REVIEW  FOR  THE  2008  SATO  AWARD Circ J 2009; 73: 2183 – 2191

Vascular Integrity Mediated by Vascular Endothelial

Cadherin and Regulated by Sphingosine 1-Phosphate
and Angiopoietin-1

Naoki Mochizuki, MD

Development of blood vessels is coordinated by angiogenesis and stabilization of vascular endothelial cells (ECs).
The vascular network is established during embryogenesis to supply oxygen and nutrients to the tissues and organs.
However, after cardiac or peripheral ischemia is caused by occlusion of the vessels, new vessels must be formed
to rescue the ischemic tissues. Many angiogenic growth factors and chemokines are produced in the ischemic
tissue to induce angiogenic sprouting of preexisting vessels. Branched vessels must be again restabilized to form
mature vessels that deliver blood to the tissues. To this end, vascular EC–cell adhesion is tightly regulated by
cell–cell adhesion molecules and extracellular stimuli that activate G protein-coupled receptors and receptor
tyrosine kinases exclusively expressed on vascular ECs. This review spotlights the recent studies of vascular
endothelial cadherin and of sphingosine 1-phosphate signaling and angiopoietin-Tie signaling.   (Circ J 2009;
73: 2183 – 2191)
Key Words: Angiogenesis; Angiopoietin; Sphingosine 1-phosphate; Stabilization; Vascular endothelial cadherin

not only relax/contract but also loosen/tighten the cell–cell

How is Vascular Integrity Regulated?
Blood Vessels Regulate Delivery of Blood and Nutrition
by Controlling Blood Flow and Permeability
Neovascularization is required for developmental (physio-
logical) and pathological angiogenesis.1 The latter is found
in the ischemic heart, peripheral arterial disease and tumors.
Thus it is important to assist neovascularization in ischemic
diseases and to block angiogenesis in cancer.2 The vascular
network of the body maintains the circulation of blood and
nutrients necessary for the peripheral tissues and organs by
relaxing and constricting autonomously or non-autono-
mously. Many of the molecules produced by endothelial cells
(ECs) and vascular smooth muscle cells (VSMCs), which
affect relaxation or contraction of blood vessels in both
an autocrine and paracrine manner, have been identified.
Among them, sphingosine 1-phosphate (S1P) released from
vascular ECs stimulates S1P receptors on the ECs to induce
vascular relaxation and those on the VSMCs to induce vas-
cular contraction.3 Vascular ECs produce nitric oxide (NO)
to induce the relaxation of VSMCs. The NO-dependent
cGMP-PKG signal modulated by phosphodiesterase in
VSMCs is central to vascular relaxation.4,5 On the other
hand, ECs synthesize vascular contraction factors, including
the metabolites of arachidonic acids.6 Vascular contraction
and relaxation are mainly regulated by the activity myosin
light-chain kinase (MLC-K) controlled by the intracellular
increase/decrease in Ca2+ and Rho kinase in the signaling
provoked by G protein-coupled receptors (GPCR).7 Recent-
ly, it has been also shown that oxidative stress affects vas-
cular cells.8 In order to deliver nutrients and blood, vessels
adhesions, which are constituted by endothelial-specific and
common adhesion molecules. Structural characteristics of
vascular ECs determine the supply of the molecules in the
blood vessels across the monolayer of ECs.9 How the mole-
cules pass through this barrier has been extensively studied.
The concentration of molecules inside or outside of the
monolayer is determined by transcellular transport and
paracellular permeability.10 The transcellular transport of
the molecules occurs in the fenestrated capillaries, whereas
the paracellular pathway is via the continuous capillaries.
The surface of the endothelial cellular that consists of
glycocalyx sieves the molecules that will be subjected to
the transcellular or paracellular pathway.11 The presence of
basement membrane beneath the endothelial monolayer
affects the diffusion of the molecules that pass the endo-
thelial layer. ECs are attached to the extracellular matrix
(ECM) via integrin receptors to form an EC–ECM adhe-
sion,12 which is involved in both the transcellular and para-
cellular pathways. In clear contrast, the cell–cell adhesions
directly control the paracellular pathway (Figure 1).
Roles of Adhesion Molecules Expressed on Vascular ECs
in Extravasation of Blood Cells
The intercellular adhesions between vascular ECs mainly
constitute adherens junctions and tight junctions. Vascular
ECs express endothelial-specific adhesion molecules: vas-
cular endothelial cadherin (VE-cad), and platelet and endo-
thelial adhesion molecule-1 (PECAM-1).13–15 In addition
to these adhesive molecules, junctional adhesion molecule
(JAM), claudin-5 and nectin are involved in the formation

Received September 8, 2009; accepted September 14, 2009; released online October 19, 2009
Department of Structural Analysis, National Cardiovascular Center Research Institute, Suita, Japan
Mailing address:  Naoki Mochizuki, MD, Department of Structural Analysis, National Cardiovascular Center Research Institute, 5-7-1
Fujishirodai, Suita 565-8565, Japan.   E-mail: nmochizu@ri.ncvc.go.jp
All rights are reserved to the Japanese Circulation Society.  For permissions, please e-mail: cj@j-circ.or.jp

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2184 MOCHIZUKI N

Figure 1.   Two types, continuous and fenestrated, of endothelial cells (ECs) regulate the paracellular and transcellular
transport of molecules across the monolayer of the endothelium. Glycocalyx expressed on both continuous ECs and
fenestrated ECs lines the luminal surface of the endothelium and limits the passage of molecules by their charge, suggest-
ing the glycocalyx is the first stage in regulation of permeability by sieving the molecules. An electron microscopic view
of the glycocalyx detected by alcian blue-lanthanium nitrate staining of rat coronary artery. Beneath the endothelium,
basement membrane (BM) forms the extracellular matrix (ECM) –endothelial cell adhesion to stabilize the endothelial
cells to the ECM. Furthermore, pericytes/vascular smooth muscle cells and podocytes in continuous ECs and fenestrated
glomerular ECs support the ECs by respectively forming cell–cell contacts and by secreting angiopoietin-1.

Figure 2.   Schema of how leukocytes extra-

vasate across the endothelium and the intercel-
lular adhesion molecules of the endothelium.
Local inflammation requires an accumulation
of leukocytes across the endothelium through
several steps: attachment of leukocytes to the
endothelium, rolling of leukocytes on the endo-
thelium, firm attachment of leukocytes to the
endothelium via integrin, and transmigration
via cell–cell adhesion molecules. Cell–cell
junction molecules not only participate in the
extravasation of blood cells but also directly
regulate loosening and tightening of the cell–
cell adhesion. Assembly of cortical actin aug-
ments the accumulation of VE-cadherin (VE-
cad), but not PECAM-1, at the cell–cell contact
by being linked to VE-cad through β-ctn and
α-ctn. JAM, junctional adhesion molecule;
PECAM-1, platelet and endothelial cell adhe-
sion molecule-1, p120ctn, p120 catenin;β-ctn,
β-catenin;α-ctn,α-catenin.

of cell–cell contacts.16–18 VE-cad constitutes adherens junc-
tion, whereas claudin-5, JAM and ESAM form tight junc-
tions. Recently, VE-cad was shown to transcriptionally
upregulate claudin-5 by inducing the nuclear export of β-
catenin (β-ctn), which is mediated by the formation of a
FoxO-1 and β-ctn complex outside of the nucleus.19
PECAM-1 localizes in the region outside of the adherens
and tight junctions20 (Figure 2).
The interendothelial cell–cell adhesions regulate the
transendothelial migration of blood cells into the intersti-

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Stabilization of Endothelial Cell–Cell Contact (2008 Sato Award) 2185

Figure 3.   Stabilization (indicated by black font) and destabilization (indicated by red font) is controlled by receptor
tyrosine kinases and G protein-coupled receptors expressed on the vascular endothelial cells. Activation of sphingosine 1-
phosphate receptor 1 (S1P1) and Tie2 enhances cell–cell adhesions, whereas that of VEGF receptor and PAR-1 receptor
destabilizes the cell–cell adhesion. Gi-mediated signal downstream of S1P1 exerts a vasculoprotective action via endo-
thelial nitric oxide synthase, while Gq- and G12/13-induced signal promotes the contraction of endothelial cells, thereby
physically dissociating cell–cell contacts. MLC-PPase, myosin light chain phosphatase; CaM-K, calmodulin kinase;
MLC-K, myosin light chain kinase.

tial cells, as well as permeability. There are several steps
observed in leukocyte recruitment to ECs: initial attachment
and rolling of leukocytes through their interaction with
integrin (leukocytes) and p- and e-selectins (ECs), then
firm attachment and subsequent recruitment of leukocytes
to the cell–cell junction mediated by the interaction between
integrins on the leukocytes and ICAM-1/VCAM-1 on the
ECs and transmigration mediated by several molecules
expressed on the interendothelial junction.21 This inter-
action has become a target for reducing inflammation.22
The homophilic association of PECAM-1 contributes to
transendothelial migration after the initial attachment of
the leukocytes to ECs.23 The engagement of integrins with
ICAM-1 results in phosphorylation of VE-cad, thereby
weakening the VE-cad-mediated cell–cell junctions.24 Thus,
VE-cad works as the gate keeper against leukocytes, which
induce inflammation (Figure 2).
In this review, I focus on vascular integrity controlled
by the VE-cad-mediated cell–cell adhesion, angiopoietin-1
(Ang1)–Tie2 signaling and the S1P-mediated signal as
representative cell adhesion molecules, tyrosine kinase
receptor-mediated signals and GPCR-mediated signals,
respectively. The atrial natriuretic peptide family of mole-
cules is essential for vascular integrity by activating their
receptors and exerting guanylate cyclase activity. The
beneficial effects of natriuretic peptides on the vasculature
have been reviewed.25,26
Control of Vascular Integrity
Permeability Regulating Factors
Increased vascular permeability is found in inflammation,
hypoxia and thrombosis. Under these conditions, vascular
permeability is either enhanced or reduced by extracellular
stimuli that activate cell surface receptors, tyrosine kinases
and GPCRs. Vascular endothelial growth factor (VEGF) is
a vascular permeability increasing factor,27 whereas Ang1
activates Tie2 receptor tyrosine kinase and counteracts the
VEGF–VEGFR-2 signal to maintain vascular integrity.28,29
Several permeability-increasing factors are induced
through GPCR activation. Thrombin is concentrated in
clots and released into the blood to activate PAR-1, which is
activated when its amino-terminus is cleaved upon throm-
bin stimulation, and it then signals to Gq and G12/13 proteins
to increase permeability.30 Gq and G12/13 activation leads to
increased intracellular Ca2+, which enhances activation of
calmodulin kinase (CaM-K)/MLC-K, leading to activation
of Rho–Rho kinase to reduce MLC phosphatase. Both
signals result in actin-myosin-dependent contraction of
vascular ECs.31 Bradykinin produced from kininogen in the
blood acts on the Gq/i protein-coupled B1 and B2 receptors
expressed on ECs to increase permeability.32 In contrast to
thrombin, bradykinin induces vascular permeability inde-
pendently of MLC-K and Rho kinase.33 Histamine released
from mast cells also transiently increases permeability, com-
pared with thrombin, by activating H1 receptors expressed
on ECs.34 Platelet-activating factor augments vascular per-
meability independently of the actin–myosin contraction,
similar to bradykinin33 (Figure 3).
VE-Cad Regulates Barrier Function in ECs
VE-cad and N-cadherin belonging to the classical cadherin
family that are expressed on the endothelium and undergo
Ca2+-dependent homophilic trans- and cis-interactions. VE-
cad accumulates at the interendothelial cell–cell junctions,
whereas N-cadherin is mostly located between ECs and
smooth muscle cells.35,36 In contrast, there is a report of
localization of N-cadherin at the interendothelial junc-
tions.37 Similar to other classical cadherins, VE-cad is a
single-membrane-spanning molecule that contains 5 extra-
cellular cadherin domains, a transmembrane domain and a
cytoplasmic domain that binds p120 catenin (p120ctn) and
β-ctn at the juxtamembrane domain and the carboxy ter-
minal domain, respectively.38
The expression of VE-cad is regulated by many steps:
shedding, endocytosis, synthesis, transport and stabiliza-
tion at the cell–cell contacts (Figure 4). The extracellular
domain of VE-cad is detected in the culture medium from
apoptotic ECs, indicating shedding of VE-cad.39 Recently,
the metalloprotease-disintegrin, ADAM9, was reported to
potentially cleave VE-cad.40
VEGF induces endocytosis of VE-cad through the phos-
phorylation of VE-cad at Ser 665. Activation of Rac by
Vav2 in VEGF-stimulated cells leads to PAK-dependent
phosphorylation of Ser at 665, resulting in endocytosis of
VE-cad in a manner dependent onβ-arrestin2.41 Src tyrosine

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2186
Figure 4.   Expression of vascular endothelial cadherin (VE-cad) at the cell–cell contact depends on shedding, endocytosis,
synthesis, transport and stabilization of VE-cad. Shedding, endocytosis and reduced transport decrease the expression of
VE-cad at the cell–cell contact, whereas accelerated transport, synthesis and stabilization results in increased expression at
the cell–cell contact. Therefore, the balance of these regulatory steps determines the expression of VE-cad and the mainte-
nance of vascular integrity.
kinase is involved in the Vav2–Rac–PAK signal-mediated
phosphorylation of VE-cad. Although phosphorylation of
the cytoplasmic domain of VE-cad is accompanied by an
increase in vascular permeability in vitro and in vivo,42,43 it
has not been explored whether tyrosine-phosphorylated
VE-cad is prone to endocytosis. Src is also involved in
tyrosine phosphorylation of VE-cad.44
VE-cad associates with VEGFR-2 (Flk-1) and reduces
the internalization of VEGFR-2.45 The significance of the
association of VE-cad with VEGFR-2 is confirmed by mice
lacking the entire VE-cad and those lacking its cytoplasmic
domain,46 although the precise mechanism of VEGF-medi-
ated internalization of VE-cad has yet to be clarified.
The VE-cad promoter has been isolated and is used as
an endothelial-specific promoter.47 There are 2 conserved
Ets binding sites in the 5’ region (within −200 bp) of the
transcription initiation site of the VE-cad gene (CDH5).
Chromatin immunoprecipitation assay confirms that Erg is
capable of binding these 2 sites. Perturbation of Erg using
antisense oligonucleotides reduces the expression of VE-
cad in HUVECs, indicating the involvement of Erg in the
transcriptional regulation of VE-cad.48 There are 6 poten-
tial and putative hypoxia response elements, suggesting
the regulation of VE-cad expression by hypoxia-inducible
factors (HIF-1α and HIF-2α).49 Although CDH5 is not
responsive to hypoxia, HIF-2α, but not HIF-1α, activates
the promoter of VE-cad. Serum response factor (SRF) is
also reported to be required for the expression of VE-cad,
as confirmed by the depletion of SRF in the vascular ECs
of mice.50 SRF binds to CArG box found in the regulatory
regions of CDH5.
The cytoplasmic domain of VE-cad is important for the
trafficking and stabilization of VE-cad because it provides
the binding sites for p120ctn and β-ctn. The amount of
MOCHIZUKI N
p120ctn determines a set point mechanism that regulates
cadherin expression levels by controlling the internaliza-
tion and degradation of VE-cad.51 Reduction of p120ctn
by siRNA results in decreased expression of endogenous
VE-cad. Forced expression of p120ctn reduces the clathrin-
mediated endocytosis of VE-cad.52 The essential role of the
association between p120ctn and VE-cad is supported by
evidence that overexpression of the juxtamembrane domain
of VE-cad reduces VE-cad expression and barrier function.53
Furthermore, p120ctn regulates the transendothelial migra-
tion of leukocytes by altering the tyrosine phosphorylation
of VE-cad.54 Conversely, phosphorylation of VE-cad at
Tyr658 and Tyr731 inhibits binding of p120ctn to VE-cad.44
Phosphorylation of the 2 tyrosines correlates with a reduc-
tion in the barrier function of ECs. Another study reports
that Golgi-associated cPLA2α regulates the transport of
VE-cad, as well as claudin-5, from the Golgi apparatus to
the cell–cell junction.55
Theβ-ctn binding to E-cadherin is well known as linking
link E-cadherin to actin via α-catenin (α-ctn) to stabilize
E-cadherin-mediated cell–cell adhesion.56 Thus, similar to
E-cadherin, VE-cad is thought to be linked to actin fibers to
maintain the integrity of endothelial adhesions. Depletion
of β-ctn in the ECs of mice results in defective vascular
patterning, suggesting the critical role of β-ctn in interen-
dothelial adhesions.57 Therefore, stabilization of VE-cad
depends on the link between VE-cad and actin. However,
this classical model is challenged by the Nelson58 and Weis
groups.59 They claim thatα-ctn does not connect actin fibers
to E-cadherin and propose a dynamic model instead of the
classical static model. In addition, phosphorylation ofβ-ctn
as well as VE-cad affects the binding of β-ctn to VE-cad.
There are several phosphatases that dephosphorylate the
molecules participating in the regulation of cell–cell adhe-
Circulation Journal Vol.73, December 2009
Stabilization of Endothelial Cell–Cell Contact (2008 Sato Award) 2187

Figure 5.   Fluorescence recovery after photobleaching technique of analyzing the mobility of vascular endothelial
cadherin (VE-cad) at the cell–cell contact. Fluorescence recovery at the region of interest (cell–cell contact marked by the
yellow circle) after photobleaching of VE-cad tagged with green fluorescent protein (VE-cad–GFP) can be detected by a
confocal microscope. The mobile and immobile fractions can be deduced from the graph (Top) of the plot of raw data
(Middle). The decrease in the mobile fraction denoted by the red arrow and red line reflects the increased stabilization of
VE-cad–GFP at the cell–cell contact. We can compare the dynamics of VE-cad to PECAM-1 by constructing plasmids
expressing VE-cad–GFP and PECAM-1 tagged with GFP (PECAM-1–GFP) and their chimeric molecules (Bottom).

sions. Thrombin induces phosphorylation of SHP2 and its
dissociation of VE-cad, resulting in the phosphorylation of
β-ctn and a reduction of the amount of β-ctn that binds to
VE-cad.60 The requirement of vascular endothelial protein
tyrosine phosphatase (VE-PTP) has been demonstrated.61
Downregulation of VE-PTP enhances EC permeability and
inhibits VE-cad adhesion. The essential substrate of VE-PTP
in VE-cad-dependent cell–cell adhesions is plakoglobin,
notβ-ctn. DEP-1/CD148 is reported to be required for VE-
cad-dependent inhibition of VEGFR-2 activation.62
The cellular actin cytoskeleton that determines cell shape
is regulated by the Rho-family GTPases (Rho, Rac and
Cdc42). Indeed, Rho-family GTPases are involved in
cadherin-mediated cell–cell adhesion by controlling the
actin-based cytoskeleton.63 Besides the Rho-family GTPases,
Rap1, a small GTPase, is well known for stabilizing the
VE-cad-mediated cell–cell adhesion.64,65
Stabilization of VE-Cad at the Cell–Cell Contact
by Rap1-Mediated Signal
We have previously shown that MAGI-1 binding to β-ctn
recruits PDZ-GEF1, a guanine nucleotide exchange factor
(GEF) for Rap1, thereby activating Rap1 at the interendothe-
lial junctions upon engagement of VE-cad.66 My and others
further demonstrate that the cAMP–Epac (another GEF for
Rap1) –Rap1 signal stabilizes the VE-cad-mediated cell–cell
adhesion.67,68 Although cAMP-increasing stimuli are known
to stabilize the cell–cell adhesion via protein kinase A (PKA),
Epac instead of PKA is critical for cAMP-induced stabili-
zation. Accumulation of VE-cad at the cell–cell contacts is
increased by an Epac-specific activator, 8-pCPT-2’-O-Me-
cAMP (known as 007), as well as forskolin in vitro and in
vivo.67,69 007 not only reduces EC permeability, but also
inhibits transendothelial migration of leukocytes.70
Although the downstream effectors of Rap1 in the stabili-
zation of VE-cad are not fully understood, several potential
signaling pathways have been suggested. Cerebral cavern-
ous malformation (CCM) is associated with defective endo-
thelial junctions. There are 3 genes related to CCM: CCM1,
CCM2 and CCM3.71,72 CCM1/KRIT1 (K-Rev1 interaction
trapped gene 1), a Rap1-binding protein, is reported to be
involved in Epac1/Rap1-regulated permeability of EC–cell
junctions.73 KRIT1 localizes at the cell–cell contacts and
associatesβ-ctn, thereby being indirectly connected to VE-
cad. CCM2 functions as a scaffold protein and forms com-
plexes with CCM1 and CCM3.74 CCM1 might connect the
heart-of-glass (Heg)-mediated signal to CCM2 to maintain
vascular structure, as well as VE-cad-β-ctn.75 Target disrup-

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2188 MOCHIZUKI N

Figure 6.   Angiopoietin-1 (Ang1) secreted from pericytes induces the trans-association of Tie2 at the cell–cell contact
and subsequently activates the Akt–MEF2–KLF2 signal. KLF2 promotes the expression of endothelial nitric oxide synthase
and reduces the expression of VCAM-1. Therefore, the Ang1–Tie2 signal is anti-inflammation and pro-quiescence (Upper).
Sphingosine 1-phosphate (S1P) is produced from sphingosine (Sph) in the plasma membrane by sphingosine kinase
(Sph-K) in the cytoplasm. Thus, to activate sphingosine 1-phosphate receptor 1 (S1P1) that governs vascular stability, S1P
is released outside the cell from the cytoplasm. Spinster2 (Spns2) functions as a S1P transporter. Thus, Spns2 is likely to
participate in S1P-mediated signaling in a paracrine or autocrine manner to control vascular integrity (Lower).

tion of Ccm2 in mice results in failure of lumen formation
and embryonic lethality. Whitehead et al showed activation
of RhoA in CCM2-depleted cells and alteration of cell–cell
junctional structure because of the activation of RhoA.74
Therefore, the Rap1-CCMs signal is proposed to regulate
the cell–cell adhesion through modification of the RhoA
signaling that controls the actin cytoskeleton.
Rap1 has been demonstrated to regulate actin cytoskeleton
via signaling to Rho-family GTPases. Rap1 is capable of acti-
vating Rac and determining the localization of GFEs, Vav2
and Tiam1 for the Rho-family GTPases.76 Cell–cell contact-
dependent engagement of nectin induces the activation of
Rap1 and subsequent activation of Rac and Cdc42 via Vav2
and FRG, respectively.15 The role of affadin, which can bind
to Rap1, nectin, and zonula occludens-1 (ZO-1), has not been
clearly investigated in the Rap1-mediated signal, although its
role in the EC–ECM has been characterized.77 Rho-family
GTPases are regulated by other molecules localized at the
cell–cell contacts. p120ctn and RhoA mutually inhibit their
functions by binding each other.78 p120ctn balances the
activity of Rho and Rac by binding to p190RhoGAP at the
cell–cell contacts when p190RhoGAP is recruited to the
cell–cell contacts upon growth factor stimulation.79
In the light of cortical actin-dependent stabilization of
VE-cad, how Rap1 regulates VE-cad stabilization at the
cell–cell contacts has been investigated. Although the static
linkage of cadherin and actin is challenged by the Nelson58
and Weis groups,59 the importance of the link between
cadherin and actin cytoskeleton has not been contradicted.
The importance of the link between VE-cad and actin can
be claimed by comparing VE-cad with PECAM-1, which is
not linked to actin. Thus, my group used a fluorescence
recovery after photobleaching (FRAP) experiment using
VE-cad or PECAM-1 tagged with green fluorescence protein
(GFP) (VE-cad-GFP and PECAM-1-GFP, respectively) to
monitor the dynamics at the junctions. We found that for-
skolin induces cortical actin bundling parallel to the cell–
cell contacts. The mobile fraction of VE-cad was decreased
upon forskolin stimulation, while that of PECAM-1 was
unchanged, suggesting a significant link between actin and
cell adhesion molecules for the stabilization of adhesion
molecules at the cell–cell contacts when the formation of
cortical actin bundling is augmented (unpublished data).
The precise molecular mechanism of how Rap1 regulates
the stabilization of VE-cad at the cell–cell contact needs to
be clarified (Figure 5).
Ang1/Tie2-Mediated Integrity of Vascular ECs
Ang1 activates Tie2 receptor tyrosine kinase and counter-
acts the VEGF-VEGFR-2 signal to maintain vascular integ-
rity.28,29 Another angiopoietin, Ang2, is thought to compete
with Ang1 and induce angiogenesis.80 Ang1 strengthens the
PECAM-1- and VE-cad-mediated intercellular junctions
by inducing the dephosphorylation of PECAM-1 and VE-
cad and by inhibiting TNF-α-induced transmigration of
leukocytes.81 We and others have demonstrated that Ang1
localizes Tie2 to the intercellular junctions by bridging
Tie2.82,83 Trans-associated Tie2 preferentially activates
AKT and reduces permeability. VE-PTP is also recruited to
the cell–cell junctions when stimulated by Ang1, suggest-
ing a potential role in reducing the permeability mediated
by enhanced accumulation of VE-cad. Antibody against
VE-PTP can mimic depletion of VE-PTP and induces acti-
vation of Tie2. Thus, the association of VE-PTP with Tie2
appears to be very important for the regulation of endothelial
stabilization and proliferation.84 We further extended our
studies of the function of trans-associated Tie2 at the cell–
cell contacts. Analysis of gene expression by cDNA array
enabled identification of the responsible molecules that
promote vascular quiescence, 1 of them being Krüppel-like
factor 2 (KLF2).82 KLF2 is a zinc finger transcription factor
that belongs to the KLF family and is expressed in vascular

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Stabilization of Endothelial Cell–Cell Contact (2008 Sato Award)
ECs.85 The importance of KLF2 for the stability of blood
vessels is confirmed by deletion of KLF2.86 One can specu-
late that KLF2 is a vascular protective transcription factor
because it upregulates endothelial NO synthase (eNOS),
natriuretic peptides and downregulates VCAM-1, E-selectin,
VEGFR-2, and endothelin-1.
My group delineated the signaling downstream of acti-
vated Tie2 at the cell–cell contacts.87 Upregulation of KLF2
is dependent on MEF2, but not ERK5, which is regulated
by the phosphatidylinositol 3-kinase (PI3K)-AKT signal.
Attachment of monocytes to cultured ECs stimulated with
VEGF was impeded by Ang1. This inhibition was reversed
by the depletion of KLF2, indicating that the inhibitory effect
of Ang1 on inflammatory cell adhesion to ECs is regulated
by KLF2. Therefore, we conclude that Ang1-dependent sta-
bility of ECs depends on KLF2 (Figure 6).
S1P Regulates Integrity of ECs
S1P is thought to be released from activated platelets and
erythrocytes.88,89 Its receptors, S1P1-S1P5, are GPCR and
expressed in various tissues, including vascular ECs and
VSMCs.90 S1P1 is coupled to Gi and induces PI3K-depen-
dent eNOS and Rac activation, whereas S1P2 and S1P3 are
coupled to Gi, Gq, and G12/13.91 Activation of Gq and that
of G12/13 results in phosphorylation of MLC and subsequent
actin–myosin-dependent contraction via the phospholipase
C-β(PLCβ)-Ca2+/CaM kinase signal and RhoA–Rho kinase
signal, respectively.91
Vascular integrity controlled by S1P is dependent on
Gi–Rac–PAK signaling that induces re-arrangement of the
actin cytoskeleton.92 S1P-mediated enhancement of the
ECs barrier function is explained by the phosphorylation of
cortactin and its binding to MLC-K.93 ZO-1 is essential for
the S1P-regulated barrier integrity of ECs, as evidenced by
the fact that depletion of ZO-1 leads to loss of the S1P-
enhanced barrier function.94 The association of VE-cad with
β-ctn-focal adhesion kinase upon S1P stimulation regulates
the cortical actin rearrangement that stabilizes the cell–cell
junction.95 Plasma S1P-less mice in which sphingosine
kinases (I and II) are knocked out show vascular leakage,
suggesting the mechanism of S1P1-regulated vascular integ-
rity of ECs in vivo.96 Thus, maintenance of vascular EC
integrity is regulated by S1P without activation of platelets.
From which cells is S1P released? S1P is synthesized
from sphingosine localized in the plasma membrane by
sphingosine kinase I and II in the cytoplasm.97 Therefore,
S1P produced in the cells must be released from inside to
outside of the cells via transporters that activate S1P recep-
tors. There are 3 ABC transporters, ABCC1, ABCA1, and
ABCA7, which might function as the S1P transporter.98–100
It is still controversial whether these transporters function in
vivo to maintain vascular integrity. My group recently iden-
tified a novel S1P transporter, Spinster2 (Spns2), by screen-
ing zebrafish mutants that show cardia bifida.101 The Spns
family consists of Spns1, Spns2 and Spns3. The mutants
showing cardia bifida harbored a R153S mutation of Spns2,
indicating that it was the gene responsible. Stainier’s group
previously reported that deletion of S1P2 causes cardia bifida
in both zebrafish102 and another mutant fish. By analyzing
the latter, they also demonstrated that the gene responsible
for the phenotype was a mutation of Spns2.103 We assumed
that Spns2 might function as a S1P transporter because it
appears to span the membrane 12 times according to its
amino acid sequence. Therefore, we investigated the export
activity of S1P using cells expressing wild-type Spns2 and
Circulation Journal Vol.73, December 2009
2189
mutant Spns2 (R153S) and found that Spns2 functioned as
a transporter of S1P. Collectively, S1P released from vas-
cular ECs via Spns2 might activates S1P1 expressed on
vascular ECs to maintain vascular integrity (Figure 6).
Conclusion
Vascular EC integrity is important for protecting the vascu-
lature from thrombosis, atherosclerosis and abnormal angio-
genesis. Because the ideal vasculature is organized during
embryonic development, established blood vessels need to
be maintained to keep blood flowing smoothly around the
body. In this review, I have focused on the molecules that
are central to the stability of vascular ECs. Other vascular
stabilization factors besides Ang1 and S1P will be identified
to extend our knowledge of vascular integrity. Destabiliza-
tion of cell–cell contacts is required for preexisting vessels
to promote the angiogenesis that should be re-stabilized
once ischemia is improved. Therefore, it is necessary to
understand the stabilization and destabilization cycle in the
vasculature under both pathological and physiological con-
ditions. We, as cardiologists, are expected to develop new
therapeutic strategies to rescue and reverse the damaged
heart by regenerative medicine. We need to think about the
regeneration of healthy vasculature, as well as the myocar-
dium, to regenerate the heart. Thus, it is important for us to
instigate understanding of how we can control both regen-
eration and maintenance of preexisting tissues.
Acknowledgments
The research described here and done by Dr Mochizuki’s group has been
supported in part by grants from the Ministry of Education, Science,
Sports and Culture of Japan; the Ministry of Health, Labour, and Welfare
of Japan; the Program for the Promotion of Fundamental Studies in Health
Sciences of the National Institute of Biomedical Innovation; Astra-Zeneca
Research Grant; Research Grants in the Natural Sciences from the
Mitsubishi Foundation; and Takeda Science Foundation.
I am grateful to emeritus professors Hisakazu Yasuda and Kazuo
Nagashima at Hokkaido University School of Medicine for teaching me
the importance of basic medical science; Professors Michiyuki Matsuda
(Kyoto University) and Kenji Kangawa (Director general at National
cardiovascular Center Research Institute) for making me rapt in signaling
and imaging; and many collaborators and friends, including Professors
Hirofumi Sawa and Naomasa Makita, for their continuous encouragement.
Finally, I thank the members of my laboratory, who study basic cardiovas-
cular medicine with me.
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