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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Redox cycling of diaspirin cross-linked hemoglobin induces G2/M arrest and
apoptosis in cultured endothelial cells
Felice D’Agnillo and Abdu I. Alayash
It is hypothesized that oxidative reactions
of hemoglobin driven by reactive oxygen
species in the vasculature lead to endothelial cell injury or death. Bovine aortic
endothelial cells were incubated with diaspirin cross-linked hemoglobin (DBBFHb), developed as a hemoglobin-based
oxygen carrier, and hydrogen peroxide
(H2O2), generated by the glucose oxidase
system. The low steady flux of H2O2 oxidizes the ferrous form of DBBF-Hb and
drives the redox cycling of ferric and
ferryl DBBF-Hb. Cells underwent rounding, swelling and detachment, and accu-
mulated in the G2/M phase of the cell
cycle. G2/M arrest preceded the onset of
apoptosis as determined by increases in
phosphatidylserine (PS) externalization
and sub-G1 events. Redox cycling of unmodified hemoglobin also led to G2/M
arrest and apoptosis. The rate and extent
of DBBF-Hb oxidation correlated with the
onset and extent of G2/M arrest and apoptosis and induced significant decreases
in soluble reduced thiols. Earlier depletion of glutathione by pretreatment with
buthionine sulfoximine rendered cells
more susceptible to G2/M arrest and apo-
ptosis. The caspase inhibitor, z-VAD-fmk,
had no effect on the induction of G2/M
arrest but completely inhibited the subsequent increases in PS externalization and
sub-G1 events. Catalase inhibited DBBF-Hb
oxidation, the loss of thiols, and the onset
of G2/M arrest and apoptosis. These data
support a causative role for the ferric–
ferryl redox cycle in the development of
endothelial cell injury. (Blood. 2001;98:
3315-3323)
© 2001 by The American Society of Hematology
Introduction
The release of hemoglobin or myoglobin from its cellular environment during certain pathologic conditions leads to hemoproteininduced vascular damage or dysfunction.1-6 Adverse microvascular
effects of cell-free hemoglobin have also hindered the development
of safe and efficacious hemoglobin-based oxygen carriers.7 These
agents are chemically stabilized forms of hemoglobin designed to
act as oxygen and volume replacement therapy in clinical settings
such as emergency resuscitation and elective surgery.7-9 Although
the molecular mechanisms of hemoprotein-mediated cytotoxicity
are not completely understood, the interactions of heme (protein or
nonprotein bound) with hydrogen peroxide (H2O2), organic peroxides (ROOH), peroxynitrite (ONOO⫺), and nitric oxide (NO) have
been implicated.1-7
The reaction of hemoglobin with H2O2 or ROOH leads to a series of
reduction-oxidation (redox) transitions between the ferrous, ferric, and
ferryl states of the protein. The oxy and deoxy forms of ferrous
hemoglobin (HbFe2⫹) and ferric hemoglobin (HbFe3⫹) can each react
with H2O2 to produce ferryl hemoglobin (HbFe4⫹) (equations 1 and
2).10-12 In the case of HbFe3⫹, this reaction also generates a proteinbased radical that can be detected by electron paramagnetic spectroscopy.12 HbFe4⫹ can, in turn, be reduced to HbFe3⫹ autocatalytically or
after substrate oxidation.10-12 Low and continuous fluxes of H2O2 or
ROOH can drive and sustain the transition between HbFe4⫹ and
HbFe3⫹ (ie, redox cycling)12:
(2)
HbFe3⫹ ⫹ H2O2 3 䡠HbFe4⫹|O ⫹ H2O
HbFe2⫹O2 ⫹ H2O2 3 HbFe4⫹|O ⫹ H2O ⫹ O2
HbFe4⫹ can initiate lipid peroxidation and oxidize other biologic molecules.13-15 HbFe4⫹ generated from the interaction of
tert-butyl hydroperoxide and hemoglobin was cytotoxic to vascular
smooth muscle cells.16 A causative role for the redox cycling of
myoglobin has been implicated in rhabdomyolysis-induced renal
failure and myocardial reperfusion injury.2,17-19 Another potential
mechanism of hemoglobin toxicity involves the oxidative degradation and release of heme or iron, which can react with H2O2 to form
the highly toxic hydroxyl radical through Fenton chemistry.
However, the significance of this pathway has been debated
because of the high oxidant concentrations required to release
heme or iron from hemoglobin.20
Endothelial cells are especially susceptible to hemoglobininduced toxicity because of their proximity to circulating blood.
Enzymes such as xanthine oxidase, NADH–NADPH oxidase, and
myeloperoxidase may play prominent roles in driving the oxidative
reactions of hemoglobin in the vasculature.21 Increased oxidant
production or antioxidant depletion associated with ischemia
reperfusion, hemorrhagic shock, and septic shock may render the
vasculature even more susceptible to hemoprotein-induced damage.22-26 These factors might have contributed to the microvascular
events associated with the preclinical and clinical use of DCLHb
(Baxter Healthcare, Deerfield, IL) and DBBF-Hb (Walter Reed
Army Institute of Research, Washington, DC), the commercial and
noncommercial analogues of diaspirin cross-linked hemoglobin,
From the Laboratory of Plasma Derivatives, Division of Hematology, Center for
Biologics Evaluation and Research, Food and Drug Administration, Bethesda,
MD.
Reprints: Abdu I. Alayash, Center for Biologics Evaluation and Research,
Food and Drug Administration, Bldg 29, Rm 112, 8800 Rockville Pike,
Bethesda, MD 20892; e-mail: [email protected].
(1)
Submitted February 15, 2001; accepted July 19, 2001.
F.D. is a recipient of a Fogarty International Fellowship from the National
Institutes of Health.
BLOOD, 1 DECEMBER 2001 䡠 VOLUME 98, NUMBER 12
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2001 by The American Society of Hematology
3315
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3316
D’AGNILLO and ALAYASH
respectively.8,27-30 Extensive in vivo and in vitro research on
DCLHb and DBBF-Hb continues to provide a useful basis for
correlating the toxicity of hemoglobin-based oxygen carriers with
the redox chemistry of hemoglobin.30
Previously, we showed that endothelial cells incubated with
DBBF-Hb and bolus amounts of H2O2 underwent apoptosis,
necrosis, or both.31 We also detected the ferryl form of DBBF-Hb in
an endothelial cell model of hypoxia and reoxygenation, and its
formation closely paralleled increases in lipid peroxidation.32,33
Recently, we demonstrated a direct correlation between the formation of DBBF-HbFe4⫹ by bolus H2O2 and the depletion of
endothelial cell glutathione (GSH).34 Furthermore, we showed that
prior depletion of cellular GSH may exacerbate the cytotoxicity of
DBBF-Hb and bolus H2O2.34 However, the addition of high bolus
amounts of oxidants by themselves causes apoptosis or necrosis;
thus, it has been difficult to distinguish the true component of
cytotoxicity mediated by hemoglobin redox reactions.34 Use of the
glucose oxidase system to enzymatically generate H2O2 has been
shown to better mimic the physiologic delivery of H2O2.35-37
Continuous generation of low levels of H2O2 can sustain the redox
cycling of HbFe3⫹ and HbFe4⫹ and can allow the determination of
hemoglobin-induced cytotoxicity without the confounding effects
of bolus oxidant addition.
In the current study, we investigated the cytotoxicity of DBBF-Hb
and glucose oxidase-generated H2O2 in cultured bovine aortic endothelial cells. We also examined the role of cellular thiols by depleting
intracellular GSH with buthionine sulfoximine, mimicking the lack of
antioxidant defenses associated with pathologic conditions. We found
that the redox cycling of DBBF-Hb by low levels of enzymatically
generated H2O2 induces G2/M cell cycle arrest, which is then followed
by apoptosis of growth-arrested cells. These findings provide new
insight into the mechanisms of hemoprotein-induced endothelial cell
injury and may have direct implications on the safety evaluation of
hemoglobin-based oxygen carriers.
BLOOD, 1 DECEMBER 2001 䡠 VOLUME 98, NUMBER 12
DBBF-Hb was aliquoted and stored at ⫺80°C. DBBF-HbFe3⫹ and its
cyanide derivative (DBBF-HbFe3⫹CN) was prepared as previously described.39 Chromatographically purified human hemoglobin, free of superoxide dismutase and catalase, was a kind gift from Hemosol (Etobicoke,
ON, Canada). Red blood cells were isolated from whole human blood by
centrifugation, washed 3 times in sterile PBS, and resuspended in PBS at a
hematocrit level of 50%.
Endothelial cell culture
Bovine aortic endothelial cells (BW-6002) were obtained from Clonetics.
Cells were grown in EGM supplemented with 10 ng/mL human recombinant epidermal growth factor, 1 ␮g/mL hydrocortisone, 10 ␮g/mL bovine
brain extract, 5% FBS, 50 ␮g/mL gentamicin, and 50 ng/mL amphotericin-B. Cells were maintained in 100-mm dishes at 37°C in a humidified
atmosphere of 95% air and 5% CO2. For experiments, cells were plated in
6-well plates and reached confluence within 2 to 3 days. For harvesting,
cells were washed with HEPES-buffered saline and incubated with 0.025%
trypsin–0.01% EDTA for 5 minutes at 37°C. After cell detachment,
trypsin-neutralizing agent was added and cells were centrifuged at 220g for
5 minutes at 4°C. Cell counts were obtained using a Coulter counter
(Hialeah, FL). Experimental data were obtained from bovine aortic
endothelial cells in the second to eighth passages.
Experimental cell treatments
Cells were grown in complete EGM for 24 hours before the medium was
replaced with fresh complete EGM with or without 1 mM BSO. After 24
hours, normal or BSO-treated cells were washed with HBSS and incubated
with 3 mL FBS-free supplemented medium or medium containing DBBFHb, GOX, or both. DBBF-Hb was added to wells just before the addition of
GOX. For caspase inhibition experiments, z-VAD-fmk (40 ␮M) was added
90 minutes before treatment and then during the treatment period. Cell
morphology was viewed by phase-contrast microscopy using an Olympus
model CK2 inverted microscope equipped with an SLR 35-mm camera
(Olympus America, Melville, NY).
Analysis of hemoglobin oxidation products
Materials and methods
Materials
Endothelial cell growth medium (EGM) containing 5.6 mM glucose and
fetal bovine serum (FBS) was obtained from Clonetics (San Diego, CA).
Phosphate-buffered saline (PBS) and Hanks balanced salt solution (HBSS)
containing 5.6 mM glucose were obtained from Life Technologies (Grand
Island, NY). Glucose oxidase (GOX, type X-S derived from Aspergillus
niger, 100 000-250 000 U/g solid), 5,5 dithio-bis (2-nitrobenzoic acid)
(DTNB), propidium iodide (PI), 5-sulfosalicylic acid, DL-buthionine-[S,
R]-sulfoximine (BSO), bovine liver catalase (catalog number C-1345),
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Odianisidine-dihydrochloride, and horseradish peroxidase (type 2) were
obtained from Sigma Chemical (St Louis, MO). Caspase inhibitor, z-VADfmk (benzyloxycarbonyl-Val-Ala-Asp(OMe)-CH2F) was purchased from
Enzyme Systems Products (Livermore, CA).
Hemoglobin solutions
DBBF-Hb, free of superoxide dismutase and catalase, was a kind gift from
the Walter Reed Army Institute of Research (Washington, DC). Hemoglobin was purified from outdated human red blood cells and reacted with
bis(3,5-dibromosalicyl)fumarate (DBBF) to intramolecularly cross-link the
␣-99 lysine residues.38 This modification prevents disassociation into ␣␤
dimers and significantly increases the circulation time of DBBF-Hb
compared to unmodified hemoglobin. The characteristics of DBBF-Hb are
similar to those of DCLHb, except that DBBF-Hb was formulated in
Ringers acetate whereas DCLHb was formulated in Ringers lactate.8
Spectral analysis of hemoglobin was performed using a Hewlett-Packard
HP-8453 rapid scanning diode array spectrophotometer (Agilent Technologies, Rockville, MD). Total hemoglobin concentration and its different
oxidation states were measured using the Winterbourn method.40 Ferryl
hemoglobin level was also determined by measuring the sodium sulfide
(Na2S)–induced formation of sulfhemoglobin.10 Samples were reacted with
2 mM Na2S, and the concentration of sulfhemoglobin was calculated using
the extinction coefficient of 10.5 mM⫺1 at 620 nm.
Hydrogen peroxide measurement
H2O2 was measured using a spectrophotometric assay based on the
peroxidase-catalyzed oxidation of O-dianisidine.35 GOX activity was
measured in reaction mixtures containing GOX, 250 ␮M O-dianisidine, and
10 U/mL horseradish peroxidase in HBSS, pH 7.4, at 25°C. The absorbance
increase at 460 nm was recorded for 15 minutes and was converted to ␮M
H2O2/min using an H2O2 calibration curve prepared in HBSS. Extracellular
H2O2 was measured by adding 950 ␮L culture samples to 1 mL cuvettes
containing O-dianisidine and horseradish peroxidase. Absorbance was
measured at 460 nm and was converted to concentration using an H2O2
calibration curve prepared in supplemented FBS-free medium.
Soluble reduced thiol determination
Soluble reduced thiols were measured using the DTNB colorimetric
assay.34 Briefly, cells were washed with 2 mL HBSS, and 0.6 mL of 2%
(wt/vol) 5-sulfosalicylic was added for solubilization and deproteinization.
After centrifuging the samples for 5 minutes at 10 000g, 500-␮L aliquots
were mixed with 500 ␮L freshly prepared DTNB solution (0.3 M sodium
phosphate, 10 mM EDTA, and 0.2 mM DTNB). After 5 minutes, the
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BLOOD, 1 DECEMBER 2001 䡠 VOLUME 98, NUMBER 12
HEMOGLOBIN REDOX CYCLE AND ENDOTHELIUM
3317
absorbance was measured at 412 nm. Calculations were based on the
extinction coefficient of 13.4 mM⫺1 cm⫺1 derived from GSH standards.
Annexin-V–propidium iodide assay
Nonadherent and adherent cells, harvested by trypsinization, were pooled,
washed twice in HBSS without calcium or magnesium, and resuspended in
1 mL assay buffer containing 10 mM HEPES/NaOH, 140 mM NaCl, 2.5
mM CaCl2, pH 7.4. Annexin V– fluorescein isothiocyanate (5 ␮L of 2
␮g/mL; PharMingen, San Diego, CA) and PI (10 ␮L of 5 ␮g/mL) were
added to a 100-␮L aliquot (approximately 1 ⫻ 105 cells) of this cell
suspension. In most cases, the remainder of the cell suspension (0.9 mL)
was immediately fixed in ethanol for later analysis of cell cycle (see below).
After a 15-minute incubation in the dark at room temperature, stained cells
were diluted with 350 ␮L assay buffer and analyzed using a FACScan flow
cytometer (Becton Dickinson, San Jose, CA) and Cellquest analysis
software. Data were collected from a minimum of 10 000 cells per sample.
Stained cell populations were defined as: R1, viable or undamaged cells
(annexin V⫺, PI⫺); R2, cells undergoing early apoptosis (annexin V⫹,
PI⫺); and R3, necrotic or late apoptotic cells, (annexin V⫹, PI⫹).
DNA content measurement and cell cycle analysis
Nonadherent and adherent cells were washed with PBS and fixed with 2 mL
ice-cold 70% ethanol for 30 minutes on ice or stored at ⫺20°C until
analysis. Fixed cells were centrifuged, washed with 10 mL PBS, and
resuspended in 0.5 mL staining solution (0.1% Triton-X, 5 ␮g/mL PI, and
50 ␮g/mL RNAse). After 25 minutes in the dark at room temperature,
samples were analyzed by flow cytometry. Data were collected from a
minimum of 20 000 events per sample, and analysis was performed on
gated populations excluding doublets, triplets, and quadruplets. Proportions
of cells in the G1, S, and G2/M phases were quantified using the ModFit LT
version 3 software (Verity Software House, Topsham, ME).
Statistical analysis
Data are represented as means ⫾ SE for replicate experiments unless
otherwise stated. Statistical analysis was performed using the unpaired
2-tailed Student t test with the SAS JMP (version 3.2) software (SAS
Institute, Cary, NC).
Results
Redox cycling of DBBF-Hb by enzymatically generated H2O2
The reaction of DBBF-Hb with GOX-generated H2O2 could be
monitored as changes in the visible spectrum of DBBF-Hb. Figure
1A shows the representative spectra of DBBF-HbFe2⫹ (the oxy
form), DBBF-HbFe3⫹, or DBBF-HbFe3⫹CN before and 2 hours
after the addition of GOX. In the case of DBBF-HbFe2⫹, the
characteristic peaks at 541 and 577 nm disappeared gradually over
the course of 2 hours. The resultant spectrum, with a shoulder at
580 nm and an absorbance increase in the 630-nm region,
suggested a mixture of ferryl and ferric species. The addition of
GOX to DBBF-HbFe3⫹ also produced a spectrum with peaks at
545 nm and a shoulder at 580 nm, indicating the formation of the
ferryl species. In contrast, GOX produced few changes in the
spectrum of DBBF-HbFe3⫹CN because cyanide prevents the
interaction of H2O2 with the heme moieties of DBBF-Hb.
Figure 1B shows the time-dependent fractional changes in the
ferrous, ferryl, and ferric states after the addition of GOX to DBBFHbFe2⫹. Before GOX was added, 98% of the total DBBF-Hb was in the
ferrous state. The addition of GOX resulted in a slow oxidation of
DBBF-HbFe2⫹ over the course of 60 minutes, accompanied by the
formation of DBBF-HbFe4⫹ and DBBF-HbFe3⫹. The ferryl species
produced during the initial 60 minutes was likely composed of the
Figure 1. Oxidation of DBBF-Hb by low levels of enzymatically generated H2O2.
(A) Reaction mixtures were prepared containing 50 ␮M DBBF-HbFe2⫹, DBBFHbFe3⫹, or DBBF-HbFe3⫹CN in HBSS, pH 7.4, 37°C. Representative visible spectra
(450-700 nm) are shown before and 2 hours after the addition of 10 mU/mL GOX.
After 2 hours, the spectra obtained with DBBF-HbFe2⫹ and DBBF-HbFe3⫹ were
similar and reflected the formation of the Fe4⫹ intermediate. Arrows indicate the
direction of change of the spectra. The characteristic spectrum of DBBF-HbFe3⫹CN
was unchanged. (B) Time-dependent changes in the redox states after GOX addition
to DBBF-HbFe2⫹. Visible absorbance spectra were collected every 5 minutes for 2
hours, and the oxidation states were calculated using the Winterbourn method. (C)
Ferryl hemoglobin formation after the addition of 500 ␮M H2O2 or 10 mU/mL GOX to
medium containing DBBF-Hb under culture conditions. Ferryl hemoglobin was
measured using the sodium sulfide method as described in “Materials and methods.”
Each point represents the mean ⫾ SE for 4 to 5 samples.
nonradical (equation 1) and radical (equation 2) forms of DBBFHbFe4⫹, which are spectrophotometrically indistinguishable.10 In previous studies, we showed that the bolus addition of H2O2, in a molar ratio
to heme ranging from 2.5 to 10, induces a rapid oxidation of DBBFHbFe2⫹ to DBBF-HbFe4⫹, which then immediately begins to autoreduce to DBBF-HbFe3⫹.34 These redox transitions of DBBF-Hb lead to
the rapid consumption of H2O2, and are consistent with the pseudoperoxidase activity of hemoglobin. In the current study, GOX was used to
generate low fluxes of H2O2 to better sustain the cycling of ferric and
ferryl species. Figure 1C shows the formation of ferryl hemoglobin after
the addition of H2O2 (500 ␮M) or GOX (10 mU/mL) to medium
containing DBBF-Hb. The results show that the peak levels of ferryl
hemoglobin are approximately the same in both systems (ie, approximately 22 ␮M for GOX and 27 ␮M for bolus H2O2). However, the
ferryl species clearly persists longer with the enzymatic system than
with the bolus addition of H2O2.
Glucose oxidase treatment
The initial rates of H2O2 production by 2 and 10 mU/mL GOX in HBSS
were 0.2 and 1.5 ␮M H2O2/min, respectively. Under culture conditions,
extracellular H2O2 concentration was maintained at approximately 7 to 9
␮M with 10 mU/mL GOX and 2 to 3 ␮M with 2 mU/mL GOX.
Preliminary experiments were carried out to determine the cytotoxicity
of GOX. We measured mitochondrial function using the MTT reduction
assay33 and plasma membrane damage by flow cytometric analysis of PI
uptake. Six-hour incubation with 2 and 10 mU/mL GOX decreased
mitochondrial function by 5% and 20%, respectively, but there was no
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3318
D’AGNILLO and ALAYASH
BLOOD, 1 DECEMBER 2001 䡠 VOLUME 98, NUMBER 12
significant increase in PI uptake over the course of 24 hours. In contrast,
20 mU/mL GOX decreased mitochondrial function by 57% and induced
significant plasma membrane damage after only 6 hours. In subsequent
experiments, 2 and 10 mU/mL GOX were used to study the effects of
DBBF-Hb redox cycling.
Morphologic examination of endothelial cells by
phase-contrast microscopy
Endothelial cell morphology was assessed by phase-contrast microscopy after incubation with FBS-free medium alone or medium
containing DBBF-Hb (50 ␮M), GOX (10 mU/mL), or both
DBBF-Hb and GOX. Figure 2A-B shows the morphology of
confluent endothelial cells incubated with FBS-free medium for 8
and 20 hours, respectively. These cells were morphologically
indistinguishable from cells treated with GOX (Figure 2C-D) or
DBBF-Hb alone. However, cells incubated with both DBBF-Hb
and GOX showed signs of cytoplasmic swelling, rounding, and
detachment from the monolayer (Figure 2E). Rounded cells
contained condensed DNA structures with no detectable nuclear
envelope, similar to cells undergoing mitosis. After a 20-hour
incubation with DBBF-Hb and GOX, many detached rounded cells
were still present but appeared smaller and were more irregularly
shaped (Figure 2F). Figure 2F also shows the accumulation of
debris from dead cells or insoluble precipitates, possibly containing
oxidized hemoglobin products.
Figure 3. Redox cycling of DBBF-Hb induces cell cycle arrest in the G2/M
phase. Cells were incubated with FBS-free medium alone (control) or medium
containing 2 mU/mL GOX with or without 50 ␮M DBBF-Hb. At each time interval,
adherent and nonadherent cells were pooled, and fixed. DNA content was determined by flow cytometry after RNase digestion and PI staining. The fraction of cells in
the (A) G1 phase, (B) S phase, and (C) G2/M phase of the cell cycle was calculated
as described in “Materials and methods.” Each point represents the mean ⫾ SE for 3
to 6 independent experiments. *P ⬍ .05 versus control. (D) Ferryl hemoglobin
formation in medium containing DBBF-Hb with or without 2 mU/mL GOX. Ferryl
hemoglobin was measured using the sodium sulfide method as described in
“Materials and methods.” Each point represents the mean ⫾ SE for 4 to 5 samples.
Redox cycling of DBBF-Hb induces G2/M cell cycle arrest
and apoptosis
Figure 2. Redox cycling of DBBF-Hb by glucose oxidase leads to morphologic
changes in endothelial cells. Endothelial cells were incubated with FBS-free
medium alone (A, B), 10 mU/mL GOX (C, D) or 50 ␮M DBBF-Hb and 10 mU/mL GOX
(E, F). Cells were viewed by phase-contrast microscopy and photographed (⫻20
objective). Photomicrographs were taken after 8 (left panels) and 20 (right panels)
hours. (E) Detached cells out of the plane of focus appear bright. Arrow indicates a
cell in the process of rounding and detaching. (F) Arrow points to cellular debris.
Figure 3 shows the proportion of cells in the G1, S, and G2/M
phases of the cell cycle after incubation with FBS-free medium,
GOX alone, or GOX and DBBF-Hb. GOX alone (2 mU/mL) had
no effect on cell cycle progression compared to control. This was
also the case with DBBF-Hb alone (5-200 ␮M) or 10 mU/mL GOX
alone. However, the combination of DBBF-Hb and GOX produced
a decrease in G1 cells from 63% ⫾ 4% at 3 hours to 52% ⫾ 6% at
9 hours (Figure 3A). The decrease in G1 cells was accompanied by
a corresponding increase in G2/M cells from 10% ⫾ 1% at 3 hours
to 25% ⫾ 4% at 9 hours (Figure 3C). During this period, no
differences in the S-phase population were found among the
different treatments (Figure 3B). These results suggest that a
portion of cells treated with DBBF-Hb and GOX undergoes cell
cycle arrest in the G2/M phase.
Figure 3A and C also show that the changes in G1 and G2/M are
transient and that there is a return to control levels during the later
stages of incubation. We investigated the possible correlation
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BLOOD, 1 DECEMBER 2001 䡠 VOLUME 98, NUMBER 12
HEMOGLOBIN REDOX CYCLE AND ENDOTHELIUM
3319
resuspended in complete growth medium, none of them re-adhered
or regained proliferative capacity. Microscopic examination of
these detached cells in the subsequent 12 hours revealed dramatic
changes in appearance that included shrinkage, denser cytoplasm,
and accumulation of cellular debris or apoptotic bodies. Alternatively, microscopic examination of the adherent monolayers after
15-hour incubation with DBBF-Hb and GOX revealed that many
of the remaining cells were able to proliferate when the treatment
was replaced with complete growth medium.
Taken together, these results suggest that redox cycling of
DBBF-Hb induces G2/M arrest and apoptosis in endothelial cells.
The causative role of DBBF-Hb redox cycling is further supported
by the fact that GOX added to DBBF-HbFe3⫹, which generates
DBBF-HbFe4⫹ (Figure 1A), also produced growth arrest and
apoptosis, whereas the addition of GOX to DBBF-HbFe3⫹CN did
not induce these effects.
Figure 4. Redox cycling of DBBF-Hb induces apoptosis. Cells were incubated
with (A) FBS-free medium alone or medium containing (B) 2 mU/mL GOX, (C) 50 ␮M
DBBF-Hb, or (D) DBBF-Hb and GOX. After 18 hours, adherent and nonadherent cells
were pooled and analyzed for PS externalization using the annexin V–PI assay (see
“Materials and methods”). Each panel shows a typical flow cytometric plot of 10 000
cells per sample from a representative experiment. Early apoptotic cells (annexin V⫹,
PI⫺) are shown in the lower right quadrant (R2), and late apoptotic–plasma
membrane-damaged cells (annexin V ⫹, PI⫹) are shown in the upper right
quadrant (R3).
between this transient cell cycle arrest and the formation of ferryl
hemoglobin. Figure 3D shows the formation of ferryl hemoglobin
in medium containing DBBF-Hb with or without 2 mU/mL GOX.
Ferryl hemoglobin was not detected in cultures with DBBF-Hb
alone. In cultures containing DBBF-Hb and GOX, the levels of
ferryl hemoglobin peaked at 5 hours and gradually declined over
the remaining 24 hours. This pattern of ferryl hemoglobin formation correlated with the transient pattern of cell cycle arrest induced
by GOX and DBBF-Hb, which further strengthened the link
between the ferric–ferryl redox cycle and the induction of cell
cycle arrest.
Because cell cycle arrest may be followed by apoptosis, we
examined the possible relation between G2/M arrest and apoptosis.
An early feature of cells undergoing apoptosis is the externalization
of phosphatidylserine (PS) from the inner leaflet of the plasma
membrane to the outer surface of the cell.41 We used flow
cytometric analysis of the binding of fluorescence-labeled annexin
V to externalized PS to quantify early apoptotic cells, and we
measured PI uptake to assess cells in the later stages of apoptosis or
cells that sustained direct plasma membrane damage. Figure 4
shows the flow cytometric plots obtained using the annexin V–PI
assay after an 18-hour incubation with medium alone or medium
containing GOX (2 mU/mL), DBBF-Hb (50 ␮M), or both DBBF-Hb
and GOX. The number of late apoptotic cells or directly damaged
cells (R3) was similar in each group (8%-10%) and partly reflects
the damage produced during the trypsin-harvesting procedure.
DBBF-Hb or GOX alone did not increase the number of early
apoptotic cells (R2) compared to control. In contrast, the number of
early apoptotic cells increased to 26% after treatment with DBBF-Hb
and GOX (Figure 4D).
In another set of experiments, detached and adherent cells were
collected separately after 15-hour treatment with DBBF-Hb (50
␮M) and GOX (10 mU/mL). More than 90% of the detached cells
were found to be early apoptotic (Annexin⫹/PI⫺), suggesting that
most detached growth-arrested cells were committed to die by
apoptosis. When detached cells were collected, washed, and
G2/M cell cycle arrest and apoptosis: unmodified hemoglobin
versus DBBF-Hb
We compared cell cycle arrest and apoptosis induced by the redox
cycling of DBBF-Hb with that induced by unmodified hemoglobin.
Figure 5A shows that the accumulation of G2/M cells after a 9-hour
incubation with 2 mU/mL GOX was similar between hemoglobin and
DBBF-Hb. The degree of PS externalization after 18 hours was also
similar between hemoglobin and DBBF-Hb (Figure 5B). Although
hemoglobin induces similar effects in this cell culture system, it is likely
that the extent or nature of these mechanisms would be different in vivo
because of the greater half-life of DBBF-Hb. We also found that the
addition of GOX to cocultures of red blood cells (0.5%) and endothelial
cells did not lead to G2/M arrest or apoptosis, which further supports the
involvement of the redox cycling of cell-free hemoglobin in the
induction of cell cycle arrest and apoptosis.
Role of intracellular thiol status in cell cycle arrest and
phosphatidylserine externalization
Figure 6 shows the effects of GOX and DBBF-Hb on the levels of
soluble reduced thiols in endothelial cells. GSH represents the main
reduced thiol measurable by this protocol because of its high intracellular concentration.34 DBBF-Hb alone had no effect on the level of soluble
reduced thiols. After a 4-hour incubation, the combination of DBBF-Hb
Figure 5. Redox cycling of unmodified hemoglobin induces G2/M arrest and
apoptosis. (A) The proportion of G2/M cells measured after a 9-hour incubation with
medium alone or medium containing 2 mU/mL GOX and 50 ␮M DBBF-Hb or
unmodified hemoglobin. Each value represents the mean ⫾ SE of 3 to 5 independent
experiments. (B) PS externalization measured after 18 hours for the same treatments
as in panel A. Each value represents the mean ⫾ SE of 3 to 5 independent
experiments.
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3320
D’AGNILLO and ALAYASH
BLOOD, 1 DECEMBER 2001 䡠 VOLUME 98, NUMBER 12
(5-200 ␮M) alone had no effect on DNA content or PS
externalization with normal or GSH-depleted cells. Treatment
with DBBF-Hb and GOX induced a similar increase in the
G2/M cells during the first 6 hours in normal and GSH-depleted
cells (Figure 7A). However, the maximum accumulation of
G2/M cells at 12 hours was greater with GSH-depleted cells
(34% ⫾ 5%) than with normal cells (20% ⫾ 2%). Figure 7B
shows that DBBF-Hb and GOX also produced a greater
accumulation of early apoptotic cells with GSH-depleted cells
than with normal cells during the later stages of incubation.
These data were further supported by microscopic examination
that showed greater morphologic alterations in GSH-depleted
cells than in normal cells. The time courses for G2/M arrest and
PS externalization in normal and GSH-depleted cells clearly
show that the accumulation of G2/M cells precedes the increase
in PS externalization. The increase in PS externalization was
also accompanied by an increase in sub-G1 events, an indirect
measure of apoptosis.
Catalase inhibits cell cycle arrest and apoptosis
Figure 8A shows the effect of catalase on the oxidation of
DBBF-HbFe2⫹ by GOX. The initial rate of DBBF-HbFe2⫹ oxidation produced by 10 mU/mL GOX in the presence of catalase was
similar to that produced by 2 mU/mL GOX. In contrast, the rate of
DBBF-HbFe2⫹ oxidation with 2 mU/mL GOX in the presence of
catalase was close to that of DBBF-Hb alone. Higher catalase
concentrations (400 U/mL) did not significantly increase inhibition. In this system, both DBBF-Hb and catalase competed for
reaction with H2O2. With the lower flux of H2O2 (2 mU/mL GOX),
the elimination of H2O2 by catalase was sufficient to prevent
significant interaction of H2O2 with DBBF-Hb. However, with the
Figure 6. Redox cycling of DBBF-Hb alters intracellular thiol status. (A) Soluble
reduced thiols in endothelial cells were measured after a 4-hour incubation with 50
␮M DBBF-Hb and GOX (2 or 10 mU/mL) in the presence or absence of 100 U/mL
catalase. Values are reported as the means ⫾ SE for 2 to 5 independent experiments
performed in triplicate. *P ⬍ .05 versus control. (B) Soluble reduced thiols were
measured after incubation with 10 mU/mL GOX and DBBF-Hb (0-100 ␮M). Values
are reported as the means ⫾ SE for 2 to 5 independent experiments performed in
triplicate. (C) Time course for reduced thiol depletion in endothelial cells by BSO, an
inhibitor of GSH synthesis. Cells were incubated in complete EGM medium with or
without 1 mM BSO. Values were recorded as the percentage of control cells (medium
alone) for each experiment and were reported as the means ⫾ SE for 3 to 5
independent experiments performed in triplicate.
and 2 or 10 mU/mL GOX decreased the levels of soluble reduced thiols
by 8% and 25%, respectively (Figure 6A). The inclusion of 100 U/mL
catalase, a scavenger of H2O2, inhibited the loss of thiols induced by
DBBF-Hb and GOX. Figure 6B shows that GOX alone induced a small
but significant increase in thiol content, possibly because of the
enhanced uptake of cystine.42 In the presence of GOX and DBBF-Hb,
the decrease in thiol content was dependent on the concentration of
DBBF-Hb (Figure 6B).
The loss of soluble thiols suggests a possible role for cellular
thiols in protecting against the adverse oxidative effects of
DBBF-Hb and GOX. To specifically study the role of GSH, we
pretreated cells with BSO, an irreversible inhibitor of GSH
production. BSO depletes 75% of reduced thiol content in endothelial cells after approximately 15 hours (Figure 6C). BSO pretreatment had no effect on the viability, cell cycle progression, or PS
externalization of endothelial cells.
Figure 7 shows the time course for the induction of G2/M
arrest and apoptosis in normal and BSO-pretreated cells
incubated with DBBF-Hb and GOX (10 mU/mL). DBBF-Hb
Figure 7. Prior depletion of GSH exacerbates G2/M arrest and apoptosis.
Normal and BSO-pretreated cells were incubated with FBS-free medium alone or
with medium containing 50 ␮M DBBF-Hb and 10 mU/mL GOX. At each time interval,
adherent and nonadherent cells were pooled and analyzed for (A) DNA content and
(B) PS externalization. No differences in DNA content or PS externalization were
observed between media-treated normal and BSO cells. Values at each time point
are reported as the mean ⫾ SE of 3 to 6 independent experiments. *P ⬍ .05 versus
DBBF-Hb and GOX in normal cells.
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BLOOD, 1 DECEMBER 2001 䡠 VOLUME 98, NUMBER 12
HEMOGLOBIN REDOX CYCLE AND ENDOTHELIUM
3321
cycling of DBBF-Hb and the onset and extent of cell cycle arrest
and apoptosis.
Role of the caspase inhibition in cell cycle arrest and apoptosis
Caspases are a group of cysteine proteases involved in the
execution of apoptosis.43 Experiments were performed with z-VADfmk, a broad-range caspase inhibitor, to further confirm the relation
between cell cycle arrest and apoptosis in this system. Figure 9
shows the effects of z-VAD-fmk on the cell cycle and PS
externalization after 12- and 24-hour treatment with DBBF-Hb and
10 mU/mL GOX. Figure 9A shows that z-VAD-fmk did not prevent
the accumulation of G2/M cells induced by DBBF-Hb and GOX at
12 or 24 hours. In fact, the fraction of G2/M cells at 24 hours was
greater in the presence of z-VAD-fmk (36%) than in the presence of
DBBF-Hb/GOX alone (21%), suggesting that z-VAD-fmk blocked
growth-arrested cells from proceeding to apoptosis. Furthermore,
z-VAD-fmk inhibited the accumulation of sub-G1 events at both
time intervals, in contrast to DBBF-Hb and GOX alone. Moreover,
z-VAD-fmk completely prevented the increase PS externalization
at 12 and 24 hours (Figure 9B). These results support a sequence of
events whereby the induction of G2/M arrest is followed by
apoptotic death of growth-arrested cells.
Figure 8. Catalase inhibits DBBF-Hb oxidation, G2/M arrest, and apoptosis. (A)
Reaction mixtures containing 50 ␮M DBBF-Hb, with or without 100 U/mL catalase,
were incubated with or without GOX (2 or 10 mU/mL). Percentages of HbFe2⫹ were
measured at 2-minute intervals for 2 hours using the Winterbourn equations.
Representative tracings of 1 of 4 experiments are shown. (B) Proportion of G2/M cells
measured after a 9-hour incubation with medium alone or 50 ␮M DBBF-Hb and GOX
(2 or 10 mU/mL) in the presence or absence of 100 U/mL catalase. Each value
represents the mean ⫾ SE of 3 to 6 independent experiments. (C) PS externalization
measured after 18 hours for the same treatments as in panel B. Each value
represents the mean ⫾ SE of 3 to 6 independent experiments. *P ⬍ .05 versus
medium alone; ⫹P ⬍ .05 versus 2 mU/mL GOX.
higher flux of H2O2 (10 mU/mL GOX), catalase was less effective
at preventing this interaction.
Figure 8B shows the accumulation of cells in the G2/M phase
after a 9-hour incubation with DBBF-Hb and 2 or 10 mU/mL of
GOX in the presence and absence of catalase. With 2 mU/mL
GOX, catalase provided near complete inhibition of growth
arrest. This inhibition corresponded with the absence of morphologic alterations and with the inhibition of DBBF-Hb oxidation
shown in Figure 8A. With 10 mU/mL GOX, catalase inhibited
the accumulation of G2/M cells, but to a lesser degree. Again,
this is explained by the fact that catalase was less effective at
inhibiting the redox cycling of DBBF-Hb in the presence of 10
mU/mL GOX (Figure 8A). We also found that catalase inhibited
PS externalization in a pattern similar to that noted for cell cycle
arrest. After an 18-hour incubation, the increase in the number
of early apoptotic cells induced by DBBF-Hb and 2 mU/mL
GOX was completely inhibited by catalase, whereas catalase
provided less protection with 10 mU/mL GOX (Figure 8C).
These results clearly suggest a close link between the redox
Figure 9. Caspase inhibitor, z-VAD-fmk, does not suppress G2/M arrest but
inhibits PS externalization. BSO-pretreated cells were analyzed 12 and 24 hours
after incubation with medium alone (control) or DBBF-Hb (50 ␮M) and GOX (10
mU/mL) with and without pretreatment with z-VAD-fmk. (A) DNA content histograms
with calculated fractions of G1, S, and G2/M from a representative experiment are
shown. The fraction of sub-G1 was calculated as a percentage of total gated events.
(B) Typical histograms of annexin V staining of the PI-negative cell population (R1 ⫹
R2). Mean fluorescence of the gated cell population is shown for each panel.
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3322
D’AGNILLO and ALAYASH
Discussion
We have established a causative role for the redox cycling of
DBBF-Hb in the inhibition of endothelial proliferation in the G2/M
phase of the cell cycle, which is followed by apoptosis of arrested
cells. We also found that cellular thiols (eg, GSH) play a significant
role in modulating the extent of cell cycle arrest and apoptosis.
These findings provide new insight into the mechanisms of
hemoprotein-induced endothelial cell damage and may explain
some of the microvascular effects of hemoglobin-based oxygen
carriers.1,7,30 They may also be factors in the etiology of cerebral
hemorrhage, myocardial injury, rhabdomyolysis, hemodialysis
injury, crush syndrome, and air blast exposure.1-7,17-19
The bolus addition of high micromolar concentrations of H2O2
can induce apoptosis or necrosis in variety of cell types.34,44
Previously, we showed that apoptosis induced by bolus H2O2 was
reduced in the presence of DBBF-Hb.34 The reaction of DBBFHbFe2⫹ with bolus H2O2, which generates its ferryl form and
rapidly autoreduces to DBBF-HbFe3⫹, does not lead to cell cycle
arrest (F.D., unpublished data, August 2000). Therefore, in our
previous studies, the bolus addition of oxidants did not sustain the
redox cycling of ferryl–ferric hemoglobin and limited our ability to
study the true cellular effects of hemoglobin redox reactions.
In the current study, GOX generates steady fluxes of H2O2,
which may better mimic the physiological setting.35-37 In vivo,
H2O2 is continuously produced and remains in a quasi-steady
state.36 The steady state H2O2 concentration in human plasma has
been reported to be 10 to 100 nM, whereas some have reported
levels as high as 5 to 35 ␮M.45 Two intravascular sources of H2O2
include NADPH oxidase of neutrophils and monocytes and circulating or membrane-bound xanthine oxidase of endothelial cells.21,45
The GOX system drives the redox cycling of DBBF-Hb and induces
G2/M cell cycle arrest. The rate of DBBF-Hb oxidation correlated with
GOX concentration, which in turn correlated with the onset time of
G2/M arrest. The fact that growth arrest was induced after the addition
of GOX to DBBF-HbFe3⫹, and not to DBBF-HbFe3⫹CN, further
supports the causative role of the ferric–ferryl redox cycle. Although the
underlying mechanism(s) are still unknown, the fact that extracellular
catalase inhibits cell cycle arrest and the loss of thiols strongly suggests
the involvement of an initial extracellular oxidative event that triggers
the subsequent cellular responses.
Oxidative stress can induce a wide array of cellular responses
ranging from growth stimulation, temporary arrest and adaptation,
permanent arrest, apoptosis, and necrosis depending on its strength
or duration.44 Cells can actively delay their proliferation after
sensing DNA damage and can activate repair pathways that
efficiently restore DNA integrity before division is resumed (ie, cell
cycle checkpoints).46,47 Permanent growth arrest and apoptosis can
occur after extensive DNA damage.44,46,47 Our data, therefore,
suggest that the redox cycling of DBBF-Hb may induce some form
of direct or indirect DNA damage. Oxidants such as hydroxyl
radical can induce significant DNA damage that includes singleand double-stranded DNA breaks, base and sugar modifications,
and DNA–protein cross-links.44,46 Because of the high oxidant
concentration needed to release heme or iron from hemoglobin,20 it
is unlikely that the extracellular generation of hydroxyl radical
mediated by free heme or iron plays a significant role in this
system. The high reactivity of hydroxyl radical also implies that for
damage to be induced, hydroxyl radical must be formed in
proximity to DNA. However, the possibility remains that intracellular oxidative stress plays a role in the induction of cell cycle arrest.
BLOOD, 1 DECEMBER 2001 䡠 VOLUME 98, NUMBER 12
The loss of reduced thiols induced by the redox cycling of
DBBF-Hb may be the result of increased S-glutathiolation or
S-thiolation of intracellular proteins. The role of S-glutathiolation
in the regulation of stress signaling pathways has recently been
reviewed.48 Oxidative stress induces the formation of proteinmixed disulfides through the reaction of GSH and other thiols with
cysteine residues. Because disulfide formation is reversible, critical
cysteines may be protected from irreversible oxidation and loss of
function.48,49 The combination of hemoprotein and peroxides is a
more effective inducer of mixed-disulfide formation compared to
H2O2 alone.49 Alternatively, the loss of GSH may be the result of
aggravated peroxidative stress induced by hemoprotein-mediated
lipid peroxidation processes or the efflux of GSH from stressed
cells.13,50 In a model of glycerol-induced rhabdomyolysis, a
condition characterized by massive muscle destruction and myoglobin release, the levels of GSH were decreased in rat kidneys within
6 hours of glycerol injection.51 Interestingly, it was recently shown
that increased lipid peroxidation with the concurrent production of
prostaglandin F2-isoprostanes occurs in animals with glycerolinduced rhabdomyolysis and in patients with rhabdomyolysis.17,52
These effects were linked to the redox cycling of myoglobin in
kidneys driven by low levels of ROOH or H2O2 and not to free
heme or iron-mediated events.
The increased susceptibility of GSH-depleted endothelial cells
to G2/M arrest and apoptosis induced by DBBF-Hb redox cycling
suggests that a GSH-dependent process may protect or help cells
adapt to or recover from this stress. Clinically, this implies that
patients with compromised vasculatures and poor antioxidant
status (eg, diabetes, hypertension, myocardial infarction, acute
ischemic stroke, and hemorrhagic shock) may be more susceptible
to the adverse effects of hemoglobin-based oxygen carriers, as may
be suggested in the clinical trials of DCLHb.27,29
Recently, it was reported that DCLHb induced necrotic lesions
in animal hearts.28 These lesions were localized to highly vascularized regions of the heart and were observed 24 to 48 hours after
infusion. Interestingly, we have observed that the redox cycling of
DBBF-Hb also induced G2/M arrest and apoptosis in human
coronary artery endothelial cells (F.D., unpublished data, August
2000). Although heme-mediated vascular injury and dysfunction
have been ongoing concerns with the use of hemoglobin-based
oxygen carriers, traditional measures of cytotoxicity (eg, LDH or
liver enzyme tests) have mainly suggested mild toxicities.53,54 In
this regard, it may be important to consider that G2/M-arrested
cells maintain plasma membrane integrity and that arrested cells
that become apoptotic would be cleared by phagocytes in vivo,
minimizing the leakage of intracellular contents. Thus, the occurrence of G2/M arrest and apoptosis in vivo may elude some of the
more traditional measures of cytotoxicity.
The increasing recognition that hemoglobin redox chemistry may
limit the safety or efficacy of first-generation hemoglobin-based therapies has prompted the design of new strategies aimed at reducing the
reactivity of hemoglobin with oxidants, limiting its binding of NO, or
both. These modifications include alterations of the heme pocket to
reduce NO reactivity and coadministration of antioxidants or antiferryl
compounds to control heme-mediated side reactions.7,9
Acknowledgments
The opinions and assertions contained herein are the scientific
views of the authors and are not to be construed as policy of the
United States Food and Drug Administration.
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BLOOD, 1 DECEMBER 2001 䡠 VOLUME 98, NUMBER 12
HEMOGLOBIN REDOX CYCLE AND ENDOTHELIUM
3323
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2001 98: 3315-3323
doi:10.1182/blood.V98.12.3315
Redox cycling of diaspirin cross-linked hemoglobin induces G2/M arrest and
apoptosis in cultured endothelial cells
Felice D'Agnillo and Abdu I. Alayash
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