|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3924-3935
By
From the Departments of Biochemistry and Molecular Biology, and
Medicine, University of Southern California, School of Medicine, Los
Angeles, CA.
The abnormal adherence of sickle red blood cells (SS RBC) to
endothelial cells has been thought to contribute to vascular occlusion,
a major cause of morbidity in sickle cell disease (SCD). We determined
whether the interaction of SS RBC with cultured endothelial cells
induced cellular oxidant stress that would culminate in expression of
cell adhesion molecules (CAMs) involved in the adhesion and diapedesis
of monocytes and the adherence of SS reticulocytes. We showed that the
interaction of SS RBC at 2% concentration in the presence of multimers
of von Willebrand factor (vWf), derived from endothelial cell-derived
conditioned medium (E-CM) with cultured human umbilical vein
endothelial cells (HUVEC), resulted in a fivefold increased formation
of thiobarbituric acid-reactive substances (TBARS) and activation of
the transcription factor NF-kB, both indicators of cellular oxidant
stress. Normal RBC show none of these phenomena. The oxidant
stress-induced signaling resulted in an increased surface expression of
a subset of CAMs, ICAM-1, E-selectin, and VCAM-1 in HUVEC. The addition
of oxygen radical scavenger enzymes (catalase, superoxide dismutase)
and antioxidant (probucol) inhibited these events. Additionally,
preincubation of HUVEC with a synthetic peptide Arg-Gly-Asp (RGD) that
prevents vWf-mediated adhesion of SS RBC reduced the surface expression of VCAM-1 and NF-kB activation. Furthermore, SS RBC-induced oxidant stress resulted in a twofold increase in the transendothelial migration
of both monocyte-like HL-60 cells and human peripheral blood monocytes,
and approximately a sixfold increase in platelet-endothelial cell
adhesion molecule-1 (PECAM-1) phosphorylation, each of which was
blocked by protein kinase C inhibitor and antioxidants. These results
suggest that the adherence/contact of SS RBC to endothelial cells in
large vessel can generate enhanced oxidant stress leading to increased
adhesion and diapedesis of monocytes, as well as heightened adherence
of SS reticulocytes, indicating that injury/activation of endothelium
can contribute to vaso-occlusion in SCD.
ENDOTHELIAL CELLS forming the lining of
the blood vessels throughout the vasculature come in continuous contact
with circulating blood cells and plasma components. Under normal
physiological conditions, blood cells are, at most, loosely attached to
endothelial cells as in the marginal pools of polymorphonuclear
leukocytes (PMN). However, under other conditions, blood cells do
adhere to localized vascular sites as part of hemostasis. For example, PMN adhere to endothelium before they migrate to sites of extravascular infection or acute inflammation, and platelets accumulate at injured sites in the vessels. These are normal processes required for host
defense and hemostasis, respectively.1 However, sickle red
blood cells (SS RBC) from patients with sickle cell anemia exhibit
increased adherence to cultured endothelial cells under both static and
flow conditions2-5 and the extent of adhesion in vitro
appears to parallel the clinical severity of vaso-occlusive events in
sickle cell disease (SCD).6 Such abnormal adhesion of SS
RBC, when compared with the behavior of normal RBC, is thought to be
important in the mechanism of vaso-occlusion, a hallmark of sickle cell
crises.6-8 In addition to adhesion events, the interaction
of SS RBC with endothelium may cause endothelial injury, culminating in
the dislodgment of cells from vessel walls9 or the altering
of endothelium characteristics, such as vascular tone.10 It
has been shown that the genes coding for vasoconstrictor endothelin-1
(ET-1)10 are induced as a result of interaction of SS RBC
with endothelium, which may affect vascular tone and thus affect blood
flow.11
Because SS RBC generate excessive amounts of reactive oxygen
metabolites due to the presence of unstable hemoglobin S and the
spontaneous autoxidation of iron in heme,12-15 we
hypothesized that the adherence of SS RBC, mediated by multimers of von
Willebrand factor (vWf), to cultured endothelial cells might induce
cellular oxidant stress, ie, generation of reactive oxygen
intermediates capable of activating transcription factor,
NF-kB.16 Numerous studies16,17 have shown that
diverse agents, eg, inflammatory cytokines, oxidative stress (hydrogen
peroxide), and endotoxins activate NF-kB by distinct intracellular
pathways that involve reactive oxygen species (ROS) as a common
messenger. The activation of NF-kB is capable of altering gene
expression of several genes including those for the cell-adhesion
molecules (ICAM-1, E-selectin,and VCAM-1).16,18 As a
consequence of the increased expression of cell-adhesion molecules, the
adherence of PMN and monocytes to vascular endothelium might be
heightened. Moreover, VCAM-1 acts as a receptor for the
To test our hypothesis, we examined whether the adhesion of SS RBC to
cultured endothelial cells generated ROS, and if so, whether it caused
activation of transcription factor NF-kB. As a consequence of
activation of NF-kB, we expect to find increased expression of VCAM-1,
which would allow adherence of sickle reticulocytes via the
Our results show that the interaction of SS RBC in the presence of
multimers of vWf, derived from endothelial cell-derived conditioned
medium (E-CM) with cultured human umbilical vein endothelial cells
(HUVEC), indeed results in an increased formation of thiobarbituric acid-reactive substances (TBARS) and the activation of the
transcription factor NF-kB, both indices of cellular oxidant
stress.16,17,21 Furthermore, we show for the first time
that the oxidant stress induced by the adhesion of SS RBC causes a
several-fold increase in the phosphorylation of PECAM-1, a
multifunctional cell-junction molecule,22-27 and
importantly, a concomitant increase in the migration of both
monocyte-like HL-60 cells and human peripheral blood monocytes (PBM)
across the endothelial cell monolayer. In SCD, the accumulation of
monocytes in large vessels, eg, cerebral vasculature in response to the
oxidant stress generated by the adhesion of SS RBC, may contribute to
the stroke syndrome.
Materials.
32P[carrier free] was obtained from ICN Biomedical Inc
(Irvine, CA); [ Isolation of RBC from whole blood.
Fresh heparinized or citrated blood was obtained from adult sickle cell
anemia patients (homozygous SS) and sickle cell trait subjects (AS) in
the adult clinic of the Comprehensive Sickle Cell Center, LAC-USC
Hospital (Los Angeles, CA); or from normal (AA) healthy
volunteers (ages 25 to 37 years) after obtaining informed consent
according to a protocol approved by the Institution Review Committee.
Sickle cell patients who had received blood transfusions within 3 months were excluded from these studies. Whole blood (8 to 10 mL) was
centrifuged at 450g for 20 minutes. Plasma and buffy coat were
removed by aspiration. The pellet was suspended in 3 volumes of
phosphate-buffered saline (PBS), pH 7.4, and passed through a column
(3.5 cm in a 30 mL syringe) of cellulose-microcrystalline cellulose
(wt/wt 1:1) as described.28 The eluted fraction was
passed twice through a new column of cellulose-microcrystalline cellulose (1:1 wt/wt) to remove platelets and white blood cells (WBC). The eluate was centrifuged at 450g for 10 minutes. The pellet of RBC was resuspended in 2 mL of PBS for differential count by
the MINOS-STX automatic cell analyzer (Roche Diagnostic Systems,
Nutley, NJ). By this method, we routinely obtained RBC preparations
that were less than 1% contaminated by platelets and WBC.
Isolation of human PBM.
Blood (80 to 100 mL) was collected by venipuncture from adult donors of
both sexes. To the whole blood collected into a heparinized 60 mL
plastic syringe was added Dextran (13.5 mL of 5% Dextran) followed by
gravity sedimentation for 40 minutes as described.29 The
supernatant containing leukocytes were fractionated into monocytes by
gradient centrifugation on Isolymph. The monocyte fraction was labeled
with MO2-FITC antibody (Coulter Diagnostics, Hialeah, FL) followed by
FACscan analysis as previously described.29 Cells obtained
by this procedure had purity in the range of 70% to 85% and a yield
of 50% to 75%.
Cell cultures.
HUVEC were harvested from umbilical cord veins by collagenase digestion
as previously described.29 The cells were evaluated for
cobblestone morphology, the binding of vWF-antibody, and the uptake of
acetylated LDL labeled with
1,1 Preparation of E-CM.
To the HUVEC (2 × 106 cells) grown to confluence,
3 mL of RPMI-1640 (devoid of fetal bovine serum), 1 mmol/L CaCl2, and 10 µmol/L A23187 calcium ionophore
were added followed by incubation for 2 hours at room
temperature.32 The supernatant was collected and
concentrated in dialysis tubing. The concentrated sample was subjected
to an agarose (2.5%) gel electrophoresis in a running buffer
containing 25 mmol/L Tris buffer, pH 8.3, 0.192 mol/L glycine, and
0.1% sodium dodecyl sulfate (SDS). The molecular sizes of vWf
multimers were estimated by comparison with Jack bean urease as a
standard (Mr. 240 and 480 kD, Sigma) after staining the gel with 0.25%
Coomassie brilliant blue dye. The analysis of conditioned medium showed
triplet bands of high-molecular weight polypeptides to be estimated in
the range of 400 to 950 kD that cross-reacted with antibody to vWf as
has been previously observed.33 We used E-CM at 100 µg/mL
for the studies described here because this concentration showed
optimal adherence of SS RBC to HUVEC (Table 1).
Measurement of lipid peroxides as thiobarbituric acid-reactive
substances (TBARS) in endothelial cells.
Confluent HUVEC (2.5 × 106 cells in 100 mm tissue
culture dishes) were washed with RPMI-1640 and incubated at 37°C in
the presence and absence of RBC (hematocrit 2%) with E-CM (100 µg/mL) in 3 mL serum-free RPMI-1640 for 4 hours. At the end of
incubation, nonadherent RBC were aspirated and plates washed three
times with 3 mL of PBS. Endothelial cells were harvested from the
culture dish by treatment with a 0.25% trypsin-0.02% EDTA solution.
The contents were centrifuged at 500g for 5 minutes at
4°C. Cell pellets were washed three times with TBS (10 mmol/L Tris-buffer, pH 7.4, containing 140 mmol/L NaCl). The content of
lipid peroxides formed was determined by the reactivity of
malondialdehyde (MDA), an end product of lipid peroxidation, with
thiobarbituric acid (TBA) as described.34 Briefly, 0.8 mL
of water was added to the pellet, followed by the addition of 0.2 mL of
8.1% sodium dodecyl sulfate (SDS), 1.5 mL of 20% acetic acid, and 1.5 mL of a 0.8% aqueous solution of TBA. The mixture was heated for 60 minutes in a boiling water bath. After cooling, 1 mL of water and 5 mL
of a mixture of n-butanol-pyridine (15:1 vol/vol) were added. Contents
were vortexed, then centrifuged at 3,000g for 10 minutes. The
upper (organic) layer was removed for measurement of absorbency at 532 nm (Hitachi U-3110 spectrophotometer; Hitachi, San Jose, CA). The TBARS
values were calculated using the molar extinction coefficient of the
MDA-TBA complex of 1.5 × 106 cm-1
M-1 as previously described.35
Static assay for the adherence of RBC to cultured HUVEC.
RBC in PBS (2 mL at Hct of 10%) were incubated with
51Cr-sodium chromate (0.5 mCi) at room temperature for 60 minutes. Labeled RBC were washed three times with 10 vol of PBS and
used at a hematocrit of 2% in PBS. HUVEC grown to confluence in 6-well
plates were washed twice with PBS (3 mL) followed by the addition of
51Cr-labeled RBC (Hct 2%) and PBS to a final volume of 1 mL. The contents were incubated for 45 minutes at room temperature.
Nonadherent RBC were removed by aspiration and monolayers were washed
three times with 1 mL of PBS containing 0.5% bovine serum albumin
(BSA). The radioactivity in the cell monolayer-containing adherent RBC was determined and the percentage of RBC adherent was
calculated.2,36
Electrophoretic mobility shift assay (EMSA) for transcription factor
NF-kB activity.
Confluent HUVEC (6 × 106 cells) were incubated at
37°C with RBC (2% hematocrit) from either sickle cell anemia (SS)
or normal (AA) subjects in 3 mL serum-free RPMI-1640 containing E-CM
(100 µg/mL) for time periods ranging from 30 to 240 minutes. At the end of the incubation period, the medium was aspirated and cells were
washed twice with 3 mL ice-cold PBS. Cells were scraped in 1 mL PBS and
nuclear extracts prepared as described.37 Briefly, cells
were suspended in 400 µL of buffer A (10 mmol/L Hepes, pH 7.9, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 1 mmol/L dithiothreitol (DTT),
0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF), 0.3 µg/mL leupeptin,
and 0.7 µg/mL pepstatin A) and incubated on ice for 10 minutes. Cells
were pelleted and resuspended in 50 µL of buffer B (20 mmol/L Hepes,
pH 7.9, 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L
Na-EDTA, 1 mmol/L DTT, 0.5 mmol/L PMSF; 0.3 µg/mL
leupeptin, and 0.7 µg/mL pepstatin A). After a 20-minute incubation
at 4°C, the homogenate was centrifuged at 8,000g for 20 minutes. The supernatant, containing the nuclear proteins, was stored
at Enzyme-linked immunosorbent assay (ELISA) for surface expression of
CAMs.
HUVEC were grown to confluence in 24-well tissue culture plates, rinsed
with RPMI-1640, and incubated with serum-free RPMI-1640 in the presence
or absence of RBC (hematocrit 2%) with E-CM at 37°C. At the end of
the indicated incubation periods, ranging from 2 to 24 hours, the
medium was aspirated and the wells washed twice with 1 mL of
PBS. To each well, 500 µL of 2.5% paraformaldehyde was added to fix
the cells. Cell-surface expression of ICAM-1, E-selectin, and VCAM-1 in
HUVEC was assayed by separate incubations with MoAb to ICAM-1,
E-selectin, and VCAM-1. Incubations were performed at room temperature
for 120 minutes at a saturating concentration of antibody (1:500
dilution in 0.5 mL of PBS). Cells were washed, then incubated with
alkaline phosphatase-conjugated goat antimouse IgG, diluted 1:1000 in
PBS, for 60 minutes. Cells were washed three times with 1 mL of PBS.
Binding of the secondary antibody was determined by the addition of 200 µL of p-nitrophenyl phosphate substrate (1 mg/mL in 0.2 mol/L Tris,
pH 9.8, containing 5 mmol/L MgCl2). Plates were incubated
for 30 minutes in the dark. The reaction was terminated by the addition
of 50 µL of 3N NaOH. The absorbency was read at 405 nm in an ELISA
plate reader (Model 7520; Cambridge Technology, Watertown, MA)
interfaced with an IBM-PC computer. The surface expression
of CAMs is shown as mean ± standard deviation (SD) of the optical
density (OD) after subtracting the blank value, that is the OD in the
absence of primary antibody.
32P Labeling of HUVEC and immunoprecipitation of
PECAM-1.
HUVEC grown to confluence in tissue culture dishes (100 × 15 mm)
were washed three times with prewarmed phosphate-free RPMI-1640 (GIBCO-BRL) and radiolabeled in 3 mL of phosphate-free medium containing 0.20 mCi of 32P (carrier-free; ICN Biomedicals,
Inc, Irvine, CA) for 4 hours at 37°C as previously
described.37 The monolayer of radiolabeled HUVEC was washed
with phosphate-free medium and incubated in 3 mL of the same medium in
the presence or absence of RBC (hematocrit 2%) and E-CM for the
indicated time intervals. At the end of incubation, the medium was
aspirated and cells washed three times with 3 mL of ice-cold PBS.
Endothelial cells were lysed in 1 mL of lysis buffer (50 mmol/L
Tris-HCl, pH 8.0; 150 mmol/L NaCl; 0.1% SDS; 50 mmol/L NaF, 10 mmol/L
EDTA, 1% Nonidet P-40; 0.1 mmol/L DTT; 0.5% sodium deoxycholate; 2 mmol/L sodium orthovanadate; 100 µg/mL PMSF; 1 µg/mL pepstatin; and
1µg/mL leupeptin). The lysate was centrifuged and immunoprecipitated
with an antibody to PECAM-1 (as ascites fluid from clone
XVD2) as previously described.37 The
immunocomplex was collected by centrifugation, washed with lysis
buffer, and the sample was subjected to electrophoresis on a 10%
SDS-polyacrylamide gel as previously described.37 Gels were
dried and exposed overnight to Kodak XR-5 film at room temperature. The
radioactivity in the band corresponding to PECAM-1 (130 kD) was
quantitated by using an Ambis radioactivity gel scanner (Model 101; San
Diego, CA).
Fluorescent labeling of HL-60 monocyte-like cells.
Vitamin D3-differentiated HL-60 cells were labeled with a
fluorescent probe, CellTracker Green BODIPY
(8-chloromethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diazaindacene) according to the manufacturer's instructions (Molecular Probes Inc,
Eugene, OR). Briefly, the medium was aspirated from Vitamin D3-differentiated HL-60 cells cultivated in tissue culture
plates, followed by the addition of the medium containing fluorescent probe (5 µmol/L). The cells were incubated for 45 minutes. The loading solution was then replaced with fresh prewarmed medium and the
cultures incubated for an additional 30 minutes at 37°C. The
fluorescently labeled HL-60 cells were used for a transendothelial migration assay.
Assay for transendothelial migration of monocytes.
HUVEC monolayers were grown to confluence on fibronectin-coated porous
membranes (Biocoat Cell culture inserts, 3.0 micron; Collaborative
Biomedical Products, Bedford, MA) for 5 to 6 days as
described.31 Transendothelial resistance was measured daily on the filters with sterilized electrodes using an EVOM voltohmmeter (World Precision Instruments, Sarasota, FL). At day 5 to 6, HUVEC monolayer exhibited total electrical resistance (endothelial cell monolayer with filter) of 58 ± 8 ohms.cm2 (n = 13) with net transendothelial electrical resistance of 30 ± 4 ohms.cm2 and were used at that time for migration
studies. The values for transendothelial electrical
resistance for HUVEC cultivated on fibronectin-coated porous membranes
are similar to literature values of 28 ± 11 ohms.cm2 for HUVEC monolayer cultured on polyethylene terephthalate
micropore membranes.38 Confluent monolayers of HUVEC were
incubated at 37°C in duplicate with aliquots (1 × 105 cells per well) of fluorescently labeled vitamin
D3-differentiated HL-60 cells, in the presence and absence
of RBC (hematocrit 2%), containing E-CM for time periods ranging from
30 minutes to 4 hours. The lower compartment of the Transwell chamber
contained 1 mL of RPMI-1640, whereas the upper compartment contained
0.5 mL of the same medium, as well as the HL-60 cells. At the indicated time points, aliquots of 50 µL were removed from the bottom
compartment of the well. Cells were counted on the coverslip using a
fluorescence microscope (Olympus IMT 2 attached with a 10 × 10 mm grid in the eyepiece; Olympus Optical Co, Tokyo, Japan).
Only those cells that were viable were counted by this procedure (dead
cells release the fluorescent marker). To keep the volume constant in
the lower compartment, an equal amount (50 µL) of medium was added at
each removal of monocytes. In inhibition experiments, antibodies and inhibitors were added 30 minutes before the addition of RBC to HUVEC.
Data analysis.
All experiments on cell-adhesion molecule expression were performed in
triplicate with at least three different batches of endothelial cell
preparations. Each treatment (incubation with RBC) was compared with
its respective control (untreated). The results for the incorporation
of 32P into PECAM-1 are expressed as the percent of control
and are mean ± SD. The statistical significance of difference in a
treatment series was determined by analysis of variance. Individual
treatments in the experimental series were compared with their controls
using Dunnet's test. Statistical analyses were performed using the
Instat-2 software program (GraphPad Software).
Effect of SS RBC on the induction of oxidant stress in HUVEC.
We determined whether the adhesion of SS RBC could induce oxidant
stress. As shown in Table 1, SS RBC were 2.5 times more adherent to the
endothelial cells than were those from normal individuals.
Additionally, SS RBC in the presence of multimers of vWf (conditioned
medium from endothelial cells, E-CM) exhibited threefold more adherence
than in the absence of E-CM, as has been previously
observed.23,39 The augmented adherence of SS RBC in the
presence of multimers of vWf (E-CM) was observed to be optimal with 100 µg protein/mL of E-CM and inhibited by antibody to vWf. As shown in
Table 1, incubation of SS RBC at a concentration approximating a
hematocrit of 2% (vol/vol) with HUVEC, in the absence and presence of
E-CM led to approximately three- and fivefold increased formation of
TBARS, respectively, relative to HUVEC coincubated with normal (AA) RBC
with and without E-CM. The adhesion of SS RBC to endothelial cells
mediated by multimeric vWF (E-CM) appears to play a role in oxidant
stress (lipid peroxide) induction because the addition of the peptide
Lys-Tyr-Arg-Gly-Asp-Ser (KYRGDS) containing the
arginine-glycine-aspartic acid (RGD) sequence motif, which prevents
multimeric vWf-mediated adhesion of SS RBC to HUVEC,23,39 completely inhibited the formation of TBARS, over and above that of SS
RBC alone, whereas a variant peptide Ala-Gly-Asp-Val (AGDV) did not
prevent either the adhesion of SS RBC or the formation of TBARS. Both
catalase (200 U/mL) and superoxide dismutase (200 U/mL) together,
scavengers of free radicals,40 completely abrogated SS RBC+
E-CM-induced TBARS formation in HUVEC, relative to HUVEC incubated
with SS RBC alone. Moreover, in the presence of superoxide dismutase
(SOD) and catalase the formation of lipid peroxides (TBARS) in HUVEC
was observed to be 50% reduced below the level of HUVEC incubated with
SS RBC alone. It is pertinent to note that incubation of SS RBC (2%
Hct) alone for 2 to 4 hours did not show the formation of TBARS because
its formation was below the detection limit of the assay. However, at a
higher concentration (10%) of SS RBC, the amount of MDA formed by SS
RBC alone was 8.78 ± 0.62 nmole MDA/g hemoglobin (n = 4) (data not
shown) in agreement with previous studies.14 Overall, these
results indicate that adhesion of SS RBC to endothelial cells, mediated
by multimers of vWf (E-CM), generates greater amounts (approximately
twofold) of ROS over and above that observed with SS RBC alone
interaction with endothelial cells.
SS RBC-induced activation of NF-kB activity in HUVEC.
We measured, in HUVEC nuclear extracts, the activation of the
transcription factor NF-kB, another index of cellular oxidant stress.16,41 As shown in Fig 1,
nuclear extracts prepared from HUVEC that had been incubated with SS
RBC and E-CM, showed a time-dependent increase in NF-kB activity. The
maximal activation of NF-kB activity occurred at 1 hour, followed by a
decrease at 2 hours and 4 hours. As shown in Fig 1, gel-shift binding
assays in the presence of excess competing oligonucleotide resulted in
a decrease in NF-kB activity, indicating specificity of interaction of
DNA binding proteins with an NF-kB nucleotide sequence. There was
minimal activation of NF-kB activity when HUVEC were incubated with AA RBC in the presence of E-CM at the 1-hour time point (Fig 1), the
optimal time period for activation of NF-kB with SS RBC plus E-CM. As
shown in Fig 2, incubation of normal red
blood cells (AA RBC) with HUVEC for a prolonged time (4 hours) with or
without E-CM did not significantly increase NF-kB activity over that of untreated HUVEC. Furthermore, SS RBC incubation with HUVEC alone for 1 hour augmented by twofold activation of NF-kB activity. However,
incubation of SS RBC with HUVEC in the presence of E-CM increased by
twofold the NF-kB activity over and above that of HUVEC incubated with
SS RBC alone. This data is qualitatively similar to the formation of
TBARS in HUVEC by SS RBC in the absence and presence of E-CM (Table 1).
As shown in Fig 2, incubation of HUVEC with E-CM alone did not affect
basal NF-kB activity.
Adherence of SS RBC to HUVEC leads to the surface expression of
adhesion molecules.
As shown in Fig 3A, incubation of SS RBC
(2% hematocrit) in the presence of vWf (E-CM), with HUVEC resulted in
a time-dependent increase in the surface expression of ICAM-1,
E-selectin, and VCAM-1, as determined by ELISA. The surface expression
of ICAM-1 continued to increase above basal level (untreated HUVEC) at
12 hours and remained unchanged up to 24 hours. Similarly, the surface expression of VCAM-1 was also optimal at 12 hours. However, E-selectin surface expression, though it increased up to 8 hours in response to
incubation with SS RBC, then declined to almost basal level by 12 to 24 hours. Moreover, there was no affect on the surface expression of
constitutively expressed ICAM-2 by SS RBC and E-CM (data not shown). To
determine whether the effect observed was specific for SS RBC (Hb SS),
HUVEC were incubated with normal RBC (Hb AA) and SC trait RBC (Hb AS)
under identical conditions and assayed for CAMs expression. As shown in
Fig 3B, there was no significant difference in the surface expression
of CAMs in response to the interaction of AA and AS RBC with
endothelial cells compared with untreated ECs.
Does adhesion of SS RBC or soluble factor released from SS RBC
mediate the surface expression of CAMs?
As shown in Fig 3C, E-CM alone (vWf), in the absence of SS RBC, did not
induce VCAM-1 surface expression. However, E-CM induced the surface
expression of VCAM-1 in the presence of SS RBC. Moreover, addition of
the KYRGDS peptide (100 µmol/L), which blocks vWf-mediated adhesion
of SS RBC,39 reduced by approximately 50% the surface expression of VCAM-1, whereas the variant-peptide AGDV failed to
inhibit VCAM-1 expression induced by SS RBC and E-CM. The VCAM-1 expression, which is not inhibitable by RGD peptide, may be due to the
basal level of oxidative stress generated by SS RBC
alone.13 It is pertinent to note that the addition of
synthetic peptides KYRGDS and AGDV directly to HUVEC, in the absence of
SS RBC, or the SS RBC-conditioned media derived from the incubation of
1 mL of SS RBC (20% Hct) for 2 hours, failed to induce the surface expression of VCAM-1 (data not shown), indicating that binding of small
peptide ligands or components present in the medium from SS RBC to the
putative receptor on HUVEC was not sufficient to induce VCAM-1
expression. Treatment of HUVEC with LPS (100 ng/mL) as a positive
control exhibited an increase in VCAM-1 expression over and above the
level observed with SS RBC in the presence of E-CM (Fig 3C). The
increase in VCAM-1 surface expression in HUVEC in response to SS RBC
E-CM was not due to the contaminating effect of endotoxin because
addition of polymyxin B (5 µg/mL), an inhibitor of endotoxin, did not
reduce VCAM-1 expression (Fig 3C). These studies indicate that
adherence of SS RBC mediated by vWf augments VCAM-1 expression, whereas
inhibition of adherence by RGD peptide reduces the VCAM-1 expression.
Effect of antioxidants on the surface expression of VCAM-1 in HUVEC.
Because probucol inhibited the activation of NF-kB activity in HUVEC in
response to the interaction of SS RBC +E-CM, we examined the effect of
antioxidants on the surface expression of VCAM-1. As shown in Fig 3D,
pretreatment of HUVEC with the antioxidants probucol (50 µmol/L) and
vitamin E (25 µmol/L) reduced by ~90% VCAM-1 expression induced by
SS RBC plus E-CM. Moreover, the addition of SOD and catalase,
completely abrogated the VCAM-1 expression over and above that observed
with SS RBC alone. The possibility of the involvement of the
glutathione redox cycle in the cellular oxidant stress generated by the
interaction of SS RBC with HUVEC was examined through the use of
agents, that affect intracellular glutathione levels.45
Elevation of intracellular glutathione (GSH) levels by
preincubation of HUVEC with the cell-permeable glutathione ester
(GSH-ethyl ester) for 24 hours completely inhibited SS RBC, plus E-CM
induced VCAM-1 expression (Fig 3D). Conversely, pretreatment of HUVEC
with butathionine sulfoximine (BSO), an agent that prevents GSH
synthesis,45 augmented VCAM-1 expression by ~40% over
and above that observed with SS RBC plus E-CM (Fig 3D).
Effect of SS RBC-induced oxidant stress on the transendothelial
migration of monocytes.
In our recent studies,44 we observed that oxidant stress
induced by tert-butylhydroperoxide promoted the transendothelial migration of monocytes; thus we examined whether ROS generated by HUVEC
in response to the adhesion of SS RBC also mediated the transendothelial migration of monocytes. First, we examined the effect
of adhesion of SS RBC on the transendothelial migration of
monocyte-like HL-60 cells. To study the migration of monocyte-like cells in the presence of RBC, we used fluorescent-labeled HL-60 cells,
so as to distinguish the migration of monocytes from that of RBC across
the endothelial cell monolayer. The migration of fluorescently labeled
monocyte-like HL-60 cells was monitored by fluorescence microscopy. As
shown in Fig 4A, the addition of SS RBC
(hematocrit 2%) with E-CM to confluent monolayers of HUVEC cultured in
Transwell chambers resulted in an approximately twofold increase in the
number of monocyte-like HL-60 cells migrating across the endothelial
cell monolayer at 2 and 4 hours posttreatment with SS RBC. Normal (AA)
RBC in the presence of E-CM failed to increase the migration of
monocyte-like HL-60 cells above basal level (Fig 4A). As previously
observed37 with 15-HPETE-induced transendothelial migration
of monocytes, an antibody to PECAM-1 inhibited by 70% ± 11% the
SS RBC-induced migration of monocyte-like HL-60 cells across HUVEC
monolayers (Fig 4B), whereas a control antibody to ICAM-2 had no effect
on transendothelial migration (Fig 4B).
Effect of SS RBC on transendothelial migration of human PBM.
Because our studies showed that incubation of HUVEC with SS RBC in
presence of E-CM increased transendothelial migration of monocyte-like
HL-60 cells (a transformed cell line); it was of interest to examine
whether freshly isolated monocytes from human peripheral blood
exhibited similar migration characteristics. As shown in Fig 4D,
incubation of HUVEC with SS RBC plus E-CM resulted in twofold increase
in the migration of human PBM at 2-hour time point as was observed for
monocyte-like HL-60 cells. Additionally, antibody to PECAM-1 inhibited
the migration of monocytes by ~80% (Fig 4D). However,
control antibody to ICAM-2 had no affect (data not shown). The addition
of KYRGDS, but not a variant peptide AGDV, blocked the transendothelial
migration of human PBM by 70%.
Phosphorylation of PECAM-1 in HUVEC induced by SS RBC.
As shown in Fig 5, incubation of
32P-labeled HUVEC with SS RBC plus E-CM resulted in a
time-dependent increase in PECAM-1 phosphorylation. We observed
approximately a sixfold (638% ± 220%) increase in the
phosphorylation of PECAM-1 at 1 hour. At the 2-hour time point; the
phosphorylation of PECAM-1 showed a fivefold increase above the basal
level. Conversely, normal (AA) RBC, under the same conditions, showed
no induction of PECAM-1 phosphorylation (data not shown). As shown in
Fig 6, SS RBC-induced phosphorylation of
PECAM-1 in HUVEC was approximately 75% inhibited by the PKC inhibitor
GF-109203X,46 but not by the adenylate cyclase inhibitor
SQ-22536.49 However, treatment of HUVEC with a protein
phosphatase inhibitor, Calyculin A,47 in the presence of SS
RBC and E-CM, increased PECAM-1 phosphorylation by ~35% above the
level induced by SS RBC plus E-CM (Fig 6). Furthermore, the
phosphorylation of PECAM-1 induced by SS RBC + E-CM was ~65% inhibited by antioxidant probucol, indicating the importance of oxidant
stress in the phosphorylation of PECAM-1. Addition of GSH-ethyl ester,
inhibited by ~85% the SS RBC plus E-CM-induced phosphorylation.
Conversely, inhibition of intracellular synthesis of GSH by
pretreatment of HUVEC with BSO elevated PECAM-1 phosphorylation by
~50%. These results indicate that redox state (GSH/GSSG levels) of
endothelial cells in response to the interaction with SS RBC can affect
the phosphorylation of PECAM-1.
Vascular occlusion leading to episodes of painful crises and damage to
various end organs is the major cause of morbidity in adults and older
children with SCD.50-52 It is recognized that abnormal
adherence of SS RBC to endothelium is a key participant in the
vaso-occlusive events,53 because studies showed that the
extent of adherence of SS RBC to cultured endothelial cells correlated
with the clinical severity of vaso-occlusive crises in
SCD.5,6 However, SCD patients with identical biochemical defects in their |
Top
Abstract
Introduction
4
1 integrin present on SS
reticulocytes
-32P]ATP (adenosine
triphosphate) was obtained from Amersham Corp (Arlington
Heights, IL). 1
-dioctadecyl-3,3,3
80°C, and used for assay of NF-kB activity within 10 days
of storage. Five nanograms of double-stranded oligonucleotide
containing a tandem repeat of the consensus sequence of the NF-kB DNA
binding site, -GGGGACTTTCC-with the following sequence:
