Elsevier

Brain Research Reviews

Volume 59, Issue 1, November 2008, Pages 22-33
Brain Research Reviews

Review
Neuroprotective properties and mechanisms of erythropoietin in in vitro and in vivo experimental models for hypoxia/ischemia

https://doi.org/10.1016/j.brainresrev.2008.04.007Get rights and content

Abstract

Besides its established function in erythropoiesis, erythropoietin (EPO) is currently also appreciated for its neuroprotective effects. The detrimental sequelae of prolonged cerebral hypoxia and ischemia have been shown to attenuate by EPO treatment. After binding to the EPO receptor, EPO is capable of initiating a cascade of events which –via different pathways–may lead to neuroprotection. The circumstances that determine which specific signalling route(s) are activated by EPO are largely unknown. We aim to provide the reader with a timely overview on the use of EPO in models of stroke and hypoxia–ischemia and to discuss the molecular events that underlie its neuroprotection.

Introduction

Erythropoietin (EPO) was originally recognized as a humoral mediator involved in the maturation and proliferation of erythroid progenitor cells (Carnot and Deflandre, 1906) but is now appreciated for its neuroprotective effects on the central nervous system as well.

In vitro and in vivo studies in adult and neonatal animal models revealed a neuroprotective role of exogenous EPO administration (Noguchi et al., 2007, Sola et al., 2005b). Clinical relevance for the use of EPO as a neuroprotective agent was enhanced when the 34 kDa glycoprotein was found to cross the blood–brain barrier (BBB) after peripheral administration (Brines et al., 2000). EPO was tested in clinical trials as a possible treatment for adult stroke and found to be both safe and beneficial (Ehrenreich et al., 2002).

Neuroprotection by EPO has been shown to associate with anti-apoptosis, neuroregeneration and anti-inflammation (Sola et al., 2005b). The biological effects of EPO mediated by the EPOR are accomplished via at least three different pathways; after binding to the EPO receptor (EPOR), EPO induces the phosphorylation of Janus kinase (JAK) 2 thereby activating 1) the phosphoinositide 3-kinase (PI3K)-serine-threonine kinase AKT, and/or 2) signal transducer and activator of transcription (STAT) 5 and/or 3) nuclear factor (NF)-κB pathway (Sola et al., 2005b). How the selection of the specific pathways is determined by the cell, is not known, although cell type, metabolic status of the cell and receptor availability will be implicated in this phenomenon.

On the other hand, results obtained from studies employing EPO variants, demonstrated that although these alternatives were less or even uncapable of binding the EPOR, neuroprotective properties were retained (Belayev et al., 2005, Coleman et al., 2006, Erbayraktar et al., 2003, Leist et al., 2004, Villa et al., 2007, Wang et al., 2004c, Wang et al., 2007). Consequently, mechanisms for neuroprotection by EPO may not be as straightforward as thought and hint towards a binding site other than the EPOR-homodimer. A receptor constellation involving the EPOR and the common beta receptor (CβR) subunit (the CβR is a known component of the IL-3, IL-5 and granulocyte/macrophage colony stimulating factor (GM-CSF) receptor) has been proposed as an alternative binding site for EPO (Brines et al., 2004).

In this review we will first discuss the data on the neuroprotective effects of EPO in vitro. Then we will evaluate the role of EPO as a neuroprotective agent against brain injury in adult and neonatal animal models for stroke and hypoxia–ischemia (HI). Next we will discuss the anti-apoptotic, neuroregenerative and anti-inflammatory routes for neuroprotection by EPO. Finally we will present recommendations for future research.

Section snippets

Endogenous production of EPO

As a consequence of hypoxia, degradation of hypoxia-inducible factor (HIF)-1α is prevented which allows the molecule to heterodimerize with HIF-1β to form HIF-1. HIF-1 induces a.o. transcription of endogenous EPO (Jones and Bergeron, 2001, Sharp et al., 2004a) (Fig. 1, top left). Especially astrocytes, but also oligodendrocytes, endothelial cells, neurons and microglia were found to produce EPO (Bernaudin et al., 2000, Bernaudin et al., 1999, Chin et al., 2000, Masuda et al., 1994, Meloni et

Neuroprotection by EPO in vitro

EPO was shown to provide protection from hypoxic and toxic insults in ex-vivo and cultured neuronal cells as well as in cultures of endothelial cells (see Table 1). For example, cellular damage induced by prolonged hypoxia in hippocampal neurons or endothelial cells was effectively counteracted by EPO (Chong et al., 2002, Lewczuk et al., 2000). Cell death as a result of serum depletion in PC12 cells (rat pheochromocytoma derived cells) was reversed by pretreatment with EPO (Koshimura et al.,

Neuroprotection by EPO in vivo

The promising neuroprotective properties by EPO as shown by initial in vitro studies were ensued by in vivo studies in which neuroprotection was demonstrated after exogenous EPO administration. Exogenous EPO includes recombinant human (rh) EPO or other EPO variants such as DarbEPO, asialoEPO and carbamylated EPO (CEPO). EPO variants differ from rhEPO by their binding capacity to the EPOR. DarbEPO contains additional oligosaccharide chains thereby extending the circulation duration compared to

Anti-apoptosis, neuroregeneration and anti-inflammation after EPO

Protective effects by EPO have been thought to result from a decrease in apoptosis, an increase in neuroregeneration and contributions to anti-inflammation. The anti-apoptotic effect of EPO after the insult is likely to be responsible for a proportion of neuronal survival as demonstrated by decreased numbers of apoptotic cells after EPO administration (Chong et al., 2002, Keller et al., 2006, Kellert et al., 2007, Lee et al., 2006, Matsushita et al., 2003, Sun et al., 2004, Wang et al., 2004c,

Signalling pathways as a result of EPO receptor stimulation

A schematic drawing of endogenous EPO formation and the capabilities of endogenous and exogenous EPO to stimulate the EPOR and activate downstream molecular pathways enabling neuroprotection is depicted in Fig. 1.

Either endogenously or exogenously applied EPO may stimulate the EPOR to induce phosphorylation of JAK2 (Kawakami et al., 2000, Kawakami et al., 2001, Sola et al., 2005a). JAK2-phosphorylation in turn activates PI3K, induces the translocation and subsequent activation of NFκB and/or

Alternative EPO signalling with EPO variants

EPO variants that do not bind the EPOR were shown to have neuroprotective properties. For example, the toxic effects of NMDA exposure of hippocampal slice cultures and NMDA-induced apoptosis in P19 (murine teratocarcinoma) cells were equally reduced by both EPO and CEPO treatment (Leist et al., 2004, Montero et al., 2007). AsialoEPO was also found to limit infarct volume after neonatal HI as potently as EPO itself (Wang et al., 2004c).

Since these non-erythropoietic variants do not bind to the

Vascular effects of EPO

In adult and neonatal animals, revascularization was enhanced by EPO treatment after stroke and HI-insult respectively (Iwai et al., 2007, Wang et al., 2004a). Consequently, in adult mice after focal cerebral ischemia, EPO was found to improve cerebral blood flow (Li et al., 2007a).

EPO treatment after permanent focal cerebral ischemia in mice increased the number of cells positive for the vessel marker glucose transporter 1 and bromo-deoxy-uridine (Li et al., 2007b). Protein levels of the

Recommendations and conclusions

The protective effects of EPO have been demonstrated in in vitro studies and in experimental animal models for cerebral injury, however, many questions still remain to be answered. It remains to be clarified as to whether EPO treatment is required at multiple time points after brain damage with respect to its anti-apoptotic and anti-inflammatory effects, its effects on the vascular system and its effects on the regeneration of neuronal progenitor cells.

The detailed mechanisms responsible for

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