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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Cell Differentiation, Institute
of Molecular Embryology and Genetics, and the Department of
Cardiovascular Medicine, Kumamoto University School of Medicine,
Kumamoto, Japan; and the Group of Developmental Neurobiology, Division
of Biological Science, Nagoya University Graduate School of Science,
Japan.
Neuropilin 1 (NP-1) is a receptor for vascular endothelial growth
factor (VEGF) 165 (VEGF165) and acts as a coreceptor that enhances VEGF165 function through tyrosine kinase VEGF
receptor 2 (VEGFR-2). Transgenic overexpression of np-1
results in an excess of capillaries and blood vessels and a malformed
heart. Thus, NP-1 may have a key role in vascular development. However,
how NP-1 regulates vascular development is not well understood. This study demonstrates how NP-1 can regulate vasculogenesis and
angiogenesis in vitro and in vivo. In homozygous np-1
mutant (np-1 The formation of blood vessels begins during early
mammalian embryogenesis with differentiation of mesodermally derived
primitive angioblasts into mature endothelial cells (ECs). At first,
those ECs form a primitive vascular structure, a process termed
vasculogenesis.1 Subsequently, ECs from these primitive
vessels proliferate and sprout to form a branching network of
capillaries during the process of angiogenesis.2 Finally,
mural cells (pericytes and vascular smooth muscle cells) and
fibroblasts are recruited around vessels and stabilized vessels are
built.3
During the past several years, much progress has been made in defining
vasculogenesis and angiogenesis by isolating factors such as a family
of vascular EC growth factors (VEGFs), angiopoietins, and
ephrins.4,5 Gene-targeting strategies revealed that these factors and their receptors were important in the development of the
vascular system. VEGFs and their receptor tyrosine kinases, tyrosine
kinase VEGF receptor 1 (VEGFR-1) and tyrosine kinase VEGF receptor 2 (VEGFR-2), are expressed specifically on the surface of ECs and were
shown to be required for both vasculogenesis and angiogenesis.6-9 More recently, neuropilin 1 (NP-1) was
found to be expressed on ECs and tumor cells10 and shown to
be the third VEGF receptor.
In contrast to VEGFR-1 and VEGFR-2, NP-1 binds the 165-amino acid form
of VEGF (VEGF165) but not the 121-amino acid form
(VEGF121). It was reported that the binding of
VEGF165 to NP-1 occurs by means of VEGF exon 7, in contrast
to VEGFR-1 and VEGFR-2, which bind VEGF165 by means of VEGF
exons 3 and 4, respectively.11-13 When NP-1 was
coexpressed on VEGFR-2-positive (VEGFR-2+) ECs, the
binding to and chemotactic activity of VEGF165 for these
cells were enhanced compared with those for ECs expressing VEGFR-2
alone.10 In vivo, overexpression of the np-1
gene in mice led to an excess of capillaries and blood vessels and
a malformed heart.14 These results suggest that NP-1 works
as a coreceptor with VEGFR-2 on ECs and enhances the function of
VEGF165.
Originally, NP-1 was identified as a receptor for the axon
chemorepellent semaphorin III.15,16 Semaphorin III can
induce neuronal growth-cone collapse and repulsion of neurites in
vitro. Studies using nonneuronal cells suggested that semaphorin III and VEGF165 bind NP-1 competitively.17
Recombinant semaphorin III inhibited the motility of porcine aortic ECs
(PAECs) expressing NP-1 alone or coexpressing VEGFR-2 and NP-1 but not
parental PAECs or PAECs expressing VEGFR-2 alone. Although it is not
clear whether VEGF165 acts on neuronal cells expressing
NP-1, a complex system that diversifies EC development using ligands,
such as VEGF; semaphorin III; and receptors, such as VEGFR-2 and NP-1,
may exist.
Np-1 Mice
Immunohistochemical analysis
Production of recombinant fusion proteins Recombinant fusion proteins of the full-length extracellular domain of murine surface molecules and the Fc part of human IgG and Flag epitope were designed (Figure 2, Shimizu et al23). NP-1-Fc, CD4-Fc, NP-1-Flag, and control Flag were produced by COS7 cells in serum-free conditioned medium as described previously.23,24 Fc fusion protein was purified over a protein A column (Bio-Rad, Richmond, CA) and Flag fusion protein was purified over an anti-Flag M2 column (Scientific Imaging System; Eastman Kodak, Rochester, NY). Their purity and disulfide-linked dimerization were assessed by Coomassie brilliant Blue staining of 6% sodium dodecyl sulfate (SDS) gels.In vitro culture of P-Sp The stromal cell line OP925 was maintained in modified minimum essential media (-MEM; Gibco BRL, Gaithersburg, MD)
supplemented with 20% fetal-calf serum (FCS; JRH Bioscience, Lenexa,
KS). P-Sp explants from E9.5 containing a part of the omphalomesenteric artery were cultured on OP9 stromal cells in 10% FCS and
10 5 M 2-mercaptoetanol (2-ME; Gibco BRL) containing RPMI
1640 (Gibco BRL) with or without full-length VEGF (Pepro Tech EC,
London, United Kingdom) and NP-1-Fc or CD4-Fc, and NP-1-Flag or
control Flag. After 14 days in culture, an anti-PECAM-1 antibody
(MEC13.3 rat antimouse mAb; Pharmingen) was used to visualize ECs.
Quantitative analysis of vascular areas Immunohistochemical analysis in the P-Sp culture using PECAM-1 antibodies was done as described above. After PECAM-1 immunohistochemical staining, images were integrated by using a color chilled 3CCD camera (Hamamatsu Photonics, Shizuoka, Japan). Image-processing software (NIH Image version 1.62 on a Power Macintosh G3; National Institutes of Health, Bethesda, MD) was used to determine alterations in the size of vascular areas. Three vascular areas from each P-Sp explant were measured under each culture condition. All values are shown as the mean ± SD. P values were calculated using 2-tailed Student t tests.Cell preparation and flow cytometry Embryos were staged by means of somite counting. Whole-mount embryos of wild-type and np-1 mutants were dissociated using Dispase II (Boehringer Mannheim, Mannheim, Germany) and drawn through a 23-gauge needle. The cell-staining procedure for the flow cytometry analysis was as described previously.26 The mAbs used in immunofluorescence staining were anti-VEGFR-2 antibody (Pharmingen) and anti-PECAM-1 antibody (Pharmingen). All mAbs were purified and conjugated with phycoerythrin (PE) or biotin. Biotinylated antibodies were visualized with PE-conjugated streptavidin (Gibco BRL) or allophycocyanin-conjugated streptavidin (Caltag, Burlingame, CA). Cells were incubated for 15 minutes on ice with CD16/32 (Fc III/II
receptor, 1:100 [Fcblock; Pharmingen]) before staining with primary
antibody. Cells were then incubated in 5% FCS and PBS (washing buffer)
with primary antibody for 30 minutes on ice and washed twice with
washing buffer. Secondary antibody was added and the cells were
incubated for 30 minutes on ice. After incubation, cells were washed
twice with washing buffer and then suspended in washing buffer for
fluorescence-activated cell-sorter (FACS) analysis. The stained cells
were analyzed and sorted using FACSvantage (Becton Dickinson, San Jose,
CA). Sorted VEGFR-2+ PECAM-1-positive
(PECAM-1+) cells were cultured on OP9 cells in 10% FCS and
105 M 2-ME containing RPMI 1640 supplemented with VEGF (1 ng/mL) and NP-1-Fc (50 µg/mL) or CD4-Fc (50 µg/mL) at 37°C in a
5% carbon dioxide (CO2) incubator.
Immunoprecipitation and immunoblotting Dissociated cells from E12.5 wild-type or np-1 mutant embryos were cultured on OP9 cells in 10% FCS and 105 M
2-ME containing RPMI 1640 supplemented with VEGF (10 ng/mL) and basic
fibroblast growth factor (bFGF [10 ng/mL]; Pepro Tech EC) at 37°C
in a 5% CO2 incubator. After 7 days, cells were harvested, labeled with PECAM-1-PE and VEGFR-2-biotin antibodies, and sorted using FACSvantage. VEGFR-2+ PECAM-1+ cells were
transferred to fibronectin-coated dishes (Iwaki Glass, Chiba, Japan)
and cultured in serum-free conditioned medium for 24 hours. ECs of
wild-type or np-1 mutants were stimulated with 1 ng/mL VEGF
in the presence or absence of 50 µg/mL NP-1-Fc for 10 minutes at
37°C. Immunoprecipitation and immunoblotting were done as described
previously.27
Injection of NP-1-Fc into mice Np-1+/ male and female mice were mated
and checked for plugs the next morning (designated as E0.5). From E9.5
to 11.5, pregnant mice were given daily intraperitoneal injections of
150 µL of a 2-mg/mL solution of NP-1-Fc or CD4-Fc (control protein).
Embryos were harvested on E12.5 and immunohistochemical analyses on
whole-mount embryos or sections was done by using PECAM-1 monoclonal
antibody as described previously.20
P-Sp explant culture system reflects the vascular defect in np-1 mutant embryos To examine the vascularity of the homozygous np-1 mutant (np-1/) embryos, E12.0
np-1/ and wild-type
(np-1+/+) embryos from the same litter were
stained with PECAM-1 mAb to visualize all ECs. No growth retardation
was observed in the mutants (data not shown). However, the vascularity
of mutant embryos compared with wild-type embryos (Figure
1A,C) was impaired in the CNS and pericardial regions (Figure 1B,D). At E12.0, vascular sprouting into
the CNS and pericardium was observed in wild-type embryos, whereas few
capillaries and branches were observed in the mutants. To examine the
vasculogenesis and angiogenesis of the np-1/
embryos more precisely, P-Sp explants from E9.5 wild-type and homozygous mutant embryos were cultured with OP9 stromal cells supplemented with interleukin (IL) 6, IL-7, stem cell factor, and
erythropoietin. We previously reported that this culture system supported the growth of both hematopoietic cells and
ECs.20,21 After 10 to 14 days, ECs in the wild-type
embryos migrated from the P-Sp explant and formed vascular beds
(sheetlike structures) and networks (cordlike structures) on OP9 cells
(Figure 1E). In contrast, in P-Sp explants from mutant embryos, neither
vascular beds nor networks formed, although a small number of ECs
developed (Figure 1F).
Structure of NP-1 recombinant proteins To analyze NP-1 function in vitro in P-Sp cultures, we produced recombinant fusion proteins containing the Fc part of human IgG1 or Flag and the full-length of the NP-1 ectodomain (NP-1-Fc and NP-1-Flag; Figure 2A). We verified the molecular size of each protein by using SDS-polyacrylamide gel electrophoresis. The molecular size of each protein was the expected size under both reducing and nonreducing conditions (Figure 2B). We then confirmed, using Western blotting (reducing and nonreducing conditions), that NP-1-Fc formed a dimer NP-1 and NP-1-Flag formed a monomer NP-1 (Figure 2C).
Effects of soluble NP-1 on vasculogenesis and angiogenesis in P-Sp cultures We previously reported that when soluble receptors, such as VEGFR-2 or Tie-2, were added to the P-Sp culture system and free ligands were saturated in the culture, EC development was suppressed.20 To test whether soluble NP-1 in P-Sp cultures prevented vascular development by inhibiting the binding of VEGF165 to VEGFR-2, excess amounts of NP-1-Flag or NP-1-Fc were added to the P-Sp cultures of the wild-type embryos. We measured the VEGF concentration in the P-Sp cultures by using enzyme-linked immunosorbent assay and confirmed that 1.2 ± 0.4 ng/mL (n = 5) VEGF was produced in wild-type cultures and 1.5 ± 0.3 ng/mL (n = 5) VEGF was produced in mutant cultures.On addition of 50 µg/mL NP-1-Flag to these cultures, vascular-bed
formation was suppressed (Figure 3C), but
unexpectedly, on addition of 50 µg/mL NP-1-Fc or NP-1-Flag plus
anti-Flag IgG (dimer form of NP-1-Flag) to this culture, vascular-bed
formation was expanded in the wild-type embryos (Figure 3E,G). In
np-1 mutants, suppressed EC development was completely
rescued by addition of NP-1-Fc or NP-1-Flag plus anti-Flag IgG
(Figure 3F,H), whereas the same amount of CD4-Fc (data not shown), Flag
(Figure 3B), or anti-Flag alone (data not shown) as a control protein
had no effect.
Effect of dimer of soluble NP-1 on the growth of sorted ECs P-Sp explants contain many types of cells, including hematopoietic cells, endothelial progenitor cells, and other mesenchymal cells. To clarify whether NP-1-Fc affects ECs directly to induce vasculogenesis and angiogenesis, we sorted ECs from E12.5 wild-type and mutant embryos using FACS and cultured them on OP9 cells in the presence or absence of NP-1-Fc. Dual staining with anti-VEGFR-2 and PECAM-1 mAbs revealed that 0.3% of cells in wild-type embryos and 0.5% of cells in mutant embryos were double positive (Figure 4A). VEGFR-2+ PECAM-1+ cells were then cultured on OP9 stromal cells in the presence of 1 ng/mL VEGF (Figure 4B). VEGFR-2+ PECAM-1+ cells from wild-type embryos formed vascular beds and networks (Figure 4Bi), whereas ECs from mutant embryos did not proliferate in the presence of 1 ng/mL VEGF (Figure 4Bii). However, 50 µg/mL NP-1-Fc in addition to 1 ng/mL VEGF promoted vascular development (Figure 4Biii) as observed in cultures of ECs from wild-type embryos (Figure 4Bi). The same amount of CD4-Fc had no effect (data not shown). These findings indicate that NP-1-Fc enhances the proliferation of individual ECs through VEGFR-2.
To examine the role of NP-1-Fc in VEGFR-2 activation, we evaluated phosphorylation of VEGFR-2 on the ECs. Whole E12.5 wild-type and mutant embryos were dissociated and then cultured on OP9 stromal cells in the presence of 10 ng/mL VEGF and bFGF for 7 days. The VEGFR-2+ PECAM-1+ ECs were then sorted with FACS, seeded on fibronectin-coated dishes, cultured for 24 hours under serum-free conditions, and subsequently challenged with VEGF. Although ECs from mutant embryos expressed slightly more VEGFR-2 than those from wild-type embryos (Figure 4A), this difference was lost after culturing in vitro (data not shown). Cell lysates were immunoprecipitated with an anti-VEGFR-2 antibody and then subjected to Western blotting using an antiphosphotyrosine mAb. Phosphorylation of VEGFR-2 was induced by addition of a low dose of VEGF and NP-1-Fc in ECs from np-1 mutants (Figure 4C; lane 4). VEGF alone did not induce phosphorylation of VEGFR-2 in ECs from np-1 mutants (Figure 4C; lane 3), whereas VEGF alone did induce phosphorylation in samples from wild-type embryos (Figure 4C; lane1). Synergistic effect of VEGF and dimer of soluble NP-1 on EC growth We next examined the synergistic effect of NP-1-Fc and VEGF in P-Sp cultures. P-Sp explants from wild-type (Figure 5Ai,iii,v,vii) and mutant (Figure 5Aii,iv,vi,viii) embryos on E9.5 were cultured on OP9 stromal cells as described above. The suppressed vasculature in the culture of np-1/ embryos (Figure 5Aii) was rescued by
addition of 50 ng/mL VEGF (Figure 5Aiv) or 50 µg/mL NP-1-Fc (Figure
5Avi). The pattern of the rescue with VEGF differed from that with
NP-1-Fc. When VEGF alone was added to P-Sp cultures of
np-1/ embryos (Figure 5Aiv), the vascular
bed was poorly formed compared with that of wild-type embryos (Figure
5Ai). In contrast, on addition of NP-1-Fc, vascular-bed and
vascular-network formation rose to a level comparable to that in
wild-type embryos (Figure 5Ai,vi). Of particular note, vascular
formation was expanded by simultaneous addition of VEGF and NP-1-Fc in
both wild-type (Figure 5Avii) and np-1 mutant explants
(Figure 5Aviii). Vascular areas from each P-Sp explant were measured
under each culture condition. Quantitative analyses showed that they
were increased in the presence of VEGF and NP-1-Fc (Figure 5B). This
suggests that VEGF and NP-1-Fc work synergistically in
vascular formation.
In vivo effect of dimer of soluble NP-1 on np-1 mutant embryos To examine the in vivo effect of NP-1-Fc, pregnant mice were given daily intraperitoneal injections of 150 µL of a 2 mg/mL solution of either NP-1-Fc or CD4-Fc from E9.5 to 11.5. Embryos were harvested on E12.5 and immunohistochemical analysis was done on whole-mount embryos or sections by using PECAM-1 mAb to observe the vascularity of the embryos. We obtained 11 np-1/ embryos and 56 np-1+/+ or np-1+/
embryos from 6 dams given injections of NP-1-Fc. Injection of CD4-Fc
into the np-1 mutants as a control had no effect (Figure 6A,C). On injection of NP-1-Fc,
vascularity in np-1+/+ and
np-1+/ embryos was slightly enhanced; however,
np-1/ embryos had substantial recovery of
vascularity, and interestingly, the caliber of the rescued capillaries
was larger than that of the wild-type embryos (Figure 1A,C and Figure
6B,D). We confirmed that NP-1-Fc bound to ECs in CNS and pericardium
by staining with antihuman IgG (data not shown). These findings
indicate that in np-1/ embryos, NP-1-Fc
affects EC proliferation and migration directly.
In this study, we showed that complexes of NP-1-Fc and
VEGF165 directly stimulate VEGFR-2 on ECs. As reported
previously, when NP-1 was coexpressed with VEGFR-2, the chemotactic
activity and mitogenicity for ECs induced by stimulation of
VEGF165 were enhanced.10 Moreover, a murine
transgenic model in which native np-1 complementary DNA was
overexpressed under the transcriptional control of the Commonly, soluble receptor proteins bind their ligands with affinities similar to those of cognate transmembrane receptors.29 Most soluble receptors for cytokines and growth factors compete with their membrane-bound counterparts for binding of the ligand and are therefore antagonists. In contrast, as in the case of NP-1-Fc, the soluble receptor works as a signal modulator, as it does in hematopoietic cells. IL-6 is one of several molecules that stimulate proliferation of primitive hematopoietic progenitors.30,31 Binding of IL-6 to IL-6 receptor (IL-6R) stimulated the signal-transducing molecule gp130.32 Exogenous soluble IL-6R-IL-6 complex stimulated gp130 on hematopoietic cells directly.33 NP-1 is composed of the a/CUB domain, b/FV/VIII domain, c/MAM domain,
transmembrane, and a short cytoplasmic domain that does not have an
obvious motif. Naturally occurring soluble NP-1 (sNP-1) contains only
the a and b domains and does not dimerize. The sNP-1 tagged with Flag
epitope (NP-1-Flag) that we constructed in this experiment contains
the a, b, and c domains. NP-1-Flag is a monomeric form and works as a
suppressor, as was observed with naturally occurring sNP-1 (Figure
7Bi). On the other hand, NP-1 tagged with the Fc part of human IgG1 (NP-1-Fc) contains the a, b, and c domains in a dimeric form. Although our soluble NP-1-Fc might not represent what occurs in vivo, this engineered protein indeed enhanced the function of VEGFR-2+ ECs (Figure 7Bii). As far as we know,
this is the first model in which a soluble receptor works as a signal
modulator for ECs. Although the molecular mechanism allowing a monomer
and dimer of NP-1 to work as an antagonist and agonist, respectively,
is not known, we think that the findings presented here may have important clinical applications.
VEGFR-2 is exclusively expressed on all ECs during embryogenesis;
however, NP-1 is not always expressed on ECs (data not shown). As we
observed in np-1 mutant embryos, NP-1 is essential for
vascular development. These results indicate that exogenous NP-1 may be required for the proliferation of VEGFR-2+
NP-1 The defective vasculogenesis and angiogenesis that we observed in np-1 mutant embryos was in restricted sites, such as the CNS and pericardium. VEGF expression has been reported to be less abundant in the CNS on E12.5.35 If we hypothesize that exogenous VEGF-NP-1 complexes on non-EC-lineage cells are required for normal vasculogenesis and angiogenesis when tissue distribution of VEGF is limited, it is logical that defective angiogenesis was observed in the CNS of np-1 mutants. In the experiment in which NP-1-Fc was injected into pregnant mice, we did not observe excess capillary formation in wild-type embryos, although defective capillary formation was rescued in np-1 mutant embryos. The effect of NP-1 might depend on the up-regulation of VEGF or VEGFR-2 protein in np-1 mutant embryos. Indeed, Northern blotting analysis showed that VEGF and VEGFR-2 expression in mutant embryos was up-regulated to levels about 1.5 to 2 times that in wild-type embryos (data not shown). Although the existence of organ-specific NP-1 functions, especially in the pericardium and CNS, cannot be ruled out, it appears that VEGF overproduced in np-1 mutants binds to NP-1-Fc and that these VEGF-NP-1 complexes stimulate excess VEGFR-2 on ECs. If such compensational up-regulation of VEGF or VEGFR-2 did not occur in np-1 mutants, more severe defects in vasculogenesis and angiogenesis might be observed.
We thank Dr Fumio Arai, Dr Takeshi Naruse, and Miss Yukari Mukoumatsu for excellent technical assistance and Dr Hiroaki Kodama for providing OP9 stromal cells.
Submitted May 22, 2000; accepted October 23, 2000.
Supported by grants-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Science, and Culture of Japan.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Toshio Suda, Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Honjo 2-2-1, Kumamoto 860-0811, Japan; e-mail: sudato{at}gpo.kumamoto-u.ac.jp.
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Top
Abstract
Introduction
80°C. The dehydrated embryos were
bleached in methanol plus 5% vol/vol hydrogen peroxide for 4 to 5 hours at 4°C. The bleached embryos were rehydrated and blocked in
PBS, milk, and Triton X-100 (PBSMT; PBS, 2% instant skim milk, and
0.1% Triton X-100) for 1 hour twice. The embryos were then incubated
with a 1:500 dilution of anti-platelet EC adhesion molecule 1 (PECAM-1) antibody (MEC13.3 rat antimouse monoclonal antibody [mAb];
Pharmingen, San Diego, CA) in PBSMT overnight at 4°C. The next day,
the embryos were washed with PBSMT at 4°C for 1 hour 5 times and
incubated at 4°C with 1 µg/mL horseradish peroxidase-conjugated
goat antirat IgG (Biosource, Camarillo, CA) in PBSMT overnight at
4°C. Embryos were then washed with PBSMT for 1 hour 5 times at 4°C
and in PBS and Triton (PBST; 0.1% Triton X-100 and PBS) for 20 minutes
3 times at room temperature. Peroxidase staining was done by incubating the embryos in 0.3 mg/mL diaminobenzidine (Dojin Chemical, Kumamoto, Japan) and 0.8 mg/mL nickel chloride (Wako Pure Chemical, Osaka, Japan)
in PBST for 20 minutes and then adding hydrogen peroxide at a final
concentration of 0.05%. Rinsing in PBST stopped the staining reaction.
-actin
promoter revealed an excess of capillaries and blood vessels, dilation
of blood vessels, and a malformed heart.