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J. Cell Biol. Vol. 185 No. 4 657–671
Correspondence to Dietmar Vestweber: firstname.lastname@example.org
Abbreviations used in this paper: ESAM, endothelial cell selective adhesion
molecule; VE-PTP, vascular endothelial protein tyrosine phosphatase.
The formation of the blood vessel system during embryonic
development requires a multitude of receptors and adhesion
molecules that regulate the creation of the first primitive vascular
plexus (vasculogenesis) and the various remodeling processes
that lead to the establishment of the mature vascular system.
Several of the receptors involved in these processes represent
tyrosine kinases such as the receptors for VEGF and the Tie-2
receptor. Whereas VEGFR-2 is essential for vasculogenesis and
sprouting of nascent blood vessels, Tie-2 is important for subse-
quent remodeling processes. Tie-2 is a receptor for the angio-
poietins, of which Ang1 promotes vascular remodeling, maturation,
and stabilization of the vasculature. Tie-2 knock-out mouse
embryos die by E10.5 due to endocardial defects, hemorrhaging,
and impaired vascular network formation (Dumont et al., 1994;
Sato et al., 1995), similar to the defects of Ang1-null mice that
die around E12.5, showing comparable deficits in vascular re-
modeling, maturation, and stabilization of blood vessels (Suri et al.,
1996). In contrast, overexpression of the Tie-2 ligand Ang2 mim-
ics the defects caused by Ang1 and Tie-2 ablation (Maisonpierre
et al., 1997). This argues for an antagonistic function of Ang2
and illustrates the need to precisely balance the activation level
of the Tie-2 receptor system during embryonic development.
Tyrosine phosphatases are obvious candidates for signal-
ing molecules that counteract the activation of tyrosine kinase
receptors. Very few receptor-type protein tyrosine phosphatases
(RPTPs) are known as regulators of angiogenesis. A mutated form
of density-enhanced phosphatase (DEP-1, CD148), with the phos-
phatase domain being replaced by the chromophore GFP caused
embryonic lethality due to vascular malformations (Takahashi
et al., 2003), and DEP-1 was found to be involved in arterial/
venous specification in zebrafish (Rodriguez et al., 2008). Sur-
prisingly, DEP-1 gene ablation in mice does not cause obvious
defects during embryonic angiogenesis or embryonic lethality
(Trapasso et al., 2006; Zhu et al., 2008).
In contrast to DEP-1, the vascular endothelial protein
tyrosine phosphatase (VE-PTP) is an endothelial-specific RPTP
(Fachinger et al., 1999). Deletion of its cytoplasmic phosphatase
domain, the transmembrane region, and the most membrane-
proximal extracellular fibronectin type III-like repeat causes em-
bryonic lethality shortly before 10 d of gestation, accompanied
VE-cadherin. VE-PTP gene disruption leads to embryonic
lethality, vascular remodeling defects, and enlargement of
vascular structures in extraembryonic tissues. We show here
that antibodies against the extracellular part of VE-PTP mimic
the effects of VE-PTP gene disruption exemplified by vessel
enlargement in allantois explants. These effects require the
presence of the angiopoietin receptor Tie-2. Analyzing the
ascular endothelial protein tyrosine phosphatase
(VE-PTP) is an endothelial-specific receptor-type ty-
rosine phosphatase that associates with Tie-2 and
mechanism we found that anti–VE-PTP antibodies trigger
endocytosis and selectively affect Tie-2–associated, but not
VE-cadherin–associated VE-PTP. Dissociation of VE-PTP trig-
gers the activation of Tie-2, leading to enhanced endothelial
cell proliferation and enlargement of vascular structures
through activation of Erk1/2. Importantly, the antibody ef-
fect on vessel enlargement is also observed in newborn mice.
We conclude that VE-PTP is required to balance Tie-2 activ-
ity and endothelial cell proliferation, thereby controlling
blood vessel development and vessel size.
VE-PTP controls blood vessel development by
balancing Tie-2 activity
Mark Winderlich,1 Linda Keller,1 Giuseppe Cagna,1 Andre Broermann,1 Olena Kamenyeva,1 Friedemann Kiefer,1
Urban Deutsch,2 Astrid F. Nottebaum,1 and Dietmar Vestweber1
1Max-Planck-Institute of Molecular Biomedicine, D-48149 Münster, Germany
2Theodor Kocher Institute, University of Bern, CH-3012 Bern, Switzerland
© 2009 Winderlich et al. This article is distributed under the terms of an Attribution–
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tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
T H E J O U R N A L O F C E L L B I O L O G Y
JCB • VOLUME 185 • NUMBER 4 • 2009 658
we found that such antibodies mimicked, when added for 22 h to
cultured allantois explants derived from wild-type mice, the ef-
fect observed in explant cultures of VE-PTP mutant allantoides.
As shown in Fig. 1 A, endothelial structures visualized by stain-
ing for VE-cadherin were enlarged upon incubation with anti–
VE-PTP antibodies, resulting in a more than 2.5-fold increase in
average diameter of endothelial cords compared with allantoides
treated with preimmune antibodies (Fig. 1 B). Determining cord
diameters is explained in LaRue et al. (2003). The number of
branching points was reduced by more than twofold (Fig. 1 B).
Endothelial cell contacts were not affected, as demonstrated by
staining for the junctional proteins VE-cadherin and endothelial
cell selective adhesion molecule (ESAM) (Fig. S1). The rat
monoclonal antibody (mAb) 109.1 against mouse VE-PTP had a
similar effect, whereas no effect was seen with a control mAb
against the endothelial antigen ESAM (Fig. S1).
Analyzing whether the antibodies against VE-PTP would
affect the expression level of VE-PTP, we found that incubation
of cultured mouse bEnd.5 endothelioma cells with antibodies
against the extracellular part of VE-PTP for 1 h strongly reduced
the amount of VE-PTP as tested in immunoblots with antibodies
against the C terminus (Fig. 1 C). Blocking endocytosis by add-
ing 0.45M sucrose to the culture medium while incubating with
the antibodies prevented the reduction of VE-PTP protein, indi-
cating that the anti–VE-PTP antibodies down-regulated the levels
of VE-PTP by triggering endocytosis (Fig. 1 C).
Endocytosis of VE-PTP induced by antibodies could be
demonstrated by immunofluorescence. As shown in Fig. 2 A, pre-
incubation of endothelioma cells with antibodies against the
extracellular part of VE-PTP for 20 min followed by fixation,
permeabilization, and incubation with a labeled secondary anti-
body indeed allowed to detect VE-PTP in granules inside the cells.
No such staining was observed when cells were not permeabilized
or when cells were preincubated with preimmune antibodies.
After 10 min of antibody preincubation, VE-PTP colocalized par-
tially with the early endosomal marker EEA1 (Fig. S2 A). After
90 min of preincubation time intracellular VE-PTP staining was
no longer detectable (Fig. S2, B and C). We conclude that anti–
VE-PTP antibodies trigger endocytosis of VE-PTP molecules.
Endocytosis of VE-PTP was also observed in allantois explant
cultures (Videos 5–7).
Antibodies against VE-PTP selectively
down-regulate the Tie-2–associated but not
the VE-cadherin–associated VE-PTP
VE-PTP associates with Tie-2 (Fachinger et al., 1999; Saharinen
et al., 2008) and with VE-cadherin (Nawroth et al., 2002). We
confirmed this by showing that VE-PTP and Tie-2, endogenously
expressed in mouse and human endothelial cells, can indeed
be coprecipitated (Fig. S3). To examine whether it would be
VE-cadherin or Tie-2 that is involved in the anti–VE-PTP effect
on the enlargement of vascular structures in allantois tissue,
we tested whether anti–VE-PTP antibodies affected Tie-2 and
VE-cadherin–associated VE-PTP molecules in the same way.
To this end, we preincubated bEnd.5 cells with antibodies against
the extracellular part of VE-PTP in order to reduce VE-PTP levels.
by dramatically enlarged blood vessels in the yolk sac, which
form large cavities (Baumer et al., 2006). Formation of the vas-
cular plexus was generally not affected throughout the embryo,
yet remodeling was defective. Explants of allantois tissue devel-
oped large endothelial sacs instead of the usual tubular vascular
network. In addition, heart development was defective (Baumer
et al., 2006). Defects essentially identical to the VE-PTP trunca-
tion mutants were observed in mice carrying a null allele of the
VE-PTP gene (Dominguez et al., 2007).
The molecular and cellular mechanisms that cause the
observed angiogenesis defects in VE-PTP mutant mice are
unknown. VE-PTP was found to associate with two endothelial
cell surface membrane proteins essential for angiogenesis. The
first one was Tie-2, which was found to bind to the cytoplasmic
phosphatase domain of VE-PTP. Co-expression with VE-PTP
in transfected cells reduced tyrosine phosphorylation of Tie-2
(Fachinger et al., 1999; Saharinen et al., 2008). Interestingly, no
such interactions were found between VE-PTP and VEGFR-2.
Whether physiological functions of Tie-2 in angiogenesis are
affected by VE-PTP has not been analyzed previously.
A second association partner of VE-PTP is the endothelial-
specific VE-cadherin (Nawroth et al., 2002). This association is
mediated via the extracellular domains of both membrane pro-
teins. We have shown that induction of VE-PTP expression in
cells cotransfected with VE-cadherin enhances the adhesive
function of VE-cadherin in these cells (Nawroth et al., 2002).
Recently, we found that silencing of VE-PTP expression in
endothelial cells indeed strongly reduced the adhesive function
of VE-cadherin, demonstrating the importance of VE-PTP in reg-
ulating VE-cadherin function in endothelial cells. (Nottebaum
et al., 2008).
Here we show that antibodies against the extracellular do-
mains of VE-PTP cause vessel enlargement in allantois explants
resembling the defects in vascular remodeling caused by VE-PTP
gene disruption. These effects required Tie-2, as they were com-
pletely abolished by lack of Tie-2. We show that mechanistically,
this is due to the anti–VE-PTP antibodies triggering selective
endocytosis of only the Tie-2–associated, but not the VE-cadherin–
associated VE-PTP protein fraction. VE-PTP dissociation from
Tie-2 led to activation of Tie-2, thus enhancing endothelial cell
proliferation and enlargement of vascular structures via activation
of Erk1/2. Importantly, the antibody effect on vessel enlargement
could be confirmed in vivo in 1–2-wk-old mice. Our results show
that VE-PTP is required to control Tie-2 activity and endothelial
cell proliferation during vascular remodeling in the embryo and
in newly born mice.
Antibodies against VE-PTP stimulate
vessel enlargement in allantois explants
We have shown recently that disruption of the VE-PTP gene
causes defects in vascular remodeling and leads to dramatically
enlarged endothelial structures in allantois explants (Baumer
et al., 2006). To elucidate the mechanism underlying this aber-
ration, we tested whether antibodies against the extracellular
domain of VE-PTP would be able to mimic this effect. Indeed,
659VE-PTP IS AN ESSENTIAL TIE-2 REGULATOR • Winderlich et al.
unchanged (Fig. 2 C). We conclude that anti–VE-PTP antibodies
selectively displace VE-PTP from Tie-2 and trigger endocytosis
of these VE-PTP molecules, whereas VE-PTP molecules that are
associated with VE-cadherin stay unaffected.
In agreement with this interpretation, we found that Tie-2
was not endocytosed together with VE-PTP upon preincubation
of living cells with anti–VE-PTP antibodies (Fig. 3 A). In addi-
tion, endocytosis of VE-PTP neither affected the expression of
VE-cadherin nor that of VE-PTP at endothelial cell contacts
(Fig. 3 B), and also the staining for the tight junction–associated
ESAM was unaffected (Fig. S2 C).
Antibodies against VE-PTP activate
tyrosine phosphorylation of Tie-2
The selective effect of the anti–VE-PTP antibodies on the Tie-2–
associated fraction of VE-PTP molecules prompted us to test
Subsequently, cells were washed and lysed and residual VE-PTP
was immunoprecipitated with antibodies against the C terminus of
VE-PTP. Precipitates were analyzed in immunoblots for VE-PTP
and for coprecipitated Tie-2 and VE-cadherin. To our surprise, we
found that the amount of VE-cadherin coprecipitated with anti–
VE-PTP antibodies was unchanged independent of whether cells
had been pretreated with anti–VE-PTP or with control antibodies
(Fig. 2 B). Thus, despite the strong down-regulation of VE-PTP, the
amount of coprecipitated VE-cadherin was not reduced. In con-
trast, the amount of coprecipitated Tie-2 was reduced to a similar
extent as the amount of VE-PTP that was still available for the
immunoprecipitation (Fig. 2 B). Analyzing the cell surface expres-
sion of VE-PTP, Tie-2, and VE-cadherin on bEnd.5 cells by FACS
revealed that cell surface levels of VE-PTP were indeed down-
regulated by the preincubation with anti–VE-PTP antibodies,
whereas the cell surface levels of Tie-2 and VE-cadherin were
Figure 1. Antibodies against VE-PTP trigger vessel enlargement in allantois explants and down-regulate VE-PTP. (A) Allantois explants from E8.5 wild-type
embryos were cultured on gelatin-coated ultrathin glass slides in the presence of polyclonal antibodies against VE-PTP (-VE-PTP) or preimmune antibodies
(control) for 22 h and subsequently stained by indirect immunofluorescence with a monoclonal antibody for VE-cadherin. Bar, 50 µm. (B) Average endo-
thelial cord diameters (top) and branching points (bottom) were determined for 5 control and 5 anti–VE-PTP treated allantois explants, with 30 randomly
chosen vessels per explant (as shown in A); ***, P < 0,001. (C) Confluent mouse bEnd.5 cells were treated with polyclonal antibodies against VE-PTP or
preimmune antibodies for 1 h either in normal culture medium or 0.45 M sucrose containing medium (as indicated) to block endocytosis. Aliquots of cell
lysates with identical protein content were immunoblotted for VE-PTP and equal loading was controlled by blotting for the endothelial antigen ESAM (as
indicated on the right). Molecular weight markers are indicated on the left.
JCB • VOLUME 185 • NUMBER 4 • 2009 660
Figure 2. Antibodies against VE-PTP trigger endocytosis and down-regulation of Tie-2–associated but not VE-cadherin–associated VE-PTP. (A) Confluent
bEnd.3 cells were treated with polyclonal antibodies against VE-PTP (-VE-PTP) or preimmune antibodies (control IgG) for 20 min. Subsequently, cells were
either fixed and permeabilized (top and bottom) or only fixed (middle) and stained with Alexa 568–conjugated secondary antibodies. Cell nuclei were
counterstained with Hoechst. Bar, 20 µm. (B) bEnd.5 cells were treated with polyclonal antibodies against VE-PTP (-VE-PTP) or preimmune antibodies
(control) for 1 h. VE-PTP was immunoprecipitated from endothelial cells and analyzed by immunoblotting for coprecipitated VE-cadherin and Tie-2, respec-
tively, or for VE-PTP (as indicated underneath). Aliquots of cell lysates with identical protein content were directly immunoblotted for VE-cadherin or Tie-2
(bottom). Quantified signal intensities are indicated. Molecular weight markers are indicated. (C) FACS analysis showing the surface expression of Tie-2,
VE-cadherin, and VE-PTP of bEnd.5 cells after 1 h pretreatment with monoclonal antibodies against VE-PTP (red) or preimmune antibodies (blue). The mean
fluorescence FACS signal for VE-PTP is indicated in percent (bottom).
661VE-PTP IS AN ESSENTIAL TIE-2 REGULATOR • Winderlich et al.
whether these antibodies stimulate tyrosine phosphorylation of
Tie-2. Incubating bEnd.5 cells for 1 h with antibodies against
VE-PTP resulted in a strong increase in tyrosine phosphoryla-
tion of Tie-2 (Fig. 4 A). This effect was rapid, because even anti–
VE-PTP antibody incubations as short as 3 min triggered Tie-2
phosphorylation (Fig. 4 B). Interestingly, anti–VE-PTP antibodies
did not enhance tyrosine phosphorylation of VE-cadherin, or
associated -catenin or plakoglobin (Fig. 5 A), although silencing
of VE-PTP by siRNA enhanced plakoglobin tyrosine phos-
phorylation (Fig. 5 B and Nottebaum et al., 2008). Again, this
suggests that the anti–VE-PTP antibodies did not affect the
VE-cadherin–associated population of VE-PTP molecules.
Figure 3. Antibodies against VE-PTP do not trigger endocytosis of Tie-2 and leave VE-cadherin and VE-PTP at endothelial cell contacts unaffected.
(A) Confluent bEnd.3 cells were treated with polyclonal antibodies against VE-PTP (-VE-PTP) or preimmune antibodies (control IgG) for 20 min. Subsequently,
fixed and permeabilized cells were stained with Alexa 568–conjugated secondary antibodies (internalized VE-PTP, internalized control) and for Tie-2 (Tie-2).
An anti–Tie-2 staining control is shown in Fig. S5. Cell nuclei were counterstained with Hoechst. Bar, 25 µm. (B) Confluent bEnd.3 cells were treated with
monoclonal antibodies against VE-PTP (-VE-PTP) or control antibodies (control IgG) for 30 min. Subsequently, fixed and permeabilized cells were stained
with Alexa 568–conjugated secondary antibodies (internalized VE-PTP, internalized control) and for VE-cadherin and VE-PTP. Cell nuclei were counter-
stained with Hoechst. Internalized VE-PTP was not detected with new antibodies against VE-PTP, probably because epitopes were masked by the antibodies
that had triggered endocytosis. Bar, 10 µm.
JCB • VOLUME 185 • NUMBER 4 • 2009 662
To obtain additional evidence for the role of VE-PTP in
the regulation of Tie-2 activity, we combined the expression of
a phosphatase-dead mutant of mouse VE-PTP in HUVEC (using
adenovirus vectors) with the use of anti–VE-PTP antibodies.
We expressed either mouse VE-PTP with an intact phosphatase
domain or in a parallel experiment the phosphatase-dead mutant
of mouse VE-PTP in HUVEC (using adenovirus) and tested the
In agreement with the anti–VE-PTP antibody effects on Tie-2
activation, similar effects were obtained after silencing VE-PTP
by siRNA in bEnd.5 (Fig. 4 C) and in HUVEC (Fig. 4 D). In addi-
tion, we found that VE-PTP gene disruption leads to tyrosine
phosphorylation of Tie-2, as was analyzed with endothelioma cells
generated from gene-disrupted mice (Baumer et al., 2006) ex-
pressing a truncated form of VE-PTP lacking the trans-membrane
region and the cytoplasmic tail with its phosphatase domain
(Fig. 4 E). Results were analyzed with three independent cell lines
for each genotype and quantified (Fig. 4 F).
Figure 4. VE-PTP expression inhibited by either antibodies, siRNA, or
gene disruption triggers Tie-2 tyrosine phosphorylation in endothelial cells.
(A) bEnd.5 cells were treated with polyclonal antibodies against VE-PTP or
preimmune antibodies for 1 h and subsequently immunoprecipitated for
Tie-2, followed by immunoblotting with anti-phosphotyrosine antibodies
(pTyr) and antibodies against Tie-2. Aliquots of cell lysates with identical
protein content were directly immunoblotted for VE-PTP and Tie-2 (bottom).
(B) Similar as in A, except that antibodies were only incubated for 3 min
(C) bEnd.5 cells were either transfected with control siRNA or with siRNA
directed against VE-PTP. 24 h later, Tie-2 was immunoprecipitated and
immunocomplexes (top) or cell lysates (bottom) were analyzed by immuno-
blotting with antibodies against phosphotyrosine (pTyr), Tie-2, VE-PTP, and
plakoglobin (as indicated). (D) HUVECs instead of bEnd.5 cells were ana-
lyzed as in C. (E) Embryonic endothelioma cells established either from
wild-type (+/+), heterozygous (+/mut), or homozygous (mut/mut) VE-PTP
mutant embryos were subjected to immunoprecipitations with antibodies
against Tie-2. Immunocomplexes were analyzed by immunoblotting with
antibodies against phosphotyrosine or Tie-2 (as indicated). (F) Quantifica-
tion of Tie-2 tyrosine phosphorylation (±SD) in wild-type (+/+; n = 2 cell
lines), heterozygous (+/mut; n = 3 cell lines), or homozygous (mut/mut; n = 3
cell lines) VE-PTP mutant endothelioma, analyzed as in E. **, P < 0,01.
Figure 5. Down-regulation of VE-PTP by siRNA, but not by anti–VE-PTP
antibodies, enhances tyrosine phosphorylation of VE-cadherin–associated
plakoglobin. (A) bEnd.5 cells were treated with polyclonal antibodies against
VE-PTP (-VE-PTP) or preimmune antibodies (control) for 1 h and subsequently
immunoprecipitated for VE-cadherin, followed by immunoblotting with anti-
bodies against phosphotyrosine (pTyr), VE-cadherin, or plakoglobin. Aliquots
of cell lysates with identical protein content were directly immunoblotted for
VE-PTP and VE-cadherin (bottom). (B) Similar as in A, except that antibody
preincubation of cells was replaced by transfection with either control siRNA
or VE-PTP siRNA.
663VE-PTP IS AN ESSENTIAL TIE-2 REGULATOR • Winderlich et al.
Vessel enlargement induced by anti–VE-PTP
antibodies requires Tie-2
Despite the essential function of Tie-2 during embryonic vascular
remodeling, we have found that allantois explants from Tie-2–null
mice develop normal vascular networks (Baumer et al., 2006).
However, the selective anti–VE-PTP antibody effect on Tie-2 acti-
vation suggested that VE-PTP deficiency, caused by either gene
disruption or by antibody-mediated down-regulation of VE-PTP,
effect of antibodies against the extracellular part of mouse
VE-PTP on Tie-2 activation in these cells. Importantly, the anti-
bodies we used did not crossreact with human VE-PTP. As we
expected, the expression of intact VE-PTP reduced tyrosine
phosphorylation of Tie-2, whereas the dominant-negative mu-
tant enhanced Tie-2 activity (Fig. 6, A and B). Incubation of the
cells with antibodies against mouse VE-PTP induced activation
of Tie-2 in cells that expressed intact mouse VE-PTP (Fig. 6 A).
Intriguingly, the same antibodies had the opposite effect in
HUVEC expressing the dominant-negative form of VE-PTP
(Fig. 6 B). This clearly rules out indirect effects of the antibodies
on Tie-2 activation and shows that it is the dissociation of the
intact phosphatase VE-PTP from Tie-2 that leads to the activation
of Tie-2, whereas the dissociation of the dominant-negative form
of VE-PTP makes it accessible again for intact human VE-PTP,
which then leads to the dephosphorylation of Tie-2.
VE-PTP counterbalances activation of Tie-2
To determine whether VE-PTP indeed counterbalances the acti-
vation of Tie-2 by Ang1, we combined silencing of VE-PTP by
siRNA in bEnd.5 cells with the stimulation with Ang1. As
shown in Fig. 7 A, a combined treatment with Ang1 and VE-PTP
siRNA stimulated Tie-2 phosphorylation to a greater extent than
a combined treatment with Ang1 and control siRNA. This sug-
gests that VE-PTP indeed counteracts the activation of Tie-2 by
Ang1. Ang2 enhanced the phosphorylation of Tie-2 in endo-
thelial cells only weakly and had no significant additive effect in
combination with silencing of VE-PTP expression (Fig. 7 A).
However, it cannot be ruled out that VE-PTP may also counter-
act small activation effects of Ang2.
Tyrosine phosphorylation analyzed in such experiments in-
deed represented proper Tie-2 activation because these results
could be verified in HUVEC for tyrosine residue 992 in the acti-
vation loop of the human Tie-2 kinase domain, the first tyrosine
phosphorylated upon activation (Shewchuk et al., 2000; Murray
et al., 2001) (Fig. 7 B). Likewise, down-regulation of VE-PTP
with antibodies also further enhanced Ang1 stimulated phosphory-
lation of tyrosine 992 of Tie-2 in mouse bEnd.5 cells (Fig. 7 C).
We conclude that antibodies against VE-PTP enhance the activa-
tion of the kinase domain of Tie-2 by Ang1.
We tested whether VE-PTP is constitutively associated
with Tie-2 or whether this would be enhanced upon activation
of Tie-2. Interestingly, we found that COMP-Ang1 stimula-
tion of Tie-2 in bEnd.3 cells clearly increased the association
of VE-PTP with Tie-2 (Fig. 7 D). A similar effect was also
seen with 600 ng/ml Ang1, but not with the same concentra-
tion of Ang2 (not depicted). This suggests that Ang1 stimu-
lates activation of Tie-2 and simultaneously triggers a negative
feed back process that limits activation by enhancing the asso-
ciation of Tie-2 with VE-PTP, and thus eventually balances
Tie-2 receptor activity.
Interestingly, antibody-stimulated endocytosis of VE-PTP
also triggered tyrosine phosphorylation of Tie-1 and this effect re-
quired the expression of Tie-2, as it was not observed in Tie-2–null
endothelioma cells (Fig. 7 E). This shows that VE-PTP specifi-
cally regulates Tie-2, whereas Tie-1 is indirectly affected.
Figure 6. VE-PTP regulates Tie-2 phosphorylation via its active phospha-
tase. (A) HUVECs either untransfected, or expressing GFP or expressing
mouse Flag-VE-PTP (as indicated above) were either untreated or treated
with polyclonal antibodies against mouse VE-PTP or preimmune antibodies
(as indicated above) for 1 h and subsequently immunoprecipitated for Tie-2,
followed by immunoblotting with anti-phosphotyrosine antibodies (pTyr)
and antibodies against Tie-2. Aliquots of cell lysates with identical protein
content were directly immunoblotted for Flag-VE-PTP and hVE-PTP (bottom).
Quantified signal intensities are indicated. (B) As in A with mouse Flag-
VE-PTP being replaced by the corresponding inactive phosphatase mutant
Flag-VE-PTP-C/S. White lines indicate that intervening lanes have been
JCB • VOLUME 185 • NUMBER 4 • 2009 664
explants had a similar effect as the anti–VE-PTP antibodies
(Fig. 8 B), further supporting the assumption that VE-PTP controls
vascular remodeling by counteracting the activation of Tie-2.
Anti–VE-PTP antibodies trigger vessel
enlargement by enhancing endothelial
cell proliferation via stimulating Tie-2
To determine how Tie-2 hyper-activation upon down-regulation of
VE-PTP leads to the enlargement of endothelial structures in
allantois explants, we directly visualized the formation of endo-
thelial structures in the explants. To this end, we performed live
imaging of allantois explants from mice expressing a VE-cadherin-
GFP fusion protein via a cDNA construct knocked into the VE-
cadherin locus, thereby replacing the endogenous VE-cadherin
allele. Mice homozygous for the VE-cadherin-GFP allele devel-
oped normally and were viable (unpublished data). Allantois
explants from these mice were cultured for 12 h and then incu-
bated for another 12 h in the presence of either anti–VE-PTP
antibodies, or preimmune antibodies or Ang1. As shown in the
respective Videos (Videos 1–3), Ang1 and anti–VE-PTP anti-
bodies, but not control antibodies, rapidly increased the area cov-
ered with endothelial structures and stimulated endothelial cell
division. Dividing endothelial cells could clearly be identified as
cells transiently rounding up. Staining of explants with phospho-
Histone 3 antibodies subsequent to live imaging revealed that round
cells still visible at the end of the observation period were indeed
positive for the mitosis marker (Fig. 9, A and B). The stimulatory
effect of anti–VE-PTP antibodies on endothelial proliferation was
quantified by costaining allantois explants for PECAM-1 and
phospho-Histone 3 and analyzing them by laser scanning confocal
microscopy with the help of 3D software (Video 4), which allowed
to unambiguously determine the endothelial nature of phospho-
Histone 3 positive nuclei. Quantification revealed that anti–VE-PTP
antibodies increased the number of mitotically active endothelial
cells by 2.8-fold in comparison to allantoides cultured in the pres-
ence of control antibodies (Fig. 9 C). In agreement with this, the
number of endothelial cells in a defined volume had increased by a
factor of 4.4, although the number of sprouts was not increased.
Thus, anti–VE-PTP antibodies stimulate endothelial cell prolifera-
tion within cord structures.
Activation of Tie-2 feeds into various signaling pathways.
Among those only Tie-2–dependent activation of Erk1/2 has been
reported to trigger proliferation of cultured endothelial cells (Kanda
et al., 2005). This prompted us to test whether anti–VE-PTP anti-
bodies stimulate Erk1/2 in mouse endothelioma cells. Indeed,
we found a rapid (10 min) and transient activation of Erk1/2
(Fig. 9 D). This activation was dependent on Tie-2 because it was
only seen in endothelioma cells established from Tie-2–expressing
mice, but not in endothelioma cells established from Tie-2/
mice (Fig. 9 E). Similar results were found with three indepen-
dently derived cell lines from wild-type and from Tie-2/
Activation of Erk1/2 with anti–mouse VE-PTP antibodies
was also seen in HUVEC that were transduced with mouse Flag-
VE-PTP (Fig. 9 G). In addition, we found that the kinase Akt
was phosphorylated at serine 473 (Fig. 9 G). Activation of Akt is
might affect vascular remodeling in the allantois through hyper-
activation of Tie-2. If this were the case, anti–VE-PTP anti-
bodies should not affect vascular remodelling in Tie-2–null
allantoides. Indeed, we found that antibodies against VE-PTP
enlarged cord structures only in Tie-2+/+ and Tie-2+/, but not in
Tie-2/ allantoides (Fig. 8 A). In agreement with this, the acti-
vating Tie-2 ligand Ang1 when added to wild-type allantois
Figure 7. VE-PTP counterbalances Ang1 activation of Tie-2 and regulates
Tie-1 tyrosine phosphorylation dependent on Tie-2. (A) bEnd.5 cells were
either transfected with control siRNAs or with siRNAs directed against VE-PTP.
24 h later, cells were stimulated either with 600 ng/ml Ang1 or 600 ng/ml
Ang2 for 10 min, or unstimulated (unstim). Immunoprecipitates of Tie-2 (top
two panels) or cell lysates (bottom two panels) were immunoblotted for the
indicated antigens. (B) HUVECs were either transfected with control siRNAs
or with siRNAs directed against hVE-PTP. 24 h later, siRNA-transfected cells
(left two lanes) and untransfected cells, stimulated either with 600 ng/ml
Ang1 for 10 min or unstimulated (right two lanes), were subjected to immuno-
precipitations for Tie-2, followed by immunoblotting with antibodies against
the phosphorylated tyrosine 992 in the active loop of the kinase (pTyr 992)
and Tie-2. Aliquots of cell lysates were immunoblotted directly for hVE-PTP
and Tie-2 (bottom two panels). White line indicates that intervening lanes
have been spliced out. (C) bEnd.5 cells were treated either with polyclonal
antibodies against VE-PTP (-VE-PTP) or preimmune antibodies (control) for
1 h, followed by either Ang1 stimulation for 10 min (Ang1) or no stimula-
tion (unstim). Phosphorylation of tyrosine 992 was analyzed as described
for B. Quantified signal intensities are indicated. (D) bEnd.5 cells were
stimulated with 200 ng/ml COMP-Ang1 for 10 min or left untreated. VE-PTP
immunoprecipitates (top three panels) and cell lysates (bottom two panels)
were analyzed by immunoblotting for Tie-2, VE-cadherin, and VE-PTP as indi-
cated on the right. (E) Embryonic endothelioma cells established either from
wild-type (+/+) or Tie-2–deficient (/) embryos were antibody pretreated
as indicated and subjected to immunoprecipitations with antibodies against
Tie-1. Immunocomplexes were analyzed by immunoblotting with antibodies
against phosphotyrosine or Tie-1 (as indicated). Aliquots of cell lysates were
immunoblotted directly for Tie-1 and Tie-2 (bottom two panels).
VE-PTP IS AN ESSENTIAL TIE-2 REGULATOR • Winderlich et al.
mice daily for 5 d with 100 µg of anti–VE-PTP antibodies.
Blood vessels were visualized in vibratome sections of the
tongue by staining for PECAM-1. As shown in Fig. 10 A, ves-
sels in the dermal papillae and draining venules were clearly en-
larged. Quantification revealed that polyclonal antibodies
against VE-PTP enlarged vessels by 82%, compared with tissue
sections from mice treated with control antibody and the mAb
109.1 against VE-PTP, which increased vessel diameters by
57% (Fig. 10 B). These effects are reminiscent of what was re-
ported for the influence of Ang1 on vessel enlargement in new-
born mice (Thurston et al., 2005). Indeed, we could show that
tyrosine phosphorylation of Tie-2 was enhanced in the animals
upon anti–VE-PTP antibody injection, as analyzed in immuno-
blots of lung tissue of these animals (Fig. 10 C). This effect was
accompanied with a fivefold increase of the number of phospho-
Histone 3 positive endothelial nuclei (Fig. 10, E and F), suggest-
ing that vessel enlargement was caused by hyperplasia. Similar
as for tongue tissue, anti–VE-PTP antibodies also enlarged
blood vessels of the trachea (Fig. 10 D). Collectively, our results
suggest that VE-PTP is required to balance the activity of Tie-2
during vascular remodeling in young mice. In addition, our re-
sults demonstrate that the described anti–VE-PTP antibody ef-
fects on vascular remodeling and Tie-2 activity are not limited to
explant cultures devoid of blood flow.
VE-PTP, the first known endothelial-specific receptor-type tyro-
sine phosphatase, associates with Tie-2 and VE-cadherin and
a step in another signaling pathway known to be stimulated by
Tie-2 (Brindle et al., 2006).
To test whether activation of Erk triggered by anti–VE-PTP
antibodies would be involved in the anti–VE-PTP induced en-
largement of endothelial structures in allantois explants, we co-
incubated explants with the antibodies together with 50 µM of
the Erk inhibitor PD98059, a concentration that blocked the
anti–VE-PTP effect on Tie-2 activation in bend.5 cells (Fig. 9 F).
As shown in Fig. 9 H, the inhibitor could strongly reduce the
antibody effect on enlargement of the vascular structures. Quan-
tification of the effect revealed a 67% inhibitory effect of the
Erk1/2 inhibitor (Fig. 9 I). Another inhibitor of Erk1/2, U0126,
also inhibited the anti–VE-PTP effect on vessel enlargement in
allantois explants (Fig. S4). In addition, this inhibitor reversed
the stimulatory effect of the anti–VE-PTP antibodies on prolif-
eration, as analyzed by counting phospho-Histone 3 positive
endothelial nuclei (control antibodies 6.25 ± 0.35; anti–VE-PTP
antibodies 14.8 ± 4.5; anti–VE-PTP antibodies plus PD98059
5.5 ± 1.24 mitotic endothelial nuclei per volume allantois;
P < 0.001). In combination with the report by Kanda et al. (2005),
these results strongly suggest that the down-regulation of VE-
PTP by antibodies leads to the activation of Tie-2, which in turn
stimulates endothelial proliferation via stimulating the MAP ki-
Antibodies against VE-PTP induce blood
vessel enlargement in 1–2-wk-old mice
To test whether antibodies against VE-PTP can indeed affect
vascular remodeling in the living animal, we injected 7-d-old
Figure 8. Vessel enlargement induced by anti–VE-PTP antibodies requires Tie-2. (A) Allantois explants from E8.5 Tie-2 +/+, Tie-2 +/, or Tie-2 /
embryos (as indicated) were cultured on gelatin-coated ultrathin glass slides in the presence of polyclonal antibodies against VE-PTP for 22 h. (B) Allantois
explants from E8.5 embryos were cultured in medium (untreated) or in the presence of 600 ng/ml Ang1. Subsequently, endothelium was visualized in all
samples in A and B by indirect immunofluorescence staining for VE-cadherin. Bars, 50 µm.
JCB • VOLUME 185 • NUMBER 4 • 2009 666
In addition, we found that the anti–VE-PTP antibodies elicit
enlargement of vascular structures in allantois explants and in
1–2-wk-old mice, accompanied by enhanced endothelial cell
proliferation. Most importantly, enlargement of vascular struc-
tures by these antibodies was eliminated in the absence of
Tie-2 and required activation of Erk1/2. Our results establish
VE-PTP as an essential negative regulator of Tie-2 during blood
vessel remodeling, providing a mechanism for how VE-PTP
is essential for vascular remodeling during embryonic develop-
ment. In this study we show that VE-PTP controls vascular re-
modeling via regulating the ability of Tie-2 to drive endothelial
cell proliferation. These mechanistic insights into the physio-
logical function of VE-PTP were enabled by antibodies against
VE-PTP, which selectively dissociate VE-PTP from Tie-2 but
not from VE-cadherin. The antibodies stimulated Tie-2 activa-
tion, as documented by increased tyrosine phosphorylation of
Tie-2 and activation of the downstream signaling target Erk1/2.
Figure 9. Anti–VE-PTP antibody treatment of
allantois explants stimulates endothelial cell
proliferation and enlargement of endothelial
cords through activation of Erk1/2. (A) Allan-
tois explants of E8.5 embryos from knock-in
mice expressing VE-cadherin-GFP from the VE-
cadherin genetic locus were cultured on gela-
tin-coated ultrathin glass slides for 12 h and
were then analyzed by live imaging during the
next 12 h (see Video 2), while they were cul-
tured in the presence of polyclonal antibodies
against VE-PTP. Subsequently, allantoides were
fixed and double stained for PECAM-1 and
phospho-Histone 3. Arrows indicate proliferat-
ing endothelial cells with a characteristic round
cell shape. Bar, 20 µm. (B) Same as in A, depict-
ing larger areas of the explants. Bar, 100 µm.
(C) Percentage of phospho-Histone 3–positive
endothelial cells per volume tissue in E8.5
allantois explants cultured with polyclonal anti-
bodies against VE-PTP or preimmune antibodies
for 12 h. *, P < 0,05. (D) bEnd.5 cells were
treated with mAb against ESAM (control) or
for indicated time periods with a mAb against
VE-PTP. Cell lysates were analyzed by immuno-
blotting with anti–phospho-Erk1/2–specific
antibodies (pT202/pY204) and antibodies
against Erk1/2. Treatment with mAb against
ESAM gave a similar result as in the absence
of antibodies (not depicted). (E) Endothelioma
cells of wild-type genotype (Tie-2 +/+) or de-
ficient for Tie-2 (Tie-2 /) were treated with
monoclonal antibodies against ESAM (control)
or against VE-PTP for 1 h, followed by immuno-
blotting cell lysates with anti–phospho-
Erk1/2–specific antibodies (pT202/pY204)
and antibodies against Erk1/2, as indicated
on the right. (F) bEnd.5 cells were treated with
50 µM of the Erk1/2 inhibitor PD98059 (PD
98059) or DMSO only (control) for 30 min,
followed by incubation with a control mAb
(control) or mAb against VE-PTP (-VE-PTP)
in the presence of the inhibitor or DMSO for
20 min. Cell lysates were analyzed by immuno-
blotting with anti–phospho-Erk1/2–specific
antibodies (pT202/pY204) and antibodies
against Erk1/2. (G) Mouse Flag-VE-PTP ex-
pressing HUVECs were treated with polyclonal
antibodies against VE-PTP or preimmune anti-
bodies for 20 min and subsequently cell lysates
were analyzed by immunoblotting with anti–
phospho-Erk1/2–specific antibodies (pT202/
pY204), anti–phospho-Akt–specific antibodies
(Ser473) and antibodies against Erk1/2 and
Akt. (H) Allantois explants from E8.5 wild-type
embryos were cultured on gelatin-coated glass
slides either in the presence of Erk1/2 inhibitor
PD 98059 (PD 98059), polyclonal antibodies against VE-PTP (-VE-PTP), or the combination of both (-VE-PTP + PD 98059) or left untreated for 22 h. Endo-
thelial structures were stained with a mAb against VE-cadherin by indirect immunofluorescence Bar, 100 µm. (I) Quantification of the experiment illustrated
in H. Average endothelial cord diameters were determined for allantois explants that were left untreated (untreated, n = 3), cultured in the presence of
Erk1/2 inhibitor PD 98059 (PD 98059, n = 3), polyclonal antibodies against VE-PTP (-VE-PTP, n = 11), or the combination of both (-VE-PTP + PD 98059,
n = 7) for 22 h; **, P < 0,01.
667 VE-PTP IS AN ESSENTIAL TIE-2 REGULATOR • Winderlich et al.
Precise balancing of positive and negative stimulation of
Tie-2 is essential for vessel remodeling and angiogenesis. A lack
of activation caused by deleting the Tie-2 gene itself or by
disrupting the gene for the agonist Ang1 leads to embryonic
vascular malformations and lethality (Dumont et al., 1994; Sato
et al., 1995; Suri et al., 1996), a similar phenotype that is ob-
served upon overexpression of the antagonistic ligand Ang2
(Maisonpierre et al., 1997). On the other hand, hyper-activation
Figure 10. Antibodies against VE-PTP induce blood vessel enlargement and endothelial proliferation in juvenile mice and lead to activation of Tie-2.
(A) 7-d-old wild-type mice were injected daily i.p. with 100 µg antibodies against VE-PTP (-VE-PTP) or preimmune antibodies (control) for a period of 7 d.
100-µm vibratome sections of tongues were immunostained for PECAM-1. Bar, 60 µm. (B) Average vessel diameter in the tongue of wild-type mice injected
daily i.p. with 100 µg polyclonal (-VE-PTP pAb), or monoclonal (a-VE-PTP mAb) antibodies against VE-PTP, preimmune antibodies (control pAb), or mono-
clonal antibodies against ESAM (control mAb), respectively, for a period of 7 d. In each case three animals were analyzed. (C) 7-d-old wild-type mice were
treated as described for A. Lungs of mice were lysed and subjected to immunoprecipitation for Tie-2 and subsequent immunoblotting with antibodies either
against phosphorylated tyrosine or against Tie-2 (as indicated). White line indicates that intervening lanes have been spliced out. (D) Mice were treated
as in A and trachea whole mounts were immunostained for PECAM-1. Arrowheads indicate enlarged blood vessels. L, lymphatic vessels. Bar, 50 µm.
(E) Mice were treated for 4 d as in A and vibratome sections of tongues were immunostained for PECAM-1 and phospho-Histone 3. Asterisk, nonendothelial
nucleus; arrowheads, endothelial nuclei. Bar, 50 µm. (F) Quantification of the experiment illustrated in E with phospho-Histone 3–positive endothelial nuclei
counted per volume tissue (38 × 200 × 500 µm). ***, P < 0,001.
JCB • VOLUME 185 • NUMBER 4 • 2009 668
Like them, we found that Tie-2–dependent proliferation was
independent from sprout formation and occurred within endothelial
Kanda et al. (2005) have shown that blocking Erk can par-
tially inhibit Ang1-stimulated endothelial cell proliferation of
cultured endothelial cells. This is in good agreement with our
finding that treatment with anti–VE-PTP antibodies feeds into
the Tie-2–signaling pathway leading to Erk activation. Impor-
tantly, the fact that the Erk1/2 inhibitors PD98059 and U0126
blocked the anti–VE-PTP effect on endothelial cell proliferation
and on enlargement of vascular structures in the allantois indi-
cates that Tie-2 triggers endothelial proliferation in the allantois
via Erk1/2 and that VE-PTP counteracts this pathway. Collec-
tively, these results indicate that anti–VE-PTP antibodies induce
enlargement of vascular structures in the allantois by stimulat-
ing endothelial cell proliferation via the Tie-2, Erk1/2 pathway.
It is intriguing that VE-PTP molecules associated with Tie-2
were selectively sensitive to anti–VE-PTP antibody-triggered
dissociation and endocytosis, whereas VE-cadherin–associated
VE-PTP molecules were not sensitive for this effect. Likewise,
anti–VE-PTP antibodies selectively affected tyrosine phosphory-
lation of Tie-2, but not the phosphorylation pattern of the com-
ponents of the VE-cadherin complex in endothelial adherens
junctions. We assume that VE-PTP complexed with VE-cadherin
may not be accessible for antibodies, possibly masked within
VE-cadherin clusters at cell contacts. Indeed, incubation of liv-
ing, intact endothelial cells or allantois explants with anti–VE-PTP
antibodies did not allow to stain endothelial cell contacts, whereas
fixing and permeabilizing the specimens rendered extracellular
epitopes of VE-PTP accessible to antibody staining (Fig. 3 and
In conclusion, our results establish VE-PTP as an essen-
tial negative regulator of Tie-2, which controls Tie-2–driven
endothelial cell proliferation, which in turn affects blood vessel
remodeling during embryonic development and determines blood
vessel size during perinatal growth. In light of the publications
analyzing the function of the Tie-receptor system in tumor
angiogenesis (Shim et al., 2007) it will be interesting to test a
potential role of VE-PTP in this pathological process. Furthermore,
because VE-PTP is an endothelial-specific transmembrane pro-
tein and antibodies against its extracellular part affect endothelial
cell proliferation and angiogenesis in vivo, VE-PTP is generally
an easily accessible, interesting novel target for pro- or anti-
angiogenic therapeutic interventions.
Materials and methods
Tie-2–deficient mice were provided by Daniel Dumont (Sunnybrook and
Women’s Research Institute, Toronto, Canada; Dumont et al., 1994). For
timed matings, mice were mated for 3 h.
Reagents and antibodies
The following reagents and antibodies were used: gelatin (Sigma-Aldrich),
mowiol (Sigma-Aldrich), fibronectin (Sigma-Aldrich), Hoechst (Invitrogen),
PD 98059 (Calbiochem), COMP-angiopoietin-1 (201–314; Qbiogene),
angiopoietin-1 (923-AN; R&D Systems), angiopoietin-2 (623-AN; R&D
Systems), mAb 109.1 against VE-PTP (Baumer et al., 2006), pAb VE-PTP-C
against VE-PTP (crossreactive to the human homologue hVE-PTP; Nawroth
et al., 2002), pAb PTP 1–8 against the extracellular fibronectin type III-like
of Tie-2 via a missense mutation in the kinase domain leads to
venous malformations in human patients (Vikkula et al., 1996),
and deletion of the gene for the antagonist Ang2 affects re-
modeling in hyaloid vasculature in the eye of newborn mice and
lymphatic patterning (Gale et al., 2002). Interestingly, defects in
Ang2-deficient mice were limited to postnatal development,
and no defects were found during embryonic vascular develop-
ment in these mice. Thus, VE-PTP represents the first negative
regulator of Tie-2 essential for embryonic angiogenesis. We can
not, of course, rule out that VE-PTP gene disruption affects ad-
ditional molecular mechanisms besides Tie-2 signaling, e.g.,
VE-cadherin or other still-unknown substrates. However, the
vascular aberrations caused by our anti–VE-PTP antibodies in
allantois explant cultures were strictly dependent on Tie-2 and
therefore identify Tie-2 as an essential substrate for VE-PTP
during embryonic vascular remodeling.
We assume that VE-PTP represents a negative feedback
control mechanism that limits the activation of Tie-2. In agree-
ment with this, stimulation of Tie-2 with Ang1 leads to increased
association of Tie-2 and VE-PTP, enabling the agonist to trigger
activation and at the same time to launch the negative mecha-
nism that ensures that the signal is switched off again. Whether,
in addition, a ligand for VE-PTP may exist that could induce
uptake of VE-PTP and thereby indirectly could enhance Ang1-
driven activation of Tie-2 is unknown, and may be an interesting
hypothesis to test in the future.
The effects of our anti–VE-PTP antibodies on blood ves-
sel enlargement in young mice are similar to the effects observed
in mice either injected with Ang1 or COMP-Ang1 (Thurston
et al., 2005; Kim et al., 2007) or overexpressing COMP-Ang1 via
adenovirus vectors (Cho et al., 2005). However, as these studies
were based on exogenously added large doses of Ang1, it could
not be determined whether physiological levels of Ang1 would
indeed play a role in the normal regulation of vessel size during
perinatal development. Because the anti–VE-PTP antibodies sim-
ply dissociate a negative regulator from Tie-2, our results sug-
gest that Tie-2–stimulating ligands are indeed acting in newborns
to determine vessel size.
It is interesting that antibodies against VE-PTP also stimu-
lated Tie-1 activation and that this only occurred in the presence
of Tie-2. This is in agreement with other studies demonstrating
that Tie-1 activation by Ang1 depends on Tie-2 (Saharinen et al.,
2005; Yuan et al., 2007).
Tie-2 activation triggers various signaling pathways and
biological activities, such as survival and protection from apop-
tosis, migration, permeability, tube formation, and sprouting, as
has been summarized in excellent reviews (Brindle et al., 2006;
Eklund and Olsen, 2006). However, studies on the ability of Ang1
to stimulate proliferation of cultured endothelial cells are contro-
versial, ranging from no effect (Davis et al., 1996; Witzenbichler
et al., 1998; Fujikawa et al., 1999) to mild effects (Koblizek
et al., 1998; Teichert-Kuliszewska et al., 2001) to substantial ef-
fects (Kanda et al., 2005). The in vivo studies with Ang1 and
COMP-Ang1 in newborns (Cho et al., 2005; Thurston et al.,
2005; Kim et al., 2007) suggest that vessel enlargement was ac-
companied by endothelial cell proliferation, establishing that
Tie-2 can stimulate proliferation of endothelial cells in vivo.
669VE-PTP IS AN ESSENTIAL TIE-2 REGULATOR • Winderlich et al.
and aliquots were set aside for direct blot analysis; for IP aliquots were in-
cubated for 2 h at 4°C with protein A– or G–Sepharose loaded with the re-
spective antibodies. Immunocomplexes were washed five times with lysis
buffer and analyzed by SDS-PAGE. Total cell lysates (3 × 105 cells/lane)
or immunoprecipitated material was separated by electrophoresis on 6%
(IP) or 8% (direct immunoblots of cell lysates) SDS-PAGE and transferred to
nitrocellulose (Whatman) by wet blotting. Blots were analyzed as described
previously (Nawroth et al., 2002) or with fluorescent dye-coupled second-
ary antibodies and Starion fluorescence Image Analyzing system (Fujifilm).
For detection of phosphotyrosine, milk powder in the blocking buffer was
replaced by 2% BSA and 200 µM Na3VO4 was added.
For RNA interference of mouse and human VE-PTP expression the following
siRNAs were used: VE-PTP-34: 5-CCUCACUGAGGGUAACAGU-3 (tar-
geting mVE-PTP) and siPTP-82: 5-GACAGUAUGAGGUGGAAGU-3 (tar-
geting hVE-PTP; Ambion). For negative controls an siRNA was used that
does not target any known mammalian gene (5-UUCUCCGAACGUGU-
CACGU-3; QIAGEN 1022076). Routinely 106 bEnd.5 cells or HUVECs
were transfected with 3 or 4 µg of siRNA, respectively, using nucleofection
(Amaxa Biosystems) according to the manufacturer’s instructions. For detec-
tion of tyrosine phosphorylation, single nucleofection reactions of bEnd.5
cells and HUVECs were scaled up fivefold and threefold, respectively.
Allantois explant cultures and immunolabeling
Allantoides were dissected from E8.0 to E8.5 C57BL/6 wild-type or Tie-2–
deficient embryos, cultured for 22 h before fixation, in the presence of
50 µg/ml mAb 109.1 (anti–VE-TP), 1G8.1 (anti-ESAM), pAb PTP 1–8 (anti–
VE-PTP), pAb VE-19 (anti-ESAM), or preimmune serum, or left untreated for
12–22 h. For stimulation with growth factors, allantoides were cultured in
the presence of 600 ng/ml COMP-Ang1 or Ang1. For inhibiting Erk1/2
activation, allantoides were pretreated with 50 µM/L PD 98059 or 10 µM
U0126 for 30 min and subsequently incubated with antibodies in the pres-
ence of 50 µM/L PD 98059 or 10 µM U0126 for 12 h. Explants were
fixed as described previously (Drake and Fleming, 2000), immunolabeled
(Baumer et al., 2006), and examined with a fluorescence microscope (Axio-
skop; Carl Zeiss, Inc.) in conjunction with a digital camera (RT KE/SE Spot;
Diagnostic Instruments, Inc.). For identifying proliferating endothelial cells,
allantoides were fixed and immunolabeled with anti–PECAM-1 and anti–
phospho-Histone 3 antibodies as described previously (Baumer et al.,
2006). Fluorescence signal was detected using a confocal laser-scan
microscope (Carl Zeiss, Inc.). Optical sections were collected along the z-axis
and 3D images were reconstructed using LSM Image Examiner software
(Video 4). Phospho-Histone 3 positive endothelial cells were counted in
four volumes (257 × 257 × 24 µm) set randomly per allantois.
Antibody treatment of 1–2-wk-old mice
For treatment of young mice (P7), pups from litters of C57BL/6 mice were
injected i.p. with 50 µg mAb 109.1 (anti–VE-TP), 1G8.1 (anti-ESAM), pAb
PTP 1–8 (anti–VE-PTP), or preimmune serum for controls daily for a period
of 7 or 4 d. Tongues from mouse pups were harvested and fixed in para-
formaldehyde (4% PFA for 3 h); lungs from the same mice were removed
and rapidly frozen, and tyrosine phosphorylation of Tie-2 was analyzed as
described previously (Thurston et al., 2005). Longitudinal vibratome sec-
tions (100 µm) of tongues and trachea whole mounts were fixed for an-
other 30 min, permeabilized with 0.5% Triton X-100, and stained with
mAbs against PECAM-1 and pAbs against phospho-Histone 3, followed by
detection with Alexa 488– and Alexa 568–coupled secondary antibodies
and mounted in mowiol (Sigma-Aldrich). Confocal fluorescence images
were collected using a confocal laser-scan microscope (Carl Zeiss, Inc.).
Measurements of vessel diameter were performed on three tongues per
group on four representative regions per tongue. Counting of phospho-
Histone 3 positive endothelial cells was performed in three tongues per
group on nine representative regions per tongue.
Live imaging of allantois explants
For live imaging, allantois explants were dissected from E8.0 to E8.5 VE-
cadherin-EGFP expressing embryos, cultured on 0.5% gelatin-coated
µ-slides (Ibidi) in a live imaging chamber (Ibidi), which guaranteed 85%
humidity, 5% CO2, and 37°C. After cultivation for 6–12 h, the EGFP fluor-
escence signal was detected using a confocal laser-scan microscope (Carl
Zeiss, Inc.). Optical sections of the cultured explants were collected along
the z-axis and collapsed into a single focal plane for each indicated time
point. After stopping confocal analysis, allantoides were fixed and immuno-
labeled with anti–PECAM-1 and phospho-Histone 3 antibodies as described
previously (Baumer et al., 2006).
domains 1–8 of VE-PTP, pAb D17 against the extracellular membrane
proximal fibronectin type III-like domain of VE-PTP, mAb 3G1 against Tie-2
(Koblizek et al., 1997), mAb against human Tie-2 (Millipore), pAb against
Tie-1 (Santa Cruz Biotechnology, Inc.), pAb against phospho-Tie-2-Tyr992,
pAb against phospho-Erk1/2-Thr202/Tyr204 pAb and mAb against
Erk1/2, pAb against phospho-Akt-Ser473, pAb against Akt (Cell Signal-
ing Technology), mAb 11D4.1 and pAb C5 against murine VE-cadherin
(Gotsch et al., 1997), pAb against human VE-cadherin (Santa Cruz Bio-
technology, Inc.), mAb 1G8 against mouse ESAM (Nasdala et al., 2002),
pAb against human ESAM (Nottebaum et al., 2008), mAb against PECAM-1
1G5.1 and 5D2.6 (Wegmann et al., 2006), mAb against plakoglobin
(BD Biosciences), mAb 4G10 against phosphotyrosine (Millipore), and
mAb and pAb against phospho-Histone H3-Ser10 (Millipore). Secondary
antibodies were purchased from Dianova; Alexa 488–, Alexa 633–, and
Alexa 568–coupled antibodies from Invitrogen; and DyLight680 and
DyLight800-coupled antibodies from Thermo Fisher Scientific.
The following cells were propagated as described: bEnd.3 and bEnd.5
(Reiss et al., 1998), Tie2+/+ and Tie2/ mouse endothelioma cells (Jones
et al., 2003), and HUVEC (Baumeister et al., 2005). Polyoma middle T im-
mortalization of embryonic endothelial cells was performed as described
previously (Reiss and Kiefer, 2004), starting from E9.5 mouse embryos.
For stimulation with angiopoietins and antibody treatment, bEnd.5 cells
were grown to confluence in DMEM, 10% FCS, starved with MCDB 131
medium (Invitrogen) containing 1% BSA overnight, and stimulated with
200 ng/ml recombinant COMP-angiopoietin-1 (COMP-Ang1) or 600 ng/ml
Ang1 and Ang2, respectively, in MCDB 131 medium (with 1% BSA) for
indicated time periods. Cells were treated with 50 µg/ml mAb 109.1 (anti-
VE-TP), 1G8.1 (anti-ESAM), pAb PTP 1–8 (anti-VE-PTP), or preimmune serum
in starvation medium for 30–90 min. HUVECs were grown to confluence
in Medium 199 (Invitrogen), 20% FCS, starved with Medium 199 contain-
ing 2% FCS for 6 h and stimulated with angiopoietins as described above
for bEnd.5 cells.
Constructs and adenoviral transfection
Flag VE-PTP (lacking the first 16 FNIII-like repeats, but containing the extra-
cellular most membrane-proximal 17th domain) and Flag VE-PTP C/S
(Fachinger et al., 1999) with a phosphatase-dead mutant (cysteine in the
active center replaced by seine) were cloned into pENTR 2B (Invitrogen).
Using the LR Recombination Reaction (Gateway Technology; Invitrogen),
the pAd-DEST vector was created and used for lipofection of 293A cells.
The viral lysate was prepared by lysis of the transfected 293A cells.
HUVECs were transduced with adFlag-VE-PTP or adFlag-VE-PTPC/S for 18 h,
washed, and starved for 4 h in Medium199, 2% FCS. Subsequently, cells
were treated with antibodies against the 17th extracellular domain of
VE-PTP for indicated time periods.
Detection of VE-PTP endocytosis by immunofluorescence staining
For detection of endocytosed VE-PTP, bEnd.3 endothelioma cells were
seeded on fibronectin-coated glass chamber slides and grown to conflu-
ence, starved with MCDB 131 medium containing 1% BSA overnight, and
treated with 50 µg/ml pAb PTP 1–8 (anti-VE-PTP), mAb 109.1 (anti-VE-PTP),
or control antibodies in starvation medium for indicated time periods. Sub-
sequently, cells were washed extensively, fixed with paraformaldehyde,
permeabilized, and blocked with 3% BSA/PBS. Endocytosed antibodies
were detected with Alexa 568–coupled secondary antibodies. For controls
cells were stained with secondary antibodies without permeabilization.
Cells were immunostained with anti–Tie-2, anti–VE-cadherin, anti-ESAM,
and anti-EEA1 antibodies for 30 min at room temperature. Subsequently,
primary antibodies were detected with Alexa 488–, Alexa 568– and Alexa
633–coupled antibodies for 30 min at room temperature. Cell nuclei were
counterstained with 10 µg/ml Hoechst in 3% BSA/PBS for 2 min and exam-
ined with a fluorescence microscope (Axioskop; Carl Zeiss, Inc.) in conjunc-
tion with a digital camera (RT KE/SE Spot; Diagnostic Instruments, Inc.) or
using a confocal laser-scan microscope (Carl Zeiss, Inc.).
Immunoprecipitation and immunoblotting
For coimmunoprecipitations (coIPs), cells were lysed in lysis buffer (20 mM
imidazole, pH 6.8, 100 mM NaCl, 2 mM CaCl2, 1% Triton X-100, 0.04%
NaN3, and 1x Complete EDTA-free protease inhibitor cocktail [Roche]).
For detection of phosphotyrosine after immunoprecipitation, cells were
lysed in lysis buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl,
2 mM CaCl2, 1 mM Na3VO4, 1% Triton X-100, 0.04% NaN3, and 1x Com-
plete EDTA-free. At no time were intact cells exposed to vanadate or per-
oxyvanadate. Lysates were centrifuged at 4°C for 30 min at 20,000 g,
JCB • VOLUME 185 • NUMBER 4 • 2009 670
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All images were acquired using a fluorescence microscope (Axioskop; Carl
Zeiss, Inc.) with 10×/NA 0.5, 20×/NA 0.75, 40×/NA 0.75, or 100×/
NA 1.3 objectives (Carl Zeiss, Inc.) in conjunction with a digital camera (RT
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The endothelial cord diameter measurements in allantois explants were
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The detailed method is illustrated by a drawing in LaRue et al. (2003).
Western blots were analyzed by measurements of pixel intensity using
Multi Gauche software (Fujifilm). P-values were calculated by using two-
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viation of the mean
Online supplemental material
Figure S1 shows that polyclonal and monoclonal antibodies against VE-PTP
trigger vessel enlargement in allantois explants and do not affect junctional
proteins. Figure S2 demonstrates that endocytosed VE-PTP colocalizes first
with early endosomes and is subsequently degraded, whereas junctional
structures stay intact. Figure S3 shows that endogenously expressed Tie-2
and VE-PTP associate in endothelial cells. Figure S4 shows allantois ex-
plants treated with anti–VE-PTP antibodies in the presence of the Erk1/2
inhibitor U0126. Figure S5 demonstrates that Tie-2 is no longer cell surface
distributed as shown in Fig. 3 A, but located at cell–cell contacts upon
stimulation with COMP-Ang1. The same monoclonal antibody against
Tie-2 was used as in Fig. 3 A. Videos 1–3 show allantois explants express-
ing VE-cadherin-GFP imaged for 10–14 h in the presence of polyclonal
antibodies against VE-PTP (Video 2), preimmune antibodies (Video 1),
or 600 ng/ml COMP-Ang1 in an Ibidi live-imaging chamber (Video 3).
Video 4 shows an animation of a 3D reconstruction of an allantois cut-
out. Allantois was treated with control antibodies and subsequently stained
for PECAM-1 and phospho-Histone 3. Videos 5 and 6 show that anti-
bodies against VE-PTP trigger endocytosis of VE-PTP in endothelial cells
of allantois explant cultures (Video 5) in comparison to preimmune serum
(Video 6). Video 7 demonstrates that VE-PTP is localized to the cell surface
and cell–cell contacts in endothelial cells of untreated allantois explant
cultures. Online supplemental material is available at http://www.jcb
We thank Ralf Adams for critically reading the manuscript.
This work was supported by the Deutsche Forschungsgemeinschaft
(SFB629) and by the Max-Planck-Society.
Submitted: 28 November 2008
Accepted: 22 April 2009
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