Department of Vascular Biology and Angiogenesis Research, Institute of Molecular Oncology, Tumor Biology Center, Breisacher Strasse 117, 79106 Freiburg, Germany
Author for correspondence (e-mail:
augustin{at}angiogenese.de)
Accepted 12 February 2003
![]() |
Summary |
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Key words: Endothelial cells, Angiogenesis, EphB4, EphrinB2, VEGF
![]() |
Introduction |
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|
|
Mice that just lack the cytoplasmic catalytic domain of ephrinB2 have an
early embryonic lethal phenotype similar to total ephrinB2-knockout mice
(Adams et al., 2001),
indicating that bidirectional forward and reverse EphB4-ephrinB2 signaling is
required for vascular morphogenesis and proper arterio-venous differentiation.
EphrinB2 binds EphB2 and EphB3 in addition to EphB4. Correspondingly, some
double EphB2- and EphB3-deficient mice die before E11.5 of embryonic
development, indicating that EphB4-ephrinB2 signaling alone cannot fully
compensate for the lack of EphB2 and EphB3
(Adams et al., 1999
).
Functionally, Eph receptors have primarily been characterized as a
repulsion-mediating signaling system. Neuronal Eph receptor activation
inhibits axonal outgrowth and leads to growth cone collapse
(Wilkinson, 2001). Repulsive
Eph receptor signaling results as the consequence of crosstalk phenomena with
integrin adhesion (Huynh-Do et al.,
1999
; Zou et al.,
1999
), as well as cytoskeletal organization involving Rho
(Carter et al., 2002
). Yet,
recent work also suggests that Eph receptors might act in a bimodal manner,
being capable of transmitting both pro-adhesive and anti-adhesive signals,
which has been demonstrated by opposing functions of different splice forms of
EphA7 (Holmberg et al., 2000
).
Similarly, reverse ephrinB signaling has been implicated in both attractive
and repulsive functions (Kullander and
Klein, 2002
).
In contrast to the dramatic vascular phenotypes of EphB- and
ephrinB-deficient mice, as well as the increasingly understood forward and
reverse signaling mechanisms, little is known about the functional
consequences of Eph and ephrin signaling in the vessel wall at the cellular
level. Activation of endothelial EphB1 receptors was shown to promote
endothelial capillary-like assembly, cell attachment and the recruitment of
low-molecular-weight phosphotyrosine phosphatase (LMW-PTP) to receptor
complexes (Stein et al.,
1998). Likewise, activation of EphB4 supposedly stimulates EC
migration and proliferation via activation of phosphoinositide 3-kinase (PI
3-kinase) (Steinle et al.,
2002
). Activation of endothelial ephrinB1 was shown to promote
integrin-mediated migration, attachment and angiogenesis
(Huynh-Do et al., 2002
), which
would imply that both endothelial EphB receptors and ephrinB ligands might be
able to act in a pro-adhesive and pro-angiogenic manner. EphB4-Fc receptor
bodies, activating ephrinB2, have been shown to act pro-angiogenicly
(Adams et al., 2001
). By
contrast, EphB4 expressed by co-cultured stromal cells has been reported to
inhibit vascular network formation of ephrinB2-expressing ECs
(Zhang et al., 2001
;
Helbling et al., 2000
).
In order to resolve these apparent discrepancies in some recent reports, the present study was aimed at specifying cellular functions mediated by forward and reverse EphB receptor and ephrinB ligand signaling during angiogenesis and vascular assembly. We applied a set of specialized 3D in vitro angiogenesis and EC differentiation assays in combination with more-conventional adhesion, lateral migration and chemotaxis experiments, and studied endogenous EphB receptor- and ephrinB ligand-expressing human umbilical vein ECs (HUVECs), as well as EphB4- and ephrinB2-overexpressing ECs. Collectively, the experiments demonstrate endothelial EphB-ephrinB signaling to act in a bidirectional antagonistic manner with EphB4 mediating anti-adhesive and repulsive signaling and ephrinB2 mediating attractive and pro-angiogenic activities.
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Materials and Methods |
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Transfection of PAE cells
Full-length ephrinB2 was cloned by RT-PCR amplification from freshly
isolated HUVECs using specific primers. EphrinB2 constructs lacking the
cytoplasmic domain were generated using primers that only included the first
266 amino acid residues and lacked the C-terminal 67 amino acids (designated
ephrinB2). Full-length murine EphB4 (mEphB4) cDNA was kindly provided
by Ralf H. Adams (CRUK, London). Sequence-controlled cDNAs were subcloned into
the pcDNA3.1+ expression vector (Invitrogen) and transfected into
PAECs by electroporation. Individual 500 µg/ml G418- (PAA, Cölbe,
Germany) resistant clones were isolated and expanded. Expression of the
transfected cDNA was confirmed by RT-PCR and corresponding receptor body
staining.
Generation of EC and EC-SMC co-culture spheroids
Spheroids of defined cell number were generated as described previously
(Korff and Augustin, 1998;
Korff et al., 2001
). In order
to generate co-culture spheroids, equal numbers of suspended HUASMCs and
HUVECs (1500 of each per spheroid) were mixed. Spheroids were cultured for at
least 24 hours and used for the corresponding experiments.
Attachment assay
Adhesive (Nunc, Wiesbaden, Germany) and non-adhesive (Greiner,
Frickenhausen, Germany) 24-well plates were coated in the center of each well
using 10 µl of either EphB4-Fc or ephrinB2-Fc (both R&D Systems,
Wiesbaden, Germany) diluted in PBS (100 ng/µl each) and were incubated for
20 minutes. Suspended HUVECs were cultured for 24 hours on top of the coated
plates, washed and fixed. Cellular attachment was quantified by measuring the
area covered by HUVECs or computer-aided cell counting of DAPI- (1:5000;
Hoechst, Frankfurt, Germany) stained nuclei analyzing at least five different
microscopic fields of view inside the coated area per experimental group and
experiment using the digital imaging software DP-Soft (Olympus, Germany)
Planar segregation assay
Equal numbers of different combinations of PAECs overexpressing ephrinB2,
ephrinB2, EphB4 or control cells (mock) were plated and allowed to grow
to confluence. Monolayers were paraformaldehyde-fixed after 3 days and
analyzed following EphB4 and ephrinB2 receptor body staining.
Lateral cell migration
Planar migration was quantitated as described previously (Augustin-Voss et
al., 1992). In brief, a silicon barrier (6 mm x 3 mm) was placed in the
middle of a tissue culture dish seeded with HUVECs. After removal of the
barrier, cells were stimulated with either EphB4-Fc or ephrinB2-Fc (1 µg/ml
dissolved in ECBM/10% FCS), or 50 ng/ml VEGF, or a combination of VEGF and
ephrinB2-Fc, for 48 hours. After this, lateral cell migration was quantitated
microscopically.
Boyden chamber assay
A modified Boyden chamber assay was performed using 48-well chambers
(Neuroprobe, Gaithersburg, MD) using polyvinylpyrrolidone-free polycarbonate
membranes with 8 µm pores (Costar, Cambridge, UK). Membranes were coated
overnight in the appropriate cell culture media (RPMI1640, 0.5% FCS, 0.1%
BSA), washed and coated for 1 hour with media containing 0.1% fibronectin
(Sigma, Deisenhofen, Germany). VEGF (20 ng/ml; R&D Systems) was loaded in
36 wells of the lower compartment, whereas 12 wells were loaded only with
media. Starved HUVECs were seeded into the upper compartment with either
medium alone (control A: medium, upper and lower compartment; control B:
medium, upper compartment, VEGF, lower compartment), ephrinB2-Fc (2 µg/ml)
or EphB4-Fc (2 µg/ml). The cells were allowed to migrate for 4 hours.
Following incubation, the cells on the upper side of the membrane were
removed, the membrane was fixed and cells were stained with DAPI. Migration of
cells across the membrane was quantified by computer-aided cell counting using
the Olympus DP-Soft analysis software.
EC alignment assay
Alignment of ECs was studied by seeding 25,000 HUVECs on 350 µl
polymerized Matrigel (B&D Biosciences, Heidelberg, Germany) in 24-well
plates. Cells were stimulated with 2 µg/ml ephrinB2-Fc or EphB4-Fc for 24
hours, after which the cells were fluorescence labeled by treatment with 1
µg/ml calceinAM (Molecular Probes, Leiden, The Netherlands) for 30 minutes.
Network formation was analyzed by automated computer-aided fluorescence
microscopic image analysis quantitating the circumference of the network
structures.
In vitro angiogenesis assay
In vitro angiogenesis in collagen gels was performed using EC spheroids as
described previously (Korff and Augustin,
1999). In vitro angiogenesis was digitally quantitated by
measuring the cumulative length of the sprouts that had grown out of each
spheroid (ocular grid at 100x magnification) using the Olympus DP-Soft
analysis software analyzing at least ten spheroids per experimental group and
experiment.
Morphological and immunohistochemical analysis
Umbilical veins were cut into slices of approximately 2 mm, washed and
incubated overnight in ECBM/10% FCS including either 2 µg/ml EphB4-Fc or
ephrinB2-Fc. Explants were fixed and processed for paraffin embedding.
Paraffin sections were stained for CD34 (1:25; Novocastra/Medac, Wedel,
Germany), secondary antibody (biotinylated goat anti-mouse immunoglobulin
antibody; Zymed, San Francisco, CA), exposed to streptavidin peroxidase,
developed with diaminobenzidine as substrate, and weakly counterstained with
hematoxylin. Positive staining was analyzed by measuring the CD34+
area using the Olympus DP-Soft analysis software.
EphB4-Fc and ephrinB2-Fc receptor body staining
Cells were fixed, blocked with 3% BSA/PBS (albumin bovine fraction V;
Serva, Heidelberg, Germany) and incubated with 1 µg/ml EphB4-Fc or
ephrinB2-Fc. Binding was detected by a goat antibody specific for anti-human
Fc conjugated to Cy3 (Sigma). Staining of the nuclei was performed using
DAPI.
Immunoprecipitation and western blotting
EphB4-Fc (4 µg/ml) and ephrinB2-Fc (4 µg/ml) were coupled to 20 µl
protein-G-agarose (Roche Diagnostics, Mannheim, Germany) in the presence of
250 µl lysis buffer. Cell lysate (1.0 ml) containing 2 mM
Na3VO4 and protease inhibitor cocktail (Sigma) were
precleared with 20 µl protein-G-agarose for 2 hours at 4°C. The cleared
cell lysates were incubated with the protein-G-agarose-coupled EphB4 and
ephrinB2. Precipitates were washed, lysed and run on a 10% SDS-PAGE gel.
Western-blotted gels were probed with a monoclonal phosphotyrosine (0.2
µg/ml; Santa Cruz, Heidelberg, Germany) and visualized by
chemiluminescence. Equal loading of gels was confirmed by stripping and
reprobing the blots with the corresponding EphB4 and ephrinB2 antibodies (0.2
µg/ml polyclonal antisera; both antibodies from R&D Systems).
Statistical analysis
All results are expressed as mean±s.d.; differences between
experimental groups were analyzed by impaired Student's t-test and
P<0.05 was considered as statistically significant.
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Results |
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|
EphrinB2-Fc acts anti-adhesively on ECs
Eph-ephrin interactions play a key role in axonal guidance by transmitting
attractive and repulsive signals
(Kullander and Klein, 2002).
Likewise, bidirectional signaling interactions of B-class ephrins with their
corresponding B-class Eph receptors have been shown to orchestrate the
invasive ingrowth of blood vessels and arterio-venous differentiation
(Adams, 2002
). In order to
define propulsive and repulsive effector functions of ephrinB2 ligands and
EphB4 receptors on ECs, HUVECs were seeded on ephrinB2-Fc- or EphB4-Fc-coated
adhesive and non-adhesive tissue culture dishes. Coating with ephrinB2-Fc
completely blocks adhesion of HUVECs to adhesive culture dishes, whereas
EphB4-Fc does not interfere with EC adhesion to tissue culture dishes. In
turn, when using EphB4-Fc-coated non-adhesive tissue culture dishes, a subset
of HUVECs quantitatively corresponding to the ephrinB2+ HUVEC
subpopulation (Fig. 1B) was
able to adhere (Fig. 2A). Similar results were obtained with microvascular EC populations, such as human
dermal microvascular ECs (HDMVECs) (data not shown). Titration experiments of
the anti-adhesive ephrinB2-Fc effect identified a steep dose-response curve
with an EC50 of 12.2 ng/mm2 ephrinB2-Fc
(Fig. 2B). Clustering of
ephrinB2-Fc receptor bodies with an Fc-specific secondary antibody prior to
coating induced a left shift of the curve and an even steeper dose-response
curve (EC50: 8.5 ng/mm2; hill slope: 2.4).
Additional adhesion/detachment experiments showed that the anti-adhesive
effect of ephrinB2-Fc on HUVECs is also exerted, albeit quantitatively
attenuated, if ephrinB2-Fc is added to adherent cells or if suspended HUVECs
are preincubated with ephrinB2-Fc prior to seeding on adhesive substrata (data
not shown). Control experiments with smooth muscle cells and fibroblasts
confirmed that the ephrinB2-Fc- and EphB4-Fc-mediated detachment-inducing and
adhesion-mediating effects are specific for ephrinB2- and EphB4-expressing ECs
(data not shown).
We have developed a spheroidal co-culture system of ECs and SMCs that
mimics the 3D assembly of the normal vessel wall with a multicellular layer of
SMCs and a surface monolayer of ECs (Korff
et al., 2001). Exposure of differentiated EC/SMC co-culture
spheroids to ephrinB2-Fc disrupts the integrity of the continuous layer of
surface ECs and induces detachment of ECs
(Fig. 3C). EphB4-Fc had no
effect on EC monolayer integrity. Corresponding to these experiments, we
performed experiments with in situ explanted fragments of umbilical cords.
Treatment of fragments of explanted umbilical cord with ephrinB2-Fc led to
detachment of ECs from their underlying extracellular matrix, which could be
visualized and quantitated after CD34 staining
(Fig. 3D,G). EphB4-Fc did not
affect ECs in co-culture spheroids or in in situ explanted fragments of the
umbilical cord (Fig. 3E,F).
Together, the in vitro and in situ experiments indicate that ephrinB2-Fc,
binding to EphB2, EphB3 and/or EphB4, is capable of acting anti-adhesively on
ECs that are in their proper organotypic context with SMCs.
|
EphrinB2-Fc inhibits EC migration and sprouting angiogenesis, whereas
EphB4-Fc stimulates migration and sprouting angiogenesis
EphrinB2 is strongly expressed by ECs during angiogenesis
(Gale et al., 2001;
Shin et al., 2001
). On the
basis of the observed anti-adhesive capacity of ephrinB2-Fc, we set out
experiments aimed at functionally manipulating Eph-ephrin interactions during
specific steps of the angiogenic cascade. A 2D lateral cell migration assay,
as well as the vertical gradient-driven Boyden chamber, was used to study EC
migration and chemotaxis. Baseline migration as well as VEGF-induced migration
of ECs was inhibited by ephrinB2-Fc (Fig.
4A). By contrast, EphB4-Fc stimulates lateral EC migration
(Fig. 4A). Correspondingly, the
capacity of ephrinB2-Fc and EphB4-Fc to modulate VEGF-mediated chemoattraction
of HUVECs was analyzed in a modified Boyden chamber assay. These experiments
showed that ephrinB2-Fc, but not EphB4-Fc, inhibits VEGF-mediated
chemoattraction (Fig. 4B). A 3D
collagen gel assay was employed to quantitate the effect of soluble dimeric
ephrin ligands and Eph receptors on sprouting angiogenesis. EphrinB2-Fc
inhibited baseline as well as VEGF-induced sprouting angiogenesis, which was
prominently stimulated by EphB4-Fc (Fig.
5A). Alignment of ECs cultured on top of Matrigel reflects some
morphogenic properties of ECs during angiogenesis. EphrinB2-Fc strongly
inhibits alignment of ECs grown on Matrigel, which is not affected by EphB4-Fc
(Fig. 5B).
|
Repulsive forward EphB4 signaling is sufficient to induce EC
segregation
The above experiments with exogenous soluble dimeric and clustered ephrinB2
and EphB4 receptor bodies had identified repulsive activities of the EphB4
receptor and propulsive activities of ephrinB2 expressed by ECs. In order to
study the functional consequences of cell-cell contact-dependent Eph-ephrin
signaling, we have generated constitutively EphB4- and ephrinB2-overexpressing
EC lines (PAECs). Wild-type or mock-transfected PAECs do not express
detectable levels of functional EphB4 or ephrinB2 as assessed by a negative
receptor body binding assay, which was strongly and uniformly positive for
EphB4- and ephrinB2-transfected cells, respectively (data not shown).
Transfected PAECs show essentially the same responses to receptor body
activation as the endogenous EphB4- and ephrinB2-expressing HUVECs. Yet, PAECs
have a high baseline sprouting activity. As a consequence, sprouting of
collagen gel-embedded spheroids of ephrinB2-transfected PAECs cannot be
further enhanced by EphB4-Fc. In turn, ephrinB2-Fc strongly and selectively
inhibits sprouting of EphB4-overexpressing PAECs (and not sprouting of
mock-transfected cells) (Fig.
6A). EphrinB2-Fc stimulation of EphB4-expressing PAE cells is
associated with strong tyrosine phosphorylation of EphB4
(Fig. 6B).
|
Cell-mixing experiments were performed with EphB4 and ephrinB2
transfectants as well as with PAECs transfected with a truncated ephrinB2
ligand that lacks the intracellular signaling domain (ephrinB2 PAECs).
Planar co-cultures of either ephrinB2,
ephrinB2, or EphB4 with
mock-transfected PAECs led to uniform mixing of the cells, as demonstrated by
the even distribution of fluorescent-labeled cells upon adhesion in tissue
culture dishes (Fig. 7A-C). By
contrast, mixing of ephrinB2- and EphB4-expressing PAECs led to the
segregation of the cells upon adhesion and formation of clusters of
ephrinB2+ and EphB4+ cells
(Fig. 7D). Unidirectional
forward EphB4 signaling was sufficient for the cellular segregation, as shown
by the segregation of EphB4-expressing cells from
ephrinB2 PAECs upon
mixing (Fig. 7E). Biochemical
analysis of the mixing experiments identified intense tyrosine phosphorylation
of EphB4 in co-cultures of both EphB4 with ephrinB2-overexpressing cells and
EphB4 with
ephrinB2-overexpressing PAE cells
(Fig. 7F,G).
|
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Discussion |
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The findings of our study support a model of endothelial propulsive and
repulsive activities that mediate endothelial guidance signals during invasive
angiogenesis as well as the positional control of EphB receptor- and ephrinB
ligand-expressing cells towards each other
(Fig. 8). This model is based
on our functional data and takes into account published data on the repulsive
guidance of ECs and neural crest cells by surrounding cells
(Helbling et al., 2000;
Krull et al., 1997
;
Oike et al., 2002
;
Wang and Anderson, 1997
). EC
guidance refers to Eph-ephrin signaling-mediated positional information
between outgrowing ECs and surrounding cells as it occurs during intersomitic
invasion of sprouting capillaries
(Helbling et al., 2000
).
Correspondingly, EphB-ephrinB interactions have been shown to regulate EC and
mural cell interactions (Oike et al.,
2002
). Likewise, mural cells (smooth muscle cells, pericytes) and
other surrounding cells such as astrocytes in the retina have been proposed to
act as guidance cells during sprouting angiogenesis
(Nehls et al., 1998
), which
may similarly involve propulsive and repulsive Eph-ephrin signaling.
|
Eph-ephrin interactions also control the spatial organization of ECs
towards each other as they are involved during vascular network formation
(Fig. 8). EphB4 forward
signaling restricts cellular intermingling and, thus, drives segregation of
EphB4- and ephrinB2-expressing ECs, which controls proper asymmetric
arterio-venous assembly of EphB4+ and ephrinB2+ ECs. The
confrontation experiments indicate an arterio-venous push and pull orientation
during angiogenesis, which supports an artery-to-vein model of sprouting
angiogenesis. Likewise, the antagonistic propulsive and repulsive functions of
ephrinB ligands and EphB receptors have led to speculations about a third
ephrin- and Eph- cell population in between the
ephrin+ and Eph+ cells
(Oike et al., 2002). This
conclusion is also supported by lacZ staining experiments suggesting that a
significant proportion of capillary ECs may express neither EphB4 nor ephrinB2
(Visconti et al., 2002
). In
turn, despite the apparently antagonistic functions of EphB4 and ephrinB2, our
experiments have shown that individual ECs can even coexpress EphB receptors
and ephrinB ligands as shown by the chimeric expression status of HUVECs.
Furthermore, we have also performed triple-cell-type intermingling experiments
(mock-transfected plus ephrinB2-transfected plus EphB4-transfected), which
have shown that EphB4 cells fail to segregate under these conditions (data not
shown). Thus, the positional orientation of EphB receptor+, ephrinB
ligand+ and Eph-ephrin- ECs in different vascular beds
awaits further analysis and might guide the identification of other EC Eph-
and ephrin-mediated functions.
The proposed model (Fig. 8)
summarizes the findings of the present study and is in line with published
data obtained in genetic mouse models. Conceptually, it also corresponds to
the propulsive and repulsive models established for Eph-ephrin-mediated
control of neuronal outgrowth (Cooke and
Moens, 2002). It is also in line with the recent identification of
EphB-receptor-mediated repulsive forces that separate proliferating and
differentiating intestinal epithelial cells. Lack of repulsive forces in
EphB2/EphB3-null mice leads to an intermingling of proliferating and
differentiating intestinal epithelial cells
(Batlle et al., 2002
). Yet, the
model also points to several key unanswered questions and unresolved
discrepancies for Eph and ephrin functions in the vascular system. Capillary
sprouting is believed to originate primarily from capillaries and venules
(Burger et al., 1983
). In turn,
the expression of ephrinB2 by arterial ECs and ECs during angiogenesis has
been considered as evidence that angiogenesis might have an arterial origin
(Shin et al., 2001
). Our data
have shown that ephrinB2-expressing ECs have a propulsive and invasive
phenotype corresponding to the properties of angiogenic ECs. Thus, it remains
to be seen if ephrinB2 expression of angiogenic ECs indicates an arterial
origin of angiogenesis or if angiogenic activation induces EC ephrinB2
expression in ephrin- cells or even a switch of the asymmetric
arterio-venous ephrinB2 and EphB4 expression towards angiogenic ephrinB2
expression. Expression profiling experiments suggest the latter, as
demonstrated by the intense upregulation of EC ephrinB2 expression following
angiogenic activation by VEGF (G. Dandekar et al., unpublished). This
observation also corresponds to the arteriolization associated with
VEGF-induced angiogenesis (Stalmans et
al., 2002
).
Forward EphB4 signaling was sufficient for the segregation of
EphB4-expressing cells as shown by the segregation-inducing effects of
ephrinB2-expressing cells. Yet, reverse ephrinB2 signaling is capable
of transducing propulsive and pro-angiogenic activities on ECs. The
cytoplasmic domain of ephrinB ligands contains five conserved tyrosine
residues and a PDZ-binding domain. Correspondingly, tyrosine
phosphorylation-dependent and phosphorylation-independent ephrinB signaling
has been reported: Src family kinases are involved in
phosphorylation-dependent signaling
(Palmer et al., 2002
) and the
SH2-SH3 domain adapter protein Grb4 acts as a downstream effector of ephrin B
ligands (Cowan and Henkemeyer,
2001
). Likewise, several PDZ-domain proteins have been reported to
interact with ephrinB ligands including GRIP1, GRIP2 and syntenin, as well as
Pick1, PDZ-RGS3 and the tyrosine phosphatase PTP-BL
(Kullander and Klein, 2002
).
The latter proteins are of particular interest as these PDZ-domain molecules
are themselves linked to a functional unit. Pick1 interacts with protein
kinase C, PTP-BL can act as a negative regulator of ephrinB phosphorylation
and Src activity (Palmer et al.,
2002
), and PDZ-RGS3 can negatively interfere with SDF-1/CXC-R4
chemokine signaling (Lu et al.,
2001
). Thus, given the different signaling pathways that may be
entertained by reverse ephrinB signaling, it is intriguing to speculate that
reverse ephrinB signaling can contribute to different angiogenic effector
functions. Further ongoing experiments with ECs expressing different ephrinB2
truncation mutants might help to shed further light onto the complexity of
endothelial EphB and ephrinB functions.
In summary, the present study has for the first time conclusively analyzed the functional consequences of EC ephrinB2 and EphB4 activation at the cellular level. The data support a model of propulsive and repulsive endothelial ephrinB2 and EphB4 signaling that contributes to providing positional cues that regulate guided EC migration and proper arterio-venous differentiation. The data support a model of artery-to-vein invasive angiogenesis and a forward push and reverse pull interaction between ephrinB2+ and EphB4+ ECs.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
Since submission of this manuscript, Hamada et al. have published a paper
on the adhesive and migratory effector functions of ephrinB2 and EphB4 on
endothelial cells (Hamada et al.,
2003). Corresponding to the findings presented in Fig. 2 and Fig. 4, Hamada et al. also
report proadhesive and promigratory functions of EphB4-Fc and antiadhesive and
antimigratory functions of ephrinB2-Fc.
* These authors contributed equally to this work
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