The FASEB Journal • Research Communication
Functional interaction of VEGF-C and VEGF-D with
neuropilin receptors
Terhi Ka¨rpa¨nen,*,1 Caroline A. Heckman,*,1 Salla Keskitalo,* Michael Jeltsch,*
Hanna Ollila,* Gera Neufeld,§ Luca Tamagnone,! and Kari Alitalo*,2
*Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Biomedicum
Helsinki, Haartman Institute and Helsinki University Central Hospital, University of Helsinki,
Helsinki, Finland; §Department of Cell Biology and Anatomy, the Rappaport Family Institute for
Research in the Medical Sciences, Technion, Israel Institute of Technology, Haifa, Israel; and
!Institute for Cancer Research and Treatment (IRCC), University of Turin Medical School, Candiolo,
Turin, Italy
ABSTRACT Lymphatic vascular development is reg-
ulated by vascular endothelial growth factor receptor-3
(VEGFR-3), which is activated by its ligands VEGF-C
and VEGF-D. Neuropilin-2 (NP2), known to be in-
volved in neuronal development, has also been impli-
cated to play a role in lymphangiogenesis. We aimed to
elucidate the mechanism by which NP2 is involved in
lymphatic endothelial cell signaling. By in vitro binding
studies we found that both VEGF-C and VEGF-D inter-
act with NP2, VEGF-C in a heparin-independent and
VEGF-D in a heparin-dependent manner. We also
mapped the domains of VEGF-C and NP2 required for
their binding. The functional importance of the inter-
action of NP2 with the lymphangiogenic growth factors
was demonstrated by cointernalization of NP2 along
with VEGFR-3 in endocytic vesicles of lymphatic endo-
thelial cells upon stimulation with VEGF-C or VEGF-D.
NP2 also interacted with VEGFR-3 in coprecipitation
studies. Our results show that NP2 is directly involved
in an active signaling complex with the key regulators of
lymphangiogenesis and thus suggest a mechanism by
which NP2 functions in the development of the lym-
phatic vasculature.—Ka¨rpa¨nen, T., Heckman, C. A.,
Keskitalo, S., Jeltsch, M., Ollila, H., Neufeld, G., Ta-
magnone, L., Alitalo, K. Functional interaction of
VEGF-C and VEGF-D with neuropilin receptors. FASEB
J. 20, 1462–1472 (2006)
Key Words: lymphatic endothelial cell ! VEGFR-3 ! NP2
Early development of the vascular system is depen-
dent on the vascular endothelial growth factor (VEGF)
family of ligands and their receptors. Deletion of VEGF
receptor-3 (VEGFR-3) is embryonic lethal due to im-
proper remodeling of the primary vascular plexus (1).
The formation of lymphatic vessels, which occurs later
in development after formation of the cardiovascular
system, is also dependent on VEGFR-3 activity (2, 3).
Missense mutations of the gene encoding VEGFR-3 that
result in impaired signaling from the receptor can lead
to hypoplasia of the lymphatic vasculature and to
congenital lymphedema (4).
Activation of VEGFR-3 is dependent on binding of its
ligands, VEGF-C and VEGF-D. Both factors are angio-
genic and lymphangiogenic, and VEGF-C is critical for
embryonic development (3, 5, 6). The full-length hu-
man VEGF-C and VEGF-D proteins are 48% homolo-
gous and share a similar structure with a VEGF homol-
ogy domain flanked by amino-terminal and carboxy-
terminal propetides. Upon secretion, the propeptides
are enzymatically cleaved, resulting in the mature sig-
naling molecules with increased affinities to both
VEGFR-2 and VEGFR-3 (7, 8).
In addition to VEGF receptors, several VEGF family
members also interact with neuropilins. Neuropilins
are transmembrane non-tyrosine kinase glycoproteins
with a short cytoplasmic domain that has limited signal-
ing capability. In the nervous system, neuropilins me-
diate axon retraction and guidance by binding class III
semaphorins and interacting with members of the
plexin receptor family, which act as the signal transduc-
ers (9). In the vascular system, neuropilin-1 (NP1) is
expressed in arteries (10) whereas NP2 expression is
restricted to the lymphatic system and at low levels to
veins (11, 12). Along with class III semaphorins, NP1
also binds VEGF165, VEGF-B167, VEGF-B186, VEGF-C,
and placenta growth factor-2 (PlGF-2) (13–16), whereas
NP2 binds VEGF165, VEGF145, PlGF-2, and VEGF-C (17,
18). NP1 was previously shown to enhance the interac-
tion of VEGF165 with VEGFR-2 and to promote endo-
thelial cell proliferation and migration (13,19). While
overexpression or deletion of NP1 in mice results in
lethal neuronal and vascular defects (20–22), mice
deficient for NP2 are viable, displaying mild neuronal
abnormalities (23, 24). In addition, NP2 null mice lack
small lymphatic vessels and capillaries at birth, while
larger lymphatic vessels, along with arteries and veins,
remain intact (12). This phenotype suggests that NP2 is
1 These authors contributed equally to this work.
2 Correspondence: Molecular/Cancer Biology Laboratory,
Biomedicum Helsinki, P.O.B. 63, FI-00014 University of Hel-
sinki, Helsinki, Finland. E-mail: kari.alitalo@helsinki.fi
doi: 10.1096/fj.05-5646com
1462 0892-6638/06/0020-1462 © FASEB

required for proper early development of the lymphatic
system, but the underlying molecular mechanisms are
still unclear.
For the present studies, we wanted to assess the
interaction of neuropilins with lymphangiogenic
growth factors and their lymphatic endothelial-specific
receptor VEGFR-3. By in vitro binding we found that
both NP1 and NP2 bind VEGF-C and VEGF-D. We also
analyzed the heparin dependency of as well as the
domains involved in these interactions. Furthermore,
we found that NP2 colocalizes to endocytic vesicles with
VEGF-C or VEGF-D after stimulation of primary lym-
phatic endothelial cells, and observed the interaction
of NP2 with VEGFR-3. Together, these results not only
indicate that VEGF-C and VEGF-D may in part promote
angiogenic signaling by interacting with NP1, but lym-
phangiogenesis could be modified by the interaction of
NP2 with VEGF-C and VEGF-D along with their recep-
tor, VEGFR-3.
MATERIALS AND METHODS
Proteins and antibodies
To produce human VEGF-C in Drosophila S2 cells, the pMT-
BiP-V5His-C vector was modified to include the hygromycin
resistance gene from pCoHygro (both Invitrogen, Carlsbad,
CA, USA). Into this resulting vector we cloned VEGF-C
nucleotides 658–996 (GenBank accession number X94216)
preceded by the melittin signal peptide and followed by a
hexahistidine tag. S2 cells were transfected using Effectene
(Qiagen, Hilden, Germany). Selection was started 3 days
post-transfection with 400 !g/ml hygromycin and the me-
dium was changed every 5 days for 3 wk, after which the
supernatant was assayed for protein. For this, the cells were
induced for 5 days with 0.5 mM CuSO4, and 15 !l of the
conditioned medium was run through 14% SDS-PAGE under
reducing conditions. The proteins were identified by Western
analysis using both pentahistidine antibody (Ab) (Qiagen)
and the specific VEGF-C antiserum 882 (7). For medium scale
production, cells were adapted to suspension culture, ex-
panded to 1 l, and induced at a density of 4 " 106 cells/ml.
The supernatant was harvested 4.5 days postinduction,
cleared by centrifugation, and dialyzed against 50 mM sodium
phosphate/300 mM NaCl, pH 6. After dialysis the pH was
adjusted to 8.0 by adding 3M Tris/HCl pH 10.5 and the
supernatant was centrifuged at 15,000 g for 30 min to
eliminate precipitate. Batch binding was performed adding 2
ml Ni2#NTA superflow resin (Qiagen), followed by gentle
agitation for 12 h at #4°C. The resin was loaded onto a
column, washed with 20 mM imidazole, and eluted with a step
gradient of 200 mM imidazole. The protein was dialyzed
against PBS and sterilized using Millex-GV filters (Millipore,
Billerica, MA, USA), then checked on silver-stained reducing
SDS-PAGE gels and quantitated using the bicinchoninic acid
protein assay (Pierce Biotechnology, Rockford, IL, USA).
Activity was determined using a bioassay as described (2).
Purified human VEGF165 and VEGF-D as well as goat
polyclonal antibodies against human VEGF-C and VEGF-D
were purchased from R&D Systems (Minneapolis, MN, USA).
Mouse monoclonal antibodies against human VEGFR-3,
9D9F9 and 2E11D11, were prepared as described (25). Rabbit
polyclonal antibodies against NP2 (H-300) and VEGFR-3
(C-20) were from Santa Cruz Biotechnology (Santa Cruz, CA,
USA), while the rabbit polyclonal antibody for EEA-1 was
from Abcam (Cambridge, UK). Rat monoclonal (1121B) and
rabbit polyclonal (RS-2) antibodies against VEGFR-2 have
been described (26, 27). AlexaFluor488 donkey antimouse,
AlexaFluor594 donkey anti-rabbit, and AlexaFluor594 donkey
anti-goat secondary antibodies were from Molecular Probes
(Eugene, OR, USA).
Cells and cell culture
293T cells were maintained in Dulbecco’s modified Eagle’s
medium (HaartBio, Helsinki, Finland) supplemented with
10% fetal calf serum (PromoCell, Heidelberg, Germany), 2
mM L-glutamine, and 0.2% penicillin/streptomycin sulfate
(HaartBio). Human dermal microvascular endothelial cells
(HDMVECs) were maintained in endothelial cell medium
(both PromoCell) and used at passages 3–7. Porcine aortic
endothelial (PAE) cells were kindly provided by Lena Claes-
son-Welsh (Uppsala University, Sweden) and cultured in F12
medium (HaartBio) supplemented as above.
PAE cells stably expressing VEGFR-3 (R3-PAE) were de-
scribed previously (28) and maintained with 0.25 !g/ml
puromycin (Sigma-Aldrich, St. Louis, MO, USA). To establish
PAE and R3-PAE cells stably expressing NP2, the pcDNA3.1z-
NP2 plasmid described below was transfected into these cells
using JetPEI (Qbiogene, Irvine, CA, USA). Selection antibi-
otics (NP2-PAE, 200 !g/ml zeocin (Invitrogen); R3/NP2-
PAE, 200 !g/ml zeocin and 0.25 !g/ml puromycin) were
added to the media 48 h after transfection and the cells
grown for 14 days, after which clones were picked. NP2 and
VEGFR-3 expression was confirmed by Western analysis.
Expression plasmids
Expression plasmids encoding full-length human VEGF-C
(29), human semaphorin 3F (SEMA3F) (30) and full-
length human VEGF-D (31) have been described. Expres-
sion plasmids coding for VEGF-C mutants were constructed
by amplifying the VEGF-C cDNA with the primers
5$-GCGGATCCGACAGAAGAGACTATAAAA-3$ and 5$-
GCGGATCCTTAGCTCATTTGTGGTCTTTTCC-3$ for
VEGF-C%N and 5$-GGGATCCGTTCGAGTCCGGACTCG-3$
and 5$-GCGGATCCTTAACGTCTAATAATGGAATG-3$ for
VEGF-C%C. Polymerase chain reaction (PCR) products were
cloned in frame with the immunoglobulin (Ig)& signal se-
quence to the BamHI site of the pMosaic vector (32). The
expression plasmid coding for VEGF-C%N%C was constructed
by inserting the BamHI-restricted PCR product obtained with
oliogonucleotide primers 5$-GGAATTCACAGAAGAGAC-
TATAAA-3$ and 5$-GCGGATCCTTAACGTCTAATAATG-
GAA-3$ into BamHI/EcoRV-opened pAdCMV (29). The result-
ing vector was opened with HindIII, blunted with Klenow
enzyme, digested with BglII, and the sequence coding for the
signal peptide of human VEGF-C inserted as an Age-
I(blunted)/BglII fragment obtained from DN VEGF-C (7).
The expression plasmid coding for human NP2 immuno-
globulin has been described (18). The cDNA encoding the
extracellular domain of human NP1 was assembled by recom-
binant PCR from Integrated Molecular Analysis of Genomes
and their Expression (IMAGE) Consortium cDNA clone
2958475 (Incyte Genomics, St. Louis, MO, USA) containing
the sequences coding for the a1a2b1b2 domains and a cDNA
fragment coding for the MAM domain obtained by RT-PCR of
RNA extracted from HDMVECs with oligonucleotide primers
5$-CCTAGCTAGCCGCAACGATAAATGTGGCGATAC-3$ and
5$-CCTGTGAGCTGGAAGTCATCACCTGTTCCACTGTG-
GCAGTTGGCCTGGTCGTC-3$ as well as 5$-GTGATGACGAC-
CAGGCCAACTGCCACAGTGGAACAGGTGATGACT-
1463NEUROPILIN INTERACTIONS WITH VEGF-C AND VEGF-D