Activated Platelets Induce Monocyte Chemotactic Protein-1 Secretion and Surface

Expression of Intercellular Adhesion Molecule-1 on Endothelial Cells

Meinrad Gawaz, Franz-Josef Neumann, Timm Dickfeld, Werner Koch, Karl-Ludwig Laugwitz,
Helmut Adelsberger, Kirsten Langenbrink, Sharon Page, Dieter Neumeier, Albert Schmig and
Korbinian Brand
Circulation. 1998;98:1164-1171
doi: 10.1161/01.CIR.98.12.1164
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright 1998 American Heart Association, Inc. All rights reserved.
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Activated Platelets Induce Monocyte Chemotactic Protein-1

Secretion and Surface Expression of Intercellular Adhesion
Molecule-1 on Endothelial Cells
Meinrad Gawaz, MD; Franz-Josef Neumann, MD; Timm Dickfeld, MD; Werner Koch, PhD;
Karl-Ludwig Laugwitz, MD; Helmut Adelsberger, PhD; Kirsten Langenbrink, BSc; Sharon Page, PhD;
Dieter Neumeier, MD; Albert Schomig, MD; Korbinian Brand, MD
BackgroundPlatelet/endothelium interaction plays an important role in the pathophysiology of inflammation and
atherosclerosis. The role of platelets for monocyte chemotactic protein-1 (MCP-1) secretion and surface expression of
intercellular adhesion molecule-1 (ICAM-1) on endothelial cells has been assessed.
Methods and ResultsMonolayers of human umbilical vein endothelial cells were incubated with nonstimulated or
ADP-activated platelets for 6 hours, and secretion of MCP-1 and surface expression of ICAM-1 were determined by
ELISA and flow cytometry, respectively. In the presence of ADP-activated platelets, both MCP-1 secretion and ICAM-1
surface expression were significantly increased compared with nonstimulated platelets (P,0.02). Activation of the
transcription factor nuclear factor-kB (NF-kB) determined by electrophoretic mobility shift assay and kB-dependent
transcriptional activity was enhanced in the presence of activated platelets. In addition, ADP-activated platelets induced
MCP-1 and ICAM-1 promoter dependent transcription. Liposomal transfection of a double-stranded kB phosphorothioate oligonucleotide, but not of the mutated form, inhibited MCP-1 secretion and surface expression of ICAM-1 on
activated endothelium (P,0.05).
ConclusionsThe present study indicates that activated platelets modulate chemotactic (MCP-1) and adhesive (ICAM-1)
properties of endothelial cells via an NF-kB dependent mechanism. Platelet-induced activation of the NF-kB system
might contribute to early inflammatory events in atherogenesis. (Circulation. 1998;98:1164-1171.)
Key Words: platelets n endothelium n proteins n cell adhesion molecules n genes

latelet/endothelium interaction plays a central role in

hemostatic and inflammatory mechanisms within the
vessel wall.13 Dysregulation of platelet/endothelium interaction might contribute to the pathophysiology of a variety of
arterial vascular disorders, including inflammation, atherosclerosis, and restenosis.3 6 Several lines of evidence indicate
that platelet-derived substances released in close proximity to
the vessel wall induce a variety of genes.6

See p 1151
Monocyte chemotactic protein-1 (MCP-1) seems to be the
major chemotactic molecule generated within the vessel
wall.7,8 MCP-1 is chemotactic for monocytes but not for
neutrophils and is found in macrophage-rich areas of atherosclerotic lesions.6 8 Next to smooth muscle cells and macrophages, endothelium represents the major source of MCP-1 in
the vessel wall. 9,10 Intercellular adhesion molecule-1
(ICAM-1) is a major adhesion receptor expressed on the
endothelium and is involved in monocyte adhesion to endothelial cells.1115

Gene expression of MCP-1 and ICAM-1 is regulated by

transcription factors of the nuclear factor-kB (NF-kB)/Rel
family.16 18 Activation of NF-kB in vascular cells can be
induced by a variety of signals, including the inflammatory
cytokines interleukin-1 (IL-1) and tumor necrosis factor
(TNF), lipopolysaccharide, and oxidative and mechanical
stress.16 18 Activation of NF-kB has been shown to play a role
in atherosclerosis.19,20 Recently, platelets have been shown to
induce NF-kBregulated gene products in leukocytes.21,22
In the present study, we investigated the effect of activated
platelets on NF-kB activation and production of the NF-kB
regulated genes MCP-1 and ICAM-1 in endothelial cells. In
addition, we assessed the feasibility and efficacy of
decoy-kB oligonucleotides transfected into endothelial
cells to modulate NF-kBregulated gene expression.

Methods
Reagents, Oligonucleotides, and Plasmids
Single-stranded oligonucleotides were purchased as phosphorothioate diester from Perkin-Elmer. For fluorescence microscopy, oligo-

Received April 13, 1998; accepted May 20, 1998.

From the 1. Medizinische Klinik und Deutsches Herzzentrum and Institut fur klinische Chemie und Pathobiologie (S.P., D.N., K.B.), Klinikum rechts
der Isar der Technischen Universitat Munchen, Germany.
Correspondence to Meinrad Gawaz, MD, 1. Medizinische Klinikum rechts der Isar und Deutsches Herzzentrum, Technische Universitat Munchen,
Lazarettstrae 36, 80636 Munchen, Germany. E-mail gawaz@dhm.mhn.de
1998 American Heart Association, Inc.

1164
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Gawaz et al
nucleotides were used as fluorescein (FITC) conjugate. Recombinant
human (rh) IL-1b was purchased from Biermann. Monoclonal
antibody anti-CD54 (ICAM-1) (clone 84H10) was from Immunotech. Apyrase, ADP, acetylsalicylic acid, and prostaglandin E1 were
from Sigma. All other reagents were of the highest purity available.
The luciferase reporter plasmids (pGL2-neo: pGL2-basic, Promega) containing 559 bp of the human MCP-1 promoter region
(pGL2 neo-MCP-1) or 1297 bp of the ICAM-1 promoter region
(pGL2 neo-ICAM-1), respectively, were kindly provided by Dr
Nigel Mackman, Scripps Research Institute, La Jolla, Calif. Construction of the luciferase reporter plasmid pGL3 neo-MCP-1-kB
containing, as a tetramer, the second k B element (59GGGAATTTCC-39) of the upstream regulatory region of the human
MCP-1 gene as enhancer23 was performed as follows. Complementary 17-mer, 59-phosphorylated oligonucleotides (Perkin-Elmer) representing the MCP-1 gene kB element were annealed and ligated into
tandem repeats. The ligated kB elements were inserted into the Nhel
site within the polylinker of the luciferase reporter plasmid pGL3promoter (Promega) containing the SV40 promoter but no enhancer
element. A recombinant clone (pGL3 neo-MCP-1-kB) containing
the tetrameric kB insertion in an orientation opposite to that existing
in the MCP-1 gene was selected for cell culture transfections. Mutant
oligonucleotides containing 3 consecutive nucleotide changes in the
MCP-1 kB element that prevented binding of NF-kB23 were used to
construct plasmid pGL3 neo-MCP-1-mut-kB serving as control
plasmid.

Cell Culture
Primary human umbilical vein endothelial cells (HUVECs) were
harvested by use of collagenase (Worthington) digestion as described.24 26 Cells were pooled from 3 to 6 prepared umbilical veins
and were grown in 24-well culture plates (Nunc) in complete
medium composed of M199 (Sigma), 10% FCS, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 mg/L streptomycin and were
used as confluent monolayers after 1 to 2 passages.

Endotoxin Assay
To eliminate endotoxin contamination, all crystalloid solutions were
ultrafiltered (U2000, Gambro), and stock solutions of proteins were
decontaminated by polymyxin columns (Pierce). To exclude endotoxin contamination, all cell suspensions at the end of each experiment were evaluated by chromogenic limulus amoebocyte lysate
assay (Schulz).

Incubation of Endothelial Monolayers

With Platelets
Platelets were isolated from acid-citrate-dextroseanticoagulated
whole blood as described.25,26 Washed platelets were resuspended in
Tyrodes solutionHEPES buffer (mmol/L: HEPES 2.5, NaCl 150,
NaHCO3 12, KCl 2.5, MgCl2 1, CaCl2 2, and D-glucose 5.5, and 1
mg/mL BSA, pH 7.4) to obtain a final platelet count of 23108/mL.
In experiments with nonstimulated platelets, whole blood was
immediately mixed with an antiactivation cocktail that contained
1 mmol/L aspirin, 1 mmol/L theophylline, and 10 nmol/L prostaglandin E1. In experiments with activated platelets, ADP in a final
concentration of 50 mmol/L was added to the platelet suspension
isolated in the absence of antiplatelet substances. Platelet suspension
(200 mL) was added to 200 mL complete medium M199 (final
platelet count, 13108/mL) containing 0.5 mmol/L apyrase and was
transferred to wells of a 24-well culture plate covered with confluent
monolayers of endothelial cells. Incubation was performed at 37C
without agitation in culture condition atmosphere for 6 hours. In
some experiments, endothelial cells were incubated with membranes
(1 mg/mL) isolated from nonstimulated or ADP-activated platelets as
described.27 Platelet releasate was obtained by removal of the
supernatant after centrifugation of suspensions of nonstimulated or
ADP-activated platelets (23108/mL).

September 22, 1998

1165

Determination of MCP-1 Secretion and Surface

Expression of ICAM-1
The supernatant of cultured endothelial cells treated with platelets or
agonists for 6 hours was aspirated, centrifuged at 4000 rpm for 10
minutes, and stored at 280C. Concentrations of MCP-1 protein
were determined by ELISA (Biermann) with a detection limit of 5
pg/mL. Surface expression of ICAM-1 was determined by FITCconjugated anti-CD54 monoclonal antibody and flow cytometry as
described.28 After aspiration of the supernatant, endothelial monolayers were incubated with anti-CD54 (50 mg/mL) and the DNAstaining fluorochrome LDS 751 (Styry 18, Exciton Inc) for 20
minutes. Thereafter, cells were mechanically detached through
repetitive pipetting, and single-cell suspension was evaluated by
flow cytometry for ICAM-1 immunofluorescence in the forward
scatter versus LDS-fluorescence scatter plot.

Electrophoretic Mobility Shift Assay

Nuclear extracts from 13106 cells were prepared and analyzed as
described.19,29 The kB oligonucleotide (59-AGAGTGGGAATTTCCACTCA-39) derived from the promoter region of the human
MCP-1 gene was used as a probe29 and labeled by annealing of
complementary primers followed by primer extension with the
Klenow fragment of DNA polymerase I (Boehringer Mannheim) in
the presence of [a-32P]dCTP (.3000 Ci/mmol; DuPont) and deoxynucleoside triphosphates (Boehringer Mannheim). Nuclear extracts (5 mg protein) were incubated with radiolabeled DNA probes
(10 ng; 105 cpm) for 30 minutes at room temperature in 20 mL of
binding buffer [20 mmol/L Tris-HCl, pH 7.9, 50 mmol/L KCl,
1 mmol/L dithiothreitol, 0.5 mmol/L EDTA, 5% glycerol, 1 mg/mL
BSA, 0.2% NP-40, and 50 ng of poly(dI-dC)/mL]. Samples were run
in 0.253TBE buffer (103TBE5890 mmol/L Tris, 890 mmol/L
boric acid, and 20 mmol/L EDTA, pH 8.0) on nondenaturing 4%
polyacrylamide gels at 125 V. To control the nuclear protein content,
the nuclear extracts were incubated with a blunt-end double-stranded
Sp-1 oligonucleotide labeled with [g-32P]ATP (.5000 Ci/mmol;
DuPont) and T4 polynucleotide kinase (Boehringer Mannheim).
Gels were dried and analyzed by autoradiography.

Liposomal Transfection of
Double-Stranded Oligonucleotides
The oligonucleotide sequence 59-AGAGTGGGAATTTCCACTCA-39
was derived from the second kB site within the human MCP-1 promoter
region, which is responsible for the enhancer activity induced by
IL-1b.23 The mutated form 59-AGAGTGGTCCTTTCCACTCA-39
served as control. Equimolar amounts of complementary oligonucleotide strands were annealed in 0.5 mol/L NaCl for 90 minutes at 80C.
Complete annealing was verified by agarose gel electrophoresis. Endothelial monolayers were treated with a liposome (1:100 lipofectamin,
Gibco)/oligonucleotide (100 nmol/L)/complete medium M199 mixture
in a total volume of 500 mL per 24-well for 3 hours.

Cotransfection of Reporter Plasmids and

Luciferase Assay
Twenty-four hours before transfection, endothelial cells were split
and seeded in 6-well plates (50 000 cells/plate). The reporter plasmid
(pGL3 neo-MCP-1-kB, pGL3 neo-MCP-1-mut-kB, pGL2 neoICAM-1, or pGL2 neo-MCP-1) was transiently cotransfected with
Renilla-luciferase control plasmid pRL-TK (Promega) by incubation
of HUVEC monolayers with liposomes (1:100 lipofectamin) at a
plasmid concentration of 1 mg/mL for 3 hours. The firefly luciferase
activity reflects gene expression under control of kB (pGL3 neoMCP-1-kB), and the Renilla luciferase activity of the cotransfected
reporter provides an internal control by which each value within the
experimental set is normalized. After the endothelial monolayer was
washed with complete medium M199, cells were incubated with
platelets or agonists as indicated for 12 hours. After aspiration of the
supernatant, cells were lysed by addition of reporter lysis buffer
(Promega). Firefly and Renilla luciferase activity was determined

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Platelets, Endothelium, and NF-kB

Figure 1. Activated platelets induce secretion of MCP-1 and

surface expression of ICAM-1 on cultured endothelium. Plots
show effect of nonstimulated (1 mmol/L theophylline, 1 mmol/L
acetylsalicylic acid, 10 nmol/L prostaglandine E1) and ADP
50 mmol/Lstimulated platelets, membranes, and releasate incubated with HUVECs for 6 hours on secretion of MCP-1 (A) and
surface expression of ICAM-1 (B). Depicted are individual data
(V, v, h, f, l), means, and SDs of 5 independent experiments.

with the dual-luciferase reporter assay system (Promega) with the

help of a luminometer (LB953 Berthold).

Statistical Analysis
Differences between groups were tested by Students t test for
unpaired values. When the Kolmogorov-Smirnov test showed that
the data were not normally distributed, we chose the Mann-WhitneyWhite U test for comparison of 2 groups. A value of P,0.05 was
considered statistically significant. Results are presented as
mean6SD.

Results
Activated Platelets Induce MCP-1 Secretion and
Surface Expression of ICAM-1 on
Endothelial Cells
To evaluate the effect of platelets on secretion of MCP-1 and
surface expression of ICAM-1, HUVECs were incubated for
6 hours with nonstimulated (pretreated with PGE1, theophylline, aspirin) or ADP 50 mmol/Lactivated platelets in the
presence of apyrase (0.5 mmol/L). Activated platelets significantly increased secretion of MCP-1 ('40% of maximal
rhIL-1b 100 pg/mLinduced secretion) (Figure 1A). Secretion of MCP-1 was dependent on activation of platelets,
because significant lower MCP-1 values were found in the
presence of nonstimulated platelets (P,0.02) (Figure 1A).
Similarly, secretion of MCP-1 was enhanced in the presence
of supernatant of ADP-activated platelets compared with

Figure 2. Activated platelets induce activation of NF-kB in endothelial cells. Confluent monolayers of HUVECs were incubated for 1 hour with ADP 50 mmol/Lactivated platelets, nonstimulated (1 mmol/L theophylline, 1 mmol/L acetylsalicylic acid,
10 nmol/L prostaglandine E1) platelets, medium, or 100 pg/mL
rhIL-1b. Thereafter, activation of NF-kB was detected in nuclear
extracts by EMSA. In same extracts, binding of nuclear proteins
to an Sp-1 oligonucleotide was monitored.

supernatant derived from nonstimulated platelets (Figure 1A)

(P,0.01). No significant change in MCP-1 secretion was
found in the presence of membranes isolated from donor
ADP-activated platelets (Figure 1A). The platelet agonist
ADP 50 mmol/L in the absence of platelets did not induce
significant MCP-1 secretion (Figure 1A).
Similarly, ADP-activated platelets induced surface expression of ICAM-1 on HUVECs (Figure 1B). Surface expression
of ICAM-1 in the presence of ADP-activated platelets was
significantly enhanced compared with experiments with nonstimulated platelets (P,0.05) and reached '50% of maximal
inducible ICAM-1 surface expression (100 pg/mL of
rhIL-1b) (Figure 1B). Again, supernatant derived from ADPactivated platelets stimulated ICAM-1 surface expression
(P,0.02), whereas no effect was found for isolated membranes (Figure 1B).

Activated Platelets Induce Activation of NF-kB

and MCP-1 or ICAM-1
Promoter-Dependent Transcription
Gene expression of MCP-1 and ICAM-1 is regulated by
transcription factor NF-kB.16 18 Thus, we asked whether
activated platelets induce activation of the NF-kB system.
HUVECs were incubated with nonstimulated or ADPactivated platelets for 1 hour, and activation of NF-kB was
determined in nuclear extracts by electrophoretic mobility
shift assay (EMSA) and kB-dependent transcriptional activity. ADP-activated platelets induced significant activation of
NF-kB over baseline values (Figure 2) that was markedly
enhanced compared with experiments with nonstimulated

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Gawaz et al

September 22, 1998

1167

for 3 hours, and secretion of MCP-1 or surface expression of

ICAM-1 was determined after stimulation with rhIL-1b for a
further 6 hours. As control, a mutated form of MCP-1kB
oligonucleotide, mut-kB, was used that does not bind to the
activated NF-kB complex.23
As shown through fluorescence microscopy, 30% to 40%
of HUVECs were transfected with kB oligonucleotides conjugated with FITC (Figure 4). kB oligonucleotides but not the
mutated form accumulated in the nucleus of rhIL-1bactivated endothelial cells (Figure 4). In nuclear extracts of
rhIL-1 b -activated and k B oligonucleotidetransfected
HUVECs, NF-kB binding activity was significantly reduced
(Figure 5). No effect on NF-kB binding activity was found in
HUVECs transfected with mut-kB oligonucleotides (Figure
5). The binding of nuclear proteins to an oligonucleotide
comprising the Sp-1 consensus sequence was not affected in
cells transfected with kB oligonucleotides (Figure 5).
In the presence of rhIL-1b 100 pg/mL, secretion of MCP-1
and ICAM-1 expression was significantly reduced, by '30%
to 40%, in kB- compared with mut-kBtransfected cells
(P,0.05) (Figure 6). Liposomal transfection of 100 nmol/L
kB oligonucleotides did not change basal secretion of MCP-1
or ICAM-1 surface expression (Figure 6).
Platelet-induced secretion of MCP-1 and ICAM-1 surface
expression of endothelial monolayers tended to be reduced in
kB oligonucleotidetransfected cells, although not to a statistically significant level (Figure 6).
Figure 3. Induction of luciferase activity in endothelial cells
transfected with a reporter construct containing MCP-1 NF-kB
site. Confluent monolayers of HUVECs were cotransfected with
pGL3 neo-MCP-1-kB/pRL-TK or pGL3 neo-MCP-1-mut-kB/
pRL-TK (A) and with pGL2 neo-MCP-1, pGL2 neo-ICAM-1, or
pGL2 Basic, and pRL-TK, as indicated (B). Transfected cell
monolayers were then incubated with medium alone, nonstimulated (1 mmol/L theophylline, 1 mmol/L acetylsalicylic acid, 10
nmol/L prostaglandin E1), or ADP 50 mmol/Lactivated platelets
or rhIL-1b 100 pg/mL for 12 hours. Ratio of firefly/Renilla luciferase activity indicates specific activity of gene expression
under control of promoter sequence. Results (mean6SD) of 4
independent experiments are shown.

platelets (Figure 2). Binding of nuclear proteins to a doublestranded Sp-1 oligonucleotide showed that protein content
was equal in all tested nuclear extracts (Figure 2). In addition,
ADP-activated platelets induced kB-dependent transcriptional activity in pGL3 neo-MCP-1-kB transiently transfected
but not in pGL3 neo-MCP-1-mut-kB transfected HUVECs
(P,0.01) (Figure 3A). Similarly, MCP-1 or ICAM-1
promoter-dependent transcription was increased in pGL2
neo-MCP-1 or pGL2 neo-ICAM-1 transfected cells coincubated with ADP-activated platelets (P,0.01) (Figure 3B).

kB Oligonucleotides Inhibit MCP-1 Secretion and

ICAM-1 Surface Expression on
Activated Endothelium

Double-stranded kB oligonucleotides have been shown to

inhibit NF-kBregulated gene expression.30,31 Thus, we asked
whether transfer of kB oligonucleotides modulates endothelial MCP-1 secretion and surface expression of ICAM-1.
HUVECs were incubated with MCP-1kB oligonucleotides

Discussion
The major findings of the present study are (1) that ADPactivated platelets induce secretion of MCP-1 and surface
expression of ICAM-1 on cultured endothelial cells through
an NF-kBregulated mechanism and (2) that transfection of
kB oligonucleotides results in reduced NF-kB activation and
decreases production of MCP-1 and ICAM-1 in activated
endothelial cells.
The results indicate that activated platelets are able to
change the chemotactic and adhesive properties of endothelial cells. This mechanism may be an important early pathophysiological event in atherogenesis or restenosis. Inhibition
of NF-kB dependent gene induction might be a potential
target in treatment of thrombosis-induced inflammatory and
proliferative reactions in atherosclerotic arteries.

Platelet/Endothelium Interaction
Dysregulation of platelet/endothelium interaction has been
implicated in atherogenesis and restenosis.1 6 On activation,
platelets release a number of biologically highly active
compounds from their granules that exert significant reactions within endothelial cells.3236 Under pathophysiological
conditions, platelets might adhere to the intact endothelial
monolayer and might change the microenvironment of the
vessel wall.1,3,26,28
The present study shows that activated platelets induce
endothelial secretion of MCP-1, the major chemotactic molecule for monocytes generated within the vessel wall.7,8
Significantly less endothelial MCP-1 secretion was shown in
the presence of nonstimulated platelets, implying that
activation-dependent release of platelet-derived products

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1168

Platelets, Endothelium, and NF-kB

Figure 4. Activation-dependent transfer of kB oligonucleotides into nucleus of endothelial cells. Photomicrographs show uptake of fluorescein (FITC)-labeled kB oligonucleotides into nucleus of cultured HUVECs. Confluent monolayers of HUVECs were incubated with
lipofectamin1100 nmol/L double-stranded FITC-kB oligonucleotides for 3 hours. rhIL-1b 100 pg/mL was then added to cells for 2
hours. Cell monolayers were evaluated by laser scanning fluorescence microscopy. Evaluation of fluorescence micrographs shows that
in a significant number of cells ('30% to 40%), oligonucleotides accumulated in nucleus on IL-1-stimulation. Fluorescence micrographs of nonstimulated (B) and IL-1bactivated (A) cell monolayers. Phase contrast and corresponding fluorescence micrographs of
IL-1bactivated (C) and nonstimulated (D) endothelial cells. Note that kB oligonucleotides accumulate exclusively in nucleus of stimulated cells.

stimulates MCP-1 production. Similarly, surface expression

of ICAM-1 on endothelial cells was markedly enhanced in
the presence of ADP-activated platelets. As a counterreceptor
for leukocytes, ICAM-1 present on the luminal aspect of
endothelium is critical for leukocyte binding to the endothelium and for concomitant extravasation to sites of inflammation or injury within the vessel wall.1115 Thus, plateletinduced secretion of MCP-1 and expression of ICAM-1 on
endothelial cells might contribute significantly to entrapment

and migration of monocytes, an early step in atherogenesis

and restenosis.6

Role of Platelets and NF-kB in Atherosclerosis

The importance of platelets in development of atherosclerosis
has been well recognized in the past.6 Activated platelets
adhere to inflamed endothelium or to subendothelial structures at the site of vascular injury.1,5 Pharmacological inhibition of platelet activation prevents ischemic complications in

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Gawaz et al

September 22, 1998

1169

Figure 5. Inhibition of NF-kB activation by kB oligonucleotides.

Confluent monolayers of HUVECs were incubated with MCP1-kB or MCP-1-mut-kB oligonucleotides and rhIL-1b as
described in Methods. Activation of NF-kB was evaluated in
nuclear extracts by EMSA. As control, binding of nuclear proteins to oligonucleotide containing Sp-1 consensus sequence
was also examined in identical nuclear extracts.

patients with coronary heart disease.37 We22 and others21 have

shown that activated platelets induce activation of transcription factor NF-kB and related gene products in leukocytes.
NF-kB is a pleiotropic regulator of gene induction involved
in immune and inflammatory responses.16 18,38 NF-kB regulates a variety of genes coding for cytokines (MCP-1,
TNF)16 18,38 and adhesion receptors (ICAM-1, vascular cell
adhesion molecule-1, endothelial-leukocyte adhesion
molecule-1)38 that mediate endothelium-leukocyte adhesion.39
NF-kBregulated gene products such as IL-1b, MCP-1, TNF,
and ICAM-1 have been found in tissue specimens of atherosclerotic lesions.6 Recently, activated NF-kB was identified
in smooth muscle cells, macrophages, and endothelial cells of
human atherosclerotic tissue specimens,19 suggesting a pathophysiological role of NF-kB in inflammatory and proliferative processes in atherosclerosis.20
Our present findings that activated platelets induce (1)
MCP-1 secretion and ICAM-1 surface expression, (2) MCP-1
or ICAM-1 promoter dependent transcription, (3) NF-kB
activation as verified by gel-shift analysis and kB-dependent
transcriptional activity, and (4) reduction of MCP-1 and
ICAM-1 production in HUVECs transfected with kB oligonucleotides provide strong evidence that activated platelets
significantly modulate NF-kBregulated inflammatory
events in endothelial cells.
Recently, it was found that double-stranded decoy oligonucleotides containing the immunoglobulin kB sequence bind
activated NF-kB and specifically inhibit NF-kBdependent
transcription of gene products.30,31 In the cited studies, however,
high micromolar concentrations of oligonucleotides were required to inhibit NF-kBinduced gene expression.30,31 Relatively
high concentrations of oligonucleotides (millimolar range) have
been reported to exert significant nonspecific and toxic effects
on transfected cells.40,41 Thus, we used a liposomal transfection
protocol that allowed us to significantly reduce concentration of
oligonucleotides in the nanomolar range to achieve effective
inhibition of NF-kB dependent gene production. Microscopic
analysis revealed no significant change in phenotype in kB-

Figure 6. Effect of kB oligonucleotides on secretion of MCP-1

and surface expression of ICAM-1 on endothelial cells. Confluent monolayers of HUVECs were incubated with lipofectamin1100 nmol/L double-stranded kB or mut-kB oligonucleotides for 3 hours. Thereafter, medium M199, 100 pg/mL
rhIL-1b, nonstimulated, or ADP 50 mmol/Lactivated platelets
were added for 6 hours to cells as indicated. Secretion of
MCP-1 into supernatant was determined by ELISA (A), and surface expression of ICAM-1 was evaluated by flow cytometry (B).
Depicted are mean6SD of 4 independent experiments.

transfected cells. Moreover, we were able to show by laser

scanning fluorescence microscopy that kB, but not mut-kB,
oligonucleotides accumulate in the nucleus exclusively of activated cells. Thus, we conclude that the liposomal transfection of
kB oligonucleotides described here allows specific inhibition of
the NF-kB system without significant cellular toxicity.

Study Limitations
The present study focuses on the effects of activated platelets
on endothelial cells but does not address the nature of
mediators released from activated platelets that might trigger

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Platelets, Endothelium, and NF-kB

NF-kBinduced gene expression. NF-kB can be activated by

many diverse agents, such as cytokines.16 18 Activated platelets may generate (eg, thrombin) or release compounds from
their granules35 that might be potential activators of the
NF-kB system in endothelial cells. Platelets contain potent
chemokines like RANTES, which is released from a-granules
and has been shown to be involved in NF-kB dependent
MCP-1 production in human monocytes.21 Moreover, activated platelets have been shown to contain IL-1like activity.42 IL-1 has been shown to be a major inducer of NF-kB and
alters adhesive and chemotactic properties of vascular
endothelium.16 18,43
Transfection of endothelial cells is limited by low efficiency and depends on the size of the DNA molecule.41
Although we show in our studies that liposomal transfection
of low-molecular-weight kB oligonucleotides into endothelial cells is feasible, successful DNA transfection in vivo
doubtless requires higher transfection efficacy. By use of
virosomes, such as Sendai virus coated liposomes,44 it may
be possible to increase transfection efficacy significantly.
Moreover, other elements of the promoter region, such as
AP-1 or Sp-1, are involved in regulation of MCP-1 gene
expression.23 Thus, combined transfection of decoy oligonucleotides containing the sequence of various promoter regions
might result in enhanced inhibition of NF-kBregulated gene
expression.

Pathophysiological Considerations and

Therapeutic Implications
The present study introduces a novel aspect of how platelets
may contribute to early stages of atherosclerosis. Contact of
the endothelial monolayer with activated platelets (eg, at the
site of high shear conditions at vascular branches) might
induce MCP-1, which might in turn enhance monocyte
chemotaxis. Alteration of adhesive properties of endothelium
through upregulated ICAM-1 expression might further support monocyte adhesion and transmigration. Thus, inhibition
of platelet activation and accumulation at the vessel wall may
be an effective strategy in downregulating atherosclerotic
mechanisms.6
Activation of vascular cells and of circulating platelets at
the injured vascular site has been suggested to contribute to
restenosis.5,6,45,46 Balloon injury can induce activation of
NF-kB,47,48 and expression of early response genes, including
MCP-1, may contribute, at least initially, to the intimal
hyperplasia observed after balloon injury.6 In addition, expression of ICAM-1 in injured tissue has been suggested to
mediate cellular inflammation processes in restenosis.49 Thus,
the NF-kB system might be a potential pharmacological
target to interfere with chemotactic and adhesive mechanisms
within the vascular wall. The present study demonstrates that
transfer of kB oligonucleotides provides one mechanism by
which activation of NF-kB in vascular cells might be specifically inhibited. Recently, it was found that in vivo transfection of cis-element decoy against NF-kB binding sites prevents myocardial infarction in animals.50 It might be of
utmost clinical interest to know whether local delivery of
antiNF-kB compounds after coronary angioplasty can modulate restenotic mechanisms.

Acknowledgments
This study was supported in part by grants from the Deutsche
Forschungsgemeinschaft (Ga 381/21 to Dr Gawaz and Br 1026/31
and SFB469 to Dr Brand). The authors appreciate the excellent
technical assistance of Caroline Bogner and Tamara Eisele.

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