Artificial Organs

36(2):139150, Wiley Periodicals, Inc.

2012, Copyright the Authors
Artificial Organs 2012, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Adenosine-5-Triphosphate-Adenosine-Glutathione
Cross-Linked Hemoglobin as
Erythropoiesis-Stimulating Agent
*Jan Simoni, *Grace Simoni, *John F. Moeller, Mario Feola, *John A. Griswold,
and Donald E. Wesson
*Department of Surgery, Texas Tech University Health Sciences Center, Lubbock; HemoBioTech, Inc., Dallas; and Texas
A&M College of Medicine and Scott and White Healthcare, Temple, TX, USA

Abstract: An effective hemoglobin (Hb)-based blood substitute that acts as a physiological oxygen carrier and volume
expander ought to stimulate erythropoiesis. A speedy
replacement of blood loss with endogenous red blood cells
should be an essential feature of any blood substitute
product because of its relatively short circulatory retention
time and high autoxidation rate. Erythropoiesis is a complex
process controlled by oxygen and redox-regulated transcription factors and their target genes that can be affected
by Hb physicochemical properties. Using an in vitro cellular
model, we investigated the molecular mechanisms of erythropoietic action of unmodified tetrameric Hb (UHb) and
Hb cross-linked with adenosine-5-triphosphate (ATP),
adenosine, and reduced glutathione (GSH). These effects
were studied under normoxic and hypoxic conditions.
Results indicate that these Hb solutions have different
effects on stabilization and nuclear translocation of
hypoxia-inducible factor (HIF)-1 alpha, induction of the
erythropoietin (EPO) gene, activation of nuclear factor

(NF)-kappa B, and expression of the anti-erythropoietic

agentstumor necrosis factor-alpha and transforming
growth factor-beta 1. UHb suppresses erythropoiesis by
increasing the cytoplasmic degradation of HIF-1 alpha
and decreasing binding to the EPO gene while inducing
NF-kappa B-dependent anti-erythropoietic genes. Crosslinked Hb accelerates erythropoiesis by downregulating
NF-kappa B, stabilizing and facilitating HIF-1 alpha binding
to the EPO gene, under both oxygen conditions. ATP and
adenosine contribute to normoxic stabilization of HIF-1
and, with GSH, inhibit the NF-kappa B pathway that is
involved in the suppression of erythroid-specific genes.
Proper chemical/pharmacological modification is required
to consider acellular Hb as an erythropoiesis-stimulating agent. Key Words: HemoglobinAdenosine-5triphosphateAdenosineGlutathione Erythropoiesis
Hypoxia-inducible factor-1 alphaNuclear factor-kappa
BErythropoietinHypoxiaNormoxia Astrocytes
Blood substitute.

To be effective oxygen-carrying plasma expanders,

hemoglobin (Hb)-based oxygen carriers (HBOCs)
must fulfill a number of requirements. In addition to
being pathogen-free, nontoxic, and nonimmunogenic,
and having an extended shelf life, these products
should have adequate oxygen-carrying capacity to
permit effective tissue oxygenation, as well as sufficient circulatory retention time. The effective HBOC,
besides being able to immediately maximize blood

flow and tissue oxygenation, should also stimulate

erythropoiesis (14).
The erythropoietic activity of HBOCs is advantageous in acute blood loss treatment as the circulatory
retention time of these products is short (half-life of
less than 24 h) and the heme oxidation rate is high (up
to 30%/day). A rapid replacement of blood loss with
endogenous red blood cells (RBCs) would seem to be
the most attractive therapeutic feature of HBOCs. In
other words, in the treatment of hemorrhage, these
products could serve as a temporary oxygen bridge
until the body would be able to produce enough RBCs
to maintain proper tissue oxygenation and avoid allogeneic blood transfusion (14).
Existing research suggests that the erythropoietic
machinery, which is controlled by oxygen tension

doi:10.1111/j.1525-1594.2011.01431.x
Received October 2011; revised November 2011.
Address correspondence and reprint requests to Dr. Jan Simoni,
Department of Surgery, Texas Tech University Health Sciences
Center, 3601 4th Street, Lubbock, TX 79430, USA. E-mail:
jan.simoni@ttuhsc.edu

139

aor_1431

139..150

140

J. SIMONI ET AL.

FIG. 1. Schematic representation of the

role of unmodified Hb (UHb), first-generation
Hb-based oxygen carriers (HBOCs) with different oxygen affinities (P50) and redox
potentials, and Hb cross-linked with ATP,
adenosine, and GSH (ATP-ADO-GSH-Hb)
in the erythropoietin (EPO) gene induction
and the mechanism of erythropoiesis mediated by HIF-1 alpha, NF-kappa B, TNFalpha, TGF-beta 1, and other factors.
Details of these interrelationships are presented in the text.

and the cellular redox state (511), can be affected

by the intrinsic properties of acellular Hb (1215),
(Fig. 1). Unmodified tetrameric Hb (UHb) and
HBOC with pro-oxidant properties, by altering the
cellular redox state, act as signaling molecules
capable of activating nuclear factor (NF)-kappa B
(16) that regulates many genes that are known to
suppress the erythropoietic response, such as tumor
necrosis factors (TNF)-alpha, interleukin (IL)
1-beta, and transforming growth factor (TGF)-beta
1 (1720). UHb and HBOC with low oxygen affinity
(high P50), by overdelivering oxygen, may degrade
hypoxia-inducible factor (HIF)-1 alpha (11,15),
which acts as a master controller of the erythropoietin (EPO) genethe most important regulator of
proliferation of committed progenitors and an antiapoptotic protector of erythroblasts (10). In normoxia or hyperoxia, hydroxylation of proline
residues by prolyl hydroxylases promotes oxygendependent degradation of HIF-1 alpha, unless it is
stabilized via Hb-mediated oxidative phosphorylation or inhibition of prolyl hydroxylases by reactive
oxygen species (ROS) (15,21,22). Conversely, UHb
and HBOC with strong pressor effects or high affinity for oxygen (low P50) that promotes hypoxia can
stabilize HIF-1 alpha, inducing erythropoiesis (23
25). Assuming that efficacious HBOCs must counteract the hypoxic and pro-oxidant environments
associated with blood loss, the stabilization of HIF-1
alpha by prolonging oxygen deprivation could be
clinically questionable (Fig. 1).
Our previously published studies have shown
that adenosine-5-triphosphate (ATP)-adenosineglutathione (GSH) cross-linked Hb (ATP-ADOGSH-Hb) is an effective stimulator of erythropoiesis
Artif Organs, Vol. 36, No. 2, 2012

while sufficiently delivering oxygen (26,27). This

product was shown to be very efficient in rapid restoration of the RBC mass in severely anemic rodents
and nonhuman primates (26). In children with sicklecell anemia, a single injection of ATP-ADO-GSH-Hb
stimulated erythropoiesis and rapidly normalized the
hematocrit (28). The molecular mechanism of ATPADO-GSH-Hbs erythropoietic action, however, was
not established.
The purpose of the present study was to determine
the actual role of UHb and ATP-ADO-GSH-Hb in
erythropoiesis. This research explores the association
between Hb and redox- and oxygen-regulated transcription factors and their target genes that control
pro- and anti-erythropoietic responses. In this study,
we evaluated these effects in human astrocytes that
are capable of producing EPO as well as cytokines
that act as anti-erythropoietic mediators. These
studies were performed under normoxic and hypoxic
conditions.
MATERIALS AND METHODS
Preparation and characterization of Hb solutions
The Hb solutions were prepared at the Texas Tech
University Health Sciences Center (TTUHSC) Blood
Substitute Manufacturing Facility (Lubbock, TX,
USA). This facility was also used for synthesis and
production of cross-linking agents. All procedures
were done under sterile, pyrogen-free conditions
(Class-100), using USP grade chemicals, water, and
buffer solutions.Blood from Hereford cattle,collected
at the TTUHSC Animal Blood Donor Facility (New
Deal, TX, USA), was used for preparation of Hb
solutions.

ATP-ADENOSINE-GSH-Hb AS ESA
The methods for the preparation and characterization of UHb, ATP-ADO-GSH-Hb, and crosslinking reagents have been described previously
(2631). In brief, Hb was extracted from washed
RBCs using the method of dialysis ultrafiltration.
The complete removal of stromal lipid contaminants
was accomplished by using a liquid/solid-phase
extraction procedure (29). Purification from nonheme proteins and peptides was achieved by using a
heat pasteurization method. The orthogonal multistep procedure that comprises nanofiltration,
membrane chromatography, solvent treatment,
and heat inactivation, was used to ensure complete
removal of viruses and prion proteins (30,31).
The removal of environmental bacterial endotoxin
was achieved with affinity chromatography, and sterility was maintained by membrane filtration (29).
The entire Hb purification process was performed
in the absence of oxygen to prevent heme oxidation (29). After dialysis against 20 mM THAM
(Abbott Laboratories, North Chicago, IL, USA) and
reoxygenation, UHb in a concentration of 10 g/dL
was stored in sterile transfer pack containers
(Fenwal, Deerfield, IL, USA) at -90C. The UHb
solution was then chemically modified according
to an earlier described and patented method (29).
The chemical/pharmacological modification procedure comprises purified bovine Hb cross-linked
intramolecularly with open ring ATP, intermolecularly with open ring adenosine, combined
with GSH, as well as enriched with oxygen free
radical scavengers, nonelectrolytes, and/or electrolytes (29). After selective nanofiltration to eliminate
tetramers and subsequent dialysis, ATP-ADOGSH-Hb in a concentration of 6.4 0.2 g/dL was
stored in sterile transfer pack containers (Fenwal) at
-90C.
Purity of Hb solutions in regard to polar and nonpolar lipids, non-heme proteins and peptides, and
endotoxin was characterized as previously described
(26,29). Molecular weight determination of Hb solutions was obtained by using a size exclusion highpressure liquid chromatography method (ProteinPak 300SW, Waters, Milford, MA, USA). The Hb
solutions surface charges were analyzed by using
isoelectric focusing gel electrophoresis (Amersham
Pharmacia PhastSystem, Piscataway, NJ, USA) and
anion exchange liquid chromatography with
Protein-Pak DEAE 5PW (Waters). The physicochemical properties of Hb solutions, including
oxygen affinity, oncotic pressure, viscosity, osmolarity, pH, and electrolyte concentration, have been
characterized by our standard quality control
methods. The total Hb concentration and level of

141

met-Hb were measured by a spectrophotometric

method (26,29).
Assessment of the effect of UHb and ATP-ADOGSH-Hb on pro-erythropoietic factors: HIF-1 alpha
and EPO in normoxia and hypoxia
In astrocytes, HIF-1 alpha regulates EPO expression that plays a neuroprotective role to shield
neurons from hypoxic/ischemic stress (32).
The initial culture of normal human astrocytes,
2nd passage, was obtained from Clonetics (BioWhittaker, A Cambrex Co., San Diego, CA, USA).
Cells were cultured in 75-cm2 tissue culture flasks
(Corning Glass Works, Corning, NY, USA) with
astrocyte growth medium (AGM) BulletKit medium
(Clonetics) in a humidified atmosphere of 5% CO2
and temperature of 37C, until they reached confluence (approximately 50 000 cells/cm2). Astrocytes
were then subcultured in 6-well cell culture plates
(Corning). Cell passage was carried out using a
trypsin reagent pack (Clonetics). During the transfer,
astrocytes were trypsinized no longer than 5 min. All
experiments were performed using fourth to sixth
cell passage. The astrocytes had previously tested
negative for HIV, hepatitis, mycoplasma, bacteria,
yeast, and fungi, and tested positive for glial fibrillary
acidic protein and stained negative for CD68 and
CNPase (Clonetics Certificate of Analysis).
The confluent astrocytes were incubated for
approximately 18 h with AGM medium supplemented with UHb or ATP-ADO-GSH-Hb in a final
concentration of 0.1, 1.0, and 1.75 g/dL, in normoxic
and hypoxic environments.
Normoxic condition was achieved by the incubation of cells in an atmosphere of 95% air and 5%
CO2. Hypoxic condition (1.5% O2, 93.5% N2, and 5%
CO2) was achieved in a humidified variable aerobic
workstation. Before experimentation, media was preequilibrated overnight at a 1.5% oxygen level. The
control astrocytes were cultured in the absence of Hb
solutions that were replaced by fetal bovine serum
(FBS, HyClone Laboratories, Inc., Logan, UT, USA).
After treatment, the cells were subjected to evaluation by various biochemical and molecular biology
methods.
The impact of Hb solutions on HIF-1 alpha
stabilization and its ability to induce the human EPO
gene in the normoxic and hypoxic conditions was
measured in cellular nuclear extracts using a highthroughput TransAM enzyme-linked immunosorbent assay (ELISA)-based assay (Active Motif,
Carlsbad, CA, USA). In this assay, a 96-well plate
was immobilized with an oligonucleotide containing
a hypoxia-responsive element (5-TACGTGCT-3)
Artif Organs, Vol. 36, No. 2, 2012

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J. SIMONI ET AL.

from a human EPO gene. Nuclear extracts were

obtained from living astrocytes using Nonidet P-40
and lysis buffer supplemented with dithiothreitol
(DDT) and a protease inhibitor cocktail (Active
Motif). Nuclear extracts were subjected for the detection of HIF-1 alpha. HIF-1 alpha that was present in
the nuclear extracts bonded to the human EPO gene
and became accessible to primary antibodies. Then,
the primary antibodies were recognized by secondary, HRP-conjugated antibodies, which provided a
sensitive colorimetric readout. The reaction was read
at 450 nm using a 3550-UV microplate reader (BioRad Laboratories, Richmond, CA, USA). Results
were expressed in optical density (OD) at 450 nm per
2.5 mg of nuclear extract. The COS-7 nuclear extract
provided by the manufacturer was used as a positive
control for HIF-1 alpha activation and binding to the
EPO gene.
The influence of Hb solutions on EPO synthesis
was assessed in the cell culture supernatants using
highly specific Quantikine In Vitro Diagnostic
Human Erythropoietin ELISA (R&D Systems, Inc.,
Minneapolis, MN, USA), according to the manufacturers protocol. The results were expressed in
mU/mL.
Assessment of the effect of UHb and ATP-ADOGSH-Hb on anti-erythropoietic factors: NF-kappa
B, TNF-alpha, and TGF-beta 1 in normoxia and
hypoxia
Human astrocytes are known to produce cytokines
(i.e., TNF, IL-1, IL-6) and growth factors (i.e., TGFbeta 1) (3335), which act as anti-erythropoietic
agents (1720).
All experiments were carried out using the human
astrocytes model and Hb concentrations as described
above. After treatment, the assessment of nuclear
activation and DNA binding of NF-kappa B was
assayed in cellular nuclear extracts using TransAM
NF-kappa B p65 transcription Factor Assay Kit
(Active Motif). This ELISA-based method detects
and quantifies NF-kappa B activation, using oligonucleotide containing the NF-kappa B consensus site
(5-GGGACTTTCC-3) immobilized on a 96-well
plate. The nuclear extracts were obtained from living
astrocytes using complete lysis buffer that contained
DTT and a protease inhibitor cocktail supplied by
the manufacturer. The complete binding buffer was
supplemented with DTT and herring sperm DNA.
After incubation, the formed DNAprotein complex
was accessible to primary antibodies, which recognized an epitope on p65, only when NF-kappa B
was activated and bound to DNA. This reaction was
then recognized with HRP-conjugated secondary
Artif Organs, Vol. 36, No. 2, 2012

antibodies against p65, and developed using a benzidine derivative and hydrogen peroxide. The reaction
was read at 450 nm using a microplate reader (BioRad Model 3550-UV). Results were expressed in OD
at 450 nm per 2.5 mg of whole-cell extract. The HeLa
whole-cell extracts, provided by the manufacturer,
were used as a positive control for NF-kappa B activation and DNA binding.
The production of factors with anti-erythropoietic
activities (TNF-alpha and TGF-beta 1) was assessed
with commercially available ELISA kits. TNF-alpha
was assayed using a TNF-alpha human EIA Kit
(Cayman Chemical,Ann Arbor, MI, USA), according
to the manufacturer. TGF-beta 1 was assessed with
Human TGF-beta 1 Quantikine Immunoassay (R&D
Systems). In this test, latent TGF-beta 1 in cell culture
supernates was transferred into the immunoreactive
form by acid activation and neutralization, then
assayed using a microplate with immobilized TGFbeta soluble receptor and expressed in pg per mL.
Data evaluation and statistical analyses
All experiments were conducted in triplicate and
results were expressed as mean standard deviation
(M SD). The differences among and between the
groups were evaluated with ANOVA, using the StatWorks statistical package (Cricket Software, Philadelphia, PA, USA).
RESULTS
Characteristics of Hb solutions
Hb was completely purified from non-heme proteins, peptides, phospholipids, viral, bacterial, and
prion contaminants. The concentration of bacterial
endotoxin in UHb and ATP-ADO-GSH-Hb was
below the Food and Drug Administration requirement of 0.25 EU/mL. Hb solutions were enriched
with electrolytes and mannitol. The final formulations were isotonic. Although UHb solution was
comprised only of 64.5 kDa tetramers, ATP-ADOGSH-Hb contained polymers below 500 kDa and less
than 5% of tetramers. Both Hb solutions had less
than 5% of met-Hb. UHb and ATP-ADO-GSH-Hb
had an isoelectric point (pI) of 6.87.0 and 6.16.3,
respectively. Both Hb solutions had a similar P50 of
23 3 mm Hg at chloride concentration of 100 mm
that provided the proper oxygen delivery index
(36,37). The AGM environment did not affect the
affinity of either Hbs for oxygen. The typical physicochemical characteristics are listed below:
Oxy-Hb: 6.4 0.2 g/dL
Met-Hb % of oxy-Hb: <5

ATP-ADENOSINE-GSH-Hb AS ESA

143

FIG. 2. The effects of unmodified Hb (UHb)

and Hb cross-linked with ATP, adenosine,
and GSH (ATP-ADO-GSH-Hb) on HIF-1
alpha stabilization in normoxia and hypoxia.
White bars, UHb under hypoxia; light gray
bars, UHb under normoxia; black bars, ATPADO-GSH-Hb under hypoxia; dark gray
bars, ATP-ADO-GSH-Hb under normoxia;
(a) significant differences at P < 0.001
between controls and among Hb group; (b)
significant differences at P < 0.01 between
controls and among Hb group; (c) significant
differences at P < 0.05 between controls
and among Hb group; (A) significant differences at P < 0.001 between Hb groups; (B)
significant differences at P < 0.01 between
Hb groups; (C) significant differences at
P < 0.05 between Hb groups (details in the
text).

CO-Hb % of oxy-Hb: <5

pH: 7.88.1 U (THAM 20 mM)
Sodium: 140 mM
Potassium: 4 mM
Chloride: 100 mM
Sodium Lactate: 27 mM
Calcium: 1.3 mM
Mannitol: 0.8 mg/mL
Colloid oncotic pressure: 2023 mm Hg
Osmolarity: 300325 mOsm/kg

Effects of UHb and ATP-ADO-GSH-Hb on

pro-erythropoietic factors
The effect of unmodified and ATP-ADO-GSH-Hb
on pro-erythropoietic factors, HIF-1 alpha, and EPO
is presented in Fig. 2 and Table 1.
As shown in Fig. 2, HIF-1 alpha can be found in the
nuclear extracts of astrocytes under hypoxic and normoxic conditions. The tested Hb solutions had a different impact on HIF-1 alpha stabilization, nuclear
translocation, and binding to the EPO gene in
hypoxic and normoxic environments.
The UHb solution increased the cytoplasmic degradation of HIF-1 alpha and generally decreased its
binding activity to the EPO gene, especially in
hypoxia. On the contrary, ATP-ADO-GSH-Hb preserved HIF-1 alpha under both oxygen conditions
(Fig. 2). This Hb solution in a dose-dependent
manner stabilized HIF-1 alpha and increased its
binding to the EPO gene, and was most effective at a
dose of 1.75 g/dL. The observed differences were statistically significant (P < 0.001).
The production of EPO under normoxic and
hypoxic conditions significantly increased following

incubation of astrocytes with ATP-ADO-GSH-Hb at

all tested concentrations (Table 1). In normoxia, this
Hb was able to increase the EPO level 11-fold at a
concentration of 0.1 g/dL, 21-fold at 1.0 g/dL, and
more than 61-fold at 1.75 g/dL. In hypoxia, the impact
of ATP-ADO-GSH-Hb on EPO synthesis was significantly enhanced at all concentrations. ATP-ADOGSH-Hb acted effectively on EPO synthesis in both
oxygen conditions. However, UHb blocked the synthesis of EPO. These effects were more pronounced
at higher Hb concentrations (Table 1).
Effects of UHb and ATP-ADO-GSH-Hb on
anti-erythropoietic factorsNF-kappa B, TGF-beta
1, and TNF-alpha
As presented in Fig. 3, ATP-ADO-GSH-Hb inhibited NF-kappa B activation at all tested concentrations and oxygen levels. On the contrary, UHb
activated NF-kappa B in a dose-dependent manner.
This effect was more pronounced in the hypoxic condition (P < 0.001).
The production of the most potent antierythropoietic factors, TGF-beta 1 and TNF-alpha, in
response to tested Hb solutions revealed that these
products affected human astrocytes differently
(Tables 2 and 3). ATP-ADO-GSH-Hb inhibited the
formation of TGF-beta 1 (Table 2) and did not
increase the production of TNF-alpha (Table 3) at all
tested concentrations and oxygen conditions. This
effect can be linked to the inability of this Hb solution to induce NF-kappa B. UHb, however, increased
the production of both TGF-beta 1 (Table 2) and
TNF-alpha (Table 3), especially at higher concentrations (1 and 1.75 g/dL).
Artif Organs, Vol. 36, No. 2, 2012

Artif Organs, Vol. 36, No. 2, 2012

Significance control
versus experimental

N.S.
N.S.
P < 0.001
N.S.
P < 0.001
P < 0.001

M SD

0.23 0.56
3.05 2.56
2.35 1.62
1.02 0.26
3.96 2.68
23.00 8.21
57.27 27.60
N.S.
P < 0.001
P < 0.001

Significance between
experimental groups
0.29 0.50
2.63 1.33
3.88 1.41
1.96 1.53
3.19 1.58
6.11 1.86
17.67 4.00

M SD

P < 0.01
P < 0.01
N.S.
P < 0.05
P < 0.001
P < 0.001

Significance control
versus experimental

N.S.
P < 0.001
P < 0.001

Significance between
experimental groups

Normoxia

P < 0.001
N.S.
N.S.
N.S.
N.S.
P < 0.001
P < 0.001

Significance hypoxia
versus normoxia

N.S.
P < 0.05
P < 0.01
P < 0.05
N.S.
N.S.

M SD

202.70 44.40
204.60 75.40
336.80 85.50
446.10 81.30
102.90 59.30
164.20 59.40
201.40 54.00

M SD, mean standard deviation; N.S., not significant.

Control
Unmodified Hb 0.1 g/dL
Unmodified Hb 1.0 g/dL
Unmodified Hb 1.75 g/dL
ATP-ADO-GSH-Hb 0.1 g/dL
ATP-ADO-GSH-Hb 1.0 g/dL
ATP-ADO-GSH-Hb 1.75 g/dL

TGF-beta 1 (pg/mL)

Significance
control versus
experimental

Hypoxia

P < 0.05
P < 0.01
P < 0.001

Significance between
experimental groups

117.70 47.90
245.70 26.60
345.50 50.40
573.10 77.80
153.80 65.90
147.50 58.40
182.90 65.90

M SD

P < 0.01
P < 0.01
P < 0.001
N.S.
N.S.
N.S.

Significance control
versus experimental

P < 0.05
P < 0.01
P < 0.001

Significance between
experimental groups

Normoxia

P < 0.05
N.S.
N.S.
P < 0.05
N.S.
N.S.
N.S.

Significance hypoxia
versus normoxia

TABLE 2. The effects of unmodified tetrameric Hb and Hb cross-linked with ATP, adenosine, and GSH (ATP-ADO-GSH-Hb) on anti-erythropoietic TGF-beta 1
production by human astrocytes under hypoxic and normoxic conditions

M SD, mean standard deviation; N.S., not significant.

Control
Unmodified Hb 0.1 g/dL
Unmodified Hb 1.0 g/dL
Unmodified Hb 1.75 g/dL
ATP-ADO-GSH-Hb 0.1 g/dL
ATP-ADO-GSH-Hb 1.0 g/dL
ATP-ADO-GSH-Hb 1.75 g/dL

EPO (mU/mL)

Hypoxia

TABLE 1. The effects of unmodified Hb and Hb cross-linked with ATP, adenosine, and GSH (ATP-ADO-GSH-Hb) on pro-erythropoietic EPO production by
human astrocytes under hypoxic and normoxic conditions

144
J. SIMONI ET AL.

N.S.
N.S.
N.S.
P < 0.05
N.S.
N.S.
N.S.

N.S.
N.S.
P < 0.05

N.S.
N.S.
P < 0.05
N.S.
N.S.
N.S.
5.47 3.31
6.46 3.51
5.65 3.26
14.92 2.69
5.55 3.20
5.30 3.06
5.27 3.04

N.S.
N.S.
P < 0.01
N.S.
N.S.
N.S.
Control
Unmodified Hb 0.1 g/dL
Unmodified Hb 1.0 g/dL
Unmodified Hb 1.75 g/dL
ATP-ADO-GSH-Hb 0.1 g/dL
ATP-ADO-GSH-Hb 1.0 g/dL
ATP-ADO-GSH-Hb 1.75 g/dL

6.35 3.66
4.87 2.82
5.42 3.13
27.04 5.06
5.61 3.24
4.72 2.74
5.39 3.12

N.S.
N.S.
P < 0.01

Significance hypoxia
versus normoxia
Significance between
experimental groups
M SD
TNF-alpha (pg/mL)

145
DISCUSSION

Significance
control versus
experimental

Significance between
experimental groups

M SD

Significance control
versus experimental

Normoxia
Hypoxia

TABLE 3. The effects of unmodified tetrameric Hb and Hb cross-linked with ATP, adenosine, and GSH (ATP-ADO-GSH-Hb) on anti-erythropoietic TNF-alpha
production by human astrocytes under hypoxic and normoxic conditions

ATP-ADENOSINE-GSH-Hb AS ESA

This study illustrates that UHb might dose dependently inhibit erythropoiesis by facilitating HIF-1
alpha degradation and NF-kappa B activation with
subsequent expression of anti-erythropoietic factors,
TGF-beta 1 that blocks differentiation of erythroid
progenitor cells and promotes apoptosis, and TNFalpha that prevents HIF-1 alpha binding to the EPO
gene (Fig. 1). However,ATP-ADO-GSH-Hb exhibits
pro-erythropoietic potential by stabilizing and
increasing HIF-1 alpha binding to the EPO gene and
downregulating NF-kappa B (Fig. 1).
We previously showed that chemical/pharmacological modification of Hb with ATP, adenosine,
and GSH produces vasodilation and a proper oxygen
delivery index (26,27). This present study revealed
that oxygen supplied by ATP-ADO-GSH-Hb does
not induce degradation of HIF-1 alpha allowing
effective induction of the EPO gene in the normoxic
condition (Fig. 2, Table 1). The earlier reported antiinflammatory and anti-apoptotic potential of ATPADO-GSH-Hb (16,23,26,3843) is mirrored in the
results of the current study, which showed that downregulation of NF-kappa B with subsequent suppression of the TGF-beta 1 and TNF-alpha genes is
essential in achieving a positive erythropoietic
response (Fig. 3, Tables 2 and 3).
These molecular findings are supported by our previous observations that ATP-ADO-GSH-Hb is an
effective inducer of erythropoiesis in vivo (26,28).
ATP-ADO-GSH-Hb, when administered in a
volume corresponding to 25% total blood volume to
pediatric sickle-cell anemia patients in aplastic crisis,
stimulated the bone marrow to a significant erythropoietic effect. The number of reticulocytes increased
from 3.7 3.09 to 49.2 6.5%, and blood Hb
increased from 6.34 2.0 to 9.54 0.72 g/dL after
3 days (28). A similar erythropoietic effect was also
observed in nonhuman primates and rodents (26).
A natural response to hypoxia is an increase in
erythropoiesis (44). Erythropoiesis is tightly regulated by oxygen tension and the cellular redox state
that involves oxygen (HIF-1 alpha) and redoxregulated transcription factors (NF-kappa B) and
many growth factors (i.e., EPO, IL-3, IL-9, stem cell
factor, granulocyte macrophage-colony stimulating
factor), and minerals, particularly iron (611,15,17
20). EPO, which is produced by Kupffer cells in the
fetal liver and peritubular interstitial cells in the adult
kidney in response to hypoxia, is the main regulator
of erythropoiesis via rescue of erythroid progenitor
cells from apoptosis (7,45). EPO can also be produced in the central nervous system by astrocytes,
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J. SIMONI ET AL.

FIG. 3. The effects of unmodified Hb (Hb)

and Hb cross-linked with ATP, adenosine,
and GSH (ATP-ADO-GSH-Hb) on NF-kappa
B induction in normoxia and hypoxia. White
bars, UHb under hypoxia; light gray bars,
UHb under normoxia; black bars, ATP-ADOGSH-Hb under hypoxia; dark gray bars,
ATP-ADO-GSH-Hb under normoxia; (a) significant differences at P < 0.001 between
controls and among Hb group; (b) significant
differences at P < 0.01 between controls
and among Hb group; (c) significant differences at P < 0.05 between controls and
among Hb group; (A) significant differences
at P < 0.001 between Hb groups; (B) significant differences at P < 0.01 between Hb
groups; (c) significant differences at
P < 0.05 between Hb groups (details in text).

shielding hypoxic neurons from apoptosis (32).

As human astrocytes are also known to produce inflammatory cytokines (3335) that act as
anti-erythropoietic agents (1720), nowadays, the
astrocyte cell culture model is used to study the
mechanism of erythropoiesis at the molecular level.
HIF-1 alpha that mediates EPO gene expression is
also a promoter of other genes important in adaptation to hypoxia, such as transferrin, vascular endothelial growth factor, nitric oxide synthase,
endothelin-1, heme oxygenase-1, glucose transporter
1, etc. (11,22,25,4649). In the hypoxic condition, a
lack of oxygen suppresses the degradation of HIF-1
alpha, which rapidly translocates from the cytoplasm
to the nucleus and acts as a master regulator of erythropoiesis (46). In the normoxic condition, oxygenmediated hydroxylation of HIF-1 alpha proline
residues initiates its rapid degradation blocking
erythropoiesis (21,22,24), (Fig. 1). There are, however, some exceptions. For instance, in oxidative
stress, ROS by changing the cellular redox equilibrium that activates NF-kappa B and induces inflammatory genes (i.e., TNF-alpha, IL-1 beta, IL-6) may
stabilize HIF-1 alpha in normoxia (Fig. 1). These
inflammatory cytokines, however, can also inhibit
HIF-1 alpha binding to the EPO gene suppressing
erythropoiesis (Fig. 1), as observed in cancer patients.
Similarly, another inflammatory mediator TGF-beta,
which is also known to stabilize HIF-1 alpha under
normoxic conditions, is capable of blocking the differentiation of erythroid progenitor cells and stimulating apoptosis (1720), as observed in end-stage
renal disease patients (19,50,51). In addition, ROS
can stabilize HIF-1 alpha in normoxia by inhibiting
prolyl hydroxylase activity (49).
Artif Organs, Vol. 36, No. 2, 2012

Hb that affects the cellular oxygen content as well

as the redox equilibrium can easily influence the
above-presented mechanisms. In fact, the involvement of Hb in erythropoietic responses is known
since Amberson in the late 1940s observed a hematocrit increase in a human receiving a crude Hb solution (52). Savitsky et al. observed a similar effect
using a stroma-free Hb solution (53). All subjects
treated with these Hb solutions showed systemic
hypertension and renal failure. At that time, the
authors were unable to explain the mechanism of
these events. By applying current knowledge, the
erythropoietic responses seen in Ambersons and
Savitskys clinical trials associated with pathological
reactions, such as hypertension, renal failure, and
even death, resulted from Hbs intrinsic toxicity
(14,13).
Several first-generation HBOCs with the typical
Hb intrinsic toxicity profile have also been reported
to possess some erythropoietic activity. In fact,
recombinant Hb, rHb1.1Optro (Somatogen, Inc.,
Boulder, CO, USA) given intravenously to mice in
extremely low doses, resulted in increased early precursors of RBC in the bone marrow and an increase
in hematocrit. Without providing the mechanism, the
authors concluded that rHb1.1 works either directly
on progenitor cells or indirectly to enhance hematopoiesis (37). However, a study with larger, clinically
relevant doses of cross-linked Hbs in rabbits revealed
that HBOCs at high concentrations did not produce a
significant variation in the generation of erythroidcommitted cells (54). While erythropoietic activity of
HemAssist (Baxter Healthcare Corporation, Deerfield, IL, USA) was not reported (55), Hemopure
(Biopure, Inc., Cambridge, MA, USA) and Poly-

ATP-ADENOSINE-GSH-Hb AS ESA
Heme (Northfield Laboratories Inc., Evanston, IL,
USA) have been characterized as products with some
erythropoietic potency largely accelerated by
supplemental recombinant human erythropoietin
(rEPO) (5658). However, Hemolink (Hemosol
Corp., Mississauga, ON, Canada) was never characterized as a product that stimulates erythropoiesis
(59,60).
Despite the mixed results, these products have in
common a very well-documented vasoconstrictive
effect (14) that perhaps promotes hypoxic HIFalpha stabilization and EPO induction (61). The literature also demonstrates that many clinically tested
HBOCs not only mediate vasoconstrictive events but
also are redox active and able to activate NF-kappa B
(14,13) and its target genes, many of which influence
the erythropoietic process (13,16,23,38,6264). Many
resulting cytokines (i.e., TNF-alpha, IL-1 beta, etc.)
might stabilize HIF-1 alpha even in normoxia;
however, they may also inhibit binding of HIF-1
alpha to the EPO gene (17,49,65), blocking
erythropoiesis. Therefore, the reported varied erythropoietic potential of UHb and tested HBOCs
appears to be the net effect between possibly competing forces of vasoconstriction and cellular redox
changes (Fig. 1).
These effects, however, are in contradiction with
the intended role of HBOCs, which is delivery of
sufficient oxygen to hypoxic tissues without causing
ischemic and inflammatory responses. Erythropoietic
effects under these circumstances should be considered pathologic.
The basic research on erythropoietic activity of Hb
is limited. It was reported that Hb under hypoxic
conditions increased the expression of HIF-1 alpha,
which was related to the loss of ferrous-Hb and accumulation of ferric-Hb (oxidation of heme). In this
study, the authors used an HBOC similar to that of
HemAssist. No connection between Hb and erythropoiesis was made (15). Other researchers have investigated HIF-1 alpha as an indicator of local hypoxia
following treatment with HBOC and liposomeencapsulated Hb (Samaja et al. and Murayama
et al.in this issue). Established evidence exists that
Hb which triggers NF-kappa B may suppress HIF-1
alpha regulated genes, particularly EPO (13,23,64
66). It was also reported that high activity of the
NF-kappa B pathway in early erythroid progenitors
is involved in the suppression of erythroid-specific
genes (65).
An evident lack of erythropoietic activity of
HBOCs under current development can be summarized by the statement of Dr. Klein from the Department of Transfusion Medicine at the National

147

Institutes of Health, Bethesda, MD. In his review

article entitled: Blood substitutes: how close to a
solution? he stated that: . . . hemoglobin-derived
red cell substitutes from human, bovine, and recombinant sources in phase III trials all have a half-life
measured in hours and are unlikely to replace transfusions or drugs that stimulate erythropoiesis for
chronic anemia, but they may play a role: (i) as a
bridge to transfusion when no compatible blood is
immediately available; (ii) as an adjunct to the
autologous hemodilution management of surgery, or
even (iii) in radiation therapy or the management of
cancer . . . (67).
As clinically tested HBOCs lack a demonstrable
physiologic erythropoietic response, the need still
exists for an artificial oxygen carrier with intrinsic
erythropoietic activity that eliminates the requirement for supplementary transfusions and/or expensive rEPO treatment.
To address these problems, we have developed
ATP-ADO-GSH-Hb that utilizes a novel concept
of pharmacologic cross-linking (26,28,29). This
chemical/pharmacologic modification with ATP,
adenosine, and GSH does not interfere with Hb respiratory function, but provides Hb molecules with
new medicinal properties, which appear to be an
effective strategy in elimination of intrinsic toxic
effects of Hb, such as vasoconstriction, oxidative
stress, and inflammation (26). In this product, ATP
stabilizes the Hb tetramer and prevents its dimerization, and adenosine allows the creation of Hb oligomers, avoiding the formation of toxic high-molecularweight polymers. ATP also produces the vasodilatory
effect via activation of P2Y receptors (26). Adenosine counteracts the vasoconstrictive and proinflammatory properties of Hb with the activation of
adenosine A2 receptors, which produce vasodilatation, moderation of inflammatory reactions, and
prevention of platelet aggregation (2629,3843).
The activation of adenosine A3 receptor provides
cytoprotection. The conjugation of Hb with GSH
shields heme from ROS and NO, thus lowering the
Hb pro-oxidant and vasoconstrictive potential
(16,26,38,43). At the same time, GSH introduces a
more electronegative charge onto the surface of the
Hb molecule that blocks Hbs transglomerular and
transendothelial passage, and makes it less accessible
to phagocytes (26,38,39,68).
This current study revealed that ATP-ADOGSH-Hb stabilizes HIF-1 alpha and facilitates its
binding to the EPO gene under both the normoxic and
hypoxic conditions (Fig. 2, Table 1). Moreover, a
downregulated NF-kappa B creates a more proerythropoietic environment, by elimination of the
Artif Organs, Vol. 36, No. 2, 2012

148

J. SIMONI ET AL.

anti-erythropoietic factors, TNF-alpha and TGF-beta

1 (Fig. 3, Tables 2 and 3). This observation provides
the molecular basis for the earlier observed in vivo
erythropoietic responses in humans and animals
(26,28).
Although the roles of individual chemical/
pharmacological elements in our HBOC need
further investigation, it is obvious that the ability of
this product to stabilize HIF-1 alpha in normoxia is
the principal mechanism behind its erythropoietic
action previously observed in humans and animals
(26,28). It has been known for more than four
decades that adenosine and its analogues stimulate
erythropoiesis (69,70).Adenosine is a key acute regulator in response to hypoxia (71). The hypoxic inhibition of adenosine deaminase prolongs adenosines
protective activity, specifically in inhibition of prolyl4-hydroxylase and generation of cyclic AMP. Cyclic
AMP that is raised following stimulation of adenosine A2 receptors by ATP-ADO-GSH-Hb, which was
evidenced in our previous studies (16,26), possesses
strong pro-erythropoietic activities. It was reported
that cyclic AMP counteracts the inhibition of the
EPO gene by inflammatory cytokines, TNF-alpha
and IL-1 alpha (72). In fact, our present study documented effective downregulation of NF-kappa B and
complete suppression of TNF-alpha by ATP-ADOGSH-Hb (Fig. 3, Table 3). Another contributory
factor toward an effective erythropoietic response
was the inevitably low pro-oxidant potential of our
HBOC maintained by GSH (16,23,26,38,43,66). The
role of heme-iron in this observed erythropoietic
response is awaiting elucidation (73).
CONCLUSION
Based on this current study and our previous
preclinical and clinical observations, it can be
concluded that the chemical/pharmacologic modification of Hb with ATP, adenosine, and GSH
resulted in an HBOC with erythropoietic activity in
normoxic and hypoxic clinical scenarios. This
HBOC, by delivering oxygen and expressing proerythropoietic potential, can serve as a primary
therapy to maintain tissue oxygenation and a secondary therapy to normalize the hematocrit through
stimulation of patients erythropoietic responses.
ATP-ADO-GSH-Hb should be considered as a
novel erythropoiesis-stimulating agent in the treatment of acute and chronic anemias.
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