The Sphingosine-1-Phosphate Receptor S1PR1 Restricts Sprouting Angiogenesis by Regulating the Interplay between VE-Cadherin and VEGFR2

Developmental Cell

Article

The Sphingosine-1-Phosphate Receptor S1PR1Restricts Sprouting Angiogenesis by Regulatingthe Interplay between VE-Cadherin and VEGFR2Konstantin Gaengel,1,8 Colin Niaudet,1,8 Kazuhiro Hagikura,1,9 Barbara Lavina,1,9 Lars Muhl,1,9 Jennifer J. Hofmann,1

Lwaki Ebarasi,1 Staffan Nystrom,2,3 Simin Rymo,1,4 Long Long Chen,1 Mei-Fong Pang,1 Yi Jin,1

Elisabeth Raschperger,1 Pernilla Roswall,1 Dorte Schulte,5 Rui Benedito,6 Jimmy Larsson,7 Mats Hellstrom,7

Jonas Fuxe,1 Per Uhlen,2 Ralf Adams,6 Lars Jakobsson,1 Arindam Majumdar,7 Dietmar Vestweber,5 Anne Uv,4

and Christer Betsholtz1,*1Department of Medical Biochemistry and Biophysics, Division of Vascular Biology2Department of Medical Biochemistry and Biophysics, Division of Molecular Neurobiology3Department of Oncology and Pathology, Cancer Center KarolinskaKarolinska Institutet, 17177 Stockholm, Sweden4Institute for Biomedicine, Gothenburg University, 41390 Gothenburg, Sweden5Department of Vascular Cell Biology6Department of Tissue MorphogenesisMax-Planck-Institute of Molecular Biomedicine and the University of Munster, D-48149 Munster, Germany7Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, 75185 Uppsala, Sweden8These authors contributed equally to this work9These authors contributed equally to this work*Correspondence: [email protected]

http://dx.doi.org/10.1016/j.devcel.2012.08.005

SUMMARY

Angiogenesis, the process by which new bloodvessels arise from preexisting ones, is critical forembryonic development and is an integral part ofmany disease processes. Recent studies haveprovided detailed information on how angiogenicsprouts initiate, elongate, and branch, but less isknown about how these processes cease. Here, weshow that S1PR1, a receptor for the blood-bornebioactive lipid sphingosine-1-phosphate (S1P), iscritical for inhibition of angiogenesis and acquisitionof vascular stability. Loss of S1PR1 leads toincreased endothelial cell sprouting and the forma-tion of ectopic vessel branches. Conversely, S1PR1signaling inhibits angiogenic sprouting and enhancescell-to-cell adhesion. This correlates with inhibitionof vascular endothelial growth factor-A (VEGF-A)-induced signaling and stabilization of vascular endo-thelial (VE)-cadherin localization at endothelial junc-tions. Our data suggest that S1PR1 signaling actsas a vascular-intrinsic stabilization mechanism, pro-tecting developing blood vessels against aberrantangiogenic responses.

INTRODUCTION

During development, the cardiovascular system faces the

complex challenge of remaining functional, while simultaneously

adapting to the growing body’s increasing need for oxygen and

Developmen

nutrients. Following de novo assembly from mesoderm-derived

endothelial precursors (vasculogenesis), a primitive vascular

network progressively develops hierarchical organization and

functional specialization through a process termed angiogen-

esis, which involves sprouting, splitting, growth, and remodeling

of vessels. Sprouting initiates by the formation of migratory

endothelial cells (tip cells), a process driven by vascular endothe-

lial growth factor (VEGF) (Gerhardt et al., 2003). A single tip cell

occupies the lead position of the sprout, whereas trailing endo-

thelial cells (stalk cells) shape the lumenized shaft. The segrega-

tion of the sprouting endothelial cells into tip cells and stalk

cells is regulated by Notch (reviewed in Eilken and Adams,

2010 and Phng andGerhardt, 2009). However, unlikemany other

Notch-dependent cell differentiation phenomena, which lock

cell differentiation to a specific fate, the tip and stalk cell

phenotypes appear dynamic and reversible, as endothelial cells

rapidly shuffle at the lead position (Jakobsson et al., 2010).

Besides tissue VEGF-A gradients, (Gerhardt et al., 2003;

Ruhrberg et al., 2002), the direction of sprout protrusion depends

on a number of other ligands and receptors that regulate tip

cell guidance mainly by repulsion, including members of the

Ephrin/EPH, Slit/ROBO, Netrin/UNC5B, and Semaphorin/Plexin

families (reviewed in Adams and Eichmann, 2010 andWeinstein,

2005).

As embryonic development proceeds, there is an increasing

need for vascular stabilization. Whereas angiogenic sprouting

and endothelial cell proliferation continue in the distal parts of

the growing vascular tree, branch patterns, identities, and hierar-

chies simultaneously need to be stabilized at proximal locations.

The term vascular stability, although commonly used in review

literature, is largely descriptive, as the molecular mechanisms

involved are poorly understood.

tal Cell 23, 587–599, September 11, 2012 ª2012 Elsevier Inc. 587

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Developmental Cell

S1P/S1PR1 Restricts Angiogenic Sprouting

Blood flow has been implicated as a vessel-stabilizing mech-

anism through the oxygenation of tissues, leading to downregu-

lation of hypoxia-sensitive pro-angiogenic factors, like VEGF-A.

Blood flow also delivers S1P, which occurs in micromolar

concentrations in plasma, whereas tissue concentrations of

S1P are usually in the nanomolar range (Hla et al., 2008). The

vast majority of circulating plasma S1P is produced from eryth-

rocytes and endothelial cells through the activity of sphingosine

kinases (SPHKs) (Pappu et al., 2007; Venkataraman et al., 2008).

S1P binds to and activates a family of five G protein–coupled

receptors in mammals, S1P receptor 1 to 5 (S1PR1-5), formerly

known as endothelial differentiation gene (EDG)-receptors (Hla

et al., 1999). Of these, S1PR1, S1PR2, and S1PR3 have been

reported to be expressed in endothelial cells (Kono et al., 2004).

S1P receptor signaling has been implicated atmultiple steps in

cardiovascular physiology, including regulation of vascular

permeability, mural cell recruitment, lymphocyte trafficking,

inflammation, coagulation, and cardiac function (reviewed in

Hla et al., 2008 and Rivera et al., 2008). Complete S1P depletion

through genetic ablation of bothSphk1 andSphk2 inmice results

in neurological and vascular defects (Mizugishi et al., 2005).

These embryos die between embryonic days (E) 11.5–13.5, dis-

playing severe hemorrhage. Their vascular defects are charac-

terized by dilated vessels and reduced vascular smooth muscle

cell (vSMC) coverage of the dorsal aorta. A similar phenotype

was observed when S1PR1, S1PR2, and S1PR3 were depleted

in combination. Single, double, or triple knockout combinations

of these receptors demonstrate that S1PR1 and S1PR3 function,

in part, redundantly during vascular development, but that

S1PR1 is the most important receptor (Kono et al., 2004). In

S1pr1 single knockout mice, embryonic lethality occurs slightly

later than in the triple knockouts (E12.5–14.5) (Liu et al., 2000).

The lethality of these mice has been attributed to incomplete

vascular maturation, due to deficient coverage of vascular mural

cells (vascular smooth muscle cells [vSMC] and pericytes).

However, early vasculogenesis and angiogenesis were reported

to proceed normally (Allende et al., 2003; Liu et al., 2000; Paik

et al., 2004). Other studies have implicated S1P in angiogenesis

through in vitro analysis, suggesting a pro-angiogenic role

(Bayless and Davis, 2003; Paik et al., 2001).

Our previous studies of pericyte-deficient platelet derived

growth factor (PDGF)-B and PDGF receptor beta (PDGFRb)

null mice demonstrate the critical importance of murals cells

for vascular development and embryonic survival. However,

this occurs at a significantly later step, and is associated with

milder vascular abnormalities compared to the S1pr1�/� mice

(Hellstrom et al., 1999; Leveen et al., 1994; Lindahl et al., 1997;

Soriano, 1994). This prompted us to further analyze the role of

S1P/S1PR1 signaling in vascular development. Here, we report

that this signaling pathway inhibits angiogenic sprouting and

promotes vascular stability in the endothelium in a cell autono-

mous manner. Based on genetic experiments in mouse and

zebrafish, as well as on the use of an S1PR1-specific agonist

and antagonist in vivo, ex vivo, and in vitro, we propose that

S1P provides essential protection of the developing vasculature

against unrestrained angiogenic sprouting and ectopic vessel

branch formation. These functions are mediated by endothelial

S1PR1, which in turn regulates the interplay between VE-

cadherin and VEGFR2.

588 Developmental Cell 23, 587–599, September 11, 2012 ª2012 Els

RESULTS

Loss of S1PR1 in Mice Leads to AngiogenicHypersprouting, whereas Mural Cell Recruitmentand Coverage Are NormalTo investigate whether the reported pericyte deficiency in

S1pr1�/� mice was a result of problems with initial recruitment

or subsequent survival/maintenance of the mural cell coat, we

analyzed the embryonic hindbrain, an organ well suited to the

study of pericyte recruitment in conjunction with embryonic

angiogenesis at midgestation (Abramsson et al., 2007; Gerhardt

et al., 2004). Much to our surprise, we observed that pericyte

recruitment and mural cell coverage of the hindbrain vasculature

was normal in S1pr1�/� embryos (Figure 1A; Figure S1A avail-

able online). However, we instead noted endothelial hyper-

sprouting from E11.5 onward, as reflected by increased

numbers of endothelial tip cells, increased protrusions of endo-

thelial filopodia, and increased overall vascular density. This

phenotype was seen in hindbrains (Figures 1B and S1B; data

not shown), as well as in other locations, including the neural

tube and the developing limbs (Figure S1C). We also studied

the effects of S1pr1 deletion on postnatal retinal angiogenesis.

In the retina, S1PR1 expression is restricted to the vascular

endothelium, as shown by S1pr1-lacZ reporter analysis (Fig-

ure 1Ci), but it differed in intensity, being strongest in arterial

branches (Figure 1Cvii–1Cix), followed by veins (Figure 1Cx)

and capillaries (Figure 1Cv). Cells at the sprouting front, including

tip cells, showed the lowest expression of S1pr1-lacZ (Fig-

ure 1Ci–1Civ). We induced S1pr1 gene deletion specifically in

endothelial cells using VE-cadherin (Cdh5) promoter-driven Cre

recombinase expression by administrating tamoxifen into

newborn Cdh5(PAC)-CreERT2 (Pitulescu et al., 2010); S1pr1fl/fl

(Allende et al., 2003); R26R-EYFP mice (Srinivas et al., 2001)

(hereafter referred to as iEC-S1pr1KO). Although tamoxifen-

induced recombination in the retinal endothelium was always

mosaic, as revealed by the R26R-YFP reporter, endothelial

hypersprouting and increased endothelial cell density was

clearly evident and was invariably associated with abundant

pericyte coverage (Figure 1D; see also Figure 4A for additional

illustration of hypersprouting). YFP+ cells, with increased likeli-

hood of also being S1pr1 null, occupied almost all tip cell posi-

tions in these retinas, in marked contrast to overall chimerism

(Figure 1E). This suggests that the effect of S1PR1 is cell

autonomous, and that loss of S1PR1 causes endothelial cells

to acquire a tip cell phenotype. In addition to the retinal micro-

vasculature, retained mural cell coverage was also observed in

retinal arteries and veins that had undergone Cre-mediated

recombination (Figure S1D).

In summary, these results demonstrate angiogenic hyper-

sprouting with retained pericyte coverage in S1pr1-deficient

mice. Our analysis also shows that the endothelial hypersprout-

ing phenotype results from loss of S1PR1 signaling in endothelial

cells.

Aortic Hyperbranching in S1pr1-Deficient Mice CausesProgressive Vascular DerangementThe most striking reported loss of mural cells in S1pr1�/� and

Sphk1/2 double knockouts occurred around the developing

aorta (Liu et al., 2000; Mizugishi et al., 2005; Paik et al., 2004).

evier Inc.

Figure 1. Genetic Ablation of S1pr1 in Mice Leads to Angiogenic Hypersprouting with Retained Mural Cell Coverage

(A and B) Vascular patterns in the E11.5mouse hindbrain. Ventricular views of the developing subventricular zone vascular plexus. Endothelial cells are stained by

isolectin B4 (IB4) and pericytes by NG2. Arrowheads in (A) point at pericytes, in (B) at ectopic filopodia.

(C) Analysis of S1pr1 expression in the developing (P5) retina shows expression in endothelial cells but not in vSMC. S1pr1-lacZ staining (blue/black intracellular

aggregates) combined with staining for endothelial cells and vSMC, as indicated. The top row of images (i–v) display a retinal whole mount (i, arrowhead pointing

at strong lacZ staining in a central large vessel, whereas the lacZ expression at the periphery (asterisk) is weaker) followed by high magnification images of the

sprouting front (ii–v). In the high magnification image (v), arrowheads point to lacZ aggregates in the retinal microvasculature. The bottom row of images (vi–x)

displays endothelial cell/vSMC staining of arteries and veins at central locations in the retina. Arrowheads in (ix) point at S1pr1-lacZ negative arterial vSMC.

(D) Vascular patterns in the postnatal retina of iEC-S1pr1KO mice. YFP expression indicates tamoxifen-induced recombination at the R26R-EYFP locus.

(E) Sprouting front of retinal vascular plexus in iEC-S1pr1KO. Note that YFP+ cells preferentially occupy the tip cell position in a background of mostly un-

recombined cells.

See also Figure S1.

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Developmental Cell 23, 587–599, September 11, 2012 ª2012 Elsevier Inc. 589

Figure 2. Ectopic Branching and Glomeru-

loid Vascular Malformations around the

Aorta in S1pr1 Knockouts

(A) Vascular phenotypes in and around the devel-

oping aorta in embryos at E11.5–13.5. Ectopic

connections between glomeruloid regions (aster-

isks) and the aorta are indicated by arrowheads.

Position of notochord (n) and aorta (ao) are indi-

cated in relevant sections.

(B) Phenotypic appearance of the aorta in

S1pr1�/� embryos at different levels along the

anterior-posterior axis. Approximate section plane

is illustrated (X0 or Y0).(C) Quantifications of aortic branch points per

cross section in three E12.5 wild-type and three

S1pr1�/� embryos.

(D) A similar but milder aortic phenotype is

observed in iEC-S1pr1KO embryos. Sections are

stained for IB4 (endothelium) NG2 (pericytes),

aSMA (vSMC), and Dapi (nuclei). All values are

mean ± SD.

See also Figure S2.

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We confirmed that the aortic vSMC coverage was abnormal in

S1pr1�/� embryos (Figures 2A and S2A), but this defect was

accompanied by a massive endothelial hyperplasia and

abnormal microvasculature around the aorta. This vasculature

formed glomeruloid lesions with numerous ectopic endothe-

lium-lined connections to the aortic lumen (Figures 2A–2C,

S2A, and S2D). At E11.5, the lesions were mainly concentrated

on the dorsal side of the aorta, whereas at E12.5 they often

occurred in lateral regions as well. By E13.5, the lesions

frequently surrounded the aortic circumference (Figure 2A).

Also, the major branches of the aorta, such as the femoral, tail,

and intercostal arteries were associated with glomeruloid lesions

590 Developmental Cell 23, 587–599, September 11, 2012 ª2012 Elsevier Inc.

(Figures S1C and S2A; data not shown).

The lesions contained abundant mural

cells and basement membrane proteins

(Figures 2A, S2A, and S2D). At E13.5,

but not earlier, we observed massive

induction of apoptosis in endothelial cells,

as well as in neighboring dorsal root

ganglia (Figure S2B; data not shown),

a phenotype that likely contributes to the

rapid demise and synchronous death

of the embryos between E13.5 and

E14. Using the S1pr1-lacZ reporter, we

confirmed that the aortic endothelium,

but not the surrounding vSMC or other

adjacent tissues, is a major site of S1pr1

expression in mouse embryos (Fig-

ure S2C). That the endothelial lesions

are likely endothelial cell autonomous

was supported by analysis of iEC-

S1pr1KO embryos obtained through

tamoxifen administration into pregnant

females. These embryos displayed an

aortic phenotype that was qualitatively

similar but milder than in S1pr1�/�

embryos (Figure 2D). The milder pheno-

type is likely explained by endothelial mosaicism for S1pr1 dele-

tion and/or by a delay in the elimination of S1PR1 protein

following genetic recombination of the S1pr1 locus.

To our knowledge, aortic hyperbranching has not been re-

ported in other vascular mutants, and we therefore considered

it specific, or even pathognomonic, for disrupted S1PR1 signal-

ing. To address the possibility that the observed aortic pheno-

type may nevertheless constitute a systemic and previously

overlooked consequence of vascular dysfunction associated

with embryonic lethality around E14, we studied Ramp2�/�

embryos, which die around E14.5 as a result of vascular abnor-

malities (Fritz-Six et al., 2008; Ichikawa-Shindo et al., 2008).

Figure 3. Effects of S1PR1-Specific Antago-

nist and Agonist In Vivo, Ex Vivo, and In Vitro

(A) Angiogenic hypersprouting in the newborn

mouse retina, as indicated by increased numbers

of tip cells and filopodia, following systemic

administration of the S1PR1 antagonist (R)-W146.

(B andC) Treatment of aortic ring explants with (R)-

W146 or SEW2871 has opposite effects on sprout

morphology and numbers of developing branches.

(B) VE-cadherin fluorescent staining of fixed aortic

rings. (C) Phase contrast microscopy and statis-

tical analysis of live aortic ring cultures at low

magnification (left), high magnification fields dis-

playing sprouts (middle, black arrowheads) and

detached cells (black arrow), and examples of how

branch numbers were counted (right; red dot

indicates branch point).

(D) MS-1 cell-covered beads allowed to sprout in

fibrin gels. Sprouts and scattered cells were

scored in phase contrast microscopy. The top

panel shows images of beads and cells, and indi-

vidual sprouts connected to the bead (red dots)

and scattered cell (blue dots). Right panel shows

the quantified results and statistics. Note that the

VEGF-induced cell detachment and scattering is

enhanced by the S1PR1 antagonist (R)-W146 and

inhibited by the agonists S1P and SEW2871. All

sprouting effects are efficiently inhibited by the

VEGFR2 tyrosine kinase inhibitor SU4516. Statis-

tically significant differences are indicated. All

values are mean ± SD. The p value is indicated

(* = p < 0.05); Student’s t test.

See also Figure S3.

Developmental Cell


Ramp2�/� mutants did not show signs of aortic hyperbranching

(Figure S2E). We also failed to observe aortic hyperbranching in

Dll4+/� embryos and embryos with induced endothelial-specific

deletion of the Notch-target RBP-J (tamoxifen-treated

Cdh5(PAC)-CreERT2; Rbp-jfl/fl) (Figure S2E), yet both of these

mutants displayed the expected angiogenic hypersprouting at

other locations (data not shown). These data also provide

evidence against the initial suspicion that S1PR1 signaling

impinges upon the Notch pathway, a conclusion further corrob-

orated by microarray (data not shown) and Q-PCR analysis of

microvascular fragments (Figure S2F), which failed to show

any consistent and significant changes in the expression of

Notch target genes in S1pr1�/� embryos. These analyses

instead revealed that the VE-cadherin target gene growth arrest

specific-1 (Gas1) (Spagnuolo et al., 2004), as well as the S1P

receptors S1pr1 and S1pr3, were significantly downregulated

Developmental Cell 23, 587–599, Se

in S1pr1�/� embryonic vasculature (Fig-

ure S2F), suggesting crosstalk with other

S1P receptors and with VE-cadherin.

Antagonists and Agonists ofS1PR1 Have Opposing Effectson Angiogenic Sprouting andEndothelial Cell-Cell AdhesionTo obtain insight into the morphogenetic

basis for the observed angiogenic

phenotypes in S1pr1 mutants, we tested

selective S1PR1 antagonists and agonists for their effects on

angiogenic sprouting in vivo, ex vivo, and in vitro. In agreement

with the gene knockout data, systemic administration of the

S1PR1-specific antagonist (R)-W146 (Sanna et al., 2006) to

newborn mice resulted in endothelial hypersprouting in the

postnatal retina (Figure 3A). Similar results were obtained when

(R)-W146 was added ex vivo to mouse aortic ring cultures.

Here, (R)-W146 led to increased density of endothelial sprouts,

whereas treatment with the S1PR1-specific agonist SEW2871

led to the formation of fewer, longer, and less branched sprouts

(Figures 3B and 3C).

We next studied in vitro assays for sprouting angiogenesis,

in which human umbilical vein endothelial cells (HUVECs) or

mouse MS-1 microvascular endothelial cells seeded on beads

were allowed to sprout in fibrin gels in the presence or absence

of exogenously added VEGF. In both the HUVEC and MS-1

ptember 11, 2012 ª2012 Elsevier Inc. 591

Figure 4. Induced Endothelial-Specific

Knockouts ofS1pr1 andCdh5 inMice Cause

Similar Angiogenic Hypersprouting in the

Postnatal Retina

Analysis of vasculature in whole-mounted retinas.

(A) P8 retinas from control, iEC-Cdh5KO and

iEC-S1pr1KO mice shows that endothelial-

specific VE-cadherin deletion, similar to S1pr1

deletion, leads to the formation of a dense vessel

network (white asterisk), delayed plexus extension

at the margins, and loss of hierarchical vessel

organization. Analysis at higher magnification

reveals angiogenic hypersprouting at the vascular

front in the iEC-Cdh5KO retina similar to the

iEC-S1pr1KO situation, as indicated by increased

numbers of tip cells and endothelial filopodia.

(B) Loss of VE-cadherin protein in iEC-Cdh5KO

retinas was verified by immunohistochemistry

against VE-cadherin. Note near complete loss of

VE-cadherin staining in the iEC-Cdh5KO retina

(yellow asterisk) and the occasional residual

expression of immuno-reactive VE-cadherin with

abnormal cellular localization (yellow arrowheads).

Collagen IV deposits were less confined to the

endothelial cells at the sprouting front of iEC-

Cdh5KO retinas, indicating increased cell migra-

tion. All values are mean ± SD. The p value is

indicated; Student’s t test.

Developmental Cell


assays, VEGF promoted the formation of sprouts led by tip

cells, an effect that was efficiently blocked by SEW2871

(Figures 3D and S3A). Conversely, (R)-W146 enhanced the

VEGF-dependent formation of sprouts and, moreover, caused

a large increase in the number of scattered cells (Figures 3D

and S3A). The sequential addition of SEW2871 and (R)-W146

to bead cultures recorded by time lapse microscopy demon-

strated that (R)-W146 strongly promoted endothelial cell scat-

tering, which was reversible following readdition of SEW2871,

leading to coalescence of the scattered endothelial cells into

vessel-like aggregates (Movie S1). The effects of (R)-W146

were neutralized by SU5416, a potent and selective inhibitor

of VEGFR2 (Fong et al., 1999), suggesting that VEGF signaling

is necessary for the angiogenic effects of S1PR1 inhibition

(Figure 3D).

In summary, experiments using S1PR1-specific pharmaco-

logical ligands show that S1PR1 inhibition promotes VEGF-


induced sprouting in vivo, ex vivo, and

in vitro, in agreement with the mouse

knockout data. In contrast, S1PR1 activa-

tion promotes endothelial cells aggrega-

tion and assembly into cords, and inhibits

VEGF-induced cellular responses.

VE-Cadherin KnockoutIn Vivo Leads to AngiogenicHypersprouting and ReducedEndothelial Cell-Cell AdhesionThe endothelial cell scattering following

S1PR1-inhibition in combination with

VEGF stimulation raised the possibility

that S1PR1 regulates angiogenic sprouting by effecting endo-

thelial cell-cell adhesion. In fact, VE-cadherin, a major endothe-

lial adherens junction protein, has previously been implicated as

a target for S1P signaling in cultured endothelial cells (Lee et al.,

1999). A role of VE-cadherin in mediating S1PR1-induced

inhibition of angiogenic sprouting would infer that ablation, or

knockdown, of VE-cadherin in vivo should phenocopy, partially

or fully, the pro-angiogenic effects of ablation or knockdown

of S1PR1. We tested this by inducing VE-cadherin deletion in

endothelial cells through administration of tamoxifen into

newborn Cdh5(PAC)-CreERT2; Cdh5fl/fl mice (hereafter referred

to as iEC-Cdh5KO). To facilitate the analysis of angiogenic

sprouting and comparison with the iEC-S1pr1KO mice, we

focused our analysis to the postnatal retina. As shown in Figures

4A and 4B, iEC-Cdh5KO led to prominent retinal angiogenic

hypersprouting, as reflected by increased numbers of tip cells

and filopodia and an overall increase in the vascular density.

Figure 5. S1PR1 Signaling Regulates VE-

Cadherin Localization at Endothelial Cell

Junctions

(A) Mislocalization of VE-cadherin at endothelial

junctions in developing P7 iEC-S1pr1KO retinas.

Whereas VE-cadherin staining is distinctly junc-

tional in controls, the staining is diffuse (yellow

asterisks), irregular and often decreased in iEC-

S1pr1 KO (yellow arrows; compare to control

vessels indicated by yellow arrowheads).

(B) siRNA-mediated knockdown of S1PR1 in MS-1

cells leads to loss of VE-cadherin and VEGFR2

from junctions, an effect that is strengthened by

VEGF. A scrambled siRNA (Scr) lacked effect.

(C) Effects of S1PR1 ligands and VEGF on VE-

cadherin and VEGFR2 junctional localization in

MS-1 cells. Note the dose-dependent down-

regulation of junctional VE-cadherin induced by

VEGF, and its efficient reversal by S1PR1 agonists,

whereas the antagonist (R)-W146 has the opposite

effect and potentiates VE-cadherin and VEGFR2

loss from junctions and intracellular pools.

(D) Time course of VEGF-induced VE-cadherin

downregulation in MS-1 cells. Note the effect of

extracellular VEGF trapping by sFlt1, indicating

that cultured MS-1 cells produce autocrine VEGF.

The endocytosis inhibitor Dynasore (Dyna) has

a similar effect as S1P, leading to an accumulation

of VE-cadherin at junctions.

(E) In MS-1 cells, the protection of VE-cadherin

from extracellular trypsin digestion, i.e., its inter-

nalization, is increased by VEGF, an effect effi-

ciently blocked by S1P.

See also Figure S4.

Developmental Cell


These phenotypes were qualitatively similar in iEC-Cdh5KO

and iEC-S1pr1KO mice, but strongest in the iEC-Cdh5KO mice

(Figure 4A). Loss of VE-cadherin protein in iEC-Cdh5KO

retinas was confirmed by immunohistochemistry (Figure 4B).

We also observed detaching tip cells that had poor cellular

connections to the vascular plexus at the angiogenic front in

iEC-Cdh5KO retinas (Figure 4B), and, to a lesser extent, in iEC-

S1pr1KO retinas (data not shown), but never in controls. In

iEC-Cdh5KO retinas, the endothelial cells, including detaching

tip cells, showed signs of increased motility in mutants, as indi-

cated by widespread collagen IV staining (Figure 4B), which

provides a record of endothelial cell movements in the tissue

(Inai et al., 2004).

Developmental Cell 23, 587–599, Se

S1PR1 Signaling RegulatesVE-Cadherin Localization atEndothelial Cell JunctionsIf the angiogenic hypersprouting ob-

served in the S1PR1-deficient situations

was mediated by reduced junctional VE-

cadherin, one would expect VE-cadherin

mislocalization to occur as a result of

S1PR1 inhibition. Indeed, we observed

abnormal VE-cadherin localization in the

retina of iEC-S1pr1KO mice (Figure 5A).

In controls, VE-cadherin decorated endo-

thelial junctions in regular patterns, yet

the S1pr1KO retinas displayed irregular VE-cadherin staining,

which was heterogeneous in intensity and less distinctly junc-

tional. The observed effects on VE-cadherin mislocalization

became evident from P7, whereas they were less clear at earlier

stages (data not shown), likely related to slow turnover of the

VE-cadherin protein. Notably, whereas tip cells in control retinas

were invariably VE-cadherin positive, many tip cells in P7

S1pr1KO retinas displayed low, irregular, or even undetectable

VE-cadherin staining (Figure 5A). The cell-autonomous nature

of this phenotype was evident in low-degree chimeras, in which

recombined single cells or cell clones were associated with

abnormal VE-cadherin staining in an otherwise normal vascular

background (Figure S4A).

ptember 11, 2012 ª2012 Elsevier Inc. 593

Figure 6. S1PR1 and VE-Cadherin Cooperate to Inhibit Angiogenic Sprouting and Promote Vascular Stabilization

(A) Analysis of zebrafish morphants. s1pr1 and cdh5 morphants display pericardial and hindbrain edema (black arrowheads). High-resolution analysis of the

developing hindbrain is focused on developing central arteries (CtAs; white). The lateral primordial hindbrain channels (PHBC) and midline basilar artery (BA) are

shown for reference (green in overview panel). Normally, developing CtAs display well-demarcated tip cells and associated filopodia (yellow arrows), whereas

s1pr1 or cdh5 knockdown leads to hypersprouting (yellow asterisks), including increased numbers of tip cells and filopodia. Middle and bottom panels show

hindbrain vasculature in sphk1, silent heart (tnnt2), and s1pr1 and cdh5 morphants, as indicated. Note that single low dose s1pr1 and cdh5 morpholinos lack

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The in vivo effects of S1PR1 depletion on VE-cadherin levels

were reproduced in vitro. SiRNA-mediated knockdown of

S1pr1 resulted in loss of VE-cadherin from junctions (Figures

5B and S4B). Conversely, and in agreement with previous results

(Lee et al., 1999), S1P stimulation led to increased junctional

VE-cadherin staining (Figure 5C). VEGF stimulation, on the

contrary, triggered a rapid decrease in junctional VE-cadherin,

recapitulating previously reported observations (Gavard and

Gutkind, 2006) (Figures 5C, 5D, and S4C). Strikingly, we found

that VEGF-mediated reduction of junctional VE-cadherin was

completely blocked by S1P or SEW2871 (Figures 5C and 7B),

suggesting a functional antagonism of S1P and VEGF signaling

on the level of VE-cadherin at cell junctions.

The S1P-induced increase in junctional VE-cadherin was also

observed when VEGFR2 signaling was inhibited either in the

presence of sFlt1 (Figure S4D) or through SU5416 (Figure S4E),

suggesting that S1P has direct effects on VE-cadherin localiza-

tion, independent of the inhibition of VEGF-induced responses,

including the ones elicited by endogenous autocrine VEGF. As

expected, the S1PR1 antagonist (R)-W146 had the opposite

effect compared to S1P or SEW2871, potentiating the VEGF-

induced internalization of VE-cadherin (Figure 5C). In agreement

with the immunofluorescence data, VEGF stimulation caused an

increase in the trypsin-insensitive (i.e., internalized) pool of VE-

cadherin, an effect efficiently blocked by S1P (Figure 5E).

Together, these data show that S1PR1 signaling positively and

directly regulates the level of VE-cadherin at endothelial junc-

tions, an effect that overrides the negative effect of VEGF on

junctional VE-cadherin concentrations.

S1PR1 and VE-Cadherin Cooperate to InhibitAngiogenic Sprouting and to Promote VascularStabilizationTo extend the comparison of phenotypic consequences of

genetic lossofVE-cadherinand lossofS1PR1 toadifferent in vivo

model system, we studied the effects of morpholino-mediated

cdh5 and s1pr1 gene knockdown in zebrafish embryos. Here,

s1pr1 knockdown produced a set of phenotypes reminiscent

of those previously reported for cdh5 morphants (Montero-

Balaguer et al., 2009). Specifically, both knockdown of cdh5

and s1pr1 resulted in reduced blood flow, pericardial, and brain

edema (Figures 6A and S5) and significant vascular hyper-

sprouting in the hindbrain (Figure 6A; Movie S2, C and D). Similar

phenotypes were observed following knockdown of the sphin-

gosine kinase 1 gene (sphk1), partially abrogating endogenous

S1Pproduction, aswell as in silent heart (tnnt2)morphants,which

lack blood flow (Figures 6A and S5; Movie S2, E and F).

Whereas the observed phenotypic similarities between s1pr1

and cdh5 morphants imply that they regulate a common bio-

logical function, they do not indicate if they cooperate molecu-

larly. A way to test if two components operate in a common

pathway is to assess possible synergy through combined inhibi-

tion of the two at levels which, by themselves, have no or minor

effects, but that their combination produced a hypersprouting response compar

quantitative data from different morphants are shown to the right.

(B) Experiments with MS-1 cells sprouting from beads. The presence or abse

pharmacological agents in different concentrations as indicated. All values are m

See also Figure S5.

Developmen

effects (Gore et al., 2008; Pham et al., 2007). Accordingly, we

titrated morpholino concentrations for s1pr1 and cdh5 to

threshold levels where they individually had only minor or no

effects, and than tested them in combination. We found that

combined injection of low dose cdh5 and s1pr1 morpholinos

reproduced the effects of single high dose morpholinos (Figures

6A and S5; Movie S2, G), suggesting that S1PR1 and VE-

cadherin could cooperate molecularly.

We obtained further evidence for a cooperation of S1PR1 and

VE-cadherin through in vitro studies, where MS-1 cells were

allowed to sprout in fibrin gels in the presence or absence of

the VE-cadherin blocking antibody BV13 (Liao et al., 2000).

In this assay,weconfirmed that BV13 treatment enhancedangio-

genic sprouting (Figure 6B), similar to the S1PR1 blocking agent

(R)-W146 (Figure 3D). We next asked if the effects of altered

S1PR1 signaling on VEGF-driven angiogenic sprouting depend

on the VE-cadherin levels. Indeed, we found that the cell scat-

tering effects, caused by S1PR1 inhibition through (R)-W146,

were enhanced in the presence of the VE-cadherin blocking anti-

body. Conversely, the inhibition of sprouting and cell scattering

caused by the S1PR1-specific agonist SEW2871 was counter-

acted by BV13 (Figure 6B). These results demonstrate that the

effects of S1PR1 signaling on VEGF-induced sprouting are

dependent on the level of functional VE-cadherin, suggesting

that S1PR1 and VE-cadherin cooperate to limit sprouting

angiogenesis.

S1PR1 Regulates VEGF-Induced VEGFR2 Signalingand Subcellular LocalizationOur data suggest that VE-cadherin and junctional stability are

critical downstream components in the negative regulation of

angiogenic sprouting exerted by S1PR1. We also show that

VEGF is a driving force for the angiogenic hypersprouting elicited

by S1PR1 inhibition, and that agonists and antagonists of S1PR1

exert strongandopposing effects onVEGF-mediated angiogenic

responses in vitro. Although, in principle, these responses could

be solely caused by changes in endothelial junctional strength

that permit or restrict the release of tip cells for sprouting, they

may also involve altered VEGF receptor signaling. To address

this issue, we studied the effects of pharmacological modulators

of S1PR1 signaling on VEGFR2 signaling in vitro and in vivo.

For the in vitro signaling experiments we used HUVECs pre-

treated with soluble VEGFR1 (sFlt1), which binds to and seques-

ters VEGF, thereby blocking endogenous autocrine/paracrine

VEGF-induced responses. This pretreatment promotes VEGFR2

accumulation at the cell surface and renders the cells sensitive to

synchronous VEGFR2 stimulation by exogenously added VEGF.

Under these conditions, VEGF promoted rapid phosphorylation

of VEGFR2, ERK1/2, and AKT, effects that were efficiently

blocked by S1P (Figure 7A). VEGF further induced relocalization

(Figures 7B and 5C) and degradation (Figure 7A) of VEGFR2,

effects that were also blocked by S1P. These data show that

S1PR1 signaling influences VEGFR2 signaling in vitro.

able to the effects of high dose single s1pr1 or cdh5 morpholinos. Normalized

nce of VE-cadherin blocking antibodies (BV13) was combined with S1PR1

ean ± SD. The p value is indicated; Student’s t test.


Figure 7. S1PR1 Regulates VEGF-Induced

VEGFR2 Signaling and Internalization

(A) VEGF-induced VEGFR2, ERK1/2, and AKT

phosphorylation, and VEGF-induced VEGFR2

degradation in HUVEC cells, are all inhibited

by S1P.

(B) VEGF-induced VEGFR2 internalization is

strongly counteracted by S1P and the S1PR1-

specific agonist SEW2871, but not by the antag-

onist (R)-W146. These effects on VEGFR2 inter-

nalization parallel the effects on junctional

VE-cadherin.

(C) In vivo administration of VEGF and/or S1PR1

pharmacological agents, as indicated, followed

by analysis of VEGFR2, ERK1/2, and AKT phos-

phorylation in lung homogenates. VEGF-induced

VEGFR2 and ERK phosphorylation are both

counteracted by SEW2871, and enhanced by (R)

W-146. Endogenous AKT levels are high but sup-

pressed by SEW2871. IP, immunoprecipitation;

WB, western blotting.

Developmental Cell


To study the effect of S1PR1 activity on VEGFR2 signaling

in vivo, we performed intracardiac administration of VEGF to

animals pretreated systemically with SEW2871 or (R)-W146

and subsequently assessed VEGFR2, ERK1/2, and AKT phos-

phorylation in lung homogenates. Under these conditions,

SEW2871 pretreatment led to a decrease in the VEGF-induced

VEGFR2 phosphorylation, whereas it was increased following

(R)-W146 treatment (Figure 7C). This result is in accordance

with the in vitro signaling and in vitro/vivo angiogenic effects of

the two drugs. (R)-W146 treatment also increased the VEGF-

induced phosphorylation of ERK1/2, whereas the opposite effect

was observed after treatment with SEW2871, in agreement with

the in vitro situation (Figure 7C). AKT phosphorylation in vivo

contrasted to the in vitro situation in that it was endogenously

high and not further increased by VEGF and/or (R)-W146.

However, it was significantly inhibited by SEW2871, in agree-

ment with the in vitro data (Figure 7C).

Combined, these data show that S1PR1-dependent inhibition

of angiogenic sprouting is connected to inhibition of VEGFR2

signaling. These effects of S1P are likely mediated by S1PR1

because the S1PR1-specific agonist SEW2871 mimicked the

S1P-induced effects on VEGFR2, whereas the S1PR1 specific

antagonist (R)-W146 had the opposite effect (Figure 7B). An

additional control for specificity in this regard was provided by

siRNA-mediated S1PR1 knockdown, which enhanced VEGF-

induced VEGFR2 internalization and degradation, and abro-


gated the S1P-induced stabilization of

VE-cadherin at cell junctions (Figure 5B).

DISCUSSION

A Revised Concept for VascularStabilizationThe recruitment of mural cells and the

deposition of basal membranes are

considered to be crucial steps to

achieve vascular stabilization (reviewed

in Potente et al., 2011). Indeed, it was

previously proposed that S1PR1 induces vessel stabilization

through mural cell recruitment, particularly around the aorta

(Liu et al., 2000). We confirmed the previously reported aortic

vSMC defect in S1pr1�/� embryos, but our observations

suggest that this phenotype occurs secondary to ectopic endo-

thelial hyperplasia and branching at this site. These ectopic

aortic branches connect to a vast glomeruloid network of capil-

laries that penetrates the aortic vSMC coat initially (around

E11.5) at the dorsal side of the aorta and subsequently spreads

ventrally to encircle the vessel before embryonic death at

E13.5–14. Although we noticed regional lack of the vSMC

marker alpha smooth muscle actin (aSMA) in the perforated

aortic wall, in agreement with previous work (Liu et al., 2000),

we found an abundance of NG2 and PDGFRb positive cells in

close association with the hyperplastic endothelial cells, sug-

gesting that S1pr1�/� endothelium is capable of recruiting asso-

ciated mural cells also around the aorta. We also found that

vascular extracellular matrix components were abundantly

deposited in S1pr1�/� embryos. Our present data thus argue

against defects in mural cell recruitment or vascular matrix

production as mechanism for vascular instability and ectopic

angiogenesis in the absence of S1PR1. The occurrence of

mural cells and matrix deposits around actively sprouting and

remodeling vessels is not unprecedented; rather, it has been

extensively documented both in retinal and tumor angiogenesis

(reviewed in Armulik et al., 2011).

Developmental Cell


Instead, our data suggest that S1P signaling via S1PR1

induces vascular stability directly in endothelial cells. Loss of

S1PR1 leads to exaggerated and ectopic endothelial sprouting

incompatible with embryonic survival. In contrast to other factors

that control angiogenic sprouting (e.g., VEGF/VEGFR2, DLL4/

NOTCH), the onset of the S1PR1 phenotype occurs at a relatively

late developmental stage, well after establishment of the major

vascular networks. Thus, S1PR1 appears to represent a central

component of a biological program that suppresses the angio-

genic responsiveness in vessels at a developmental stage

when vascular stabilization is essential, such as after the onset

of flow and intraluminal pressure. We postulate that blood flow

delivers S1P, which subsequently activates S1PR1 on endothe-

lial cells to restrict their angiogenic responsiveness and to stabi-

lize already established and newly perfused vessels. At the

angiogenic front, this blockade may be inefficient due to

restricted blood flow and low S1pr1 expression.

Mechanism of S1PR1-Induced Vascular Stabilization—Balancing the Opposing Functions of VE-Cadherinand VEGFR2Key insights into themechanism(s) leading to the hypersprouting

phenotype in the S1PR1-inhibited situation were obtained by

studying underlying morphogenetic events. Observations of

endothelial cell sprouting in vitro suggest that S1PR1 signaling

inhibits VEGF-induced sprouting and branching and promotes

the formation of fewer, but consolidated, sprouts. Conversely,

the combination of VEGF stimulation and S1PR1 antagonism

led to an almost complete dissociation of sprouts and extensive

endothelial cell scattering. Time-lapse recordings of sequential

inhibition and activation of S1PR1 further showed that the

scattering phenotype was reversible. Together, these results

demonstrate that S1PR1 activation counteracts VEGF function

and positively regulates endothelial cell adhesion.

A central mediator of endothelial cell adhesion is VE-cadherin,

which becomes strongly increased at endothelial junctions in

response to S1P (Lee et al., 1999). VEGF, on the other hand,

causes junctional VE-cadherin destabilization and triggers its

subsequent internalization (Gavard and Gutkind, 2006). When

costimulating cells with VEGF and S1P, we observed that VE-

cadherin remained stabilized at endothelial junctions and insen-

sitive to the VEGF-induced internalization. This result establishes

a functional antagonism between S1PR1 and VEGFR2 at the

levels of VE-cadherin and junctional stability. Partial inhibition

of VE-cadherin through blocking antibodies showed that the

amount of functional VE-cadherin is critical for S1PR1 to exert

its antiangiogenic effects, demonstrating that VE-cadherin is

a critical downstream mediator of S1PR1 function.

Further evidence for VE-cadherin operating downstream of

S1PR1 comes from the observation that the junctional localiza-

tion of VE-cadherin is disturbed in cultured cells and the mouse

retina as a result of S1PR1 ablation. Furthermore, both in mouse

and zebrafish, S1PR1 ablation causes angiogenic hypersprout-

ing. Our zebrafish data are in agreement with recently published

work, suggesting an inhibitory role for VE-cadherin during angio-

genic sprouting in zebrafish (Montero-Balaguer et al., 2009) and

in vitro (Abraham et al., 2009). In addition, our data show that low

dose morpholinos against S1PR1 and VE-cadherin, which by

themselves had minor effects, resulted in strong phenotypes

Developmen

when combined, suggesting that S1PR1 and VE-cadherin oper-

ate in a common pathway.

We also propose a second layer of regulation through which

S1PR1 activity inhibits VEGFR2 signaling. Following S1P stimu-

lation, we observed a decrease in VEGF-induced phosphoryla-

tion of VEGFR2, ERK1/2, and AKT, suggesting that VEGFR2

signaling events are inhibited by S1P. VE-cadherin has also

been reported to antagonize VEGF signaling through recruitment

of phosphatases to the VEGFR2 signaling complex, as well as

through inhibition of VEGFR2 internalization (Lampugnani et al.,

2006; Zanetti et al., 2002). It remains to be clarified if the inhibi-

tory effect of S1P on VEGFR2 signaling is direct, or mediated

through VE-cadherin.

In summary, although further work is required to sort out the

causal relationships between trafficking and signaling of

VEGFR2 downstream of S1PR1 and its relationship to VE-

cadherin stabilization at junctions, our data establish two critical

means by which S1PR1 signaling suppresses endothelial hyper-

sprouting—through stabilization of junctional VE-cadherin and

through inhibition of VEGFR2 phosphorylation and downstream

signaling.

Among the many vascular beds affected by S1pr1 gene dele-

tion, the hyperbranched aorta provides a good illustration of the

critical relevance of S1PR1-dependent stabilization mecha-

nisms in vascular development. The aorta normally displays

a highly stereotyped branching pattern, and deviations would

be expected to have deleterious effects on inner organ anatomy

and function. Whereas angiogenic hypersprouting has been

observed in other genetic mutants, a similarly hyperbranched

aorta has to our knowledge not been described before. Our

observation that ectopic branches initially extend from the dorsal

part of the aortic circumference and in close spatial correlation

with intercostal arteries is noteworthy, given that VEGF

produced by the somites is thought to drive formation of interso-

mitic (prospective intercostal) vessels (Coultas et al., 2005). S1P

distributed via the blood stream may hence work in order to

hinder illegitimate vascular sprouting and branching in major

vessels in response to VEGF.

EXPERIMENTAL PROCEDURES

Detailed protocols and description of reagents used are provided in Supple-

mental Experimental Procedures. The following information is intended for

brief orientation. Animal housing, as well as the experiments performed,

were in accordance with Swedish legislation and were approved by the local

animal ethics committees prior to experimentation.

Mouse Experiments

S1pr1 null and flox alleles and the Cdh5(PAC)-CreERT2 and R26R-EYFP trans-

genic lines have been published previously, whereas the Cdh5 flox allele,

generated by the Dietmar Vestweber Laboratory, is presently unpublished.

For induced gene knockout in embryos and pups, tamoxifen was administered

to the mother and transferred to the offspring via the placenta or the milk. For

pharmacological experiments (R)-W146, SEW2871, VEGF, or vehicle were

administered intraperitoneally (IP) to newborn pups. Histological examinations

of whole-mounted tissues and tissue sections were done using standard tech-

niques essentially as described previously (Gerhardt et al., 2003; Ruhrberg

et al., 2002), using commercially available reagents. For each analysis,

a minimum of three mutants and control littermates were compared. VEGF

signaling in vivo was assessed in lung homogenates after exposure to

SEW2871 or (R)-W146 administered IP, followed by VEGF exposure through

intracardiac injections under full anesthesia.


Developmental Cell


Zebrafish Experiments

The Tg(fli:eGFP)y1 transgenic line was used. One-to-two cell stage embryos

were injected with morpholino oligonucleotides at different concentrations,

raised at 28�C using standard methods, manually dechorionated at 1 dpf

and incubated at 28�C before analysis. Prior to scoring or live microscopy,

the morphant embryos were anesthetized. For live imaging of zebrafish hind-

brain development, anesthetized embryos (52–58 hpf) were embedded in low

melting point agarose and analyzed in an upright 2-photon laser scanning

microscope equipped with a 403/1.0 water dipping lens and a Ti:Sapphire

tunable Chameleon Ultra-II infrared laser. For statistical analysis of endothelial

sprouting, embryos were fixed and the hindbrain region was dissected to re-

move surrounding tissues. Filopodia and tip cell numbers were counted in

hindbrains of at least six fish for each group.

Ex Vivo Experiments

Rings (1–2 mm) of the mouse thoracic aorta were embedded between two

disks of collagen type I and cultivated in MCDB131 medium and 10% fetal

calf serum (FCS). Dose-response experiments identified 25 mg/ml for (R)-

W146 and 10 mg/ml for SEW2871 as optimal concentrations for further anal-

ysis, which included the scoring of angiogenic responses and immunohisto-

chemical analysis of vascular cell markers. A minimum of three aortic ring

cultures were analyzed for each condition and experiments were repeated

four times. For each culture, three sampled areas, each of 0.75 mm 3

0.45 mm, at the sprouting front were quantified.

Cell Culture Experiments

Human umbilical vein endothelial cells (HUVECs; passage 5-10) and mouse

pancreatic islet-derived microvascular endothelial cells (MS-1; passage

8-15) were routinely cultured in gelatin-coated tissue culture flasks in EGM-

MV or EGM-II medium, respectively. Normal human skin fibroblasts

(NHSF; passage 2-10) were cultured in DMEM with 5% FCS. For fibrin bead

assays HUVECs or MS-1 cells were mixed with dextran-coated Cytodex 3

microbeads in heparin-containing medium. Cell-coated beads were cultured

over night, re-suspended in fibrinogen, aprotinin and thrombin and allowed

to clot in 24-well tissue culture plates. A feeder layer of NHDFs was seeded

on top of the fibrin gel. Medium was changed regularly with new addition of

drugs (SEW2871, (R)-W146, SU5416), VEGF and/or VE-cadherin blocking

antibody. Bead assays were monitored for 6 days and scored for filopodia,

angiogenic sprouts and dissociated endothelial cells. MS-1 cells stably ex-

pressing a GFP-like protein (copGFP) or mCherry under the CMV promoter

were generated through lentiviral infections and used for time-lapse experi-

ments. For immunofluorescence, MS-1 and HUVEC were growth factor

starved prior to the experiments, then treated with VE-cadherin blocking anti-

body, (R)-W146, S1P, SEW2871, or dynasore hydrochlorate, and finally with

hVEGF165, sFlt1, or SU5416. Cells were stained overnight using VEGFR2

and VE-cadherin antibodies.

Molecular Biology

Real-time quantitative PCR (RTQ-PCR) was performed on RNA from E13.5

S1pr1�/� or control embryo microvascular fragments using TaqMan or

Roto-Gene Q commercial reagents and standard protocols. The comparative

CTmethod was used to calculate fold differences. For VE-cadherin and S1PR1

silencing by siRNA, HUVECs, or MS-1 cells were incubated with commercial

reagents according to published protocols. Cells were subsequently stimu-

lated with VEGFR2 and S1PR1 agonists (hVEGF165, SEW2871, S1P) and pro-

cessed for immunohistochemistry.

Biochemistry

As a general approach, growth factor-starved HUVECs and MS-1 cells were

preincubated with S1P or vehicle and processed further according to experi-

ment. For trypsin digestion assays, MS-1 cells were further incubated with

VEGF for 30 min, exposed to trypsin-EDTA, transferred to a protease inhibitor

cocktail and frozen in liquid nitrogen. For VEGF pathway activation, HUVECs

were stimulated with VEGF, rinsed, and frozen. For VEGFR2 inhibition,

HUVECs and MS-1 cells were exposed to SU5416 before stimulation with

VEGF and freezing. For western blot analysis, proteins from frozen cells or

organs were extracted and 10 mg of total proteins separated by SDS-PAGE,

transferred to PVDF membranes and blotted against relevant antibody. For

598 Developmental Cell 23, 587–599, September 11, 2012 ª2012 Els

immunoprecipitation, tissue debris was removed from cell or tissue lysates

by centrifugation and protein concentration determined. Lysates were further

precleared by protein-G-Sepharose, and then exposed to the relevant

antibody precoupled to protein-G-Sepharose beads. Immunocomplexes

were washed and separated by SDS-PAGE, transferred to nitrocellulose

membranes and blotted against relevant antibody. Signals were detected

using horseradish peroxidase-coupled secondary antibodies and commercial

reagents. For multiple probing, membranes were stripped with stripping buffer

and re-probed.

Statistical Analysis

Statistical significancewas determined by using unpaired two-tailed Student’s

t test. Differences were considered significant with a p value <0.05. Quantified

data are presented as mean ± SD.

SUPPLEMENTAL INFORMATION

Supplemental Information includes, five figures, Supplemental Experimental

Procedures, and two movies and can be found with this article online at

http://dx.doi.org/10.1016/j.devcel.2012.08.005.

ACKNOWLEDGMENTS

We thank Richard Proia (NIDDK, NIH) for S1pr1 knockout and loxP mice,

Elisabetta Dejana (IFOM, Milan) and Lena Claesson-Welsh (IGP, Uppsala) for

advice on protocols, Holger Gerhardt (CRUK, London) and Alexandra

Abramson (Gothenburg University) for initial contributions to the project, and

Sara Kamph, Helene Leksell, Malin Pettersson, Cecilia Olsson, and the CLICK

and zebrafish core facilities at MBB, Karolinska for technical assistance.

This study was supported by grants from the Swedish Cancer Society, the

Soderberg, Wallenberg, Wenner-Gren, and Lundberg Foundations, the

Leducq Foundation, The Swedish Research Council, Karolinska Institutet’s

Strategic Cardiovascular Programme, Stockholm County Council, and the

Max-Planck Society.

Received: November 28, 2011

Revised: May 25, 2012

Accepted: August 8, 2012

Published online: September 10, 2012

REFERENCES

Abraham, S., Yeo, M., Montero-Balaguer, M., Paterson, H., Dejana, E.,

Marshall, C.J., and Mavria, G. (2009). VE-Cadherin-mediated cell-cell interac-

tion suppresses sprouting via signaling to MLC2 phosphorylation. Curr. Biol.

19, 668–674.

Abramsson, A., Kurup, S., Busse, M., Yamada, S., Lindblom, P., Schallmeiner,

E., Stenzel, D., Sauvaget, D., Ledin, J., Ringvall, M., et al. (2007). Defective

N-sulfation of heparan sulfate proteoglycans limits PDGF-BB binding and peri-

cyte recruitment in vascular development. Genes Dev. 21, 316–331.

Adams, R.H., and Eichmann, A. (2010). Axon guidance molecules in vascular

patterning. Cold Spring Harb. Perspect. Biol. 2, a001875.

Allende, M.L., Yamashita, T., and Proia, R.L. (2003). G-protein-coupled

receptor S1P1 acts within endothelial cells to regulate vascular maturation.

Blood 102, 3665–3667.

Armulik, A., Genove, G., and Betsholtz, C. (2011). Pericytes: developmental,

physiological, and pathological perspectives, problems, and promises. Dev.

Cell 21, 193–215.

Bayless, K.J., and Davis, G.E. (2003). Sphingosine-1-phosphate markedly

induces matrix metalloproteinase and integrin-dependent human endothelial

cell invasion and lumen formation in three-dimensional collagen and fibrin

matrices. Biochem. Biophys. Res. Commun. 312, 903–913.

Coultas, L., Chawengsaksophak, K., and Rossant, J. (2005). Endothelial cells

and VEGF in vascular development. Nature 438, 937–945.

Eilken, H.M., and Adams, R.H. (2010). Dynamics of endothelial cell behavior in

sprouting angiogenesis. Curr. Opin. Cell Biol. 22, 617–625.

evier Inc.


Developmental Cell


Fong, T.A.T., Shawver, L.K.L., Sun, L.L., Tang, C.C., App, H.H., Powell, T.J.T.,

Kim, Y.H.Y., Schreck, R.R., Wang, X.X., Risau, W.W., et al. (1999). SU5416 is

a potent and selective inhibitor of the vascular endothelial growth factor

receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vasculariza-

tion, and growth of multiple tumor types. Cancer Res. 59, 99–106.

Fritz-Six, K.L., Dunworth,W.P., Li, M., and Caron, K.M. (2008). Adrenomedullin

signaling is necessary for murine lymphatic vascular development. J. Clin.

Invest. 118, 40–50.

Gavard, J., and Gutkind, J.S. (2006). VEGF controls endothelial-cell perme-

ability by promoting the beta-arrestin-dependent endocytosis of VE-cadherin.

Nat. Cell Biol. 8, 1223–1234.

Gerhardt, H., Golding, M., Fruttiger, M., Ruhrberg, C., Lundkvist, A.,

Abramsson, A., Jeltsch, M., Mitchell, C., Alitalo, K., Shima, D., and

Betsholtz, C. (2003). VEGF guides angiogenic sprouting utilizing endothelial

tip cell filopodia. J. Cell Biol. 161, 1163–1177.

Gerhardt, H., Ruhrberg, C., Abramsson, A., Fujisawa, H., Shima, D., and

Betsholtz, C. (2004). Neuropilin-1 is required for endothelial tip cell guidance

in the developing central nervous system. Dev. Dyn. 231, 503–509.

Gore, A.V., Lampugnani, M.G., Dye, L., Dejana, E., andWeinstein, B.M. (2008).

Combinatorial interaction between CCM pathway genes precipitates hemor-

rhagic stroke. Dis. Model. Mech. 1, 275–281.

Hellstrom, M., Kalen, M., Lindahl, P., Abramsson, A., and Betsholtz, C. (1999).

Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle

cells and pericytes during embryonic blood vessel formation in the mouse.

Development 126, 3047–3055.

Hla, T., Lee, M.J., Ancellin, N., Liu, C.H., Thangada, S., Thompson, B.D., and

Kluk, M. (1999). Sphingosine-1-phosphate: extracellular mediator or intracel-

lular second messenger? Biochem. Pharmacol. 58, 201–207.

Hla, T., Venkataraman, K., and Michaud, J. (2008). The vascular S1P gradient-

cellular sources and biological significance. Biochim. Biophys. Acta 1781,

477–482.

Ichikawa-Shindo, Y., Sakurai, T., Kamiyoshi, A., Kawate, H., Iinuma, N.,

Yoshizawa, T., Koyama, T., Fukuchi, J., Iimuro, S., Moriyama, N., et al.

(2008). The GPCR modulator protein RAMP2 is essential for angiogenesis

and vascular integrity. J. Clin. Invest. 118, 29–39.

Inai, T., Mancuso, M., Hashizume, H., Baffert, F., Haskell, A., Baluk, P., Hu-

Lowe, D.D., Shalinsky, D.R., Thurston, G., Yancopoulos, G.D., and McDonald,

D.M. (2004). Inhibition of vascular endothelial growth factor (VEGF) signaling

in cancer causes loss of endothelial fenestrations, regression of tumor vessels,

and appearance of basement membrane ghosts. Am. J. Pathol. 165, 35–52.

Jakobsson, L., Franco, C.A., Bentley, K., Collins, R.T., Ponsioen, B., Aspalter,

I.M., Rosewell, I., Busse, M., Thurston, G., Medvinsky, A., et al. (2010).

Endothelial cells dynamically compete for the tip cell position during angio-

genic sprouting. Nat. Cell Biol. 12, 943–953.

Kono, M., Mi, Y., Liu, Y., Sasaki, T., Allende, M.L., Wu, Y.P., Yamashita, T., and

Proia, R.L. (2004). The sphingosine-1-phosphate receptors S1P1, S1P2, and

S1P3 function coordinately during embryonic angiogenesis. J. Biol. Chem.

279, 29367–29373.

Lampugnani, M.G., Orsenigo, F., Gagliani, M.C., Tacchetti, C., and Dejana, E.

(2006). Vascular endothelial cadherin controls VEGFR-2 internalization and

signaling from intracellular compartments. J. Cell Biol. 174, 593–604.

Lee, M.J., Thangada, S., Claffey, K.P., Ancellin, N., Liu, C.H., Kluk, M., Volpi,

M., Sha’afi, R.I., and Hla, T. (1999). Vascular endothelial cell adherens junction

assembly and morphogenesis induced by sphingosine-1-phosphate. Cell 99,

301–312.

Leveen, P., Pekny, M., Gebre-Medhin, S., Swolin, B., Larsson, E., and

Betsholtz, C. (1994). Mice deficient for PDGF B show renal, cardiovascular,

and hematological abnormalities. Genes Dev. 8, 1875–1887.

Liao, F., Li, Y., O’Connor, W., Zanetta, L., Bassi, R., Santiago, A., Overholser,

J., Hooper, A., Mignatti, P., Dejana, E., et al. (2000). Monoclonal antibody to

vascular endothelial-cadherin is a potent inhibitor of angiogenesis, tumor

growth, and metastasis. Cancer Res. 60, 6805–6810.

Developmen

Lindahl, P., Johansson, B.R., Leveen, P., and Betsholtz, C. (1997). Pericyte

loss and microaneurysm formation in PDGF-B-deficient mice. Science 277,

242–245.

Liu, Y., Wada, R., Yamashita, T., Mi, Y., Deng, C.X., Hobson, J.P., Rosenfeldt,

H.M., Nava, V.E., Chae, S.S., Lee, M.J., et al. (2000). Edg-1, the G protein-

coupled receptor for sphingosine-1-phosphate, is essential for vascular matu-

ration. J. Clin. Invest. 106, 951–961.

Mizugishi, K., Yamashita, T., Olivera, A., Miller, G.F., Spiegel, S., and Proia,

R.L. (2005). Essential role for sphingosine kinases in neural and vascular devel-

opment. Mol. Cell. Biol. 25, 11113–11121.

Montero-Balaguer, M., Swirsding, K., Orsenigo, F., Cotelli, F., Mione, M., and

Dejana, E. (2009). Stable vascular connections and remodeling require full

expression of VE-cadherin in zebrafish embryos. PLoS ONE 4, e5772.

Paik, J.H., Skoura, A., Chae, S.S., Cowan, A.E., Han, D.K., Proia, R.L., and

Hla, T. (2004). Sphingosine 1-phosphate receptor regulation of N-cadherin

mediates vascular stabilization. Genes Dev. 18, 2392–2403.

Paik, J.H., Chae, S.S., Lee,M.J., Thangada, S., and Hla, T. (2001). Sphingosine

1-phosphate-induced endothelial cell migration requires the expression of

EDG-1 and EDG-3 receptors and Rho-dependent activation of alpha vbeta3-

and beta1-containing integrins. J. Biol. Chem. 276, 11830–11837.

Pappu, R., Schwab, S.R., Cornelissen, I., Pereira, J.P., Regard, J.B., Xu, Y.,

Camerer, E., Zheng, Y.W., Huang, Y., Cyster, J.G., and Coughlin, S.R.

(2007). Promotion of lymphocyte egress into blood and lymph by distinct

sources of sphingosine-1-phosphate. Science 316, 295–298.

Pham, V.N., Lawson, N.D., Mugford, J.W., Dye, L., Castranova, D., Lo, B., and

Weinstein, B.M. (2007). Combinatorial function of ETS transcription factors in

the developing vasculature. Dev. Biol. 303, 772–783.

Phng, L.K., and Gerhardt, H. (2009). Angiogenesis: a team effort coordinated

by notch. Dev. Cell 16, 196–208.

Pitulescu, M.E., Schmidt, I., Benedito, R., and Adams, R.H. (2010). Inducible

gene targeting in the neonatal vasculature and analysis of retinal angiogenesis

in mice. Nat. Protoc. 5, 1518–1534.

Potente, M., Gerhardt, H., and Carmeliet, P. (2011). Basic and therapeutic

aspects of angiogenesis. Cell 146, 873–887.

Rivera, J., Proia, R.L., and Olivera, A. (2008). The alliance of sphingosine-1-

phosphate and its receptors in immunity. Nat. Rev. Immunol. 8, 753–763.

Ruhrberg, C., Gerhardt, H., Golding, M., Watson, R., Ioannidou, S., Fujisawa,

H., Betsholtz, C., and Shima, D.T. (2002). Spatially restricted patterning cues

provided by heparin-binding VEGF-A control blood vessel branching morpho-

genesis. Genes Dev. 16, 2684–2698.

Sanna, M.G., Wang, S.K., Gonzalez-Cabrera, P.J., Don, A., Marsolais, D.,

Matheu, M.P., Wei, S.H., Parker, I., Jo, E., Cheng, W.C., et al. (2006).

Enhancement of capillary leakage and restoration of lymphocyte egress by

a chiral S1P1 antagonist in vivo. Nat. Chem. Biol. 2, 434–441.

Soriano, P. (1994). Abnormal kidney development and hematological disor-

ders in PDGF beta-receptor mutant mice. Genes Dev. 8, 1888–1896.

Spagnuolo, R., Corada, M., Orsenigo, F., Zanetta, L., Deuschle, U., Sandy, P.,

Schneider, C., Drake, C.J., Breviario, F., and Dejana, E. (2004). Gas1 is

induced by VE-cadherin and vascular endothelial growth factor and inhibits

endothelial cell apoptosis. Blood 103, 3005–3012.

Srinivas, S., Watanabe, T., Lin, C.S., William, C.M., Tanabe, Y., Jessell, T.M.,

and Costantini, F. (2001). Cre reporter strains produced by targeted insertion

of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1, 4.

Venkataraman, K., Lee, Y.M., Michaud, J., Thangada, S., Ai, Y., Bonkovsky,

H.L., Parikh, N.S., Habrukowich, C., and Hla, T. (2008). Vascular endothelium

as a contributor of plasma sphingosine 1-phosphate. Circ. Res. 102, 669–676.

Weinstein, B.M. (2005). Vessels and nerves: marching to the same tune. Cell

120, 299–302.

Zanetti, A., Lampugnani, M.G., Balconi, G., Breviario, F., Corada, M.,

Lanfrancone, L., and Dejana, E. (2002). Vascular endothelial growth factor

induces SHC association with vascular endothelial cadherin: a potential feed-

back mechanism to control vascular endothelial growth factor receptor-2

signaling. Arterioscler. Thromb. Vasc. Biol. 22, 617–622.


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