Human Sickle Cell Blood Modulates Endothelial Heme
Oxygenase Activity
Effects on Vascular Adhesion and Reactivity
Sandip K. Bains, Roberta Foresti, Jo Howard, Sangeeta Atwal, Colin J. Green, Roberto Motterlini
Downloaded from http://atvb.ahajournals.org/ by guest on June 18, 2017
Objective—Sickle cell disease (SCD) is characterized by extensive hemolysis, increased cellular adhesion, and
vaso-occlusion. Tissues from sickle patients express heme oxygenase-1 (HO-1), the enzyme that degrades free
heme/hemoglobin to the signaling molecule carbon monoxide, and the antioxidants biliverdin/bilirubin. Here, we
examined the HO response in endothelial cells exposed to human sickle blood and determined whether this response is
beneficial for SCD.
Methods and Results—We measured HO activity in human and bovine aortic endothelial cells incubated with human
sickle or normal blood. Sickle blood increased HO activity, which was enhanced by hypoxia and was caused mainly by
the red cell components of sickle blood. Oxidized hemoglobin was higher in sickle blood and increased markedly over
time. Interestingly, HO activity correlated inversely with patients’ hemoglobin levels and positively with bilirubin and
lactate dehydrogenase. HO-1 induction, exogenous biliverdin, or carbon monoxide markedly decreased adhesion of
sickle blood to the endothelium, and sickle red cells partially inhibited relaxation mediated by carbon monoxide in
isolated aortas.
Conclusions—Our results highlight important associations between SCD and HO byproducts, which may counteract
vascular complications of SCD. (Arterioscler Thromb Vasc Biol. 2010;30:305-312.)
Key Words: adhesion 䡲 carbon monoxide 䡲 endothelium 䡲 heme oxygenase 䡲 sickle cell disease
S
ickle cell disease (SCD) is caused by a mutation in the
␤-globin gene, resulting in abnormal hemoglobin
(HbS), either in a homozygous or in a compound heterozygous form with another atypical hemoglobin. The mutated
HbS polymerizes in the deoxygenated state, leading to
unusually shaped red cells. Sickle erythrocytes hemolyze
easily, causing chronic anemia and small vessel occlusion,
triggering acute painful episodes and other complications
of SCD, such as acute chest syndrome, avascular necrosis,
leg ulcers, and renal failure.1,2 Presently, hydroxyurea is
the only drug widely used for treating SCD. Hydroxyurea
stimulates production of fetal hemoglobin and lowers the
concentration of HbS;3 however, most patients receiving it
still exhibit hemolysis and an underlying inflammatory
state,4 indicating the need for new therapeutic strategies to
help patients with SCD.
Despite a well-characterized genetic origin, SCD may be
viewed as a chronic inflammatory condition,4 – 6 with studies
revealing abnormally high levels of leukocytes in subjects
with SCD.7 Circulating endothelial cells from SCD patients
also display an activated state with expression of adhesion
molecules,8 and a genetic profile of blood mononuclear cells
from 27 patients in steady-state SCD showed upregulation of
genes related to oxidative stress and inflammation and global
upregulation of proinflammatory markers.4 Interestingly,
genes of heme metabolism, including heme oxygenase-1
(HO-1) and biliverdin reductase, were also increased, as
already reported for circulating endothelial cells and kidneys
of SCD subjects.9
HO-1 is the inducible isoform of HO, the enzyme that
converts heme to iron, carbon monoxide (CO), and biliverdin.
Biliverdin is subsequently reduced to bilirubin by biliverdin
reductase.10 Given that sickle erythrocytes release large
amounts of hemoglobin after hemolysis, endothelial HO-1
induction may serve to control excessive heme overload.
However, the antioxidant and antiinflammatory action attributed to the HO system11 suggest that HO-1 upregulation in
SCD may attenuate ischemia-reperfusion injury induced by
vaso-occlusive crises. Few published reports addressing this
possibility indicate contrasting effects. For instance, a recent
investigation in transgenic sickle mice showed that HO-1
induction (via administration of hemin or HO-1 adenovirus)
Received September 20, 2008; revision accepted November 19, 2009.
From Department of Surgical Research (S.K.B., R.F., C.J.G., R.M.), Northwick Park Institute for Medical Research, Harrow, Middlesex, United
Kingdom; Department of Haematology (J.H.), St Thomas’ Hospital, London, United Kingdom; Department of Haematology (S.A.), Central Middlesex
Hospital, Brent, Middlesex, United Kingdom; Department of Drug Discovery and Development (R.F., R.M.), Italian Institute of Technology, Genova,
Italy.
Correspondence to Dr Roberta Foresti or Dr Roberto Motterlini, Department of Drug Discovery and Development, Italian Institute of Technology, Via
Morego 30, Genova, 16163, Italy. E-mail [email protected] or [email protected]
© 2010 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
305
DOI: 10.1161/ATVBAHA.109.196360
306
Table.
Arterioscler Thromb Vasc Biol
February 2010
Patient Characteristics
Control
All SCD
HbSS
HbSC
HbS␤Thal
Other
P*
N of patients
20
196
136
43
13
4
…
Age,
mean⫾SD (%)
36.6⫾4.56
34⫾13
33⫾12
40⫾16
36⫾12
26.5⫾6
…
Adult/child ratio
(%)
20 (100%)/0 (0%)
187 (95%)/9 (5%)
128 (94%)/8 (6%)
42 (98%)/1 (2%)
13 (100%)/0 (0%)
4 (100%)/0 (0%)
…
Gender,
female/male
ratio (%)
13 (65%)/7 (35%)
130 (66%)/66 (34%)
88 (65%)/48 (35%)
32 (74%)/11 (26%)
9 (69%)/4 (31%)
4 (50%)/4 (50%)
…
Hb, mg/dL,
mean⫾SD
13.71⫾1.07
10.1⫾4.9
9.6⫾4.9
11.3⫾1.6
10.2⫾1.8
10.6⫾3.9
⬍0.0001
BR, ␮mol/L,
mean⫾SD
7.82⫾2.7
50.5⫾42.3
60.4⫾46.0
29.7⫾21.4
23.6⫾9.3
36.5⫾14.8
⬍0.0001
189⫾71.21
424⫾238
501⫾237
333⫾149
237⫾75
341⫾176
0.0335
White blood
cells ⫻109/L,
mean⫾SD
6.198⫾1.169
10.33⫾4.6
11.01⫾4.9
8.7⫾3.2
9.9⫾3.7
4.4⫾0.9
0.0001
Reticulocytes
⫻109/L,
mean⫾SD
…
204.9⫾96.5
226⫾101
162⫾52
165⫾84
…
…
Patients
administered
hydroxyurea
…
27 (14%)
25 (18%)
0 (0%)
2 (15%)
1 (25%)
…
Patients
administered
transfusion
…
6 (3%)
6 (4%)
0 (0%)
0 (0%)
0 (0%)
…
Disease state,
acute crisis/
steady-state
…
27 (14%)/169 (86%)
21 (15%)/115 (85%)
5 (12%)/38 (88%)
1 (8%)/12 (92%)
0 (0%)/4 (100%)
…
LDH, U/L,
mean⫾SD
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BR indicates bilirubin; Hb, hemoglobin; HbSS, homozygous sickle Hb; HbSC, sickle Hb C; HbS␤Thal, HbS␤ thalassemia.
*P value is for the comparison of all SCD patients vs control subjects.
reduced inflammation and vaso-occlusion, partly by decreasing expression of the adhesion molecules vascular cell adhesion molecule-1 and intercellular adhesion molecule-1.12
Furthermore, exogenous CO or biliverdin afforded similar
protection. Conversely, inhibiting HO activity protected kidneys of sickle mice against ischemic-mediated injury,13 suggesting that a better characterization of the HO system in
SCD is required.
In the present study, we investigated the response of human
and bovine aortic endothelial cells and rat aortic tissue on
exposure to human sickle blood. We measured HO activity
and correlated the values with patients’ blood parameters.
The effect of HO-1 upregulation, biliverdin, or CO (liberated
by CO-releasing molecules14) on red blood cell (RBC)
adhesion to the endothelium was also examined. Finally, we
studied the vasorelaxant properties of CO in the presence of
sickle blood in rat isolated aortic rings.
Materials and Methods
Blood Collection and Patients Characteristics
Blood was collected from 196 SCD patients and 20 healthy
controls; detailed information about patients’ characteristics is
reported in the Table. Patients and controls were matched for age
and gender; however, the ethnicities of the groups were not
matched because of the difficulty in recruiting healthy volunteers.
Subjects gave written informed consent and the protocol was
approved by the National Research Ethics Service (formerly
COREC) in England. Patient eligibility criteria included all SCD
forms (homozygous HbSS, HbSC, HbS␤ thalassemia), age older
than 12 months, and receiving hydroxyurea, blood transfusion, or
no treatment. Current disease status (steady-state or acute crisis),
date of last transfusion or last crisis, and number and date of
crises in the previous 2 years were recorded. An acute crisis was
defined as acute sickle pain requiring analgesics and hospital
admission of the patient, whereas patients with steady-state were
asymptomatic. Blood samples were analyzed for hemoglobin,
bilirubin, and lactate dehydrogenase (LDH) by the hematology
laboratory. Of the 196 blood samples collected, 141 were used for
the HO assay (102 samples used with bovine aortic endothelial
cells [BAEC] and 39 with human aortic endothelial cells
[HAEC]), 37 were used for the cell adhesion assay, and 18 were
used for vasorelaxation experiments.
Cell Culture and Experimental Protocol
BAEC were purchased from Coriell Cell Repositories (Camden, NJ)
and HAEC were purchased from Promocell (Heidelberg, Germany).
Blood was collected in EDTA-coated tubes, stored at 4°C, and used
within 24 hours for all experiments. Blood components were
separated by centrifugation of whole blood at 100g for 5 minutes;
RBC were washed with phosphate-buffered saline 3 times before
use, and plasma not immediately used for experiments was stored at
⫺80°C.
We first investigated whether sickle blood affected endothelial HO
activity at 21% oxygen. BAEC and HAEC were incubated for 6 or
Bains et al
Sickle Blood Induces Endothelial HO
307
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Figure 1. Sickle blood induces endothelial
HO activity. HO activity was measured in
BAEC (A) and HAEC (B) after exposure for
6 or 18 hours to 20% whole sickle (SCD)
or control blood (normal) (n⫽52 [6 hours]
and n⫽14 [18 hours] for SCD and n⫽6 [6
hours] and n⫽10 [18 hours] for normal).
Parallel experiments were performed with
HAEC (B) (n⫽29 [6 hours] and n⫽10 [18
hours] for SCD and n⫽5 [hours] and n⫽5
[18 hours] for normal). In further experiments with BAEC (C), cells were exposed
to 10% normal or sickle plasma (n⫽6 [6
hours] and n⫽5 [18 hours] for normal or
n⫽16 [6 hours] and n⫽8 [18 hours] for
sickle plasma). D, BAEC were also treated
with 10% normal or sickle RBC; n⫽6 (6
hours) and n⫽8 (18 hours) for normal or
n⫽8 (6 hours) and n⫽8 (18 hours) for
sickle RBC. Cells were also incubated in
medium alone; n⫽9 (6 hours) and n⫽14
(18 hours). E, Cells were exposed for 18
hours to 20% whole sickle blood (SCD) or
control blood (normal) at 1% O2 (hypoxia)
or 21% O2 (normoxia) (n⫽19 [SCD] or
n⫽6 [normal] for hypoxic experiments and
n⫽5 [SCD] or n⫽19 [normal] for normoxic
experiments). Bars represent the
mean⫾SEM. *P⬍0.05 vs medium alone
(medium).
18 hours with medium containing 20% whole sickle or control blood.
BAEC were also exposed to 20% plasma or washed RBC for 6 and
18 hours; 20% sickle blood was chosen because this was the minimal
dilution affecting HO in preliminary experiments (not shown). We
also examined endothelial HO activation by whole sickle blood at
1% O2, because lack of oxygen stimulates HbS polymerization with
deformation of erythrocytes and hemolysis.
Assay for Endothelial HO Activity
Cellular HO activity was determined as previously described by our
group.15
Statistical Analysis
Data were analyzed for normal distribution using the KolmogorovSmirnov test, and data sets fulfilling the normality criteria were
further analyzed by ANOVA or unpaired t test to determine
significance. For data not normally distributed (data sets in Figure 2),
nonparametric tests (Mann-Whitney) were applied. Pearson correlation coefficients examined the relationship between patients’ hemoglobin values (n⫽47), plasma bilirubin (n⫽48), LDH (n⫽26), and
age (n⫽43) with HO activity. Results were expressed as
means⫾SEM and considered significant at P⬍0.05.
Measurement of Oxidized Hemoglobin
Oxidized hemoglobin (methemoglobin, oxidized hemoglobin)
was determined in the culture medium after incubation of cells
with 20% sickle or normal blood for different times as described
previously.16
RBC Adhesion to the Endothelium
Adhesion of RBC was measured using an in vitro static assay
(gravity sedimentation method) adapted from a previously published
method.17 BAEC and HAEC were exposed to either 5% sickle or
normal blood for 45 minutes before measurement of RBC adhesion.
Furthermore, BAEC were treated with hemin (1, 10, 30 ␮mol/L) for
6 hours to induce HO-1 before addition of 5% whole sickle blood. To
assess the contribution of HO byproducts, cells were also treated
with biliverdin or CO using 2 water-soluble CO-releasing molecules
(CO-RMs), CORM-3 and CORM-A1.18,19 For this purpose, cells
were incubated with biliverdin (0.1, 0.5, 1 ␮mol/L), CORM-3 (1, 10,
30 ␮mol/L), or CORM-A1 (1, 5, 10 ␮mol/L) 30 minutes before
exposure to sickle blood. Alternatively, sickle blood treated with
biliverdin, CORM-A1, or CORM-3 for 30 minutes was added to
endothelial cells for the adhesion assay.
Isolated Rat Aortic Ring Model
Transverse ring sections of rat aortas were prepared as previously
described.20,21 Aortic rings were incubated with 1% sickle or normal
blood components and challenged with CORM-A1 (please see
http:/atvb.ahajournals.org).
Results
Sickle Blood Induces HO in Endothelial Cells
As shown in Figure 1A (BAEC) and B (HAEC), 20% whole
normal blood did not affect cellular HO activity after 6 or 18
hours of incubation. In contrast, 20% whole sickle blood
significantly increased the activity, with the increase being
more pronounced at 18 hours than at 6 hours. Interestingly,
20% normal or sickle plasma incubated for 6 hours or 18
hours did not change activity levels (Figure 1C), whereas
sickle RBC caused a significant increase in HO compared
with normal red cells (Figure 1D). We note that HO induction
was more pronounced with whole sickle blood than sickle
RBC. Compared to normoxic conditions, hypoxia further
enhanced (⬇2-fold) the increase in HO activity elicited by
sickle blood after 18 hours of incubation (Figure 1E), whereas
normal whole blood caused no changes.
Basal levels of methemoglobin were higher in sickle than
normal blood and further increased during incubation time,
following a pattern that paralleled HO induction by sickle
blood (please see http:/atvb.ahajournals.org).
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Arterioscler Thromb Vasc Biol
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Figure 2. HO activity levels grouped
according to patients’ treatment or disease
state. HO activity was measured 6 hours
after incubation with (A) 20% whole sickle
blood from patients receiving (⫹HU, n⫽7)
or not receiving (⫺HU, n⫽46) hydroxyurea
treatment; (B) 20% whole sickle blood from
patients receiving transfusion (⫹transfusion,
n⫽5) or not (⫺transfusion, n⫽46), or (C)
20% whole sickle blood from patients
experiencing an acute crisis (acute crisis,
n⫽10) or in steady-state SCD (steady-state,
n⫽48). D, HO activity measured after
6-hour incubation with 20% whole sickle
blood from patients who experienced sickle
crises within the previous year (1 year,
n⫽23), 1 to 2 years (2 years, n⫽2), and ⬎2
years before (⬎2 years, n⫽6). The group
“normal” represents cells incubated with
control blood. Bars represent the
mean⫾SEM. *P⬍0.05 vs normal. †P⬍0.05
vs sickle crises within the previous year.
HO Activity Levels Analyzed According to Patient
Treatment Regimen and Time Elapsed Since Last
Sickle Crisis
Considering that patients were experiencing different
stages of SCD and were undergoing different treatment
regimens when the blood was collected, we hypothesized
that these differences could be reflected in the levels of HO
activity stimulated by sickle blood. The sickle sample
population included HbSS, HbSC, and HbS␤ thalassemia
traits and, as reported in Figure 2, blood from patients
receiving hydroxyurea (n⫽7; Figure 2A), transfusion
(n⫽5; Figure 2B), or in acute crisis (n⫽10; Figure 2C) did
not elicit any changes in endothelial HO upregulation
when compared with samples from patients not receiving
treatment and stable. Conversely, HO levels grouped
according to time elapsed from the last crisis indicated that
blood from SCD subjects who experienced vaso-occlusive
crises in the previous year (n⫽23) elicited a significantly
higher activity (P⬍0.05) compared to blood from subjects
experiencing crises ⬎2 years before (n⫽6; Figure 2D). It
is acknowledged that the small number of subjects in
certain subgroups is a limitation that reduces the power of
the statistical analysis of interpatient variability.
Correlations of HO Activity With Patients’
Hemoglobin, Bilirubin, LDH Levels, and Age
Correlation analysis performed comparing HO activity with
patient variables revealed that: (1) HO activity correlated inversely with hemoglobin levels (n⫽47; r⫽⫺0.39;
Figure 3. HO activity correlates with
patients’ hemoglobin, bilirubin, and LDH
levels. The graphs show the relationships
between HO activity stimulated by sickle
blood with hemoglobin (A, n⫽47), bilirubin
(B, n⫽48), LDH (C, n⫽26), and age (D,
n⫽43), respectively. r⫽Pearson correlation.
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Sickle Blood Induces Endothelial HO
309
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Figure 4. Pretreatment of endothelial
cells with HO-1 byproducts: effect on
adhesion of sickle blood to the endothelium. A, Adhesion was measured in
BAEC incubated for 45 minutes with 5%
dilution of sickle (SCD, n⫽6) or normal
blood (n⫽10). B, Adhesion was determined in BAEC pretreated for 6 hours
with hemin (1, 10, 30 ␮mol/L, n⫽5 for
each concentration) or 30 ␮mol/L hemin
in the presence of 60 ␮mol/L tin protoporphyrin (SnPPIX; n⫽10) before exposure to sickle blood. C, Cells were preincubated for 30 minutes with biliverdin
(0.1, 0.5, 1 ␮mol/L, n⫽5 for each concentration). D and E, Adhesion was measured in cells preincubated for 30 minutes with CORM-3 (1, 10, 30 ␮mol/L,
n⫽5 for each concentration) or
CORM-A1 (1, 5, 10 ␮mol/L, n⫽5 for
each concentration), respectively, before
exposure to sickle blood. iCORM-3
(30 ␮mol/L, n⫽5) and iCORM-A1
(10 ␮mol/L, n⫽5) were used as
CO-depleted negative controls. Bars
represent the mean⫾SEM. *P⬍0.05 vs
normal in (A); *P⬍0.05 vs SCD in the
other graphs. †P⬍0.05 vs hemin alone.
P⫽0.007; Figure 3A), indicating that higher activity was
stimulated by sickle blood containing progressively lower
hemoglobin; (2) there was a positive relationship between
HO and plasma bilirubin levels (n⫽48; r⫽0.58; P⬍0.001;
Figure 3B), implying that the in vivo production of bilirubin
in SCD subjects significantly matched the activity levels
induced by blood in vitro; (3) HO correlated positively with
LDH (n⫽26; r⫽0.45; P⫽0.02; Figure 3C), a marker of cell
injury, suggesting that higher vascular damage in patients was
accompanied by greater HO activation; and (4) HO activity
tended to decline with increasing age of patients (n⫽43;
r⫽⫺0.34; P⫽0.03; Figure 3D).
HO-1 Induction, Biliverdin, and CO Attenuate the
Adhesion of Sickle RBC to the Endothelium
As expected, normal blood did not adhere to the BAEC in
culture, whereas sickle blood adhered extensively (Figure 4A;
P⬍0.05; for data on HAEC, see http:/atvb.ahajournals.org).
Pretreatment with hemin (1, 10, and 30 ␮mol/L), which leads
to upregulation of HO-1, reduced sickle cell adhesion in a
concentration-dependent manner, and inhibition of HO activity with tin protoporphyrin IX reversed the protection elicited
by hemin (Figure 4B). Pretreating cells with biliverdin (0.1,
0.5, 1 ␮mol/L) also decreased sickle cell adhesion (Figure
4C). To examine whether CO influenced RBC adhesion, we
used 2 CO-RMs recently developed in our laboratory.
CORM-3 and CORM-A1 have distinguished chemical characteristics affecting their kinetic of CO release in physiological buffers (CORM-3 half life⫽3.6 minutes; CORM-A1
half-life⫽21 minutes) and their interactions with cells and
tissues,22,23 Pretreating endothelial cells with CORM-3 had
little effect on sickle cell adhesion (Figure 4D), whereas
CORM-A1 reduced adhesiveness in a concentrationdependent manner (Figure 4E). Neither iCORM-3 nor
iCORM-A1 influenced the adhesion response. Furthermore,
longer or shorter preincubation times did not change the
outcome of the experiment. Importantly, direct incubation of
sickle blood with biliverdin, CORM-3, or CORM-A1 before
addition to cells markedly decreased adhesion (Figure 5A–C).
Adhesion was also lower when sickle blood was pretreated
with i-CORM-A1. Thus, induction of endothelial HO-1
prevents sickle red cell adhesion, as does treatment of either
endothelial cells or sickle blood with HO-1-derived products.
The kinetics of CO release by CORM-3 and CORM-A1, and
possibly their diverse chemical nature, explain their different
effects.
Sickle RBC Modulate CORM-A1 Vasodilation in
Isolated Aortas
In experiments studying aortic relaxation by CORM-A1, we
found that sickle blood components decreased CORM-A1
relaxation, whereas normal blood did not (please see
http:/atvb.ahajournals.org).
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Figure 5. Pretreatment of blood with HO-1 byproducts: effect
on adhesion of sickle blood to the endothelium. A, Results of
the adhesion assay when sickle blood was pretreated for 30
minutes with biliverdin (0.1, 0.5, 1 ␮mol/L, n⫽5 for each concentration) before incubation with cells. B and C, Results of the
adhesion assay when sickle blood was pretreated for 30 minutes with CORM-3 (1, 10, 30 ␮mol/L) or CORM-A1 (1, 5,
10 ␮mol/L) (n⫽5 for each concentration of CO-releasing molecule), respectively, before incubation with cells. iCORM-3
(30 ␮mol/L, n⫽5) and iCORM-A1 (10 ␮mol/L, n⫽5) were used
as CO-depleted negative controls. Bars represent the
mean⫾SEM. *P⬍0.05 vs SCD.
Discussion
Chronic hemolysis in SCD stimulates vascular tissue to
counteract the pro-oxidative and proinflammatory environment created by free heme/hemoglobin. HO-1, the inducible
protein responsible for heme degradation, is an essential
enzyme recruited by the vasculature in the active response to
high heme exposure.24 Here, we examined whether human
sickle blood affects HO activation in HAEC and BAEC, and
whether HO-1– derived products exert antiadhesive and vasodilatory properties in the presence of sickle blood. We
found that sickle blood increased endothelial HO activity in a
time-dependent manner, and hypoxia further amplified this
effect. The RBC fraction of sickle blood seemed the major
contributor to HO induction. Sickle blood exhibited higher
basal levels of oxidized hemoglobin when compared to
normal blood, and these changes correlated closely with the
ability of sickle blood to induce HO. Analysis of patient
profiles revealed that the time elapsed from the last vasoocclusive crisis influenced the extent of HO activity. In
addition, HO values correlated with patients’ hemoglobin,
bilirubin, and LDH levels, indices of hemolysis, and vascular
damage. Most specifically, sickle blood taken from patients
with the most severe SCD complications elicited the highest
endothelial HO response, perhaps suggesting a homeostatic
mechanism. Upregulation of HO-1, exogenous biliverdin, or
CO markedly decreased adhesion of sickle erythrocytes to the
endothelium. Furthermore, sickle red cells partially inhibited
the relaxation elicited by CORM-A1 in precontracted aortic
rings. Our results indicate an important association between
SCD and HO, suggesting that heme degradation products
may reduce vascular complications elicited by sickle blood.
We expected to find that both sickle red cells (a source of
heme) and plasma (containing extraerythrocytic factors such
as free heme and inflammatory molecules) would contribute
to inducing HO in our experiments; however, increased HO
activity was only stimulated by RBC. We do not exclude that
plasmatic components may synergize with erythrocytes to
upregulate HO because the activity was higher with whole
blood than RBC. Accordingly, reports25,26 have shown that
sickle plasma modifies endothelial gene expression toward a
proangiogenic and anti-inflammatory phenotype. We also
found that hypoxia enhanced HO activity, potentially mimicking the pathological scenario occurring during vasoocclusive crises and ischemia-reperfusion episodes. It is
known that oxidized hemoglobin is susceptible to heme loss,
and heme is a strong inducer of HO-1.27 Therefore, our data
detecting high basal levels of oxidized hemoglobin in sickle
blood and its continuous increase during the incubation with
cells strongly implicate that heme derived from oxidized
hemoglobin contributes HO activation. Altogether, these
results point to sickle red cell hemolysis, which is stimulated
under hypoxia, as one of the causes of endothelial HO
induction. Nevertheless, the participation of factors such as
heme-induced oxidative stress is possible and we are currently exploring this hypothesis.
Differences within the SCD population in the present study
were analyzed retrospectively, allowing us to explore variables that could influence the extent of HO induction. The
analysis and discussion of these results are made with
acknowledgement of some limitations concerning our approach. Because data were evaluated subsequently to testing
blood samples, we could explore only a restricted number of
variables. For example, fetal hemoglobin levels, an important
determinant of SCD severity, were not routinely measured in
our patients, and therefore we could not examine their
influence on HO induction. Furthermore, despite our total
study group consisting of 196 patients and 20 controls, the
statistical analysis of interpatient variability was underpowered because only few patients represented some subgroups
investigated. Nevertheless, the analysis uncovered interesting
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trends that future exploration in a larger number of patients
may corroborate. For instance, although not statistically
significant, blood from patients receiving hydroxyurea or
transfusion elicited lower HO compared to blood from
patients not receiving treatment. Hydroxyurea, usually prescribed to patients with severe and recurrent chest crises,
stimulates fetal hemoglobin production and ameliorates clinical symptoms.28 Regular blood transfusion is performed to
improve anemia and prevent complications in SCD patients
who already had strokes.29 Thus, induction of HO may reflect
disease severity and a better clinical management of SCD
may diminish the factor(s) in blood responsible for enzyme
activation. The results showing that blood from patients
experiencing a crisis within the previous year stimulated
higher HO than blood from subjects whose last crisis was ⱖ2
years before support this idea. Therefore, the clinical and
biochemical stress imposed by vaso-occlusive crises may be
linked to HO expression in SCD.
In our analysis correlating patients’ blood parameters with
HO activity values, the decline in patients’ hemoglobin levels
correlated with increasing endothelial HO in vitro. This again
suggests a relationship between the severity of the disease
and HO response, because higher free hemoglobin/heme
liberated during hemolysis and vaso-occlusive crises could
increase the activity. Accordingly, patients’ bilirubin levels
also correlated with HO, suggesting that the stimuli inducing
production of heme metabolites in SCD subjects also promoted HO upregulation in vitro. Moreover, LDH correlated
positively with HO, linking the levels of activity to the extent
of vascular damage. In this study, we also found that HO-1
overexpression attenuated adhesiveness of sickle blood to the
endothelium. This effect was reversed by an inhibitor of the
enzyme activity, potentially implicating all heme degradation
products. In fact, exogenous application of biliverdin or CO
significantly decreased adhesion, presumably via antioxidant30 and antiinflammatory31 mechanisms that remain to be
investigated. Preincubation of blood with biliverdin,
CORM-3, or CORM-A1 inhibited blood adhesion more than
preincubation of cells, suggesting that sickle blood plays a
predominant role in stimulating adhesion and may be a target
for therapeutic intervention. However, vascular endothelial
cells of SCD subjects exhibit a proinflammatory/activated
phenotype8,32 and might affect the adhesion process more
than our cells in culture. Collectively, our results support
recent findings showing that biliverdin and CO gas inhibited
vascular stasis and expression of adhesion molecules in a
transgenic mice model of SCD.12 It was also interesting that
sickle erythrocytes inhibited CORM-A1–mediated relaxation
in precontracted aortas, suggesting that defective sickle erythrocytes bind CO with different affinity, thus scavenging CO
and preventing its vascular activities more effectively than
normal RBC.
Conclusion
In conclusion, human sickle blood enhances endothelial HO
and the positive effect of HO-1 induction, biliverdin, and
CO-RMs in reducing sickle blood adherence and in promoting vasodilation, indicating the need to further explore the
Sickle Blood Induces Endothelial HO
311
therapeutic potentials of the HO pathway in the treatment of
SCD.
Acknowledgments
The authors thank Professor Brian Mann and Professor Roger
Alberto for synthesis of CO-RM.
Sources of Funding
This research was supported by a grant from the Dunhil Medical trust
(R.M., R.F).
Disclosure
R.M. is a shareholder, co-founder, and member of the Board of
Directors of hemoCORM. C.J.G. is co-founder and member of the
Board of Directors of hemoCORM. Northwick Park Institute for
Medical Research and R. Motterlini have financial interests
with Alfama.
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Human Sickle Cell Blood Modulates Endothelial Heme Oxygenase Activity: Effects on
Vascular Adhesion and Reactivity
Sandip K. Bains, Roberta Foresti, Jo Howard, Sangeeta Atwal, Colin J. Green and Roberto
Motterlini
Arterioscler Thromb Vasc Biol. 2010;30:305-312; originally published online December 3,
2009;
doi: 10.1161/ATVBAHA.109.196360
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Supplement Material
MetHb (% Total Hb)
Figure I
*
30
*
20
10
Normal
Sickle
*
0
0h
6h
18 h
Figure I. Hemoglobin from sickle blood is more susceptible to oxidation than hemoglobin from
normal blood. Human aortic endothelial cells were incubated with medium containing 20% whole
sickle (n=5) or normal blood (n=5) for 6 or 18 h. Aliquots of medium were collected at 0, 6 and 18
h and the levels of methemoglobin (MetHb) determined. As shown in the graph, sickle blood
exhibited at basal conditions (0 h) higher levels of MetHb than normal blood. Furthermore,
hemoglobin oxidation in sickle blood rose progressively and dramatically over the time of
incubation while MetHb levels remained steady and unchanged in normal blood. Data represent the
mean±SEM of independent experiments. * P<0.05vs normal blood.
Figure II
A
*
No. of adherent
RBC/mm2
12.5
10.0
7.5
5.0
2.5
0.0
SCD
Normal
No. of adherent RBC/mm 2
B
15
*
10
5
0
SCD
BV
(1 µmol/L)
CORM-A1
CORM-3
(10 µmol/L) (10 µmol/L)
No. of adherent RBC/mm 2
C
15
10
*
5
*
0
SCD
BV
(1 µmol/L)
*
CORM-A1
CORM-3
(10 µmol/L) (10 µmol/L)
Figure II. Biliverdin and carbon monoxide inhibit sickle red cell adhesion in human aortic
endothelial cells. A, adhesion of red blood cells determined after exposure of HAEC to normal
(n=5) or sickle blood (n=10). B, the adhesion assay was also performed in HAEC incubated with
biliverdin (BV, 1 µmol/L, n=5), CORM-A1 (10 µmol/L, n=5) or CORM-3 (30 µmol/L, n=5) for 30
min before exposure to sickle blood (SCD). C, alternatively, sickle blood pre-treated with
biliverdin, CORM-A1 or CORM-3 (n=5 for each treatment) for 30 min was added to endothelial
cells for the adhesion assay. As observed with BAEC, pre-incubation of endothelial cells with
biliverdin, CORM-A1 or CORM-3 resulted in a reduction in the adhesion of sickle red blood cells
to the endothelium; sickle red cell adhesion was further and significantly diminished when sickle
blood was pre-incubated with the compounds (p<0.001). Data represent the mean±SEM of
independent experiments. * P<0.05 vs normal blood in graph A; * P<0.05vs SCD in graphs B and
C.
Figure III
Whole blood
A
% Contraction
100
CORM-A1
75
Normal blood+CORM-A1
*
50
Sickle blood+CORM-A1
25
0
0
10
20
30
40
50
60
70
Time (min)
B
RBC
% Contraction
100
*
CORM-A1
*
75
Normal RBC+CORM-A1
50
Sickle RBC+CORM-A1
25
0
0
10
20
30
40
50
60
70
Time (min)
C
Plasma
% Contraction
100
*
75
50
CORM-A1
*
Normal plasma+CORM-A1
Sickle plasma+CORM-A1
*
25
*
0
0
10
20
30
40
Time (min)
50
60
70
Figure III. Sickle red blood cells modulate CORM-A1 vasodilation in isolated aortas. Transverse
ring sections of aortas from male Sprague Dawley rats were suspended under 2 g tension in
oxygenated Krebs Henseleit buffer. A 1% solution of blood was warranted in this model as higher
concentrations of blood would lead to scavenging of CO by hemoglobin. The protocol used was
based on previous studies performed in a similar model but assessing the effect of nitric oxide 19.
(A) Aortic rings in Krebs buffer (CORM-A1, n=10), 1% whole sickle (Sickle blood+CORM-A1,
n=5) or normal blood (Normal blood+CORM-A1, n=5) were challenged with 1 µmol/L
phenylephrine and 100 µmol/L CORM-A1 was added at the peak of phenylephrine contraction.
Contraction was followed for 60 min from addition of CORM-A1. (B) Results of similar
experiments conducted in the presence of 0.5% sickle (Sickle RBC+CORM-A1, n=7) or normal
(Normal RBC+CORM-A1, n=4) RBCs or (C) 0.5% sickle (Sickle plasma+CORM-A1, n=7) or
normal (Normal plasma+CORM-A1, n=4) plasma. We found that whole sickle blood only
marginally reduced CORM-A1-mediated dilation with an overall response similar to that of normal
blood or buffer. However, sickle RBCs significantly decreased CORM-A1 relaxation, while normal
red cells did not. Moreover, normal and sickle plasma inhibited the dilation elicited by CORM-A1
to a similar extent.Data represent the mean±SEM. * P<0.05 vs. CORM-A1 and normal blood.