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.

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 ␤ receptor (␤cR), the signal-transducing subunit shared by the granulocyte-macrophage colony stimulating factor, and the IL-3 and IL-5 receptors. However, because ␤cR knockout mice exhibit normal erythrocyte maturation, ␤cR is not required for erythropoiesis. We hypothesized that ␤cR in combination with the EpoR expressed by nonhematopoietic cells constitutes a tissueprotective 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 ␤cR. Further, antibodies against EpoR coimmunoprecipitated ␤cR 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 ␤cR and EpoR. Finally, as predicted by the hypothesis, neither Epo nor CEpo was active in cardiomyocyte or spinal cord injury models performed in the ␤cR knockout mouse. These data support the concept that EpoR and ␤cR comprise a tissue-protective heteroreceptor.

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.

Cerebral vasospasm and ischemic damage are important causes of mortality and morbidity in patients affected by aneurysmal subarachnoid hemorrhage (SAH). Recently, i.p. administration of recombinant human erythropoietin (r-Hu-EPO) has been shown to exert a neuroprotective effect during experimental SAH. The present study was conducted to evaluate further the effect of r-Hu-EPO administration after SAH in rabbits on neurological outcome, degree of basilar artery spasm, and magnitude of neuronal ischemic damage. Experimental animals were divided into six groups: group 1 (n ‫؍‬ 8), control; group 2 (n ‫؍‬ 8), control plus placebo; group 3 (n ‫؍‬ 8), control plus r-Hu-EPO; group 4 (n ‫؍‬ 8), SAH; group 5 (n ‫؍‬ 8), SAH plus placebo; group 6 (n ‫؍‬ 8), SAH plus r-Hu-EPO. r-Hu-EPO, at a dose of 1,000 units͞kg, and placebo were injected i.p. starting 5 min after inducing SAH and followed by clinical and pathological assessment 72 h later. Systemic administration of r-Hu-EPO produced significant increases in cerebrospinal fluid EPO concentrations (P < 0.001), and reduced vasoconstriction of the basilar artery (P < 0.05), ischemic neuronal damage (P < 0.001), and subsequent neurological deterioration (P < 0.05). These observations suggest that r-Hu-EPO may provide an effective treatment to reduce the post-SAH morbidity.

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 μg/g versus 181.3 ± 21 μg/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.