*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
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.
151
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 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.
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W. ZHANG ET AL.
154
200
150
100
*
**
50
**
0
0
10
20
40
60
80
100
150
10000
1000
**
100
10
1
RBC
pSFHb
pPolyHb
pHb
100000
10000
1000
**
100
10
1
RBC
pSFHb
pPolyHb
pHb
155
B
100 M pSFHb and 200 M H2O2
100 M pPolyHb and 200 M H2O2
60
40
20
0
0
15
30
Time (min)
45
60
C
100 M pSFHb and 200 M H2O2
100 M pPolyHb and 200 M H2O2
50
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.
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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 pPolyHb
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).
157
**
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.
(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
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