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VEGFR-3 controls tip to stalk conversion at vessel fusion sites
by reinforcing Notch signalling
Citation for published version:
Tammela, T, Zarkada, G, Nurmi, H, Jakobsson, L, Heinolainen, K, Tvorogov, D, Zheng, W, Franco, CA,
Murtomäki, A, Aranda, E, Miura, N, Ylä-Herttuala, S, Fruttiger, M, Mäkinen, T, Eichmann, A, Pollard, JW,
Gerhardt, H & Alitalo, K 2011, 'VEGFR-3 controls tip to stalk conversion at vessel fusion sites by reinforcing
Notch signalling', Nature Cell Biology, vol. 13, no. 10, pp. 1202-13. https://doi.org/10.1038/ncb2331
Digital Object Identifier (DOI):
10.1038/ncb2331
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Download date: 09. Aug. 2022
VEGFR-3 controls tip to stalk conversion at vessel fusion sites
by reinforcing Notch signalling
Tuomas Tammela
1,9
, Georgia Zarkada
1,9
, Harri Nurmi
1
, Lars Jakobsson
2,10
, Krista
Heinolainen
1
, Denis Tvorogov
1
, Wei Zheng
1
, Claudio A. Franco
2
, Aino Murtomäki
1
, Evelyn
Aranda
3
, Naoyuki Miura
4
, Seppo Ylä-Herttuala
5
, Marcus Fruttiger
6
, Taija Mäkinen
1,10
, Anne
Eichmann
7
, Jeffrey W. Pollard
3
, Holger Gerhardt
2,8
, and Kari Alitalo
1,11
1
Molecular/Cancer Biology Laboratory, Institute for Molecular Medicine Finland, Research
Programs Unit and Department of Pathology, Haartman Institute, Biomedicum Helsinki, PO Box
63 (Haartmaninkatu 8), 00014 University of Helsinki, Finland
2
Vascular Biology Laboratory,
London Research Institute—Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3PX,
UK
3
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine,
New York, New York 10461, USA
4
Department of Biochemistry, Hamamatsu University School of
Medicine, 431-3192 Hamamatsu, Japan
5
A. I. Virtanen Institute and Department of Medicine,
University of Kuopio, PO Box 1627, 70211 Kuopio, Finland
6
Institute of Ophthalmology, University
College London, London EC1V 9EL, UK
7
Institut National de la Santé et de la Recherche
Médicale U833, Collège de France, 11 Place Marcelin Berthelot, 75005 Paris, France
8
Vascular
Patterning Laboratory, Vesalius Research Center, VIB, Campus Gasthuisberg, B-3000 Leuven,
Belgium
Abstract
Angiogenesis, the growth of new blood vessels, involves specification of endothelial cells to tip
cells and stalk cells, which is controlled by Notch signalling, whereas vascular endothelial growth
factor receptor (VEGFR)-2 and VEGFR-3 have been implicated in angiogenic sprouting.
Surprisingly, we found that endothelial deletion of Vegfr3, but not VEGFR-3-blocking antibodies,
postnatally led to excessive angiogenic sprouting and branching, and decreased the level of Notch
signalling, indicating that VEGFR-3 possesses passive and active signalling modalities.
© 2011 Macmillan Publishers Limited. All rights reserved.
11
Correspondence should be addressed to K.A. (Kari.Alitalo@Helsinki.Fi).
9
These authors contributed equally to this work.
10
Present addresses: Vascular Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles Väg 2,
SE171 77 Stockholm, Sweden (L.K.); Lymphatic Development Laboratory, Cancer Research UK London Research Institute, 44
Lincoln’s Inn Fields, London WC2A 3PX, UK (T.M.).
Note: Supplementary Information is available on the Nature Cell Biology website
AUTHOR CONTRIBUTIONS
T.T. and G.Z. designed, directed and carried out experiments and data analysis, as well as interpreted results, and wrote the paper;
H.N. designed and carried out cell culture and biochemistry experiments, and analysed data; L.J. carried out three-dimensional
embryoid body sprouting experiments and analysed data; K.H. carried out cell culture, morphometry of retinal vessels and qRT-PCR,
and analysed data; D.T. carried out biochemistry experiments and analysed data; W.Z. produced and validated Notch ligand and
inhibitor proteins; C.A.F. carried out three-dimensional embryoid body sprouting experiments and analysed data; A.M. carried out
retina experiments and analysed data; E.A. provided op/op retinas and carried out genotyping; N.M. generated FoxC2 antibodies; S.Y-
H. generated adenoviral vectors; M.F. generated PdgfbCreER
T2
mice; T.M. generated Vegfr3
flox/flox
mice; A.E. analysed retinas of
Vegfr3
+/LacZ
mice; J.W.P. provided op/op retinas; H.G. directed experiments, interpreted results and helped write the paper; K.A.
designed and directed experiments, interpreted results and wrote the paper.
COMPETING FINANCIAL INTERESTS
K.A. is the chairman of the Scientific Advisory Board of Circadian.
Reprints and permissions information is available online at http://www.nature.com/reprints
NIH Public Access
Author Manuscript
Nat Cell Biol. Author manuscript; available in PMC 2012 January 19.
Published in final edited form as:
Nat Cell Biol
. ; 13(10): 1202–1213. doi:10.1038/ncb2331.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Furthermore, macrophages expressing the VEGFR-3 and VEGFR-2 ligand VEGF-C localized to
vessel branch points, and Vegfc heterozygous mice exhibited inefficient angiogenesis
characterized by decreased vascular branching. FoxC2 is a known regulator of Notch ligand and
target gene expression, and Foxc2
+/−
; Vegfr3
+/−
compound heterozygosity recapitulated
homozygous loss of Vegfr3. These results indicate that macrophage-derived VEGF-C activates
VEGFR-3 in tip cells to reinforce Notch signalling, which contributes to the phenotypic
conversion of endothelial cells at fusion points of vessel sprouts.
During late embryogenesis and in the adult, blood vessels form primarily by angiogenesis,
that is by sprouting from pre-existing vessels. Vascular endothelial growth factor (VEGF)
potently promotes angiogenesis, and is indispensable for vascular development
1,2
, and
VEGFR-2 tyrosine kinase is the primary receptor transmitting VEGF signals in endothelial
cells
3,4
. VEGFR-3 is activated by the VEGF homologues VEGF-C and VEGF-D, which,
when fully proteolytically processed, also stimulate VEGFR-2 (ref. 5) and induce the
formation and activation of VEGFR-2–VEGFR-3 heterodimers
6,7
. Inactivation of the Vegfr3
gene (also known as Flt4) leads to marked defects in arterial–venous remodelling of the
primary vascular plexus, resulting in lethality by embryonic day (E) 10.5 (ref. 8) or to
defective segmental artery morphogenesis
9
in mice or zebrafish, respectively.
As the lymphatic vessels begin to develop at around E10.5, the level of Vegfr3 expression
gradually decreases in the blood vessels, and from E16.5 onwards it is found nearly
exclusively in the lymphatic vascular endothelium
10,11
. However, VEGFR-3 is induced in
angiogenic endothelial cells for example in tumours, wounds and in maturing ovarian
follicles
12–14
. Homozygous gene-targeting of Vegfc leads to embryonic lethality at E16.5
due to a complete failure in lymphatic vessel formation, whereas Vegfc heterozygous mice
survive with lymphatic vessel hypoplasia and lymphedema, but do not exhibit blood
vascular defects as adults
15
. Conversely, Vegfd gene-targeted mice are viable and normal
16
.
Interestingly, compound deletion of both Vegfc and Vegfd phenocopies the homozygous loss
of Vegfc, but these mice survive past the time point of critical requirement for Vegfr3
(ref.
17
), implicating other as yet unknown ligands or ligand-independent signalling for
VEGFR-3.
Angiogenic sprouting involves specification of subpopulations of endothelial cells into tip
cells that respond to VEGF guidance cues, and stalk cells that follow the tip cells and
proliferate to form the vascular network
18
. Recent evidence indicates that VEGF induces the
membrane-bound Notch ligand delta-like 4 (Dll4) in the tip cells, which leads to the
induction of the stalk-cell phenotype in adjacent endothelial cells through activation of
Notch-1 (refs 10,19–21). The angiogenic sprouts fuse at intervals
18
, followed by the
formation of a vessel lumen to form a functional microcirculatory loop
22,23
. The fusion of
migrating tip cells is chaperoned by Tie2- and neuropilin-1-positive macrophages
24
, which
express a variety of growth factors and proteolytic enzymes
24–26
. However, the molecular
players regulating sprout fusion and vessel anastomosis have remained unknown.
We recently demonstrated that VEGFR-3 is expressed at a high level in endothelial tip cells,
and that blocking VEGFR-3 with antibodies results in decreased angiogenesis during
postnatal development and in tumours
14
. Stimulation of VEGFR-3 augments VEGF-induced
angiogenesis and sustains blood vessel growth even in the presence of VEGFR-2 inhibitors,
whereas antibodies against VEGFR-3 and VEGFR-2 in combination produce additive
inhibition of angiogenesis and tumour growth
14
. Consistent with the concept of high levels
of VEGFR-3 activity in the tip cells, genetic or pharmacological disruption of the Notch
signalling pathway in vivo leads to widespread endothelial Vegfr3 expression and excessive
sprouting
14,27,28
.
Tammela et al. Page 2
Nat Cell Biol. Author manuscript; available in PMC 2012 January 19.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Here, we show that genetic inactivation of Vegfr3 in endothelial cells surprisingly resulted in
increased numbers of tip cells and vessel hyperplasia, which closely resembled loss of Notch
signalling, whereas haploinsufficiency of Vegfc led to disruption of tip cell fusion points and
inefficient angiogenesis. Our results implicate a bimodal role for VEGFR-3 in regulating
angiogenesis, and indicate that the VEGF-C–VEGFR-3 signalling pathway controls the
branching morphogenesis of blood vessels.
RESULTS
Endothelial deletion of Vegfr3 results in excessive angiogenesis
To study the consequences of homozygous endothelial-specific loss of Vegfr3 during
angiogenesis, we mated Vegfr3
flox/flox
mice with PdgfbiCreER
T2
mice that express
tamoxifen-activated Cre recombinase in endothelial cells
29
. Complete deletion of Vegfr3 in
the retinal endothelium was achieved by 24 h following administration of 4-
hydroxytamoxifen (4-OHT; Supplementary Fig. S1a–d). Some residual Vegfr3 expression
was detected by quantitative real-time (qRT) PCR (Supplementary Fig. S1e), presumably
originating from retinal oligodendrocytes
30
or from monocytic cells
31
.
Surprisingly, when Cre was induced in PdgfbiCreER
T2
; Vegfr3
flox/flox
(Vegfr3
iΔEC
) mice for
48 h from postnatal day (P) 3 to P5, marked excessive branching, filopodia projection and
hyperplasia of the nascent vascular plexus were observed (Fig. 1a–e). There was a
significant increase in the proliferation of retinal endothelial cells (Fig. 1f and
Supplementary Fig. S2). Increased branching and vascular hyperplasia were also observed in
hindbrains of Vegfr3
iΔEC
embryos at E11.5 (Fig. 1g–k and Supplementary Fig. S3).
We sought to validate these findings in other models outside the developing central nervous
system. Excessive angiogenesis and sprouting were also detected in syngeneic
subcutaneously implanted Lewis lung carcinomas (LLC) and B16-F10 melanomas in the
Vegfr3
iΔEC
mice (Fig. 1l,m and data not shown). Furthermore, when ears of adult
Vegfr3
iΔEC
mice were transduced with AdVEGF, we observed a more robust angiogenic
response, characterized by increased vascular tortuosity, enlargement and surface area (Fig.
1n and Supplementary Fig. S4).
VEGFR-3 tyrosine kinase activity is crucial for lymphatic vessel growth
32
, but its role in
angiogenesis is not known. To determine whether VEGFR-3 is tyrosine phosphorylated in
blood vascular endothelial cells in vivo, we injected recombinant VEGF, VEGF-C or BSA
control protein into the outflow tract of wild-type embryos at E10.5, a stage when lymphatic
vessels have not yet developed (Fig. 2a–c). VEGF did not promote tyrosine phosphorylation
of VEGFR-3, unlike VEGF-C, but a faint phosphorylation signal was detected in both
VEGF- and BSA-stimulated embryos, indicating a baseline level of VEGFR-3
phosphorylation (Fig. 2b). As expected, VEGF and VEGF-C both stimulated VEGFR-2
phosphorylation (Fig. 2c).
We have previously shown that VEGFR-3-blocking antibodies suppress angiogenesis
14
,
whereas our results surprisingly showed that genetic targeting of Vegfr3 produced excessive
angiogenic sprouting, indicating the possibility of ligand-independent sprouting. We found
that VEGFR-3 was phosphorylated in the absence of its ligands by stimulation with collagen
I in cultured human dermal blood vascular endothelial cells (hBECs) even in the presence of
blocking monoclonal antibodies or a VEGFR tyrosine kinase inhibitor, whereas the Src
inhibitor PP2 blocked collagen-I-induced phosphorylation of VEGFR-3 (Fig. 2d), indicating
that VEGFR-3 can be phosphorylated independently of its ligands
33
.
Tammela et al. Page 3
Nat Cell Biol. Author manuscript; available in PMC 2012 January 19.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
We addressed the role of VEGFR-3 kinase activity in angiogenesis in vivo by studying the
retinas of Chy mice, which harbour a heterozygous kinase-inactivating point mutation in the
tyrosine kinase domain (Vegfr3
KD/+
), leading to a decreased level of VEGFR-3 signalling
and severe lymphatic vessel hypoplasia
32
. The retinas of mice harbouring one kinase-dead
(KD) and one deleted Vegfr3 allele (Vegfr3
iΔEC/KD
) showed an increase in the vascular area,
branching and filopodia projection that was comparable to homozygous loss of Vegfr3
(Vegfr3
iΔEC/iΔEC
; Fig. 2e–i), indicating that VEGFR-3 hypophosphorylation can trigger the
phenotype. In contrast, Vegfr3
KD/+
and Vegfr3
iΔEC/+
single heterozygotes were
indistinguishable from wild-type retinas (Fig. 2e–i).
The administration of VEGFR-3-blocking antibodies to Vegfr3
iΔEC
mice did not affect the
hypervascular phenotype (Fig. 3a,b). In contrast, VEGFR-2-blocking antibodies rescued the
increase in vascular area in the Vegfr3
iΔEC
mice (Fig. 3a,b). However, the nascent vessels
appeared abnormally thick in the Vegfr3
iΔEC
retinas following administration of VEGFR-2-
blocking antibodies (arrowheads in Fig. 3a), indicating that the phenotypic rescue was not
complete. Furthermore, the expression level of VEGFR-1, a negative regulator of VEGF,
was decreased in the Vegfr3
iΔEC
retinas (Fig. 3c), indicating an increased level of VEGF–
VEGFR-2 signalling. Consistently, we detected a minor increase in the level of VEGFR-2
phosphorylation following stimulation of cultured human umbilical vein endothelial cells
(HUVECs) with VEGF when VEGFR-3 expression was silenced using siRNA
oligonucleotides (Fig. 3d). Antibodies blocking human VEGFR-3 had no effect on
VEGFR-2 phosphorylation in response to VEGF in HUVECs (Supplementary Fig. S5a).
Loss of Vegfr3 in endothelial cells leads to a decreased level of Notch target gene
expression
The phenotype resulting from endothelial Vegfr3 deletion closely resembled the hyperplastic
vascular pattern resulting from inhibition of Dll4/Notch signalling between tip and stalk
cells. Indeed, we detected a marked decrease in the level of Notch target gene transcripts and
the Notch ligand Dll4 in the Vegfr3
iΔEC
retinas (Fig. 4a), indicating a decreased level of
Notch signalling in the endothelium, resulting in tip cell dominance over stalk cells. In
contrast, no changes in Notch targets could be observed in pups treated with VEGFR-3-
blocking antibodies (Supplementary Fig. S5b), indicating that the perturbations to VEGFR-3
by blocking antibodies and genetic targeting are qualitatively different.
To investigate the responsiveness of the Vegfr3-deficient endothelium to exogenous Notch
activation, we administered Jagged1, a small peptide Notch agonist, to Vegfr3
iΔEC
pups, and
observed a rescue of the hypervascular phenotype (Fig. 4b,c). Notably, the vasculature was
normalized also in terms of morphology, unlike after anti-VEGFR-2 antibody administration
(Fig. 4c), indicating that decreased Notch signalling underlies the phenotype in Vegfr3
iΔEC
retinas.
According to our results, VEGFR-3 contributes to the activation of Notch that is known to
promote a phenotypic switch from a tip cell to a stalk cell. We chose to test this hypothesis
in mosaic embryoid bodies consisting of both Vegfr3
+/LacZ
heterozygous and wild-type
embryonic stem cells
34
. Vegfr3
+/LacZ
endothelial cells preferentially localized to the tips of
VEGF-induced vascular sprouts (Fig. 4d,g), whereas inhibiting Notch cleavage with the γ-
secretase inhibitor DAPT abrogated the competitive advantage of the Vegfr3
+/LacZ
endothelial cells (Fig. 4h). Vegfr3
+/LacZ
endothelial cells preferentially localized to the tips
of vascular sprouts also in mosaic retinas at P5 (Fig. 4i), indicating increased tip cell
competence for the Vegfr3 haploinsufficient cells, which further implicates a decreased level
of Notch signalling in endothelial cells with targeted Vegfr3 loss-of-function.
Tammela et al. Page 4
Nat Cell Biol. Author manuscript; available in PMC 2012 January 19.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript