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Cytokine 32 (2005) 206e212

Upregulation of neuropilin-1 by basic fibroblast growth factor enhances

vascular smooth muscle cell migration in response to VEGF
Wenbiao Liu a, Alexander A. Parikh b, Oliver Stoeltzing a, Fan Fan a, Marya F. McCarty a,
Jane Wey a, Daniel J. Hicklin c, Lee M. Ellis a,b,*
a
Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030-4009, USA
b
Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030-4009, USA
c
Department of Experimental Therapeutics, ImClone Systems, 180 Varick Street, New York, NY 10014, USA
Received 9 May 2005; received in revised form 18 August 2005; accepted 8 September 2005

Abstract

Neuropilin-1 (NRP-1) is a co-receptor for vascular endothelial growth factor (VEGF). During neovascularization, vascular smooth muscle
cells (VSMCs) and pericytes modulate the function of endothelial cells. Factors that mediate NRP-1 in human VSMCs (hVSMCs) remain to
be elucidated. We studied various angiogenic cytokines to identify factors that increase NRP-1 expression in hVSMCs. Treatment of hVSMCs
with basic fibroblast growth factor (b-FGF) induced expressions of NRP-1 mRNA and protein whereas epidermal growth factor, insulin-like
growth factor-1, and interleukin-1b did not. b-FGF induced phosphorylation of Erk-1/2 and JNK. MEK1/2 and nuclear factor kappa B (NF-kB)
inhibitors (U0126 and TLCK, respectively) blocked the ability of b-FGF to induce NRP-1 mRNA expression, but inhibition of JNK
(SP600125) or PI3-kinase activity (wortmannin) did not. Further, the increase in NRP-1 expression by b-FGF enhanced hVSMCs migration
in response to VEGF165. This effect was dependent on the binding of VEGF165 to VEGFR-2, as blocking antibodies to VEGFR-2, but not
VEGFR-1, inhibited VEGF165-induced migration. In conclusion, b-FGF increased NRP-1 expression in hVSMCs that in turn enhance the effect
of VEGF165 on cell migration. The enhanced migration of hVSMCs was mediated through binding of VEGF165 to both NRP-1 and VEGFR-2, as
inhibition of VEGFR-2 on these cells blocked the effect of VEGF-mediated cell migration.
Ó 2005 Elsevier Ltd. All rights reserved.

Keywords: Neuropilin-1; b-FGF; Angiogenesis; Pericytes; VEGF

1. Introduction namely VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), and

Vascular endothelial growth factor (VEGF), the best char-
acterized of the angiogenic factors, has been associated with
increased tumor angiogenesis, tumor growth, and poor progno-
sis in various human tumor systems. VEGF mediates tumor
angiogenesis via three high-affinity tyrosine kinase receptors,
* Corresponding author. Department of Surgical Oncology, Unit 444, The
University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boule-
vard, P.O. Box 301402, Houston, TX 77230-1402, USA. Tel.: C1 713 792
6926; fax: C1 713 792 4689.
E-mail address: lellis@mdanderson.org (L.M. Ellis).
VEGFR-3 (Flt-4) [1].
Neuropilin-1 (NRP-1) was originally characterized as a re-
ceptor for class III semaphorin/collapsin family members,
which are involved in vasculogenesis and neuronal guidance
in the nervous system [2,3]. NRP-1 was subsequently identi-
fied as a co-receptor for specific isoforms of VEGF. NRP-1
has been shown to regulate tumor angiogenesis via binding
the VEGF isoform VEGF165 [4] and as well as other VEGF
family members such as VEGF-B, VEGF-E, and placental
growth factor 2 [5e7]. In endothelial cells (ECs), NRP-1 binds
VEGF165 and enhances its binding to VEGFR-2 [4,8]. More
recently, NRP-1 was demonstrated to be expressed on non-
endothelial cells, including various tumor cells (prostate,

1043-4666/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cyto.2005.09.009
 W. Liu et al. / Cytokine 32 (2005) 206e212
breast, melanoma, colorectal, and pancreatic cell lines [9e13])
and tumor cells in pathologic specimens. We recently found
that human vascular smooth muscle cells (hVSMCs) also ex-
press NRP-1. VSMCs/pericytes are components of the vascu-
lature and play important roles during vascular remodeling and
maturation. hVSMCs stabilize the vasculature and maintain
the integrity of newly formed vessels. We previously found
that specific cytokines led to the induction of NRP-1 in human
tumor cell lines. In the present study, we investigated whether
NRP-1 expression by hVSMCs is regulated by the pro-angio-
genic factor basic fibroblast growth factor (b-FGF) and sought
to clarify the signaling pathways involved in this regulation.
We also examined whether the induction of NRP-1 in
hVSMCs affects their biological function by assessing cell
migration and proliferation.
2. Materials and methods
2.1. Cell and cell culture
hVSMCs were obtained from the American Type Culture
Collection (ATCC, Manassas, VA) and were cultured in Hanks
modified Dulbecco’s minimum essential medium (DMEM)/
F12 medium supplemented with 10% fetal bovine serum and
2 U/ml penicillinestreptomycin (Life Technologies, Inc.,
Grand Island, NY). hVSMCs were grown at 37  C in 5%
CO2 and 95% air.
2.2. Materials and antibodies
b-FGF, VEGF121, and VEGF165 were purchased from R&D
Systems, Inc. (Minneapolis, MN). Antibodies to phosphorylated
Erk-1/2 (Thr202/Tyr204), JNK (Thr183/Tyr185), P38
(Thr180/Tyr182) MAPK, and Akt (Ser473) and antibodies to
total Erk-1/2, JNK, P38 MAPK, and Akt were obtained from
Cell Signaling Technology, Inc. (Beverly, MA). SP600125
(JNK inhibitor) was purchased from Calbiochem Co. (La
Jolla, CA). U0126 (MEK1/2 inhibitor) was purchased from
New England Biolabs, Inc. (Beverly, MA). Actin antibody,
actinomycin (Act D), wortmannin (PI3K inhibitor), and
TLCK (NF-kB inhibitor) were purchased from Sigma Chemi-
cal Company (St. Louis, MO). NRP-1 antibody was purchased
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Neu-
tralizing antibodies against human VEGFR-1 and VEGFR-2
were provided by Daniel J. Hicklin, Ph.D., of ImClone Systems
(New York, NY). As a negative control, a non-specific mouse
immunoglobulin G (Calbiochem Co.) was used at the same
dose as the neutralizing antibodies.
2.3. Western blot hybridization
Cells were lysed with protein lysis buffer (20 mM sodium
phosphate (pH 7.4), 150 mM sodium chloride, 1% Triton
X-100, 5 mM EDTA, 5 mM phenylmethylsulfonyl fluoride,
1% aprotinin, 1 mg/ml leupeptin, and 500 mM Na3VO4). The
protein was quantitated spectrophotometrically with a bicin-
choninic acid assay (Pierce, Rockford, IL). Aliquots (50 mg)
207
of protein were subjected to electrophoresis on 10% polyacryl-
amide gels and then transferred to a nylon membrane (Millipore
Corp., Bedford, MA) by electrotransfer. After being blocked
with 5% milk in 0.1% Tween-20 in phosphate-buffered saline,
the membranes were probed with the primary antibodies
(1:1000 dilutions of rabbit polyclonal anti-phosphospecific
Erk-1/2 MAPK antibody, anti-phosphospecific P38 MAPK
antibody, anti-phosphospecific Akt antibody or anti-phospho-
specific JNK antibody, or 1:100 dilution of polyclonal rabbit
anti-NRP-1). The membranes were then washed and treated
with the secondary antibody labeled with horseradish peroxi-
dase (anti-rabbit, anti-mouse, or anti-goat immunoglobulin at
1:3000 dilutions (Amersham Biosciences, Piscataway, NJ)).
Protein bands were visualized with a chemiluminescence kit
(Amersham Biosciences). For assaying total protein levels,
membranes were washed with stripping solution (100 mM
2-mercaptoethanol, 2% sodium dodecyl sulfate, and
62.5 mM TriseHCl (pH 6.7)) for 30 min at 50  C and
reprobed with rabbit polyclonal anti-Erk-1/2, anti-P38, anti-
Akt, anti-JNK (all at 1:1000 dilution) or anti-actin antibody
(at 1:3000 dilution).
2.4. Isolation of mRNA and Northern blot analysis
Total RNA was extracted from cell cultures at 80% conflu-
ence with TRIzol reagent according to the manufacturer’s pro-
tocol (Life Technologies, Inc.). Northern blot analysis was
performed as previously described [14]. Each cDNA probe
was radiolabeled with (a-32P) deoxyribonucleotide triphos-
phate with a random-primer technique from a commercially
available kit (Amersham Biosciences). After the blots were
prehybridized for 3e4 h at 65  C in rapid hybridization buffer
(Amersham Biosciences), the membranes were hybridized
overnight at 65  C with the cDNA probe for NRP-1 or glycer-
aldehyde-phosphate dehydrogenase (GAPDH (ATCC) as an
internal control). The probed nylon membranes were washed
at 65  C with 30 mM NaCl, 3 mM sodium citrate (pH 7.2),
and 0.1% sodium dodecyl sulfate. Autoradiography was then
performed.
The cDNA probes used were a human NRP-1 450-bp
cDNA probe derived from the RT-PCR product of PC-3 human
prostate cancer cells (purchased from ATCC) using the follow-
ing primers (forward 5#-ACGATGAATGTGGCGATACT-3#
and reverse 5#-AGTGCATTCAAGGCTGTTGG-3#) and a
GAPDH probe (from ATCC). Each cDNA probe was purified
by agarose gel electrophoresis, recovered with a QIAEX gel
extraction kit (QIAGEN Inc., Valencia, CA), and radiolabeled
with a random-primer technique from a commercially avail-
able kit (Amersham Biosciences).
2.5. Transcriptional activity and mRNA half-life studies
To determine whether the increase in NRP-1 mRNA in
hVSMCs resulted from an increase in transcription, hVSMCs
were incubated in the presence or absence of Act D (0.5 mg/ml)
for 2 h before being exposed to b-FGF, and total RNA was ex-
tracted from the cells after treatment with b-FGF for 2 h.
208 W. Liu et al. / Cytokine 32 (2005) 206e212

Control cells were treated with Act D without b-FGF. To in- A

vestigate the effect of b-FGF on the half-life of NRP-1 Time (hrs) 4 24 48
mRNA, hVSMCs were incubated in the presence or absence b-FGF (10 ng/ml) - + - + - +
of b-FGF (10 ng/ml) for 24 h. Further transcription was
NRP-1
blocked by adding Act D (0.5 mg/ml). Total RNA was ex-
tracted from the cells at 0, 0.5, 1, 2, and 4 h after the addition
GAPDH
of Act D, and Northern blot analysis was done. The half-life of
NRP-1 mRNA was determined by plotting relative NRP-1
mRNA expression levels on a semilogarithmic axis versus B
Time (hrs) 4 24 48
time (Cricket Software Inc., Malvern, PA).
b-FGF (10 ng/ml) - + - + - +
NRP-1
2.6. Cell migration assay
Actin
hVSMC migration was investigated by using Boyden
chambers (BD Biosciences, Bedford, MA) with uncoated in- Fig. 1. Time course for induction of NRP-1 mRNA and protein by b-FGF in
serts (8.0-mm pores). hVSMCs were pretreated with and with- hVSMCs. hVSMCs were incubated in the presence of b-FGF (10 ng/ml) for
out b-FGF (10 ng/ml) for 24 h in flasks, and then the cells 4, 24, or 48 h. (A) NRP-1 mRNA levels increased after incubation of cells
were trypsinized and plated into migration inserts (50,000/ with b-FGF at 24 h and remained elevated for at least 48 h. (B) NRP-1 protein
insert in 1% DMEM/F12) on 24-well plates; 1% DMEM/ levels increased after incubation of cells with b-FGF by 24 h and remained
elevated for at least 48 h.
F12 supplemented with solvent, VEGF121, or VEGF165 at a fi-
nal concentration of 10 ng/ml was placed in the bottom wells.
The plated inserts were incubated for 24 h at 37  C. Migrated 3.2. Effect of b-FGF on signaling pathways involved
cells were stained with DiffQuick (Dade Behring, Newark, in NRP-1 induction in hVSMCs
DE) and five random fields were counted per insert at 100!
magnification. To determine whether the effect of VEGF in- To identify the signaling pathways induced by b-FGF in
duction of cell migration was mediated through NRP-1, hVSMCs, Western blot analyses were performed after cells
VEGFR-1, or VEGFR-2 (or binding to both NRP-1 and had been incubated with b-FGF for various times. A signifi-
VEGF tyrosine kinase receptors), hVSMCs on migration in- cant increase in the phosphorylation of Erk-1/2 was observed
serts were pretreated for 1 h with antibodies to human within 5 min of b-FGF treatment, and an increase was still
VEGFR-1 or -2 (1 ug/ml), after which the migration assay evident at 60 min (Fig. 3). b-FGF also produced a moderate
was performed with VEGF ligands as described above. increase in JNK phosphorylation as early as 5 min of in-
cubation, which peaked at about 10e15 min, and the effect
2.7. Statistical analyses continued for at least 60 min. b-FGF did not increase phos-
phorylation of Akt/PKB or P38 (data not shown). The relative
All statistical analyses were done using InStat Statistical expression levels of total Erk-1/2, Akt, P38 and JNK were not
Software V2.03 (GraphPad Software, San Diego, CA), with significantly altered after b-FGF treatment.
P values of less than 0.05 considered to be statistically We next selectively blocked the Erk-1/2, JNK, Akt, or NF-kB
significant. pathways to determine which pathway was involved in the

3. Results A
b-FGF (ng/ml) 0 0.1 1 5 10
3.1. Effect of b-FGF on NRP-1 mRNA and protein
expression in hVSMCs NRP-1

hVSMCs were treated with b-FGF for 4, 24, or 48 h. GAPDH

Expression of both NRP-1 mRNA (Fig. 1A) and protein
(Fig. 1B) was induced by b-FGF; induction levels were highest
B
at 24 h, but NRP-1 mRNA and protein levels remained elevated b-FGF (ng/ml) 0 0.1 1 5 10
for at least 48 h. To test whether this induction was dose-
NRP-1
dependent, hVSMCs were treated with increasing doses of
b-FGF (0e10 ng/ml) for 24 h. The induction of NRP-1
Actin
mRNA appeared to peak between 5 and 10 ng/ml (Fig. 2A),
and protein levels remained relatively stable between 1 and
Fig. 2. b-FGF induces NRP-1 mRNA and protein in hVSMCs. hVSMCs were
10 ng/ml (Fig. 2B). Western blotting showed that b-FGF had incubated with 0.1e10 ng/ml of b-FGF for 24 h. (A) NRP-1 mRNA levels in-
no effect on VEGFR-1 (flt-1) or VEGFR-2 (KDR) protein ex- creased after incubation with b-FGF at 5 ng/ml, with no further increases seen
pression in hVSMCs (data not shown). at 10 ng/ml. (B) NRP-1 protein was induced at as low as 1 ng/ml of b-FGF.

Time (min) 0 5 10 15 30
b-FGF (10 ng/ml) - + + + + +
Fig. 3. b-FGF increases the phosphorylation of Erk-1/2 and JNK in hVSMCs.
Cells were exposed to b-FGF (10 ng/ml) for various times and lysed for West-
ern blot analyses of signaling intermediates. The presence of b-FGF led to in-
creased levels of phosphorylated Erk and JNK as early as 5 min after exposure.
induction of NRP-1 mRNA expression in hVSMCs by b-FGF.
U0126 (MEK1/2 inhibitor) and TLCK (an NF-kB inhibitor)
nearly completely inhibited NRP-1 induction by b-FGF,
whereas wortmannin (PI3K inhibitor) and SP600125 (JNK in-
hibitor) had minimal or no effect on NRP-1 induction (Fig. 4).
3.3. Effects of b-FGF on NRP-1 transcriptional activity
and mRNA half-life
To determine the mechanism by which b-FGF induced
NRP-1 mRNA expression, we used Act D to block transcrip-
tion in hVSMCs before adding b-FGF. This blockade of tran-
scription completely abolished induction of NRP-1 mRNA by
b-FGF (Fig. 5). To determine the effect of b-FGF on the half-
life of the NRP-1 mRNA, hVSMCs were incubated in the
presence or absence of b-FGF for various times, and further
transcription was blocked with Act D. The half-life of NRP-1
mRNA was similar in b-FGF-treated cells and in controls
(data not shown).
3.4. Effects of NRP-1 upregulation by b-FGF on VEGF
induction of cell migration and proliferation
To examine whether increasing NRP-1 with b-FGF can me-
diate the biological activity of hVSMCs, cells were pretreated
U0126 (50 µM)
WT (200 nM)
SP (25 µM)
Inhibitors
-
b-FGF (10 ng/ml) - + + + + +
Fig. 4. NRP-1 induction by b-FGF is dependent on Erk-1/2 and NF-kB signal-
ing pathways in hVSMCs. hVSMCs were pretreated with different kinase
inhibitors (U0126-MEK1/2 inhibitor, SP600125 (SP)-JNK inhibitor, and wort-
mannin (WT)-PI3K inhibitor) or an NF-kB inhibitor (TLCK) for 1 h before
being treated with b-FGF (10 ng/ml) for 24 h. Inhibition of MEK1/2 and
NF-kB blocked induction of NRP-1 by b-FGF.
W. Liu et al. / Cytokine 32 (2005) 206e212 209
60 b-FGF (10 ng/ml) - + - +
Act D (0.5 µg/ml) - - + +
Phospho-Erk-1/2 NRP-1
Total-Erk-1/2 GAPDH
Phospho-JNK Fig. 5. b-FGF induction of NRP-1 is transcriptionally regulated. hVSMCs
were incubated in the presence or absence of Act D (0.5 mg/ml) 2 h before
Total-JNK exposure to b-FGF. Act D completely blocked the induction of NRP-1
mRNA expression by b-FGF.
for 24 h with or without b-FGF (10 ng/ml) in 1% DMEM/F12.
Migration assays were then performed with VEGF121 (does
not bind NRP-1) and VEGF165 (binds NRP-1) as chemoattrac-
tants. Upregulation of NRP-1 in hVSMCs after pretreatment
with b-FGF led to a significant increase in hVSMC migration
in response to VEGF165 as compared with VEGF121 or control
(Fig. 6A). To determine whether VEGFR-1 or -2 participated
in this induction of hVSMCs migration, we treated cells with
VEGFR-1 or -2 blocking antibodies prior to performing the
migration assay. Pretreatment with VEGFR-2 antibody com-
pletely blocked VEGF165-induced migration, whereas anti-
VEGFR-1 antibody had no effect. This data demonstrate that
the effect of VEGF165 on hVSMC migration was mediated
through an NRP-1/VEGFR-2 interaction (Fig. 6B).
4. Discussion
Neuropilins are receptors for two different classes of extra-
cellular ligands, the semaphorins 3Ae3F and specific VEGF
isoforms [15e17]. NRP-1 and NRP-2 mediate axon growth
and guidance in developing neurons [16,18,19]. In angiogene-
sis, the NRPs enhance the effects of specific VEGF isoforms
on endothelial cell proliferation by serving as a co-receptor
for VEGFR-2 and increasing the binding affinity of VEGF
to VEGFR-2 [4]. NRP-1 is also expressed in tumor cells and
the level of NRP-1 was found significantly higher in tumor tis-
sues than in nonmalignant tissues in several types of cancer
[4,10,11,20,21]. In human prostate carcinoma specimens,
NRP-1 overexpression correlated with Gleason score, suggest-
ing that NRP-1 overexpression is a marker of tumor aggres-
TLCK (300 µM)
siveness and metastatic potential [11]. In both in vitro and in
vivo experimental studies, overexpression of NRP-1 by human
SP (50 µM)
colon cancer cells led to significant increases in tumor growth
and angiogenesis [22]. Other studies have shown that NRP-1
might be involved in the induction of tumor invasion and an-
+ giogenesis in human breast carcinoma [23]. These findings
NRP-1 suggest that NRP-1 plays important roles in tumor progression
and metastasis.
GAPDH In the tumor neovasculature, VSMCs/pericytes are impor-
tant components of the vasculature in that they provide both
anatomic and paracrine support of the associated ECs [24].
Several studies have suggested that VSMCs/pericytes regulate
ECs proliferation, survival and migration, differentiation, and
vascular branching in tumor vessels [25e31]. Pericyte cover-
age of ECs (indicating ‘‘mature’’ vessels) may prevent ECs
210 W. Liu et al. / Cytokine 32 (2005) 206e212

A expression of NRP-1, but not that of other VEGFRs

15 (VEGFR-1 or -2) (data not shown) in hVSMCs. We then de-
(-)
termined that b-FGF led to activation of the MAPKs Erk-1/2
and JNK in hVSMCs, and that the induction of NRP-1
12 b-FGF
* mRNA caused by b-FGF was abolished by pretreatment
with U0126 (MEK1/2 inhibitor) but not by blockade of
Migrated Cells/HPF

9 JNK. Because the nuclear transcriptional factor NF-kB is

known to be involved in b-FGF-induced hVSMC proliferation
[34], we also investigated whether blocking its activity could
6 block b-FGF induction of NRP-1. TLCK, a chemical NF-kB
inhibitor, nearly completely blocked NRP-1 mRNA induction
by b-FGF. Further studies are necessary to determine whether
3
other transcription factors are also involved in the induction.
Neuropilin-1 is expressed by ECs and binds with high affin-
0 ity to VEGF165 acting as a co-receptor for VEGFR-2 [3,35].
(-) VEGF121 VEGF165 The binding of VEGF165 to NRP-1 is mediated by amino acids
residing at the carboxyl-terminal region of the exon 7-encoded
B sequence of VEGF165 [36]. In contrast, the binding of
20 (-)
VEGF121 and VEGF165 to VEGFR-1 (Flt-1) and VEGFR-2
VEGF165
(KDR/Flk-1) occurs via the peptide sequence encoded by
* exons 3 and 4 [36,37], thus enabling VEGF165 to bind to
15 both NRP-1 and VEGFR-1 or -2 simultaneously. VEGF121,
Migrated Cells/HPF

* NRP-1), does not bind to NRP-1. Thus, we used VEGF121
10
5
0
IgG VEGFR-1 AB VEGFR-2 AB
Fig. 6. NRP-1 induction enhances migration of hVSMCs in response to VEGF.
hVSMCs were pretreated with b-FGF for 24 h to upregulate NRP-1, and their
migrations were studied in response to the NRP-1 ligand VEGF165. VEGF121,
a ligand that does not bind NRP-1, and solvent alone were used as negative
controls. (A) Pretreating hVSMCs with b-FGF enhanced the effect of
VEGF165 on cell migration (*P Z 0.0002) but did not enhance migration in
response to VEGF121. (B) VEGF165 stimulated the migration of hVSMCs,
and this effect was blocked in the presence of an antibody to VEGFR-2 but
not VEGFR-1 (*P ! 0.05). There was no statistical difference in migration
between cells treated with control antibody (IgG) and VEGFR-1 antibody.
This finding demonstrates that VEGF165 mediated migration occurred via an
NRP-1/VEGFR-2 interaction.
apoptosis secondary to agents or genetic attempts to inhibit
VEGF activity [32,33]. Mature vessels are associated with
quiescent ECs with few associated sprouts [32].
It is apparent that both NRP-1 and VSMCs/pericytes seem
to have important roles in tumor growth, progression, metasta-
sis, and angiogenesis. However, NRP-1 expression and regula-
tion in hVSMCs have not been well characterized. In this
study, we observed that NRP-1 was expressed by hVSMCs
and then explored potential growth factors or cytokines that
may regulate NRP-1 expression. We then sought to determine
in vitro studies if upregulation of NRP-1 had any functional
consequences. Our data indicate that b-FGF induces the
which lacks the exon 7-encoded domain (the binding site for
and VEGF165 to determine whether the VEGF-induced migra-
tion of hVSMCs was due to the induction of NRP-1 by b-FGF.
We demonstrated that the induction of NRP-1 by b-FGF,
followed by treatment with VEGF165, led to induction of
migration. In contrast, VEGF121 did not prompt a significant
increase in migration, suggesting that the upregulation of
NRP-1 and response to VEGF were mediated by NRP-1. Re-
cently, many studies have shown that NRP-1 (as a co-receptor
for VEGF) complexes with VEGFR-2 and NRP-1 increasing
the binding affinity of VEGF165 to VEGFR-2 [4,38e42]. There-
fore, we sought to determine if VEGFR-1 or -2 (b-FGF had
no effect on VEGFR-1 or VEGFR-2 protein expression in
hVSMCs) was involved in the effect of NRP-1 on hVSMC mi-
gration. Using neutralizing antibodies to VEGFR-1 or 2, we
found that only VEGFR-2 antibody blocked VEGF165-induced
migration, suggesting that VEGF165, but not VEGF121, mediates
hVSMC migration via an NRP-1/VEGFR-2 interaction. These
findings are supported by two other reports showing that
VEGF promoted the migration (but not the proliferation) of
VSMCs [43,44].
In summary, our results demonstrate that b-FGF upregu-
lates NRP-1 via the Erk-1/2 and NF-kB pathways in hVSMCs.
This induction of NRP-1 by b-FGF can ‘‘prime’’ hVSMCs to
respond to VEGF165 to enhance migration through an NRP-1/
VEGFR-2 interaction.
Acknowledgments
The authors thank Christine Wogan from the Department of
Scientific Publications and Rita Hernandez from the Depart-
ment of Surgical Oncology at The University of Texas
M. D. Anderson Cancer Center for editorial assistance.
 W. Liu et al. / Cytokine 32 (2005) 206e212
This work was supported in part by grants from the Lock-
ton Fund for Pancreatic Cancer Research (L. M. E.), NIH
Grant T-32 CA09599 (A. A. P., J. W.), and NIH Cancer Center
Support Grant CA16672.
References
[1] Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z. Vascular endothelial
growth factor (VEGF) and its receptors. The FASEB Journal 1999;13:9.
[2] Fujisawa H, Kitsukawa T. Receptors for collapsin/semaphorins. Current
Opinion in Neurobiology 1998;8:587.
[3] Miao HQ, Klagsbrun M. Neuropilin is a mediator of angiogenesis.
Cancer and Metastasis Reviews 2000;19:29.
[4] Soker S, Takashima S, Miao HQ, Neufeld G, Klagsbrun M. Neuropilin-1
is expressed by endothelial and tumor cells as an isoform-specific recep-
tor for vascular endothelial growth factor. Cell 1998;92:735.
[5] Makinen T, Olofsson B, Karpanen T, Hellman U, Soker S, Klagsbrun M,
et al. Differential binding of vascular endothelial growth factor B splice
and proteolytic isoforms to neuropilin-1. The Journal of Biological
Chemistry 1999;274:21217.
[6] Migdal M, Huppertz B, Tessler S, Comforti A, Shibuya M, Reich R, et al.
Neuropilin-1 is a placenta growth factor-2 receptor. The Journal of
Biological Chemistry 1998;273:22272.
[7] Wise LM, Veikkola T, Mercer AA, Savory LJ, Fleming SB, Caesar C,
et al. Vascular endothelial growth factor (VEGF)-like protein from of vi-
rus NZ2 binds to VEGFR2 and neuropilin-1. Proceedings of the National
Academy of Sciences of the United States of America 1999;96:3071.
[8] Fuh G, Garcia KC, de Vos AM. The interaction of neuropilin-1 with
vascular endothelial growth factor and its receptor flt-1. The Journal of
Biological Chemistry 2000;275:26690.
[9] Miao HQ, Lee P, Lin H, Soker S, Klagsbrun M. Neuropilin-1 expression
by tumor cells promotes tumor angiogenesis and progression. The
FASEB Journal 2000;14:2532.
[10] Fakhari M, Pullirsch D, Abraham D, Paya K, Hofbauer R, Holzfeind P,
et al. Selective upregulation of vascular endothelial growth factor recep-
tors neuropilin-1 and -2 in human neuroblastoma. Cancer 2002;94:258.
[11] Latil A, Bieche I, Pesche S, Valeri A, Fournier G, Cussenot O, et al.
VEGF overexpression in clinically localized prostate tumors and neuro-
pilin-1 overexpression in metastatic forms. International Journal of
Cancer 2000;89:167.
[12] Bachelder RE, Crago A, Chung J, Wendt MA, Shaw LM, Robinson G,
et al. Vascular endothelial growth factor is an autocrine survival factor
for neuropilin-expressing breast carcinoma cells. Cancer Research
2001;61:5736.
[13] Parikh AA, Liu W, Fan F, Stoeltzing O, Reinmuth N, Bruns CJ, et al.
Expression and regulation of the novel vascular endothelial growth factor
receptor neuropilin-1 by epidermal growth factor in human pancreatic
carcinoma. Cancer 2003;98:720.
[14] Liu W, Reinmuth N, Stoeltzing O, Parikh AA, Tellez C, Williams S, et al.
Cyclooxygenase-2 is up-regulated by interleukin-1 beta in human colo-
rectal cancer cells via multiple signaling pathways. Cancer Research
2003;63:3632.
[15] Kolodkin AL, Levengood DV, Rowe EG, Tai YT, Giger RJ, Ginty DD.
Neuropilin is a semaphorin III receptor. Cell 1997;90:753.
[16] He Z, Tessier-Lavigne M. Neuropilin is a receptor for the axonal chemo-
repellent semaphorin III. Cell 1997;90:739.
[17] Nakamura F, Kalb RG, Strittmatter SM. Molecular basis of semaphorin-
mediated axon guidance. Journal of Neurobiology 2000;44:219.
[18] Chen H, Chedotal A, He Z, Goodman CS, Tessier-Lavigne M. Neuropi-
lin-2, a novel member of the neuropilin family, is a high affinity receptor
for the semaphorins sema E and sema IV but not sema III [erratum
appears in Neuron 1997 Sep; 19(3):559]. Neuron 1997;19:547.
[19] Fujisawa H, Kitsukawa T, Kawakami A, Takagi S, Shimizu M, Hirata T.
Roles of a neuronal cell-surface molecule, neuropilin, in nerve fiber fas-
ciculation and guidance. Cell and Tissue Research 1997;290:465.
211
[20] Lantuejoul S, Constantin B, Drabkin H, Brambilla C, Roche J,
Brambilla E. Expression of VEGF, semaphorin SEMA3F, and their com-
mon receptors neuropilins NP1 and NP2 in preinvasive bronchial le-
sions, lung tumours, and cell lines. The Journal of Pathology 2003;
200:336.
[21] Ding H, Wu X, Roncari L, Lau N, Shannon P, Nagy A, et al. Expression
and regulation of neuropilin-1 in human astrocytomas. International
Journal of Cancer 2000;88:584.
[22] Parikh AA, Fan F, Liu WB, Ahmad SA, Stoeltzing O, Reinmuth N, et al.
Neuropilin-1 in human colon cancer: expression, regulation, and role
in induction of angiogenesis. American Journal of Pathology
2004;164:2139.
[23] Stephenson JM, Banerjee S, Saxena NK, Cherian R, Banerjee SK.
Neuropilin-1 is differentially expressed in myoepithelial cells and vascu-
lar smooth muscle cells in preneoplastic and neoplastic human breast:
a possible marker for the progression of breast cancer. International
Journal of Cancer 2002;101:409.
[24] Hirschi KK, Rohovsky SA, Beck LH, Smith SR, D’Amore PA. Endothe-
lial cells modulate the proliferation of mural cell precursors via platelet-
derived growth factor-BB and heterotypic cell contact. Circulation
Research 1999;84:298.
[25] Nicosia RF, Villaschi S. Rat aortic smooth muscle cells become pericytes
during angiogenesis in vitro. Laboratory Investigation 1995;73:658.
[26] Beck Jr L, D’Amore PA. Vascular development: cellular and molecular
regulation. The FASEB Journal 1997;11:365.
[27] Benjamin LE, Golijanin D, Itin A, Pode D, Keshet E. Selective ablation
of immature blood vessels in established human tumors follows vascular
endothelial growth factor withdrawal [see comment]. The Journal of
Clinical Investigation 1999;103:159.
[28] Benjamin LE, Hemo I, Keshet E. A plasticity window for blood vessel
remodelling is defined by pericyte coverage of the preformed endothelial
network and is regulated by PDGF-B and VEGF. Development
1998;125:1591.
[29] Nehls V, Schuchardt E, Drenckhahn D. The effect of fibroblasts, vascular
smooth muscle cells, and pericytes on sprout formation of endothelial
cells in a fibrin gel angiogenesis system. Microvascular Research
1994;48:349.
[30] Hellstrom M, Gerhardt H, Kalen M, Li X, Eriksson U, Wolburg H, et al.
Lack of pericytes leads to endothelial hyperplasia and abnormal vascular
morphogenesis. The Journal of Cell Biology 2001;153:543.
[31] Hirschi KK, Rohovsky SA, D’Amore PA. PDGF, TGF-beta, and hetero-
typic cellecell interactions mediate endothelial cell-induced recruitment
of 10T1/2 cells and their differentiation to a smooth muscle fate [erratum
appears in J Cell Biol 1998 Jun 1; 141(5):1287]. The Journal of Cell
Biology 1998;141:805.
[32] Gee MS, Procopio WN, Makonnen S, Feldman MD, Yeilding NM,
Lee WM. Tumor vessel development and maturation impose limits on
the effectiveness of anti-vascular therapy. American Journal of Pathology
2003;162:183.
[33] McCarty MF, Takeda A, Stoeltzing O, Liu W, Fan F, Reinmuth N, et al.
ZD6126 inhibits orthotopic growth and peritoneal carcinomatosis in
a mouse model of human gastric cancer. British Journal of Cancer
2004;90:705.
[34] Hoshi S, Goto M, Koyama N, Nomoto K, Tanaka H. Regulation of vas-
cular smooth muscle cell proliferation by nuclear factor-kappaB and its
inhibitor, I-kappaB. The Journal of Biological Chemistry 2000;275:883.
[35] Soker S, Fidder H, Neufeld G, Klagsbrun M. Characterization of novel
vascular endothelial growth factor (VEGF) receptors on tumor cells
that bind VEGF165 via its exon 7-encoded domain. The Journal of Bio-
logical Chemistry 1996;271:5761.
[36] Soker S, Gollamudi-Payne S, Fidder H, Charmahelli H, Klagsbrun M.
Inhibition of vascular endothelial growth factor (VEGF)-induced
endothelial cell proliferation by a peptide corresponding to the exon 7-
encoded domain of VEGF165. The Journal of Biological Chemistry
1997;272:31582.
[37] Keyt BA, Nguyen HV, Berleau LT, Duarte CM, Park J, Chen H, et al.
Identification of vascular endothelial growth factor determinants for
binding KDR and FLT-1 receptors. Generation of receptor-selective
212 W. Liu et al. / Cytokine 32 (2005) 206e212
VEGF variants by site-directed mutagenesis. The Journal of Biological
Chemistry 1996;271:5638.
[38] Poltorak Z, Cohen T, Neufeld G. The VEGF splice variants: properties,
receptors, and usage for the treatment of ischemic diseases. Herz
2000;25:126.
[39] Gluzman-Poltorak Z, Cohen T, Shibuya M, Neufeld G. Vascular endothe-
lial growth factor receptor-1 and neuropilin-2 form complexes. The
Journal of Biological Chemistry 2001;276:18688.
[40] Soker S, Miao HQ, Nomi M, Takashima S, Klagsbrun M. VEGF165 me-
diates formation of complexes containing VEGFR-2 and neuropilin-1 that
enhance VEGF165-receptor binding. Journal of Cellular Biochemistry
2002;85:357.
[41] Neufeld G, Kessler O, Herzog Y. The interaction of neuropilin-1 and
neuropilin-2 with tyrosine-kinase receptors for VEGF. Advances in
Experimental Medicine and Biology 2002;515:81.
[42] Rollin S, Lemieux C, Maliba R, Favier J, Villeneuve LR, Allen BG, et al.
VEGF-mediated endothelial P-selectin translocation: role of VEGF
receptors and endogenous PAF synthesis. Blood 2004;103:3789.
[43] Grosskreutz CL, Anand-Apte B, Duplaa C, Quinn TP, Terman BI, Zetter B,
et al. Vascular endothelial growth factor-induced migration of vascular
smooth muscle cells in vitro. Microvascular Research 1999;58:128.
[44] Wang H, Keiser JA. Vascular endothelial growth factor upregulates the
expression of matrix metalloproteinases in vascular smooth muscle cells:
role of flt-1. Circulation Research 1998;83:832.