ATVB In Focus
Vascular Adhesion Molecules
Series Editor:
Dietmar Vestweber
Previous Brief Reviews in this Series:
• van Buul JD, Kanters E, Hordijk PL. Endothelial signaling by Ig-like cell adhesion molecules. Arterioscler Thromb
Vasc Biol. 2007;27:1870 –1876.
• Bradfield PF, Nourshargh S, Aurrand-Lions M, Imhof BA. JAM family and related proteins in leukocyte migration.
Arterioscler Thromb Vasc Biol. 2007;27:2104 –2112.
• Galkina E and Ley K. Vascular adhesion molecules in atherosclerosis. Arterioscler Thromb Vasc Biol. 2007;27:
2292–2301.
• Jalkanen S, Salmi M. VAP-1 and CD73, endothelial cell surface enzymes in leukocyte extravasation. Arterioscler
Thromb Vasc Biol. 2008;28:18 –26.
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VE-Cadherin
The Major Endothelial Adhesion Molecule Controlling
Cellular Junctions and Blood Vessel Formation
Dietmar Vestweber
Abstract—Vascular endothelial (VE)-cadherin is a strictly endothelial specific adhesion molecule located at junctions
between endothelial cells. In analogy of the role of E-cadherin as major determinant for epithelial cell contact integrity,
VE-cadherin is of vital importance for the maintenance and control of endothelial cell contacts. Mechanisms that
regulate VE-cadherin–mediated adhesion are important for the control of vascular permeability and leukocyte
extravasation. In addition to its adhesive functions, VE-cadherin regulates various cellular processes such as cell
proliferation and apoptosis and modulates vascular endothelial growth factor receptor functions. Consequently,
VE-cadherin is essential during embryonic angiogenesis. This review will focus on recent new developments in
understanding the role of VE-cadherin in controlling endothelial cell contacts and influencing endothelial cell behavior
by various outside-in signaling processes. (Arterioscler Thromb Vasc Biol 2008;28:223-232)
Key Words: vascular permeability 䡲 VE-cadherin 䡲 leukocyte extravasation 䡲 cell adhesion
B
esides providing a complex system of passively transporting tubes, the endothelium of blood vessels controls actively the entry of leukocytes and substances into tissue. The
control of endothelial cell contacts is of vital importance for this
process. VE-cadherin is the major determinant of endothelial cell
contact integrity and regulation of its activity or its presence at
cell contacts is an essential step that controls the permeability of
the blood vessel wall for cells and substances.
Cadherins represent one of the major families of cell adhesion
molecules. They are defined by the typical extracellular cadherin
domains (EC-domain) and mediate adhesion via homophilic,
Ca2⫹-dependent interactions. Most cadherins typically possess 5
extracellular cadherin domains. Based on sequence comparison
they can be divided into different subfamilies of which 2 are the
classical type I cadherins such as E- N- P- and C-cadherin, and
the type II cadherins, which lack the HAV motif, a classical
cadherin binding motif in the EC1 domains of type I cadherins.
VE-cadherin belongs to the type II cadherins.
Optimal adhesive function of cadherins requires association of their C terminus with cytoplasmic proteins: the
Original received October 17, 2007; final version accepted December 1, 2007.
From the Max-Planck-Institute of Molecular Biomedicine, Münster, Germany.
Correspondence to Dr Dietmar Vestweber, Max-Planck-Institute of Molecular Biomedicine, Röntgenstr. 20, D-48149 Münster, Germany. E-mail
[email protected]
© 2008 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
223
DOI: 10.1161/ATVBAHA.107.158014
224
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catenins. Cadherins bind directly to ␤-catenin (alternatively
to plakoglobin) and to p120. Plakoglobin and ␤-catenin can
bind to ␣-catenin, an actin binding protein. For many years it
has been the generally accepted view that linkage of the
cadherins via the catenins to the actin cytoskeleton is the
mechanism by which catenins strengthen cadherin-mediated
adhesion. This view has recently been challenged in studies
that were unable to verify actin binding to a preformed
E-cadherin–␤-catenin–␣-catenin complex.1 Nevertheless,
even if binding of the catenins to a cadherin would not
provide linkage to the actin cytoskeleton, it is still commonly
accepted that the lack of catenin association destabilizes
cadherin-mediated cell adhesion. Consequently the cadherincatenin association is still the major target of many studies
aiming at understanding the mechanism by which cadherin
function is regulated. Various intracellular signaling molecules as well as phosphorylation of tyrosine and serine
residues of catenins or cadherins have been reported to play
a role in cadherin regulation.
Similar to integrins, cadherins are also known for
outside-in signaling, a term that describes receptor functions
of cell adhesion molecules. VE-cadherin has been desribed to
mediate contact inhibition of cell growth, thereby negatively
interfering with VEGFR-2–stimulated cell proliferation. In
contrast to this effect, VE-cadherin seems to support VEGFR2–stimulated antiapoptotic effects. This review will focus on
recent progress in understanding the regulation of VEcadherin mediated adhesion and current concepts for the
function of VE-cadherin as a signaling receptor.
Identification of VE-Cadherin and Its
Requirement for Endothelial
Junction Integrity
VE-cadherin was originally identified and cloned as one of 8
new cadherins using an RT-PCR approach.2 These cadherins
were originally named cadherin-4 to11, and cadherin-5, later
renamed as VE-cadherin, was the only endothelial cadherin
among them. Its complete human cDNA sequence was cloned
revealing clear homology to the other known cadherins with
clear differences within the cytoplasmic tail.2 The same
cadherin was identified by a monoclonal antibody (mAb)
approach as an endothelial cell contact protein.3 Its cell
adhesion function was demonstrated by the ability of transfected CHO cells to aggregate in a Ca2⫹-dependent way and
the molecule was renamed VE-cadherin.4 Cloning of mouse
VE-cadherin allowed to show its endothelial specificity at the
very earliest stages of vascular development in embryonic
tissue.5 This has been confirmed for VE-cadherin in zebrafish
embryos.6 The genomic structure and chromosomal mapping
of mouse VE-cadherin were published.7 A roughly 2.5-kb
region of the promotor was characterized to direct endothelial
expression of reporter genes in vivo, although expression was
not found in all endothelial cells.8,9,10 Stronger endothelial
expression was observed, when another 4 kb from the 5⬘ half
of the first intron were added to the regulatory sequences.11
The first evidence for the function of VE-cadherin in vivo
was established with antibodies. A mAb that disrupted
endothelial cell contacts in vitro enhanced neutrophil accumulation in inflamed peritoneum of mice on intravenous
injection,12 and another hybridoma against mouse VEcadherin caused subcutaneous hemorrhage and death of mice
when intraperitoneally injected.13 These studies were confirmed in vitro with polyclonal anti–VE-cadherin antibodies14
and in vivo with yet another mAb against mouse VE-cadherin
that induced increased vascular permeability.15 Adhesion of
human VE-cadherin could be blocked with antibodies against
EC1 as well as with antibodies against EC3.16
Structural Basis of Homophilic Interactions
of VE-Cadherin
The structural basis of homophilic interactions of cadherins
has been extensively analyzed in recent years. Type I and
type II cadherins have 5 EC domains and a conserved
tryptophan (W2) within their N-terminal EC-1 domain,
whereas type II cadherins have an additional tryptophan (W4)
within this domain. It was shown for type I cadherins that
binding of the W2-residues into a hydrophobic pocket of the
adjacent EC1 domain in trans position supports homophilic
adhesive contacts between cadherins.17,18,19 In addition, lateral (cis) dimerization of cadherins is believed to provide the
interface that supports homophilic interactions of cadherins.18,19,20 It has been suggested that this cis interaction is
based on the binding between EC1 and EC2 domains.19
For VE-cadherin 2 different modes of interactions have
been suggested. One model was proposed based on work with
a recombinant form of VE-cadherin expressed in bacteria and
containing domains EC-1 to EC-4. This protein was found to
form hexameric complexes that were analyzed biochemically
and by cryoelectron microscopy.21 3D reconstructions of the
electron micrographs were used to suggest a homology model
(24 Å resolution) based on the known crystal structure of
C-cadherin.22 The arrangement of the 6 VE-cadherin molecules resembled 2 VE-cadherin “triple helices” with each
helix containing 3 parallel VE-cadherin monomers and the 2
trimers being connected in trans via “adhesive” interactions
between 3 pairs of EC-1 domains. The proposed “triple helix”
was supported by trimeric interactions between the EC4
domains.
The second model was based on a recombinant soluble
form of mouse VE-cadherin, expressed in mammalian cells,
and containing all 5 EC domains connected to the N terminus
of the coiled-coil domain of cartilage matrix protein (CMP).23
This protein formed Ca2⫹-dependent ring-like and double
ring–like arrangements that suggested interactions between
domains 1 and 2 of the ectodomains. This model perfectly
agrees with the predicted interactions between adjacent EC1
domains and EC1 and EC2 domains that were suggested
based on the crystal structure for C-cadherin.19 Importantly,
only dimers but no hexamers were found in these studies.
Both models reflect on the elongated but substantially
curved structure of the crystal data for C-cadherin,19 although
only the studies by the Engel laboratory directly showed this
curved shapes in the electron microscope for E-, N-, and
VE-cadherin.18,23 The second model is quite convincing,
reflecting the prominent involvement of the EC1 and EC2
domains reported for C-cadherin.19 The molecular shapes
observed in the electron microscope for analogous multimeric
E- and N-cadherin fusion proteins was principally identical,
Vestweber
suggesting similar interaction modes for VE-cadherin and
“classical” type I cadherins.
The relevance of the hexameric structure has been questioned, because the bacterial recombinant form used in these
studies lacked glycosylation and the EC5 domain. In fact, the
EC2 to EC5 domains of C-cadherin do contain N and
O-glycosylation sites (almost all on EC3 and EC4) that may
well prevent association between these domains,19 and VEcadherin contains potential glycosylation sites at similar
locations. N-linked glycosylation of VE-cadherin has been
demonstrated.24
Some biophysical parameters of the homophilic VE-cadherin
interaction have been determined by atomic force microscopy
using a VE-cadherin-Fc fusion protein.25 Trans association of
VE-cadherin dimers was shown to be a low affinity reaction
with a KD of 10⫺3 to 10⫺5 mol/L. Association required low
affinity Ca2⫹-binding sites (KD⫽1.15 mmol/L).
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Regulation of VE-Cadherin Adhesiveness:
A Role for Tyrosine Phosphorylation?
Leukocytes can transmigrate through the endothelial barrier
via a junctional as well as a transcellular pathway, with the
latter one being much less frequently used.26 It is generally
accepted that endothelial cell contacts need to be opened to
let leukocytes pass through the junctions of the endothelial
barrier. With VE-cadherin being the major adhesive mechanism for the integrity of endothelial cell contacts, it is
reasonable to assume that it needs to be locally downregulated to allow a leukocyte to pass the barrier. Initially,
docking of neutrophils to human endothelial cell monolayers
was reported to lead to degradation of components of the
VE-cadherin/catenin complex by mechanisms within endothelial cells.27,28 However, extending these studies revealed
that the observed effects had been attributable to proteases
released during specimen preparations.29
Nevertheless, when the experimental set up was changed
and monocyte interactions with endothelial cell monolayers
were analyzed under flow, it was shown that staining for the
VE-cadherin– catenin complex was focally lost at endothelial
contacts where monocytes were transmigrating.30 This finding was confirmed by video microscopy and VE-cadherin–
GFP transfected endothelial cells31 or antibody-labeled VEcadherin and PECAM-1.32 It was concluded that VE-cadherin
would “move aside like a curtain” to let the leukocyte pass
through the junction. An alternative explanation would be
endocytosis. Either way, it is likely that VE-cadherin adhesive interactions would open before VE-cadherin molecules
move away from sites of transmigration. How this is triggered
is not entirely understood.
Several publications report on correlations between
changes in the stability of VE-cadherin adhesion and changes
in the tyrosine phosphorylation of the VE-cadherin catenin
complex. Increasing cell confluence of endothelial cells
correlated with increasing tyrosine phosphorylation of VEcadherin and ␤-catenin, but not of ␣-catenin.33 Neutrophils
stimulated by C5a were reported to enhance endothelial cell
permeability for albumin, and this correlated with increased
tyrosine phosphorylation of VE-cadherin and ␤-catenin.34
VE-Cadherin in Vascular Biology
225
It has been suggested that tyrosine phosphorylation of
VE-cadherin itself might affect VE-cadherin functions. Based
on permeability studies of transfected CHO cells, expressing
point mutated forms of VE-cadherin with tyrosine residues
replaced by either glutamate or phenylalanine, tyrosine residues 731 and 658 were suggested to participate in the
regulation of the adhesive function of VE-cadherin.35 However, CHO cells expressing these mutants bound as well to
immobilized VE-cadherin-Fc as CHO cells expressing nonmutated VE-cadherin.34 Intriguingly, overexpression of tyrosine/phenylalanine replacement mutants of VE-cadherin for
either tyrosine 731 or 658 in primary human umbilical vein
endothelial cells (HUVECs) inhibited transendothelial migration of leukocytes, suggesting for the first time that phosphorylation of specific residues is involved in leukocyte extravasation.35 In addition, crosslinking of intercellular adhesion
molecule-1 (ICAM-1) triggered tyrosine phosphorylation of
VE-cadherin, a process that required Src and the proline-rich
tyrosine kinase 2 (Pyk-2).36 Essentially similar results were
shown by Turowsky et al for mouse VE-cadherin, although in
this report several more tyrosine mutants of VE-cadherin
were analyzed and only the Y731F point mutated form of
VE-cadherin inhibited leukocyte diapedesis.37
Various factors are known to enhance vascular permeability and to affect phosphorylation of the VE-cadherin– catenin
complex. The receptor type protein tyrosine phosphatase
(RPTP)-␮ is not restricted to endothelial cells and has been
described to affect the function of various cadherins.38,39 It
associates directly with the cytoplasmic tail of VE-cadherin
(Table 1) and silencing its expression in human endothelial
cells enhanced their permeability for albumin.40
Recently, the endothelial specific RPTP, called VE-PTP,
was found to associate specifically and selectively with
VE-cadherin.41 Expression of VE-PTP in triple-transfected
CHO cells reversed the tyrosine phosphorylation of VEcadherin elicited by vascular endothelial growth factorreceptor 2 (VEGFR-2). Expression of VE-PTP under an
inducible promotor in CHO cells, transfected with VEcadherin and VEGFR-2, increased VE-cadherin–mediated
barrier integrity of a cellular monolayer. The highly specific
interaction of VE-PTP with VE-cadherin further supports the
hypothesis that tyrosine phosphorylation of the VE-cadherin/
catenin complex or of factors in its vicinity is involved in the
regulation of VE-cadherin–mediated cell contacts. Indeed,
knocking down VE-PTP expression in endothelial cells leads
to increased permeability across the endothelial cell layer as
well as to increased transendothelial migration of neutrophils
(Nottebaum et al, unpublished data, 2004). In addition, the
docking of neutrophils to the apical surface of endothelial
cells triggers the dissociation of VE-cadherin from VE-PTP
(Nottebaum et al, unpublished data, 2005). Thus, VE-PTP is
required for the maintenance of VE-cadherin mediated endothelial cell contacts and is likely to participate in neutrophil
transmigration.
Vascular endothelial gowth factor (VEGF) was found to
enhance the permeability of HUVEC monolayers and to
increase tyrosine phosphorylation of VE-cadherin, ␤-catenin,
and plakoglobin, although the time courses of both events
were not similar.42 In mice, intravenous injection of VEGF
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was reported to lead within 2 to 5 minutes to the dissociation
of a preexisting complex of the VEGF-receptor 2 with
VE-cadherin and ␤-catenin as well as tyrosine phosphorylation of VE-cadherin and ␤-catenin.43 These effects required
the kinase Src and were already reversed after 15 minutes.
Repeated VEGF injections lead to vascular damages including edema formation.
Association between the VE-cadherin/catenin complex and
VEGFR-2 seems to be important for outside in (Table 2) as
well as for inside out signaling events (Table 3) concerning
VE-cadherin. In cultured cells, and in contrast to the above
cited report by Weis et al,43 VEGF usually leads to transient
complex formation (not dissociation) between VE-cadherin
and VEGFR-2, an interaction that requires expression of
␤-catenin and of the scaffold protein IQGAP1.44,45 On the one
hand this complex is probably important for the counterregulation of VEGF stimulated proliferation44,46 and the
regulation of apoptosis47 as described in more detail below.
On the other hand, this complex is most likely important for
the regulation of VE-cadherin mediated adhesion. VEGFstimulated tyrosine phosphorylation of VE-cadherin was
reported to require Src, and this kinase was found constitutively associated with VE-cadherin.48 Furthermore, Src was
described to exclusively phosphorylate tyrosine 685 on VEcadherin.49 It is yet unknown whether phosphorylation of this
tyrosine would affect the adhesive function of VE-cadherin,
although it has been reported that inhibition of Src inhibitis
VEGF-stimulated vascular permeability.50
A correlation between an increase of VE-cadherin/catenin
phosphorylation and a decrease in cell contact integrity has
not always been verified. In a thorough kinetic analysis,
Seebach et al found that 2 different factors, VEGF and the
phosphatase inhibitor pervanadate, each led to a rapid increase (minutes after stimulation) of the tyrosine phosphorylation of VE-cadherin, ␤-catenin, and plakoglobin, whereas at
the same time the transendothelial electrical resistance increased.51 A decrease in electrical resistance occurred only 40
to 60 minutes later.
An alternative mechanism for the downregulation of VEcadherin function during VEGF-induced permeability could
be based on the phosphorylation of serine 665 in the cytoplasmic tail of VE-cadherin, leading to endocytosis.52 According to this report VEGF-stimulation leads via Srcdependent phosphorylation of the guanine exchange factor
Vav2, to the activation of the GTPase Rac and subsequent
phosphorylation of serine 665 of VE-cadherin. This phopshorylation results in the recruitment of ␤-arrestin2, thereby
promoting internalization of VE-cadherin.52 Intriguingly,
serine 665 is located on VE-cadherin directly adjacent to the
p120-catenin binding site. Because it has been shown that
p120 regulates clathrin-dependent endocytosis,53–55 it would
be interesting to know whether p120 binding could affect
serine 665 phosphorylation.
Partly contradictory results were published for the effects
of thrombin on the VE-cadherin catenin complex. Thrombininduced increase of endothelial permeability was found to go
along with the transient dissociation of all catenins from
VE-cadherin.56 Others found no overall dissociation of
␤-catenin from VE-cadherin on thrombin stimulation.57 In-
stead, they observed that the phosphorylation of p120 was
upregulated and that of ␤-catenin was downregulated. A third
study demonstrated that thrombin dissociated the phosphatase
SHP2 from ␤-catenin and at the same time increased transiently the tyrosine phosphorylation of ␤-catenin, plakoglobin, and p120.58 For histamine it was reported that tyrosine
phosphorylation of VE-cadherin, ␤-catenin, and plakoglobin
was rapidly stimulated going along with a better extractability
of the complex with detergent.59 Histamine had no effect on
the N-cadherin/catenin complex. These data were confirmed
by others.60
Small GTPases and Other Molecules Affecting
VE-Cadherin Adhesiveness
Small GTPases have been reported to influence the function
of various cadherins.61 In epithelial cells, Rac, Rho, and
Cdc42 have all been implicated in the formation and control
of E-cadherin–mediated cell– cell adhesion.62,63,64,65 The
functional link between VE-cadherin and Rho-like GTPases
in endothelial cells is less clear. Dominant negative Rac
(RacN17) has been described as blocking66 as well as
promoting67 thrombin-induced permeability in HUVECs. In
agreement with the latter report, flow-induced increase of
endothelial barrier function and VE-cadherin clustering were
both inhibited by dominant-negative Rac.68 Interestingly and
in contrast with the generally reported correlations, increase
of barrier function correlated with an increase of tyrosine
phosphorylation of the VE-cadherin/catenin complex.68
Cdc42 was suggested to stimulate the interaction between
␣-catenin and ␤-catenin thereby preventing the increase of
permeability in lung endothelium caused by a dominant
negative form of VE-cadherin.69
On the other hand it was found that VE-cadherin localization in primary endothelial cells was independent of Rac and
Rho activity.65 However, VE-cadherin expression in VEcadherin null endothelial cells induced actin rearrangement,
augmented the level of active Rac, decreased active Rho, and
stimulated expression of the Rac-specific guanosine exchange factor Tiam-1.70
An increasing effect of Rac on endothelial permeability
was described using a cell-penetrating constitutively active
form of Rac (Tat-RacV12) in HUVECs.71 Tat-RacV12 also
induced reactice oxygen species in endothelial cells and
induced tyrosine phosphorylation of ␣-catenin. This is one of
the very rare cases that shows ␣-catenin as target of tyrosine
phosphorylation. In a detailed analysis the dissociation of
endothelial cell contacts with anti–VE-cadherin antibodies
was investigated.72 It was found that loss of cell contacts was
preceded by and dependent on the rapid activation of Rac1
and increased production of reactive oxygen species. In
addition, VE-cadherin–associated ␤-catenin became tyrosine
phosphorylated. Interestingly, a dominant negative form of
redox-sensitive proline-rich tyrosine kinase 2 (Pyk2) inhibited tyrosine phosphorylation of ␤-catenin as well as the loss
of endothelial cell contacts.
Other mechanisms that could control VE-cadherin mediated endothelial cell contacts rely on the cAMP-triggered
activation of the guanine nucleotide exchange factor Epac1
that in turn activates the small GTPase Rap1, which supports
Vestweber
Table 1.
VE-Cadherin in Vascular Biology
227
Proteins That Are Directly or Indirectly Associated With VE-Cadherin
Molecule
Details About the Interaction
References
Intracellular binding
partners
VEGFR-2
Stimulation with VEGF triggers
Association of VEGFR-2 and VE-cadherin in vitro
44,45
Dissociation of VEGFR-2 and VE-cadherin in vivo
43
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SHP-2
Binds to ␤-catenin, thrombin triggers dissociation
58
RPTP-␮
Binds directly to the cytoplasmic tail of VE-cadherin
40
DEP-1
Phophatase trapping mutants bind ␤-catenin, plakoglobin and
p120 from epithelial cell lysates, however, co-precipitation
with VE-cadherin has not yet been analysed
102
Csk
Binding to phosphorylated tyrosine 685 of VE-cadherin
100
PAR-3
This polarity protein binds via its third PDZ doman to the
C-terminal 5 amino acids of VE-cadherin, no binding to N- or
E-cadherin
103
PAR-6
This polarity protein binds to a region between aa 621–689
and does not compete with p120 binding
103
Extratracellular binding
partners
VE-PTP
This receptor type tyrosine phosphatase associates via
extracellular domains with VE-cadherin
Fibrin
NDSK-II, a natural fragment of fibrin, and the fibrin derived
peptide (␤ 15–42) bind to VE-cadherin, binding requires
domains EC3 and EC4 and affects leukocyte extravasation
Fragment of
t-RNA-synthetase
A natural fragment of tryptophanyl tRNA synthetase with anti
angiogenic activity binds to VE-cadherin
41
104,105,106
107
Binding of ␤-catenin, ␣-catenin, and p120 is similar to other cadherins and has not been listed.
the function of VE-cadherin.73,74 How Rap1 improves the
adhesive function of the cadherin is not known, but it has
been proposed for E-cadherin, that Rap-1 is necessary for
proper targeting of E-cadherin to maturing cell contacts.75
Sphingosin 1-phophate (S1P), a phospholipid that binds to
a class of G protein– coupled receptors (GPCR) was shown to
stabilize endothelial cell contacts and enhance the expression
of VE-cadherin at cell contacts of recently confluent endothelial cells, a process that required Rho and Rac.76,77 Knocking down the expression of the S1P-receptor 1 resulted in a
loss of VE-cadherin and PECAM-1 expression at cell contacts.78 Interestingly, early effects of S1P on endothelial
barrier enhancement were found to be independent of the
presence of VE-cadherin.79 In general, vascular permeability
is affected by a complex set of physiological and pathophysiological factors of which many act via different targets
including VE-cadherin. An excellent overview on this topic is
given in a recent comprehensive review.80
Two adhesion molecules at endothelial tight junctions that
are involved in leukocyte extravasation or the regulation of
endothelial cell contacts could potentially affect VE-cadherin
function. The junctional adhesion molecule JAM-C, one of a
small subgroup of tight junction-located Imunoglobulinsuperfamily proteins involved in leukocyte extravasation, was
recently found to affect junction stability in endothelial cells.
Downregulation of its expression by siRNA in cultured
endothelial cells led to a decrease of endothelial cell permeability and enhanced VE-cadherin adhesive function.81 Both
effects were dependent on Rap1. A structurally related
protein to JAM-C, the endothelial cell-selective adhesion
molecule (ESAM), was recently found to play a role in vivo
in neutrophil extravasation and in the regulation of endothelial cell contacts.82 Gene-deficient mice displayed a delay in
leukocyte extravasation and a delay in the increase of
vascular permeability induced by VEGF.82 In the context of
these effects, it is intriguing that MAGI-1, a PDZ protein that
binds to ESAM,83 is required for Rap-1 activation and for the
enhancement of VE-cadherin–mediated cell contacts.84
VE-Cadherin Is an Essential Adhesion
Molecule in Angiogenesis
Disrupting the VE-cadherin gene in mouse embryonic stem
(ES) cells allowed to investigate its role in the development
of vascular structures. Analyzing embryoid bodies developed
from such ES cells revealed that the differentiation of
endothelial cells was not impaired, yet they remained dispersed and failed to organize a vessel-like pattern.85 Mice
deficient for VE-cadherin died at midgestation of vascular
malformations.86 Defects were more severe in the extraembryonic vasculature. No capillary plexus was formed in the
allantois, although interendothelial junctions did form as
shown by electron microscopy. Thus VE-cadherin was dispensable for initial vasculogenesis but was required for
subsequent remodeling and morphogenesis.86 Another study
showed similar results analyzing VE-cadherin null mice as
well as mice lacking the C-terminal 82 amino acids of
VE-cadherin, containing the ␤-catenin binding site.47 Mutants
had increased numbers of apoptotic endothelial cells. In
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Table 2. Outside-In Signaling, Receptor-Like Function
of VE-Cadherin
Factors and Proposed Mechanisms
References
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Prosurvival signals (antiapoptotic effects)
VE-cadherin/␤-catenin forms a complex with VEGFR-2
and PI3-kinase that is required for VEGF-triggered
phosphorylation of protein kinase Akt and induction of
Bcl2, both mediators of the antiapoptotic machinery.
Sensing of shear forces
PECAM-1 acts as a sensor for shear and transmits
signals via VE-cadherin (acting as an adaptor) and
VEGFR-2, which activate PI3-kinase.
Antiproliferative effects, contact inhibition of cell growth
VE-cadherin engagement reduces VEGF-triggered
tyrosine phosphorylation of VEGFR-2 and expression
of VE-cadherin extends the half life of VEGFR-2.
Lack of VE-cadherin enhances VEGF-triggered
VEGFR-2 phosphorylation, accelerates uptake and
extends internalization times of VEGFR-2. Silencing of
DEP-1 had the same effects.
Increased cell density triggers binding of C-terminal
src kinase (Csk) to phosphorylated tyrosine 685 of
VE-cadherin, Csk slows down proliferation and a
VE-cadherin-Y685F mutant partially abolishes contact
inhibition of cell growth.
47
108
97,90
44,42
100
endothelioma cells the C-terminal truncation of VE-cadherin
reduced complex formation with VEGF-receptor 2,
␤-catenin, and phosphoinositide 2 (PI3)-kinase and abolished
survival signals by VEGF-A to Akt kinase.47 Another detailed
analysis revealed a temporal correlation linking failure in
vessel morphogenesis to a specific step in vasculogenesis.87
Those vessels forming first—yolk sac, allantoic, and endocardial vessels— deteriorated first. In allantois explants, an
adhesion-blocking antibody against VE-cadherin did not
block the formation of nascent blood vessels but disassembled nascent vessels, supporting the idea that VE-cadherin is
required for the maintenance of nascent vessels.
It is unknown which other adhesive mechanisms mediate
endothelial cell contact formation in the absence of VEcadherin. N-cadherin would be an obvious candidate, because
it is also expressed in endothelial cells. In transfected CHO
cells it was found that VE-cadherin competes with
N-cadherin for junctional localization and drives it out of
junctions.88 It has not been analyzed whether N-cadherin is
located in interendothelial cell contacts of nascent vessels
from VE-cadherin null mice. Surprisingly, it was recently
reported that N-cadherin is expressed at endothelial cell
contacts of healthy mice and that endothelial specific ablation
of N-cadherin in mice results in embryonic lethality at
mid-gestation because of vascular defects.89 Surprisingly,
loss of N-cadherin caused a decrease in VE-cadherin expression, suggesting that N-cadherin might regulate angiogenesis,
in part, by controlling VE-cadherin expression at cell membranes.89 Contrary to these results it was found that embryoid
bodies derived from ES-cells deficient for N-cadherin could
form normal vessel like structures.90 The only defect observed was a lack of pericyte covering of endothelial
outgrowths.
It is intriguing, that gene disruption of the VE-cadherin–
associated receptor type tyrosine phosphatase, VE-PTP, also
Table 3. Inside-Out Signaling, Stimuli and Mechanisms That Affect the Adhesive Function of
VE-Cadherin
Stimulus
Proposed Mechanism
References
Thrombin
Enhanced endothelial cell layer permeability
dissociation of SHP-2 from ␤-catenin, increased P-Y of catenin
Dissociation of catenins from VE-cadherin
No dissociation of cadherin/catenin complex, but upregulation of
P-Y on p120, and dowregulation on ␤-catenin
Enhanced endothelial cell layer permeability and Increased P-Y of
VE-cadherin/catenin complex
Enhanced endothelial permeability and P-Y of VE-cadherin/catenin
complex
Opposite effect: enhanced endothelial cell contact integrity and
increased P-Y of VE-cadherin/catenin complex
Enhanced endothelial permeability and increased endocytosis of
VE-cadherin
Enhanced transendothelial electrical resistance and increased P-Y
of VE-cadherin/catenin complex
Increased association of VE-PTP with VE-cadherin enhances
VE-cadherin mediated adhesion; correlation with reduced
VE-cadherin and ␤-catenin P-Y
Knocking down RPTP-␮ enhances endothelial cell layer
permeability, overexpression reduces P-Y on VE-cadherin
Increase of cAMP levels activates the GTPase Rap1 and enhances
VE-cadherin mediated adhesion
58
Histamine
VEGF
Shear
force
Unknown
Unknown
Unknown
56
57
59,60
42,48,49
51
52
68
41
40
74
It should be noted that none of these studies (except for74) directly analysed the adhesive function of VE-cadherin,
but rather endothelial cell contact integrity. The detected effects on tyrosine phosphorylation and protein–protein
interactions correlated with changes in cell contact stability, however causal relationships have not yet been strictly
established. P-Y indicates phospho-tyrosine.
Vestweber
VE-Cadherin in Vascular Biology
229
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VE-Cadherin Mediated Signals Inhibit
Endothelial Cell Proliferation
regulation of this phosphatase enhanced VEGFR-2 phosphorylation and cell proliferation. The same group showed46 in a
later report that VEGF triggered clathrin-dependent internalization of VEGFR-2, and this internalization was more rapid
and VEGFR-2 remained longer in endosomal compartments
if VE-cadherin was absent or cells were sparsely confluent.
Endocytosed VEGFR-2 was still phosphorylated and inhibition of internalization reestablished contact inhibition of
growth. Silencing DEP-1 enhanced internalization and signaling of VEGFR-2. The same group reported that VEGF
triggered the association of the adaptor protein Shc with
VE-cadherin, an association that required the C-terminal 82
amino acids of VE-cadherin.99 Phosphorylation of Shc lasted
longer in VE-cadherin null endothelial cells compared with
VE-cadherin expressing cells.
An alternative mechanism for VE-cadherin–mediated contact inhibition of growth was recently suggested by the
finding that the C-terminal Src kinase (Csk), a kinase that
phosphorylates and thereby inhibits Src, binds specifically to
phosphorylated tyrosine 685 of VE-cadherin.100 This is the
same tyrosine that was later described as exclusively phosphorylated by Src49 (see above). Binding of Csk increased
with higher cell density and silencing Csk expression enhanced endothelial cell proliferation, suggesting that Cskbinding to VE-cadherin may be involved in VE-cadherin–
mediated contact inhibition of cell growth.100 Although Csk
binding to tyrosine 685 was not found to affect VE-cadherin
adhesive activities in transfected cells, a function of tyrosine
685 in the regulation of VE-cadherin adhesion in endothelium
should not be excluded.
Another molecule suggested to be involved in VE-cadherin–mediated contact inhibition of growth is surviving
because it was found to be slightly upregulated in angiogenically active endothelial cells and downregulated in culture by
cell confluence and VE-cadherin expression.101
Similar to other cadherins, VE-cadherin has antiproliferative
effects that mediate cell contact– driven inhibition of cell
growth in cultured endothelial cells.96 This effect requires the
presence of the last 82 amino acids at the C terminus of
VE-cadherin,96 the same part of the protein that is required to
support survival of endothelial cells.47 Thus, surprisingly, the
same part of VE-cadherin mediates effects that in one case
support VEGFR-2 signaling (antiapoptosis, prosurvival effects) and in the other case inhibit VEGFR-2 signaling
(proliferative effects). Indeed VE-cadherin–mediated inhibition of VEGFR-2 signaling had already been published
around the same time.97 This report demonstrated that VEGFtriggered phosphorylation of VEGFR-2 was reduced at high
cell density and was enhanced if confluent endothelial cell
layers were pretreated with an adhesion blocking antibody
against VE-cadherin.97 The same group found that overexpression of VE-cadherin in endothelial cells increases the
halflife of VEGFR-2.98 In agreement with these studies, it
was described that the lack of VE-cadherin enhanced VEGFtriggered phosphorylation of VEGFR-2.44 It was suggested
that VEGF stimulation leads to the association of the receptor
with VE-cadherin, a process that requires the presence of
␤-catenin. This association would trigger dephosphorylation
of the receptor, possibly mediated by DEP-1 because down-
Investigating VE-cadherin over the last 15 years has established it as one of the 2 to 3 most intensely studied cadherins
known today. Its accessibility from within the vasculature, its
prominent role as the dominant adhesion molecule responsible for the maintenance and control of endothelial cell
contacts, and its essential role during morphogenesis of the
blood vessel system has made VE-cadherin a prime research
target among adhesion molecules in the vasculature. Much
progress has been made in identifying signaling partners
(Table 2) and factors that influence VE-cadherin function
(Table 3). Based on these findings several major goals and
questions have emerged that will be important in the future.
As for other cadherins, it will be important to understand in
detail, how phosphorylation of the cadherin/catenin complex
and of other substrates in its close vicinity translate into
changes in adhesive strength. Furthermore, how do other
adhesion mechanisms at endothelial junctions (ESAM,
JAM-C, or maybe PECAM-1) modify the function of VEcadherin? How do small GTP binding proteins such as Rap1
affect VE-cadherin? To what extent is the regulation of the
VE-cadherin–mediated barrier key to the process of leukocyte extravasation in inflammation and lympohcyte homing?
leads to embryonic lethality before E10 because of a defect in
angiogenesis.91 Although blood vessels were initially formed,
the intraembryonic vascular system soon deteriorated. Blood
vessels in the yolk sac developed into dramatically enlarged
cavities. In explant cultures of mutant allantoides, endothelial
cells were found next to vessel structures growing as cell
layers. No obvious signs for enhanced endothelial apoptosis
or proliferation were observed. A subsequent study essentially confirmed these results.92 Although VE-cadherin could
have a role in mechanisms that causes the observed vascular
defects, VE-PTP also associates with Tie-2 and may have yet
other substrates that could be involved.
Based on the idea that VE-cadherin transmits antiapoptotic
signals and thereby supports the survival signals of VEGFR2,47 antibodies against VE-cadherin were screened for their
ability to block this function to obtain a reagent that could
possibly drive tumor endothelium into apotosis. Indeed an
anti-mouse VE-cadherin adhesion blocking antibody reduced
tumor growth in a Lewis lung metastasis model.93 However,
because of its potent antiadhesive effects, slightly higher
doses than those that efficiently inhibited tumor growth were
lethal because they caused increased vascular permeability
and edema in the lung. To circumvent this problem antibodies
were screened that would block VE-cadherin functions during tumor angiogenesis without affecting vascular permeability. An antibody was described (E4G10) that specifically only
bound to endothelial cells in a subset of tumor vasculature but
not to normal vasculature.94 It was speculated that the epitope
would only be accessible in recently formed adherens junctions of tumor endothelium while masked in resting vasculature. In a similar study, another mAb was described, which
had a similar function and bound to domain EC4.95
Concluding Remarks
230
Arterioscler Thromb Vasc Biol
February 2008
Which of the many different factors that influence vascular
permeability do indeed affect VE-cadherin function and
which ones act indirectly via other molecular mechanisms?
Which endothelial adhesion mechanisms mediate the formation of nascent blood vessels in VE-cadherin– deficient mice?
How do mechanisms that control endothelial cell contacts and
VE-cadherin function affect or even control endothelial cell
sprouting, proliferation and branching? These are only some
of the most fascinating questions connected to the central role
of VE-cadherin in vascular biology. Studying them will
reveal important insights into basic mechanisms of angiogenesis, inflammation, and vascular biology as a whole.
Sources of Funding
This work was supported by The European Consortium (NoE MAIN
502935), The Max-Planck-Gesellschaft, and the Deutsche
Forschungsgemeinschaft.
Downloaded from http://atvb.ahajournals.org/ by guest on June 18, 2017
Disclosures
None.
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VE-Cadherin: The Major Endothelial Adhesion Molecule Controlling Cellular Junctions
and Blood Vessel Formation
Dietmar Vestweber
Arterioscler Thromb Vasc Biol. 2008;28:223-232; originally published online December 27,
2007;
doi: 10.1161/ATVBAHA.107.158014
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