What Evidence Supports Use of Erythropoietin as a Novel Neurotherapeutic?

For nearly a century, it was known that a substance within the systemic circulation regulates the serum hemoglobin concentration of mammals. Following many years of futile attempts to isolate this factor, which is present in only trace amounts in the plasma, the glycoprotein erythropoietin(Drug information on erythropoietin) was finally isolated from the urine of patients with aplastic anemia.[1] Erythropoietin DNA probes were constructed from tryptic fragments of the isolate and the gene cloned in 1985.[2,3] In the normal adult, the kidney produces erythropoietin for control of the circulating erythrocyte mass in a classic hormonal feedback loop responding to low tissue oxygenation.[4,5] The targets of this renal erythropoietin are the colony-forming units-erythroid (CFUe) that reside within the bone marrow for which erythropoietin acts as a survival and differentiation factor.

Determination of the structure of erythropoietin and its receptor identified these molecules as members of a large family of related macromolecules with pleiotropic activities involving cell survival, growth, differentiation, and inflammation.[5] Structurally, erythropoietin is most closely related to growth hormone and prolactin and more distantly to several interleukins— important molecules in the modulation of inflammation.[6] Following identification of the erythropoietin and the erythropoietin-receptor genes, tissue surveys showed surprisingly that these proteins were also expressed in other tissues,[7] including the brain.[8] These findings clearly suggested that the target(s) of erythropoietin extend beyond the bone marrow where its action is likely not hormonal in nature.

Erythropoietin Action Within the Nervous System

Effects of Direct Administration Into the Brain

The first studies evaluating the function of brain-derived erythropoietin suggested that it possessed trophic activities. Specifically, recombinant human erythropoietin supported the growth and differentiation of septal cholinergic forebrain neurons in vitro, reminiscent of its activity in the bone marrow. However, this erythropoietic therapy also enhanced the survival of injured neurons in vivo after intrathecal injection.[9] Initial study of the cell-type specific expression of these proteins concluded that astrocytes were the source of "brain" erythropoietin and the erythropoietin-receptor-expressing neurons were the presumed target.[10,11]

Primary rodent astrocytes maintained in vitro produce erythropoietin as a response to hypoxic stress.[12] Notably, when these astrocytes are cocultured with neurons, the erythropoietin induced by hypoxic stress protects adjacent neurons from injury.[10] More extensive study of the cell-type-specific expression patterns of the erythropoietin/erythropoietin-receptor genes in the nervous system has clarified that a much more complicated relationship exists as both neurons and astrocytes can express either protein (or both), with a pronounced variation depending upon the anatomic region and the developmental stage that is examined.[13]

The relevance of these findings for the adult mammalian brain was confirmed by the results of in vivo experiments performed using rodent models. These findings clearly demonstrated that ischemic neuronal injury could be impressively reduced by the direct administration of erythropoietin into the brain or the lateral ventricles.[14-16] Immunocytochemical studies additionally showed that the central nervous system (CNS) responds to hypoxic insults by up-regulating the expression of erythropoietin and erythropoietin receptor by cells within the border zone (penumbra) surrounding the necrotic core over a period of 12 to 24 hours.[15-17] These changes are adaptive, as neutralizing the endogenous erythropoietin produced by this up-regulation markedly amplifies injury.[14]

The mechanism(s) of action of recombinant erythropoietin in tissue protection could involve either the protection of neurons from death, the reduction of inflammatory responses, or both. These possibilities have not been adequately investigated to date. Erythropoietin has also been shown to affect stem cell recruitment and differentiation,[18] which could provide important restorative benefits. In the case of ischemic brain injury, acute neuroprotection appears similar to the role of erythropoietin in erythropoiesis. Specifically, in the control of red cell production, erythropoietin does not function strictly as a growth factor, but rather, prevents death of erythrocyte precursors by inhibiting programmed cell death or apoptosis.[4] To accomplish this, erythropoietin induces a gene expression program that includes activation of members of the bcl-2 family of antiapoptotic proteins, especially the long form of BCLx (Bcl-xL).

Regardless of type, brain injury commonly initiates apoptosis. Using an in vitro model, prior study has demonstrated that the neuroprotective effects of recombinant erythropoietin following hypoxic or neurotoxin stress requires protein synthesis,[10] which is required for modulation of programmed cell death. Erythropoietin protects neurons from chemical hypoxia and cerebral ischemic injury by up-regulating Bcl-xL expression.[19] However, in addition to its acute effects on neuronal programmed cell death, erythropoietin also provides beneficial effects through modulation of the inflammation reaction to brain injury. This action appears to account for a major component of its neuroprotective activity (see below).

One way erythropoietin could affect inflammation is by modulation of members of the NFkB family that are principal regulators of inflammatory genes.[20,21] For example, previous work has shown that NFkB itself is strongly up-regulated following traumatic spinal cord injury, a product of macrophages/microglia, endothelial cells, and neurons.[22] One NFkB-dependent gene product is inducible nitric oxide synthase, which has been shown to increase the first day after injury, and peaks by day 7.[23] Erythropoietin is known to modulate nitric oxide production in the brain,[24] as well in other tissues.[25] Recently, erythropoietin has been shown to signal through the NFkB pathway,[26] as well as by the Jak2/STAT5 system.[27-29] Further study will be necessary to determine whether the prominent effects of recombinant erythropoietin on both primary and secondary injury depend in part upon regulation of NFkB or other pathways.

In addition to neurons and astrocytes, capillary endothelial cells forming the blood-brain barrier (BBB) also express erythropoietin receptor at high levels.[17,30] Similar to the response of brain cells to hypoxia, the capillary endothelium responds to hypoxia by up-regulating erythropoietin receptor. In experimental models, administration of erythropoietin not only can prevent endothelial cell death after hypoxic injury, but also is effective in preventing apoptosis of the endothelium following nonischemic injuries.[31] The protective effects of erythropoietin on the vasculature may not be limited to capillaries, however, as erythropoietin also potently protects vascular smooth muscle cells from injury-induced apoptosis.[32]

In sum, the results of multiple studies of animal models have definitively established that an endogenous erythropoietin/erythropoietin-receptor system functions within the CNS outside of hormonal (feedback) control. This local system is activated by hypoxic stressors, is protective of neurons and endothelial cells, and can be beneficially activated by the direct intrathecal administration of erythropoietin (Figure 1).