|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Cell Differentiation, Institute
of Molecular Embryology and Genetics, Kumamoto University, Japan; and
Regeneron Pharmaceuticals, Tarrytown, NY.
Ephrin-B2 is a transmembrane ligand that is specifically expressed
on arterial endothelial cells (ECs) and surrounding cells and interacts
with multiple EphB class receptors. Conversely, EphB4, a specific
receptor for ephrin-B2, is expressed on venous ECs, and both ephrin-B2
and EphB4 play essential roles in vascular development. The
bidirectional signals between EphB4 and ephrin-B2 are thought to be
specific for the interaction between arteries and veins and to regulate
cell mixing and the making of particular boundaries. However, the
molecular mechanism during vasculogenesis and angiogenesis remains
unclear. Manipulative functional studies were performed on these
proteins in an endothelial cell system. Using in vitro stromal cells
(OP9 cells) and a paraaortic splanchnopleura (P-Sp) coculture system,
these studies found that the stromal cells expressing ephrin-B2
promoted vascular network formation and ephrin-B2+ EC
proliferation and that they also induced the recruitment and proliferation of During embryogenesis, vascular development consists
of 2 processes: vasculogenesis, whereby endothelial cell (EC)
precursors differentiate and proliferate to form the initial vascular
plexus,1-3 and angiogenesis, in which the initial vascular
plexus forms mature vessels by sprouting, branching, pruning,
differential growth of ECs, and recruitment of supporting cells, such
as pericytes and smooth muscle cells (SMCs).1,3,4 To date,
3 growth factor systems VEGFs and their receptors VEGFR-2/Flk-1, VEGFR-1/Flt-1, and
VEGFR-3/Flt-4 are key regulators of vasculogenesis and angiogenesis. VEGF and Flk-1 are required to grow and establish an endothelial lineage,7-10 whereas VEGF and Flt-1 are involved in the
organization of ECs into tubelike structures.11 Flt-4 also
contributes to angiogenesis and lymphangiogenesis.12
Angiopoietins and their EC-specific receptors, TIEs, have been
suggested to be important for vascular remodeling and cardiac
development.13-15 Platelet-derived growth factor (PDGF)
and tumor growth factor (TGF)- One of the most important events in vascularization is the development
of arteries and veins. Generally, the differences between arteries and
veins have been defined by function, anatomy, pressure, and blood flow
direction. Recent studies have shown that the arterial and venous ECs
are molecularly distinct from the earliest types of blood vessels. This
distinction is revealed by a tyrosine kinase receptor, EphB4, that is
dominantly expressed on venous ECs and whose cognate ligand, ephrin-B2,
is expressed on arterial ECs.20,21 The Eph receptor and
ephrin ligand families play important roles in embryonic
development.22 They can be grouped into 2 subclasses based
on structural homology and binding specificity: ephrin-A ligands
(ephrin-A1 to -A5), which are tethered to the cell surface via a
glycosylphosphatidylinositol anchor and bind to the EphA receptor
subfamily, and ephrin-B ligands (ephrin-B1 to -B3), which are inserted
into the plasma membrane via a transmembrane region along with
conserved cytoplasmic tyrosine residues and bind to the corresponding
EphB receptor subfamily.23-25 These ephrins need to be
membrane bound to activate their receptors but do not function in a
soluble form.26 Notably, signaling between ligands and receptors in the B subfamily appears to be reciprocal because ephrin-B
ligands not only activate their bound receptor but in return are
activated by their receptors in a neighboring cell.25,27
A functional analysis of Eph-ephrin signals has been conducted in the
circulatory and nervous systems. Targeted disruption of the ephrin-B2
gene leads to embryonic lethality at around E11 because of a defect in
both arterial and venous vessel remodeling. This defect was accompanied
by a failure of intercalation between the arteries and
veins.20 The EphB4 homozygous mutants have a symmetric
phenotype with ephrin-B2 homozygous embryos in the cardiovascular
system.21 Thus, signaling between arteries and veins
mediated by EphB4 and ephrin-B2 may be required for proper morphogenesis of the intervening capillary beds and network as well as
for interdigitation and differential growth of arterial and venous
vessels.20,28
Other Eph receptors and ephrin ligands are also involved in
angiogenesis. Ephrin-B1 promotes the formation of EC capillarylike structures, cell attachment, and sprouting angiogenesis in
vitro.29 An in vitro sprouting assay also shows that
purified ephrin-B1-Fc induces a significant increase in the number of
sprouts in adrenal cortex-derived microvascular endothelial cells, and
this activity is completely blocked by EphB1-Fc and
EphB2-Fc.30 Ephrin-B1 is coexpressed with ephrin-B2 in
ECs. EphB3 and ephrin-B3 are coexpressed with EphB4 in venous ECs.
EphB3 is also expressed in some arteries, and EphB2 and ephrin-B2 are
coexpressed in mesenchymal cells adjacent to ECs. But this overlapping
of expression is unable to compensate for the lack of ephrin-B2 or
EphB4. No vascular defects are found in either EphB2 or EphB3
homozygous mutant mice, whereas EphB2 and EphB3 double-mutant mice have
shown vascular defects only with 30% penetrance, which is similar but
not identical to that of ephrin-B2 mutants.30 It has been
suggested that the complex cell-to-cell interaction via Eph receptors
and ephrin ligands on ECs is restricted mainly by EphB4/ephrin-B2 but
also involves other Eph receptors and ephrin ligands.
In the nervous system, Eph and their ligands regulate topographic
map formation in the visual system.31-34 The bidirectional activation of Eph receptor and ephrin-B ligands is important for the patterning of the embryonic structure of the brain and
somites24,27,35-37; is implicated in the
repulsion that guides the migration of cells and growth cones to
specific destinations; and maintains the boundaries between cell groups
in the neuronal system.38,39 Recent studies have revealed
that a clear border is formed between the Eph- and ephrin-B-expressing
cell populations and that bidirectional signaling between Eph/ephrin-B
restricts the intermingling of adjacent cell populations and
maintenance of the boundaries.40,41
However, the molecular mechanism and how such ligands and receptors
work for vasculogenesis and angiogenesis is not clearly understood. To
clarify this, we have generated ephrin-B2- and EphB4-overexpressing
stromal cell lines OP9 and cocultured P-Sp (paraaortic splanchnopleural
mesoderm) explants with transfected OP9 cells. This system provides us
with opportunities to observe the interaction of ECs and surrounding
cells during vasculogenesis and angiogenesis.42-44
Cell line and animals
Transfections
Western blot analysis
In vitro coculture of P-Sp explants or single-cell suspensions on stromal cells The method of P-Sp dissection was performed as described previously.44 Embryos were dissected from pregnant females at 9.0 to 9.5 days postcoitum. Ephrin-B2LacZ/+ embryos were generated from crosses of ephrin-B2LacZ/+ and wild-type mouse. By convention, the morning the vaginal plug was detected was defined as embryonic day 0.5 (E0.5). Ephrin-B2 genotyping was confirmed by LacZ staining in yolk sacs and reverse transcriptase-polymerase chain reaction (RT-PCR) using a LacZ-specific primer. Then the wild-type or ephrin-B2LacZ/+ P-Sp explants were cultured on OP9/ephrin-B2, OP9/EphB4, or OP9/vector stromal cells in RPMI 1640 (Gibco) containing 10% FCS and 10 5 M 2-mercaptoethanol
(2ME) (Sigma) and supplemented with interleukin (IL)-6 (20 ng/mL), IL-7
(20 U/mL) (a gift from Dr T. Sudo, Toray Industries, Kamakura, Japan),
stem cell factor (SCF, 50 ng/mL) (a gift from Chemo-Sero-Therapeutic,
Kumamoto, Japan), and erythropoietin (2 U/mL) (a gift from Snow-Brand
Milk Product, Tochigi, Japan) in 12-well plates; the plates were then
incubated at 37°C in a humidified 5% CO2 atmosphere for
4 to 14 days.
After isolating the E9.0-9.5 P-Sp regions, we prepared single-cell
suspensions with 2.4 U/mL Dispase (Gibco, Grand Island, NY) and
passed the tissues through a 23G needle. The single cells were
cocultured with OP9/ephrin-B2 or OP9/vector stromal cells in RPMI
1640 medium containing 10% FCS, 10 Immunohistochemistry Vascular formation from P-Sp explants was analyzed on OP9/ephrin-B2, OP9/EphB4, and OP9/vector feeder layers. After coculturing the P-Sp with OP9/ephrin-B2, OP9/EphB4, or OP9/vector stromal cells for 4, 7, 10, and 14 days, immunohistochemistry was performed as described.42,48 In brief, the cultures were fixed in situ with 4% paraformaldehyde in PBS for 10 minutes at 4°C, washed twice with PBS, and incubated with 0.3% H2O2 in PBS at room temperature for 30 minutes to block endogenous peroxidase activity. To block nonspecific reactions, cultures were incubated with 1% normal goat serum and 0.2% bovine serum albumin (Sigma) in PBS-T at room temperature for 30 minutes. Then the fixed dishes were incubated with platelet endothelial cell adhesion molecule (PECAM)-1 monoclonal antibody (MoAb) (MEC13.3, rat antimouse monoclonal, PharMingen, San Diego, CA) overnight at 4°C, washed with PBS-T 3 times, and incubated with peroxidase-conjugated antirat immunoglobulin G (BioSource, Camarillo, CA) for 1 hour at room temperature. After 3 more washes, the samples were visualized by using the AEC substrate system (Nichirei, Tokyo, Japan). Alternatively, they were soaked in PBS-T containing 300 µg/mL diaminobenzidine (Dojin Chemical, Kumamoto, Japan) in the presence of 0.05% NiCl2 for 10 to 30 minutes, hydrogen peroxide was added to 0.01%, and the color reactions were visualized. To detect the SMCs, anti- -smooth muscle actin (-SMA) antibody (Dako, Glostrup,
Denmark) was used for immunohistochemical staining in this
culture system.
Immunofluorescence staining, EC sorting, and single EC culture Single-cell suspensions for cell sorting were prepared from the E9.0-9.5 P-Sp regions. The cells were incubated for 30 minutes on ice with biotin-anti-Flk-1 MoAb (AVAS12, rat antimouse monoclonal, a gift from Dr S.-I. Nishikawa, Kyoto University, Japan) and washed twice with washing buffer (5% FCS/PBS). The cells were subsequently incubated with phycoerythrin-conjugated PECAM-1 antibody (PharMingen) and allophycocyanin-conjugated streptavidin (Caltag Laboratories, South San Francisco, CA) for 30 minutes on ice. Then the cells were washed twice with washing buffer and suspended for cell sorting. The stained cells were sorted by FACSVantage (Becton Dickinson Immunocytometry Systems, San Jose, CA) to obtain Flk-1+PECAM-1(R2) and
Flk-1+PECAM-1+(R3) EC cell populations. Then
the sorted R2 and R3 cells were cocultured with OP9/ephrin-B2 or
OP9/vector stromal cells. The cell cultures were performed at 300 cells
per well in 24-well plates in RPMI 1640 medium containing 10% FCS,
105 M 2ME, and 5 ng/mL VEGF. Immunohistochemistry with
anti-PECAM-1 MoAb was performed on day 10 to detect the ECs, and the
sheetlike or cordlike structures were enumerated.
RT-PCR analysis Total RNA was isolated from whole yolk sac (as a control), R2 and R3 cells sorted from E9.0-9.5 P-Sp regions using the RNeasy mini kit (Qiagen). Then the RNA was subjected to reverse transcription with an Advantage RT for PCR kit (Clontech Laboratories, Palo Alto, CA). After reverse transcription, the cDNA was amplified using an Advantage polymerase mix PCR kit (Clontech) in a GeneAmp PCR system model 9700 (Perkin-Elmer, Norwalk, CT) for 25 to 30 cycles. To detect the expression of ephrin-B2 and EphB4, the following primers were used: ephrin-B2, 5'-TCTGTGTGGAAGTACTGTTGGGGACTTT-3' (sense), 5'-TGTACCAGCTTCTAGCTCTGGACGTCTT-3' (antisense); EphB4, 5'-CGTCCTGATGTCACCTATACCTTTGAGG-3' (sense), 5'-GAGTACTCAACTTCCCTCCCATTGCTCT-3' (antisense), for amplification.Preparation of recombinant fusion protein To generate cDNA encoding the full-length EphB4 extracellular domain, an expression plasmid containing full-length mouse EphB4 cDNA (pRK5-EphB4) was used as a template for PCR using the Advantage 2 PCR kit (Clontech). Sense and antisense primers that had SalI and EcoRI restriction enzyme sites at their 5' ends for reconstruction were designed: 5'-ACGCGTCGACATGGAGCTCCGAGCGCTGCTG-3' (sense), 5'-CGGAATTCTGCT CCCGCCAGCTCTCGCTCTC-3' (antisense). Amplified DNA fragments were sequenced (ABI Prism 310) and subcloned into the expression vector pCAGneo-human Fc (Fc part of human immunoglobulin G1, EphB4-Fc). EphB4-Fc or CD4-Fc was prepared from COS7 cell supernatant in GIT medium (Wako Pure Chemical, Osaka, Japan) as previously described.49LacZ staining For culture plate LacZ staining, the P-Sp cultures were fixed in a solution containing 2% formaldehyde and 0.2% glutaraldehyde in PBS for 10 minutes at room temperature, washed with PBS 3 times, and then stained at room temperature for 30 to 60 minutes in a solution containing 1 mg/mL X-Gal (Nacalai Tesque, Kyoto, Japan), 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, and 2 mM MgCl2 in PBS (X-Gal staining solution). For whole mount LacZ staining of the yolk sac, specimens were fixed in 4% paraformaldehyde in PBS for 5 to 10 minutes at 4°C, washed twice with PBS, and stained with X-Gal staining solution at room temperature for 30 to 60 minutes. For in vivo LacZ/ephrin-B2 expression analysis, the venae cava and aortas, along with surrounding tissues, were removed from adult ephrin-B2LacZ/+ or wild-type mice, and ephrin-B2LacZ/+ or wild-type embryos were removed at E9.0-9.5. The samples were fixed in 4% paraformaldehyde/PBS for 30 to 60 minutes for adult tissues and 5 to 10 minutes for embryos, respectively, at 4°C and washed twice with PBS. The tissues were then stained from 4 hours to overnight at room temperature in a LacZ staining solution. After LacZ staining, the tissues were postfixed in 4% formaldehyde/PBS for 4 hours at 4°C, embedded in polyester wax, and transverse sectioned 8 µm thick. The immunohistochemical staining for PECAM-1 and-SMA in sections was carried out as previously
described.48
Quantitative analysis of vascular network areas After PECAM-1 staining, the images were integrated using a color camera (Hamamatsu Photonics, Shizuoka, Japan). Image-processing (NIH Image 1.62/Power Macintosh G3, National Institutes of Health, Bethesda, MD) was used to determine alterations in the size of vascular networks.50 In P-Sp cultures, PECAM-1+ ECs formed a sheetlike structure (vascular beds) beside the P-Sp explant. Subsequently, PECAM-1+ ECs sprouted from the sheet and formed a cordlike structure (vascular networks). Because the outside border of the vascular network is not in a regular pattern, we delimited the outside border with curved lines. Then we measured the areas between the outside border of vascular network and the boundary line of vascular bed and network as a vascular network area. Three vascular networks from each P-Sp explant were measured under 12.5 × magnification. The average value and SD for each period was calculated, and the statistical significance was tested using an unpaired t test in Startview.
Function of ephrin-B2 in vasculogenesis and angiogenesis The OP9 stromal cell line was established from the newborn calvaria of the (C57BL/6xC3H) F2-op/op mouse, which lacks a functional macrophage colony-stimulating factor.51,52 According to previous studies,42-44 OP9 stromal cells are suitable for analysis for angiogenesis compared with other stromal cells such as NIH3T3, BALB/c3T3, and PA6. To examine the function of ephrin-B2 in angiogenesis, we transfected full-length ephrin-B2 cDNA into OP9 stromal cells, which did not express ephrin-B2. The predicted molecular weight for ephrin-B2 is 37 kd, but 3 bands of 38 kd, 46 kd, and 48 kd were detected in OP9/ephrin-B2 stromal cells by Western blot analysis. Consistent with a previous paper, this slow electrophoretic mobility may be due to the modification of glycosylation (Figure 1A).27P-Sp explants from E9.0-9.5 embryos were cocultured with the OP9/vector
or OP9/ephrin-B2 stromal cells in the presence of IL-6, IL-7, SCF, and
erythropoietin. Immunohistochemical staining with PECAM-1 MoAb to
detect ECs was performed after culturing for 4, 7, 10, and 14 days. In
P-Sp culture,
PECAM-1+Flk-1+TIE2low/ Effect of EphB4 during angiogenesis in P-Sp culture system Although it has been reported that mesenchymal cells do not express EphB4 in vivo,30 to clarify the interactions between environmental EphB4+ cells and ECs, we transfected EphB4 into OP9 cells and cloned OP9 cells that expressed high levels of EphB4 (OP9/EphB4), while parent OP9 cells expressed little endogenous EphB4 (Figure 2A). To confirm the function of EphB4 in angiogenesis in the OP9 culture system, E9.0-9.5 P-Sp explants were cocultured with OP9/EphB4 or OP9/vector stromal cells as above. After 14 days of coculture, immunohistochemistry with the anti-PECAM-1 antibody was performed to detect the ECs. The level of vascular network formation in all 3 of the EphB4-overexpressing clones was severely suppressed compared with that seen on OP9/vector stromal cell clones; however, the vascular bed formation was not changed (Figure 2B).When the chimeric protein EphB4-Fc was added to the OP9/ephrin-B2 cocultures, vascular network formation was also inhibited (Figure 2B); however, CD4-Fc as a control did not affect vascular formation (data not shown). On the other hand, when 20 µg/mL ephrin-B2-Fc was added to this coculture system, vascular EC sprouting/remodeling from the P-Sp explant increased in both OP9/EphB4 and OP9/vector stromal cells and was strongly increased in OP9/ephrin-B2 stromal cells (Figure 2C). In contrast, monomer ephrin-B2 chimeric protein (ephrin-B2-FLAG) did not affect vascular formation (data not shown). These results indicate that, contrary to OP9/ephrin-B2, OP9/EphB4 inhibited vascular network formation from P-Sp explants but did not affect development of vascular beds. The signals from a soluble form of ephrin-B2-Fc also promoted EC sprouting/remodeling, but soluble EphB4-Fc inhibited it. Effect of OP9/ephrin-B2 stromal cells on P-Sp single-cell suspension cultures To analyze the effect of ephrin-B2 on endothelial precursor cells, dissociated cells of E9.0-9.5 P-Sp were seeded on OP9/ephrin-B2 or OP9/vector stromal cells in the medium containing VEGF and SCF. ECs proliferated by 2 different means in this condition. One consisted of spreading EC networks and the other of aggregated ECs. We named the former a "cordlike" and the latter a "sheetlike" structure. In the OP9/ephrin-B2 stromal cells, most of the PECAM-1+ ECs formed a cordlike structure, whereas in OP9/vector stromal cells ECs formed a sheetlike structure (Figure 3A). The number of sheetlike structures in OP9/vector stromal cells was about 5 times greater than that in OP9/ephrin-B2 stromal cells. On the other hand, the number of cordlike structures was about 2.5 times higher in OP9/ephrin-B2 stromal cells than in the OP9/vector stromal cells (Figure 3B). These observations further indicated that OP9/ephrin-B2 stromal cells support EC sprouting.
P-Sp explants contain various kinds of cells, such as endothelial
precursors, hematopoietic cells, and mesenchymal cells. To examine
whether OP9/ephrin-B2 stromal cells affect EC precursors directly, ECs
from the E9.5 P-Sp regions were sorted by FACSVantage (Figure
4A).
Flk-1+PECAM-1
Effect of ephrin-B2 and EphB4 on ephrin-B2+ cells Vascular ECs contain arterial ECs and venous ECs. Recent studies have shown that the 2 kinds of ECs are molecularly distinct, which is revealed by a ligand ephrin-B2 that is dominantly expressed on arterial ECs; and EphB4, a receptor specific for ephrin-B2, is expressed on venous ECs.20,21,47 We analyzed in vivo ephrin-B2 expression on arteries and veins, conducted LacZ and anti-PECAM-1 or anti--SMA double staining using wild-type or
ephrin-B2LacZ/+ (in which LacZ expression is under the
transcriptional control of an ephrin-B2 promoter) embryos, and examined
adult abdominal/thoracic aorta and superior/inferior vena cava
containing the surrounding tissues. Serial staining of tissue sections
revealed that in E9.5 embryos the ephrin-B2 was absolutely expressed in
the dorsal aorta ECs and surrounding cells but not in the anterior
cardinal vein (Figure 5). Also, in the
adult, ephrin-B2 is expressed on arterial ECs and surrounding SMCs. By
contrast, no ephrin-B2 was detected in the venous cells
(Figure 5).
Next, we sought to determine which type of EC (ephrin-B2+
or EphB4+ ECs) were supported or inhibited by ephrin-B2 or
EphB4 on OP9 cells and how they interact with each other in this
system. The ephrin-B2LacZ/+ embryos were obtained from a
cross of wild-type and ephrin-B2LacZ/+ mice. After
genotypying of the embryo by LacZ staining of the yolk sac (Figure
6A), the E9.0-9.5
ephrin-B2+/+ or ephrin-B2LacZ/+ P-Sp was
cocultured with OP9/ephrin-B2, OP9/EphB4, or OP9/vector stromal cells.
After culturing for 10 to 14 days, we examined the formation of
vascular networks by immunohistochemical staining with PECAM-1 MoAb and
LacZ staining. Ephrin-B2+ ECs were detected as PECAM-1 and
LacZ++ cells. As shown in Figure 6B, the OP9/vector stromal
cells form a fine vascular network, which consisted of both
PECAM-1+ cells and LacZ/ephrin-B2-PECAM-1++
cells. But the vascular networks were formed mainly by LacZ and PECAM-1++ cells on OP9/ephrin-B2 stromal cells. In
contrast, OP9/EphB4 stromal cells did not support vascular network
formation and almost no LacZ/ephrin-B2+ cells were
observed. This indicates that OP9/ephrin-B2 promotes vascular network
formation and supports the proliferation and sprouting of
ephrin-B2+ cells but that OP9/EphB4 inhibits
ephrin-B2+ cell proliferation.
Function of ephrin-B2 and EphB4 in SMC recruitment During vascular development, an important event is the formation of vessel walls by recruitment of mural cell precursors, especially in the artery. To investigate SMC recruitment in this culture system, we performed immunohistochemical staining with anti--SMA antibody. On
day 14 of culturing, the number of -SMA+ SMCs increased
in the surrounding ECs formed on OP9/ephrin-B2 stromal cells.
Nevertheless, no -SMA+ cells were detected in OP9/EphB4
stromal cells, whereas a small number of -SMA+ cells
were detected in OP9/vector stromal cells (Figure
7A,B). It has been reported that SMCs
develop from mesodermal cells or neural crest cells or
transdifferentiate from other cells.53,54 We have further
confirmed that the SMA+ cells in the vascular network were
derived from P-Sp explants, but not from OP9 stromal cells, by using
P-Sp obtained from green mice harboring GFP ubiquitously under the
transcriptional control of a CAG promoter in this coculture system
(Figure 7C). This result suggested that the ephrin-B2+ ECs
and stromal cells have the ability to promote SMC recruitment and
proliferation. However, EphB4+ EC might be unable to
promote SMC recruitment. This further suggests that ephrin-B2/EphB4
signaling is important for the interaction between the ECs and
surrounding cells.
In this paper, we examined the function of EphB4 receptors and
ephrin-B2 ligands in the interaction of ECs with mesenchymal cells
using an in vitro P-Sp and stromal cell coculture system. To address
this issue, we analyzed angiogenesis using OP9 stromal cells that were
transfected with ephrin-B2 or EphB4. Using these sublines of OP9 cells,
we observed several pieces of evidence: (1) Environmental ephrin-B2
supports the proliferation of ephrin-B2+ ECs and suppresses
the proliferation of ephrin-B2 The EphB4 receptor and its cognate ligand, ephrin-B2, are crucial for
successful cardiovascular development during
embryogenesis.20,21,28 To elucidate the interactions
between the ECs and surrounding cells, which express ephrin-B2 and
EphB4, we established an in vitro OP9 stromal cell and P-Sp coculture
assay for analyses of angiogenesis. Ephrin-B2-transfected stromal
cells enhanced the vascular network formation from P-Sp, whereas
EphB4-transfected stromal cells inhibited it. RT-PCR analyses have
shown that ephrin-B2, ephrin-B1, EphB4, EphB3, and EphB2 are expressed
in the PECAM-1+CD45 In this culture system, we found that both ephrin-B2+ and
ephrin-B2 During angiogenesis, one important event is the formation of the
vascular wall by the recruitment of pericytes and SMCs from mesenchymal
progenitor cells and neural crest cells.56,57 Progress in
elucidating the mechanism of SMC recruitment, proliferation, and
differentiation was achieved by identification of a number of smooth
muscle-specific proteins and their expression in SMC lineage
cells.58,59 ECs can modulate phenotypic change, regulate proliferation, and induce migration of SMCs.60,61 These
processes are regulated by growth factors such as PDGF and TGF- Vascular SMCs produce and organize extracellular matrix molecules
within the developing vessel wall. In our in vitro assays, OP9/ephrin-B2 promoted not only proliferation and sprouting of ephrin-B2+ ECs but also recruitment and proliferation of
In summary, blood vessels are formed by the recruitment and migration
of mesenchymal cells outside the endothelial layer. Therefore, from our
results and others, it is reasonable to conclude that the initial
commitment of ephrin-B2+ or EphB4+ ECs from
angioblasts is the trigger for determining vessels that become arteries
or veins.20,21,47 During this process, along with other
factors,16-19,64,65 Ephs/ephrins and, in particular, EphB4/ephrin-B2 are the key regulators in the recruitment and migration
of SMC precursors in vivo and maintain the balance of proliferation
activity. Based on previous studies and our own findings, we propose a
model (Figure 8) to account for the role of EphB4/ephrin-B2 signaling in the interaction between endothelial and
mesenchymal cells during vasculogenesis, angiogenesis, and vascular
morphologic change.
The authors thank Dr Jon K. Moon for critical reading of this manuscript.
Submitted August 24, 2000; accepted April 4, 2001.
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, Dept of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan; e-mail: sudato{at}gpo.kumamoto-u.ac.jp.
1. Risau W. Differentiation of endothelium. FASEB J. 1995;9:926-933[Abstract].
2.
Hanahan D.
Signaling vascular morphogenesis and maintenance.
Science.
1997;277:48-50 3. Risau W. Mechanisms of angiogenesis. Nature. 1997;386:671-674[CrossRef][Medline] [Order article via Infotrieve]. 4. Folkman J, D'Amore PA. Blood vessel formation: what is its molecular basis? Cell. 1996;87:1153-1155[CrossRef][Medline] [Order article via Infotrieve]. 5. Beck LJ, D'Amore PA. Vascular development: cellular and molecular regulation. FASEB J. 1997;11:365-373[Abstract].
6.
Gale NW, Yancopoulos GD.
Growth factors acting via endothelial cell-specific receptor tyrosine kinases: VEGF, angiopoietins, and ephrins in vascular development.
Genes Dev.
1999;13:1055-1066 7. Shalaby F, Rossant J, Yamaguchi TP, et al. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature. 1995;376:62-66[CrossRef][Medline] [Order article via Infotrieve]. 8. Shalaby F, Ho J, Stanford WL, et al. A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis. Cell. 1997;89:981-990[CrossRef][Medline] [Order article via Infotrieve]. 9. Ferrara N, Carver-Moore K, Chen H, et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature. 1996;380:439-442[CrossRef][Medline] [Order article via Infotrieve]. 10. Carmeliet P, Ferreira V, Breier G, et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature. 1996;380:435-439[CrossRef][Medline] [Order article via Infotrieve]. 11. Fong GH, Rossant J, Gertsenstein M, Breitman ML. Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature. 1995;376:66-70[CrossRef][Medline] [Order article via Infotrieve].
12.
Dumont DJ, Tussila L, Lymboussaki A, et al.
Cardiovascular failure in mouse embryos deficient in VEGF receptor-3.
Science.
1998;282:946-949
13.
Dumont DJ, Gradwohl G, Fong GH, et al.
Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo.
Genes Dev.
1994;8:1897-1909 14. Suri C, Jones PF, Patan S, et al. Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell. 1996;87:1171-1180[CrossRef][Medline] [Order article via Infotrieve]. 15. Vikkula M, Boon LM, Carraway KL III, et al. Vascular dysmorphogenesis caused by an activating mutation in the receptor tyrosine kinase TIE2. Cell. 1996;87:1181-1190[CrossRef][Medline] [Order article via Infotrieve]. 16. Holmgren L, Glaser A, Pfeifer-Ohlsson S, Ohlsson R. Angiogenesis during human extraembryonic development involves the spatiotemporal control of PDGF ligand and receptor gene expression. Development. 1991;113:749-754[Abstract].
17.
Leveen P, Pekny M, Gebre-Medhin S, Swolin B, Larsson E, Betsholtzm C.
Mice deficient for PDGF B show renal, cardiovascular, and hematological abnormalities.
Genes Dev.
1994;8:1875-1887
18.
Heimark RL, Twardzik DR, Schwartz SM.
Inhibition of endothelial regeneration by type-beta transforming growth factor from platelets.
Science.
1986;233:1078-1080 19. Dickson MC, Martin JS, Cousins FM, Kulkarni AB, Karlsson S, Akhurst RJ. Defective haematopoiesis and vasculogenesis in transforming growth factor-beta 1 knock out mice. Development. 1995;121:1845-1854[Abstract]. 20. Wang HU, Chen ZF, Anderson DJ. Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell. 1998;93:741-753[CrossRef][Medline] [Order article via Infotrieve]. 21. Gerety SS, Wang HU, Chen ZF, Anderson J. Symmetrical mutant phenotypes of the receptor ephB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development. Mol Cell. 1999;4:403-414[CrossRef][Medline] [Order article via Infotrieve]. 22. Holder N, Klein R. Eph receptors and ephrins: effectors of morphogenesis. Development. 1999;126:2033-2044[Abstract]. 23. Brambilla R, Schnapp A, Casagranda F, et al. Membrane-bound LERK2 ligand can signal through three different Eph-related receptor tyrosine kinases. EMBO J. 1995;14:3116-3126[Medline] [Order article via Infotrieve]. 24. Gale NW, Holland SJ, Valenzuela DM, et al. Eph receptors and ligands comprise two major specificity subclasses and are reciprocally compartmentalized during embryogenesis. Neuron. 1996;17:9-19[CrossRef][Medline] [Order article via Infotrieve].
25.
Bruckner K, Pasquale EB, Klein R.
Tyrosine phosphorylation of transmembrane ligands for Eph receptors.
Science.
1997;275:1640-1643
26.
Davis S, Gale NW, Aldrich TH, et al.
Ligands for EPH-related receptor tyrosine kinases that require membrane attachment or clustering for activity.
Science.
1994;266:816-819 27. Holland SJ, Gale NW, Mbamalu G, Yancopoulos GD, Henkemeyer M, Pawson T. Bidirectional signalling through the EPH-family receptor Nuk and its transmembrane ligands. Nature. 1996;383:722-725[CrossRef][Medline] [Order article via Infotrieve]. 28. Yancopoulos GD, Klagsbrun M, Folkman J. Vasculogenesis, angiogenesis, and growth factors: ephrins enter the fray at the border. Cell. 1998;93:661-664[Medline] [Order article via Infotrieve].
29.
Stein E, Lane AA, Cerretti DP, et al.
Eph receptors discriminate specific ligand oligomers to determine alternative signaling complexes, attachment, and assembly responses.
Genes Dev.
1998;12:667-678
30.
Adams RH, Wilkinson GA, Weiss C, et al.
Roles of ephrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/venous domains, vascular morphogenesis, and sprouting angiogenesis.
Genes Dev.
1999;13:295-306 31. Cheng HJ, Nakamoto M, Bergemann AD, Flanagan JG. Complementary gradients in expression and binding of ELF-1 and Mek4 in development of the topographic retinotectal projection map. Cell. 1995;82:371-381[CrossRef][Medline] [Order article via Infotrieve]. 32. Drescher U, Kremoser C, Handwerker C, Loschinger J, Noda M, Bonhoeffer F. In vitro guidance of retinal ganglion cell axons by RAGS, a 25 kDa tectal protein related to ligands for Eph receptor tyrosine kinases. Cell. 1995;82:359-370[CrossRef][Medline] [Order article via Infotrieve]. 33. Nakamoto M, Cheng HJ, Friedman GC, et al. Topographically specific effects of ELF-1 on retinal axon guidance in vitro and retinal axon mapping in vivo. Cell. 1996;86:755-766[CrossRef][Medline] [Order article via Infotrieve]. 34. Frisen J, Yates PA, McLaughlin T, Friedman GC, O'Leary DD, Barbacid M. Ephrin-A5 (AL-1/RAGS) is essential for proper retinal axon guidance and topographic mapping in the mammalian visual system. Neuron. 1998;20:235-243[CrossRef][Medline] [Order article via Infotrieve]. 35. Xu Q, Alldus G, Holder N, Wilkinson DG. Expression of truncated Sek-1 receptor tyrosine kinase disrupts the segmental restriction of gene expression in the Xenopus and zebrafish hindbrain. Development. 1995;121:4005-4016[Abstract]. 36. Xu Q, Alldus G, Macdonald R, Wilkinson DG, Holder N. Function of the Eph-related kinase rtk1 in patterning of the zebrafish forebrain. Nature. 1996;381:319-322[CrossRef][Medline] [Order article via Infotrieve].
37.
Durbin L, Brennan C, Shiomi K, et al.
Eph signaling is required for segmentation and differentiation of the somites.
Genes Dev.
1998;12:3096-3109 38. Wang HU, Anderson DJ. Eph family transmembrane ligands can mediate repulsive guidance of trunk neural crest migration and motor axon outgrowth. Neuron. 1997;18:383-396[CrossRef][Medline] [Order article via Infotrieve]. 39. Brennan C, Monschau B, Lindberg R, et al. Two Eph receptor tyrosine kinase ligands control axon growth and may be involved in the creation of the retinotectal map in the zebrafish. Development. 1997;124:655-664[Abstract]. 40. Xu Q, Mellitzer G, Robinson V, Wilkinson DG. In vivo cell sorting in complementary segmental domains mediated by Eph receptors and ephrins. Nature. 1999;399:267-271[CrossRef][Medline] [Order article via Infotrieve]. 41. Mellitzer G, Xu Q, Wilkinson DG. Eph receptors and ephrins restrict cell intermingling and communication. Nature. 1999;400:77-81[CrossRef][Medline] [Order article via Infotrieve]. 42. Takakura N, Huang XL, Naruse T, et al. Critical role of the TIE2 endothelial cell receptor in the development of definitive hematopoiesis. Immunity. 1998;9:677-686[CrossRef][Medline] [Order article via Infotrieve].
43.
Hirashima M, Kataoka H, Nishikawa S, Matsuyoshi N, Nishikawa SI.
Maturation of embryonic stem cells into endothelial cells in an in vitro model of vasculogenesis.
Blood.
1999;93:1253-1263 44. Takakura N, Watanabe T, Suenobu S, et al. A role for hematopoietic stem cell in promoting angiogenesis. Cell. 2000;102:199-209[CrossRef][Medline] [Order article via Infotrieve].
45.
Nakano T, Kodama H, Honjo T.
Generation of lymphohematopoietic cells from embryonic stem cells in culture.
Science.
1994;265:1098-1101 46. Okabe M, Ikawa M, Kominami K, et al. `Green mice' as a source of ubiquitous green cells. FEBS Lett. 1997;407:313-319[CrossRef][Medline] [Order article via Infotrieve]. 47. Gale NW, Baluk P, Pan L, et al. Ephein-B2 selectively marks arterial vessels and neovascularization sites in the adult, with expression in both endothelial and smooth muscle cells. Dev Biol. 2001;230:151-160[CrossRef][Medline] [Order article via Infotrieve].
48.
Takakura N, Yoshida H, Ogura Y, Kataoka H, Nishikawa S, Nishikawa S.
PDGFR alpha expression during mouse embryogenesis: immunolocalization analyzed by whole-mount immunohistostaining using the monoclonal anti-mouse PDGFR alpha antibody APA5.
J Histochem Cytochem.
1997;45:883-893
49.
Yano M, Iwama A, Nishio H, Suda J, Takada G, Suda T.
Expression and function of murine receptor tyrosine kinases, TIE and TEK, in hematopoietic stem cells.
Blood.
1997;89:4317-4326
50.
Hamada K, Oike Y, Takakura N, et al.
VEGF-C signaling pathways through VEGFR-3 in vasculoangiogenesis and hematopoiesis.
Blood.
2000;96:3793-3800 51. Yoshida H, Hayashi S, Kunisada T, et al. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature. 1990;345:442-444[CrossRef][Medline] [Order article via Infotrieve]. 52. Kodama H, Nose M, Niida S, Nishikawa S, Nishikawa S. Involvement of the c-kit receptor in the adhesion of hematopoietic stem cells to stromal cells. Exp Hematol. 1994;22:979-984[Medline] [Order article via Infotrieve]. 53. Hungerford JE, Little CD. Developmental biology of the vascular smooth muscle cell: building a multilayered vessel wall. J Vasc Res. 1999;36:2-27[CrossRef][Medline] [Order article via Infotrieve]. 54. Yamashita J, Itoh H, Hirashima M, et al. Flk1-positive cells derived from embryo stem cells serve as vascular progenitors. Nature. 2000;408:92-96[CrossRef][Medline] [Order article via Infotrieve]. 55. Helbling PM, Saulnier DM, Brandli AW. The receptor tyrosine kinase EphB4 and ephrin-B ligands restrict angiogenic growth of embryonic veins in Xenopus laevis. Development. 2000;127:269-278[Abstract].
56.
Kirby ML, Waldo KL.
Neural crest and cardiovascular patterning.
Circ Res.
1995;77:211-215 57. Hungerford JE, Owens GK, Argraves WS, Little CD. Development of the aortic vessel wall as defined by vascular smooth muscle and extracellular matrix markers. Dev Biol. 1996;178:375-392[CrossRef][Medline] [Order article via Infotrieve].
58.
Skalli O, Ropraz P, Trzeciak A, Benzonana G, Gillessen D, Gabbiani GA.
Monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation.
J Cell Biol.
1986;103:2787-2796 59. Lee SH, Hungerford JE, Little CD, Iruela-Arispe ML. Proliferation and differentiation of smooth muscle cell precursors occurs simultaneously during the development of the vessel wall. Dev Dyn. 1997;209:342-352[CrossRef][Medline] [Order article via Infotrieve]. 60. Powell RJ, Carruth JA, Basson MD, Bloodgood R, Sumpio BE. Matrix-specific effect of endothelial control of smooth muscle cell migration. J Vasc Surg. 1996;24:51-57[CrossRef][Medline] [Order article via Infotrieve]. 61. Powell RJ, Cronenwett JL, Fillinger MF, Wagner RJ, Sampson LN. Endothelial cell modulation of smooth muscle cell morphology and organizational growth pattern. Ann Vasc Surg. 1996;10:4-10[CrossRef][Medline] [Order article via Infotrieve].
62.
Westermark B, Siegbahn A, Heldin CH, Claesson-Welsh L.
B-type receptor for platelet-derived growth factor mediates a chemotactic response by means of ligand-induced activation of the receptor protein-tyrosine kinase.
Proc Natl Acad Sci U S A.
1990;87:128-132 63. Shah NM, Groves AK, Anderson DJ. Alternative neural crest cell fates are instructively promoted by TGF-beta superfamily members. Cell. 1996;85:331-343[CrossRef][Medline] [Order article via Infotrieve].
64.
Sato Y, Tsuboi R, Lyons R, Moses H, Rifkin DB.
Characterization of the activation of latent TGF-beta by cocultures of endothelial cells and pericytes or smooth muscle cells: a self-regulating system.
J Cell Biol.
1990;111:757-763
65.
Hirschi KK, Rohovsky SA, D'Amore PA.
PDGF, TGF-beta, and heterotypic cell-cell interactions mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate.
J Cell Biol.
1998;141:805-814
© 2001 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  A. Pennisi, W. Ling, X. Li, S. Khan, J. D. Shaughnessy Jr, B. Barlogie, and S. Yaccoby The ephrinB2/EphB4 axis is dysregulated in osteoprogenitors from myeloma patients and its activation affects myeloma bone disease and tumor growth Blood, August 27, 2009; 114(9): 1803 - 1812. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Schwarz, L. Caldwell, D. Cafasso, and H. Zheng Emerging pulmonary vasculature lacks fate specification Am J Physiol Lung Cell Mol Physiol, January 1, 2009; 296(1): L71 - L81. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Zhang, H. Dong, B. Wang, S. Zhu, and B. A. Croy Dynamic Changes Occur in Patterns of Endometrial EFNB2/EPHB4 Expression During the Period of Spiral Arterial Modification in Mice Biol Reprod, September 1, 2008; 79(3): 450 - 458. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Huang, Y. Yamada, H. Kidoya, H. Naito, Y. Nagahama, L. Kong, S.-Y. Katoh, W.-l. Li, M. Ueno, and N. Takakura EphB4 Overexpression in B16 Melanoma Cells Affects Arterial-Venous Patterning in Tumor Angiogenesis Cancer Res., October 15, 2007; 67(20): 9800 - 9808. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Schmidt, K. Brixius, and W. Bloch Endothelial Precursor Cell Migration During Vasculogenesis Circ. Res., July 20, 2007; 101(2): 125 - 136. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Red-Horse, M. Kapidzic, Y. Zhou, K.-T. Feng, H. Singh, and S. J. Fisher EPHB4 regulates chemokine-evoked trophoblast responses: a mechanism for incorporating the human placenta into the maternal circulation Development, September 15, 2005; 132(18): 4097 - 4106. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. O. Zamora, M. H. Davies, S. R. Planck, J. T. Rosenbaum, and M. R. Powers Soluble Forms of EphrinB2 and EphB4 Reduce Retinal Neovascularization in a Model of Proliferative Retinopathy Invest. Ophthalmol. Vis. Sci., June 1, 2005; 46(6): 2175 - 2182. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-i. Hayashi, T. Asahara, H. Masuda, J. M. Isner, and D. W. Losordo Functional Ephrin-B2 Expression for Promotive Interaction Between Arterial and Venous Vessels in Postnatal Neovascularization Circulation, May 3, 2005; 111(17): 2210 - 2218. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Eichmann, T. Makinen, and K. Alitalo Neural guidance molecules regulate vascular remodeling and vessel navigation Genes & Dev., May 1, 2005; 19(9): 1013 - 1021. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Yuan, T.-M. Hong, J. J. W. Chen, W. H. Tsai, and M. T. Lin Syndecan-1 up-regulated by ephrinB2/EphB4 plays dual roles in inflammatory angiogenesis Blood, August 15, 2004; 104(4): 1025 - 1033. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Tsunematsu, K. Nakayama, Y. Oike, M. Nishiyama, N. Ishida, S. Hatakeyama, Y. Bessho, R. Kageyama, T. Suda, and K. I. Nakayama Mouse Fbw7/Sel-10/Cdc4 Is Required for Notch Degradation during Vascular Development J. Biol. Chem., March 5, 2004; 279(10): 9417 - 9423. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Fuller, T. Korff, A. Kilian, G. Dandekar, and H. G. Augustin Forward EphB4 signaling in endothelial cells controls cellular repulsion and segregation from ephrinB2 positive cells J. Cell Sci., June 15, 2003; 116(12): 2461 - 2470. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Hamada, Y. Oike, Y. Ito, H. Maekawa, K. Miyata, T. Shimomura, and T. Suda Distinct Roles of Ephrin-B2 Forward and EphB4 Reverse Signaling in Endothelial Cells Arterioscler Thromb Vasc Biol, February 1, 2003; 23(2): 190 - 197. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Oike, Y. Ito, K. Hamada, X.-Q. Zhang, K. Miyata, F. Arai, T. Inada, K. Araki, N. Nakagata, M. Takeya, et al. Regulation of vasculogenesis and angiogenesis by EphB/ephrin-B2 signaling between endothelial cells and surrounding mesenchymal cells Blood, July 30, 2002; 100(4): 1326 - 1333. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
vascular endothelial growth factors (VEGFs),
angiopoietins, and ephrins
have been reported to be associated
with pericyte proliferation and movement.
, OP9/ephrin-B2; 
,
OP9/vector; *P < .05; **P < .01.
) or OP9/vector (
