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Artificial Organs

36(2):151160, Wiley Periodicals, Inc.


2011, Copyright the Authors
Artificial Organs 2011, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

A Novel Hemoglobin-Based Oxygen Carrier, Polymerized


Porcine Hemoglobin, Inhibits H2O2-Induced Cytotoxicity of
Endothelial Cells
1

*Wei Zhang, 1*Kunping Yan, *Penggao Dai, *Jingjing Tian, *Hongli Zhu,
and *Chao Chen

*College of Life Science and National Engineering Research Center for Miniaturized Detection Systems, Northwest
University; Shaanxi Lifegen Co. Ltd, Xian; and Department of Biochemistry, Jilin Medical College, Jilin, China

Abstract: Hemoglobin-based oxygen carriers (HBOCs),


with their capacity for delivering oxygen, could potentially function as red blood cell substitutes or primary
resuscitation solutions. However, there has been some
concern regarding redox-related safety issues of HBOCs.
The present study describes a novel function of polymerized porcine hemoglobin (pPolyHb) in protecting a
human umbilical vein endothelial cell line from H2O2induced cytotoxicity. Through the examination of H2O2
consumption and ferrylhemoglobin formation, we found
that pPolyHb exhibits antioxidant activity, suggesting that

pPolyHb may protect cells from free radical-induced cell


damage. Additionally, we investigated the effect of
pPolyHb on H2O2-induced cell cytotoxicity, and found
that pPolyHb significantly inhibits H2O2-mediated endothelial cell damage as well as apoptosis. Thus, pPolyHb
may be developed as a new HBOC in the future. Key
Words: Hemoglobin-based oxygen carriersPolymerized
porcine hemoglobinHydrogen peroxideCytotoxicity
Human umbilical vein endothelial cell lineSuperoxide
dismutaseCatalase.

Hemoglobin-based oxygen carriers (HBOCs) are a


potential replacement for red blood cells (RBCs)
when transfusion is needed (1). Over the past several
decades, various hemoglobin (Hb) modifications
have been developed, studied, and brought to clinical
trials in humans. Unfortunately, progress to date has
not fulfilled early expectations because of unanticipated and unwanted adverse clinical effects. A significant impediment to progress is the oxygen-binding
affinity and cytotoxicity of HBOCs (2).
Hydrogen peroxide (H2O2) has been implicated in
the redox regulation of several physiological processes, including signal transduction (3,4), response to

oxidative stress (57), development (7), cell proliferation (810), and apoptosis (7,11). H2O2, the product
of spontaneous superoxide dismutation or direct
enzymatic reaction (e.g. amine oxidase or glucose
oxidase reactions), can be generated by a variety of
mammalian cells, including neutrophils, macrophages, vascular smooth muscle, and endothelial cells.
The yield of H2O2 may increase under certain conditions, such as ischemiareperfusion (12,13).
It has been reported that H2O2 is able to induce
both apoptotic and necrotic cell death (3,1416), and
to generate hydroxyl radicals when reacting with free
ferrous iron (Fe2+), oxidizing it to ferric iron (Fe3+) in
a process called the Fenton reaction. H2O2 can also
convert ferrous Hb (containing bound Fe2+) into
ferric Hb (Fe3+), which in its oxidized form cannot
deliver O2 and is toxic to cells (17). Under some
conditions, H2O2 reacts with ferric Hb as well as other
heme proteins to produce an even higher oxidation
state of the iron (ferryl, Fe4+), which can cause lipid
peroxidation, carbohydrate degradation, and protein
cross-linking (1820). Ferrylhemoglobin can also

doi:10.1111/j.1525-1594.2011.01305.x
Received July 2010; revised May 2011.
Address correspondence and reprint requests to Dr. Chao Chen
or Dr. Hongli Zhu, College of Life Science, Northwest University,
No. 229, Taibai Northroad, Xian, Shaanxi 710069, China. E-mail:
cchen898@nwu.edu.cn or zhuyjw1971@nwu.edu.cn
1
These two authors contributed equally to this work.

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aor_1305

151..160

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W. ZHANG ET AL.

generate stable F2-isoprostanes through the peroxidation of arachidonic acid. F2-isoprostanes are
potent vasoconstrictors, a fact that is particularly
interesting, given the vasopressor effects associated
with most HBOCs (21).
An important concern in this field is the inherent
redox activity of Hb and its potentially deleterious
consequences. Recently, it has been reported that
Hb can attenuate H2O2-induced oxidative stress by
acting under certain circumstances as an antioxidative peroxidase (22), suggesting promise for the practical use of HBOCs in the future.
Polymerized porcine hemoglobin (pPolyHb), a
newly developed HBOC with superoxide dismutase
(SOD) and catalase (CAT) activities, was obtained by
glutaraldehyde cross-linking of porcine Hb. In previous studies, both rat exchange transfusion and
shock models were used to investigate the effect of
pPolyHb on rat tissues and organ recovery. After
90% of the blood was replaced with either pPolyHb
or hydroxyethyl starch (a clinically used volume
expander), it was shown that the survival time for
the pPolyHb group was much longer than for the
hydroxyethyl starch group. The blood gas and hemodynamic results, as well as physiological indicators
such as blood pressure and heart rate, were also much
better in the pPolyHb group than in the hydroxyethyl
starch group. Similar results were also observed in
the shock model (unpublished data).
In the present study, we found for the first time that
pPolyHb exhibits high antioxidative activity against
H2O2-induced cytotoxicity in human blood vessel
endothelial cells (EVC-304), and that H2O2-mediated
ferrylhemoglobin formation was inhibited by
pPolyHb. The anti-apoptotic effect of pPolyHb was
also investigated.

Preparation of porcine stroma-free hemoglobin


(pSFHb) and purified porcine hemoglobin (pHb)
Porcine blood was freshly collected from a slaughterhouse and stored at 4C with a citric acid/sodium
citrate buffer as the anticoagulant. Erythrocytes were
thoroughly washed and lysed. Membrane-associated
lipids and Hb were separated by filtration to obtain
pSFHb (used only for positive control purposes).
Subsequent pasteurization of the deoxyhemoglobin
solution was performed at 60C for 10 h, which
significantly reduced macromolecular impurities
and viral contamination. The average phospholipid
content of pHb was lower than 1.0 mg/mL. The
content of endotoxin was lower than 0.2 EU/mL, and
the final purity of pHb was greater than 99.5%
according to high-performance size-exclusion chromatography analysis. A 0.22-mm filter membrane was
used for final sterilization. This ultrapure Hb (used
only for negative control purposes) has been characterized extensively and is free of antioxidants.

MATERIALS AND METHODS

Measurement of SOD and CAT activities


SOD activity was determined using a commercially
available kit. The perborate titration method was

Reagents and animals


RPMI 1640 Medium and fetal bovine serum (FBS)
were purchased from Gibco (Grand Island, NY,
USA. Propidium iodide (PI), 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium
bromide
(MTT),
horseradish peroxidase (HRP), 4-aminoantipyrine,
3,5-dichloro-2-hydroxybenzenesulfonic acid, sodium
sulfide (Na2S), H2O2, and SOD kits were purchased
from Sigma (Sigma-Aldrich Co., St. Louis, MO,
USA). Hoechst 33258 kits were purchased from
Keygen of Nanjing. All experimental procedures in
our lab were approved by the Human Subjects and
Animal Care Committee at Northwest University
(China).
Artif Organs, Vol. 36, No. 2, 2012

Preparation of pPolyHb
The porcine Hb cross-linked by glutaraldehyde
(Lifegen 100) was prepared as described previously,
with certain modifications (23,24), and was a kind
gift from Lifegen Co. Ltd. (Xian, Shaanxi, China)
Briefly, Hb from fresh porcine blood was purified
through specific steps, and then cross-linked by
glutaraldehyde. Small molecules, including excess
glutaraldehyde and tetrameric hemoglobin, were
removed by ultrafiltration. The polymerization technique allowed the retention of SOD and CAT
activities. The physiochemical characteristics and
structural properties of pPolyHb are listed in Table 1.
The protein sample was stored at 4C under nitrogen
gas until use. Additional details regarding pPolyHb
are currently being withheld because of pending
patents.

TABLE 1. Physiochemical and structural characteristics


of pPolyHb
Polymerized porcine hemoglobin
Methemoglobin
Endotoxin
Osmolality
pH
P50
Average molecular weight of pPolyHb
64 kD tetramer
Na+
K+
Ca2+
Cl-

10.5 0.5 g/dL


<5%
<1.0 EU/mL
300330 mOsm
7.4 0.05
28 3 mm Hg
600 50 kD
<2%
135155 mmol/L
3.05.0 mmol/L
13 mmol/L
140160 mmol/L

pPolyHb INHIBITS H2O2-INDUCED CYTOTOXICITY OF ENDOTHELIAL CELLS


used to measure CAT activity (25). CAT units were
expressed as milliequivalents of perborate decomposed per milligram of blood sample, and then converted to units per gram of Hb.
H2O2 measurement
Reaction volumes (3 mL) containing the HRP/4aminoantipyrine/3,5-dichloro- 2-hydroxybenzenesul
fonic acid reagent solution (1.2 mL), pPolyHb, water,
and H2O2 were prepared (26). The final concentrations of HRP, 4-aminoantipyrine, 3,5-dichloro-2hydroxybenzenesulfonic acid were 500 kU/L, 0.99
mM, and 0.6 mM, respectively. Identical mixtures containing additional water instead of H2O2 served as
controls. The mixture stood for 2 min at 22C, and
then, the absorbance at 510 nm was recorded. H2O2
and the reagent solution participated in a peroxidasecatalyzed reaction to form a dye which can be measured at this wavelength. The molar ratio between
heme/H2O2 ranged from 1:10 to 3:1.
Ferric and ferrylhemoglobin concentration
measurement
Spectral analysis of Hb was performed using a
SPD-10AVP plus UV-VIS spectrophotometer at
37C. The concentration of ferrichemoglobin was calculated according to Chinese National Clinical Laboratory Procedures (27). Twenty microliters of each
sample was added in cyanmethemoglobin (HiCN)
reagent, mixed thoroughly, and allowed to stand for
5 min. Absorbance was determined by spectrophotometry (wavelength 540 nm, optical path: 1.0 cm),
adjusting to zero with HiCN before determination.
The Hb concentration was calculated from the
standard curve, using the equation: hemoglobin
(M) = A 5.0 10-3. The level of ferrylhemoglobin
was detected by its reaction with Na2S to form
sulfhemoglobin. The conversion of ferrylhemoglobin
to sulfhemoglobin was monitored by determining the
absorbance of the reaction mixture at 620 nm. The
concentration of sulfhemoglobin was calculated
using the extinction coefficient of sulfheme (e620
604 nm = 10.5 mM-1 cm-1).
Cell culture
EVC-304 cells were a gift from the Fourth Military
Medical University, and the cells were harvested with
permission from the Institutional Review Board for
the Protection of Human Subjects. Cells were grown
in RPMI 1640 Medium with 10% FBS and maintained in 100-mm dishes at 37C in a humidified
atmosphere of 95% air and 5% CO2. For experiments, cells were plated in 60 mm dishes and reached
confluence within 2 to 3 days. For harvesting, cells

153

were washed with N-2-hydroxyethylpiperazine-N-2ethanesulfonic acid (HEPES)-buffered saline and


incubated with 0.025% trypsin0.01% ethylenediaminetetraacetic acid for 5 min at 37C. After detachment, a trypsin-neutralizing agent was added and
cells were centrifuged at 800 g for 5 min at 4C. Cell
counts were obtained using a Coulter counter.
Experimental cell treatments
Early passage EVC-304 cells were grown in complete RPMI 1640 for 24 h before the medium was
replaced with fresh complete RPMI 1640. Cells were
subsequently washed with Hanks buffered salt solution (HBSS) and incubated with 3 mL FBS-free
supplemented medium or medium containing H2O2,
or with three types of Hb solution combined with
H 2 O2 .
After 12 h incubation, cell morphology was viewed
by phase contrast microscopy using an Olympus
model Bx61/Bx62 inverted microscope equipped
with a MCA-85001 camera (Olympus, Tokyo, Japan).
MTT assay
Cells (~8 104/well) were seeded in 96-well cell
culture plates. After treatment, 20 mL MTT (5 mg/
mL) was added to the media and incubated for 3 h at
37C. The MTT solution was replaced by 150 mL dimethyl sulfoxide (DMSO), and the cells were agitated
for 10 min. The absorbance was then read at 570 nm.
Fluorescence microscopy
Cells were treated with 150 mM pPolyHb, 50 mM
H2O2, or both. After 12 h incubation, they were
washed twice with PBS, and then treated according to
the Hoechst 33258 kit instructions. Three hours later,
the cells were viewed by phase contrast microscopy
using an Olympus model Bx61/Bx62 inverted microscope equipped with a MCA-85001 camera.
Annexin VPI assay
Cells were treated with 50 mM H2O2, or 150 mM
pPolyHb and 50 mM H2O2, for 12 h. The media was
then removed and cells were washed with HBSS
without calcium or magnesium and trypsinized. Cells
were suspended in 1 annexin V binding buffer
containing 10 mM HEPES/NaOH, 140 mM NaCl,
2.5 mM CaCl2, pH 7.4. Cells were transferred to a
culture tube, and annexin V and PI were added. After
gentle vortexing, the cells were incubated for 15 min
at room temperature in the dark. After adding
350 mL assay buffer to each tube, cells were analyzed
by flow cytometry (Beckman Coulter-Elite, Fullerton, CA, USA). Data (collected from a minimum of
Artif Organs, Vol. 36, No. 2, 2012

W. ZHANG ET AL.

10 000 cells per sample) were analyzed using Expo32


(AV) analysis software (Beckman Coulter, Miami,
FL, USA). Stained cell populations were defined as
either B2, that is, necrotic or late apoptotic cells
(annexin V+, P+), or B4, that is, cells undergoing early
apoptosis (annexin V+, PI).
Statistical analysis
Data were represented as mean SD for replicate
experiments. The differences between treatment
groups were assessed by one-way ANOVA followed
by unpaired Students t-test. Statistical significance
was defined as P < 0.05 to reject a null hypothesis. All
statistical calculations were performed with JMP
version 3.2 for the Macintosh (SAS Institute, Cary,
NC, USA).

H2O2 concentration (M)

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200
150
100

*
**

50

**

0
0

10

20

40

60

80

100

150

pPolyHb concentration (M)


FIG. 2. Dose-dependent H2O2 consumption by pPolyHb. Serial
concentrations of pPolyHb were incubated with 200 mM H2O2
at room temperature for 2 min. The amount of H2O2 remaining
was measured by reaction with a HRP/4-aminoantipyrine/3,
5-dichloro-2-hydroxybenzenesulfonic acid solution. Data shown
are mean SD of one representative experiment of at least five
independent experiments. Statistical significance is indicated by
*P < 0.05 for the 80-mM pPolyHb reaction, and ** P < 0.01 for the
100 and 150 mM pPolyHb reactions.

RESULTS AND DISCUSSION


Results
SOD and CAT activity of pPolyHb
Superoxide radicals play a key role in conditions of
oxidative stress. To investigate the degradation of
superoxide radicals by pPolyHb, the activity of SOD
in pPolyHb was assayed. SOD catalyzes the dismutation of superoxide radicals into H2O2 and elemental
oxygen, thus providing an important defense against
the toxicity of the superoxide radical. As shown in
Fig. 1A, the SOD activity of pPolyHb was comparable to that of RBC and pSFHb, a product originating from RBCs. However, its SOD activity is much
higher than that of pHb. We also compared the CAT
activity of pPolyHb with RBC and pSFHb (Fig. 1B),
and the results showed that pPolyHb CAT activity is
comparable to that of RBC and pSFHb.

SOD activity (U/g Hb)

10000
1000

**
100
10
1
RBC

pSFHb

pPolyHb

pHb

Type of hemoglobin solution

CAT activity (U/g Hb)

100000
10000
1000

**

100
10
1
RBC

pSFHb

pPolyHb

pHb

Type of hemoglobin solution


FIG. 1. Determination of pPolyHb SOD and CAT activities. (A)
SOD activity in RBC lysate, pSFHb, pPolyHb, and pHb; (B) CAT
activity in RBC lysate, pSFHb, pPolyHb, and pHb. Data represent
mean SD of one representative experiment of at least five
independent experiments. Statistical significance indicated by
**P < 0.001.
Artif Organs, Vol. 36, No. 2, 2012

H2O2 consumption by pPolyHb


H2O2 is an endogenous oxidant, which is believed
to be cytotoxic when it reacts with Hb under conditions of oxidative stress. To determine the H2O2 consumption, or peroxidase activity, of pPolyHb, we
added a series of amounts of pPolyHb to 200 mM
H2O2 solution and then measured the remaining
H2O2 (Fig. 2). These results showed that 80 mM
pPolyHb can consume a significant amount of H2O2
and that the H2O2 level decreased to a basal physiological level when 150 mM pPolyHb was added to
the solution.
Formation of ferrylhemoglobin intermediate was
inhibited by pPolyHb
Ferrylhemoglobin is a cytotoxic agent produced by
the interaction of ferrous Hb or ferric Hb with H2O2

pPolyHb INHIBITS H2O2-INDUCED CYTOTOXICITY OF ENDOTHELIAL CELLS

155

B
100 M pSFHb and 200 M H2O2
100 M pPolyHb and 200 M H2O2

Concentration of ferrichemoglobin (M)

60

100 M pHb and 200 M H2O2

40
20
0
0

15
30
Time (min)

45

60

Concentration of ferrylhemoglobin (M)

C
100 M pSFHb and 200 M H2O2
100 M pPolyHb and 200 M H2O2

50

100 M pHb and 200 M H2O2

40
30
20
10
0
0

15

30

45

60

Time (min)
FIG. 3. pPolyHb inhibition of ferrichemoglobin or ferrylhemoglobin formation. pSFHb, pPolyHb, or pHb solutions were incubated with
H2O2 at room temperature for 60 min. (A) Spectral analysis of hemoglobin with a SPD-10AVP plus UV-VIS spectrophotometer; (B) and
(C) Ferrichemoglobin and ferrylhemoglobin formation. Each curve represents measurements taken every 2 min for the first 10 min,
followed by every 5 min for the next 50 min.

under conditions of oxidative stress. It can be


detected by reaction with Na2S to produce sulfhemoglobin, which absorbs light at 620 nm (28). To further
confirm the antioxidant activity of pPolyHb, H2O2
and either pSFHb, pPolyHb, or purified Hb were
incubated for up to 60 min; after which, the concentration of ferrylhemoglobin was measured. The spectrometry results revealed no significant changes in

the absorbance spectrum of pPolyHb with H2O2 over


a time course of 60 min. However, the pHb caused a
visible spectrum change, as seen in Fig. 3A. Consistent with the spectrometry results, no ferrylhemoglobin was formed from the incubation of pSFHb or
pPolyHb and H2O2 (Fig. 3C), as measured by a ferrylhemoglobin concentration assay.These results suggested that pPolyHb exhibits antioxidant activity and
Artif Organs, Vol. 36, No. 2, 2012

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W. ZHANG ET AL.
A

B
1.4
1.2

OD Value

1
0.8

**

0.6
0.4
0.2
0
Untreated

200 M H2O2

150 M pSFH and


200 M H2O2

150 M pPolyHb
and 200 M H2O2

150 M pHb and


200 M H2O2

Culture conditions
FIG. 4. pPolyHb inhibition of H2O2-induced cell damage. Cells were incubated with FBS-free medium, H2O2, pSFHb, pPolyHb, or pHb
combined with H2O2 as indicated. (A) Cell morphology, as detected by phase contrast microscopy; (B) MTT assay analysis of living cells.
Statistical significance indicated by **P < 0.01 for pPolyHb vs. H2O2; #P < 0.05 for pPolyHb vs. pHb.

does not produce the cytotoxic agent ferrylhemoglobin when exposed to H2O2. The accumulation of ferrichemoglobin was also investigated, and the results
showed that the formation of ferrichemoglobin was
significantly inhibited by pPolyHb, relative to pHb
(Fig. 3B).

pPolyHb plays a protective role in H2O2-induced


cell damage
In order to verify that pPolyHb has a potential
protective role against cell damage induced by H2O2,
two different assays were used to examine the effect
Artif Organs, Vol. 36, No. 2, 2012

of pPolyHb on cell viability. Initially, a cell viability


and cytotoxicity assay was performed using a MTT
cell viability kit. EVC-304 cells were treated with
H2O2, or H2O2 combined with pSFHb, pPolyHb, or
pHb, and the cell viability was determined. The molar
ratio of heme/H2O2 was 3:1. The results of the cell
viability assays from EVC-304 cells were confirmed
using phase contrast microscopy. The drastic decline
in viability was associated with visible rounding of
the cells and detachment when H2O2 alone was
added to the cells (Fig. 4). In contrast, the cells
treated with pSFHb and H2O2, or pPolyHb and H2O2,
did not show visible changes in cell morphology and

pPolyHb INHIBITS H2O2-INDUCED CYTOTOXICITY OF ENDOTHELIAL CELLS

157

Early apoptosis ratio (%)

**

30
20
10
0
Control

50 M H2O2

50 M H2O2 and
150 M pPolyHb

Culture conditions
FIG. 5. pPolyHb inhibition of H2O2-induced cell apoptosis. Cells were incubated with FBS-free medium, H2O2, or pPolyHb combined with
H2O2, as indicated. (A) Fluorescence microscopic examination of nuclear staining with Hoechst 33258; (B) flow cytometric analysis of
annexin V-stained cells; (C) quantitative analysis of data in (B). Statistical significance indicated by **P < 0.01.

cell number (Fig. 4), indicating that pSFHb and


pPolyHb protect against H2O2-induced cell damage.
Figure 4A also shows the accumulation of debris
from dead cells or insoluble precipitates in cells
treated with pHb with H2O2, which possibly contain
oxidized Hb products.
pPolyHb inhibited H2O2-induced cell apoptosis
The oxidative stress exerted by H2O2 exposure
eventually induces cell death by apoptosis or necrosis

(10). To confirm that pPolyHb plays a role in preventing H2O2-induced cell apoptosis, we tracked cell
viability using nuclear staining and annexin V
staining. EVC-304 cells were incubated with H2O2
alone or H2O2 together with pPolyHb. Nuclei were
stained with Hoechst 33258, a cell-permeable blue
fluorescent DNA dye, to detect nuclear condensation and fragmentation, which are characteristics of
apoptosis. As shown in Fig. 5A, H2O2-treated cells
were characterized by condensed bright Hoechst
Artif Organs, Vol. 36, No. 2, 2012

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W. ZHANG ET AL.

staining, indicating that the cells underwent apoptosis, while the combination of pPolyHb and H2O2 prevented the apoptotic phenomena. These cells showed
a normal Hoechst staining pattern, similar to the
control cells. To confirm this result, we stained the
cells with annexin V, an early apoptotic marker, and
PI for detection of late apoptosis. Flow cytometry
was used to quantify fluorescent cells, and showed
that pPolyHb significantly reduced the percentage of
cells in both early and late stages of apoptosis (Fig.
5B,C).
DISCUSSION AND CONCLUSIONS
HBOCs have been extensively studied as potential
blood substitutes, and several products have been
brought to clinical trials in humans (29). However,
redox-related safety issues are still among the major
concerns in this field. As mentioned above, most
HBOC products have been shown to be cytotoxic to
cells through a mechanism that involves the oxidant
activity of H2O2 (30,31). pHb or HBOC preparations
in their reduced forms are not, by themselves, cytotoxic to endothelial cells (32,33). However, when oxidized by H2O2, the accumulation of highly reactive
Fe4+ can induce significant cytotoxicity through a
mechanism that involves the production of toxic
oxygen intermediates such as ferryl-Hb (HbFe4+ = O)
and a globin tyrosyl-based radical (HbFe4+ = O).
OH and Hb Fe4+ = O can initiate lipid peroxidation,
while HbFe4+ = O is an effective modifier of lowdensity lipoproteins. Ferryl-Hb species may also contribute to many other pathological events, including
damage to large molecules and apoptosis (20,3436).
The molar ratio between H2O2 and Hb was shown to
be a critical factor for the formation of ferryl-Hb.
Simoni et al. reported that a high molar ratio of H2O2
to Hb increased ferryl-Hb formation by about 15%,
and would likely increase the damage to human
endothelium (37).
However, we have shown here that pPolyHb has a
cytoprotective function and thus is a potential solution to the problems mentioned above. Several lines
of evidence support our conclusion. First, an H2O2
consumption assay showed that pPolyHb is able
to neutralize exogenously added H2O2, indicating
that pPolyHb can decrease H2O2-mediated cytotoxicity. Second, ferrylhemoglobin formation was not
observed as a result of incubation of pPolyHb and
H2O2, suggesting a lack of toxic product formation by
pPolyHb. Third, MTT and cell morphology assays
showed that cell death caused by H2O2 could be prevented by the presence of pPolyHb. Fourth, flow
cytometry and immunostaining with an apoptotic
Artif Organs, Vol. 36, No. 2, 2012

marker revealed that pPolyHb inhibited apoptosis


induced by H2O2. Consistent with the involvement of
the pPolyHb in preventing H2O2-induced cell cytotoxicity, the SOD and CAT activities of pPolyHb are
much higher than those of purified Hb, which may
result from the polymerization technique used in the
preparation of pPolyHb.
Redox-related safety issues underlie some of the
adverse effects of Hb. pPolyHb is able to overcome
these adverse effects due to its antioxidant properties, which are similar to those of RBCs. In the case of
blood exchange or hemorrhagic shock, colloid or
crystal fluid resuscitation will dilute the RBCs and
decrease the antioxidant effects. In contrast, resuscitation of pPolyHb with antioxidant properties should
allow retention of most antioxidant capability, and
thus be superior to other resuscitation fluids in this
respect.
The increasing awareness that the toxicity of Hb
may limit the safety or efficacy of first-generation
Hb-based therapies has prompted the design of new
strategies aimed at reducing this toxicity (38). Currently, several strategies have been explored, including chemical cross-linking, alteration of the heme
pocket to reduce nitric oxide (NO) reactivity, and
coadministration of antioxidants or antiferryl compounds to limit HBOC toxicity (20,39). The strategy
for preparing the pPolyHb used in this experiment
results in a product that retains relatively high SOD
and CAT activities. These enzymes convert superoxide radicals into H2O2, which in turn is converted
into water and oxygen. Thus, H2O2 is consumed by
pPolyHb, and cellular toxicity is lowered by pPolyHb.
Although the mechanisms underlying these activities
are still unknown, inhibition of cell cycle arrest by
extracellular CAT and the loss of thiols strongly
suggest the involvement of an initial extracellular
oxidative event that triggers the subsequent cellular
responses (19,32).
Our present study showed that pPolyHb is nontoxic in vitro. While it is not yet clear whether this
product will yield benefits in vivo, additional in vivo
studies, such as ischemiareperfusion experiments,
should be performed to examine the effects of
pPolyHb in the circulatory blood. pPolyHb is being
developed for eventual use in humans. Some HBOCs
(PolyHeme, Hemolink, Hemospan) use outdated
human blood as Hb sources. However, due to the
severe limitation of human Hb sources, other Hb
sources have also been developed. For example,
Biopure Co. Ltd. uses bovine blood as the raw material for Hemopure. pPolyHb is derived from porcine
sources. Due to its abundant supply and high homology with human Hb, as well as its freedom from

pPolyHb INHIBITS H2O2-INDUCED CYTOTOXICITY OF ENDOTHELIAL CELLS


contamination with human viruses such as HIV and
hepatitis virus, porcine Hb could be a potential
material in large-scale production of HBOCs. More
studies will need to be performed on pPolyHb in the
areas of immunogenicity and pathogen removal prior
to its use in humans.
Ferrylhemoglobin has been shown to be toxic to a
number of different cell types, including endothelial
cells (19,40). It is known to be formed via the reaction of oxy- and methemoglobin with H2O2,
although we still do not know which cellular oxidants react with Hb to produce the ferryl-Hb oxidation state. Under our experimental conditions,
ferrylhemoglobin formation after the addition of
pPolyHb and H2O2 was not observed, suggesting
that pPolyHb has no potential cytotoxicity. We are
currently investigating ferrylhemoglobin formation
in animal models, which will provide us with a
physiological condition for understanding the
potential toxicity of pPolyHb.
H2O2 can induce apoptosis or necrosis in a variety
of cell types (7). H2O2 treatment caused toxic effects
to EVC-304 cells as previously reported, while the
addition of pPolyHb significantly increased the
number of live cells. This effect was shown to be dose
dependent (unpublished data). In the presence of
pPolyHb, we do not detect H2O2-induced apoptosis,
indicating either that H2O2 is immediately consumed
by the peroxidase activity of pPolyHb or that apoptosis signaling is impaired by the addition of
pPolyHb. The bolus addition of pPolyHb to high
micromolar concentrations of H2O2 decreased the
amount of H2O2 detected, suggesting that the protective effect of pPolyHb is due to H2O2 consumption.
We do not yet have any evidence to show that
pPolyHb participates in the apoptotic signaling
pathway. The investigation of pPolyHb involvement
in other chemically induced apoptotic pathways will
provide further evidence for the protective mechanism of pPolyHb.
Although the exact molecular mechanism by
which pPolyHb prevents H2O2-induced cell death
has yet to be determined, the demonstration that
pPolyHb is involved in cellular protection suggests a
direction for further studies. The fact that polymerized placental Hb significantly inhibits the expression of cleaved caspase-3 as well as the ratio of
Bax/Bcl-2 points to a potential anti-apoptotic
mechanism for the cytoprotective function of
pPolyHb (41). While this mechanism is attractive,
currently, we do not have evidence indicating
pPolyHb involvement in the caspase-3 signaling
pathway, although related experiments are underway. Regardless of whether pPolyHb participates in

159

this signaling pathway, the present study has not


only uncovered a novel function of pPolyHb, but
has also set the direction for the development of a
new generation of blood substitutes.
Acknowledgments: This work was supported by
grants from the National 863 Program of China
(grant nos. 2006AA02A143 and 2009AA022710) and
the Special Research Foundation of the Education
Department of Shaanxi Province (grant nos. 08JK469
and 09JK784) as well as grants from Shaanxi Science
and Technology Department (grant nos. 2011K12-0310). We would like to express our gratitude to Dr.
Ning Dan for his help and discussion in experiment
design and Dr. Simoni for his constructive advice.

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