Circulation Research. 2000;87:92-98
(Circulation Research. 2000;87:92.)
© 2000 American Heart Association, Inc.
Heparin Blockade of Thrombin-Induced Smooth Muscle Cell Migration Involves Inhibition of Epidermal Growth Factor (EGF) Receptor Transactivation by Heparin-Binding EGF-Like Growth Factor
Presented in part at the Experimental Biology 99 meeting, Washington DC, April 1721, 1999, and published in abstract form (FASEB J. 1999;13:A35, A136).
Andreas Kalmes,
Beatrice R. Vesti,
Günter Daum,
Judith A. Abraham,
Alexander W. Clowes
From the Department of Surgery (A.K., B.R.V., G.D., A.W.C.), University
of Washington (Seattle); and Scios Inc (J.A.A.), Sunnyvale, Calif.
Correspondence to Andreas Kalmes, PhD, University of Washington School of Medicine, Department of Surgery, Box 356410, 1959 NE Pacific St, Seattle, WA 98195-6410. E-mail kalmes{at}u.washington.edu
 |
Abstract
|
|---|
AbstractAgonists of G
proteincoupled receptors,
such as thrombin, act in part by
transactivating the epidermal
growth factor (EGF) receptor (EGFR).
Although at first a ligand-independent
mechanism for EGFR
transactivation was postulated, it has recently
been shown that this
transactivation by various G proteincoupled
receptor agonists can
involve heparin-binding EGF-like growth
factor (HB-EGF). Because
thrombin stimulation of vascular smooth
muscle cell migration is
blocked by heparin and because heparin
can displace HB-EGF, we
investigated the possibility that thrombin
stimulation of smooth muscle
cells (SMCs) depends on EGFR activation
by HB-EGF. In rat SMCs, EGFR
phosphorylation and extracellular
signal-regulated
kinase (ERK) activation in response to thrombin
are inhibited not only
by the EGFR inhibitor AG1478 and by EGFR
blocking antibody
but also by heparin and by neutralizing HB-EGF
antibody.
HB-EGFdependent signaling induced by thrombin
is inhibited by
batimastat, which suggests a requirement for
pro-HB-EGF shedding by a
metalloproteinase. We further demonstrate
that this novel pathway is
required for the migration of rat
and baboon SMCs in response to
thrombin. We conclude from these
data that the inhibitory
effect of heparin on SMC migration
induced by thrombin relies, at least
in part, on a blockade
of HB-EGFmediated EGFR transactivation.
(Circ Res. 2000;87:92-98.)
Key Words: heparin growth factors muscle, smooth migration
 |
Introduction
|
|---|
The accumulation of vascular smooth muscle cells
(SMCs) in the
arterial intima contributes to pathological
disorders such as
atherosclerosis and the development
of stenotic lesions after
arterial injury (reviewed
in Ross
1 and Schwartz et al
2 ). Heparin,
a
glycosaminoglycan, inhibits SMC proliferation and
migration
in vivo and in vitro.
3 4 5 6 7 It is not known for
certain
how heparin blocks SMC function. The effects of heparin on SMCs
include
the inhibition of the expression of early genes,
8
as well as
matrix-degrading proteases
9 10 11 and certain
matrix molecules.
12 13 We and others have previously shown
that heparin suppresses
activation of the extracellular
signal-regulated kinases, ERK1
and ERK2, in response to serum and
thrombin and other agonists
of G protein-coupled receptors
(GPCRs).
14
Signaling through ERK1 and ERK2 in response to various GPCR agonists
relies on a transactivation of the epidermal growth factor (EGF)
receptor (EGFR) (reviewed in Zwick et al15 ). Thrombin and
the thrombin receptor agonist peptide SFLLRN (thrombin receptor agonist
peptide) have been shown to transactivate the EGFR in
Rat-1 fibroblasts, keratinocytes, primary
astrocytes, and COS-7 cells.16 17 EGFR transactivation had
first been suggested to be independent of EGFR ligands, based on the
rapid onset of EGFR tyrosine phosphorylation and the
failure to detect EGFR ligands, in particular EGF, in conditioned
medium of stimulated cells.16 17 18 19 Recently, it has been
shown that EGFR transactivation can be mediated by heparin-binding
EGF-like growth factor (HB-EGF).20 HB-EGF is a member of
the EGF family and is synthesized as a transmembrane precursor that is
proteolytically processed into the mature, soluble growth factor
(reviewed in Raab and Klagsbrun21 ). HB-EGF is a mitogen as
well as a stimulator of cell migration for fibroblasts and
SMCs.21 We report here that in rat and baboon SMCs,
thrombin-stimulated SMC migration relies on HB-EGFdependent EGFR
transactivation, a mechanism that is blocked with heparin.
 |
Materials and Methods
|
|---|
Materials
Antibodies against phosphorylated ERK1/2 were
obtained from
New England Biolabs. ERK1/2 antiserum (7884) was a
generous
gift from Rony Seger. EGFR antibodies (Abs) were from Santa
Cruz
Biotechnology and Upstate Biotechnology, respectively.
Rat-specific
EGFR blocking monoclonal antibody (mAb)
(mAb151-IgG,
22 ) was
obtained from the Developmental
Studies Hybridoma Bank, University
of Iowa. Human-specific EGFR
blocking Ab (mAb 225
23 ) was a
generous gift from Stephen
Prescott. Neutralizing HB-EGF Abs
24 25 26 were raised by
injecting goats with recombinant human
HB-EGF (Ab 197) or rat HB-EGF
(Ab 19). Batimastat (BB94) was
from British Biotech. Plasmid pGEX-RBD
was a generous gift from
David Shalloway. Antibodies against Shc and
H-Ras were from
Transduction Laboratories. Phosphotyrosine Ab (clone
4G10) was
from Upstate Biotechnology. EGF and the tyrphostins AG1478
and
AG1296 were from Calbiochem. Protein A and protein Gagarose
were
from Roche Molecular Biochemicals. Human

-thrombin was from
American
Diagnostica. Platelet-derived growth factor
(PDGF)-BB was a
generous gift from Zymogenetics. Heparin (porcine
intestinal
mucosa) was from Sigma Chemical Co.
Cell Culture
Aortic SMCs were prepared from Fischer rats and baboons as
described previously.27 28 Human embryonic kidney (HEK)
293 cells were from American Type Culture Collection. SMCs (passages 3
to 15) and HEK 293 cells were cultured in DMEM supplemented with 10%
fetal bovine serum, 200 U/mL penicillin, and 200 µg/mL streptomycin.
Before stimulation, cells were starved for 2 to 3 days in medium
without fetal bovine serum (SMCs) or overnight in medium containing
0.3% (HEK 293 cells).
Ras Assay
Ras activity was determined as described
previously29 through affinity purification of Ras-GTP with
a GST-fusion protein containing the Ras binding domain of Raf-1
(GST-RBD), followed by Western blot analysis with an H-Ras
Ab.
Immunoprecipitations
Cells from 100-mm culture dishes were harvested in 1 mL ice-cold
buffer HEB28 (25 mmol/L HEPES-NaOH, pH 7.5, 10%
glycerol, 150 mmol/L NaCl, 5 mmol/L EDTA, 5 mmol/L EGTA, 100 mmol/L
sodium pyrophosphate, 50 mmol/L NaF, 1 mmol/L sodium vanadate, 1 mmol/L
benzamidine, 0.1% 2-mercaptoethanol, 1% Triton X-100, 1 µmol/L
pepstatin A, 2 µg/mL leupeptin, and 20 kallikrein inhibitor units/mL
aprotinin) and lysed for 10 minutes on ice. The lysates were cleared
with centrifugation at 10 000g for 10
minutes in a microfuge. One microgram of Abs and 10 µL protein A or
protein Gagarose slurry were added to the supernatants and incubated
overnight at 4°C with constant agitation. The beads were washed 3
times in HEB and boiled in 20 µL Laemmlis sample buffer.
Western Blots
Total protein from cell lysates or immunocomplexes were
subjected to SDS-PAGE and transferred to nitrocellulose membranes with
electroblotting. Immunodetection was performed with the enhanced
chemiluminescence kit (Amersham) according to the manufacturers
protocol.
Migration Assay for SMCs In Vitro
Modified Boyden chamber assays were performed as
described30 for 5 hours at 37°C under 5%
CO2 with 48-well microchemotaxis chambers (Neuro
Probe) and polycarbonate filters (10-µm pores; Nucleopore Corp)
coated with collagen (Vitrogen 100; Collagen Corp). Chemoattractants or
serum-free DMEM as a control was added to the lower chamber.
Statistical Analysis
All experiments were repeated at least twice with similar
results. Statistical analysis of the migration data shown in
the Table
was performed with the
Wilcoxon signed rank test (SPSS/PC+).
Statistical significance was accepted at P<0.05.
 |
Results
|
|---|
Heparin Inhibits EGFR Transactivation by Thrombin
We found previously that in SMCs, heparin blocks the activation
of
ERK and its activator MEK by thrombin and other agonists
of
GPCRs.
14 Because thrombin has been reported to cause EGFR
transactivation
in other cell types, we investigated whether this
pathway is
also used in SMCs and whether it is affected by heparin.
Thrombin causes an increase in tyrosine phosphorylation
of the EGFR (Figure 1
) that is detectable
within 2 minutes of the exposure of rat SMCs to thrombin (data not
shown). As expected, thrombin-induced EGFR tyrosine
phosphorylation in SMCs is blocked by an
inhibitor of the intrinsic EGFR kinase activity, the
tyrphostin AG1478 (Figure 1
). EGFR transactivation is also
inhibited by heparin (Figure 1
). In addition, thrombin-induced
signaling events upstream of ERK, such as tyrosine
phosphorylation of the adapter protein Shc and GTP
binding of the small GTPase Ras, as well as ERK
phosphorylation, are inhibited by both AG1478 and
heparin (Figure 2
). Activation of the
same signaling elements by EGF is inhibited by AG1478 but not by
heparin (Figure 2
and data not shown). On the other hand,
thrombin-induced Shc and ERK phosphorylations are not
inhibited by a PDGF receptor inhibitor, the tyrphostin
AG1296, at concentrations that completely abolish PDGF-induced
signaling (Figures 2A
and 2C
). We conclude from these results
that thrombin induces EGFR transactivation in rat SMCs and that the
inhibitory effect of heparin on thrombin-induced ERK
activation is due to a blockade of this transactivation.

View larger version (30K):
[in this window]
[in a new window]
|
Figure 1. Thrombin-induced EGFR transactivation requires the
intrinsic EGFR kinase activity and is inhibited by heparin. Quiescent
rat SMCs were stimulated with 10 nmol/L thrombin for 15 minutes in the
presence or absence of heparin (100 µg/mL) or AG1478 (100 nmol/L,
preincubated for 30 minutes). EGFR was immunoprecipitated and Western
blots of the immunoprecipitated material were probed with
anti-phosphotyrosine Ab (PY, top); blots were reprobed with anti-EGFR
Ab (bottom). IB indicates immunoblot.
|
|

View larger version (44K):
[in this window]
[in a new window]
|
Figure 2. Thrombin-induced signaling through Shc, Ras, and
ERK is EGFR dependent and is inhibited by heparin. Quiescent rat SMCs
were stimulated with 10 nmol/L thrombin, 10 ng/mL EGF, or 10 ng/mL
PDGF-BB for 15 minutes in the presence or absence of heparin (100
µg/mL) or after preincubation for 30 minutes with AG1478 (100 nmol/L)
or AG1296 (25 µmol/L), as indicated. A, Shc proteins were
immunoprecipitated and Western blots of the immunoprecipitated material
were probed with anti-phosphotyrosine (PY) Ab (top) and reprobed with
anti-Shc Ab (bottom). Arrows indicate the position of the 3 Shc
isoforms. B, Activation of Ras was determined as described in Materials
and Methods. C, ERK activation was analyzed by probing Western
blots of total protein extracts with anti-phospho-ERK Ab (top) or
anti-ERK Ab (bottom). IB indicates immunoblot.
|
|
EGFR Transactivation by Thrombin Is Mediated by HB-EGF
Because EGFR transactivation has been reported to be either ligand
independent16 17 18 19 or dependent on HB-EGF,20
we further investigated the mechanism involved in thrombin signaling by
using an EGFR blocking Ab (mAb 151-IgG). This Ab binds to the
extracellular portion of rat EGFR, thereby competing with ligand
binding. The preincubation of SMCs with this Ab suppresses ERK
activation by thrombin as well as by EGF. The Ab has no effect when
PDGF-BB is used for stimulation (Figure 3
), demonstrating its specificity. This
result suggested that an EGFR ligand might be involved in EGFR
transactivation. Because heparin blocks EGFR transactivation, we
examined whether HB-EGF is involved. HB-EGF requires cell
surfaceassociated heparan sulfate proteoglycans (HSPGs) as
coreceptors for the EGFR,21 raising the possibility that
exogenously added heparin could inhibit HB-EGF by competing with cell
surface HSPGs for HB-EGF-binding. Consistent with this model,
heparin at the concentration used here (100 µg/mL) blocks
HB-EGFinduced EGFR activation (Figure 4
). In addition, neutralizing Ab against
rat HB-EGF (Ab 19) prevents EGFR phosphorylation and
ERK activation by HB-EGF as well as by thrombin (Figure 4
) but
has no effect when EGF is used as the stimulant (data not shown). We
conclude from these findings that HB-EGF mediates EGFR transactivation
by thrombin in SMCs.

View larger version (29K):
[in this window]
[in a new window]
|
Figure 3. ERK activation by thrombin is inhibited by an EGFR
blocking Ab. Quiescent rat SMCs were stimulated with 10 ng/mL EGF, 10
ng/mL PDGF-BB, or 10 nmol/L thrombin for 15 minutes with or without
preincubation for 30 minutes with anti-EGFR blocking Ab (151-IgG, 15
µg/mL). Total protein extracts were analyzed on Western blots
for ERK phosphorylation as described in Figure 2 . IB indicates immunoblot.
|
|
EGFR Transactivation by Thrombin Receptor Agonist Peptide in HEK
293 Cells Is Mediated by HB-EGF
Because thrombin is a protease, we wanted to exclude the
possibility that it might act on the HB-EGF precursor to release the
mature growth factor in a thrombin receptorindependent manner. The
peptide SFLLRN (thrombin receptor agonist peptide [TRAP])
activates the proteinase-activated receptor (PAR)1
thrombin receptor in the absence of thrombin.31 In SMCs,
however, TRAP is only a weak ERK activator14
and does not cause detectable EGFR transactivation (data not shown).
HEK 293 cells express endogenous PAR1 thrombin receptor
and, in contrast to SMCs, respond to TRAP with EGFR transactivation
(Figure 5
). Therefore, we examined
whether TRAP causes EGFR activation in these cells in an
HB-EGFdependent manner. Both EGFR phosphorylation and
ERK activation stimulated by TRAP are inhibited by the tyrphostin
AG1478, by a human-specific EGFR blocking Ab (mAb 225), and by a
human-specific neutralizing HB-EGF Ab (Ab 197; Figure 5
).
Because human pro-HB-EGF acts as the cellular receptor for diphtheria
toxin, we used its nontoxic analog, CRM197, which has been shown to
block HB-EGF signaling.32 In HEK 293 cells, CRM197
abolishes EGFR phosphorylation and ERK activation
induced by TRAP (Figure 5
), as well as by HB-EGF, but has no
effect on ERK activation in response to EGF (data not shown).

View larger version (30K):
[in this window]
[in a new window]
|
Figure 5. In HEK 293 cells, EGFR transactivation by TRAP
requires HB-EGF. Quiescent cells were stimulated with 30 µmol/L
TRAP for 15 minutes after preincubation for 30 minutes with the
indicated reagents: neutralizing antiHB-EGF (Ab 197, 100 µg/mL),
control IgG (normal goat IgG, 100 µg/mL), AG1478 (100 nmol/L), EGFR
blocking Ab (mAb 225, 25 µg/mL), and CRM197 (10 µg/mL). EGFR
tyrosine phosphorylation (A) and ERK activation (B)
were analyzed as described in the legends for Figures 1 and 2 . Data in B were obtained by scanning autoradiographs and
are presented as a percentage of ERK activation achieved with
TRAP stimulation alone (mean±SD, n=3 or 4). IB indicates
immunoblot; PY, phosphotyrosine.
|
|
HB-EGFDependent EGFR Transactivation Requires Matrix
Metalloproteinase Activity
HB-EGF shedding in different cell types has been reported to be
matrix metalloproteinase (MMP) dependent.20 33 34 35 To
examine whether MMP activity is involved in thrombin-induced EGFR
transactivation, we used the MMP inhibitor batimastat
(BB94). Pretreatment of cells with batimastat inhibits ERK activation
induced by thrombin in rat SMCs and by TRAP in HEK 293 cells (Figure 6
); in contrast, ERK activation by EGF or
recombinant HB-EGF is not affected (Figure 6
).

View larger version (35K):
[in this window]
[in a new window]
|
Figure 6. HB-EGFdependent EGFR transactivation in response
to thrombin and TRAP requires MMP activity. A, Quiescent rat SMCs were
stimulated with 10 nmol/L thrombin, 1 ng/mL rat HB-EGF, or 10 ng/mL EGF
for 15 minutes after preincubation with 5 µmol/L batimastat
(BB94) for 30 minutes. B, Quiescent HEK 293 cells were stimulated with
30 µmol/L TRAP (SFLLRN), 1 ng/mL human HB-EGF, or 10 ng/mL EGF
for 15 minutes after preincubation with 5 µmol/L (BB94) for 30
minutes. ERK activation was analyzed as described in the legend
to Figure 2 . IB indicates immunoblot.
|
|
SMC Migration Induced by Thrombin Requires HB-EGF
To address whether this novel signaling pathway plays a role in
SMC migration, we determined the ability of SMCs to migrate toward
thrombin in a modified Boyden chamber assay. In rat SMCs, thrombin
causes an average 1.75-fold stimulation of migration. The inhibition of
EGFR kinase activity by AG1478, the inhibition of ligand binding by
EGFR blocking Ab, and the sequestration of HB-EGF by heparin or
neutralizing HB-EGF Ab all completely block thrombin-induced migration
(Table
).
To examine whether the same pathway is used in primate cells, we used
baboon SMCs. Under the conditions tested, we did not observe
significant differences between the 2 species: migration in response to
thrombin is activated to the same extent (Figure 7A
) and is inhibited by blocking EGFR Abs
as well as neutralizing HB-EGF Abs (Figure 7B
). Because
PDGF-BBinduced SMC migration is not inhibited by
heparin,36 HB-EGF should not be involved. PDGF-BB is an
equally potent chemoattractant for rat and baboon SMCs (Figure 7A
) and migration toward PDGF-BB is not affected by Abs against
EGFR or HB-EGF (Figure 7B
).

View larger version (20K):
[in this window]
[in a new window]
|
Figure 7. Migration of both rat and baboon SMCs in response
to thrombin, but not PDGF-BB, depends on EGFR transactivation by
HB-EGF. Migration of SMCs was induced in a Boyden chamber by the
addition of 10 nmol/L thrombin or 10 ng/mL PDGF-BB to the lower
chamber. Where indicated, anti-EGFR Abs (25 µg/mL) and antiHB-EGF
Abs (100 µg/mL) were added together with the cells to the upper
chamber. Migration was measured as the number of cells per high-power
field migrating across the membrane between the 2 chambers. Data are
presented as mean±SD (corresponding numbers of independent
experiments [n] are indicated in the figure). Filled columns
represent rat SMCs; open columns, baboon SMCs. A, Induction of
migration by PDGF-BB and thrombin is shown as percent migration of
unstimulated controls. B, Effect of anti-EGFR and antiHB-EGF Abs on
migration in response to PDGF-BB and thrombin. Migration induced in the
controls (PDGF-BB or thrombin alone, minus background of unstimulated
cells) was set to 100%, and the activity in the Ab groups is shown as
a percentage of control.
|
|
We conclude from these data that EGFR transactivation by HB-EGF is
required for rat and baboon SMC migration stimulated by thrombin and
that the inhibitory effect of heparin is based, at least in
part, on a blockade of HB-EGF.
 |
Discussion
|
|---|
EGFR transactivation is necessary for intracellular signaling
by a
variety of GPCR agonists, including thrombin.
16 17 18 19 37 38
We show here that SMC migration in response to thrombin
depends on EGFR
activation and that this mechanism is blocked
by heparin. This is to
our knowledge the first report that describes
a requirement of EGFR
transactivation by a GPCR agonist for
cell migration. In addition, our
data demonstrate that EGFR
transactivation by thrombin relies on a
novel mechanism that
has recently been described in nonvascular cells
that overexpress
various receptor molecules.
20 These
investigators have shown
that EGFR activation by GPCR agonists in
genetically engineered
Rat-1 fibroblasts, COS-7 cells, and HEK 293
cells requires MMP-mediated
cleavage of the pro form of HB-EGF.
Our data demonstrate that
this mechanism is used in normal vascular
SMCs to regulate migration.
Because SMC migration is a crucial process
in the development
of pathological disorders of the vessel wall that
involve neointima
formation, our data indicate an important
physiological role
for this novel signaling
pathway. Consistent with this hypothesis,
HB-EGF expression has
been shown to be upregulated in human
arteriosclerotic
plaques
39 40 41 and in
neointimal cells of rat carotid arteries
in response to
balloon injury.
42
HB-EGF requires cell surface HSPGs as coreceptors (reviewed in
Raab and Klagsbrun21 ). HB-EGF binding to EGFR is
antagonized in a dose-dependent manner by heparin.43 In
our experiments, heparin blocked EGFR activation by HB-EGF as well as
EGFR transactivation and SMC migration induced by thrombin. Thus, the
inhibitory effect of heparin on thrombin-induced ERK
activation and SMC migration may be accounted for by a blockade of
soluble HB-EGF.
Interestingly, the PAR1 thrombin receptor agonist peptide TRAP
causes HB-EGFdependent EGFR transactivation in HEK 293 cells but not
in rat SMCs. Consistent with this failure to activate a
pathway that, as shown here, is necessary for migration, TRAP does not
act as a chemoattractant for rat SMCs (data not shown). In SMCs, TRAP
is also a weak ERK activator that is not inhibited by
heparin.14 Although we cannot completely rule out that
TRAP-activated PAR1 does not fully duplicate the signaling
induced by the proteolytically processed receptor, it may also be
possible that thrombin-induced EGFR transactivation in SMCs is mediated
by a different receptor. Recently, 2 novel members of the family of
protease-activated receptors, PAR3 and PAR4, were cloned as
additional thrombin receptors from human and mouse
cells.44 45 The presence of PAR4 as a second thrombin
receptor in vascular and gastric SMCs has been
suggested.46
Ectodomain shedding provides an important mechanism for HB-EGF
signaling, although the pro form may also have biological activity
(reviewed in Raab and Klagsbrun21 ). Because the MMP
inhibitor batimastat inhibits thrombin receptorinduced
EGFR transactivation in both SMCs and HEK 293 cells, we conclude that
an MMP is involved and that the EGFR is activated by soluble
HB-EGF. These data are consistent with the recent observation
that batimastat inhibits signaling by lysophosphatidic acid, carbachol,
and phorbol-12-myristate-13-acetate in COS-7 cells and by
bombesin and phorbol-12-myristate-13-acetate in PC-3 prostate
carcinoma cells.20 The identity of the protease or
proteases that mediate GPCR-induced HB-EGF shedding in SMCs remains to
be determined.
Taken together, our data demonstrate that EGFR transactivation is an
essential step in thrombin-induced SMC migration. Furthermore, they
reveal that a novel mechanism of EGFR transactivation, which involves
HB-EGF as an EGFR ligand, is used in normal vascular SMCs. This
mechanism appears to account, at least in part, for the
inhibitory effects of heparin on signaling by GPCR
agonists.
 |
Acknowledgments
|
|---|
This work was supported by grants from the National Institutes
of
Health (HL-18645 and HL-30946) and a fellowship of the Swiss
National
Foundation (Dr Vesti). We thank Jessie Deou for technical
help;
Debby Damm, Brett Garrick, and Peter Strathis for the
production
of recombinant HB-EGF and HB-EGF Abs; and Michael
Klagsbrun
and Zeev Gechtman for helpful discussions. We also thank
David
Shalloway for the GST-RBD construct, Stephen Prescott for EGFR
mAb
225, Rony Seger for ERK Ab 7884, and Zymogenetics for
PDGF-BB.
Received April 28, 2000;
accepted May 25, 2000.
 |
References
|
|---|
-
Ross R. The pathogenesis of
atherosclerosis: a perspective for the 1990s.
Nature. 1993;362:801809.[Medline]
[Order article via Infotrieve]
-
Schwartz SM, deBlois D, OBrien ER. The intima; soil
for atherosclerosis and restenosis. Circ
Res. 1995;77:445465.[Free Full Text]
-
Clowes AW, Karnowsky MJ. Suppression by heparin of
smooth muscle cell proliferation in injured arteries.
Nature. 1977;265:625626.[Medline]
[Order article via Infotrieve]
-
Clowes AW, Karnovsky MJ. Suppression by heparin of
injury-induced myointimal thickening. J Surg Res. 1978;24:161168.[Medline]
[Order article via Infotrieve]
-
Guyton JR, Rosenberg RD, Clowes AW, Karnovsky
MJ. Inhibition of rat arterial smooth muscle cell
proliferation by heparin: in vivo studies with anticoagulant and
nonanticoagulant heparin. Circ Res. 1980;46:625634.[Free Full Text]
-
Majack RA, Clowes AW. Inhibition of vascular smooth
muscle cell migration by heparin-like
glycosaminoglycans. J Cell Physiol. 1984;118:253256.[Medline]
[Order article via Infotrieve]
-
Clowes AW, Clowes MM. Regulation of smooth muscle
proliferation by heparin in vitro and in vivo. Int Angiol. 1987;6:4551.[Medline]
[Order article via Infotrieve]
-
Pukac LA, Castellot JJ Jr, Wright TC Jr, Caleb BL,
Karnovsky MJ. Heparin inhibits c-fos and c-myc mRNA expression in
vascular smooth muscle cells. Cell Regul. 1990;1:435443.[Medline]
[Order article via Infotrieve]
-
Au YP, Kenagy RD, Clowes MM, Clowes AW. Mechanisms of
inhibition by heparin of vascular smooth muscle cell proliferation and
migration. Haemostasis. 1993;1:177182.
-
Kenagy RD, Nikkari ST, Welgus HG, Clowes AW. Heparin
inhibits the induction of three matrix metalloproteinases (stromelysin,
92-kD gelatinase, and collagenase) in primate
arterial smooth muscle cells. J Clin
Invest. 1994;93:19871993.
-
Kenagy RD, Clowes AW. Regulation of baboon
arterial smooth muscle cell plasminogen
activators by heparin and growth factors. Thromb
Res. 1995;77:5561.[Medline]
[Order article via Infotrieve]
-
Nikkari ST, Jarvelainen HT, Wight TN, Ferguson M,
Clowes AW. Smooth muscle cell expression of extracellular matrix genes
after arterial injury. Am J Pathol. 1994;144:13481356.[Abstract]
-
Snow AD, Bolender RP, Wight TN, Clowes AW. Heparin
modulates the composition of the extracellular matrix domain
surrounding arterial smooth muscle cells. Am J
Pathol. 1990;137:313330.[Abstract]
-
Hedin U, Daum G, Clowes AW. Heparin inhibits
thrombin-induced mitogen-activated protein kinase signaling in
arterial smooth muscle cells. J Vasc Surg. 1998;27:512520.[Medline]
[Order article via Infotrieve]
-
Zwick E, Hackel PO, Prenzel N, Ullrich A. The EGF
receptor as central transducer of heterologous signalling systems.
Trends Pharmacol Sci. 1999;20:408412.[Medline]
[Order article via Infotrieve]
-
Daub H, Weiss FU, Wallasch C, Ullrich A. Role of
transactivation of the EGF receptor in signalling by G-protein-coupled
receptors. Nature. 1996;379:557560.[Medline]
[Order article via Infotrieve]
-
Daub H, Wallasch C, Lankenau A, Herrlich A, Ullrich A.
Signal characteristics of G protein-transactivated EGF
receptor. EMBO J. 1997;16:70327044.[Medline]
[Order article via Infotrieve]
-
Eguchi S, Numaguchi K, Iwasaki H, Matsumoto T, Yamakawa
T, Utsunomiya H, Motley ED, Kawakatsu H, Owada KM, Hirata Y, Marumo F,
Inagami T. Calcium-dependent epidermal growth factor receptor
transactivation mediates the angiotensin II-induced
mitogen-activated protein kinase activation in vascular smooth
muscle cells. J Biol Chem. 1998;273:88908896.[Abstract/Free Full Text]
-
Murasawa S, Mori Y, Nozawa Y, Gotoh N, Shibuya M,
Masaki H, Maruyama K, Tsutsumi Y, Moriguchi Y, Shibazaki Y, Tanaka Y,
Iwasaka T, Inada M, Matsubara H. Angiotensin II type 1
receptor-induced extracellular signal-regulated protein kinase
activation is mediated by
Ca2+/calmodulin-dependent
transactivation of epidermal growth factor receptor. Circ
Res. 1998;82:13381348.[Abstract/Free Full Text]
-
Prenzel N, Zwick E, Daub H, Leserer M, Abraham R,
Wallasch C, Ullrich A. EGF receptor transactivation by
G-protein-coupled receptors requires metalloproteinase cleavage of
proHB-EGF. Nature. 1999;402:884888.[Medline]
[Order article via Infotrieve]
-
Raab G, Klagsbrun M. Heparin-binding EGF-like growth
factor. Biochim Biophys Acta. 1997;1333:F179F199.[Medline]
[Order article via Infotrieve]
-
Chandler LP, Chandler CE, Hosang M, Shooter EM. A
monoclonal antibody which inhibits epidermal growth factor binding has
opposite effects on the biological action of epidermal growth factor in
different cells. J Biol Chem. 1985;260:33603367.[Abstract/Free Full Text]
-
Masui H, Kawamoto T, Sato JD, Wolf B, Sato G,
Mendelsohn J. Growth inhibition of human tumor cells in athymic mice by
anti-epidermal growth factor receptor monoclonal antibodies.
Cancer Res. 1984;44:10021007.[Abstract/Free Full Text]
-
McCarthy SA, Samuels ML, Pritchard CA, Abraham JA,
McMahon M. Rapid induction of heparin-binding epidermal growth
factor/diphtheria toxin receptor expression by Raf and Ras oncogenes.
Genes Dev. 1995;9:19531964.[Abstract/Free Full Text]
-
Leslie CC, McCormick-Shannon K, Shannon JM, Garrick B,
Damm D, Abraham JA, Mason RJ. Heparin-binding EGF-like growth
factor is a mitogen for rat alveolar type II cells. Am J
Respir Cell Mol Biol. 1997;16:379387.[Abstract]
-
Abramovitch R, Neeman M, Reich R, Stein I, Keshet E,
Abraham J, Solomon A, Marikovsky M. Intercellular communication between
vascular smooth muscle and endothelial cells mediated
by heparin-binding epidermal growth factor-like growth factor and
vascular endothelial growth factor. FEBS
Lett. 1998;425:441447.[Medline]
[Order article via Infotrieve]
-
Clowes MM, Lynch CM, Miller AD, Miller DG, Osborne WR,
Clowes AW. Long-term biological response of injured rat carotid artery
seeded with smooth muscle cells expressing retrovirally introduced
human genes. J Clin Invest. 1994;93:644651.
-
Daum G, Hedin U, Wang Y, Wang T, Clowes AW. Diverse
effects of heparin on mitogen-activated protein
kinase-dependent signal transduction in vascular smooth muscle cells.
Circ Res. 1997;81:1723.[Abstract/Free Full Text]
-
Taylor SJ, Shalloway D. Cell cycle-dependent activation
of Ras. Curr Biol. 1996;6:16211627.[Medline]
[Order article via Infotrieve]
-
Koyama N, Hart CE, Clowes AW. Different functions of
the platelet-derived growth factor-
and -ß receptors for
the migration and proliferation of cultured baboon smooth muscle cells.
Circ Res. 1994;75:682691.[Abstract/Free Full Text]
-
Grand RJ, Turnell AS, Grabham PW. Cellular consequences
of thrombin-receptor activation. Biochem J. 1996;313:353368.
-
Mitamura T, Higashiyama S, Taniguchi N, Klagsbrun M,
Mekada E. Diphtheria toxin binds to the epidermal growth factor
(EGF)-like domain of human heparin-binding EGF-like growth
factor/diphtheria toxin receptor and inhibits specifically its
mitogenic activity. J Biol Chem. 1995;270:10151019.[Abstract/Free Full Text]
-
Goishi K, Higashiyama S, Klagsbrun M, Nakano N,
Umata T, Ishikawa M, Mekada E, Taniguchi N. Phorbol ester induces the
rapid processing of cell surface heparin-binding EGF-like growth
factor: conversion from juxtacrine to paracrine growth factor activity.
Mol Biol Cell. 1995;6:967980.[Abstract]
-
Suzuki M, Raab G, Moses MA, Fernandez CA, Klagsbrun M.
Matrix metalloproteinase-3 releases active heparin-binding EGF-like
growth factor by cleavage at a specific juxtamembrane site.
J Biol Chem. 1997;272:3173031737.[Abstract/Free Full Text]
-
Dethlefsen SM, Raab G, Moses MA, Adam RM, Klagsbrun M,
Freeman MR. Extracellular calcium influx stimulates metalloproteinase
cleavage and secretion of heparin-binding EGF-like growth factor
independently of protein kinase C. J Cell Biochem. 1998;69:143153.[Medline]
[Order article via Infotrieve]
-
Geary RL, Koyama N, Wang TW, Vergel S, Clowes AW.
Failure of heparin to inhibit intimal hyperplasia in injured baboon
arteries: the role of heparin-sensitive and -insensitive pathways in
the stimulation of smooth muscle cell migration and proliferation.
Circulation. 1995;91:29722981.[Abstract/Free Full Text]
-
Luttrell LM, Della Rocca GJ, van Biesen T,
Luttrell DK, Lefkowitz RJ. Gbetagamma subunits mediate Src-dependent
phosphorylation of the epidermal growth factor
receptor: a scaffold for G protein-coupled receptor-mediated Ras
activation. J Biol Chem. 1997;272:46374644.[Abstract/Free Full Text]
-
Cunnick JM, Dorsey JF, Standley T, Turkson J, Kraker
AJ, Fry DW, Jove R, Wu J. Role of tyrosine kinase activity of epidermal
growth factor receptor in the lysophosphatidic acid-stimulated
mitogen-activated protein kinase pathway. J Biol
Chem. 1998;273:1446814475.[Abstract/Free Full Text]
-
Miyagawa J, Higashiyama S, Kawata S, Inui Y, Tamura S,
Yamamoto K, Nishida M, Nakamura T, Yamashita S, Matsuzawa Y, Taniguchi
N. Localization of heparin-binding EGF-like growth factor
in the smooth muscle cells and macrophages of human
atherosclerotic plaques. J Clin Invest. 1995;95:404411.
-
Nakata A, Miyagawa J, Yamashita S, Nishida M, Tamura R,
Yamamori K, Nakamura T, Nozaki S, Kameda Takemura K, Kawata S,
Taniguchi N, Higashiyama S, Matsuzawa Y. Localization of
heparin-binding epidermal growth factorlike growth factor in human
coronary arteries: possible roles of HB-EGF in the formation of
coronary atherosclerosis.
Circulation. 1996;94:27782786.[Abstract/Free Full Text]
-
Reape TJ, Wilson VJ, Kanczler JM, Ward JP, Burnand KG,
Thomas CR. Detection and cellular localization of heparin-binding
epidermal growth factor-like growth factor mRNA and protein in human
atherosclerotic tissue. J Mol Cell Cardiol. 1997;29:16391648.[Medline]
[Order article via Infotrieve]
-
Igura T, Kawata S, Miyagawa J, Inui Y, Tamura S, Fukuda
K, Isozaki K, Yamamori K, Taniguchi N, Higashiyama S, Matsuzawa Y.
Expression of heparin-binding epidermal growth factorlike growth
factor in neointimal cells induced by balloon injury in rat
carotid arteries. Arterioscler Thromb Vasc Biol. 1996;16:15241531.[Abstract/Free Full Text]
-
Besner GE, Whelton D, Crissman-Combs MA, Steffen CL,
Kim GY, Brigstock DR. Interaction of heparin-binding EGF-like growth
factor (HB-EGF) with the epidermal growth factor receptor: modulation
by heparin, heparinase, or synthetic heparin-binding HB-EGF fragments.
Growth Factors. 1992;7:289296.[Medline]
[Order article via Infotrieve]
-
Ishihara H, Connolly AJ, Zeng D, Kahn ML,
Zheng YW, Timmons C, Tram T, Coughlin SR. Protease-activated
receptor 3 is a second thrombin receptor in humans. Nature. 1997;386:502506.[Medline]
[Order article via Infotrieve]
-
Xu WF, Andersen H, Whitmore TE, Presnell SR,
Yee DP, Ching A, Gilbert T, Davie EW, Foster DC. Cloning and
characterization of human protease-activated receptor 4.
Proc Natl Acad Sci U S A. 1998;95:66426646.[Abstract/Free Full Text]
-
Hollenberg MD, Saifeddine M, Al-Ani B, Gui Y.
Proteinase-activated receptor 4 (PAR4): action of
PAR4-activating peptides in vascular and gastric tissue and lack of
cross-reactivity with PAR1 and PAR2. Can J Physiol
Pharmacol. 1999;77:458464.[Medline]
[Order article via Infotrieve]
This article has been cited by other articles:

|
 |

|
 |
 
M.-C. Lauzier, E. L. Page, M. D. Michaud, and D. E. Richard
Differential Regulation of Hypoxia-Inducible Factor-1 through Receptor Tyrosine Kinase Transactivation in Vascular Smooth Muscle Cells
Endocrinology,
August 1, 2007;
148(8):
4023 - 4031.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
B. H. Rauch, G. A. Scholz, D. Baumgartel-Allekotte, P. Censarek, J. W. Fischer, A.-A. Weber, and K. Schror
Cholesterol Enhances Thrombin-Induced Release of Fibroblast Growth Factor-2 in Human Vascular Smooth Muscle Cells
Arterioscler. Thromb. Vasc. Biol.,
April 1, 2007;
27(4):
e20 - e25.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
A. C. Snider and K. E. Meier
Receptor transactivation cascades. Focus on "Effects of {alpha}1D-adrenergic receptors on shedding of biologically active EGF in freshly isolated lacrimal gland epithelial cells"
Am J Physiol Cell Physiol,
January 1, 2007;
292(1):
C1 - C3.
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
Y. V. Mukhin, M. Gooz, J. R. Raymond, and M. N. Garnovskaya
Collagenase-2 and -3 Mediate Epidermal Growth Factor Receptor Transactivation by Bradykinin B2 Receptor in Kidney Cells
J. Pharmacol. Exp. Ther.,
September 1, 2006;
318(3):
1033 - 1043.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
H. Ohtsu, P. J. Dempsey, G. D. Frank, E. Brailoiu, S. Higuchi, H. Suzuki, H. Nakashima, K. Eguchi, and S. Eguchi
ADAM17 Mediates Epidermal Growth Factor Receptor Transactivation and Vascular Smooth Muscle Cell Hypertrophy Induced by Angiotensin II
Arterioscler. Thromb. Vasc. Biol.,
September 1, 2006;
26(9):
e133 - e137.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
D. Chansel, M. Ciroldi, S. Vandermeersch, L. F Jackson, A.-M. Gomez, D. Henrion, D. C. Lee, T. M. Coffman, S. Richard, J.-C. Dussaule, et al.
Heparin binding EGF is necessary for vasospastic response to endothelin
FASEB J,
September 1, 2006;
20(11):
1936 - 1938.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
H. Cao, N. Dronadula, F. Rizvi, Q. Li, K. Srivastava, W. T. Gerthoffer, and G. N. Rao
Novel Role for STAT-5B in the Regulation of Hsp27-FGF-2 Axis Facilitating Thrombin-Induced Vascular Smooth Muscle Cell Growth and Motility
Circ. Res.,
April 14, 2006;
98(7):
913 - 922.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
H. Cao, N. Dronadula, and G. N. Rao
Thrombin induces expression of FGF-2 via activation of PI3K-Akt-Fra-1 signaling axis leading to DNA synthesis and motility in vascular smooth muscle cells
Am J Physiol Cell Physiol,
January 1, 2006;
290(1):
C172 - C182.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
L. Yu, D. A. Quinn, H. G. Garg, and C. A. Hales
Cyclin-Dependent Kinase Inhibitor p27Kip1, But Not p21WAF1/Cip1, Is Required for Inhibition of Hypoxia-Induced Pulmonary Hypertension and Remodeling by Heparin in Mice
Circ. Res.,
October 28, 2005;
97(9):
937 - 945.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
S. Zhuang, Y. Yan, J. Han, and R. G. Schnellmann
p38 Kinase-mediated Transactivation of the Epidermal Growth Factor Receptor Is Required for Dedifferentiation of Renal Epithelial Cells after Oxidant Injury
J. Biol. Chem.,
June 3, 2005;
280(22):
21036 - 21042.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
B. H. Rauch, E. Millette, R. D. Kenagy, G. Daum, J. W. Fischer, and A. W. Clowes
Syndecan-4 Is Required for Thrombin-induced Migration and Proliferation in Human Vascular Smooth Muscle Cells
J. Biol. Chem.,
April 29, 2005;
280(17):
17507 - 17511.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
S. Fasciano, R. C. Patel, I. Handy, and C. V. Patel
Regulation of Vascular Smooth Muscle Proliferation by Heparin: INHIBITION OF CYCLIN-DEPENDENT KINASE 2 ACTIVITY BY p27kip1
J. Biol. Chem.,
April 22, 2005;
280(16):
15682 - 15689.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
Z. Liu, C. Zhang, N. Dronadula, Q. Li, and G. N. Rao
Blockade of Nuclear Factor of Activated T Cells Activation Signaling Suppresses Balloon Injury-induced Neointima Formation in a Rat Carotid Artery Model
J. Biol. Chem.,
April 15, 2005;
280(15):
14700 - 14708.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
E. Millette, B. H. Rauch, O. Defawe, R. D. Kenagy, G. Daum, and A. W. Clowes
Platelet-Derived Growth Factor-BB-Induced Human Smooth Muscle Cell Proliferation Depends on Basic FGF Release and FGFR-1 Activation
Circ. Res.,
February 4, 2005;
96(2):
172 - 179.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
T. Chiu, C. Santiskulvong, and E. Rozengurt
EGF receptor transactivation mediates ANG II-stimulated mitogenesis in intestinal epithelial cells through the PI3-kinase/Akt/mTOR/p70S6K1 signaling pathway
Am J Physiol Gastrointest Liver Physiol,
February 1, 2005;
288(2):
G182 - G194.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
N. Dronadula, Z. Liu, C. Wang, H. Cao, and G. N. Rao
STAT-3-dependent Cytosolic Phospholipase A2 Expression Is Required for Thrombin-induced Vascular Smooth Muscle Cell Motility
J. Biol. Chem.,
January 28, 2005;
280(4):
3112 - 3120.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
B. Schafer, B. Marg, A. Gschwind, and A. Ullrich
Distinct ADAM Metalloproteinases Regulate G Protein-coupled Receptor-induced Cell Proliferation and Survival
J. Biol. Chem.,
November 12, 2004;
279(46):
47929 - 47938.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
H. Zhang, D. Chalothorn, L. F. Jackson, D. C. Lee, and J. E. Faber
Transactivation of Epidermal Growth Factor Receptor Mediates Catecholamine-Induced Growth of Vascular Smooth Muscle
Circ. Res.,
November 12, 2004;
95(10):
989 - 997.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
I. Neeli, Z. Liu, N. Dronadula, Z. A. Ma, and G. N. Rao
An Essential Role of the Jak-2/STAT-3/Cytosolic Phospholipase A2 Axis in Platelet-derived Growth Factor BB-induced Vascular Smooth Muscle Cell Motility
J. Biol. Chem.,
October 29, 2004;
279(44):
46122 - 46128.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
Z. Liu, N. Dronadula, and G. N. Rao
A Novel Role for Nuclear Factor of Activated T Cells in Receptor Tyrosine Kinase and G Protein-coupled Receptor Agonist-induced Vascular Smooth Muscle Cell Motility
J. Biol. Chem.,
September 24, 2004;
279(39):
41218 - 41226.
[Abstract]
|