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VASCULAR BIOLOGY
Notch restricts lymphatic vessel sprouting induced by vascular endothelial
growth factor
Wei Zheng,1 Tuomas Tammela,1 Masahiro Yamamoto,1 Andrey Anisimov,1 Tanja Holopainen,1 Seppo Kaijalainen,1
Terhi Karpanen,1 Kaisa Lehti,1,2 Seppo Ylä-Herttuala,3 and Kari Alitalo1
1Molecular/Cancer Biology Laboratory, Research Programs Unit, Institute for Molecular Medicine Finland and Helsinki University Central Hospital, Helsinki,
Finland; 2Genome-Scale Biology Program, Research Programs Unit, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland; and 3Department of
Biotechnology and Molecular Medicine, University of Eastern Finland, Kuopio, Finland
Notch signaling plays a central role in
cell-fate determination, and its role in
lateral inhibition in angiogenic sprouting
is well established. However, the role of
Notch signaling in lymphangiogenesis,
the growth of lymphatic vessels, is poorly
understood. Here we demonstrate Notch
pathway activity in lymphatic endothelial
cells (LECs), as well as induction of deltalike ligand 4 (Dll4) and Notch target genes
on stimulation with VEGF or VEGF-C.
Suppression of Notch signaling by a
soluble form of Dll4 (Dll4-Fc) synergized
with VEGF in inducing LEC sprouting in
3-dimensional (3D) fibrin gel assays. Expression of Dll4-Fc in adult mouse ears
promoted lymphangiogenesis, which was
augmented by coexpressing VEGF. Lymphangiogenesis triggered by Notch inhibition was suppressed by a monoclonal
VEGFR-2 Ab as well as soluble VEGF and
VEGF-C/VEGF-D ligand traps. LECs transduced with Dll4 preferentially adopted the
tip cell position over nontransduced cells
in 3D sprouting assays, suggesting an
analogous role for Dll4/Notch in lymphatic and blood vessel sprouting. These
results indicate that the Notch pathway
controls lymphatic endothelial quiescence, and explain why LECs are poorly
responsive to VEGF compared with
VEGF-C. Understanding the role of the
Notch pathway in lymphangiogenesis provides further insight for the therapeutic
manipulation of the lymphatic vessels.
(Blood. 2011;118(4):1154-1162)
Introduction
The Notch pathway is a highly conserved signaling system that
controls cell-fate determination. Recently, Notch has been shown
to control endothelial cell (EC) proliferation, motility, filopodia
formation, adhesion, and vessel stabilization.1,2 Among the Notch
receptors, the endothelium expresses mainly Notch1 and Notch4,
which are activated by Delta-like or Jagged family ligands
presented in trans by the neighboring cells. This leads to cleavage
of the receptor at the juxtamembrane domain by ␥-secretase to
release the Notch intracellular domain (NICD), which translocates
to the nucleus to regulate downstream gene expression.1,2 Loss-offunction studies using inhibitors of Dll4 or Notch1, heterozygous
Dll4 gene deletion, or inhibition of Notch1 cleavage with ␥secretase inhibitors all demonstrated excessive sprouting of blood
vascular endothelial cells.1,2 Interestingly, Dll4 inhibitors produced
excessive angiogenesis also in tumors, but the perfusion of the
newly formed vessels was compromised and thus tumor growth
was retarded.3-5
Lymphatic vessels are critical for the maintenance of tissue fluid
balance, immune responses, and absorption of hydrophobic nutrients in the gut.6 Lymphangiogenesis, the growth of new lymphatic
vessels, is an essential process during embryonic development.6,7
Although usually quiescent in adults, lymphatic vessels can sprout
from preexisting vessels and anastomose to form new vessels in
pathologic conditions, such as inflammation and tumor progression, where production of lymphangiogenic factors is induced.
VEGF-C and VEGF-D, acting through VEGF receptor 3 (VEGFR3), are key inducers of lymphangiogenesis.6 Loss of Vegfc leads to
complete aplasia of the lymphatic vessels and embryonic lethality
because of edema,8 whereas VEGF-D is dispensable for lymphatic
development in mice.6,9
VEGF-C and VEGF-D are also capable of activating VEGFR-2
after proteolytic processing in the extracellular space.6 Although
VEGFR-3 signals are sufficient for inducing lymphangiogenesis,10
VEGFR-2, the key receptor driving angiogenesis, is also expressed
in the lymphatic endothelium and signaling via this receptor
induces circumferential hyperplasia, but not sprouting of lymphatic
vessels in vivo, as assessed by adenoviral expression of human or
murine VEGF, or a VEGFR-2–specific ligand, VEGF-E, derived
from the Orf virus.11-13
VEGF, the major ligand for VEGFR-2, is critical for angiogenesis,14,15 but its role in physiologic and pathologic lymphangiogenesis is not well understood. Heterozygous loss of Vegf or homozygous inactivation of Vegfr2 leads to death of mice at around
embryonic day (E) 8.5 because of failure to form blood vessels.14-16
As the lymphatic vessels begin to develop considerably later, at
around E10.5, the contribution of the VEGF/VEGFR-2 signaling
pathway to lymphatic vessel development has not been addressed
by direct gene-targeting studies.6 Overexpression of VEGF was
shown to promote peritumoral lymphangiogenesis and metastasis
to distal lymph nodes,17,18 but blocking VEGFR-2 in a prostate
tumor model, although inhibiting tumor growth and angiogenesis,
failed to suppress lymph node metastasis.19 Nevertheless, combinational silencing of both VEGF-C and VEGF showed synergistic
benefit in blocking lymph node and lung metastases.20 However, it
Submitted November 5, 2010; accepted April 22, 2011. Prepublished online as
Blood First Edition paper, May 12, 2011; DOI 10.1182/blood-2010-11-317800.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
An Inside Blood analysis of this article appears at the front of this issue.
The online version of this article contains a data supplement.
1154
© 2011 by The American Society of Hematology
BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
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BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
is noteworthy that VEGF can recruit VEGFR-1–positive macrophages that produce VEGF-C and VEGF-D,21,22 which makes it
difficult to assess the direct role of VEGF in lymphangiogenesis.
VEGF has thus been implicated as a weak lymphangiogenic
factor despite VEGFR-2 expression in the lymphatic endothelium.
Although Dll4-Notch interactions play an important role in angiogenesis, it is not known whether similar mechanisms are at play
during lymphatic vessel sprouting. For example, the mechanisms
regulating tip-versus-stalk cell specification in lymphatic endothelial cells (LECs) have remained enigmatic. Here we show that
suppression of Notch signaling, in synergy with VEGF, induces
lymphangiogenesis both in vitro and in vivo. We also demonstrate
that the Notch ligand Dll4 determines the tip/stalk fate during
lymphangiogenesis in analogy to angiogenesis.
Methods
Production and purification of Dll4-Fc
293T cells were transfected with Dll4-Fc or HSA (Fugene6 Transfection
Reagent; Roche Diagnostics), and cultured in serum-free medium 24 hours
after transfection. The supernatant was collected on the following day,
concentrated 10 times with Centrifugal Filter Units (membrane size:
10 kDa; Millipore), aliquoted and frozen. Dll4-Fc was purified from the
supernatant by protein G chromatography (GE Healthcare) according to the
manufacturer’s instructions. Protein concentration was determined by the
Pierce BCA Protein Assay Kit (Thermo Scientific).
Immunoprecipitation and Western blotting
Cells were lysed in RIPA buffer (150mM NaCl, 1% Nonidet P40 [NP40],
0.5% sodium deoxycholate, 0.1% SDS, 50mM Tris, 1 ␮g/mL aprotinin,
1 ␮g/mL leupeptin, 1mM NaF, 1mM NaVO4, 1mM PMSF). Cell lysates
were separated by SDS-PAGE, transferred to polyvinylidene fluoride
(PVDF) membranes, and incubated with Abs directed against the cleaved
Notch1 intracellular domain (cNICD; Cell Signaling Technology), VEGFR-2
(R&D Systems), VEGFR-3 (Millipore), phospho-Akt (Cell Signaling
Technology), phospho-Erk1/2 (Cell Signaling Technology), phosphotyrosine (Upstate Biotechnology), Prox-1 (R&D Systems), HSC70 (Santa
Cruz Biotechnology), or ␤-actin (Cell Signaling Technology), followed by
appropriate HRP-conjugated secondary Abs. For immunoprecipitation, cell
lysates were incubated with VEGFR-2 Ab (R&D Systems) overnight and
1 hour with protein G Sepharose (GE Healthcare) at ⫹4°C, and subjected to
Western blot analysis.
Quantitative PCR analysis
LECs were treated, as indicated in the figure legends, in EC medium
containing 0.5% FCS. Total RNA was isolated with the RNAeasy Mini Kit
(Qiagen) and reverse-transcribed with the DyNAmo cDNA Synthesis Kit
(Finnzymes). Quantitative PCR (qPCR) was performed in technical triplicates using the DyNAmo HS SYBR Green qPCR kit or the Probe qPCR kit
(Finnzymes). Gene expression was normalized to ␤-actin or GAPDH.
Relative fold changes were calculated by the formula, 2⫺⌬⌬Ct, where Ct
stands for threshold cycles. Primer information can be found in supplemental Table 1 (available on the Blood Web site; see the Supplemental Materials
link at the top of the online article).
Notch RESTRICTS VEGF-INDUCED LYMPHANGIOGENESIS
1155
VEGF-C (100 ng/mL), Dll4-Fc– or HSA-conditioned medium, Compound
X (30mM),23 VEGFR-2 blocking Ab (7 ␮g/mL; Imclone), VEGFR1-Fc24
(7 ␮g/mL), VEGFR3-Fc,24 VEGFR-3 blocking Ab (3C5, 10 ␮g/mL; Imclone), human IgG (7 ␮g/mL, 10 ␮g/mL, or 20 ␮g/mL; Sigma-Aldrich),
purified Dll4-Fc (20 ␮g/mL), or their indicated combinations. The cultures
were maintained for 6-9 days by changing the medium every other day
before fixation with 4% paraformaldehyde (PFA) for 1 hour at room
temperature (RT). Bright field images were captured with Axiovert
200 (Zeiss) at 5⫻ magnification and sprout lengths were measured with
NIH ImageJ.
3D spheroid sprouting assay
Three thousand LECs were cultured in round-bottom 96-well plates (Nunc)
precoated with 0.8% agarose for 1 day for spheroid formation. The
spheroids were then collected and embedded in 20% Matrigel-containing
(BD Biosciences) fibrin gels (3 mg/mL fibrinogen, 2 U/mL thrombin, and
200 ␮g/mL aprotinin), treated with hIgG (Sigma-Aldrich), Dll4-Fc, human
VEGF165 (R&D Systems), VEGF-C, LY29200 (Calbiochem), ephrinB2
blocking peptide (see supplemental Methods for details) or their combinations in EC medium (PromoCell) containing 1% FCS at final concentrations
indicated in the figure legends for 48 hours. The spheroids were then fixed
in 4% PFA for 1 hour at room temperature (RT).
The tip cell competition assay was performed similarly, except that the
LECs were transduced with vehicle or mDll4-ECTM-EGFP, prelabeled
with Orange and Green CellTracker (Invitrogen), respectively, according to
the manufacturer’s protocol and mixed 1:1 for spheroid formation. A
fraction of the mixed cells were plated on a coverslip and stained for mDll4
to evaluate the percentage of mDll4-ECTM-EGFP–transduced cells in the
total population.
In vivo use of the viral vectors
The Provincial State Office of Southern Finland approved all animal
experiments. Details of viral transduction experiments are found in
supplemental Methods.
Immunofluorescent staining
Details of the whole-mount staining of ears, beads, and spheroids are found
in supplemental Methods. For cleaved NICD staining, LECs cultured on
coverslips were treated with Dll4-Fc or hIgG (10 ␮g/mL) in 0.5%
FCS-containing medium overnight and fixed with 4% PFA for 5 minutes at
RT. The cells were then stained with an Ab specifically targeting the cleaved
NICD (Cell Signaling Technology) using the TSA system (PerkinElmer) to
amplify the signal, according to the manufacturer’s protocol, along with
PECAM-1 (Dako) Ab, and mounted in mounting medium containing DAPI
(Vector Laboratories).
Fluorescent microscopy
All the whole-mount stained samples were imaged using a confocal
microscope (Zeiss LSM 510, air objectives: 10⫻ with numerical aperture
[NA] 0.5; oil objectives: 40⫻ with NA 1.3) at RT with LSM AIM software.
3D projections were digitally reconstructed from Z-stacks. For measurement of VEGFR-3 and Prox1-positive vessel area per microscopic area,
color images were converted to grayscale images using Adobe Photoshop
and analyzed with ImageJ. Measurement of NICD and DAPI intensity was
performed similarly. Brightness and contrast of the images were adjusted
using Adobe Photoshop.
Three-dimensional bead sprouting assay
Cytodex 3 microcarrier beads (GE Healthcare) were coated with endothelial cells (mixed at 400 cells per bead) in Microvascular Endothelial Cell
Growth Medium-2 MV (Lonza), and embedded in 2 mg/mL fibrin gels in
48-well plates by mixing 2 mg/mL fibrinogen (Calbiochem) in HBSS,
1 U/mL thrombin (Sigma-Aldrich), and 150 ␮g/mL aprotinin (SigmaAldrich). Endothelial Cell Growth Medium-2 (Lonza) containing WI-38
cells (11 000 per well) was added to each well in the presence of human
Quantitative analysis and statistics
Images for quantitation were coded with numbers and evaluated blindly
to the treatments. Data were presented as mean ⫾ SEM except for the
tip cell-competition assay, where percentages were used. For comparison of means, 2-way ANOVA without interaction followed by the
Holm-Sidak test was used. For comparison of proportions, the 2-tailed
binomial test was used. Statistical analyses were carried out with SPSS
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1156
ZHENG et al
BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
Figure 1. Notch pathway components, the soluble inhibitor of Notch
and its effects on the downstream Notch targets in LECs. (A) Comparison of mRNA expression levels of Notch signaling molecules between
cultured human BECs and LECs. (B) qRT-PCR analysis of DLL4,
NOTCH1, NOTCH4, HES1, HEY1, and NRARP in LECs treated with
VEGF (100 ng/mL) or VEGF-C (100 ng/mL) for 1 hour. The dashed line
indicates the nontreated control levels, which are taken as 1. (C) Diagram
of the domain structures of human and mouse full-length Dll4 and
mDll4-Fc. The latter comprises the extracellular domain (ECD) of mDll4
and the Fc domain of human IgG. (D) Western blot analysis of cleaved
N1ICD in LECs treated with Dll4-Fc or hIgG (10 ␮g/mL) overnight. ␤-actin
served as a loading control. (E) Immunofluorescent staining for cleaved
N1ICD (red), PECAM-1 (green), and DAPI (blue) of LECs treated with
Dll4-Fc (10 ␮g/mL) overnight or untreated. Arrows indicate diminished
N1ICD staining in the nuclei and arrowheads the active N1ICD. Scale bar,
50 ␮m. (F) Quantification of N1ICD in the nuclei in panel E. (G) qRT-PCR
analysis showing inhibition of Notch target gene expression by Dll4-Fc in
LECs 24 hours after treatment. (H) qPCR analysis of DLL4 expression in
LECs treated with Dll4-Fc– or HSA-conditioned medium for 24 hours.
Data represent means ⫾ SEM of at least 3 independent experiments;
*P ⬍ .05; **P ⬍ .01; ***P ⬍ .001.
17.0 software or manually. Statistical significance is indicated in the
figures by asterisks: *P ⬍ .05, **P ⬍ .01, and ***P ⬍ .001.
More information on the methods and materials can be found in
supplemental Methods.
Results
Characterization of Notch pathway components, the soluble
inhibitor of Notch, and its effects on the downstream Notch
targets in LECs
To study the role of Notch signaling in LECs, we first examined the
expression levels of several main components of the pathway.
qPCR analysis of cultured human blood vascular endothelial cells
(BECs) and LECs indicated that Notch receptors, ligands, and
downstream targets were expressed at comparable levels in these
2 cell types (Figure 1A), suggesting an active role for Notch
signaling in the LECs. To understand whether Notch signaling is
regulated by VEGF or VEGF-C signaling, which are involved in
LEC activation,6 we analyzed the expression of DLL4, NOTCH1,
and NOTCH4 in LECs in response to VEGF and VEGF-C.
Whereas NOTCH1 and NOTCH4 expression were essentially
unchanged, DLL4 as well as the downstream Notch target genes
HES1, HEY1, and NRARP were up-regulated by VEGF or VEGF-C
treatment (Figure 1B). Dll4 was also induced in the lymphatic
endothelium by VEGF or VEGF-C expression in vivo, although
weak expression was also detected in resting vessels (supplemental
Figure 1).
We produced a soluble, competitive Notch inhibitor, comprising
the extracellular domain of mouse Dll4 and the Fc domain of
human IgG, designated as Dll4-Fc (Figure 1C). Dll4-Fc effectively
inhibited the cleavage and nuclear localization of NICD, using
an Ab specifically recognizing the cleaved form (Figure 1D-F),
and the expression of Notch target genes, including HES1,
HEY2, and NRARP, in LECs (Figure 1G). Interestingly, inhibition of Notch signaling with Dll4-Fc also suppressed the
expression of DLL4 in LECs (Figure 1H), indicating that in
addition to blocking the ligand-receptor interaction, Dll4-Fc
further inhibited Notch signaling at the gene expression level of
the pathway by down-regulating DLL4.
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BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
Notch RESTRICTS VEGF-INDUCED LYMPHANGIOGENESIS
1157
Dll4-Fc potentiates VEGF-induced lymphangiogenesis in vivo
To assess the concomitant effects of Notch inhibition and VEGF or
VEGF-C stimulation in vivo, we injected recombinant adenoassociated virus (rAAVs) encoding Dll4-Fc, VEGF, VEGF-C, or
HSA intradermally into mouse ears (Figure 4A-L). As in the bead
sprouting assay, Dll4-Fc alone (Figure 4C,I) induced lymphangiogenesis as quantified by both number of branch points and vessel
area (Figure 4M-N), whereas no lymphangiogenesis was observed
in ears transduced with the HSA control virus (Figure 4F,L). The
addition of rAAV-VEGF to Dll4-Fc increased the vessel area
compared with Dll4-Fc alone, although we did not observe
increased branching in the combination experiment (Figure 4A-N).
In line with our previous results,11 expression of human VEGF
alone did not induce sprouting or branching, but only circumferential hyperplasia of the lymphatic vessels (Figure 4J,L-N). As in the
bead sprouting assay and the spheroid sprouting assay with
VEGF-C (Figures 2A-B and 3C-D), Dll4-Fc did not significantly
Figure 2. Blockade of Notch signaling results in LEC sprouting. (A) Immunofluorescent PECAM-1 staining of LEC-coated microbeads subjected to Dll4-Fc– or
HSA-conditioned medium with or without VEGF-C (100 ng/mL). (B) Quantification of
the cumulative sprout lengths and number per bead in the experiment of panel A.
(C) PECAM-1 staining of LEC-coated beads treated with the ␥-secretase inhibitor
Compound X (X, 30mM) or DMSO (vehicle). Scale bars, 100 ␮m. Data summarized
from 2 independent experiments with conditioned media and a third independent
experiment with purified Dll4-Fc (20 ␮g/mL), with similar results (n ⫽ 8-15 per group
in each experiment) in panel B and 3 independent experiments in panel C. Error bars,
SEM; ***P ⬍ .001; N.S., not significant.
Blocking Notch promotes LEC sprouting in vitro
To study whether endogenous Notch signals are involved in
inhibiting LEC sprouting, as has been demonstrated for BECs, we
applied Dll4-Fc to LEC-coated beads embedded in 3D fibrin gels
that were covered by WI-38 human fibroblasts in the presence or
absence of VEGF-C (Figure 2A). Conditioned medium containing
Dll4-Fc or purified protein stimulated robust sprouting of the
LECs, whereas beads stimulated with the control medium or hIgG
did not form sprouts (Figure 2A-B). Compound X, a ␥-secretase
inhibitor that inhibits Notch signaling,23 also induced LEC sprouting (Figure 2C). Taken together, these results indicate an important
role for Notch signaling in the regulation of lymphatic sprouting.
Notch blockade enhances lymphatic sprouting induced
by VEGF
Because VEGF is an integral component of the medium in the
bead-sprouting assay, and the cocultured fibroblasts also produce
various growth factors, we resorted to an EC spheroid-sprouting
assay, which does not require coculture of fibroblasts, and allows
the titration of VEGF and VEGF-C. As shown in Figure 3A and C,
inhibition of Notch by Dll4-Fc synergized with VEGF in inducing
lymphatic sprouting in a dose-dependent fashion, whereas VEGF
alone was less effective. Although Dll4-Fc also increased the
sprouting induced by low concentration of VEGF-C (10 ng/mL),
this effect was lost when the concentration of VEGF-C increased to
50 ng/mL (Figure 3B,D). Taken together, these data suggest that
Notch signaling inhibits VEGF-induced lymphatic sprouting,
whereas VEGF-C can override this restriction at sufficient doses.
Figure 3. Notch inhibition and VEGF stimulation synergize in lymphatic
sprouting in vitro. (A) LEC spheroids were treated with Dll4-Fc (20 ␮g/mL), VEGF
(10 or 50 ng/mL), or their combination as indicated and stained for PECAM-1. Note
the synergistic induction of LEC sprouting by Dll4-Fc and VEGF in a dose-dependent
manner, as quantified in panel C. GFs indicates growth factors. (B) Similar titration of
VEGF-C in the presence or absence of Dll4-Fc (20 ␮g/mL). (D) Quantification of the
sprout number in the experiment shown in panel B. Scale bar, 100 ␮m. Data
represent means ⫾ SEM from 2-3 independent experiments with n ⫽ 6-13 per group
per experiment; *P ⬍ .05; **P ⬍ .01; ***P ⬍ .001.
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ZHENG et al
BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
of VEGFR-3 signaling by a blocking Ab against VEGFR-3 or VEGFR3Fc, a VEGFR-3 trap, did not significantly suppress LEC sprouting in
response to Dll4-Fc (Figure 5B,D).
In vivo, the lymphangiogenesis induced by Dll4-Fc in vivo was
suppressed by VEGFR1-Fc and VEGFR-3(domains 1-3)-Fc (also
called VEGF-C/D trap), a competitive inhibitor of VEGFR-3, but
not by an inactive VEGFR-3(domains 4-7)-Fc, which does not
have ligand-binding activity and served as a control (Figure 6),
indicating that VEGFR-2 and VEGFR-3 signaling regulate the
lymphangiogenesis induced by Notch inhibition in vivo.
Regulation of VEGFR-2, VEGFR-3, and ephrinB2 signaling by
Notch signaling in LECs
To dissect the mechanism of how Notch signaling regulates
lymphatic sprouting, we determined the expression levels of
VEGFR-2 and VEGFR-3. Neither VEGFR-2 nor VEGFR-3 expression at the mRNA or protein level was significantly altered by
exposure to Dll4-Fc in LECs cultured in a 2-dimensional monolayer (supplemental Figure 2A-C). Furthermore, the phosphorylation of VEGFR-2 stimulated by VEGF was not affected by Dll4-Fc
treatment (supplemental Figure 2D). The serine-threonine kinase
Akt is known to regulate EC migration and angiogenesis,25 and Akt
Figure 4. Notch inhibition and VEGF cooperate to induce lymphangiogenesis in
vivo. (A-F) Lymphatic vessels of mouse ears were visualized by VEGFR-3 wholemount staining (red) 2 weeks after transduction with rAAVs encoding the indicated
factors. White dots indicate branchpoints. (G-L) Higher magnifications of panels A
through F. Note the increased branching and circumferential hyperplasia of lymphatic
vessels in the VEGF and Dll4-Fc combination shown in panels A and G. Scale bars
indicate 200 ␮m (A-F) and 50 ␮m (G-L). Quantification of the branchpoints (M), as
indicated by the white dots in panels A through F, and vessel area (N) per microscopic
field. A indicates VEGF; C, VEGF-C, and HSA, human serum albumin. Shown are
means ⫾ SEM summarized from 2 independent experiments with n ⫽ 4-6 per group
per experiment; *P ⬍ .05; **P ⬍ .01; ***P ⬍ .001.
increase the VEGF-C induced lymphatic vessel area or number of
branch points (Figure 4B,E,M-N).
Role of VEGFR-2 and VEGFR-3 signaling in lymphangiogenesis
induced by Notch inhibition
As LECs express both VEGFR-3 and VEGFR-2, which can both
stimulate lymphangiogenesis,6 we wondered whether the sprouting induced by Notch blockade is dependent on the activation of these receptors.
In the bead-sprouting assay, where VEGF was contained in the medium,
either a blockingAb against VEGFR-2 or a soluble VEGFR-1 (VEGFR1Fc), serving as a VEGF trap, almost completely blocked the effect of
Dll4-Fc (Figure 5A,C), indicating that VEGF/VEGFR2 signals control
the lymphatic sprouting induced by Notch inhibition. However, blockade
Figure 5. Involvement of VEGFR-2 and VEGFR-3 in the lymphatic sprouting
induced by Notch inhibition in vitro. (A-B) LEC-coated beads were treated with
a blocking Ab against VEGFR-2 (5 ␮g/mL) or VEGFR-3 (10 ␮g/mL), a soluble
VEGFR-1-Fc (5 ␮g/mL) or VEGFR3-Fc (5 ␮g/mL) or control hIgG, in the presence
of Dll4-Fc (20 ␮g/mL) and stained for PECAM-1. Scale bars, 100 ␮m. (C-D) Quantification
of the sprouting in panels A and B. anti-R2 indicates anti–VEGFR-2; anti-R3,
anti-VEGFR-3; R1-Fc, VEGFR-1-Fc; and R3-Fc, VEGFR-3-Fc. Results represent
means ⫾ SEM summarized from 2 independent experiments with n ⫽ 6-12 per
group per experiment; ***P ⬍ .001 compared with the last bar in panel C.
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BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
Notch RESTRICTS VEGF-INDUCED LYMPHANGIOGENESIS
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whether ephrinB2 was also regulated by Notch in LECs and
accounted for the LEC sprouting induced by Notch inhibition.
Indeed, ephrinB2 expression was suppressed by Dll4-Fc, both at
the basal level and in the presence of VEGF (supplemental Figure
4A). However, blocking the interaction between ephrinB2 and
EphB4 using a blocking peptide did not enhance LEC sprouting
induced by VEGF or VEGF-C, but rather inhibited the sprouting
(supplemental Figure 4B-C).
Inhibition of Notch signaling does not affect Prox1 expression
in adult mouse tissues or in vitro
Prox1 is a master regulator of lymphatic development, whose
deficiency results in failure of lymphatic vessel formation.28 We
studied Prox1 expression in our in vivo model, as it was recently
suggested that overactivation of the Notch signaling pathway can
suppress Prox1 expression in cultured LECs.29 We found that
inhibiting Notch by Dll4-Fc did not affect Prox1 expression in
adult lymphatic vessels (supplemental Figure 5A-B), nor did it alter
Prox1 expression in cultured LECs (supplemental Figure 5C).
Notch signaling regulates tip-stalk specification in LECs
In vitro and in vivo studies have shown that Notch is cell
autonomously required for stalk cell specification in BECs, and that
cells with a low degree of Notch signaling and abundant Dll4
expression become selected as tip cells.30,31 We therefore hypothesized that the LECs expressing more Dll4 win the competition for
the tips. To address this question, a retrovirus expressing Dll4
extracellular and transmembrane domains fused to EGFP (DLL4ECTM-EGFP, Figure 7A) was constructed and used to transduce
cultured LECs. The cells were labeled with green CellTracker and
mixed with empty retrovirus-transduced LECs that were labeled
with orange CellTracker to form mosaic spheroids. A fraction of the
mixed cells were plated on coverslips and stained for mDll4 (30%
positive in the total population; Figure 7H). When stimulated with
VEGF or VEGF-C, the percentage of Dll4-positive cells in the tip
cell position was significantly higher (increased to 43% and 50%,
respectively) than expected (30%; Figure 7B-H), indicating that the
tip cell position is determined by Dll4/Notch signals during
lymphangiogenesis in analogy to BEC sprouting.
Figure 6. Requirements for VEGFR-2 and VEGFR-3 in Notch inhibition-induced
lymphatic sprouting in vivo. (A-F) Lymphatic vessels stained by VEGFR-3 2 weeks
after transduction of mouse ears with the indicated rAAVs. (G-M) Close-up views of
panels A through F. (N) Quantification of the ear lymphatic vessel branchpoints in
mice transduced with the indicated rAAVs. R1-Fc indicates VEGFR-1-Fc; and R3-Fc,
VEGFR-3(domain1-3)-Fc. Ctrl-Fc indicates VEGFR-3(domain 4-7)-Fc, used as a
negative control. Scale bars, 100 ␮m. Results represent means ⫾ SEM summarized
from 2 independent experiments with n ⫽ 4-8 per group per experiment; *P ⬍ .05;
***P ⬍ .001 compared with the first bar in panel N.
is phosphorylated on VEGF and VEGF-C stimulation in LECs.26
We therefore asked whether Akt was important in lymphatic
sprouting induced by VEGF and Notch inhibition. Blocking Akt
activity with LY292002, an inhibitor of PI3K, efficiently abrogated
LEC sprouting induced by combined VEGF and Dll4-Fc treatment
(supplemental Figure 3A-B). However, treatment with Dll4-Fc did
not enhance VEGF-induced Akt phosphorylation (supplemental
Figure 3C), suggesting that while Akt is important for VEGFinduced LEC sprouting, it is not directly regulated by the Notch
pathway. Similarly, phosphorylation of the MAPK Erk1/2 was not
altered by Dll4-Fc in the presence or absence of VEGF (supplemental Figure 3C).
In BECs, ephrinB2 has been shown to be a target of Notch
signaling and involved in EC sprouting,27 we therefore asked
Discussion
In this study we demonstrate that Notch signaling molecules are
expressed at comparable levels in LECs and BECs. Interestingly,
inhibition of Notch worked in synergy with VEGF to induce
lymphatic sprouting both in vitro and in vivo. The induction of
lymphangiogenesis in response to Notch pathway inhibition seems
to depend on VEGFR-2 and VEGFR-3 signaling. Furthermore,
forced expression of Dll4 in LECs promoted adoption of the tip
cell position. These results suggest that the Notch pathway
negatively regulates lymphatic sprouting and directs stalk cell
specification in LECs.
Role of Notch signaling in LECs
In cultured LECs, the key components of the Notch pathway were
expressed at levels comparable with, or even higher than those in
BECs, indicating that Notch signaling is important for controlling
lymphangiogenesis. Notch1 and Notch4, the 2 Notch receptors
expressed in the vascular endothelium,1,2 have been shown to be
expressed in lymphatic vessels in normal and tumor tissues,32
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BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
Notch in inducing cell differentiation and suppressing cell growth,
which may be differently regulated in embryonic development and
in adults. It is conceivable that, in the embryos, Notch signaling is
important for the formation, remodeling, or maturation of the
lymphatic vasculature, whereas in adults its role is shifted to
maintenance of the quiescence of the established vessels. This
certainly seems to be the case for blood vessels in adults, as chronic
inhibition of Dll4/Notch signaling was recently shown to lead to
spontaneous hyperproliferation of BECs and the formation of
hemangiomas,36 and Notch1 haploinsufficiency resulted in vascular tumors.37 In addition, the differences between the endothelial
Notch receptors in zebrafish (Notch1b and Notch6) and in mammals (Notch1 and Notch4) may also contribute to the discrepancy.
Notch inhibition results in nonproductive blood vessels that are
poorly perfused.1 Whether the sprouting lymphatic vessels induced
by Notch inhibition are functional remains enigmatic. We and
others have previously shown that the lymphatic vessels are poorly
functional during sprouting and growth, but they gradually stabilize and become functional within a few months after growth factor
stimulation.38,39 The lymphatic vessels induced by Dll4-Fc likely
undergo a similar course of events.
Interaction between Notch signaling and the VEGF family
Figure 7. Lymphatic endothelial cells expressing Dll4 show an advantage in
competition for the tip cell position. (A) Schematic representation of the mouse
Dll4-EGFP fusion protein. (B-G) LECs transduced with mDll4-ECTM-EGFP or empty
retrovirus were prelabeled with Green (green) or Orange (red) CellTracker (CT),
respectively, and mixed in a 1:1 ratio to form the spheroids, which were treated with
VEGF (100 ng/mL) or VEGF-C (100 ng/mL) for 48 hours. The spheroids were
subsequently stained for mDll4 (white) and DAPI (blue). The arrows indicate Dll4⫹ tip
cells. (D,G) Low-power magnification of the spheroids in panels B,C,E and F (䡺).
Scale bars, 25 ␮m in panels B,C,E and F and 100 ␮m in panels D and G. (H) The
percentage of mDll4-positive tip cells (white cells in panels A-D) out of the total tip
cells was quantified and plotted against their expected values. The figure summarizes data from 2-3 independent experiments with a total of n ⫽ 122 for VEGF and
n ⫽ 229 for VEGF-C treatments. A 2-tailed binomial test was used for statistical
analysis. **P ⬍ .01; ***P ⬍ .001.
indicating that Notch signaling is active in the lymphatic endothelium in both physiologic and pathologic conditions.
Several papers showed that the Notch pathway was important
for blood vessel remodeling and ischemia-induced angiogenesis,
whereas blockade of Notch signaling resulted in impaired migration, proliferation, and survival of BECs and impaired the recovery
of blood flow in response to ischemia.33,34 However, the principal
role of endothelial Notch signaling appears to rather lie in the
regulation of blood vessel quiescence and lateral inhibition of new
sprout formation during angiogenesis: studies from mouse retina
models, tumor models, and in vitro 3D sprouting assays have
established that Notch signaling serves as a negative regulator of
VEGF-induced angiogenesis, and that inhibition of the Notch
pathway leads to excessive sprouting of blood vessels and nonproductive angiogenesis.1
The role of Notch signaling in LECs is less well understood. A
recent study reported that genetic targeting of Notch impaired LEC
migration during embryonic development in a zebrafish model.35
However, results from our mammalian model using adult mice
indicated that inhibition of Notch signaling rather induced lymphangiogenesis. This was further supported by 3D in vitro sprouting
assays using human LECs subjected to a Notch inhibitor, consistent
with the negative role of Notch in regulating the sprouting of the
blood vessels.1 The discrepancy between the study by Geudens et
al35 and our current work may be explained by the dual function of
Although VEGF potently promotes angiogenesis and sprouting of
BECs in vivo, it does not stimulate LEC sprouting, but rather
promotes circumferential enlargement of the lymphatic vessels
accompanied by LEC proliferation.11 Here we show that VEGF
up-regulates Dll4 expression in LECs, which activates Notch to
suppress the lymphatic sprouting in response to VEGF. Our gene
expression analysis indicated that LECs have a higher baseline
expression of several Notch pathway components compared with
BECs. Thus the weaker effects of VEGF on LECs compared with
BECs could be explained by the higher activity of Notch in the
LECs at baseline. The VEGF-C/VEGFR-3 pathway also activated
Notch signaling in LECs, which seemed to negatively regulate
VEGF-C–induced lymphatic sprouting when VEGF-C levels were
low, but once VEGFR-3 was fully activated by VEGF-C, it was
able to overcome the restriction imposed by Notch and potently
induced LEC sprouting. However, the grip on VEGF by Notch was
tighter, as Notch blockade could still enhance VEGF-induced
sprouting when VEGF was used at high concentrations.
In vivo, combinational treatment with VEGF and Dll4-Fc
induced both circumferential enlargement and sprouting of lymphatic vessels, whereas Dll4-Fc did not increase VEGF-C–induced
sprouting, consistent with the in vitro bead-sprouting assay. Thus,
the lymphatic sprouting induced by VEGF-C/VEGFR-3 is essentially unrestrained by Notch, whereas the lymphatic sprouting
activity of VEGF/VEGFR-2 is rather weak because of the tight
restriction imposed by Notch signaling. This is consistent with the
fact that the VEGFR-2 and VEGFR-3 signals in the ECs, although
generally overlapping, show some differences. For example in
angiogenesis, VEGFR-3 activation seems to potently promote EC
migration rather than proliferation.40,41 It should also be noted that
in the LECs, VEGFR-3 is expressed much more abundantly than
VEGFR-2,42 which may explain why Notch does not restrict
VEGFR-3–induced sprouting.
The lymphatic sprouting after Notch inhibition seemed to
depend on the VEGF/VEGFR-2 pathway, as Dll4-Fc by itself did
not induce sprouting in the spheroid assay of pure LECs, but
potentiated VEGF-induced sprouting to a similar extent as was
achieved by VEGF-C alone. The dependence on VEGF/VEGFR-2
signals was further supported by the fact that both a VEGFR-2
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BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
blocking Ab and VEGF trapping by VEGFR-1-Fc ablated the
Dll4-Fc–induced sprouts both in vivo and in vitro. This is not
surprising, given that the increased endothelial filopodia in the
developing retinas of Dll4⫹/⫺ mice were also suppressed by an Ab
targeting VEGFR-2 or by soluble VEGFR-1.43 The requirement for
VEGFR-3 is more complicated. Whereas VEGFR-3 is dispensable
for Dll4-Fc–induced LEC sprouting in vitro, blocking VEGFR-3
signaling suppressed Dll4-Fc–induced lymphangiogenesis in vivo.
The importance of VEGFR-3 signaling for lymphangiogenesis in
vivo is evidenced by the fact that activation of VEGFR-2 could not
rescue the lymphatic regression caused by prolonged systemic
VEGFR3-Fc expression.11 Importantly, Notch inhibition did not
enhance LEC sprouting induced by VEGF-C in any of the assays.
Thus, it is possible that in vivo, a low level of VEGF-C or
VEGFR-3 activity is required to prime LECs to become responsive
to VEGF, for example, by controlling a critical step in sprout
initiation.44 However in the downstream, lymphatic sprouting
induced by a high dose of VEGF, but not of VEGF-C, is restricted
by Notch signaling, as inhibition of Notch signaling enhanced
sprouting in response to VEGF, but not to VEGF-C. In our in vivo
model of adult mouse ears, inhibition of Notch alone induced
lymphangiogenesis. It therefore seems that blocking Notch was
able to sensitize the lymphatic endothelium to endogenous growth
factor signals emanating from the surrounding microenvironment.
Similar endothelial hyperreactivity was observed in certain blood
vascular beds of rats and cynomolgus monkeys that were exposed
to Notch inhibitors for prolonged periods of time.36
Mechanism of lymphatic sprouting induced by Notch inhibition
The exact mechanism of how Notch inhibits sprouting has not been
fully elucidated. In vivo and in vitro studies show that Notch
activation down-regulates VEGFR-2 expression in the BECs, thus
making them less responsive to VEGF.1 Although cultured BECs
up-regulate VEGFR-3 on NICD overexpression,32 inhibition of
Notch in vivo resulted in widespread VEGFR-3 expression and
sprouting of blood vessels.40 In the LECs, a recent study showed
that endothelial overexpression of the Notch1 intracellular domain
(N1ICD) up-regulated VEGFR-2 expression, whereas the Notch
targets Hey1 and Hey2, but not Hes1 or NICD, could downregulate VEGFR-3.29 Another study using N1ICD overexpression
did not report changes in the percentage of LECs positive for
VEGFR-3.32 We observed neither significant alteration in the
expression of VEGFR-2 or VEGFR-3, nor in VEGF-induced
VEGFR-2 phosphorylation when blocking Notch signaling in
LECs. Actually, whereas activation of Notch signaling suppressed
VEGFR-2 expression, Notch blockade by ␥-secretase inhibitor did
not alter VEGFR-2 levels in cultured BECs.45 Similarly, in vivo,
Notch inhibition failed to increase VEGFR-2 expression in the
retinal vessels.46 Thus, although forced activation of Notch signaling may tilt the balance of gene expression to one side, in the
loss-of-function condition the balance can be maintained by
alternative pathways. Taken together, these results imply that
Notch signaling probably does not restrict VEGF-induced lymphangiogenesis at the VEGF receptor level, but rather acts on a
downstream signaling cascade that would otherwise contribute to
the sprouting.
EphrinB2 expression was reduced by Notch signaling targeting,
as previously shown in BECs,27 but ephrinB2 blockade did not
reproduce the stimulatory effects of Dll4-Fc, and rather inhibited
the LEC sprouting induced by VEGF and VEGF-C, suggesting that
Notch regulates ephrinB2 in a negative feedback loop to control
LEC sprouting. In fact, the effects of the ephrinB2 blocking peptide
Notch RESTRICTS VEGF-INDUCED LYMPHANGIOGENESIS
1161
are most likely because of modulation of VEGFR-2 and VEGFR-3
signaling.47,48 Thus, in addition to the interplay between VEGF and
Notch signaling, a third signal may be also integrated in the
signaling network to determine the final outcome of vessel
quiescence/sprouting. Other Notch targets that account for LEC
sprouting induced by Notch inhibition remain to be explored.
Role of Notch in the LEC differentiation
Mosaic analysis of BECs in mice and zebrafish demonstrated that
Notch controls tip-stalk specification in growing sprouts, as
evidenced by the dominant tip cell adoption of the Notch-deficient
cells and, conversely, exclusion of NICD overexpressing cells from
the tip position.31,49 According to this “tug-of-war” concept,
neighboring ECs compete for the tip position through contactdependent Dll4-Notch signaling.1 Here, we demonstrate that similar cell-fate control by Notch also holds true in the LECs: the cells
with forced Dll4 expression preferentially adopt the tip cell
position, presumably through a similar mechanism as in BECs.
Regulation on the transcription factor Prox1, the master gene
specifying lymphatic cell fate,28 by Notch signaling has been
studied in early embryonic development and in vitro. Endothelial
deletion of Rbpj, a transcriptional coactivator acting in concert with
NICD to activate Notch target genes, did not interfere with Prox1
expression or lymphatic development when analyzed at E10.0.50
Our in vivo data suggest that Notch signaling does not influence
Prox1 expression in adult lymphatic vessels, although this conclusion needs to be confirmed by investigation of later embryonic and
postnatal stages of development. In cultured LECs, overexpression
of NICD suppressed Prox1 expression,29 whereas in our experiments Notch suppression did not alter Prox1 levels.
Taken together, we established a negative role of Notch
signaling in controlling lymphatic sprouting induced by VEGF, and
provided an explanation for the weak lymphangiogenic activity of
VEGF despite ample levels of VEGFR-2 expression in the LECs.
This finding should have considerable biological significance, as it
provides a further mechanistic explanation for the remarkable
specificity of VEGF for stimulating angiogenesis. Furthermore, our
results caution the possible future use of Notch pathway inhibitors
in the treatment of cancer, as they may sensitize tumor-associated
lymphatic vessels to VEGF, promote tumor lymphangiogenesis,
and drive metastatic spreading of the malignancy.
Acknowledgments
The authors thank Dr Michael Jeltsch for the VEGF-C protein, Denis
Tvorogov and Ralf Adams for helpful discussions, Tapio Tainola for
expert technical assistance, the Molecular Imaging Unit for microscopy
support, and the staff at the Biomedicum Helsinki and the Haartman
Institute Animal Facilities for excellent animal husbandry.
This work was supported by grants from the Finnish Academy,
the Finnish Cancer Research Organizations, the European Community’s Seventh Framework Program FP7/2007-2011 grant 201279,
the Association for International Cancer Research, and the Helsinki
University Research Fund.
Authorship
Contribution: W.Z. and T.T. designed and performed the research,
analyzed the results, and wrote the paper; M.Y. and A.A. constructed
AAV vectors and produced the viruses; T.H. performed serologic
evaluation of AAV transduction; S.K. generated the Dll4 plasmids;
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1162
BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
ZHENG et al
T.K. generated VEGFR3(D4-7)-Fc; K.L. contributed to the spheroidsprouting assay; S.Y.-H. provided the adenoviruses; and K.A.
designed the research, interpreted the data, and wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Kari Alitalo, Molecular/Cancer Biology
Laboratory, Research Programs Unit, Institute for Molecular
Medicine Finland and Helsinki University Hospital Biomedicum Helsinki, PO Box 63 (Haartmaninkatu 8), Mail Code
00014, Helsinki, Finland; e-mail: [email protected].
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2011 118: 1154-1162
doi:10.1182/blood-2010-11-317800 originally published
online May 12, 2011
Notch restricts lymphatic vessel sprouting induced by vascular
endothelial growth factor
Wei Zheng, Tuomas Tammela, Masahiro Yamamoto, Andrey Anisimov, Tanja Holopainen, Seppo
Kaijalainen, Terhi Karpanen, Kaisa Lehti, Seppo Ylä-Herttuala and Kari Alitalo
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