (absent in rodent EPO), which does not appear to be necessary for EPO activity . Nonsialated EPO is rapidly cleared from the circulation via the galactose receptor in the liver . EPO shares structural similarities with growth hormone and other members of the hematopoietic class 1 cytokine superfamily that include several interleukins (e,g, IL-2, -3, -4, -6), granulocyte-colony-stimulating factor, thrombopoietin, prolactin, oncostatin M, ciliary neurotrophic factor, and leukocyte inhibitory factor [6, 7]. The corresponding receptors for the hematopoietic class 1 cytokines are single transmembrane polypeptides that associate as homodimers, as is the case with EpoR, heterodimers, or heterotrimers. These receptors have no intrinsic catalytic domains and their cytoplasmic regions associate with Janus kinases (JAK) such as JAK2 for EPO signaling . The extracellular domain of EpoR has a short α-helix preceding two seven-stranded β-sheet domains, five cysteine residues, and a Trp-Ser-X-Trp-Ser motif proximal to the membrane and characteristic of receptors for the hematopoietic class 1 cytokines . The human EpoR gene product is encoded from 8 exons spanning over 6.5 kb, creating a 508 amino acid protein . The single transmembrane domain is encoded in exon 6 with much of the cytoplasmic domain encoded in exon 8 [9, 11]. JAK2 binds to EpoR at its Box1/Box2 regions, which allows JAK2 to move from an inactive state when not in contact with EpoR to an active state following EPO stimulation . EPO binding to EpoR on the surface of early erythroid progenitor cells causes a conformational change in the cytoplasmic domain bringing the two associated JAK2 proteins in close proximity leading to transphosphorylation of JAK2 and EpoR and activation of downstream signal transduction pathways (Figure 1) . Notably, the extent of cell surface EpoR expression determines EPO response.
EPO is produced in the fetal liver in hepatocytes surrounding central veins and in fibroblast-like Ito cells . Meanwhile, EPO production in the kidney begins before birth, increasing sharply after 30 weeks in human and localizes to the peritubular interstitial cells with neural characteristics [21, 23, 24]. Although primary EPO production switches to the adult kidney after birth, hepatic EPO mRNA can increase to 20%–50% of total body EPO mRNA in response to hypoxic-challenge [25, 26]. Hypoxia inducible factor (HIF) is the primary regulatory factor for hypoxic induction of EPO resulting in increased production of 150-fold or more. An HIF motif that regulates induction by hypoxia in the liver is located immediately 3′ of the EPO gene, and regulatory elements necessary for hypoxic induction in the kidney are located 6–14 kb 5′ [22, 27]. In the kidney, hypoxic induction results in increased number of cells expressing EPO, while hypoxic induction in the liver increases the amount of EPO expression per hepatocyte [23, 28].
EPO production is regulated primarily at the transcription level. The HIF binding site located within the highly conserved 3′ hypoxia-responsive element (HRE) of the EPO gene is necessary for hypoxia induction in hepatocytes [27, 42–44]. HIF is an important regulatory factor for the activation of genes in response to physiologic stress or hypoxia. The HRE also provides for hypoxic regulation by transcription factors including hepatocyte nuclear factor 4, an orphan nuclear receptor expressed in kidney and liver . HIF is a heterodimer that consists of a constitutive β-subunit (also called aryl hydrocarbon receptor nuclear translocator protein; ARNT) that resides in the nucleus and an α-subunit (HIF-1α, HIF-2α, and HIF-3α) localized to the cytoplasm, which is transported to the nucleus for HIF activation [46, 47]. Hypoxia induction of EPO was initially thought to be regulated primarily by HIF-1α, but increasing evidence indicates that HIF-2α is primarily responsible for hypoxia EPO induction [48–52]. Deletion of HIF-2α, but not HIF-1α, in adult mice gives rise to anemia, indicating its requirement for EPO regulation in physiologic and stress conditions . In contrast, HIF-1α plays a critical role in regulation of hypoxic induction of vascular endothelial growth factor (VEGF) . The α-subunit of HIF in the cytoplasm under normoxia is proline hydroxylated and ubiquitinated by the von Hippel-Lindau protein leading to rapid degradation by the proteasome. Hypoxia stabilizes the α-subunit of HIF that is then transported to the nucleus and heterodimerizes with the β-subunit for transactivation of target genes. Patients with genetic mutations in proline hydroxylase, von Hippel-Lindau protein, or HIF-2α have been identified and associated with familial erythrocytosis . Regulation of EPO production by other transcription factors includes negative regulation by the GATA transcription factors  and activation by GATA-4 in fetal liver . The Wilms tumor suppressor (Wt1) can upregulate EPO gene expression, and its colocalization with EPO in developing mice has led to the suggestion that Wt1 contributes to EPO gene regulation in hepatocytes and neuronal cells of the dorsal root ganglia .
Expression of the erythroid form of EpoR in nonhematopoietic tissues was first detected in endothelial cells providing for a mitogenic and chemotactic response to EPO [59, 60]. In mice, deletion of EPO (Epo−/−) or EpoR (EpoR−/−) leads to angiogenic defects detectable at day E10.5, two days prior to the onset of severe anemia . Although EpoR expression is not HIF responsive, expression in endothelial cells can be induced in culture by the combination of EPO treatment and reduced oxygen tension [62, 63]. Interestingly, the endothelial response appears to contribute to the cardioprotective effects of EPO in animal models [64, 65]. Furthermore, EPO was found to enhance reendothelialization and prevent neointimal hyperplasia , and promote survival of primary human endothelial cells .
EpoR expression in neuronal cells, and the high level of EpoR expression in mouse brain midgestation, provided early evidence for potential EPO activity in brain (Figure 2(a)) [68, 70]. As with erythroid tissue, the extent of EpoR expression regulates EPO response in brain . Mouse EpoR expression in the developing brain appears in regions associated with neurogenesis. For instance, EpoR localizes to the neural tube midgestation and is expressed at a high level comparable to adult hematopoietic tissue in mice but becomes subsequently downregulated by about 100-fold at birth . EpoR expression and hence Epo signaling in brain decrease as development progresses in human. For instance, EpoR expression in adult brain is two orders of magnitude lower than the adult erythropoietic organ (bone marrow) . Observations in the developing midbrain show EPO expression at day E11 in neurons attached to radial glial cells which transiently express EpoR . At day E12.5, EpoR appears to shift to EPO expressing neurons adjacent to apoptotic bodies, and at day E14.5 apoptotic bodies appear without EPO expression in bands along the rostrocaudal length of the midbrain, collectively suggesting a role for EPO in brain morphogenesis. EPO expression in developing spinal cord and dorsal root ganglia follow EpoR expression in radial glial cells, providing more evidence that EPO may contribute to interaction among neurons and between neurons and radial glial cells, and promote differentiation or survival of specific subsets of neurons . Both EPO and the small fraction of nonglycosylated EPO are downregulated during brain development. A heterodimer complex between the classical hematopoietic form of EpoR and the common β chain has been proposed as the receptor binding EPO in neural cells, but the common β chain does not appear to localize with EpoR or EPO in the rat brain and is below the level of detection in EPO responsive neuronal cell lines, SH-Sy5y and PC12 [76–78].
EPO stimulates proliferation of neural progenitor cells in culture. Moreover, the increased proliferation at modest hypoxia is mimicked by EPO treatment and is blocked by an EPO neutralizing antibody [69, 80–83]. Interestingly, EPO-stimulated proliferation in neural progenitor cultures is comparable to but less than FGF, and when added in combination does not increase proliferation beyond that of FGF alone . Additionally, EPO was found to mimic in part the increase in dopaminergic neurons in day E12 rat mesencephalic precursor cells cultured at low oxygen tension . Embryonic rat hippocampal and cortical neuron cultures demonstrate a protective effect of EPO from glutamate toxicity, and EPO promotes survival in the absence of trophic factors [69, 82]. EPO also provides protection to embryonic and postnatal hippocampal neurons from hypoxia-induced cell death [69, 84]. In the retina, EPO is inducible by HIF and is protective against light-induced damage , where hypoxia was found to increase EPO response and EpoR expression [69, 85].
Mice with targeted deletion of EpoR (EpoR−/−) exhibit increased apoptosis in the brain as early as day E10.5, thinning of the neuroepithelium, and smaller brain size prior to death around day E13.5 due to severe anemia (Figure 2(b)) [69, 74]. EpoR expression in neural progenitor cells is downregulated with differentiation and persists at a lower level in differentiated neurons . A role for endogenous EPO in neuron maintenance and survival is suggested by cultures of neural cells lacking endogenous EpoR that exhibit reduced proliferative capacity and increased susceptibility to hypoxia and glutamate toxicity [69, 83]. A human EpoR transgene rescues the EpoR−/− genotype, normalizes the erythroid potential and brain development, and rescues the increased apoptosis observed in the EpoR−/− embryonic brain . EpoR−/− mice rescued with an erythroid restricted EpoR transgene survive to adulthood with normalized hematocrit and without any gross abnormal organ morphology . However, adult mice that lack EpoR expression in the brain exhibit reduced neurogenesis in the subventricular zone and dentate gyrus and increased susceptibility to glutamate toxicity . During ischemic stroke, mice that lack EpoR in neural cells show defective neural cell migration to the peri-infarct cortex . Collectively, the increased apoptosis in embryonic brain, reduced neurogenesis in adult brain, and increased sensitivity to hypoxia and glutamate toxicity exhibited in mice with loss of EpoR expression in brain provide evidence that endogenous EPO signaling is neuroprotective throughout mouse development and adulthood.
EPO stimulation of neuronal cells activates JAK2, STAT5, AKT, and MAPK signaling pathways [77, 94]. Increased EPO production in brain by transgene expression during ischemic injury resulted in activation of JAK2, ERK1/2, and AKT required for EPO-mediated neuroprotection [95, 96]. In hippocampal neurons, EPO was protective in free radical injury and maintained mitochondrial membrane potential . The PI3K signaling pathway was also implicated in EPO protection to ischemic challenge . Targeted deletion of STAT5 did not affect the EPO neuroprotective activity in hippocampal neurons but abrogated the EPO neurotrophic activity . NF-κB is associated with EPO neural protection but not with EPO erythroid activity (Figure 1) .
Brain capillary endothelial cells express EpoR, where Epo acts directly as a competence factor . EPO has been reported to regulate not only neurogenesis but also angiogenesis [80, 89]. Angiogenesis is a tightly controlled multistep process through which new blood vessels are formed by sprouting from the preexisting vasculature in the presence of VEGF and its receptor (VEGF-R). It is suggested that EPO may play a role in stimulating angiogenesis in response to ischemic injury in the brain possibly via VEGF upregulation . Indeed, EpoR in microvascular/capillary endothelial cells is induced during evolution of cerebral infarct following permanent cerebral ischemia in mice and is further enhanced with EPO treatment [102, 103]. EPO treatment has been reported to have an antiapoptotic effect in cerebral vascular cells , and EPO-mediated neurovascular response has been suggested to occur via proangiogenic effects and through the regulation of cerebral blood flow. For instance, treatment with EPO was shown to upregulate EpoR expression in cerebral vascular endothelial cells, which in turn was suggested to drive neurovascular protection and angiogenesis and restore local cerebral blood flow in a mouse model of focal ischemia (Figure 3) .
In the adult rodent brain, neuronal progenitor cells are localized adjacent to endothelial cells in the subventricular zone and the dentate gyrus . Interestingly, cerebral ischemia has been reported to induce angiogenesis and neurogenesis [89, 106, 107]. Moreover, angiogenesis was shown to be coupled with neurogenesis [105, 108], whereby suppressing angiogenesis reduces neuroblast migration toward the ischemic cortex , thus indicating that interactive networks exist between endothelial and neuronal cells. The migration of new-born neurons toward the ischemic boundary region was found to be promoted by EPO-enhanced angiogenesis through matrix metalloproteinase-2 and -9 released by cerebral endothelial cells , thereby further highlighting the involvement of endothelial cell responses in regulation of neuronal cell responses. Neuronal progenitor cells can directly promote angiogenesis , and EPO treated neuronal progenitor cells induce angiogenesis through the production of VEGF and upregulation of VEGF-R2 in cerebral endothelial cells . VEGF, in turn, was proposed to mediate neurogenesis by augmenting proliferation and neuronal differentiation of neural progenitor cells [106, 113].
Basal production of NO by endothelial nitric oxide synthase (eNOS) is believed to play pivotal roles in the regulation of cerebral blood flow, vascular tone, vascular resistance, and vascular growth under resting conditions in various mammals [114, 115]. In addition, vasodilation is suppressed with specific inhibition of neuronal NOS (nNOS) and in mice that lack nNOS [116, 117]. Interestingly, EPO has been reported to exert neuroprotective effects in vivo, by regulating neurovascular response resulting in vasodilation and increased blood flow through increasing NO production . It is noteworthy that NO can induce EpoR expression in neuronal cell cultures , can improve O2 supply by means of vasodilation, and thus can provide tissue protective effects, although excessive production of NO is neurotoxic . Overproduction of NO by inducible NOS (iNOS) during inflammation has been implicated in various pathological processes, including tissue injury and cell apoptosis caused by ischemia and inflammation [121, 122]. Separate eNOS-, nNOS- or iNOS-deficient mouse models have been useful in demonstrating that the eNOS isoform is protective against focal cerebral ischemic injury, while the nNOS and the iNOS isoforms play roles in early and later stages of ischemic injury, respectively [122–124]. nNOS isoform was found to be heavily involved in hemodynamic response to local neuronal activity, a process called neurovascular coupling . In relation to the neuroprotective effects of EPO, using primary dorsal root ganglion cultures, NO administration was found to protect against axonal degeneration in a manner dependent on HIF-mediated transcription of EPO in glial cells . These findings collectively underscore the link between the EPO responses in brain and NO.
Studies using animal models provide ample evidence for the neuroprotective activity of EPO. EPO stimulation of neural and endothelial cells and EPO production in brain have been suggested to contribute to neuroprotection, with the latter being of particular importance since only low levels of EPO appear to cross the blood-brain barrier when administered at high dose intravenously . EPO has been reported to inhibit neuronal cell apoptosis, stimulate cell survival and differentiation, and promote neurogenesis and neurotrophic functions. To date, EPO-mediated neuroprotection has been shown to occur via (i) antiapoptotic response in neurons, (ii) endothelial response by increased blood flow and oxygen delivery through increased vascular relaxation and angiogenesis, and (iii) anti-inflammatory effects.
Direct brain injection of EPO provided protection in a neonatal rat model of hypoxic-ischemic injury and reduced the size and extent of brain damage . Pretreatment with intraperitoneally injected EPO in neonatal mice was also neuroprotective in hypoxic-ischemic injury . In neonatal rats, EPO neuroprotection was found to provide a long-term effect in the developing brain that was greater in females than in males . EPO treatment in neonatal hypoxic-ischemic brain injury in rats increased brain weight, decreased apoptosis, protected dopamine neurons, and improved functional outcomes including long-term spatial memory deficits [147–150]. EPO administration in ischemic injury in the developing brain favored neurogenesis and decreased gliogenesis . EPO also improved auditory processing and sensorimotor function in hypoxia-ischemic injury [152, 153].
Treatment with recombinant human EPO has been reported to stimulate anti-inflammatory signaling, which was suggested to contribute to its direct neuroprotective effect during cerebral ischemia . Inflammation plays a critical role in the pathogenesis of cerebral ischemia, where the influx of leukocytes from the blood into the brain and activation of resident microglial cells to secrete inflammatory mediators and cytokines result in barrier damage, microvascular occlusion, and, thus, aggravate injury [169, 170]. The administration of EPO to animals with experimental cerebral ischemia resulted in the reduction of the local production of TNF-α, IL-6, and the chemokine MCP-1, subsequently leading to a marked reduction of infarct size . Although EPO-mediated reduction of ischemia-induced inflammation has been proposed to occur via reducing neuronal death rather than by direct effects upon EpoR-expressing inflammatory cells , it remains unknown whether or not EPO can play a direct role in regulating inflammatory cell responses. In an experimental model of multiple sclerosis, an autoimmune disease of the CNS, administration of EPO inhibited the inflammatory response, delayed the onset of the disease, and decreased its severity [171–174]. Similarly, in a rat model of optic neuritis, systemic administration of EPO significantly increased the survival of retinal ganglion cells . Although the exact mechanisms underlying the anti-inflammatory effects of EPO remain unknown, EPO might act by reducing leukocyte transmigration through endothelial cells, since it enhances the resistance of endothelial cells toward ischemia  and could be mediated by activating immune suppressive lymphocyte response . In addition, EPO-mediated oligodendrogenesis and the inhibition of oligodendrocyte cytotoxicity, induced by inflammatory stimuli, could also contribute to the observed neuroprotection in experimental multiple sclerosis [171, 176].
As previously mentioned, the neuroprotective effect of EPO extends to the retina and provides protection to mouse photoreceptor cells against UV light damage . During human development, EPO mRNA in the retina and EPO protein in the vitreous at levels greater than in serum were observed between 12 and 24 weeks gestation and increased with gestational age . However, concerns about the angiogenic properties of EPO remain. Association between elevated EPO in the vitreous and proliferative retinopathy was observed in adult diabetic patients exhibiting increased levels of VEGF and EPO in the vitreous fluid compared with nondiabetic patients with ocular disease . EPO was found to be more strongly associated with diabetic retinopathy than VEGF, and the level of EPO in the vitreous did not correlate with plasma level, suggesting local production of EPO. Indeed, a single nucleotide polymorphism in the human EPO gene promoter is associated with increased EPO in the vitreous and severe microvascular complications in diabetes, proliferative diabetic retinopathy, and end-stage renal disease .
Attention was drawn to the potential for EPO therapy in neurological diseases by the initial pilot study of 40 adult stroke patients that suggested high dose EPO administration was well tolerated in acute ischemic stroke and was associated with improved clinical outcome . Subsequently, an expanded trial of 460 patients revealed that a high number of stroke patients received tissue plasminogen activator (TPA) and that there was a significant increase in overall death rate with EPO treatment (42/256) compared with the placebo group (24/266) . Further animal studies revealed that in rodents treated with TPA after middle cerebral artery occlusion, EPO administered up to 6 hours after reperfusion showed no reduction in the volume of ischemic injury and a significant increase in the incidence of brain hemorrhage. Moreover, the expected EPO-associated reduction in brain swelling was abolished with TPA, underscoring again the importance of timing in EPO therapy [185, 186]. Of note, delayed EPO treatment was neuroprotective in a rat model of traumatic brain injury .
Recently, several metabolic effects for EPO have been described. For example, EPO treatment in obese mice decreased body weight, fat mass and blood glucose in a dose-dependent manner [200–203] and provided protection in mouse models of type 1 and type 2 diabetes . These EPO activities were attributed to EPO response in multiple tissues including pancreatic beta cells, skeletal muscle, adipose tissue and brain, independent of changes in hematocrit [200, 203, 204]. Indeed, the significant decrease in food consumption in response to high-dose EPO treatment in association with the decreased body weight and fat mass in obese animals correlated with EPO activity in the hypothalamus [200, 201]. EpoR expression in hypothalamus was found to localize to proopiomelanocortin (POMC) neurons that regulate food intake, and EPO treatment increased POMC production in the hypothalamus . Conversely, no increase in POMC production or decrease in fat mass was observed with EPO treatment in mice with EPO signaling restricted to erythroid tissue that exhibit an obese phenotype . These observations suggest a direct link between EPO and POMC neurons in the hypothalamus during energy and metabolic regulation.
Identification of the spatial and temporal expression of EPO and EpoR during brain development has facilitated understanding of the potential range of EPO response in brain during ischemic/hypoxic stress or disease and provided insight on the utility of EPO treatment. The pleiotropic activity of EPO evident in culture and animal studies remain of continuing interest, particularly its potential treatment for neurological disease. EpoR expression in neuronal and endothelial cells and hypoxic-induction of EPO production in brain likely drive the cytoprotective outcome, through antiapoptotic, vascular, and anti-inflammatory responses. In addition, the hypoxic induction of EPO in the kidney drives the erythroid response to increase blood cell production in the bone marrow leading to increased oxygen delivery and neuroprotection. Contributing to increased oxygen delivery is endothelial response to EPO, which leads to elevated NO production, vasodilation, and increased blood flow. In conclusion, the high level of EpoR expression in the developing brain including neural progenitor cells and neurons, and continued EpoR expression in adult brain can support direct proliferative and antiapoptotic responses to EPO, suggesting that local EPO production and/or availability can directly promote a neuroprotective response involving neural and endothelial cells collaboration to promote the brain response to EPO (see Figure 3).