Nonerythropoietic, tissue-protective peptides derived
from the tertiary structure of erythropoietin
Michael Brines*†‡, Nimesh S. A. Patel§, Pia Villa¶?, Courtenay Brines*, Tiziana Mennini¶, Massimiliano De Paola¶,
Zubeyde Erbayraktar**, Serhat Erbayraktar**, Bruno Sepodes††, Christoph Thiemermann§, Pietro Ghezzi†¶,
Michael Yamin*, Carla C. Hand†, Qiao-wen Xie*†, Thomas Coleman*†‡‡, and Anthony Cerami*†‡
*Warren Pharmaceuticals, Ossining, NY 10562;†The Kenneth S. Warren Institute, Ossining, NY 10562;§Centre for Translational Medicine and Therapeutics,
William Harvey Research Institute, Barts and London School of Medicine and Dentistry, Queen Mary, University of London, London EC1M 6BQ, England;
¶Mario Negri Institute for Pharmacological Research, 20-20156 Milan, Italy; **Dokuz Eylu ¨l University, Izmir 35340, Turkey;††Faculty of Pharmacy, University
of Lisbon, 1600 Lisbon, Portugal; and?National Research Council, Institute of Neuroscience, 20129 Milan, Italy
Contributed by Anthony Cerami, June 10, 2008 (sent for review May 22, 2008)
plays a critical hormonal role regulating erythrocyte production as
well as a paracrine/autocrine role in which locally produced EPO
protects a wide variety of tissues from diverse injuries. Signifi-
cantly, these functions are mediated by distinct receptors: hema-
a heterocomplex composed of the EPO receptor and CD131, the ?
common receptor. In the present work, we have delimited tissue-
protective domains within EPO to short peptide sequences. We
demonstrate that helix B (amino acid residues 58–82) of EPO,
which faces the aqueous medium when EPO is bound to the
receptor homodimer, is both neuroprotective in vitro and tissue
protective in vivo in a variety of models, including ischemic stroke,
diabetes-induced retinal edema, and peripheral nerve trauma.
Remarkably, an 11-aa peptide composed of adjacent amino acids
forming the aqueous face of helix B is also tissue protective, as
confirmed by its therapeutic benefit in models of ischemic stroke
and renal ischemia–reperfusion. Further, this peptide simulating
the aqueous surface of helix B also exhibits EPO’s trophic effects by
accelerating wound healing and augmenting cognitive function in
rodents. As anticipated, neither helix B nor the 11-aa peptide is
erythropoietic in vitro or in vivo. Thus, the tissue-protective activ-
simulate a portion of EPO’s three-dimensional structure.
cognition ? cytoprotection ? excitotoxicity ? ischemia–reperfusion injury ?
present on the cell membrane of erythrocytic progenitors.
Subsequently, a molecular cascade begins with the phosphory-
lation of Janus tyrosine kinase 2 and ultimately results in
inhibition of programmed cell death, fostering the survival and
maturation of erythroid precursors to erythrocytes [reviewed by
Fisher (1)]. However, over the last 15 years, it has been discov-
ered that EPO is also synthesized locally by many tissues,
especially in response to metabolic stress. This pool of EPO acts
as a multifunctional protective molecule [reviewed by Brines and
Cerami (2)]. In this paracrine/autocrine role, EPO inhibits
apoptosis in a wide variety of cell types and activates multiple
mechanisms to protect stressed tissues, e.g., reducing inflamma-
tion and local edema. EPO also plays crucial roles during
development (3). Therefore, it is not surprising that in the adult
organism, EPO mediates multiple trophic effects, leading to
accelerated healing and tissue regeneration. Finally, EPO has
been shown to enhance cognition in normal (4) as well as
diseased (5) human subjects.
The molecular interaction of EPO with the erythropoietic
receptor (EPOR)2has been studied intensively, such that the
regions of EPO that interact with (EPOR)2have been identified
n its hormonal role, the cytokine erythropoietin (EPO) is
released by the kidney into the circulation in response to
(Fig. 1A). These include portions of helices A and C (site 2), as
well as helix D and the loop connecting helices A and B (site 1)
(6–9). Chemical or mutational modifications of amino acid
residues within these two regions of EPO abolish its binding to
(EPOR)2and, therefore, these modified EPOs are not erythro-
poietic in vivo or in vitro. Remarkably, a number of these
modified EPOs retain potent tissue-protective properties (10).
Clearly, sites 1 and 2, which are essential for erythropoiesis, are
not required for tissue protection.
These observations suggest that an additional receptor for
EPO mediates tissue protection. This receptor is pharmacolog-
ically distinct from that of erythropoiesis, because it exhibits a
lower affinity for EPO and forms distinct molecular species in
cross-linking experiments (11). In prior studies, we have pro-
vided evidence that the receptor that promotes tissue protection
is a heteromer composed of EPOR and CD131, the ? common
receptor (?cR) (12). CD131 also forms receptor complexes with
the ? receptor subunits specific for GM-CSF, IL-3, and IL-5 and
has been termed the ‘‘common’’ receptor [reviewed by Murphy
and Young (13)].
Results from experiments showing that chemical modification
of lysine residues or amino acid substitutions made within sites
1 and/or 2 do not affect tissue protection suggest that other
regions of EPO contain the recognition site for the tissue-
protective receptor. Notably, in aqueous media, EPO’s tertiary
structure is relatively well defined because of the interaction of
the hydrophobic content of its four ?-helices, constraining the
molecule into a compact, relatively rigid, globular structure.
When EPO is bound to the hematopoietic receptor (14), helix B
and parts of the AB and CD loops face the aqueous medium,
away from the homodimer binding sites [Protein Data Bank
(PDB) ID code 1EER; Fig. 1]. These regions do not contain
lysine and therefore are not modified by carbamylation of EPO,
a procedure that produces a selectively tissue-protective com-
pound (10). In view of these observations, we hypothesized that
tissue protection, as distinct from erythropoiesis, depends on a
region within helix B and/or loop AB within the EPO molecule.
Author contributions: M.B., N.S.A.P., C.T., P.G., T.C., and A.C. designed research; M.B.,
and C.C.H. contributed new reagents/analytic tools; M.B., N.S.A.P., P.V., C.B., T.M., M.D.P.,
B.S., Q.-w.X., and T.C. analyzed data; and M.B., N.S.A.P., C.T., M.Y., T.C., and A.C. wrote the
Conflict of interest statement: M.B., C.B., M.Y., Q.-w.X., T.C., and A.C. were employees of
Warren Pharmaceuticals when this work was performed. Warren Pharmaceuticals is
developing erythropoietin analogues and tissue-protective compounds for potential clin-
‡To whom correspondence may be addressed: firstname.lastname@example.org or acerami@
‡‡Present address: Feinstein Institute for Medical Research, Manhasset, NY 11030.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
August 5, 2008 ?
vol. 105 ?
no. 31 ?
Results and Discussion
Based on data available from PDB ID code 1EER, we began the
study by synthesizing helix B peptide (HBP; residues 58–82). As
expected, this molecule, like carbamylated EPO [CEPO (10)],
was not erythropoietic in the UT-7 EPO cell assay or in vivo (see
supporting information (SI) Materials and Methods and Fig. S1).
However, HBP possessed potent neuroprotective activity com-
parable to EPO (Fig. 2A) and to CEPO (15) in a rat motoneuron
model in vitro. In this model, the neurotoxic effects of the
glutamate receptor agonist kainic acid were blocked by either
HBP (1.8 nM) or EPO (3.3 nM). Because HBP is small and does
not contain features designed to resist proteolysis or decrease
clearance, its plasma half-life is presumably very short. It was
therefore of great interest to determine whether HBP exhibited
protective properties in vivo.
HBP was protective in a rat model of middle cerebral artery
occlusion that has previously shown large protective effects for
EPO (16), asialo-EPO (17), and CEPO (10) (Fig. 2B). In this
experiment, HBP administered as a single i.v. dose [1.5 nmol/kg
of body weight (bw)] reduced infarct volume as determined by
tetrazolium salt staining 24 h after reperfusion. HBP was also
associated with an improved behavioral outcome (foot faults of
the saline group 24.6 ? 2 versus 16.4 ? 2 in the single dose HBP
group and 14.5 ? 0.9 in the four-dose HBP group; P ? 0.05
between saline and treated groups). Notably, additional doses of
HBP administered i.p. at 2-h intervals for three additional doses
did not further improve the extent of neuroprotection.
Confirming that HBP was neuroprotective in vitro and in vivo,
we then assessed whether HBP possessed other properties
consistent with EPO’s nonerythropoietic activities. For example,
EPO reduces injury-related local edema in a number of tissues
(18–21), including the retina (22). Specifically, in models of
diabetic retinopathy, hyperglycemia produces endothelial injury,
leading to vascular leakage and retinal edema. Further, it is
notable that in a small, retrospective study of diabetic patients
with macular edema, EPO treatment was associated with an
increase in visual acuity and a decrease in retinal exudates (23).
To determine whether HBP could inhibit diabetes-related ret-
inal edema, rats were administered streptozotocin. After the
confirmation of the diabetic state, HBP (1.5 nmol/kg of bw) or
saline was administered i.p. 5 days each week. After 3 weeks of
hyperglycemia, retinal leakage in the HBP group (as assessed by
extravasation of Evans blue dye) was not different from animals
without diabetes (Fig. 2C). In contrast, retinas from animals that
received only saline exhibited significant edema.
The results of these experiments showed that a peptide
fragment of EPO comprising the amino acid sequence corre-
sponding to helix B exhibited tissue-protective effects similar to
EPO and its nonerythropoietic derivatives in a variety of in vitro
and in vivo models. Previous study has shown that peptides can
Q Q A V E V W Q G L A L L S E A V L R G Q A L L V N S S
(A) Schematized drawing of EPO bound to the hematopoietic receptor dimer
hydrophobic interactions to form a compact, globular structure. Sites 1 and 2
(indicated by dashed boxes) within the topography of the EPO molecule bind
with high affinity to each EPOR monomer. The aqueous face of helix B faces
away from the interior of the receptor, as indicated by the dashed ellipse. (B)
acids 58–82: boxed region; single-letter code). Circled residues show those
amino acid residues on the aqueous face of the 4-3 ?-helix B. Leucine in
residues was synthesized as HBSP. Residues 83–85 are relatively rigidly ori-
ented because of the associated helices. Glutamine in the N-terminal position
can spontaneously undergo cyclization into pyroglutamate (U), forming pH-
BSP (bottom peptide).
Infarct Volume [mm3]
HBP 1.8 nM
µ µ µ
µ µ µ
HBP 1.8 nM + KA 5
µ µ µ
EPO 3.3 nM + KA 5
HPB 1.5 nmole/kg
HBP 1.5 nmole/kg: 4 doses
diabetes + PBS
diabetes + HBP 1.5 nmole/kg
evans blue leakage
[compared to normal]
Mixed anterior horn cultures obtained from the ventral horn of the spinal cord of 14-day rat embryos were treated on the sixth day in vitro by incubation for
48 h with kainic acid (5 ?M) alone or in cotreatment with EPO or HBP. Data are means and SEM;***, P ? 0.001 compared with kainic acid alone. (B) HBP is
neuroprotective in a stroke model. A single dose of HBP administered i.v. immediately after a 1-h arterial occlusion significantly reduces infarct volume
the development of retinal edema in a rat model of diabetes (PBS; n ? 14). Results of three experiments are shown. Evans blue extravasation in normal retinas
(n ? 12) was 6 ? 0.2 ng/mg of dry retina. Data are means and SEM;**, P ? 0.01.
Helix B peptide (HBP) is tissue protective in vitro and in vivo. (A) HBP protects against kainic acid (KA)-induced motoneuron excitotoxic death in vitro.
www.pnas.org?cgi?doi?10.1073?pnas.0805594105Brines et al.
be synthesized to mimic the helical structure of a protein that
interacts with its receptor to reproduce the biological activities
of the full molecule (24). Upon further consideration, however,
we reasoned that because helix B is amphipathic and of the 4-3
?-helix type, specific amino acid residues within the hydrophilic
portion face the external, aqueous face (i.e., every fourth and
third residue in the b and f position, respectively). Spatially (but
not linearly) adjacent residues therein could constitute a recog-
nition site for the tissue-protective receptor.
Data obtained from crystallographic studies of EPO bound to
(EPOR)2show that the aqueous face of helix B consists of amino
acids QEQLERAL (PDB ID code 1EER; Fig. 1B). Thus, a
peptide derived from surface-simulation analysis of EPO should
possess the biological activities of helix B. To test this hypothesis,
a peptide was synthesized to include these surface amino acids
as well as the three residues within the proximal portion of the
BC loop that are relatively constrained by the rigid structure of
the associated helices. The resulting 11-mer helix B surface
peptide (HBSP: QEQLERALNSS), unrelated in primary se-
quence to EPO, was thus intended to mimic a particular feature
of EPO’s three-dimensional structure, notwithstanding the pos-
sible steric constraints of spatially but not linearly adjacent
residues bonded directly together. We subsequently assessed
whether this peptide was a nonerythropoietic, tissue-protective
HBSP, which does not contain site 1 or 2, was, as predicted,
not erythropoietic in vitro (UT7-EPO cells) or in vivo in the rat
(data not shown). However, HBSP was highly active in reducing
the degree of injury observed in a sciatic nerve crush injury
model to a degree identical on a molar basis to EPO and
chemical derivatives of EPO that are not erythropoietic (10, 17).
In this model, the sciatic nerve was reversibly compressed by
using a ligature for a duration of 1 min and single doses of HBP
or HBSP (0.3 nmol/kg of bw) administered i.v. immediately after
removal of the constriction. The tissue-protective potency of
previously reported for asialo-EPO (17) and CEPO (10) and
were found to be equivalent (Fig. 3). In contrast, an equimolar
amount of a 20-mer fragment of pigment epithelium-derived
factor (amino acids 102–121), derived from a biologically active
region of this molecule (25), was inactive.
Thus, a peptide designed to mimic the external, aqueous face
of helix B resembled EPO sufficiently to activate tissue-
protective pathways. In the past, successful surface-simulation
synthesis has been reported for antigenic determinants in pro-
teins (26), as well as the surface of the ?-helix of HIV-1 virus
(27). Although there have also been claims that peptides exhib-
iting enzymatic activity can be synthesized from surface simu-
lation analysis of the catalytic site of an enzyme (28), these have
not been substantiated (29). In retrospect, it seems very unlikely
that a small peptide could effectively reproduce the complex
three-dimensional structure required for enzymatic activity,
because binding sites for proper orientation of the substrate to
the catalytic site require a rigid, three-dimensional scaffold not
structurally attainable by using a small peptide. In the case of
receptor-mediated biological activity, however, binding and ac-
tivation of a receptor can topologically be much simpler. Hence,
a number of examples exist of small peptides that reproduce the
biological activity of a larger protein. For example, the clinically
useful parathyroid hormone fragment (1–34) possesses the same
be noted that a 17-mer peptide derived from a portion of EPO’s
AB loop (residues 30–47) has been reported to possess neuro-
trophic activity (31). It is currently unclear whether this peptide
interacts with the tissue-protective receptor subtype or mediates
it biological effects by a different mechanism.
With respect to the primary structure of HBSP, however, it is
well known that N-terminal glutamine residues can undergo a
spontaneous, irreversible cyclization (particularly at room tem-
perature under acidic conditions) into pyroglutamate (32). In
confirmation of this fact, amino acid analysis of production
batches of HBSP revealed that ?90% of the product possessed
a free N-terminal glutamine, whereas the remainder was cy-
clized. Thus, HBSP was actually a mixture of two peptides. To
determine whether pyroglutamate HBSP (pHBSP) was biolog-
ically active, it was synthesized de novo (Fig. 1B).
pHBSP (which was nonerythropoietic; see Figs. S2–S5) was
evaluated in a rodent model of renal ischemia–reperfusion
groups and administered either vehicle or various amounts of
pHBSP as an i.p. bolus at 1 min, 6 h, and again at 12 h after
reperfusion. Twenty-four hours later, plasma creatinine and
PEDF, pigment epithelium-derived factor. Compounds were administered at
a dose of 0.3 nmol/kg of bw i.v. immediately after a 1-min compression of the
sciatic nerve at the level of the mid-thigh. Data are means and SEM plotted as
the negative of the static sciatic index (SSI). n ? 6–8 for each group;***, P ?
0.001 compared with PBS.
HBSP is equipotent to HBP and EPO in a sciatic nerve injury model.
pHBSP 0.08 nmole/kg
pHBSP 0.8 nmole/kg
pHBSP 8 nmole/kg
pHBSP 0.08 nmole/kg
pHBSP 0.8 nmole/kg
pHBSP 8 nmole/kg
pHBSP 0.08 nmole/kg
pHBSP 0.8 nmole/kg
pHBSP 8 nmole/kg
(C) were measured from mice (n ? 12 each group) as biochemical markers of renal dysfunction and injury subsequent to sham-operation or renal ischemia–
reperfusion injury (bilateral renal pedicle occlusion for 30 min). PBS or pHBSP (8.0 nmol/kg of bw) was administered i.p. 1 min, 6 h, and 12 h into reperfusion.
Data represent mean and SEM;***, P ? 0.001 versus PBS.
Brines et al.
August 5, 2008 ?
vol. 105 ?
no. 31 ?
urea were obtained to estimate renal function and aspartate
aminotransferase to assess injury. The results show a dose-
dependent renoprotective effect, with the lowest dose adminis-
tered (0.08 nmol/kg of bw) ineffective (Fig. 4). The degree of
in this model (33).
Results of administering pHBSP in a stroke model as a single
i.v. dose (1.5 nmol/kg of bw) upon reperfusion after 1 h of
demonstrated a significant reduction in infarct volume at 24 h
[225 ? 20 mm3for pHBSP (n ? 8) compared with 291 ? 23 mm3
for saline (n ? 7); P ? 0.05] and an improvement in neurological
function (saline group foots faults 20.2 ? 0.8 versus 11.2 ? 1.1
in the pHBSP group; P ? 0.001). In contrast, a scrambled version
of HBSP (LSEQARNQSEL; n ? 6) was biologically inactive
(20.1 ? 2.1 foot faults; P ? 0.05 versus the pHBSP group). This
observation provides additional support that the surface struc-
ture of helix B is specific for tissue-protective activities of EPO.
As noted above, EPO has also been observed to mediate other
biological activities in addition to purely tissue-protective effects
(reviewed in refs. 2 and 34). Among these pleiotropic effects,
EPO accelerates wound healing and modulates cognitive func-
tion. For example, EPO has been observed to promote incisional
wound closure in rodent models by reducing ischemic and
reperfusion injury, mobilizing endothelial progenitor cells, aug-
menting angiogenesis, and decreasing inflammation (35). To
determine whether pHBSP could also provide benefits in wound
healing, we examined its effect in the healing of punch biopsy
In this experiment, 3.5-mm-diameter full-thickness skin
wounds were placed at the corners of a 3-cm-wide square on the
shaved and depilated scapular region of the rat. pHBSP (24
nmol/kg of bw) or PBS was administered s.c. daily for 10 days.
The area of open wound, measured in a blinded fashion from
serial digital photographs, exhibited faster healing in animals
that received peptide compared with saline controls (Fig. 5).
Previous studies using systemically administered EPO have
shown acceleration of wound healing in association with in-
creased expression of inducible NO synthase (iNOS) and in-
creased angiogenesis, vascular endothelial growth factor, and
wound collagen content (reviewed in ref. 34).
Historically, one of the first nonerythropoietic activities noted
for EPO was a strong neurotrophic effect (36). EPO and its
nonerythropoietic tissue-protective derivatives have been shown
to possess these properties in vitro, as well as in vivo for injured
(37–39) and normal (40) animals. Recently, by using functional
MRI, EPO has also been shown to enhance cognition in normal
human volunteers (4, 41, 42). To determine whether pHBSP
modulates cognitive function, we used the novel object recog-
nition paradigm in rats, in which memory recall for previously
experienced objects is evaluated. Specifically, rats were exposed
to novel test objects and then reexposed to them 24 h later.
Animals having received galantamine (3 mg/kg i.p. 1 h before
testing; a positive control) displayed enhanced memory reten-
tion of previously experienced objects (Fig. 6). Similarly, animals
receiving pHBSP (24 nmol/kg of bw i.p.) 3 h after the first
exposure to the objects to be learned, or receiving twice daily
doses 5 days before training and continued through the day
immediately after training, showed enhanced memory for the
objects. In contrast, animals that received pHBSP 1 h before the
first object exposure did not show enhancement. Because pH-
BSP was effective only when administered after training, this
molecule likely acts by intensifying the consolidation phase of
In summary, using a variety of in vitro and in vivo models, we
have shown that helix B of EPO has tissue-protective activities
representative of the full molecule. Further, a peptide con-
structed to mimic the external, aqueous surface of EPO without
primary sequence similarity recapitulates EPO’s tissue-
protective, neurotrophic, and reparative properties. Peptide
doses that exhibited tissue protection were similar on a molar
basis to those observed for EPO and are higher than those
required for EPO-mediated erythropoiesis. For example, in the
renal ischemia model, 0.08 nmol/kg of bw (equivalent to ?300
units/kg of bw of EPO) was ineffective, whereas a 10-fold higher
dose elicited strong tissue protection.
Finally, pharmacokinetic studies confirm that pHBSP pos-
sesses a plasma half-life of ?2 min in the rat and rabbit (see SI
Materials and Methods, Figs. S6 and S7, and Tables S1 and S2).
It is especially notable that, similar to asialo-EPO (17), an agent
present within the circulation for only a short time after i.v.
dosing elicits protective effects equivalent to EPO or CEPO with
plasma half-lives of 4–6 h. Tissue-protective peptides may
therefore be of use as pharmacological reagents to delineate
aspects of timing in tissue protection and trophic effects, in
addition to potentially being of therapeutic benefit in a wide
variety of clinical scenarios.
Materials and Methods
The animal protocols followed in this study were approved by the respective
Animal Use and Care Committees of each institution in accordance with the
directives of the Guide for the Care and Use of Laboratory Animals of the
National Research Council or the Home Office Guidance on the operation of
Office or in compliance with national (D.L. n. 116, G.U., suppl. 40, Feb. 18,
1992) and international laws and policies (EU Council Directive 86/609, OJ L
358, 1, Dec. 12, 1987).
Materials. Peptides were obtained from commercial manufacturers. The UT-7
EPO hematopoietic assay (10, 17), motoneuron excitotoxicity study (15, 43),
Wound Area [mm2]
of a rat heal more rapidly after pHBSP (24 nmol/kg of bw) administered daily
via the s.c. route. n ? 9 animals each group. Curves differ at the P ? 0.05 level
by repeated-measures analysis.
Full-thickness punch biopsy wounds placed over the scapular region
tamine (3 mg/kg of bw) on novel object recognition memory test performed
24-h retention test. n ? 8 each group;*, P ? 0.05 versus saline.
Effect of pHBSP (24 nmol/kg of bw) and the positive control galan-
www.pnas.org?cgi?doi?10.1073?pnas.0805594105 Brines et al.
middle cerebral artery occlusion model (44), and sciatic nerve compression
injury (10) were performed as previously reported.
Diabetic Retinal Edema. Fasting male Sprague–Dawley rats weighing ?250 g
were administered streptozotocin (60 mg/kg of bw) i.p., and diabetes was
confirmed by a fasting blood glucose of ?250 mg/dl 2 days later. Diabetic
animals were administered HBP (1.5 nmol/kg of bw) or saline i.p. 5 days a
week, while a third group of normal animals received saline. After 3 weeks,
animals were anesthetized by using isoflurane, and Evans blue solution (45
later, the animals were reanesthetized, a small blood sample was obtained to
determine plasma concentration of Evans blue, and each rat was perfused
with pH 7.4 citrate buffer at 120 mmHg for 2 min and, thereafter, both eyes
were immediately removed. Under an operating microscope, the eyes were
bisected along the equator and the retinas were removed. The retinas were
desiccated at 60°C overnight in a vacuum, weighed, crushed in 120 ?l of
formamide, and incubated at 70°C for 18 additional hours. The retinal form-
amide solution was filter-centrifuged at 15,000 ? g for 30 min to remove
retinal debris. Evans blue concentration was determined by a background-
subtracted absorbance at wavelengths of 620 nm (maximum) and 740 nm
(minimum). Fluid extravasation was calculated as Evans blue (?g)/retina dry
weight (g). Data were analyzed by analysis of variance (ANOVA) followed by
Dunnett’s post hoc test comparison.
Renal Ischemia–Reperfusion Model. Sixty male C57/BL6 mice (?25 g; Charles
River Laboratories) were anesthetized with ketamine (150 mg/kg) and xyla-
zine (15 mg/kg) i.p. Each animal was placed on a homeothermic blanket set at
37°C, and after a mid-line laparotomy, the renal pedicles were clamped for 30
min by using nontraumatic microvascular clamps. pHBSP was administered at
the indicated dose via i.p. injection at 1 min, 6 h, and 12 h after reperfusion.
Twenty-four hours later, mice were reanesthetized and blood was obtained
by cardiac puncture. Plasma urea and creatinine were used as indicators of
renal dysfunction and aspartate aminotransferase was used as an indicator of
renal injury. Data were analyzed by ANOVA followed by Dunnett’s post hoc
Wound Healing. Methods were adapted from the protocol of Padgett et al.
the procedure. Under isoflurane anesthesia, a 5 ? 5 cm region of skin was
shaved on the dorsum in the subscapular region and washed with povidone
was then infiltrated with 1% lidocaine solution and lidocaine-saturated gel
foam was attached with adhesive tape. Wound assessment was obtained by
serial digital photographs that included a 3.5-mm diameter standard. Area
was determined by using digital planimetry and the four measurements were
averaged. Data were analyzed by using a repeated-measures analysis.
Novel Object Recognition in Rats. This model is based on the greater sponta-
neous exploration of a novel object, compared with a familiar object, shown
by rodents (46). Male Wistar rats were assessed for cognitive ability in a test
under dim lighting.
the presence of two identical plastic shapes, and the time spent actively
in the test arena for 5 min (T2) in the presence of one of the familiar objects
and a novel object, and the time spent exploring each object was again
recorded. A recognition index for each object, the ratio of the time spent
exploring either the familiar object or the novel object over the total time
spent exploring both objects (during retention session T2), was used to
measure cognitive (memory) function.
Rats (n ? 8 each group) were treated with the test compounds before the
test period (T1), after T1, or chronically for 5 days before T1, via the i.p. route.
Groups consisted of those that received vehicle, galantamine (3 mg/kg of bw)
administered 1 h before the first 5-min exposure to the two identical objects
to be learned, pHBSP (24 nmol/kg of bw) administered 1 h before the first
5-min exposure to the two identical objects to be learned, pHBSP (24 nmol/kg
of bw) administered 3 h after the first 5-min exposure to the two identical
5 days before training and then 12 and 24 h after training (the last dose was
administered 1 h before the novel object exposure). Data were analyzed by
ANOVA followed by Dunnett’s post hoc test comparison.
ACKNOWLEDGMENTS. We thank Annie Zhu and Deborah Gomez for expert
technical assistance. This work was funded in part by the William Harvey
Research Foundation (N.S.A.P. and C.T.).
1. Fisher JW (2003) Erythropoietin: Physiology and pharmacology update. Exp Biol Med
Biology and clinical promise. Kidney Int 70:246–250.
4. Miskowiak K, O’Sullivan U, Harmer CJ (2007) Erythropoietin enhances hippocampal
response during memory retrieval in humans. J Neurosci 27:2788–2792.
patients by recombinant human erythropoietin. Mol Psychiatry 12:206–220.
6. Boissel JP, Lee WR, Presnell SR, Cohen FE, Bunn HF (1993) Erythropoietin structure-
function relationships. Mutant proteins that test a model of tertiary structure. J Biol
7. Cheetham JC, et al. (1998) NMR structure of human erythropoietin and a comparison
with its receptor bound conformation. Nat Struct Biol 5:861–866.
8. Elliott S, Lorenzini T, Chang D, Barzilay J, Delorme E (1997) Mapping of the active site
of recombinant human erythropoietin. Blood 89:493–502.
9. Wen D, Boissel JP, Showers M, Ruch BC, Bunn HF (1994) Erythropoietin structure-
function relationships. Identification of functionally important domains. J Biol Chem
10. Leist M, et al. (2004) Derivatives of erythropoietin that are tissue protective but not
erythropoietic. Science 305:239–242.
11. Masuda S, et al. (1993) Functional erythropoietin receptor of the cells with neural
characteristics. Comparison with receptor properties of erythroid cells. J Biol Chem
12. Brines M, et al. (2004) Erythropoietin mediates tissue protection through an erythro-
poietin and common beta-subunit heteroreceptor. Proc Natl Acad Sci USA 101:14907–
13. Murphy JM, Young IG (2006) IL-3, IL-5, and GM-CSF signaling: Crystal structure of the
human beta-common receptor. Vitam Horm 74:1–30.
14. Syed RS, et al. (1998) Efficiency of signalling through cytokine receptors depends
critically on receptor orientation. Nature 395:511–516.
15. Mennini T, et al. (2006) Nonhematopoietic erythropoietin derivatives prevent mo-
toneuron degeneration in vitro and in vivo. Mol Med 12:153–160.
16. Brines ML, et al. (2000) Erythropoietin crosses the blood-brain barrier to protect
against experimental brain injury. Proc Natl Acad Sci USA 97:10526–10531.
17. Erbayraktar S, et al. (2003) Asialoerythropoietin is a nonerythropoietic cytokine
with broad neuroprotective activity in vivo. Proc Natl Acad Sci USA 100:6741–
II collagen in the mouse. Arthritis Rheum 52:940–950.
19. Okutan O, Turkoglu OF, Gok HB, Beskonakli E (2008) Neuroprotective effect of
erythropoietin after experimental cold injury-induced vasogenic brain edema in rats.
Surg Neurol, in press.
brain. J Cereb Blood Flow Metab 27:1369–1376.
21. Wu H, et al. (2006) Pretreatment with recombined human erythropoietin attenu-
ates ischemia-reperfusion-induced lung injury in rats. Eur J Cardiothorac Surg
22. Zhang J, et al. (2008) Intravitreal injection of erythropoietin protects both retinal
vascular and neuronal cells in early diabetes. Invest Ophthalmol Vis Sci 49:732–
23. Friedman EA, L’Esperance FA, Brown CD, Berman DH (2003) Treating azotemia-
induced anemia with erythropoietin improves diabetic eye disease. Kidney Int Suppl
24. D’Andrea LD, et al. (2005) Targeting angiogenesis: Structural characterization and
Sci USA 102:14215–14220.
25. Liu H, et al. (2004) Identification of the antivasopermeability effect of pigment
epithelium-derived factor and its active site. Proc Natl Acad Sci USA 101:6605–6610.
26. Kazim AL, Atassi MZ (1980) Antibody combining sites can be mimicked synthetically.
Surface-simulation synthesis of the phosphorylcholine-combining site of myeloma
protein M-603. Biochem J 187:661–666.
27. Dong XN, Chen Y, Chen YH (2007) Surface simulation synthesis: A new strategy to spy
alpha-helix structure. Vaccine 25:6569–6571.
28. Atassi MZ, Manshouri T (1993) Design of peptide enzymes (pepzymes): Surface-
simulation synthetic peptides that mimic the chymotrypsin and trypsin active sites
exhibit the activity and specificity of the respective enzyme. Proc Natl Acad Sci USA
29. Matthews BW, Craik CS, Neurath H (1994) Can small cyclic peptides have the activity
and specificity of proteolytic enzymes? Proc Natl Acad Sci USA 91:4103–4105.
30. Tregear GW, et al. (1973) Bovine parathyroid hormone: Minimum chain length of
synthetic peptide required for biological activity. Endocrinology 93:1349–1353.
31. Campana WM, Misasi R, O’Brien JS (1998) Identification of a neurotrophic sequence in
erythropoietin. Int J Mol Med 1:235–241.
32. Yu L, et al. (2006) Investigation of N-terminal glutamate cyclization of recombinant
monoclonal antibody in formulation development. J Pharm Biomed Anal 42:455–
Brines et al.
August 5, 2008 ?
vol. 105 ?
no. 31 ?
by ischemia/reperfusion in the mouse kidney in vivo. Kidney Int 66:983–989.
34. Arcasoy MO (2008) The non-haematopoietic biological effects of erythropoietin. Br J
35. Buemi M, et al. (2002) Recombinant human erythropoietin influences revasculariza-
tion and healing in a rat model of random ischaemic flaps. Acta Derm Venereol 82:
36. Konishi Y, Chui DH, Hirose H, Kunishita T, Tabira T (1993) Trophic effect of erythro-
poietin and other hematopoietic factors on central cholinergic neurons in vitro and in
vivo. Brain Res 609:29–35.
37. Gonzalez FF, et al. (2007) Erythropoietin enhances long-term neuroprotection and
neurogenesis in neonatal stroke. Dev Neurosci 29:321–330.
38. Lu D, et al. (2005) Erythropoietin enhances neurogenesis and restores spatial memory
in rats after traumatic brain injury. J Neurotrauma 22:1011–1017.
39. Wang L, Zhang Z, Wang Y, Zhang R, Chopp M (2004) Treatment of stroke with
erythropoietin enhances neurogenesis and angiogenesis and improves neurological
function in rats. Stroke 35:1732–1737.
40. Ransome MI, Turnley AM (2007) Systemically delivered erythropoietin transiently
enhances adult hippocampal neurogenesis. J Neurochem 102:1953–1965.
41. Miskowiak K, et al. (2008) Differential effects of erythropoietin on neural and cogni-
tive measures of executive function 3 and 7 days post-administration. Exp Brain Res
42. Miskowiak K, Inkster B, Selvaraj S, Goodwin G, Harmer C (2007) Erythropoietin has no
effect on hippocampal response during memory retrieval 3 days post-administration.
Psychopharmacology (Berlin) 195:451–453.
43. De Paola M, et al. (2008) Chemokine MIP-2/CXCL2, acting on CXCR2, induces mo-
tor neuron death in primary cultures. Neuroimmunomodulation 14:310–316.
44. Villa P, et al. (2007) Reduced functional deficits, neuroinflammation, and secondary
tissue damage after treatment of stroke by nonerythropoietic erythropoietin deriva-
tives. J Cereb Blood Flow Metab 27:552–563.
45. Padgett DA, Marucha PT, Sheridan JF (1998) Restraint stress slows cutaneous wound
healing in mice. Brain Behav Immun 12:64–73.
46. Ennaceur A, Delacour J (1988) A new one-trial test for neurobiological studies of
memory in rats. 1: Behavioral data. Behav Brain Res 31:47–59.
www.pnas.org?cgi?doi?10.1073?pnas.0805594105Brines et al.