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What Evidence Supports Use of Erythropoietin as a Novel Neurotherapeutic?

What Evidence Supports Use of Erythropoietin as a Novel Neurotherapeutic?

ABSTRACT: In its hormonal role, erythropoietin is produced by the kidney in response to hypoxic stress and signals the bone marrow to increase the number of circulating erythrocytes. It has become clear in recent years, however, that erythropoietin and its receptor are members of a cytokine superfamily that mediates diverse functions in nonhematopoietic tissues. Nonhormonal erythropoietin actions include a critical role in the development, maintenance, protection, and repair of the central nervous system (CNS). Our group has found serendipitously that recombinant human erythropoietin administered into the systemic circulation is not strictly excluded from the brain. Human recombinant erythropoietin appears within the cerebrospinal fluid in neuroprotective concentrations, probably by translocation initiated by binding to the erythropoietin receptor on the luminal surface of the endothelium. This observation suggested that recombinant human erythropoietin could be therapeutic for CNS diseases, a possibility further supported by positive findings in a model of ischemic stroke. Recombinant human erythropoietin administered systemically either in advance of, or up to 3 hours after, a cerebral arterial occlusion in rats prevents apoptosis of neurons within the ischemic penumbra and reduces infarction volume by 75%. Erythropoietin also dramatically reduces postinfarct inflammation in this model. Other brain and spinal cord injuries such as mechanical trauma, experimental autoimmune encephalitis or subarachnoid hemorrhage also respond favorably to erythropoietin administered within a similar window of time. In addition to ameliorating neuronal injury, erythropoietic therapy also directly modulates neuronal excitability and acts as a trophic factor for neurons in vivo and in vitro. Erythropoietin may therefore provide benefit in epileptic or degenerative neurologic diseases. Given the outstanding safety record for recombinant human erythropoietin after more than a decade in widespread clinical use, the results of multiple preclinical investigations suggest that this cytokine or its derivatives may be useful for treatment of a variety of nervous system diseases. [ONCOLOGY 16(Suppl 10):79-89, 2002]

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
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).

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