simoni-2012-adenosine-5'-triphos

aor_1431 139..150

Adenosine-5′-Triphosphate-Adenosine-GlutathioneCross-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 ‡TexasA&M College of Medicine and Scott and White Healthcare, Temple, TX, USA

Abstract: An effective hemoglobin (Hb)-based blood sub-stitute that acts as a physiological oxygen carrier and volumeexpander ought to stimulate erythropoiesis. A speedyreplacement of blood loss with endogenous red blood cellsshould be an essential feature of any blood substituteproduct because of its relatively short circulatory retentiontime and high autoxidation rate.Erythropoiesis is a complexprocess controlled by oxygen and redox-regulated tran-scription factors and their target genes that can be affectedby Hb physicochemical properties. Using an in vitro cellularmodel, we investigated the molecular mechanisms of eryth-ropoietic action of unmodified tetrameric Hb (UHb) andHb cross-linked with adenosine-5′-triphosphate (ATP),adenosine, and reduced glutathione (GSH). These effectswere studied under normoxic and hypoxic conditions.Results indicate that these Hb solutions have differenteffects on stabilization and nuclear translocation ofhypoxia-inducible factor (HIF)-1 alpha, induction of theerythropoietin (EPO) gene, activation of nuclear factor

(NF)-kappa B, and expression of the anti-erythropoieticagents—tumor necrosis factor-alpha and transforminggrowth factor-beta 1. UHb suppresses erythropoiesis byincreasing the cytoplasmic degradation of HIF-1 alphaand decreasing binding to the EPO gene while inducingNF-kappa B-dependent anti-erythropoietic genes. Cross-linked Hb accelerates erythropoiesis by downregulatingNF-kappa B,stabilizing and facilitating HIF-1 alpha bindingto the EPO gene, under both oxygen conditions. ATP andadenosine contribute to normoxic stabilization of HIF-1and, with GSH, inhibit the NF-kappa B pathway that isinvolved in the suppression of erythroid-specific genes.Proper chemical/pharmacological modification is requiredto consider acellular Hb as an erythropoiesis-stimu-lating agent. Key Words: Hemoglobin—Adenosine-5′-triphosphate—Adenosine—Glutathione—Erythropoiesis—Hypoxia-inducible factor-1 alpha—Nuclear factor-kappaB—Erythropoietin—Hypoxia—Normoxia—Astrocytes—Blood substitute.

To be effective oxygen-carrying plasma expanders,hemoglobin (Hb)-based oxygen carriers (HBOCs)must fulfill a number of requirements. In addition tobeing pathogen-free, nontoxic, and nonimmunogenic,and having an extended shelf life, these productsshould have adequate oxygen-carrying capacity topermit effective tissue oxygenation, as well as suffi-cient circulatory retention time.The effective HBOC,besides being able to immediately maximize blood

flow and tissue oxygenation, should also stimulateerythropoiesis (1–4).

The erythropoietic activity of HBOCs is advanta-geous in acute blood loss treatment as the circulatoryretention time of these products is short (half-life ofless than 24 h) and the heme oxidation rate is high (upto 30%/day). A rapid replacement of blood loss withendogenous red blood cells (RBCs) would seem to bethe most attractive therapeutic feature of HBOCs. Inother words, in the treatment of hemorrhage, theseproducts could serve as a temporary “oxygen bridge”until the body would be able to produce enough RBCsto maintain proper tissue oxygenation and avoid allo-geneic blood transfusion (1–4).

Existing research suggests that the erythropoieticmachinery, 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 SciencesCenter, 3601 4th Street, Lubbock, TX 79430, USA. E-mail:[email protected]

Artificial Organs36(2):139–150, Wiley Periodicals, Inc.© 2012, Copyright the AuthorsArtificial Organs © 2012, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

139

and the cellular redox state (5–11), can be affectedby the intrinsic properties of acellular Hb (12–15),(Fig. 1). Unmodified tetrameric Hb (UHb) andHBOC with pro-oxidant properties, by altering thecellular redox state, act as signaling moleculescapable of activating nuclear factor (NF)-kappa B(16) that regulates many genes that are known tosuppress the erythropoietic response, such as tumornecrosis factors (TNF)-alpha, interleukin (IL)1-beta, and transforming growth factor (TGF)-beta1 (17–20). UHb and HBOC with low oxygen affinity(high P50), by overdelivering oxygen, may degradehypoxia-inducible factor (HIF)-1 alpha (11,15),which acts as a master controller of the erythropoi-etin (EPO) gene—the most important regulator ofproliferation of committed progenitors and an anti-apoptotic protector of erythroblasts (10). In nor-moxia or hyperoxia, hydroxylation of prolineresidues by prolyl hydroxylases promotes oxygen-dependent degradation of HIF-1 alpha, unless it isstabilized via Hb-mediated oxidative phosphoryla-tion or inhibition of prolyl hydroxylases by reactiveoxygen species (ROS) (15,21,22). Conversely, UHband HBOC with strong pressor effects or high affin-ity for oxygen (low P50) that promotes hypoxia canstabilize HIF-1 alpha, inducing erythropoiesis (23–25). Assuming that efficacious HBOCs must coun-teract the hypoxic and pro-oxidant environmentsassociated with blood loss, the stabilization of HIF-1alpha by prolonging oxygen deprivation could beclinically questionable (Fig. 1).

Our previously published studies have shownthat adenosine-5′-triphosphate (ATP)-adenosine-glutathione (GSH) cross-linked Hb (ATP-ADO-GSH-Hb) is an effective stimulator of erythropoiesis

while sufficiently delivering oxygen (26,27). Thisproduct was shown to be very efficient in rapid res-toration of the RBC mass in severely anemic rodentsand nonhuman primates (26). In children with sickle-cell anemia, a single injection of ATP-ADO-GSH-Hbstimulated erythropoiesis and rapidly normalized thehematocrit (28). The molecular mechanism of ATP-ADO-GSH-Hb’s erythropoietic action, however, wasnot established.

The purpose of the present study was to determinethe actual role of UHb and ATP-ADO-GSH-Hb inerythropoiesis. This research explores the associationbetween Hb and redox- and oxygen-regulated tran-scription factors and their target genes that controlpro- and anti-erythropoietic responses. In this study,we evaluated these effects in human astrocytes thatare capable of producing EPO as well as cytokinesthat act as anti-erythropoietic mediators. Thesestudies were performed under normoxic and hypoxicconditions.

MATERIALS AND METHODS

Preparation and characterization of Hb solutionsThe Hb solutions were prepared at the Texas Tech

University Health Sciences Center (TTUHSC) BloodSubstitute Manufacturing Facility (Lubbock, TX,USA). This facility was also used for synthesis andproduction of cross-linking agents. All procedureswere done under sterile, pyrogen-free conditions(Class-100), using USP grade chemicals, water, andbuffer solutions.Blood from Hereford cattle,collectedat the TTUHSC Animal Blood Donor Facility (NewDeal, TX, USA), was used for preparation of Hbsolutions.

FIG. 1. Schematic representation of therole of unmodified Hb (UHb), first-generationHb-based oxygen carriers (HBOCs) with dif-ferent oxygen affinities (P50) and redoxpotentials, and Hb cross-linked with ATP,adenosine, and GSH (ATP-ADO-GSH-Hb)in the erythropoietin (EPO) gene inductionand the mechanism of erythropoiesis medi-ated by HIF-1 alpha, NF-kappa B, TNF-alpha, TGF-beta 1, and other factors.Details of these interrelationships are pre-sented in the text.

J. SIMONI ET AL.140

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

The methods for the preparation and character-ization of UHb, ATP-ADO-GSH-Hb, and cross-linking reagents have been described previously(26–31). In brief, Hb was extracted from washedRBCs using the method of dialysis ultrafiltration.The complete removal of stromal lipid contaminantswas accomplished by using a liquid/solid-phaseextraction procedure (29). Purification from non-heme proteins and peptides was achieved by using aheat pasteurization method. The orthogonal multi-step procedure that comprises nanofiltration,membrane chromatography, solvent treatment,and heat inactivation, was used to ensure completeremoval of viruses and prion proteins (30,31).The removal of environmental bacterial endotoxinwas achieved with affinity chromatography, and ste-rility was maintained by membrane filtration (29).The entire Hb purification process was performedin the absence of oxygen to prevent heme oxid-ation (29). After dialysis against 20 mM THAM(Abbott Laboratories, North Chicago, IL, USA) andreoxygenation, UHb in a concentration of 10 g/dLwas stored in sterile transfer pack containers(Fenwal, Deerfield, IL, USA) at -90°C. The UHbsolution was then chemically modified accordingto an earlier described and patented method (29).The chemical/pharmacological modification proce-dure comprises purified bovine Hb cross-linkedintramolecularly with open ring ATP, intermo-lecularly with open ring adenosine, combinedwith GSH, as well as enriched with oxygen freeradical scavengers, nonelectrolytes, and/or electro-lytes (29). After selective nanofiltration to eliminatetetramers and subsequent dialysis, ATP-ADO-GSH-Hb in a concentration of 6.4 � 0.2 g/dL wasstored in sterile transfer pack containers (Fenwal) at-90°C.

Purity of Hb solutions in regard to polar and non-polar lipids, non-heme proteins and peptides, andendotoxin was characterized as previously described(26,29). Molecular weight determination of Hb solu-tions was obtained by using a size exclusion high-pressure liquid chromatography method (Protein-Pak 300SW, Waters, Milford, MA, USA). The Hbsolutions’ surface charges were analyzed by usingisoelectric focusing gel electrophoresis (AmershamPharmacia PhastSystem, Piscataway, NJ, USA) andanion exchange liquid chromatography withProtein-Pak DEAE 5PW (Waters). The physico-chemical properties of Hb solutions, includingoxygen affinity, oncotic pressure, viscosity, osmolar-ity, pH, and electrolyte concentration, have beencharacterized by our standard quality controlmethods. The total Hb concentration and level of

met-Hb were measured by a spectrophotometricmethod (26,29).

Assessment of the effect of UHb and ATP-ADO-GSH-Hb on pro-erythropoietic factors: HIF-1 alphaand EPO in normoxia and hypoxia

In astrocytes, HIF-1 alpha regulates EPO expres-sion that plays a neuroprotective role to shieldneurons from hypoxic/ischemic stress (32).

The initial culture of normal human astrocytes,2nd passage, was obtained from Clonetics (Bio-Whittaker, A Cambrex Co., San Diego, CA, USA).Cells were cultured in 75-cm2 tissue culture flasks(Corning Glass Works, Corning, NY, USA) withastrocyte growth medium (AGM) BulletKit medium(Clonetics) in a humidified atmosphere of 5% CO2

and temperature of 37°C, until they reached conflu-ence (approximately 50 000 cells/cm2). Astrocyteswere then subcultured in 6-well cell culture plates(Corning). Cell passage was carried out using atrypsin reagent pack (Clonetics). During the transfer,astrocytes were trypsinized no longer than 5 min. Allexperiments were performed using fourth to sixthcell passage. The astrocytes had previously testednegative for HIV, hepatitis, mycoplasma, bacteria,yeast, and fungi, and tested positive for glial fibrillaryacidic protein and stained negative for CD68 andCNPase (Clonetics Certificate of Analysis).

The confluent astrocytes were incubated forapproximately 18 h with AGM medium supple-mented with UHb or ATP-ADO-GSH-Hb in a finalconcentration of 0.1, 1.0, and 1.75 g/dL, in normoxicand hypoxic environments.

Normoxic condition was achieved by the incuba-tion 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 aerobicworkstation. Before experimentation, media was pre-equilibrated overnight at a 1.5% oxygen level. Thecontrol astrocytes were cultured in the absence of Hbsolutions that were replaced by fetal bovine serum(FBS, HyClone Laboratories, Inc., Logan, UT, USA).

After treatment, the cells were subjected to evalu-ation by various biochemical and molecular biologymethods.

The impact of Hb solutions on HIF-1 alphastabilization and its ability to induce the human EPOgene in the normoxic and hypoxic conditions wasmeasured in cellular nuclear extracts using a high-throughput TransAM enzyme-linked immunosor-bent assay (ELISA)-based assay (Active Motif,Carlsbad, CA, USA). In this assay, a 96-well platewas immobilized with an oligonucleotide containinga hypoxia-responsive element (5′-TACGTGCT-3′)

ATP-ADENOSINE-GSH-Hb AS ESA 141


from a human EPO gene. Nuclear extracts wereobtained from living astrocytes using Nonidet P-40and lysis buffer supplemented with dithiothreitol(DDT) and a protease inhibitor cocktail (ActiveMotif). Nuclear extracts were subjected for the detec-tion of HIF-1 alpha. HIF-1 alpha that was present inthe nuclear extracts bonded to the human EPO geneand became accessible to primary antibodies. Then,the primary antibodies were recognized by second-ary, HRP-conjugated antibodies, which provided asensitive colorimetric readout. The reaction was readat 450 nm using a 3550-UV microplate reader (Bio-Rad Laboratories, Richmond, CA, USA). Resultswere expressed in optical density (OD) at 450 nm per2.5 mg of nuclear extract. The COS-7 nuclear extractprovided by the manufacturer was used as a positivecontrol for HIF-1 alpha activation and binding to theEPO gene.

The influence of Hb solutions on EPO synthesiswas assessed in the cell culture supernatants usinghighly specific Quantikine In Vitro DiagnosticHuman Erythropoietin ELISA (R&D Systems, Inc.,Minneapolis, MN, USA), according to the manufac-turer’s protocol. The results were expressed inmU/mL.

Assessment of the effect of UHb and ATP-ADO-GSH-Hb on anti-erythropoietic factors: NF-kappaB, TNF-alpha, and TGF-beta 1 in normoxia andhypoxia

Human astrocytes are known to produce cytokines(i.e., TNF, IL-1, IL-6) and growth factors (i.e., TGF-beta 1) (33–35), which act as anti-erythropoieticagents (17–20).

All experiments were carried out using the humanastrocytes model and Hb concentrations as describedabove. After treatment, the assessment of nuclearactivation and DNA binding of NF-kappa B wasassayed in cellular nuclear extracts using TransAMNF-kappa B p65 transcription Factor Assay Kit(Active Motif). This ELISA-based method detectsand quantifies NF-kappa B activation, using oligo-nucleotide containing the NF-kappa B consensus site(5’-GGGACTTTCC-3’) immobilized on a 96-wellplate. The nuclear extracts were obtained from livingastrocytes using complete lysis buffer that containedDTT and a protease inhibitor cocktail supplied bythe manufacturer. The complete binding buffer wassupplemented with DTT and herring sperm DNA.After incubation, the formed DNA–protein complexwas accessible to primary antibodies, which recog-nized an epitope on p65, only when NF-kappa Bwas activated and bound to DNA. This reaction wasthen recognized with HRP-conjugated secondary

antibodies against p65, and developed using a benzi-dine derivative and hydrogen peroxide. The reactionwas read at 450 nm using a microplate reader (Bio-Rad Model 3550-UV). Results were expressed in ODat 450 nm per 2.5 mg of whole-cell extract. The HeLawhole-cell extracts, provided by the manufacturer,were used as a positive control for NF-kappa B acti-vation and DNA binding.

The production of factors with anti-erythropoieticactivities (TNF-alpha and TGF-beta 1) was assessedwith commercially available ELISA kits. TNF-alphawas assayed using a TNF-alpha human EIA Kit(Cayman Chemical,Ann Arbor, MI, USA), accordingto the manufacturer. TGF-beta 1 was assessed withHuman TGF-beta 1 Quantikine Immunoassay (R&DSystems). In this test, latent TGF-beta 1 in cell culturesupernates was transferred into the immunoreactiveform by acid activation and neutralization, thenassayed using a microplate with immobilized TGF-beta soluble receptor and expressed in pg per mL.

Data evaluation and statistical analysesAll experiments were conducted in triplicate and

results were expressed as mean � standard deviation(M � SD). The differences among and between thegroups were evaluated with ANOVA, using the Stat-Works statistical package (Cricket Software, Phila-delphia, PA, USA).

RESULTS

Characteristics of Hb solutionsHb was completely purified from non-heme pro-

teins, peptides, phospholipids, viral, bacterial, andprion contaminants. The concentration of bacterialendotoxin in UHb and ATP-ADO-GSH-Hb wasbelow the Food and Drug Administration require-ment of 0.25 EU/mL. Hb solutions were enrichedwith electrolytes and mannitol. The final formula-tions were isotonic. Although UHb solution wascomprised only of 64.5 kDa tetramers, ATP-ADO-GSH-Hb contained polymers below 500 kDa and lessthan 5% of tetramers. Both Hb solutions had lessthan 5% of met-Hb. UHb and ATP-ADO-GSH-Hbhad an isoelectric point (pI) of 6.8–7.0 and 6.1–6.3,respectively. Both Hb solutions had a similar P50 of23 � 3 mm Hg at chloride concentration of 100 mmthat provided the proper oxygen delivery index(36,37). The AGM environment did not affect theaffinity of either Hbs for oxygen. The typical physi-cochemical characteristics are listed below:

• Oxy-Hb: 6.4 � 0.2 g/dL• Met-Hb % of oxy-Hb: <5

J. SIMONI ET AL.142


• CO-Hb % of oxy-Hb: <5• pH: 7.8–8.1 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: 20–23 mm Hg• Osmolarity: 300–325 mOsm/kg

Effects of UHb and ATP-ADO-GSH-Hb onpro-erythropoietic factors

The effect of unmodified and ATP-ADO-GSH-Hbon pro-erythropoietic factors, HIF-1 alpha, and EPOis presented in Fig. 2 and Table 1.

As shown in Fig. 2, HIF-1 alpha can be found in thenuclear extracts of astrocytes under hypoxic and nor-moxic conditions. The tested Hb solutions had a dif-ferent impact on HIF-1 alpha stabilization, nucleartranslocation, and binding to the EPO gene inhypoxic and normoxic environments.

The UHb solution increased the cytoplasmic deg-radation of HIF-1 alpha and generally decreased itsbinding activity to the EPO gene, especially inhypoxia. On the contrary, ATP-ADO-GSH-Hb pre-served HIF-1 alpha under both oxygen conditions(Fig. 2). This Hb solution in a dose-dependentmanner stabilized HIF-1 alpha and increased itsbinding to the EPO gene, and was most effective at adose of 1.75 g/dL. The observed differences were sta-tistically significant (P < 0.001).

The production of EPO under normoxic andhypoxic conditions significantly increased following

incubation of astrocytes with ATP-ADO-GSH-Hb atall tested concentrations (Table 1). In normoxia, thisHb was able to increase the EPO level 11-fold at aconcentration of 0.1 g/dL, 21-fold at 1.0 g/dL, andmore than 61-fold at 1.75 g/dL. In hypoxia, the impactof ATP-ADO-GSH-Hb on EPO synthesis was sig-nificantly enhanced at all concentrations.ATP-ADO-GSH-Hb acted effectively on EPO synthesis in bothoxygen conditions. However, UHb blocked the syn-thesis of EPO. These effects were more pronouncedat higher Hb concentrations (Table 1).

Effects of UHb and ATP-ADO-GSH-Hb onanti-erythropoietic factors—NF-kappa B, TGF-beta1, and TNF-alpha

As presented in Fig. 3, ATP-ADO-GSH-Hb inhib-ited NF-kappa B activation at all tested concentra-tions and oxygen levels. On the contrary, UHbactivated NF-kappa B in a dose-dependent manner.This effect was more pronounced in the hypoxic con-dition (P < 0.001).

The production of the most potent anti-erythropoietic factors, TGF-beta 1 and TNF-alpha, inresponse to tested Hb solutions revealed that theseproducts affected human astrocytes differently(Tables 2 and 3). ATP-ADO-GSH-Hb inhibited theformation of TGF-beta 1 (Table 2) and did notincrease the production of TNF-alpha (Table 3) at alltested concentrations and oxygen conditions. Thiseffect can be linked to the inability of this Hb solu-tion to induce NF-kappa B. UHb, however, increasedthe production of both TGF-beta 1 (Table 2) andTNF-alpha (Table 3), especially at higher concentra-tions (1 and 1.75 g/dL).

FIG. 2. The effects of unmodified Hb (UHb)and Hb cross-linked with ATP, adenosine,and GSH (ATP-ADO-GSH-Hb) on HIF-1alpha stabilization in normoxia and hypoxia.White bars, UHb under hypoxia; light graybars, UHb under normoxia; black bars, ATP-ADO-GSH-Hb under hypoxia; dark graybars, ATP-ADO-GSH-Hb under normoxia;(a) significant differences at P < 0.001between controls and among Hb group; (b)significant differences at P < 0.01 betweencontrols and among Hb group; (c) significantdifferences at P < 0.05 between controlsand among Hb group; (A) significant differ-ences at P < 0.001 between Hb groups; (B)significant differences at P < 0.01 betweenHb groups; (C) significant differences atP < 0.05 between Hb groups (details in thetext).



TAB

LE

1.T

heef

fect

sof

unm

odifi

edH

ban

dH

bcr

oss-

linke

dw

ithA

TP,

aden

osin

e,an

dG

SH(A

TP

-AD

O-G

SH-H

b)on

pro-

eryt

hrop

oiet

icE

PO

prod

uctio

nby

hum

anas

troc

ytes

unde

rhy

poxi

can

dno

rmox

icco

nditi

ons

Hyp

oxia

Nor

mox

ia

EP

O(m

U/m

L)

M�

SDSi

gnifi

canc

eco

ntro

lve

rsus

expe

rim

enta

lSi

gnifi

canc

ebe

twee

nex

peri

men

talg

roup

sM

�SD

Sign

ifica

nce

cont

rol

vers

usex

peri

men

tal

Sign

ifica

nce

betw

een

expe

rim

enta

lgro

ups

Sign

ifica

nce

hypo

xia

vers

usno

rmox

ia

Con

trol

0.23

�0.

56—

0.29

�0.

50—

P<

0.00

1U

nmod

ified

Hb

0.1

g/dL

3.05

�2.

56N

.S.

—2.

63�

1.33

P<

0.01

—N

.S.

Unm

odifi

edH

b1.

0g/

dL2.

35�

1.62

N.S

.—

3.88

�1.

41P

<0.

01—

N.S

.U

nmod

ified

Hb

1.75

g/dL

1.02

�0.

26P

<0.

001

—1.

96�

1.53

N.S

.—

N.S

.A

TP

-AD

O-G

SH-H

b0.

1g/

dL3.

96�

2.68

N.S

.N

.S.

3.19

�1.

58P

<0.

05N

.S.

N.S

.A

TP

-AD

O-G

SH-H

b1.

0g/

dL23

.00

�8.

21P

<0.

001

P<

0.00

16.

11�

1.86

P<

0.00

1P

<0.

001

P<

0.00

1A

TP

-AD

O-G

SH-H

b1.

75g/

dL57

.27

�27

.60

P<

0.00

1P

<0.

001

17.6

7�

4.00

P<

0.00

1P

<0.

001

P<

0.00

1

M�

SD,m

ean

�st

anda

rdde

viat

ion;

N.S

.,no

tsi

gnifi

cant

.

TAB

LE

2.T

heef

fect

sof

unm

odifi

edte

tram

eric

Hb

and

Hb

cros

s-lin

ked

with

AT

P,ad

enos

ine,

and

GSH

(AT

P-A

DO

-GSH

-Hb)

onan

ti-er

ythr

opoi

etic

TG

F-b

eta

1pr

oduc

tion

byhu

man

astr

ocyt

esun

der

hypo

xic

and

norm

oxic

cond

ition

s

Hyp

oxia

Nor

mox

ia

TG

F-b

eta

1(p

g/m

L)

M�

SD

Sign

ifica

nce

cont

rolv

ersu

sex

peri

men

tal

Sign

ifica

nce

betw

een

expe

rim

enta

lgro

ups

M�

SDSi

gnifi

canc

eco

ntro

lve

rsus

expe

rim

enta

lSi

gnifi

canc

ebe

twee

nex

peri

men

talg

roup

sSi

gnifi

canc

ehy

poxi

ave

rsus

norm

oxia

Con

trol

202.

70�

44.4

0—

117.

70�

47.9

0—

P<

0.05

Unm

odifi

edH

b0.

1g/

dL20

4.60

�75

.40

N.S

.—

245.

70�

26.6

0P

<0.

01—

N.S

.U

nmod

ified

Hb

1.0

g/dL

336.

80�

85.5

0P

<0.

05—

345.

50�

50.4

0P

<0.

01—

N.S

.U

nmod

ified

Hb

1.75

g/dL

446.

10�

81.3

0P

<0.

01—

573.

10�

77.8

0P

<0.

001

—P

<0.

05A

TP

-AD

O-G

SH-H

b0.

1g/

dL10

2.90

�59

.30

P<

0.05

P<

0.05

153.

80�

65.9

0N

.S.

P<

0.05

N.S

.A

TP

-AD

O-G

SH-H

b1.

0g/

dL16

4.20

�59

.40

N.S

.P

<0.

0114

7.50

�58

.40

N.S

.P

<0.

01N

.S.

AT

P-A

DO

-GSH

-Hb

1.75

g/dL

201.

40�

54.0

0N

.S.

P<

0.00

118

2.90

�65

.90

N.S

.P

<0.

001

N.S

.

M�

SD,m

ean

�st

anda

rdde

viat

ion;

N.S

.,no

tsi

gnifi

cant

.

J. SIMONI ET AL.144


DISCUSSION

This study illustrates that UHb might dose depen-dently inhibit erythropoiesis by facilitating HIF-1alpha degradation and NF-kappa B activation withsubsequent expression of anti-erythropoietic factors,TGF-beta 1 that blocks differentiation of erythroidprogenitor cells and promotes apoptosis, and TNF-alpha that prevents HIF-1 alpha binding to the EPOgene (Fig. 1). However,ATP-ADO-GSH-Hb exhibitspro-erythropoietic potential by stabilizing andincreasing HIF-1 alpha binding to the EPO gene anddownregulating NF-kappa B (Fig. 1).

We previously showed that chemical/pharma-cological modification of Hb with ATP, adenosine,and GSH produces vasodilation and a proper oxygendelivery index (26,27). This present study revealedthat oxygen supplied by ATP-ADO-GSH-Hb doesnot induce degradation of HIF-1 alpha allowingeffective induction of the EPO gene in the normoxiccondition (Fig. 2, Table 1). The earlier reported anti-inflammatory and anti-apoptotic potential of ATP-ADO-GSH-Hb (16,23,26,38–43) is mirrored in theresults of the current study, which showed that down-regulation of NF-kappa B with subsequent suppres-sion of the TGF-beta 1 and TNF-alpha genes isessential in achieving a positive erythropoieticresponse (Fig. 3, Tables 2 and 3).

These molecular findings are supported by our pre-vious observations that ATP-ADO-GSH-Hb is aneffective inducer of erythropoiesis in vivo (26,28).ATP-ADO-GSH-Hb, when administered in avolume corresponding to 25% total blood volume topediatric sickle-cell anemia patients in aplastic crisis,stimulated the bone marrow to a significant erythro-poietic effect. The number of reticulocytes increasedfrom 3.7 � 3.09 to 49.2 � 6.5%, and blood Hbincreased from 6.34 � 2.0 to 9.54 � 0.72 g/dL after3 days (28). A similar erythropoietic effect was alsoobserved in nonhuman primates and rodents (26).

A natural response to hypoxia is an increase inerythropoiesis (44). Erythropoiesis is tightly regu-lated by oxygen tension and the cellular redox statethat involves oxygen (HIF-1 alpha) and redox-regulated transcription factors (NF-kappa B) andmany growth factors (i.e., EPO, IL-3, IL-9, stem cellfactor, granulocyte macrophage-colony stimulatingfactor), and minerals, particularly iron (6–11,15,17–20). EPO, which is produced by Kupffer cells in thefetal liver and peritubular interstitial cells in the adultkidney in response to hypoxia, is the main regulatorof erythropoiesis via rescue of erythroid progenitorcells from apoptosis (7,45). EPO can also be pro-duced in the central nervous system by astrocytes,

TAB

LE

3.T

heef

fect

sof

unm

odifi

edte

tram

eric

Hb

and

Hb

cros

s-lin

ked

with

AT

P,ad

enos

ine,

and

GSH

(AT

P-A

DO

-GSH

-Hb)

onan

ti-er

ythr

opoi

etic

TN

F-a

lpha

prod

uctio

nby

hum

anas

troc

ytes

unde

rhy

poxi

can

dno

rmox

icco

nditi

ons

Hyp

oxia

Nor

mox

ia

TN

F-a

lpha

(pg/

mL

)M

�SD

Sign

ifica

nce

cont

rolv

ersu

sex

peri

men

tal

Sign

ifica

nce

betw

een

expe

rim

enta

lgro

ups

M�

SDSi

gnifi

canc

eco

ntro

lve

rsus

expe

rim

enta

lSi

gnifi

canc

ebe

twee

nex

peri

men

talg

roup

sSi

gnifi

canc

ehy

poxi

ave

rsus

norm

oxia

Con

trol

6.35

�3.

66—

5.47

�3.

31—

N.S

.U

nmod

ified

Hb

0.1

g/dL

4.87

�2.

82N

.S.

—6.

46�

3.51

N.S

.—

N.S

.U

nmod

ified

Hb

1.0

g/dL

5.42

�3.

13N

.S.

—5.

65�

3.26

N.S

.—

N.S

.U

nmod

ified

Hb

1.75

g/dL

27.0

4�

5.06

P<

0.01

—14

.92

�2.

69P

<0.

05—

P<

0.05

AT

P-A

DO

-GSH

-Hb

0.1

g/dL

5.61

�3.

24N

.S.

N.S

.5.

55�

3.20

N.S

.N

.S.

N.S

.A

TP

-AD

O-G

SH-H

b1.

0g/

dL4.

72�

2.74

N.S

.N

.S.

5.30

�3.

06N

.S.

N.S

.N

.S.

AT

P-A

DO

-GSH

-Hb

1.75

g/dL

5.39

�3.

12N

.S.

P<

0.01

5.27

�3.

04N

.S.

P<

0.05

N.S

.



shielding hypoxic neurons from apoptosis (32).As human astrocytes are also known to pro-duce inflammatory cytokines (33–35) that act asanti-erythropoietic agents (17–20), nowadays, theastrocyte cell culture model is used to study themechanism of erythropoiesis at the molecular level.HIF-1 alpha that mediates EPO gene expression isalso a promoter of other genes important in adapta-tion to hypoxia, such as transferrin, vascular endo-thelial growth factor, nitric oxide synthase,endothelin-1, heme oxygenase-1, glucose transporter1, etc. (11,22,25,46–49). In the hypoxic condition, alack of oxygen suppresses the degradation of HIF-1alpha, which rapidly translocates from the cytoplasmto the nucleus and acts as a master regulator of eryth-ropoiesis (46). In the normoxic condition, oxygen-mediated hydroxylation of HIF-1 alpha prolineresidues initiates its rapid degradation blockingerythropoiesis (21,22,24), (Fig. 1). There are, how-ever, some exceptions. For instance, in oxidativestress, ROS by changing the cellular redox equilib-rium that activates NF-kappa B and induces inflam-matory genes (i.e., TNF-alpha, IL-1 beta, IL-6) maystabilize HIF-1 alpha in normoxia (Fig. 1). Theseinflammatory cytokines, however, can also inhibitHIF-1 alpha binding to the EPO gene suppressingerythropoiesis (Fig. 1), as observed in cancer patients.Similarly, another inflammatory mediator TGF-beta,which is also known to stabilize HIF-1 alpha undernormoxic conditions, is capable of blocking the dif-ferentiation of erythroid progenitor cells and stimu-lating apoptosis (17–20), as observed in end-stagerenal disease patients (19,50,51). In addition, ROScan stabilize HIF-1 alpha in normoxia by inhibitingprolyl hydroxylase activity (49).

Hb that affects the cellular oxygen content as wellas the redox equilibrium can easily influence theabove-presented mechanisms. In fact, the involve-ment of Hb in erythropoietic responses is knownsince Amberson in the late 1940s observed a hemat-ocrit increase in a human receiving a crude Hb solu-tion (52). Savitsky et al. observed a similar effectusing a stroma-free Hb solution (53). All subjectstreated with these Hb solutions showed systemichypertension and renal failure. At that time, theauthors were unable to explain the mechanism ofthese events. By applying current knowledge, theerythropoietic responses seen in Amberson’s andSavitsky’s clinical trials associated with pathologicalreactions, such as hypertension, renal failure, andeven death, resulted from Hb’s intrinsic toxicity(1–4,13).

Several first-generation HBOCs with the typicalHb intrinsic toxicity profile have also been reportedto possess some erythropoietic activity. In fact,recombinant Hb, rHb1.1—Optro (Somatogen, Inc.,Boulder, CO, USA) given intravenously to mice inextremely low doses, resulted in increased early pre-cursors of RBC in the bone marrow and an increasein hematocrit. Without providing the mechanism, theauthors concluded that rHb1.1 works either directlyon progenitor cells or indirectly to enhance hemato-poiesis (37). However, a study with larger, clinicallyrelevant doses of cross-linked Hbs in rabbits revealedthat HBOCs at high concentrations did not produce asignificant variation in the generation of erythroid-committed cells (54). While erythropoietic activity ofHemAssist (Baxter Healthcare Corporation, Deer-field, IL, USA) was not reported (55), Hemopure(Biopure, Inc., Cambridge, MA, USA) and Poly-

FIG. 3. The effects of unmodified Hb (Hb)and Hb cross-linked with ATP, adenosine,and GSH (ATP-ADO-GSH-Hb) on NF-kappaB induction in normoxia and hypoxia. Whitebars, UHb under hypoxia; light gray bars,UHb under normoxia; black bars, ATP-ADO-GSH-Hb under hypoxia; dark gray bars,ATP-ADO-GSH-Hb under normoxia; (a) sig-nificant differences at P < 0.001 betweencontrols and among Hb group; (b) significantdifferences at P < 0.01 between controlsand among Hb group; (c) significant differ-ences at P < 0.05 between controls andamong Hb group; (A) significant differencesat P < 0.001 between Hb groups; (B) signifi-cant differences at P < 0.01 between Hbgroups; (c) significant differences atP < 0.05 between Hb groups (details in text).

J. SIMONI ET AL.146


Heme (Northfield Laboratories Inc., Evanston, IL,USA) have been characterized as products with someerythropoietic potency largely accelerated bysupplemental recombinant human erythropoietin(rEPO) (56–58). However, Hemolink (HemosolCorp., Mississauga, ON, Canada) was never charac-terized as a product that stimulates erythropoiesis(59,60).

Despite the mixed results, these products have incommon a very well-documented vasoconstrictiveeffect (1–4) that perhaps promotes hypoxic HIF-alpha stabilization and EPO induction (61). The lit-erature also demonstrates that many clinically testedHBOCs not only mediate vasoconstrictive events butalso are redox active and able to activate NF-kappa B(1–4,13) and its target genes, many of which influencethe erythropoietic process (13,16,23,38,62–64). Manyresulting 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-1alpha to the EPO gene (17,49,65), blockingerythropoiesis. Therefore, the reported varied eryth-ropoietic potential of UHb and tested HBOCsappears to be the net effect between possibly com-peting forces of vasoconstriction and cellular redoxchanges (Fig. 1).

These effects, however, are in contradiction withthe intended role of HBOCs, which is delivery ofsufficient oxygen to hypoxic tissues without causingischemic and inflammatory responses. Erythropoieticeffects under these circumstances should be consid-ered pathologic.

The basic research on erythropoietic activity of Hbis limited. It was reported that Hb under hypoxicconditions increased the expression of HIF-1 alpha,which was related to the loss of ferrous-Hb and accu-mulation of ferric-Hb (oxidation of heme). In thisstudy, the authors used an HBOC similar to that ofHemAssist. No connection between Hb and erythro-poiesis was made (15). Other researchers have inves-tigated HIF-1 alpha as an indicator of local hypoxiafollowing treatment with HBOC and liposome-encapsulated Hb (Samaja et al. and Murayamaet al.—in this issue). Established evidence exists thatHb which triggers NF-kappa B may suppress HIF-1alpha regulated genes, particularly EPO (13,23,64–66). It was also reported that high activity of theNF-kappa B pathway in early erythroid progenitorsis involved in the suppression of erythroid-specificgenes (65).

An evident lack of erythropoietic activity ofHBOCs under current development can be summa-rized by the statement of Dr. Klein from the Depart-ment of Transfusion Medicine at the National

Institutes of Health, Bethesda, MD. In his reviewarticle entitled: “Blood substitutes: how close to asolution?” he stated that: “. . . hemoglobin-derivedred cell substitutes from human, bovine, and recom-binant sources in phase III trials all have a half-lifemeasured in hours and are unlikely to replace trans-fusions or drugs that stimulate erythropoiesis forchronic anemia, but they may play a role: (i) as abridge to transfusion when no compatible blood isimmediately available; (ii) as an adjunct to theautologous hemodilution management of surgery, oreven (iii) in radiation therapy or the management ofcancer . . .” (67).

As clinically tested HBOCs lack a demonstrablephysiologic erythropoietic response, the need stillexists for an artificial oxygen carrier with intrinsicerythropoietic activity that eliminates the require-ment for supplementary transfusions and/or expen-sive rEPO treatment.

To address these problems, we have developedATP-ADO-GSH-Hb that utilizes a novel conceptof “pharmacologic cross-linking” (26,28,29). Thischemical/pharmacologic modification with ATP,adenosine, and GSH does not interfere with Hb res-piratory function, but provides Hb molecules withnew medicinal properties, which appear to be aneffective strategy in elimination of intrinsic toxiceffects of Hb, such as vasoconstriction, oxidativestress, and inflammation (26). In this product, ATPstabilizes the Hb tetramer and prevents its dimeriza-tion, and adenosine allows the creation of Hb oligo-mers, avoiding the formation of toxic high-molecular-weight polymers. ATP also produces the vasodilatoryeffect via activation of P2Y receptors (26). Adeno-sine counteracts the vasoconstrictive and pro-inflammatory properties of Hb with the activation ofadenosine A2 receptors, which produce vasodilata-tion, moderation of inflammatory reactions, andprevention of platelet aggregation (26–29,38–43).The activation of adenosine A3 receptor providescytoprotection. The conjugation of Hb with GSHshields heme from ROS and NO, thus lowering theHb pro-oxidant and vasoconstrictive potential(16,26,38,43). At the same time, GSH introduces amore electronegative charge onto the surface of theHb molecule that blocks Hb’s transglomerular andtransendothelial passage, and makes it less accessibleto phagocytes (26,38,39,68).

This current study revealed that ATP-ADO-GSH-Hb stabilizes HIF-1 alpha and facilitates itsbinding to the EPO gene under both the normoxic andhypoxic conditions (Fig. 2, Table 1). Moreover, adownregulated NF-kappa B creates a more pro-erythropoietic environment, by elimination of the



anti-erythropoietic factors, TNF-alpha and TGF-beta1 (Fig. 3, Tables 2 and 3). This observation providesthe molecular basis for the earlier observed in vivoerythropoietic responses in humans and animals(26,28).

Although the roles of individual chemical/pharmacological elements in our HBOC needfurther investigation, it is obvious that the ability ofthis product to stabilize HIF-1 alpha in normoxia isthe principal mechanism behind its erythropoieticaction previously observed in humans and animals(26,28). It has been known for more than fourdecades that adenosine and its analogues stimulateerythropoiesis (69,70).Adenosine is a key acute regu-lator in response to hypoxia (71). The hypoxic inhi-bition of adenosine deaminase prolongs adenosine’sprotective activity, specifically in inhibition of prolyl-4-hydroxylase and generation of cyclic AMP. CyclicAMP that is raised following stimulation of adenos-ine A2 receptors by ATP-ADO-GSH-Hb, which wasevidenced in our previous studies (16,26), possessesstrong pro-erythropoietic activities. It was reportedthat cyclic AMP counteracts the inhibition of theEPO gene by inflammatory cytokines, TNF-alphaand IL-1 alpha (72). In fact, our present study docu-mented effective downregulation of NF-kappa B andcomplete suppression of TNF-alpha by ATP-ADO-GSH-Hb (Fig. 3, Table 3). Another contributoryfactor toward an effective erythropoietic responsewas the inevitably low pro-oxidant potential of ourHBOC maintained by GSH (16,23,26,38,43,66). Therole of heme-iron in this observed erythropoieticresponse is awaiting elucidation (73).

CONCLUSION

Based on this current study and our previouspreclinical and clinical observations, it can beconcluded that the chemical/pharmacologic modifi-cation of Hb with ATP, adenosine, and GSHresulted in an HBOC with erythropoietic activity innormoxic and hypoxic clinical scenarios. ThisHBOC, by delivering oxygen and expressing pro-erythropoietic potential, can serve as a primarytherapy to maintain tissue oxygenation and a sec-ondary therapy to normalize the hematocrit throughstimulation of patients’ erythropoietic responses.ATP-ADO-GSH-Hb should be considered as anovel erythropoiesis-stimulating agent in the treat-ment of acute and chronic anemias.

REFERENCES

1. Guidance for Industry. Criteria for Safety and Efficacy Evalu-ation of Oxygen Therapeutics as Red Cell Substitutes. U.S.

Department of Health and Human Services, Food andDrug Administration, Center for Biologics Evaluation andResearch, Rockville, MD. October 2004, pp. 1–19.

2. Public Workshop. Safety of Hemoglobin-Based Oxygen Car-riers (HBOCs), April 29–30, 2008. The Food and Drug Admin-istration (FDA), the National Heart, Lung and Blood Institute,National Institutes of Health; and the Department of Healthand Human Service’s Office of the Secretary and Office ofPublic Health and Science (OS/OPHS), Natcher ConferenceCenter, Bethesta, MD.

3. Silverman TA, Weiskopf RB; Planning Committee and theSpeakers. Hemoglobin-based oxygen carriers: current statusand future directions. Anesthesiology 2009;111:946–63.

4. Natanson C, Kern SJ, Lurie P, Banks SM, Wolfe SM. Cell-freehemoglobin-based blood substitutes and risk of myocardialinfarction and death: a meta-analysis. J Am Med Assoc2008;299:2304–12.

5. Hillman RS, Finch CA. Red Cell Manual, 7th Edition. Phila-delphia: F.A. Davis, 1996;viii, 190.

6. Adamson JW. Erythropoietin: its role in the regulation oferythropoiesis and as a therapeutic in humans. Biotechnology1991;19:351–61.

7. Sasaki R. Pleiotropic functions of erythropoietin. Intern Med2003;42:142–9.

8. Lacombe C, Mayeux P. Biology of erythropoietin. Haemato-logica 1998;83:724–32.

9. Socolovsky M, Fallon AE, Wang S, Brugnara C, Lodish HF.Fetal anemia and apoptosis of red cell progenitors in Stat5a-/-5b-/- mice: a direct role for Stat5 in BcL-X(L) induction. Cell1999;98:181–91.

10. Dolznig H, Habermann B, Stangel K, et al. Apoptosis protec-tion by the EPO target Bcl-X(L) allows factor-independentdifferentiation of primary ertythroblasts. Curr Biol 2002;12:1076–85.

11. Semenza GL, Wang GL. A nuclear factor induced by hypoxiavia de novo protein synthesis binds to the human erythropoi-etin gene enhancer at a site required for transcriptionalactivation. Mol Cell Biol 1992;12:5447–54.

12. Kluger R. Red cell substitutes from hemoglobin—do we startall over again? Curr Opin Chem Biol 2010;14:538–43.

13. Simoni J, Simoni G, Moeller JF. Intrinsic toxicity of hemoglo-bin: how to counteract it. Artif Organs 2009;33:100–9.

14. Buehler PW, D’Agnillo F. Toxicological consequences ofextracellular hemoglobin: biochemical and physiologicalperspectives. Antioxid Redox Signal 2010;12:275–91.

15. Yeh LH, Alayash AI. Effects of cell-free hemoglobin onhypoxia-inducible factor (HIF-1 alpha) and heme oxygenase(HO-1) expression in endothelial cells subjected to hypoxia.Antioxid Redox Signal 2004;6:944–53.

16. Simoni J, Simoni G, Lox CD, Prien SD, Shires GT. Modifiedhemoglobin solution, with desired pharmacological properties,does not activate nuclear transcription factor NF-kappa B inhuman vascular endothelial cells. Artif Cells Blood SubstitImmobil Biotechnol 1997;25:193–210.

17. Hellwing-Burgel T, Rutkowski K, Metzen E, Fanderey J,Jelkman W. Interleukin-1 beta and tumor necrosis factor-alpha stimulate DNA binding of hypoxia-inducible factor-1.Blood 1999;94:1561–7.

18. Linch DC. The regulation of erythropoiesis in man. SchweizMed Wochenschr 1989;119:1327–8.

19. Yuen D, Richardson RMA, Fenton SSA, McGrath-ChongME, Chan CT. Quotidian nocturnal hemodialysis improvescytokine profile and enhances EPO responsiveness. ASAIO J2005;51:236–41.

20. Trey JE, Kushner I. The acute phase response and the hemato-poietic system: the role of cytokines. Crit Rev Oncol Hematol1995;21:1–8.

21. Kallio PJ, Wilson WJ, O’Brien S, Makino Y, Poellinger L.Regulation of the hypoxia-inducible transcription factor 1alpha by the ubiquitin-proteasome pathway. J Biol Chem1999;274:6519–25.

J. SIMONI ET AL.148


22. Wenger RH. Cellular adaptation to hypoxia: O2-sensingprotein hydrolases, hypoxia-inducible transcription factors,and O2-regulated gene expression. FASEB J 2002;16:1151–62.

23. Simoni J, Simoni G, Griswold JA, Moeller JF, Wesson DE.Hemoglobin, transcriptional activator and suppressor. How totip the balance? Artif Blood 2003;11:69.

24. Wang GL, Semenza GL. Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity byhypoxia. J Biol Chem 1993;268:21513–8.

25. Gleadle JM, Ratcliffe PJ. Hypoxia and the regulation of geneexpression. Mol Med Today 1998;4:122–9.

26. Simoni J, Simoni G, Wesson DE, Feola M. ATP-adenosine-glutathione cross-linked hemoglobin as clinically usefuloxygen carrier. Curr Drug Discov Technol 2011 Sep 9. [Epubahead of print].

27. Simoni J, Simoni G, Newman G, Feola M. An improved bloodsubstitute. In vivo evaluation of its hemodynamic effects.ASAIO J 1996;42:M773–M782.

28. Feola M, Simoni J, Angelillo R, et al. Clinical trial of a hemo-globin based blood substitute in patients with sickle cellanemia. Surg Gynecol Obstet 1992;174:379–86.

29. Feola M, Simoni J, Canizaro PC. Blood substitute. 1995. U.S.Patent No. 5,439,882.

30. Simoni J, Simoni G, Moeller JF. Orthogonal method for theremoval of transmissible spongiform encephalopathy agentsfrom biological fluids. 2011. Patent ApplicationUS20110097746. Publication Date: 04/28/2011.

31. Simoni J, Simoni G, Moeller JF. Orthogonal method for theremoval of prions and viruses from hemoglobin solution. TheXIIIth International Symposium on Blood Substitutes andOxygen Therapeutics. Massachusetts General Hospital andHarvard Medical School, Boston, MA, July 27–29, 2011.Abstracts Book 2011;69(P-28).

32. Siren AL, Knerlich F, Poser W, Gleiter CH, Bruck W, Ehren-reich H. Erythropoietin and erythropoietin receptor in humanischemic/hypoxic brain. Acta Neuropathol (Berl) 2001;101:271–6.

33. Van Wagoner NJ, Oh JW, Repovic P, Benveniste EN.Interleukin-6 (IL-6) production by astrocytes: autocrine regu-lation by IL-6 and the soluble IL-6 receptor. J Neurosci1999;19:5236–44.

34. Oh JW, Schweibert LM, Beneviste EN. Cytokine regulation ofCC and CXC chemokine expression by human astrocytes. JNeurovirol 1999;5:82–94.

35. Flanders KC, Ren RF, Lippa CF. Transforming growth factor-betas in neurodegenerative disease. Prog Neurobiol 1998;54:71–85.

36. Alayash AI, D’Agnillo F, Buehler PW. First-generationblood substitutes: what have we learned? Biochemical andphysiological perspectives. Expert Opin Biol Ther 2007;7:665–75.

37. Rosenthal GJ, Gerber MJ. Method of stimulating hematopoie-sis with hemoglobin. 1997. U.S. Patent No. 5,631,219.

38. Simoni J, Simoni G, Martinez-Zaguilan R, et al. Improvedblood substitute: evaluation of its effects on human endothelialcells. ASAIO J 1998;44:M356–M367.

39. Simoni J, Simoni G, Wesson DE, Griswold JA, Feola M. Anovel hemoglobin adenosine-glutathione based blood substi-tute: evaluation of its effects on human blood ex vivo. ASAIOJ 2000;46:679–92.

40. Simoni J, Simoni G, Wesson DE, Griswold JA, Feola M. Theresponse of human brain capillary endothelial cells to a novelhemoglobin-adenosine-glutathione based red cell substitute.Artif Cells Blood Substit Biotechnol 2002;31:486–7.

41. Simoni J, Simoni G, Wesson DE, Griswold JA, Feola M.Attenuation of hemoglobin neurotoxic potential by cross-linking with adenosine and conjugation with reducedglutathione. Artif Cells Blood Substit Immobil Biotechnol2001;29:128.

42. Simoni J, Simoni G, Wesson DE, Griswold JA, Moeller JF,Feola M. Combining hemoglobin with adenosine and reduced

glutathione attenuates its direct and indirect neurotoxicpotential. Artif Blood 2003;11:96.

43. Simoni J, Simoni G, Wesson DE, Griswold JA, Feola M. Oxi-dative reactions of hemoglobin cross-linked with adenosineand conjugated with reduced glutathione. Artif Cells BloodSubstit Immobil Biotechnol 2001;29:164.

44. Campbell K. Pathophysiology of anemia. Nurs Times2004;100:40–3.

45. Variano M, Dello Russo C, Pozzoli G, et al. Erythropoietinexerts anti-apoptotic effects on rat microglial cells in vitro. EurJ Neurosci 2002;16:684–92.

46. Gleadle JM, Ratcliffe PJ. Induction of hypoxia-induciblefactor-1, erythropoietin, vascular endothelial growth factor,and glucose transporter-1 by hypoxia: evidence against a regu-latory role for Src kinase. Blood 1997;89:503–9.

47. Brines M, Cerami A. Emerging biological roles for erythro-poietin in the nervous system. Nat Rev Neurosci 2005;6:484–94.

48. Wang GL, Semenza GL. Purification and characterizationof hypoxia-inducible factor 1. J Biol Chem 1995;270:1230–7.

49. Kobliakov VA. Mechanisms of tumor promotion by reactiveoxygen species. Biochemistry (Mosc) 2010;75:675–85.

50. Sirken G, Kung SC, Raja R. Decreased erythropoietin require-ments in maintenance hemodialysis patients with statintherapy. ASAIO J 2003;49:422–5.

51. Simoni J, Simoni G, Moeller JF, et al. Anemia in chronic renalfailure (CRF): role of oxidative stress and inflammation.ASAIO J 2006;52:75A.

52. Amberson WR, Jennings JJ, Rhode CM. Clinical experiencewith hemoglobin-saline solutions. J Appl Physiol 1949;1:469–89.

53. Savitsky J, Doci J, Black J, Arnold J. A clinical safety trialof stroma-free hemoglobin. Clin Pharmacol Ther 1978;23:73–80.

54. Lutton JD, Jiang S, Drummond GS, Abraham NG, LevereRD, Kappas A. Pharmacologic effects of the red blood cellsubstitutes cross-linked and non-crosslinked hemoglobinson hematopoiesis in rabbits. Pharmacology 1999;58:319–24.

55. Sloan EP, Koenisberg M, Gens D Jr, et al. Diaspirin cross-linked hemoglobin (DCLHb) in the treatment of severe trau-matic hemorrhagic shock, a randomized controlled efficacytrial. JAMA 1999;282:1857–64.

56. Gawryl MS. Hemopure: clinical development and experience.The IX ISBS, March 2–5, 2003, Tokyo, Japan. Abstract in: ArtifBlood 2003;11:46.

57. Gannon CJ, Napolitano LM. Severe anemia after gastrointes-tinal hemorrhage in a Jehovah’s Witness: new treatmentstrategies. Crit Care Med 2002;30:1893–5.

58. Allison G, Feeney C. Successful use of a polymerized hemo-globin blood substitute in a critically anemic Jehovah’sWitness. South Med J 2004;97:1257–8.

59. Alayash AI. Oxygen therapeutics: can we tame hemoglobin?Nature 2004;3:152–9.

60. Riess JG. Oxygen carriers (“blood substitutes”)-raison d’etre,chemistry, and some physiology. Chem Rev 2001;101:2797–919.

61. Manalo DJ, Buehler PW, Baek JH, Butt O, D’agnillo F,Alayash AI. Acellular haemoglobin attenuates hypoxia-inducible factor-1alpha (HIF-1alpha) and its target genes inhaemodiluted rats. Biochem J 2008;414:461–9.

62. Simoni J, Simoni G, Lox CD, McGunegle DE, Feola M. Cytok-ines and PAF release from human monocytes and macroph-ages: effect of hemoglobin and contaminants. Artif Cells BloodSubstit Immobil Biotechnol 1994;22:525–34.

63. Pahl HL. Activators and target genes of Rel/NF-kappa B tran-scriptional factors. Oncogene 1999;18:6853–66.

64. Simoni J, Simoni G, Griswold JA, Moeller JF, Wesson DE.Activatory and suppressive roles of hemoglobin-based bloodsubstitutes at transcriptional level. ASAIO J 2003;49:181.



65. Liu JJ, Hou SC, Shen CK. Erythroid gene suppression byNF-kappa B. J Biol Chem 2003;278:19534–40.

66. Simoni J. Endothelial cell response to hemoglobin basedoxygen carriers. Is the attenuation of pathological reactionspossible? In: Kobayashi K, Thuchida E, Horinouchi H, eds.Artificial Oxygen Carrier. Its Front Line. Tokyo: Springer-Verlag, 2005;75–126.

67. Klein HG. Blood substitutes: how close to solution? Dev Biol(Basel) 2005;120:45–52.

68. Simoni J, Simoni G, Hartsell A, Feola M. An improved bloodsubstitute. In vivo evaluation of its renal effects. ASAIO J1997;43:M714–M725.

69. Keighley G, Cohen NS. Stimulation of erythropoiesis in miceby adenosine 3′,5′-monophosphate and prostaglandin E1.J Med 1978;9:129–38.

70. Suzuki Y, Takagi R, Kawasaki I, Matsudaira T, Yanagisawa H,Shimizu H. The micronucleus test and erythropoiesis: effectsof cyclic adenosine monophosphate (cAMP) on micronucleusformation. Mutat Res 2008;655:47–51.

71. Colgan SP, Eltzschig HK. Adenosine and hypoxia-induciblefactor signaling in intestinal injury and recovery. Annu RevPhysiol 2012;74:7.1–7.23.

72. Batmunkh C, Krajewski J, Jelkmann W, Hellwig-Bürgel T.Erythropoietin production: molecular mechanisms of theantagonistic actions of cyclic adenosine monophosphate andinterleukin-1. FEBS Lett 2006;580:3153–60.

73. Chepelev NL, Willmore WG. Regulation of iron pathwaysin response to hypoxia. Free Radic Biol Med 2011;50:645–66.

J. SIMONI ET AL.150


simoni-2012-adenosine-5'-triphos

Documents