Vascular endothelial tyrosine phosphatase (VE-PTP)-null mice undergo vasculogenesis but die embryonically because of defects in angiogenesis.
ABSTRACT Development of the vascular system depends on the highly coordinated actions of a variety of angiogenic regulators. Several of these regulators are members of the tyrosine kinase superfamily, including VEGF receptors and angiopoietin receptors, Tie1 and Tie2. Tyrosine kinase signaling is counter-regulated by the activity of tyrosine phosphatases, including vascular endothelial protein tyrosine phosphatase (VE-PTP), which has previously been shown to modulate Tie2 activity. We generated mice in which VE-PTP is replaced with a reporter gene. We confirm that VE-PTP is expressed in endothelium and also show that VE-PTP is highly expressed in the developing outflow tract of the heart and later is expressed in developing heart valves. Vasculogenesis occurs normally in mice lacking VE-PTP; however, angiogenesis is abnormal. Angiogenic defects in VE-PTP-null mice were most pronounced in the yolk sac and include a complete failure to elaborate the primitive vascular scaffold into higher-order branched arteries, veins, and capillaries. VE-PTP continues to be expressed into adulthood in the vasculature and heart valves, suggesting later roles in vascular development or homeostasis. VE-PTP is also expressed in the vasculature of growing tumors, suggesting that VE-PTP may be a new potential target for angiogenic therapies.
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ABSTRACT: The endothelial layer of blood vessels controls the passage of cells and solutes from the blood into the surrounding tissue. Crucial for this regulation is the integrity of endothelial cell-cell junctions. Various molecular mechanisms control junctional integrity of the endothelial layer including GTPases, modulation of the actomyosin cytoskeleton and phosphorylation and dephosphorylation of junctional proteins. Several kinases and phosphatases have been identified that are good candidates for the regulation of the endothelial barrier function. For some of them, in vivo evidence has recently been presented that highlights their importance in either the regulation of vascular permeability or leukocyte extravasation. This review will summarize current knowledge about the regulation of endothelial junctions by kinases and phosphatases. In particular, the role of the endothelial specific phosphatase VE-PTP in the context of endothelial cell contact stability will be highlighted.Cell and Tissue Research 02/2014; · 3.68 Impact Factor
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ABSTRACT: Leukocyte extravasation is regulated and mediated by a multitude of adhesion and signaling molecules. Many of them enable the capturing and docking of leukocytes to the vessel wall. Others allow leukocytes to crawl on the apical surface of endothelial cells to appropriate sites of exit. While these steps are well understood and the adhesion molecules mediating these interactions are largely identified, a still growing number of adhesion receptors mediate the diapedesis process, the actual migration of leukocytes through the endothelial cell layer, and the underlying basement membrane. In most cases, it is not known which molecular processes they actually mediate, whether they enable the migration of leukocytes through the endothelial cell layer or whether they are involved in the destabilization of endothelial junctions. In addition, leukocytes are able to circumvent junctions and transcytose directly through the body of endothelial cells. While this latter route indeed exists, recent work has highlighted in vivo the junctional pathway as the prevalent way of leukocyte exit in various inflamed tissues. Recent work elucidating molecular mechanisms that regulate endothelial junctions and thereby leukocyte extravasation and vascular permeability will be discussed.Seminars in Immunopathology 03/2014; · 5.38 Impact Factor
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ABSTRACT: Angiosarcoma is an aggressive malignancy that arises spontaneously or secondarily to ionizing radiation or chronic lymphoedema. Previous work has identified aberrant angiogenesis, including occasional somatic mutations in angiogenesis signaling genes, as a key driver of angiosarcoma. Here we employed whole-genome, whole-exome and targeted sequencing to study the somatic changes underpinning primary and secondary angiosarcoma. We identified recurrent mutations in two genes, PTPRB and PLCG1, which are intimately linked to angiogenesis. The endothelial phosphatase PTPRB, a negative regulator of vascular growth factor tyrosine kinases, harbored predominantly truncating mutations in 10 of 39 tumors (26%). PLCG1, a signal transducer of tyrosine kinases, encoded a recurrent, likely activating p.Arg707Gln missense variant in 3 of 34 cases (9%). Overall, 15 of 39 tumors (38%) harbored at least one driver mutation in angiogenesis signaling genes. Our findings inform and reinforce current therapeutic efforts to target angiogenesis signaling in angiosarcoma.Nature Genetics 03/2014; · 35.21 Impact Factor
Vascular endothelial tyrosine phosphatase
(VE-PTP)-null mice undergo vasculogenesis but die
embryonically because of defects in angiogenesis
Melissa G. Dominguez, Virginia C. Hughes, Li Pan, Mary Simmons, Christopher Daly, Keith Anderson,
Irene Noguera-Troise, Andrew J. Murphy, David M. Valenzuela, Samuel Davis, Gavin Thurston,
George D. Yancopoulos*, and Nicholas W. Gale*
Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591
Contributed by George D. Yancopoulos, December 28, 2006 (sent for review December 21, 2006)
Development of the vascular system depends on the highly coordi-
nated actions of a variety of angiogenic regulators. Several of these
regulators are members of the tyrosine kinase superfamily, including
VEGF receptors and angiopoietin receptors, Tie1 and Tie2. Tyrosine
kinase signaling is counter-regulated by the activity of tyrosine
phosphatases, including vascular endothelial protein tyrosine phos-
activity. We generated mice in which VE-PTP is replaced with a
reporter gene. We confirm that VE-PTP is expressed in endothelium
and also show that VE-PTP is highly expressed in the developing
outflow tract of the heart and later is expressed in developing heart
valves. Vasculogenesis occurs normally in mice lacking VE-PTP; how-
ever, angiogenesis is abnormal. Angiogenic defects in VE-PTP-null
mice were most pronounced in the yolk sac and include a complete
failure to elaborate the primitive vascular scaffold into higher-order
branched arteries, veins, and capillaries. VE-PTP continues to be
expressed into adulthood in the vasculature and heart valves, sug-
gesting later roles in vascular development or homeostasis. VE-PTP is
VE-PTP may be a new potential target for angiogenic therapies.
gene targeting ? tyrosine kinase ? Tie2
bryonic tissue, by a process termed vasculogenesis (1). The prim-
itive heart, paired dorsal aortas, and vitelline vessels also form
differentiates from a primitive undifferentiated plexus into a hier-
archy of arteries, veins, and capillaries by the process known as
angiogenesis. Significant disruption of vasculogenesis or angiogen-
in vascular development are perturbed. Several families of mole-
cules, which are expressed in vascular endothelium, are critical to
these processes. These include VEGF and its receptors VEGF-R1,
-R2, and -R3, which have been shown to be critical in the earliest
stages of vasculogenesis, as well as later in angiogenesis, and the
angiopoietin/Tie, ephrin/Eph, TGF-?/TGF-? receptor and Delta/
Notch families, shown to be critical for angiogenesis (summarized
in ref. 2).
Several of these critical molecular players in vasculogenesis and
angiogenesis are members of the receptor tyrosine kinase super-
family, which mediates signal transduction by tyrosine phosphory-
lation of signaling intermediates and effector molecules. Transduc-
tion of signals by tyrosine phosphorylation is counter-regulated by
a variety of transmembrane and cytoplasmic protein tyrosine
phosphatases (PTPs), a few of which are known to be expressed in
ECs. One of these PTPs, CD148/DEP-1/PTP?, causes vascular
defects and embryonic lethality when mutated, although its expres-
sion is not restricted to ECs (3). The only known PTP whose
expression appears to be restricted to vascular ECs in embryos is
uring embryonic development, endothelial cells (ECs) form
the first blood vessels, a primary capillary plexus in extraem-
vascular endothelial PTP (VE-PTP) (4). VE-PTP, the mouse
homologue of receptor-type human PTP-? (5), has been shown to
associate with and dephosphorylate Tie2 (4) and VE-cadherin (6)
but was shown to not interact with VEGF-R2 (4).
To study the expression pattern in embryonic and adult tissues
and to elucidate the functional role of VE-PTP in vascular devel-
opment, we generated mice in which the VE-PTP gene was
confirms that VE-PTP is expressed in both arterial and venous
vessels. We also show that, early in development, VE-PTP is highly
expressed in the outflow tract and later in the valves of the heart.
VE-PTP continues to be expressed in adult vascular endothelium
and is also expressed in tumor vasculature.
VE-PTP-null mice die embryonically because of a variety of
failure of remodeling of the vascular plexus into large veins and
branched vascular networks, which is clear throughout the embryo
but is most prominent in the yolk sac. Our findings show that
VE-PTP is essential for cardiovascular development, and its con-
tinued expression in the adult suggests it may also have a role in
vascular homeostasis and may be involved in the pathological
angiogenesis of tumors.
Although the genetic deletion in VE-PTP described here is
distinct, the vascular phenotype is essentially the same as that
described for another VE-PTP mutant published during the prep-
in VE-PTP that resulted in a soluble extracellular form of VE-PTP
However, the symmetry of our phenotype with that of Baumer et
al. (7) suggests the phenotypes resulting from both mutations are a
consequence of loss of VE-PTP function.
VE-PTP Homozygous Mutant Mice Die Embryonically. VelociGene
technology (9) was used to create a targeting vector in which exons
2 (except for the first 26 nucleotides, retaining the endogenous
signal peptide) to 3 were replaced with a transmembrane-LacZ
reporter gene and a loxP-flanked neomycin selection cassette (Fig.
1A). Correct gene targeting (frequency ? 3.5%) in F1H4 (C57BL/
Author contributions: M.G.D., G.T., and N.W.G. designed research; M.G.D., V.C.H., L.P.,
M.S., I.N.-T., and N.W.G. performed research; K.A., I.N.-T., D.M.V., and N.W.G. contributed
new reagents/analytic tools; M.G.D., V.C.H., L.P., C.D., K.A., A.J.M., S.D., G.T., G.D.Y., and
N.W.G. analyzed data; and M.G.D. and N.W.G. wrote the paper.
Conflict of interest statement: All authors are employees of Regeneron Pharmaceuticals, Inc..
Freely available online through the PNAS open access option.
Abbreviations: EC, endothelial cell; PTP, protein tyrosine phosphatase; VE-PTP, vascular
endothelial PTP; En, embryonic day n; PECAM-1, platelet/endothelial cell adhesion mole-
regeneron.com or email@example.com.
© 2007 by The National Academy of Sciences of the USA
www.pnas.org?cgi?doi?10.1073?pnas.0611510104 PNAS ?
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6:129 hybrid) ES cell clones was scored by using the loss of native
allele assay (data not shown, and see ref. 9). Male chimeric mice
(complete transmitters of ES cell-derived sperm) from two inde-
pendent ES cell clones were bred to C57BL/6 mice to generate F1
offspring heterozygous for the modified VE-PTP allele (VE-
PTPLz/?). When F1 animals were intercrossed to generate F2
offspring, no pups homozygous for the modified VE-PTP allele
(VE-PTPLz/Lz) were born out of 25 litters (174 mice; 62% VE-
PTPLz/?, 37% WT), leading to the suspicion that homozygous
deletion of VE-PTP results in embryonic death. Timed matings of
VE-PTPLz/?mice were used to generate embryos 8.5–10.5 days
VE-PTPLz/?, and WT littermate embryos were obtained at ap-
proximate Mendelian ratios. However, by E9.5, the VE-PTPLz/Lz
embryos exhibited obvious abnormalities (see below). The VE-
were both used as normal controls throughout this study.
To confirm that the loss of VE-PTP alleles results in the loss of
VE-PTP mRNA expression and acquisition of LacZ expression,
quantitative RT-PCR analysis was performed on E9.5 embryos
(Fig. 1B). As expected, WT embryos express VE-PTP, but not
LacZ, whereas VE-PTPLz/?embryos express LacZ and have a
the LacZ reporter gene but lack VE-PTP transcripts.
from their WT littermates, either by size or appearance (data not
shown). However, by E9.5, the VE-PTPLz/Lzembryos start dying,
and no surviving embryos were recovered beyond E11. Compared
with control littermates, VE-PTPLz/Lzembryos appear develop-
mentally delayed starting at E9.0 and do not appear to progress
beyond the E9.0 to early E9.5 stage, based on several objective
criteria, including somite number and hallmarks of forelimb devel-
opment. As such, they are significantly smaller than control litter-
mates (compare Fig. 1 D with C). VE-PTPLz/Lzembryos have
pericardial edema (Fig. 1D), which varies in severity. VE-PTPLz/Lz
embryos collected at E10.0 and E10.5 are mostly dead, as assessed
by the lack of heartbeat, or the appearance of necrotic tissue, and
can have pronounced pericardial edema (data not shown).
Angiogenic Defects in Embryos. The time of embryonic death,
developmental delay, and appearance of pericardial edema ob-
served in the VE-PTPLz/Lzembryos are hallmarks of defective
cardiovascular development. To evaluate the vascular system of
VE-PTP mutant animals, CD-31 [platelet/EC adhesion molecule 1
(PECAM-1)] immunohistochemistry was used to visualize the
vasculature in whole mounts and sections of VE-PTPLz/Lzand
control littermate embryos and yolk sacs. Because we observed an
overall developmental retardation in VE-PTPLz/Lzembryos, their
vascular system was compared with stage-matched control em-
bryos. At E8.5, when the vasculature consists primarily of a simple
plexus, there is no obvious difference between VE-PTPLz/Lzem-
bryos and their normal littermates (data not shown). However, by
E9.5, when the vasculature of control littermates includes clearly
defined large veins and networks of branching vessels (Fig. 2A),
remodeling should take place (Fig. 2B). In control embryos, the
vascular system has undergone extensive remodeling, as seen in the
dorsal midbrain, where cephalic veins (Fig. 2C, blue arrowheads)
and major branches of the mesencephalic artery (Fig. 2C, red
arrowheads) can be readily distinguished. In contrast, the cephalic
plexus in VE-PTPLz/Lzembryos fails to remodel into a hierarchical
branched vascular network and instead remains as a plexus with
only minor pruning (compare Fig. 2 D with C). In the lateral aspect
of the developing control embryo, the cardinal veins are well
defined from the head to the heart (Fig. 2E). In VE-PTPLz/Lz
embryos (Fig. 2F), there is PECAM-1 staining in the correct
location; however, the edges are lacy and not clearly delineated,
especially in the more caudal region. Occasionally, the cardinal
veins are absent in VE-PTPLz/Lzembryos (data not shown). Cross
sections of PECAM-1-immunostained E9.0 embryos show that the
dorsal aortas in VE-PTPLz/Lzembryos are much smaller and have
G), and the cardinal veins are revealed in cross section to be poorly
flanking homology sequence (HBox) was used for bacterial homologous recombination with VelociGene technology (9). A TM-?-gal/PGK-Neo reporter cassette
was inserted, deleting from 27 nucleotides (in exon 2) through exon 3 in the targeted allele. (B) Quantitative RT-PCR results of transcript levels of VE-PTP and
on separate scales. (C and D) Freshly dissected E9.5 embryos, showing the growth retardation, large heart, and pericardial edema phenotypes seen in the
VE-PTPLz/Lzembryo (D) compared with VE-PTPLz/?(C).
www.pnas.org?cgi?doi?10.1073?pnas.0611510104Dominguez et al.
Angiogenic Defects in Yolk Sacs. Beginning at E9.0, homozygous
embryos, there are no visible blood-filled vessels, and the yolk sac
surface appears dimpled in VE-PTPLz/Lzembryos (data not
shown). At E9.5, there are still no visible vessels, and the VE-
PTPLz/Lzyolk sac phenotype is even more obvious, because the
surface is now dimpled and wrinkled (compare Fig. 3 B and A)
because of a dramatic separation of the endoderm and mesoderm
layers resulting from the disproportionate distention of the outer
endodermal layer relative to the inner mesodermal layer (compare
Fig. 3 F with E) . The severity of this yolk sac phenotype is variable,
ranging from the endoderm and mesoderm layers still being fairly
well connected by multiple adhesion sites, to barely being held
embryos staged E9.0 or older have yolk sac defects, the severity of
which was highly correlated with the severity of the vascular
remodeling defects observed in the embryo proper.
To study the morphology of the developing vasculature of the
yolk sac in detail, whole-mount PECAM-1 immunostaining was
to be remodeled in the yolk sacs of WT and VE-PTPLz/?embryos
and by E9.5, the yolk sacs have an organized vascular network of
branching vessels (Fig. 3C), which are lined with PECAM-1-
VE-PTPLz/Lzyolk sacs, PECAM-1 immunostaining confirms the
lack of any identifiable mature or remodeled blood vessels in
VE-PTPLz/Lzyolk sacs (Fig. 3D), as observed during dissection.
Instead, the vessels of the primitive plexus appear to merge to-
gether. These hyperfused vessels continue to expand, to the point
of almost completely separating the endoderm and mesoderm
layers, except for a few remaining adhesion sites (Fig. 3 D and F).
In the VE-PTPLz/Lzyolk sacs with the most severe manifestation of
these defects, the two layers are completely separated and held
together only by sparse PECAM-1-positive strands (data not
shown). However, even with this severe morphological defect, the
EC layers appear intact, lining the entire surfaces of the mesoderm
and endoderm layers of the yolk sac (Fig. 3 D and F), essentially
making VE-PTPLz/Lzyolk sacs one large endoderm- and meso-
derm-enveloped blood vessel.
VE-PTP Is Expressed in Embryonic Arterial and Venous Endothelium.
To determine the sites of VE-PTP expression with high resolution,
VE-PTP promoter activity was followed in embryos, yolk sacs, and
adult tissues of VE-PTPLz/?mice by using the ?-gal reporter.
Consistent with the observed cardiovascular defects, VE-PTP
promoter activity is restricted to the developing cardiovascular
system and in particular to the vascular endothelium. A careful
a VE-PTPLz/Lzlittermate (B, D, and F). (C and D) A closeup of the head vascu-
lature shows the normal remodeling pattern of veins (blue arrows) and
arteries (red arrows) in VE-PTPLz/?embryos (C). In VE-PTPLz/Lzembryos, the
venous plexus (blue arrows) undergoes only minor remodeling (D). (E and F)
Closeup of the cardinal vein (blue arrows), which does not form correctly in
VE-PTPLz/Lzembryos (F). (G and H) PECAM-1 immunostaining of E9.0 embryo
cross sections at heart level. The dorsal aortas (red arrows) are small and
collapsed in VE-PTPLz/Lzembryos (H) compared with WT (G), and the cardinal
veins (blue arrows) are diffuse and not clearly delineated.
Defects in vascular remodeling in VE-PTPLz/Lzembryos. (A–F) Whole-
dissected E9.5 VE-PTPLz/Lzyolk sacs (B) can be clearly distinguished from
VE-PTPLz/?littermates (A), because of a wrinkled appearance and a lack of
visible vessels, which can be seen in the yolk sacs from VE-PTPLz/?littermates
The endoderm and mesoderm layers in VE-PTPLz/Lzyolk sacs are barely con-
nected (D, blue arrows), and the entire surface is lined with PECAM-1-positive
ECs (red arrows). The VE-PTPLz/?yolk sacs have an organized branched net-
work of vessels (C, red arrows). (E and F) PECAM-1/hematoxylin/eosin staining
of E9.5 yolk sac cross sections. In VE-PTPLz/?yolk sacs (E), the vessels are well
formed (red arrows). In VE-PTPLz/Lzyolk sacs (F), the vessels are so large (red
arrows) that the endoderm and mesoderm layers are only sparsely connected
Vascular defects in yolk sacs of VE-PTPLz/Lzembryos. (A and B) Freshly
Dominguez et al.PNAS ?
February 27, 2007 ?
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to E15.5. At E9.5, the time when the VE-PTPLz/Lzcardiovascular
defects become apparent, the highest sites of expression are in the
shown), with limited or weak expression in smaller-caliber vessels.
endothelium of the heart, including the atrioventricular canal,
aortic sac, and outflow tract (Fig. 4 C and D), dorsal aortas (Fig. 4
A and C–E), and umbilical arteries (Fig. 4A), but it is also strongly
expressed in the endothelium of vessels of all sizes, including
arteries, capillaries, and somewhat more lightly in veins (Fig. 4
A–C). Expression is also observed in the vitelline vessels of the yolk
valves (Fig. 5 E and F) and continues to be expressed in the
endothelium of the major arteries and veins and in the arterioles
and venules of organs, with expression being higher in arteries (Fig.
4F). This expression in organs correlates with previous results from
adult, expression is very high in the vasculature of lung, spleen, and
kidney, as well as in the heart valves, and is also present in the
endothelium of arterioles and venules (data not shown).
VE-PTP Heart Defects. In addition to pericardial edema, VE-
PTPLz/Lzembryos have hearts that are relatively enlarged com-
pared with control littermates. At E9.5, although VE-PTPLz/Lz
embryos are significantly smaller than their control littermates,
immunostaining in VE-PTPLz/?hearts shows the developing ven-
tricles, atria, and connecting atrioventricular canal (Fig. 5C). In
contrast, VE-PTPLz/Lzlittermates have limited PECAM-1 staining
in the relative position of the developing atria (Fig. 5D, dotted
developmentally delayed. As previously shown, the outflow tract
and the atrioventricular canal is the site of highest expression of the
?-gal reporter (Fig. 4C). Interestingly, this expression corresponds
to the precursors of the developing cardiac valves, and VE-PTP
reporter gene expression continues to remain high in all of the
developing heart valves (Fig. 5 E and F) at E15.5. In addition, in
adult heart, VE-PTP expression remains high in endothelium
covering all of the cardiac valves (data not shown).
VE-PTP Is Expressed in Tumor Vasculature. Subcutaneous implanta-
tumor growth and induces angiogenesis. ?-Gal staining shows that
6 A–C). The tumor invades the overlying skin and coopts the
resident vasculature, which leads to the dramatic induction of
VE-PTP expression in the coopted vessels (Fig. 6 B, D, and E).
Deletion of the VE-PTP gene causes severe cardiovascular
defects and embryonic lethality by E10. Because the vascular
disruption begins at E9.0, after the primary vasculature is
established during vasculogenesis, it appears VE-PTP is not
necessary for this process. However, it is required for angiogen-
esis in embryos, and its absence results in lethality due to
defective remodeling. This finding is consistent with previous
biochemical studies in which VE-PTP was shown to interact with
of the cross sections in D and E. (B) Head vasculature showing reporter
expression in arterial endothelium (red arrows), especially the aortic sac,
outflow tract, and the atrioventricular canal, and lower expression in the
cardinal vein (blue arrows). (D) Cross section just above the heart, showing
expression in the dorsal aortas, third brachial arch artery, aortic sac, outflow
tract (red arrows), and anterior cardinal veins (blue arrows). (E) Cross section
just above the forelimbs, showing expression in the fusing dorsal aortas (red
arrow) and cardinal veins (blue arrows). (F) Whole-mount ?-gal staining in
E15.5 VE-PTPLz/?lung, clearly showing expression in veins (blue arrows) and
the stronger expression in arteries (red arrows).
VE-PTP LacZ reporter gene expression in embryos. (A–E) Whole-
(A and B) Freshly dissected E9.5 embryos. VE-PTPLz/Lzembryos have pericardial
edema (blue arrow) (B). Although knockout embryos are smaller and devel-
opmentally delayed compared with their VE-PTPLz/?littermates (A), their
hearts tend to have similar lengths (dotted line is the same length in A and B).
(C and D) Whole-mount PECAM-1 staining of E9.5 hearts. The pattern in the
VE-PTPLz/Lzheart (D), there is only staining in the ventricle (blue arrow) and
atrioventricular canal (black arrow), but not in the atrium (dotted circle). (E
sion in arteries (red arrows), veins (blue arrows), and high expression in all
heart valves (black arrows). Heart valves also shown in thick cross section (F).
Cardiac defects and reporter gene expression in VE-PTPLz/Lzembryos.
www.pnas.org?cgi?doi?10.1073?pnas.0611510104Dominguez et al.
and dephosphorylate Tie2 (which, like VE-PTP, is critical for
angiogenesis but not vasculogenesis) but not VEGF-R2 (4),
which has been clearly shown to be critical for vasculogenesis,
and in fact, the formation of ECs themselves (10) (see below for
During development, the vascular plexus, formed during vascu-
cence, sprouting angiogenesis (11), and intussusception (12). In
VE-PTP mutant embryos, the vascular plexus is established, but it
does not remodel correctly. Defects include failure to form
possibly because of improper pruning and intussusception. Also,
indiscernible in VE-PTP mutant embryos, except for partial as-
sembly at its most anterior segment. Instead, the portion of the
vascular plexus destined to be cardinal veins remains as a collection
of vessels that fail to reshape into a cohesive vessel. The dorsal
aortas, which formed normally during vasculogenesis, become
stenotic in VE-PTPLz/Lzembryos. Because the mutant embryos do
not have a normal vascular framework, normal blood flow, which
is a key component in arterio-venous differentiation (13), is pre-
vented. The VE-PTPLz/Lzembryos are also developmentally de-
layed and have pericardial edema, which are indicators of cardio-
with other gene deletions that cause midgestation lethality because
of cardiovascular defects [e.g., VEGF-R3 (14), Dll4 (15–17), and
develop properly, which may be due in part to inappropriate
vascularization and underdevelopment.
The vascular defects in VE-PTP mutants are most striking in the
yolk sac, which completely lacks a remodeled vascular network.
Instead, the yolk sac vascular plexus essentially becomes one large
vessel fully lined with PECAM-1-positive cells. There is a range in
the amount of endodermal and mesodermal detachment in differ-
ent VE-PTPLz/Lzyolk sacs. In those that are less detached, the yolk
sac is formed of large interconnected channels, and in those more
detached, only sparse PECAM-1-positive strands connect the
endoderm and mesoderm layers. This variation shows the progres-
sion of this vascular defect as a case of unchecked coalescence,
the two yolk sac layers, suggesting that VE-PTP plays a role in
controlling that remodeling process. Other genes, when deleted,
also cause a similar phenotype in yolk sacs (summarized in ref. 18),
which has been termed vascular ‘‘hyperfusion’’ (19). Similar hyper-
fusion defects were described in quail embryos when excessive
VEGF was administered, suggesting that this phenomenon can be
VEGF driven (19). However, VE-PTP does not appear to play a
direct role in the modulation of VEGF-R2, as suggested by prior
normally in VE-PTP mutant embryos. In addition, expression of
VE-PTP was low during vasculogenesis and became most highly
expressed during angiogenic stages (data not shown and Fig. 4).
However, it remains possible that VE-PTP indirectly regulates
production of VEGF itself, a possibility not explored in the current
study. Thus, the mechanistic role that VE-PTP plays in the hyper-
fusion process remains unclear.
In embryos, VE-PTP is expressed in both arterial and venous
endothelium with the highest apparent levels in arterial vessels and
components of the heart. VE-PTP continues to be expressed in
maintenance. To explore whether VE-PTP is involved in adult
cells were xenografted into the flanks of VE-PTPLz/?mice, and
VE-PTP expression was examined in the collected tumors. VE-
of the skin overlying the tumors, as compared with the control skin
on the contralateral flank. These results suggest VE-PTP may be
important in tumor angiogenesis, in addition to embryonic angio-
genesis, and may in fact represent a novel therapeutic target.
angiogenesis will be necessary to test this hypothesis.
The cardiovascular defects described here for VE-PTP are
reminiscent of those reported for the CD148/DEP-1/PTP? mutant
mouse, the only other known receptor-type phosphatase expressed
(although not exclusively) in ECs (3). In addition, a recently
published report (7) describes a distinct VE-PTP mutation, in
which an insertion is made between exons 17 and 19, and which
results in mice in which the majority of the extracellular region of
VE-PTP remains intact and is in fact found to be secreted. It was
speculated that this secreted form of VE-PTP ectodomain may
represent a neomorphic allele with the potential to retain some
function or possibly function in an antagonistic form (7, 8). How-
ever, despite the differences in knockout strategy, the described
exon 17–19 insertion/deletion and our exon 2–3 deletion mutants
appear to have essentially the same spectrum of vascular pheno-
types, suggesting that both represent loss of function rather than
Tie2 (4) and VE-cadherin (6) have been shown to associate with
VE-PTP and as such should be considered, along with any other
substrates, as having a role in the mechanism that causes the
observed vascular defects. The VE-PTP mutants share many
common characteristics with the Tie2 and Ang1 loss-of-function
phenotype (20–22). However, deletion of a phosphatase that
negatively regulates this signaling pathway would be presumed to
have a gain-of-function phenotype rather than to mimic the loss of
signaling in either direction would lead to similar lethal conse-
quences. In allantois explant studies done with ECs from various
E8.5 homozygous mutants, it was found that Tie2?/?had normal
networks of endothelial cords compared with VE-PTP?/?(7).
Further biochemical analysis of the signaling of Tie2 and other
VE-PTP targets in VE-PTP-null ECs, as well as the generation of
a temporally controlled VE-PTP deletion later in development or
in the adult vasculature, will be necessary to further elucidate many
of these issues.
planted in VE-PTPLz/?mice. Whole-mount ?-gal staining of tumors is shown.
(A) Thick cross section of the tumor. Boxes depict the areas taken at higher
magnification in B and C. (B) Closeup of tumor blood vessels (blue arrows),
a high concentration of blood vessels (blue arrows). (D and E) View of the skin
surface directly over the tumor (E) and from the contralateral side of the
VE-PTP reporter expression in Lewis lung carcinoma tumors im-
Dominguez et al. PNAS ?
February 27, 2007 ?
vol. 104 ?
no. 9 ?