From the National Creative Research Initiatives
Center for Cardiac Regeneration, and the § Department of
Urology, Chonbuk National University School of Medicine,
Chonju, 560-180, Republic of Korea
Received for publication, October 24, 2000, and in revised form, November 29, 2000
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ABSTRACT |
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Vascular endothelial growth factor (VEGF)
induces adhesion molecules on endothelial cells during inflammation.
Here we examined the mechanisms underlying VEGF-stimulated expression
of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion
molecule 1 (VCAM-1), and E-selectin in human umbilical vein endothelial cells. VEGF (20 ng/ml) increased expression of ICAM-1, VCAM-1, and E-selectin mRNAs in a time-dependent manner. These
effects were significantly suppressed by Flk-1/kinase-insert domain
containing receptor (KDR) antagonist and by inhibitors of phospholipase
C, nuclear factor (NF)- The adhesive properties of the endothelium, the single-cell lining
of the cardiovascular system, are central to its physiology and
pathophysiology (1, 2). In health, the luminal endothelial cell surface
is a relatively nonadhesive and nonthrombogenic conduit for the
cellular and macromolecular constituents of the blood. The
extracellular matrix holds the basal endothelial cell surface in a well
arranged array. In certain diseases, various adhesive interactions
between endothelial cells and the constituents of the blood or
extracellular matrix are changed. These diseases include inflammation,
atherosclerosis, pathologic angiogenesis, and vascular injury. During
these disease processes, adhesion molecules are closely involved (1,
2). To date, four families of cell adhesion molecules have been
described: integrins, immunoglobulin superfamily members, cadherins,
and selectins. Members of each family have been detected in blood
vessels during angiogenesis and inflammation (3).
Vascular endothelial growth factor
(VEGF)1 is a potent
angiogenic factor whose activities include endothelial cell survival, proliferation, migration, and tube formation (4). VEGF also acts as a
proinflammatory cytokine by increasing endothelial permeability and
inducing adhesion molecules that bind leukocytes to endothelial cells
(5, 6). The distinct signal transduction mechanisms by which VEGF
induces survival, proliferation, migration, and nitric oxide (NO)
production in endothelial cells have been identified (7-17). However,
the signal transduction mechanisms leading to the induction of adhesion
molecules are little known.
In this study, we examined signal transduction mechanisms by which VEGF
induces adhesion molecules in human umbilical vein endothelial cells
(HUVECs). We demonstrate that VEGF-stimulated expression of
intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion
molecule-1 (VCAM-1), and E-selectin mRNAs is mediated mainly
through nuclear factor- Materials and Cell Culture--
Recombinant human vascular
endothelial growth factor165 (VEGF165),
placenta growth factor, and tumor necrosis factor- RNase Protection Assay (RPA) for Expression Analysis of ICAM-1,
VCAM-1, and E-selectin mRNA Transcripts--
The partial cDNAs
of human ICAM-1 (nucleotides 859-1225, GenBank accession
NM_000201), human VCAM-1 (nucleotides 538-816, GenBank
accession M60335), and human E-selectin (nucleotides 783-989, GenBank
accession M30640) were amplified by polymerase chain reaction and
subcloned into pBluescript II KS+ (Stratagene). After linearizing with
EcoRI, 32P-labeled antisense RNA probes were
synthesized by in vitro transcription using T7 polymerase
(Ambion Maxiscript kit) and gel purified. RPA was performed on total
RNAs using the Ambion RPA kit. An antisense RNA probe of human
cyclophilin (nucleotides 135-239, GenBank accession X52856) was used
as an internal control for RNA quantification.
Electrophoretic Gel Mobility Shift Analysis--
HUVECs were
incubated with the indicated agents for the indicated times and then
washed twice with phosphate-buffered saline. Nuclear proteins were
extracted as follows. The cells were scraped into buffer A (10 mmol/liter HEPES, 1.5 mmol/liter MgCl2, 10 mmol/liter KCl)
and centrifuged briefly. The cell pellet was resuspended in buffer A
plus 0.1% Nonidet P-40. After centrifugation at 14,000 rpm for 10 min,
the nuclear pellet was resuspended in buffer B (20 mmol/liter HEPES,
1.5 mmol/liter MgCl2, 0.42 mol/liter NaCl, 0.2 mmol/liter
EDTA, 25% glycerol, dithiothreitol, phenylmethylsulfonyl fluoride, and leupeptin). After centrifugation at 14,000 rpm for 10 min, the supernatant, which contains the nuclear proteins, was diluted
with buffer C (20 mmol/liter HEPES, 50 mmol/liter KCl, 0.2 mmol/liter
EDTA, 20% glycerol, dithiothreitol, phenylmethylsulfonyl fluoride, and
leupeptin). The protein concentrations were measured using Coomassie
Plus Protein Assay Reagent (Pierce). The binding reaction was a 30-min
incubation of 10 µg of nuclear protein with a 32P
end-labeled, double-stranded oligonucleotide containing the NF- Western Blot Analysis--
For Western blot analysis, samples
were mixed with sample buffer, boiled for 10 min, separated by
SDS-polyacrylamide gel electrophoresis under denaturing conditions, and
electroblotted to nitrocellulose membranes. The nitrocellulose
membranes were blocked by incubation in blocking buffer, incubated with
anti-VCAM-1 polyclonal antibody (Santa Cruz Biotechnology) or
anti-ICAM-1 monoclonal antibody (Santa Cruz Biotechnology), washed, and
incubated with horseradish peroxidase-conjugated secondary antibody.
Signals were visualized by chemiluminescent detection according to the
manufacturer's protocol (Amersham Pharmacia Biotech). The membrane was
reblotted with anti-actin antibody to verify equal loading of protein
in each lane.
NOS Activity--
HUVECs were cultured in 24-well plates. At
subconfluence, the medium was replaced with medium without phenol red
in the presence or absence of VEGF, L-NAME, and
D-NAME. After a 30-min incubation, this medium was
collected, and total NO was measured with a nitrate/nitrite colorimetric assay kit (Cayman Chemical) according to the
manufacturer's instruction. The measured value was normalized to the
number of HUVECs in the well from which the medium was collected.
Flow Cytometry Analysis--
HUVECs were stimulated with VEGF or
TNF- Adhesion Assay--
Leukocyte-endothelial adhesion was measured
by fluorescent labeling of leukocytes according to the methods of
Akeson and Woods (20). Peripheral blood leukocytes were
separated from heparinized peripheral blood of healthy volunteers by
Histopaque-1077 density gradient centrifugation. The cells were labeled
with Vybrant DiD (5 µM, 20 min, 37 °C, Molecular
Probes) in phenol red-free RPMI containing 5% fetal bovine serum. The
viability after labeling was always >95% as judged by trypan blue
exclusion. Cells were washed twice and resuspended in adhesion medium
(RPMI containing 2% fetal bovine serum and 20 mM HEPES).
The leukocytes were added (1.5 × 106/ml, 200 µl/well) to confluent monolayers of HUVECs that had been grown in
24-well plates and treated with various reagents and blocking
antibodies. The amount of labeled cells added was assessed by recording
the fluorescence signal (total signal) using a fluorescence spectrometer equipped with a microplate reader (Molecular Device). After incubation for 60 min at 37 °C, nonadherent cells were removed by washing four times with prewarmed RPMI. The fluorescent signal was
reassessed by the microplate reader (adherent signal). The percentage
of leukocytes adhering to HUVECs was calculated by the formula: % adherence = (adherent signal/total signal) × 100.
Densitometric Analyses and Statistics--
All signals were
visualized and analyzed by densitometric scanning (LAS-1000, Fuji Film,
Tokyo). Data are expressed as mean ± S.D. Statistical
significance was tested using one-way analysis of variance followed by
the Student-Newman-Keuls test. Statistical significance was set at
p < 0.05.
VEGF Increased Expression of ICAM-1, VCAM-1, and E-selectin
mRNAs in HUVECs--
We developed a method of RPA by which we can
detect the mRNA levels of ICAM-1, VCAM-1, E-selectin, and
cyclophilin simultaneously. The addition of 20 ng/ml VEGF increased the
expression of ICAM-1, VCAM-1, and E-selectin mRNAs as early as
2 h and produced a maximal effect at 4 h (Fig. 1,
A and B). The
higher expression levels declined thereafter, but the level of ICAM-1
and VCAM-1 continued to be higher than control for up to 8 h. The
maximum mean increases in ICAM-1, VCAM-1, and E-selectin were 5.2-, 9.8-, and 2.2-fold, respectively (Fig. 1B). As a positive
control, the addition of 1 ng/ml TNF- Inhibitors Changed VEGF-stimulated Expression of ICAM-1, VCAM-1,
and E-selectin mRNAs--
To examine the receptor/second messenger
mechanisms leading to induction of adhesion molecules by VEGF, a
receptor antagonist and various intracellular kinase inhibitors were
added to 20 ng/ml VEGF-treated HUVECs. A specific KDR antagonist
(SU1498, 20 µM) completely inhibited VEGF-stimulated
expression of the adhesion molecule mRNAs (Fig. 2,
A and B). Placenta
growth factor is known to be a specific Flt-1 ligand (21). 10-500
ng/ml placenta growth factor did not produce any effect on expression
of the adhesion molecules (data not shown). MEK 1/2 inhibitor (PD98059,
50 µM) did not produce any changes, whereas PLC inhibitor
(U73122, 1 µM), NF- VEGF-induced Expression of ICAM-1, VCAM-1, and E-selectin Was
Correlated with NF- VEGF-induced Expression of ICAM-1, VCAM-1, and E-selectin Was
Independent of NO but Was Suppressed by Activation of PI
3'-Kinase--
The addition of NOS inhibitor L-NAME (3 mM), but not its inactive D isomer D-NAME (3 mM), markedly suppressed basal and VEGF-stimulated NOS
activity (Table I). Under these
conditions, basal and VEGF-stimulated expression of ICAM-1, VCAM-1, and
E-selectin was not changed (Fig. 4,
A and B). The
addition of PI 3'-kinase inhibitor wortmannin (30 nM)
markedly suppressed basal and VEGF-stimulated NOS activity (Table I).
Under these conditions, both the basal and the VEGF-stimulated expression of the three adhesion molecules was enhanced (Fig. 4,
A and B). Inhibition of PI 3'-kinase activity
with 100 nM LY294002 produced a similar effect (data not
shown). Alternatively, activation of PI 3'-kinase with 50 microunits of
insulin suppressed basal and VEGF-stimulated expression of the three
adhesion molecules (Fig. 4, A and B).
VEGF Increased the Protein Levels of ICAM-1 and VCAM-1, and
Inhibitors Changed This Effect--
Because ICAM-1 and VCAM-1 showed
the strongest response to VEGF among the three molecules we examined,
we looked further at the protein levels of ICAM-1 and VCAM-1 in HUVECs
treated with VEGF. The addition of 20 ng/ml VEGF increased protein
levels of ICAM-1 as early as 2 h and produced a maximal effect at
6-12 h (Fig. 5A, upper
panels). These effects declined but continued to be higher than
control levels up to 18 h. The maximum mean increase in ICAM-1 was
8.7-fold. The addition of 20 ng/ml VEGF increased protein levels of
VCAM-1 as early as 2 h and produced a maximal effect at 4-6 h
(Fig. 5A, lower panels). These effects declined
but continued to be higher than control levels up to 12 h. The
maximum mean increase in VCAM-1 was 6.5-fold. 1 ng/ml TNF- VEGF-induced Leukocyte Adhesiveness Was Correlated with
VEGF-induced Expression of Adhesion Molecules--
Because the
induction of adhesion molecules in endothelial cells induces leukocyte
adhesiveness, we examined whether VEGF induces leukocyte adhesion to
HUVECs. Accordingly, the addition of 20 ng/ml VEGF produced ~3.1-fold
increases in leukocyte adhesiveness after 8 h compared with the
addition of control buffer (Fig. 6, A and B). Tje
Flk-1/KDR antagonist (SU1498, 20 µM), MEK 1/2 inhibitor (PD98059, 50 µM) PLC inhibitor (U73122, 1 µM), NF- VEGF exerts its action by binding to two cell surface receptors,
Flk-1/KDR and Flt-1 (25). In Flk-1/KDR null mutant mice, development of
endothelial and hematopoietic cells is impaired (26). Flt-1 null mutant
mice have an apparent overgrowth of endothelial cells, accompanied by
blood vessel disorganization (27). The distinct phenotypes of the
Flk-1/KDR and Flt-1 knockout animals show that these receptors have
different biological functions. Therefore, it is likely that the
two VEGF receptors signal through different transduction pathways. Our
results indicate that a specific Flk-1/KDR antagonist completely
blocked VEGF-induced expression of ICAM-1, VCAM-1, and E-selectin and
blocked VEGF-induced NF-B, sphingosine kinase, and protein kinase C,
but they were not affected by inhibitors of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1/2 or
nitric-oxide synthase. Unexpectedly, the phosphatidylinositol (PI) 3'-kinase inhibitor wortmannin enhanced both basal and
VEGF-stimulated adhesion molecule expression, whereas insulin, a PI
3'-kinase activator, suppressed both basal and VEGF-stimulated
expression. Gel shift analysis revealed that VEGF stimulated NF-
B
activity. This effect was inhibited by phospholipase C, NF-
B, or
protein kinase C inhibitor. VEGF increased VCAM-1 and ICAM-1 protein
levels and increased leukocyte adhesiveness in a
NF-
B-dependent manner. These results suggest that
VEGF-stimulated expression of ICAM-1, VCAM-1, and E-selectin mRNAs
was mainly through NF-
B activation with PI 3'-kinase-mediated
suppression, but was independent of nitric oxide and MEK. Thus, VEGF
simultaneously activates two signal transduction pathways that have
opposite functions in the induction of adhesion molecule expression.
The existence of parallel inverse signaling implies that the induction
of adhesion molecule expression by VEGF is very finely regulated.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B (NF-
B) activation with
phosphatidylinositol (PI) 3'-kinase-mediated suppression but is
independent of NO and mitogen-activated protein/extracellular
signal-regulated kinase (ERK) kinase (MEK).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TNF-
) were purchased from R&D Systems. Flk-1/kinase-insert domain containing receptor (KDR) antagonist SU1498, nitric-oxide synthase (NOS) inhibitor, NG-nitro-L-arginine
methyl ester (L-NAME) and its inactive isomer, NG-nitro-D-arginine methyl ester
(D-NAME) were purchased from Calbiochem. PI 3'-kinase
inhibitors wortmannin and LY294002 were purchased from RBI, Inc. MEK
1/2 inhibitor PD98059 was obtained from New England Biolabs. PLC
inhibitor U73122 was purchased from Biomol Research Laboratory Inc.
Sphingosine kinase inhibitor
N,N-dimethylsphingosine (DMS) was purchased from
ICN Pharmaceuticals. NF-
B inhibitor pyrrolidine dithiocarbamate
(PDTC) and protein kinase C (PKC) inhibitor chelerythrine chloride were
purchased from Sigma. Media and sera were obtained from Life
Technology, Inc. Functional blocking antibodies for ICAM-1 (clone
P2A4), VCAM-1 (clone P3C4), and E-selectin (clone P2H3) were purchased
from Chemicon, Inc. Most other biochemical reagents were purchased from
Sigma, unless otherwise specified. HUVECs were prepared from human
umbilical cords by collagenase digestion and maintained as described
previously (18).
B
binding site on the human VCAM-1 promoter (5'-CCTTGAAGGGATTTCCCTCC-3') (19). Cold competition controls were performed by preincubating the
nuclear proteins with unlabeled 20-fold molar excess of the NF-
B
double-stranded oligonucleotide for 20 min before the addition of the
32P-labeled oligonucleotide. As a negative control, a cold
competition was also performed with an irrelevant octamer transcription
factor (Oct)-1 oligonucleotide (5'-TAGAGGATCCATGCAAATGCCGGGTACC-3'). In
antibody supershift experiments, nuclear extracts were incubated for 30 min at room temperature with 2 µg of polyclonal rabbit antibodies to
human NF-
B proteins (p65, p50, p52, RelB, and c-Rel; Santa Cruz
Biotechnology) and then incubated with labeled oligonucleotide. The
mixtures were resolved on native 5% polyacrylamide gels, which were
dried and autoradiographed.
for 8 h. Then, cells were washed twice with cold
phosphate-buffered saline, removed by careful trypsinization, and
washed again with Ca2+/Mg2+-free
phosphate-buffered saline before incubating with 20% fetal bovine
serum for 30 min. After two washes, cells were incubated with an
antibody against human VCAM-1 or ICAM-1 (Santa Cruz Biotechnology) for
1 h at 4 °C. Cells were then washed twice with
phosphate-buffered saline/fetal bovine serum and incubated for 1 h
at 4 °C with a fluorescein isothiocyanate-conjugated secondary
antibody. Cells were then fixed with 2% paraformaldehyde and analyzed
by flow cytometry in a fluorescence-activated cell sorter
cytofluorometer (Becton Dickinson). The results were gated for mean
fluorescence intensity above the fluorescence produced by the secondary
antibody alone.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
for 1 h also markedly
increased the expression of ICAM-1, VCAM-1, and E-selectin (Fig.
1A).
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Fig. 1.
RPA of adhesion molecule mRNAs in
VEGF-stimulated HUVECs. Panel A, HUVECs were incubated
with 20 ng/ml VEGF165 for the indicated times. Total RNAs
(10 µg) were subjected to multiplex RPA probed with antisense ICAM-1,
antisense VCAM-1, and antisense E-selectin RNA probes. Equivalent
loading was confirmed by probing the same reactions with an antisense
cyclophilin RNA probe (105 base pairs). To clarify the identity of the
bands, ICAM-1 (I), VCAM-1 (V), and E-selectin
(E) probes were applied individually to the total RNA from
HUVECs treated with VEGF for 4 h to reveal protected bands of 367, 279, and 187 base pairs, respectively. The positive control was total
RNA (2 µg) from HUVECs that had been incubated with 1 ng/ml TNF-
(T) for 1 h and subjected to the same assay conditions.
Panel B, densitometric analyses are presented as the
relative ratio of ICAM-1, VCAM-1, or E-selectin mRNA to cyclophilin
mRNA. The relative ratio measured at time 0 h is arbitrarily
presented as 1. Results were similar in three independent experiments.
Bars represent the mean ± S.D. from three experiments.
*, p < 0.05 versus time 0.
B inhibitor (PDTC, 50 µg/ml),
sphingosine kinase inhibitor (DMS, 5 µM), and PKC
inhibitor (chelerythrine chloride, 5 µM) suppressed
VEGF-induced expression of ICAM-1, VCAM-1, and E-selectin (Fig. 2,
A and B). Unexpectedly, the PI 3'-kinase
inhibitor (wortmannin, 30 nM) enhanced VEGF-induced
expression of the three adhesion molecule mRNAs (Fig. 2,
A and B). These results suggested that VEGF-stimulated expression of ICAM-1, VCAM-1, and E-selectin mRNAs may be mediated mainly through activation of PLC
and NF-
B, along with PI 3'-kinase-mediated suppression. The process appears to be
independent of the MEK/ERK pathway.
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Fig. 2.
RPA of adhesion molecule mRNAs in
VEGF-stimulated HUVECs cotreated with inhibitors. Panel
A, HUVECs were incubated with 20 ng/ml VEGF165 for
4 h in the presence of control buffer (CB), 20 µM SU1498 (SU), 30 nM wortmannin
(WT), 50 µM PD98059 (PD), 1 µM U73122 (U7), 50 µg/ml PDTC
(PT), 5 µM DMS (DM), or 5 µM chelerythrine chloride (CC). Total RNAs (10 µg) isolated from the cells were subjected to RPA as described in
Fig. 1. Panel B, densitometric analyses are presented as the
relative ratio of ICAM-1, VCAM-1, or E-selectin mRNA to cyclophilin
mRNA. The relative ratio measured after the addition of control
buffer is arbitrarily presented as 1. Results were similar in three
independent experiments. Bars represent the mean ± S.D. from three experiments. *, p < 0.05 versus control buffer plus 20 ng/ml VEGF.
B Activity--
Because the expression of
adhesion molecules is mainly regulated by NF-
B (22-24), we examined
NF-
B activity in HUVECs treated with VEGF in the absence or presence
of various intercellular kinase inhibitors. The addition of 20 ng/ml
VEGF increased NF-
B activity as early as 0.5 h and produced a
maximal effect at 1 h (Fig. 3,
A and D). These
effects declined but continued to be higher than control levels up to
6 h. The maximum mean increase in NF-
B activity was 5.8-fold.
As a positive control, the addition of 1 ng/ml TNF-
for 1 h
increased NF-
B activity. A 20-fold molar excess of unlabeled
competitor almost completely blocked the NF-
B binding site, whereas
the irrelevant oligonucleotide, Oct-1, did not produce any effect on
the binding site. The MEK 1/2 inhibitor (PD98059, 50 µM)
did not produce any change in VEGF-induced NF-
B activity, whereas
KDR antagonist (SU1498, 20 µM), PLC inhibitor (U73122, 1 µM), NF-
B inhibitor (PDTC, 50 µg/ml), sphingosine kinase inhibitor (DMS, 5 µM), and PKC inhibitor
(chelerythrine chloride, 5 µM) suppressed VEGF-induced
NF-
B activity (Fig. 3, B and D). PI 3'-kinase
inhibitor (wortmannin, 30 nM) enhanced VEGF-induced NF-
B
activity (Fig. 3, B and D). Overall, VEGF-induced NF-
B activity was correlated with the expression of adhesion molecules by VEGF. We performed supershift experiments using specific antibodies to p65 (RelA), RelB, c-Rel, p50, and p52 to reveal the
identities of the proteins in the VEGF-induced NF-
B binding complex.
Incubation with antibody to p65 or p50, but not with antibody to RelB,
c-Rel, or p52, shifted the protein·DNA complexes (Fig.
3C). These data indicate that VEGF activates NF-
B in the form of a p65·p50 heterodimer in HUVECs.
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Fig. 3.
Activation of NF- B
binding by VEGF and TNF-
in HUVECs.
Panel A, HUVECs were incubated with 20 ng/ml
VEGF165 and 1 ng/ml TNF-
for the indicated hours.
Nuclear extracts were incubated in the absence or presence of a 20-fold
molar excess of cold human VCAM-1 NF-
B oligonucleotide (lane
CE) or cold Oct-1 oligonucleotide (lane O1) before the
addition of radiolabeled human VCAM-1 NF-
B oligonucleotide.
Panel B, HUVECs were incubated with 20 ng/ml
VEGF165 for 1.5 h in the presence of control buffer
(CB) or inhibitors, as described in Fig. 2. NF-
B binding
activities in nuclear extracts were assayed by gel shift assay.
Panel C, nuclear extracts from HUVECs treated with 20 ng/ml
VEGF165 for 1.5 h were incubated with control buffer,
antibody specific for p65 (65), RelB (Rb), c-Rel
(cR), p50 (50), or p52 (52) followed
by the addition of radiolabeled human VCAM-1 NF-
B oligonucleotide.
p65/p50, p65·p50 heterodimer; SS, supershift
band; NS, nonspecific bands. Panel D,
densitometric analyses are presented as the relative ratio of NF-
B
activity. The relative ratio measured at time 0 h or from the
addition of control buffer is arbitrarily presented as 1. Results were
similar in three independent experiments. Bars represent the
mean ± S.D. from three experiments. *, p < 0.05 versus time 0 or control buffer. #,
p < 0.05 versus control buffer plus 20 ng/ml VEGF.
Effect of VEGF on production of NO metabolites in HUVECs
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Fig. 4.
RPA of adhesion molecule mRNAs in
VEGF-stimulated HUVECs cotreated with NOS inhibitor, PI 3'-kinase
inhibitor, and insulin. Panel A, HUVECs were incubated
with 20 ng/ml VEGF165 for 4 h in the absence or
presence of 3 mM L-NAME, 3 mM
D-NAME, 50 microunits of insulin, or 30 nM
wortmannin (WT). Total RNAs (10 µg) isolated from the
cells were subjected to RPA as described in Fig. 1. Panel B,
densitometric analyses are presented as the relative ratio of ICAM-1,
VCAM-1, or E-selectin mRNA to cyclophilin mRNA. The relative
ratio measured after the addition of control buffer is arbitrarily
presented as 1. Results were similar in three independent experiments.
Bars represent the mean ± S.D. from three experiments.
*, p < 0.05 versus control buffer.
#, p < 0.05 versus control
buffer plus 20 ng/ml VEGF.
, used as
a positive control, increased protein levels of ICAM-1 and VCAM-1
markedly at 6 h. The effect of various inhibitors on VEGF-induced
protein levels of ICAM-1 and VCAM-1 was similar to their effect on
VEGF-induced mRNA levels. MEK 1/2 inhibitor did not produce any
changes, whereas inhibitors of PLC, NF-
B, and PKC suppressed
VEGF-induced protein levels of ICAM-1 and VCAM-1 (Fig. 5B).
PI 3'-kinase inhibitor enhanced VEGF-induced protein levels of ICAM-1
and VCAM-1 (Fig. 5B). Using flow cytometry, we also
confirmed that the protein levels of VCAM-1 and ICAM-1 on the
endothelial cell surface increased after treatment of 20 ng/ml VEGF for
8 h (data not shown).
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Fig. 5.
Western blot analyses of VCAM-1 and ICAM-1
protein in VEGF-stimulated HUVECs. Panel A, HUVECs were
incubated for the indicated times with 20 ng/ml VEGF165.
Each lane contains 50 µg of cellular protein. Panel
B, HUVECs were incubated for 6 h with 20 ng/ml
VEGF165 in the presence of control buffer (CB)
or inhibitors, as described in Fig. 2. The Western blot was probed with
an anti-VCAM-1 antibody or an anti-ICAM-1 antibody (upper
panels) and reprobed with an anti-actin antibody (lower
panels) to verify equal loading of protein in each
lane. Molecular mass markers shown were used to
estimate masses. Results were similar in three independent
experiments.
B inhibitor (PDTC, 50 µg/ml), and PKC
inhibitor (chelerythrine chloride, 5 µM) all suppressed
basal and VEGF-induced leukocyte adhesiveness (Fig. 6, A and
B). However, the PI 3-kinase inhibitor (wortmannin, 30 nM) produced a profoundly variable effect on VEGF-induced
leukocyte adhesiveness (Fig. 6, A and B).
Although functional blocking antibodies to ICAM-1, VCAM-1, and
E-selectin did not produce significant changes in basal leukocyte
adhesiveness, they reduced VEGF-induced leukocyte adhesiveness (Fig. 6,
A and B). A triple combination of these
antibodies produced marked suppression of VEGF-induced leukocyte
adhesiveness (Fig. 6, A and B).
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Fig. 6.
Leukocyte adhesiveness in VEGF-stimulated
HUVECs. Panel A, representative phase-contrast
photographs of leukocytes adhesion to HUVECs. Note that there are more
leukocytes in VEGF-treated HUVECs than in control buffer-treated
HUVECs. This effect is changed by cotreatment with various reagents.
Bar, 50 µm. Panel B, quantification of the
leukocyte adhesion to HUVECs. Leukocytes were labeled with the Vybrant
DiD and added to confluent monolayers of HUVECs that were treated with
and without 20 ng/ml VEGF165 for 8 h and were also
treated with control buffer (CB), inhibitors (as described
in Fig. 2), and 10 µg/ml anti-ICAM-1 antibody (IA), 10 µg/ml anti-VCAM-1 antibody (VA), 10 µg/ml
anti-E-selectin antibody (SA), or a triple combination of
these antibodies (TA). Then, leukocyte adhesion was measured
as described under "Experimental Procedures." The percentage of
leukocytes adhering to HUVECs was calculated by the following formula:
% adherence = (adherent signal/total signal) × 100. Bars represent the mean ± S.D. from five experiments.
*, p < 0.05 versus control buffer.
#, p < 0.05 versus control
buffer plus VEGF (20 ng/ml).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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B activity. However, a specific Flt-1
ligand, placenta growth factor, did not produce any effect on the
expression of the adhesion molecules. Thus, VEGF-induced expression of
adhesion proteins in endothelial cells occurs through VEGF binding to
the Flk-1/KDR receptor, but not to the Flt-1 receptor (Fig.
7).
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Fig. 7.
Second messenger pathways in VEGF-stimulated
expression of ICAM-1, VCAM-1, and E-selectin in endothelial cells.
After the binding of VEGF to the VEGF receptor (VEGFR,
Flk-1/KDR), PLC , PI 3'-kinase, and MEK/ERK were activated. NF-
B
was activated through activation of PLC
-sphingosine
kinase-PKC cascade. This cascade is the main pathway that
induces the transcription of ICAM-1, VCAM-1, and E-selectin.
Unidentified pathways through activation PI 3'-kinase or PI
3'-kinase/Akt may suppress the transcription of ICAM-1, VCAM-1, and
E-selectin. The induction of NO and the activation of MEK/ERK by VEGF
may not be involved in the regulation of ICAM-1, VCAM-1, and E-selectin
expression.
Upon activation of the Flk-1/KDR receptor in endothelial cells, three
major second messenger pathways elicit cell proliferation, migration,
survival, and NO production (7-17). These pathways are: PI
3'-kinase/serine-threonine protein kinase/Akt cascade, the tyrosine
phosphorylation of PLC, and the MEK/ERK cascade (7-17).
VEGF-induced activation of PI 3'-kinase results in phosphorylation of
Akt in endothelial cells (9, 14, 15, 17). This phosphorylated Akt
results in phosphorylation of Bad and endothelial NOS, resulting in cell survival, NO production and migration (9, 14, 15, 17).
Pharmacological inhibition of PI 3'-kinase with wortmannin and LY294002
completely inhibited these VEGF-induced cellular effects in endothelial
cells (9, 14, 15). Consistent with previous reports (14, 15), we found
that pharmacological inhibition of PI 3'-kinase with wortmannin and
LY294002 inhibited basal and VEGF-induced NO production. However,
unexpectedly, our data indicated that under PI 3'-kinase inhibition,
the basal expression levels of ICAM-1, VCAM-1, and E-selectin mRNA
were higher. Furthermore, under PI 3'-kinase inhibition, VEGF-induced
expression levels were higher. Alternatively, insulin, an activator of
PI 3'-kinase, decreased basal and VEGF-induced ICAM-1, VCAM-1, and
E-selectin expression. These data strongly suggest that PI 3'-kinase
could be an intracellular suppressor for the expression of ICAM-1,
VCAM-1, and E-selectin through yet unidentified signaling pathways
(Fig. 7). To our knowledge, these results are the first to demonstrate an additional role of PI 3'-kinase in suppressing the expression of
adhesion molecules. Thus, selective activation of PI 3'-kinase suppresses the induction of ICAM-1, VCAM-1, and E-selectin in endothelial cells. Therefore, PI 3'-kinase may decrease inflammatory responses, and a selective activator of PI 3'-kinase could be considered as a therapeutic agent for reducing a VEGF-induced inflammation in endothelial cells.
A previous report indicated that VEGF induces expression of monocyte
chemoattractant protein-1, a chemokine that is involved in recruiting
leukocytes to sites of inflammation, mainly through activation of
NF-B and AP-1 in retinal endothelial cells (28). The MEK/ERK system
is not involved in VEGF-induced activation of NF-
B, but it is
involved in VEGF-induced activation of AP-1 in the VEGF-induced
expression of monocyte chemoattractant protein-1 (28). VEGF/Flk-1/KDR
binding triggers a signaling cascade that results in tyrosine
phosphorylation of PLC
(7, 11, 13). Phosphorylation of PLC
increases intracellular levels of inositol 1,4,5-triphosphate and
diacylglycerol. Inositol 1,4,5-triphosphate elevates intracellular
calcium through an efflux from the endoplasmic reticulum. The increase
in intracellular calcium also can activate sphingosine kinase to
produce sphingosine 1-phosphate (29). In turn, the increase in
intracellular sphingosine 1-phosphate activates PKC. In addition,
activated PLC
also activates PKC by increasing diacylglycerol.
Activated PKC is known to be a strong activator of NF-
B (30). There
is ample evidence that activation of NF-
B stimulates expression of
ICAM-1, VCAM-1, and E-selectin mRNAs in endothelial cells (22-24).
Thus, VEGF-induced activation of PLC
and PKC is an essential step
for induction of these adhesion molecule mRNAs in endothelial
cells, and the induction occurs through NF-
B activation (Fig. 7).
Upon activation of the Flk-1/KDR receptor, increased intracellular
calcium and the activation of PKC or Akt result in activation of
endothelial NOS and thus increased production of NO (11,
14-16). Although previous reports (31, 32) indicate that NO modulates
the protein levels of VCAM-1 or ICAM-1 differently in endothelial
cells, our results indicated that NO is not involved in VEGF-induced
mRNA expression of ICAM-1, VCAM-1, and E-selectin. Thus, NO may
modulate expression of ICAM-1 and VCAM-1 at the translational level but
not at the transcriptional level. Upon activation of Flk-1/KDR, MEK/ERK
signal messenger transduction pathways are activated and lead to
cellular proliferation (10, 12, 13). Pharmacological inhibition of
MEK/ERK pathways with PD98059 did not have any effect on the expression
of ICAM-1, VCAM-1, and E-selectin mRNAs. Thus, NO and the MEK/ERK
system are not involved in VEGF-stimulated expression of adhesion
molecules (Fig. 7).
Induction of adhesion molecules is an initial step in inflammation mediated by leukocyte adhesion. Previous reports have shown that VEGF did not affect the expression of ICAM-1 and VCAM-1 in human dermal microvascular endothelial cells (33), whereas VEGF increased the expression of ICAM-1, but not VCAM-1 and E-selectin, in vivo in retinal capillary endothelial cells (34). Our results indicate that VEGF increased the expression of ICAM-1, VCAM-1, and E-selectin in HUVECs. Endothelial cells from different areas have different characteristics and different responses to growth factors (35, 36). Thus, the expression of adhesion molecules in response to VEGF may be different between large vessel endothelial cells and microvascular endothelial cells. Our results clearly indicated that VEGF increased VCAM-1 and ICAM-1 protein in a time-dependent manner. Accordingly, VEGF increased leukocyte adhesion in endothelial cells. Leukocyte adhesion to endothelial cells requires multiple cellular steps and intracellular second messenger signaling systems. Although the kinase inhibitors used in this study could be involved in multiple downstream effects in the response of the endothelial cells to VEGF, there were close relationships between induction of adhesion molecules and leukocyte adhesiveness. In addition, a combination of specific blocking antibodies to ICAM-1, VCAM-1, and E-selectin significantly inhibited VEGF-induced leukocyte adhesiveness to endothelial cells. Thus, VEGF-induced adhesion molecules in endothelial cells is closely involved in VEGF-induced leukocyte adhesiveness.
In summary, the present results explain how VEGF stimulates the
expression of adhesion molecules in HUVECs. Our results show that
VEGF-stimulated expression of ICAM-1, VCAM-1, and E-selectin mRNAs
was mainly through activation of PLC and NF-
B. The induction was
suppressed by a PI 3'-kinase-mediated pathway but was independent of NO
and MEK/ERK. Thus, VEGF simultaneously activates two signal tranduction
pathways that have opposite functions in the induction of adhesion
molecule expression.
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ACKNOWLEDGEMENT |
---|
We thank Jennifer Macke for help in preparing the manuscript.
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FOOTNOTES |
---|
* This work was supported by the Creative Research Initiatives of the Korean Ministry of Science and Technology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: National Creative Research Initiatives Center for Cardiac Regeneration, Chonbuk National University School of Medicine, San 2-20, Keum-Am-Dong, Chonju, 560-180, Republic of Korea. Tel.: 82-63-270-3080; Fax: 82-63-270-4071; E-mail: gykoh@moak.chonbuk.ac.kr.
Published, JBC Papers in Press, December 6, 2000, DOI 10.1074/jbc.M009705200
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ABBREVIATIONS |
---|
The abbreviations used are:
VEGF, vascular
endothelial growth factor;
NO, nitric oxide;
HUVECs, human umbilical
vein endothelial cells;
ICAM-1, intercellular adhesion molecule-1;
VCAM-1, vascular cell adhesion molecule-1;
NF-B, nuclear
factor-
B;
PI 3'-kinase, phosphatidylinositol 3'-kinase;
ERK, extracellular signal-regulated kinase;
MEK, mitogen-activated
protein/extracellular signal-regulated kinase kinase;
TNF-
, tumor
necrosis factor-
;
KDR, kinase-insert domain containing receptor;
NOS, nitric-oxide synthase;
L-NAME, NG-nitro-L-arginine methyl ester;
D-NAME, NG-nitro-D-arginine methyl ester;
DMS, N,N-dimethylsphingosine;
PDTC, pyrrolidine
dithiocarbamate;
RPA, RNase protection assay;
PLC, phospholipase C;
PKC, protein kinase C.
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