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Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3924-3935
Interaction of Sickle Erythrocytes With Endothelial Cells in the
Presence of Endothelial Cell Conditioned Medium Induces Oxidant Stress
Leading to Transendothelial Migration of Monocytes
By
Chand Sultana,
Yamin Shen,
Vinod Rattan,
Cage Johnson, and
Vijay
K. Kalra
From the Departments of Biochemistry and Molecular Biology, and
Medicine, University of Southern California, School of Medicine, Los
Angeles, CA.
 |
ABSTRACT |
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.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
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
 4 1 integrin present on SS
reticulocytes19,20 thus promoting the additional adherence
of SS RBC. As a consequence of the additional adherence of SS RBC, PMN,
and monocytes, blood flow could be further obstructed, which may
contribute to the recurrent vaso-occlusive events in SCD.
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
41 integrin. The activation of NF-kB
would also lead to increased expression of CAMs, which participate in
the adhesion of monocytes to endothelial cells. Thus, adhesion of both
reticulocytes and monocytes to the vascular endothelium could cause
further obstruction and participate in vaso-occlusive phenomenon.
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.
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MATERIALS AND METHODS |
Materials.
32P[carrier free] was obtained from ICN Biomedical Inc
(Irvine, CA); [ -32P]ATP (adenosine
triphosphate) was obtained from Amersham Corp (Arlington
Heights, IL). 1, 25-dihydroxyvitamin D3, Calyculin A,
and SQ-22536 were obtained from Biomol Research Laboratories (Plymouth
Meeting, PA); GF-109203X was obtained from Calbiochem-Novabiochem International (San Diego, CA). Acetylated low-density lipoprotein (LDL) labeled with
1,1 -dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate was obtained from Biomedical Technologies, Inc (Stoughton, MA). Monoclonal antibodies (MoAb) to ICAM-1, ICAM-2, E-selectin, VCAM-1, and PECAM-1 were obtained from Immunotech Inc (Westbrook, MN).
A MoAb to bovine PECAM-1, a purified ascites fluid from clones XVD2, was prepared as described.22 All other
reagents were obtained from Sigma (St Louis, MO) unless otherwise
specified.
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-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate. Cells were used from passages two through six. The human
promyelocytic cell line HL-60 (American Type Culture Collection, Rockville, MD) was cultured in RPMI-1640 containing 20%
heat-inactivated fetal calf serum (Gemini Bioproducts, Calabasas, CA).
HL-60 cells differentiate to a monocyte-like phenotype by incubation in
the presence of 1 × 10-7 mol/L
1-25-dihydroxyvitamin D3 after 3 to 4 days under culture conditions, as previously described.30,31
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  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:
was end labeled with 100 µCi [-32P] ATP using T4
polynucleotide kinase as suggested in the manufacturer's kit
(GIBCO-BRL, Gaithersburg, MD). DNA-protein-binding reactions were
performed by preincubating 3 to 5 µg nuclear extract protein on ice
for 15 minutes in a total volume of 25 µL containing 10 mmol/L Tris,
pH 7.6, 50 mmol/L NaCl , 1 mmol/L DTT, 0.02 µmol/L ATP, 5 µg BSA,
and 10% glycerol in the presence and absence of excess
specific-competitor oligonucleotide. This was followed by the addition
of the double-stranded 32P-labeled oligonucleotide (1 × 105 cpm) and a second incubation at room
temperature for 20 minutes. The samples were subjected to
electrophoresis on 6% nondenaturing polyacrylamide gels as previously
described37 using 0.25 × TBE running buffer
containing Tris (25 mmol/L; pH 8.0), borate (22.5 mmol/L), and EDTA
(0.025 mmol/L) at 150 V for 2 to 3 hours. Gels were then dried and
exposed to Kodak X-Omat AR film (Eastman Kodak, Rochester,
NY) with intensifying screens at 80°C.
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).
| |
RESULTS |
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.
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| Fig 1.
Effect of incubation of RBC and inhibitors on NF-kB
activity in HUVEC nuclear extracts by gel-shift assay. HUVEC were
incubated with RBC (2% Hct) in the presence of E-CM (100 µg/mL) for
60 minutes, unless otherwise indicated in the presence and absence of
inhibitors (genistein, 25 µg/mL; KYRGDS, 100 µmol/L; HFPA, 10 µmol/L; and probucol, 50 µmol/L). Nuclear extracts were prepared
and incubated with a double-stranded 32P-labeled NF-kB
oligonucleotide probe. Where indicated, excess cold competitor
oligonucleotide was added to the nuclear extracts and incubated for 10 minutes before addition of radiolabeled DNA probe. The data are
representative of four independent experiments.
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| Fig 2.
Relative NF-kB activation in HUVEC in response to
interaction of RBC. HUVEC were incubated with either AA RBC (n = 3;
two different donors ) for 1 hour and 4 hours or with SS RBC (n = 4;
three different donors and one replicate) for 1 hour in the absence and
presence of E-CM. Nuclear extracts were probed for NF-kB activity. The
NF-kB activity in the gel (not shown) was quantified by densitometric
scan of the autoradiograph by Alpha Image 2000 Documentation and
Analysis system. The data shown is relative change in NF-kB activity
compared to untreated HUVEC. NF-kB activity increased to 365% ± 34% (range, 320% to 425%) on incubation of HUVEC with SS RBC
and E-CM. Replicate of the same donor sample showed less than 10%
variation.
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As shown in Fig 1, SS RBC + E-CM induced activation of NF-kB was
inhibited by a tyrosine kinse inhibitor, genistein42; and by antioxidant, probucol.40 Other antioxidants,
pyrrolidinedithiocarbamate (PDTC)43 and vitamin E, also
inhibited NF-kB activity (data not shown). Additionally, the activation
of NF-kB in HUVEC nuclear extracts induced by SS RBC in the presence of
E-CM was inhibited when HUVEC were preincubated with the synthetic
peptide KYRGDS (Fig 1). We have recently observed that
tert-butylhydroperoxide, an inducer of oxidant stress, causes
activation of the transcription factor NF-kB as a result of activation
of p21ras 44; thus we examined whether the
NF-kB activation that was induced by SS RBC could be blocked by an
inhibitor of p21ras activity. As shown in Fig 1, treatment
of HUVEC with a p21ras farnesyltransferase
inhibitor41 (-hydroxyfarnesylphosphonic acid, HFPA),
before the addition of SS RBC + E-CM inhibited NF-kB activity. These
results suggest that interaction of SS RBC with HUVEC, in the presence
of multimers of vWf (E-CM), causes a greater activation of NF-kB over
and above that observed with SS RBC alone. Thus, we chose to study the
effect of adhesion of SS RBC, mediated by multimers of vWf (E-CM), on
the cellular signaling in HUVEC leading to the expression of CAMs and
migration of monocytes across the endothelial cell monolayer in the
following studies.
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.
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| Fig 3.
Cell adhesion molecule expression in HUVEC in response to
interaction with RBC. HUVEC grown to confluence in 24-well plates were
incubated with RBC (2% Hct) and E-CM (100 µg/mL) for 4-hour time
periods, unless otherwise indicated. ICAM-1, E-selectin, and VCAM-1
expression was determined by ELISA. (A) Time period of expression of
CAMs in response to SS RBC plus E-CM; results are expressed as mean ± SD of OD at 405 nm of triplicate determinations, (n = 5; including
three different donors). (B) Effect of AA RBC (n = 3), AS RBC (n = 3), and SS RBC (n = 6; including four different donors) on CAMs
expression at 4 hours. (C) HUVEC were preincubated for 30 minutes with
peptides (KYRGDS, 100 µmol/L; AGDV, 100 µmol/L) and polymyxin
sulfate (5 µg/mL) before incubation with SS RBC plus E-CM or E-CM
(100 µg/mL) alone for 4 hours. Incubation with LPS (100 ng/mL) was
performed for 4 hours. (D) HUVEC were preincubated for 30 minutes with
inhibitors (Probucol, 50 µmol/L; vitamin E, 25 µmol/L; SOD, (200 U/mL); catalase, (200 U/mL); GSH-EE, 0.5 mmol/L; and BSO (100 µmol/L)
before incubation with SS RBC for 4 hours, followed by measurement of
VCAM-1 expression.
|
|
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).
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| Fig 4.
Migration of monocyte-like HL-60 cells and PBM across
HUVEC monolayer in response to incubation with RBC. HUVEC were grown to
confluence on fibronectin-coated porous membranes (Transwell, Cat.
#40492, Biocoat cell culture inserts, Becton-Dickinson). To the upper
compartment, RPMI-1640 containing FCS was added, followed by RBC (2%
Hct) and E-CM (100 µg/mL) and fluorescently labeled vitamin
D3-differentiated HL-60 cells or PBM (0.5 × 106 cells per well). At the indicated time points, aliquots
were removed from the lower compartment of the Transwell chamber for
counting of monocyte-like HL-60 cells. Data are expressed as mean ± SD of SS RBC, n = 4 (three different donors), and three independent
determinations for AA RBC. (A) Time course of HL-60 cells
transendothelial migration. (B) HUVEC were incubated with inhibitors
(MoAb to human PECAM-1 (10 µg/mL); MoAb to ICAM-2 (10 µg/mL); GF
109203X (20 nmol/L); and Calyculin A, (2 nmol/L) for 45 minutes before
the addition of SS RBC (2% Hct) and E-CM. (C) HUVEC were preincubated
with either antioxidant (probucol, 50 µmol/L) for 45 minutes or GSH
effectors (GSH-EE, 0.5 mmol/L and BSO, 100 µmol/L) for 24 hours,
before the addition of SS RBC (2% Hct) and HL-60 cells. (D) HUVEC were
incubated with MoAb to human PECAM-1 (10 µg/mL); KYRGDS, (100 µmol/L); and AGDV, (100 µmol/L) for 45 minutes before the addition
of SS RBC (2% Hct) and E-CM (100 µg/mL). This was followed by the
addition of fluorescently labeled PBM. The transmigrated monocyte cells
were counted at 2 hours. Data are expressed as mean ± SD for three
different donors RBC.
|
|
Our previous studies37 showed that 15-HPETE induced the
phosphorylation of PECAM-1 and, concomitant with it, increased the transendothelial migration of monocytes; thus we investigated whether
the transendothelial migration of monocyte-like HL-60 cells induced by
SS RBC was linked to PECAM-1 phosphorylation. As shown in Fig 4B,
pretreatment of HUVEC with the PKC inhibitor GF-109203X (20 nmol/L)46 for 30 minutes, followed by incubation with SS
RBC and E-CM, resulted in an approximate 60% decrease in the
transendothelial migration of monocyte-like HL-60 cells at the 2-hour
time point. As predicted, augmentation of PECAM-1 phosphorylation with
the protein phosphatase inhibitor Calyculin A (2 nmol/L)47
increased by ~70% (P < .001) the transendothelial migration of monocyte-like HL-60 cells, relative to endothelial cells
incubated with SS RBC and E-CM (Fig 4B). These studies indicate that
there may be a direct or causal relationship between phosphorylation of
PECAM-1 and transendothelial migration of monocytes.
Treatment of HUVEC with antioxidant probucol (50 µmol/L) reduced by
~70% the migration of monocyte-like HL-60 cells (Fig 4C). As shown
in Fig 4C, pretreatment of HUVEC with GSH-ethylester, which causes
elevation of intracellular GSH levels,48 for 24 hours
reduced by ~90% the migration of monocyte-like HL-60 cells. However,
treatment of HUVEC with BSO to prevent GSH synthesis48 increased by 50% the migration of monocyte-like HL-60 cells induced by
SS RBC plus E-CM (Fig 4C).
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.
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| Fig 5.
Time course of phosphorylation of PECAM-1 in
HUVEC in response to incubation with SS RBC. HUVEC were labeled with
32P and incubated with SS RBC (2% Hct) for the indicated
time period and then processed for immunoprecipitation with PECAM-1
antibody. The immunoprecipitate was subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioactivity
quantitated in the gel lane corresponding to PECAM-1 (130 kD) by Ambis
radiogel scanner as described in Materials and Methods. The data is
presented as percent increase in the incorporation of 32P
in PECAM-1, assigning the value of 100% for 32P
incorporated into PECAM-1 in untreated HUVEC. Data are mean ± SD of n
= 3, with each experiment run in duplicate for indicated time points,
except for 1-hour period (n = 7). 32P incorporated
into PECAM-1 was 638% ± 220% at 1-hour time point, with a range of
410% to 850%. The replicate data for the same donor sample showed
less than 15% difference in the 32P incorporation into
PECAM-1.
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| Fig 6.
Effect of inhibitors on the 32P incorporation
in PECAM-1 in HUVEC on incubation with RBC. 32P-labeled
HUVEC were incubated with inhibitors either for 45 minute (GF 109203X,
20 nmol/L; SQ 22536, 2 µmol/L; calyculin A, 2 nmol/L; probucol, 50 µmol/L; or for 24 hours (GSH-EE, 0.5 mmol/L and BSO, 100 µmol/L),
followed by 1-hour incubation with SS RBC (2% Hct) and E-CM. Data are
expressed as mean ± SD for three different SS RBC donors, in
duplicate determinations, relative to untreated HUVEC
(none).
|
|
| |
DISCUSSION |
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 -globin genes show a wide variability in the frequency and clinical severity of vasoocclusive crises, and can remain
asymptomatic for prolonged periods,51 indicating that additional factors must contribute to the pathophysiology of
vaso-occlusion.8 Previous studies have shown that
biochemical properties of sickle cells, including membrane
characteristics, microvascular dynamics,4,54 tissue
hypoxia,55 and perturbation of vascular
endothelium,9 contribute to the pathogenesis of the
vaso-occlusive process. We hypothesized that the adherence of SS RBC to
endothelium may cause injury or activation of endothelium leading to
the surface expression of CAMs that can lead to the increased adherence
of PMN and monocytes to the damaged/activated endothelium through counter ligands, as well as to the additional adherence of SS reticulocytes through the 41 ligand to
VCAM-1.19,20,55 The adhesion of SS RBC and leukocytes to
injured/activated microvascular endothelium can adversely affect the
blood flow in the capillary. Such flow conditions will favor the direct
contact of SS RBC with neutrophils and concomitant attachment of PMN to
SS RBC, promoting the risk of vessel occlusion. We have recently
observed that such recognition of SS RBC by PMN leads to activation of
PMN,56 which can directly damage the vascular endothelium
through the generation and release of superoxide radicals. The injury
or activation of vascular endothelium in vivo has been observed in
patients with sickle cell anemia, wherein one observes the presence of
increased number of activated circulating endothelial cells in blood
samples.9,57
In this study, we examined the mechanism(s) by which the adherence of
SS RBC to cultured endothelial cells, proximal sites of stasis of SS
RBC in the vasculature, potentially augments the adherence of SS
reticulocytes, and promotes the adherence and migration of monocytes
across the endothelial cell monolayer. SS RBC, because of the presence
of unstable SS hemoglobin and spontaneous auto-oxidation of iron in
sickle heme13 generate excessive (two- to threefold)
amounts of ROS (O2., OH-, and
H2O2) (ROS) compared with control normal
RBC.58 We show here that HUVEC incubated with SS RBC
generates threefold increased amount of lipid peroxides (measured as
thiobarbituric acid reactive substances, TBARS). However, HUVEC
incubated with SS RBC plus vWf (E-CM) resulted in an additional twofold
TBARS formation. The formation of TBARS by HUVEC is inhibited by the
addition of free radical scavenging enzymes, superoxide dismutase, and
catalase. Moreover, blocking the adherence of SS RBC to endothelial
cells, reduced TBARS formation. We also measured the activation of the transcription factor NF-kB in HUVEC, another indicator of cellular oxidant stress.16 Our results show that the
adherence/contact of SS RBC to endothelial cells, mediated by multimers
of vWf, led to activation of NF-kB. This activation of NF-kB in HUVEC required the adhesion/contact of SS RBC and was also inhibited by
antioxidants. These results suggest that adherence/contact of SS RBC
mediated by multimers of vWf, localizes the oxidant stimulus generated
by SS RBC to the membrane of endothelial cells and generates
intracellular ROS in HUVEC to activate NF-kB. The intracellular
generation of ROS (oxidative stress) could occur as a result of
activation of NADH-oxidase associated with the plasma membrane of
endothelial cells.59
We show that incubation of HUVEC with SS RBC, in the presence of
multimers of vWf, results in a time-dependent increase in the surface
expression of a subset of CAMs, ICAM-1, E-selectin, and VCAM-1 without
affecting the surface expression of constitutively expressed ICAM-2.
The augmented expression of VCAM-1, examined in this study, was
observed on the adhesion of SS RBC to HUVEC but was not observed when
vWf (E-CM) alone was added to HUVEC. Our studies show that the cellular
oxidant stress generated in HUVEC induced by SS RBC adherence involves
a glutathione redox step because agents that restore intracellular GSH
levels prevent VCAM-1 expression, whereas an opposite effect occurs
when intracellular GSH levels are reduced by preincubation of HUVEC
with BSO, an inhibitor of GSH synthesis. Consequently, agents that
restore intracellular levels of GSH levels may prevent VCAM-1
expression and thus could have beneficial effects on microvascular
occlusion. Our results are consonant with previous studies wherein the
transcription of VCAM-1 in human vascular endothelial cells, induced by
cytokine IL-1, has also been shown to be regulated through an
antioxidant-sensitive mechanism.18 Moreover, our studies
show that the addition of free radical scavenger enzymes (superoxide
dismutase and catalase), abrogated SS RBC plus E-CM-induced VCAM-1
expression. Our studies indicate that ROS formed by SS RBC or SS RBC
ligand-receptor interaction (juxtacrine intercellular
signaling)60 and/or both result in the generation
of reactive oxygen intermediates (oxidant stress) intracellularly in
endothelial cells, which activate signaling pathways leading to the
activation of redox-sensitive transcription factor NF-kB16.
Our studies show here, for the first time, that interaction of SS RBC
with HUVEC, in the presence of vWf causes an increase in the migration
of monocyte-like HL-60 cells. We used vitamin D3-differentiated human HL-60 cells (monocyte-like HL-60
cells), because this cell line was previously used as a reliable model of in vivo monocyte function and transendothelial migration
studies.30,31 Similarly, the transendothelial migration of
human PBM was augmented by the cellular oxidant stress generated by the
adhesion of SS RBC to endothelial cells. The increase in the
transendothelial migration of both monocyte-like HL-60 cells and PBM is
inhibited by an antibody to PECAM-1, as has been previously observed
for the transendothelial migration of monocytes induced by lipoxygenase metabolites.37 We show here that the adherence of SS RBC to HUVEC, mediated by vWf, leads to PECAM-1 phosphorylation. Both the SS
RBC plus E-CM-induced phosphorylation of PECAM-1 and the transendothelial migration of monocyte-like HL-60 cells are inhibited by protein kinase C inhibitor, indicating the direct or causal involvement of PECAM-1 phosphorylation in mediating the flux of monocytes across the endothelial cell monolayer, as has been previously observed for the lipoxygenase metabolite-induced transendothelial migration of monocytes.37 The importance of PECAM-1
phosphorylation to transendothelial migration is further supported by
findings that agents such as the phosphatase inhibitor Calyculin A,
which increases the phosphorylation of PECAM-1, also augment monocyte migration over and above the level of SS RBC alone. As expected, antioxidants and modulators of intracellular GSH levels, which affect
SS RBC-induced cellular signals, also affect phosphorylation of PECAM-1
and, concomitant with it, the transendothelial migration of monocytes.
In conclusion, our studies show that interaction of oxygenated SS RBC
with HUVEC in the presence of multimers of vWf, generates reactive
oxygen species (oxidant stress) in endothelial cells. The
adherence/contact of SS RBC to endothelial cells helps in localizing
the oxidant stimulus to the cell surface to initiate cellular
signaling. As a consequence of the cellular signaling, activation of
transcription factor NF-kB occurs in HUVEC leading to the binding of
activated NF-kB to the consensus sites in the regulatory regions of DNA
for several genes including a subset of CAMs, thereby bringing about
the cell-surface expression of a subset of CAMs, ICAM-1,
E-selectin, and VCAM-1. The increased expression of CAMs in
response to oxidative stress is not unique to the adhesion of SS RBC to
endothelium, but can occur in response to endotoxins or hypoxia,
conditions eg, bacterial infection and tissue hypoxemia commonly
associated with SCD. The increased surface expression of VCAM-1 in
endothelial cells, as a result of the adherence of SS RBC, will promote
additional adherence of SS reticulocytes expressing the
41 ligand via VCAM-1. These CAMs also act
as receptors/adhesive agents for PMN and monocytes expressing the appropriate counter-receptors. The adhesion of PMN and monocytes at the
sites of thrombotic occlusion by SS RBC or in small venules in which
adhesion of SS RBC is maximal8 may cause further
obstruction in the flow of blood and promote vasoocclusion.
Furthermore, our studies show that the cellular oxidant stress,
generated by the adhesion of SS RBC to HUVEC brings about increased
migration of monocytes across the endothelial cell monolayer. In SCD,
it is possible that the accumulation of monocytes may participate in the stroke syndrome in which arterial narrowing and thrombosis resemble
the events observed in cerebrovascular atherosclerosis.
Thus the abnormal adherence of SS RBC to endothelial cells from large
vessels can generate enhanced oxidant stress leading to increased
adhesion and diapedesis of monocytes, as well as heightened adherence
of SS reticulocytes. Although the studies presented have been conducted
in endothelial cells derived from large vessels, we suspect that a
similar phenomenon may also occur in microvascular endothelium in which
the adhesion of SS RBC has been shown to be mediated by thrombospondin.
Such studies should provide an insight into the process of obstruction
or intermittent flow of blood observed in the microcirculation of
patients with SCD. Further studies should be performed to determine
whether density-fractionated less-dense (enriched in reticulocytes), or most-dense fraction of sickle blood, with and without deoxygenation, are involved in the generation of cellular oxidant stress and hence
activation of endothelium.
| |
ACKNOWLEDGMENT |
We deeply appreciate the assistance of Pat Corley, RN, for obtaining
the blood specimens from patients. The assistance of Anne Erwin, MS, in
technical editing of the manuscript is greatly appreciated.
| |
FOOTNOTES |
Submitted March 24, 1997;
accepted July 2, 1998.
Supported by National Institute of Health, Heart, Lung, and Blood
Institute Grant No. P60-HL-48484.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address correspondence to Vijay K. Kalra, PhD, University
of Southern California, School of Medicine, 2250 Alcazar St, CSC/1 GM
204, Los Angeles, CA 90033.
| |
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Abstract
Introduction