ATVB In Focus

Developmental Biology in the Vasculature

Series Editor: Mark Majesky, PhD

Mechanisms of Vessel Branching

Filopodia on Endothelial Tip Cells Lead the Way
Frederik De Smet, Inmaculada Segura, Katrien De Bock, Philipp J. Hohensinner, Peter Carmeliet

Abstract—Filopodia, “the fingers that do the walking,” have been identified on endothelial cells at the tip of sprouting
vessels for half a century, but the key role of the tip cell in vessel branching has been recognized only in the past few
years. A model is emerging, whereby tip cells lead the way in a branching vessel, stalk cells elongate the sprout, and
a very recently discovered phalanx cell ensures quiescence and perfusion of the newly formed branch. Recent genetic
studies have shed light on the molecular signature of these distinct endothelial phenotypes; this provides a novel
conceptual framework of how vessel morphogenesis occurs. Here, we will discuss the molecular candidates that
participate in the decision of endothelial cells to adapt these distinct fates and highlight the emerging insights on how
these cells send out filopodia while navigating. (Arterioscler Thromb Vasc Biol. 2009;29:639-649.)
Key Words: endothelial tip cells 䡲 stalk cells 䡲 phalanx cells 䡲 filopodia 䡲 sprouting angiogenesis

M uch has been learned about the importance of angio-

genesis in health and disease, but our understanding of
how vessels branch remains incomplete. An exciting break-
through in the field has been the recognition that several types
of specialized endothelial cells (ECs), each with distinct
cellular fate specifications, are required to build a functional
branch.1 The first and, to a certain extent, the most undertak-
ing cell in a vessel branch is the “tip cell,” which leads the
way. With their continuously searching filopodia, these tip
cells sense and respond to guidance cues in their microenvi-
ronment, similar to how an axonal growth cone in the nervous
system2 or an epithelial tip cell in the fruitfly airway system3
explores its surroundings. Even in the sea squirt, tip cells use
filopodia to build a primitive vascular network.4 It is therefore
not surprising that several classes of molecules and principles,
used by navigating axons or epithelial cells, are evolutionary
conserved and shared, and even might have been coopted by the
migrating endothelial tip cell.1,2 “Stalk cells” trail behind the tip
cell and elongate the stalk of the sprout; they proliferate, form
junctions, lay down extracellular matrix, and form a lumen.
“Phalanx cells” are the most quiescent ECs, lining vessels once
the new vessel branches have been consolidated; they form a
smooth cobblestone monolayer and are aligned as in a phalanx
formation of the ancient Greek soldiers, are covered by peri-
cytes, stick to each other via tight junctions, are embedded in a
thick basement membrane, and stay foot. These cells are
engaged in optimizing blood flow, tissue perfusion, and oxygen-
ation.5 Tip, stalk, and phalanx ECs each have a specialized
function in vessel branching. How these cells execute their job
depends, to a substantial extent, on the organization of their
cytoskeleton; for instance, for an EC to migrate, it needs to form
spike-like filopodia, fan-like lamellipodia, and polarize its actin
cytoskeleton in the direction of migration.
Endothelial Tip Cells
Even though the existence of “filliform processes” at the tip
of endothelial sprouts was already observed in the brain
almost half a century ago,6 the concept and importance of
vessel guidance was not fully appreciated until recently.7,8
Classical studies have documented the existence of “seamless
ECs” in vivo and postulated the existence of different
endothelial fates (trunk cells with lumen and tip cells without
lumen) on growing sprout capillaries.9 Electron microscopy
studies further described the existence of filopodia at the
leading edge of growing capillaries (Figure 1a and 1b).10
Tip cells have now been observed in various models of
sprouting angiogenesis. Key features of this cell are their
location at the forefront of vessel branches, highly polarized
nature, and numerous filopodia probing the environment,
while migrating toward an angiogenic stimulus.7,11 They do
not form a lumen and, with some exceptions, proliferate
minimally (Figure 1c and 1d).7,12,13 Tip cells have a specific
molecular signature, characterized by the expression of
VEGF receptor (VEGFR) 2, VEGFR3, platelet-derived
growth factor (PDGF)-BB, Unc5B, Delta-like ligand-4
(Dll4), neuropilin-1 (NRP1), and others (see below).7,14 –18
These cells detect gradients of navigatory cues and integrate
combinatorial molecular codes into directional migration.

Received January 23, 2009; revision accepted February 23, 2009.

From the Vesalius Research Center, VIB, K.U. Leuven, Belgium.
Correspondence to Peter Carmeliet, MD, PhD, Vesalius Research Center, VIB, K.U. Leuven, Campus Gasthuisberg, Herestraat 49, B-3000, Leuven,
Belgium. E-mail peter.carmeliet@vib-kuleuven.be
© 2009 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.109.185165

639
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640 Arterioscler Thromb Vasc Biol May 2009

lularly, generating the Notch intracellular domain (NICD)

Figure 1. Vascular sprouts are guided by endothelial tip cells. a,
Original picture showing an embryonic endothelial tip cell
obtained by electron microscopy (reprinted with permission
from10). b, Confocal micrograph showing filopodia extensions at
the leading tip cell (reprinted with permission from7). c, Scheme
from 1972 proposing several endothelial subtypes in the angio-
genic sprout (reprinted with permission from9). d, Schematic
representation of a tip cell (green) extending filopodia toward an
angiogenic stimulus (red gradient), followed by stalk cells (pur-
ple), while phalanx cells (gray) remain quiescent.
Central to their phenotype is the formation of filopodia. We
will first describe how tip cells are selected in the nascent
vessel branch, and explain why ECs do not move as a sheet
but form a sprout in response to an angiogenic signal.
Induction of Tip Cell Formation by VEGFR2
and Dll4
Genetic studies show that a VEGF gradient is important in the
process of selection and induction of the endothelial tip cell.7
Via binding to VEGFR2, VEGF induces a signaling cascade,
which enables one EC to take the lead and become a tip cell,
while its neighbors are prevented from doing so and, instead,
are instructed to become stalk cells. Such lateral inhibition
relies on tip-to-stalk cell communication by Dll4/Notch
signaling (Figure 2a).18 –20 ECs express various Notch recep-
tors (Notch1, 3, 4) and ligands (Dll1, Dll4, Jagged1,
Jagged2).21 After ligand binding, Notch is cleaved intracel-
that acts as a transcriptional regulator.21 Tip cells are exposed
to the highest levels of VEGF, which induces the expression
of Dll4 in these cells.21 Dll4 binds to Notch on neighboring
(future stalk) ECs and downregulates VEGFR2 signaling; this
dampens the VEGF-induced expression of Dll4 in these cells,
thereby establishing a self-reinforcing feedback that allows
the leading cell to gain and retain its tip position, while
preventing the follower neighbors from leaving their position
in the stalk. Genetic mosaic studies reveal that Notch regu-
lates EC specification by actively suppressing the tip cell
phenotype in stalk cells.19
A number of questions about the mechanisms of Dll4 and
Notch remain unanswered. For instance, it is unknown
whether Dll4 reverse-signaling prevents the tip cell from
becoming a stalk cell by inactivating Notch signaling in this
cell through internalization of the receptor.22 Another ques-
tion is whether Dll4 induces cytoskeletal rearrangements in
the tip cell. Notch ligands such as Dll4, Dll1, and Jagged1
contain a PDZ-binding motif, which facilitates interactions
with adaptor proteins, thereby mediating adhesion and migra-
tion in the ligand-expressing cell.23 Such adaptor molecules
include Dlg1, MAGI proteins, and syntenin, which interact
with the cytoskeleton.23
Lamellipodia and Filopodia Lead the Way
The key job of endothelial tip cells is to navigate, a process
that relies on correct probing of microenvironmental cues,
and translating them into a dynamic process of adhesion (at
the front) and deadhesion (at the rear), that ultimately leads to
cell movement. Therefore, the tip cell forms lamellipodia and
filopodia. Surprisingly, however, relatively little is known
about the processes regulating the assembly of these cellular
protrusions in ECs. We will therefore briefly overview some
key insights of this process, as deduced from studying the
axon growth cone, and thereafter discuss our current under-
standing of these processes in ECs.
Lamellipodia are short veil-like structures in close prox-
imity to the plasma membrane that contain a highly branched
actin network. Filopodia, on the other hand, consist of long
spiky plasma membrane protrusions containing tight parallel
bundles of filamentous actin (F-actin), which usually extend
from lamellipodia (Figure 3a).24 –26 Filopodia and lamellipo-

a Role of VEGF and Dll4 b Cytoskeletal rearrangements c Inflammatory cytokines

in tip cell formation induce tip cell formation
VEGF VEGF
Figure 2. Molecular pathways of tip cell signaling.
VEGFR2 VEGFR2
FGF2 Angiostatin TNFα a, VEGF signaling via VEGFR2 enables tip cell for-
VEGFR3 FGFR mation (green), whereas Dll4 signaling induces
NRP1 Amot Netrin Bradykinin stalk cells (purple). VEGFR3 is upregulated,
Integrins TNFR
whereas Neuropilin 1 (NRP1) is maintained. b, Dif-
Unc5B ferent signaling cascades converge to small
SRF CD13
MMP GTPases activation, thereby regulating filopodia
Dll4 induction Jagged1 and lamellipodia formation in endothelial cells. c,
active GTPase induction
active Inflammatory cytokines (blue gradient), including
Filopodia, GTPase tumor necrosis factor ␣ (TNF␣) and bradykinin,
Lamellipodia, also induce a tip cell phenotype.
?
Cell migration
Dll4 Jagged1
Notch Notch

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 De Smet et al Mechanisms of Vessel Branching 641

a Stress fibers Lamellipodia Filopodia

active
small GTPase RhoA Rac1 Cdc42

Exta Cellular
Matrix

Focal
contact point

b Figure 3. Overview of the filopodia/la-

mellipodia machinery during cell migra-
G-actin
F-actin tion. a, Schematic representation of fila-
Negative end Positive end mentous actin in filopodia (red),
(-) Cofilin Profilin, lamellipodia (blue), and stress fibers
(+)
(green). Cellular protrusions anchor in
Formin,
the extracellular matrix by focal contact
ARP2/3 Ena/Vasp points (brown). b, Regulation of filamen-
Wasp tous (F-actin) and globular (G-actin) actin
Retraction through Elongation through
dynamics. c, Guanine nucleotide
de-polymerization and Branching of F-actin to polymerization
exchange factors (GEFs) and GTPase
retrograde flow generate meshworks for filopodia extension activating proteins (GAPs) regulate GTP-
bound (active) and GDP-bound (inactive)
RhoA, Rac1, and Cdc42 states, respec-
c Guanine nucleotide d RhoA Rac1 Cdc42 tively. d, RhoA, Rac1, and Cdc42 down-
exchange factor stream signaling regulates actin
(GEF) dynamics.
+GTP ROCK PAK WASP-
ARP2/3
small GTPase small GTPase
LIMK
GDP GTP
Pi branching
GTPase Cofilin
activating protein
(GAP)
de-polymerization
inactive active

dia are highly dynamic structures, generated within minutes
after stimulation.27 Both structures are capable of probing the
environment, thereby sensing the presence of attractive or
repulsive guidance cues.26 An attractive cue will induce
F-actin polymerization thereby extending filopodia, whereas
a repulsive cue causes depolymerization and retrograde actin
flow, resulting in retraction.26 Filopodia and lamellipodia also
adhere and form focal contact points to connect the cytoskel-
eton to the extracellular matrix (ECM).27 This allows stress
fibers of actin/myosin filaments to pull the cell toward these
anchors, and induce forward movement.27
F-actin fibers are elongated at their ‘barbed’ or positive end
by proteins of the Profilin, Ena/Vasp, and Formin families,
which promote polymerization of G-actin into long F-actin
strings in proximity of the plasma membrane (Figure 3b).24,25
Whereas Profilin and Formin directly bind to G-actin and
present it to the extending fiber, Ena/Vasp functions as an
anticapping protein keeping the barbed end clear of inhibitory
cap-proteins.25–27 Elongation of these filaments pushes the
leading edge forward and promotes cell migration. At the
“pointed” or negative end of the actin-filament (pointing
toward the inside of the cell), Cofilin depolymerizes and
shortens the actin string.25–27 Branching of F-actin filaments
is mediated by the Actin-related protein-2/3 (ARP2/3) com-
plex and members of the Wiscott-Aldrich Syndrome protein
(WASP) family.25,26 Myosin X groups these growing fila-
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ments into parallel bundles, which are cross-linked by mem-
bers of the Fascin protein family.24 Coincidently, several
other proteins, including members of the Inverse (I)-BAR
domain such as Insulin receptors substrate p53 (IRSp53),
prepare the plasma membrane by inducing bulging of its
surface.24
The main regulators of filopodia and lamellipodia forma-
tion are members of the Rho small GTPases, of which RhoA,
Rac1, and Cdc42 have been most extensively studied (Figure
3c).25,26 These molecules are activated by binding of GTP
nucleotides, supplied by Guanine nucleotide Exchange Fac-
tors (GEFs). Inactivation occurs via their intrinsic GTPase
activity, which converts GTP into GDP; this process is
stimulated by GTPase Activating Proteins (GAPs). RhoA,
Rac1, and Cdc42 are activated in response to stimulation of
many membrane receptors, including receptor tyrosine
kinases and G protein– coupled receptors.26 Cdc42 and Rac1
regulate filopodia and lamellipodia formation, respectively,
through activation of p21-activated kinase (PAK); RhoA is
involved in adhesion and forward movement through regula-
tion of stress fiber formation via the Rho-associated serine-
threonine protein kinase (ROCK; Figure 3d).25–27 PAK and
ROCK activate LIM-kinase, which, through inhibition of
Cofilin, blocks F-actin depolymerization.26 Activated Cdc42
and Rac1 also interact with the WASP:ARP2/3 complex,
thereby inducing F-actin branching.25 Filopodia extension is
642 Arterioscler Thromb Vasc Biol May 2009
also regulated by targeting Ena/Vasp proteins to the mem-
brane at the leading edge of migrating cells.26 Once extended,
filopodia make contact with ECM components such as
laminin, fibronectin, and collagen through integrins on their
surface that initiate the formation of focal contact points.27 An
important regulator of this process is the focal adhesion
kinase (FAK).28 Hence, signals from integrins and growth
factor receptors are transduced to the cytoskeleton in these
filopodia.28
Formation and Regulation of Filopodia in
Endothelial Tip Cells
To navigate, tip cells become polarized, so that their leading
front extends filopodia, whereas their rear maintains contact
with trailing stalk cells to avoid branch desintegration. How
VEGF induces these processes is poorly understood. Based
on experiments in fibroblasts and neurons, Rho small GTPase
proteins have been identified as key regulators of cell
migration and morphogenesis.25–27 Increasing evidence sug-
gests that these molecules also act downstream of VEGFR2
to regulate the formation of filopodia, lamellipodia, and stress
fibers, and consolidate adhesion in ECs as well. Indeed, in
response to VEGF, Cdc42 triggers the formation of filopodia
and regulates cell polarization through microtubule organiza-
tion, whereas Rac1, together with PAK, controls lamellipodia
formation in response to VEGF.29,30 Loss of Rac1 results in
early embryonic lethality attributable to defective vessel
branching.31 RhoA induces stress fiber formation and medi-
ates EC permeability, migration, and stabilization of capillary
tubes in response to VEGF.30,32,33 Shear stress also increases
RhoA activity, and the formation of stress fibers, focal
adhesions, and junctional complexes.34 Inhibition of RhoA
impairs EC migration and tube formation, while a dominant
negative RhoA protein inhibits angiogenesis.35 As in other
cells, Cdc42 activates Rac1 in ECs.30
For Rac1 to promote EC migration, it needs to be activated
by the GEF Vav2.36 In vitro, silencing of Vav2 impairs
VEGF-induced EC migration, which is in line with its
function in neurite outgrowth,36 whereas Vav2 knockout mice
have cardiovascular defects.37 Wave2, a member of the
WASP proteins, is also required for actin reorganization and
EC movement38: loss of Wave2 impairs vessel branching and
the formation of EC lamellipodia. Single deficiency of each
of the Ena/VASP proteins, which promote actin polymeriza-
tion, results in relatively subtle phenotypes, but triple defi-
ciency disrupts vessel integrity and endothelial barrier func-
tion.39 Several components of the machinery responsible to
generate and consolidate focal adhesion points in other cells
is also conserved in ECs, and is, in part, mediated by
integrins.28,40 Moreover, FAK regulates EC migration in
response to VEGF, whereas deficiency of FAK in ECs leads
to vessel defects and regression.40
Genetic studies reveal that Aquaporin-1 (AQP1), a water
channel protein increasing water permeability in ECs, regu-
lates EC migration.41 As a consequence of local actin depo-
lymerization and transmembrane ionic fluxes, the cytoplasm
adjacent to the leading edge of migrating cells undergoes
rapid changes in osmolality. AQPs, polarized to lamellipodia,
facilitate osmotic water flow across the plasma membrane in
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lamellipodia. Water entry increases local hydrostatic pres-
sure, producing cell membrane expansion to form a protru-
sion, thereby enhancing lamellipodia dynamics.41 According
to this model, actin repolymerization thereafter stabilizes the
protrusion.
Modulation of VEGF Signaling in Tip Cells
Besides VEGFR2, tip cells also express other VEGF recep-
tors, including VEGFR3 (also known as Flt4) and NRP1. In
development, VEGFR3 is present in endothelia, but becomes
largely restricted to the lymphatic endothelium in adult-
hood.16 However, in active vascular endothelia, VEGFR3
reappears42 and its expression is mainly confined to filopodial
extensions on tip cells at the sprouting front.16 Blocking
VEGFR3 reduces the number of sprouts and branch points
and EC proliferation. VEGFR2 signaling induces VEGFR3
expression in tip cells, whereas Notch downregulates its
expression in stalk cells.16 As is the case for VEGFR2, tip
cells express higher levels of VEGFR3 than stalk cells.
VEGFR2 and -3 are able to form heterodimers, and may
transmit distinct signals than receptor homodimers.43 An
intriguing possibility is that such VEGFR2/3 heterodimers
may be involved in tip cell functions. VEGFR3 also regulates
vessel integrity, though it is unknown whether this is an effect
mediated via tip or, more likely, via stalk cells.42
NRPs function as endothelial receptors for members of the
Sema3 and VEGF families.2,44 As VEGF receptors, NRPs are
essential for cardiovascular development and tumor angio-
genesis.44 Both NRP1 and NRP2 are detected in the devel-
oping vasculature. The use of an anti-NRP1 antibody engi-
neered to bind solely VEGF, and not Sema3s, has revealed
that Sema3s have little effect on VEGF/NRP1-driven vascu-
lar development.44 However, Sema3F is a potent inhibitor of
tumor angiogenesis, progression, and metastasis.44 NRPs
either form receptor complexes with VEGFR1, -2, or -344,45
or induce direct signaling through synectin (also known as
NIP), a PDZ domain-containing protein that acts as a scaffold
for downstream signaling cascades. Genetic studies show that
NRP1 and synectin regulate vessel branching and morpho-
genesis.44,46 In the absence of NRP1, sprouts in the embryonic
hindbrain do not branch and fuse into a vascular plexus,15
while blocking the binding of VEGF to NRP1 prevents
vascular remodeling.44 The defective vessel branching and
fusion were not caused by the absence of tip cells or filopodia
but were the result of defects in lateral filopodia extension,
which is critical for turning and fusion of tip cells.15 How
NRP1 regulates tip cell filopodia and in particular their lateral
extensions remains to be further explored. NRP1 induces
filopodial extension and navigation of tumor cells and neu-
rons, though this activity is context-dependent. In tumor cells,
filopodia formation in response to NRP1/VEGFR2 signaling
is mediated via Cdc42 activation.47
Novel Regulators of Tip Cell Formation
Only a few other molecules have been identified to induce
filopodia formation in ECs (Figure 2c). Inflammation is
known to activate ECs and stimulate vessel branching. Brief
exposure to the inflammatory cytokine TNF␣ primes ECs for
angiogenic sprouting by inducing a tip cell phenotype and
 De Smet et al
expression of the tip cell markers PDGF-BB and VEGFR2.48
Furthermore, the Notch ligand Jagged1 was induced in tip
cells via a NF-kB dependent mechanism, raising the question
whether Jagged1 might have a similar role in tip cell
induction in pathological (inflammatory) angiogenesis as
Dll4 has in physiological conditions.48 Another inflammatory
mediator, bradykinin, induces filopodia formation in ECs
through activation of the Cdc42 pathway, a process which is
modulated by the transmembrane peptidase CD13. 49
Sphingosine-1-phosphate (S1P), an antiinflammatory phos-
pholipid derivative which has angiogenic and chemo-
attractant properties,50 has also been involved in the forma-
tion lamellipodia on ECs. It is released from activated
platelets, and exerts its function on ECs via binding to its
S1P-G protein– coupled receptors (S1P1–3). S1P1 signaling
is involved in the translocation of the Arp2/3 complexes from
an intracellular location to the cellular migratory front,
resulting in the formation of lamellipodia.50 This process is
dependent on Cdc42 and Rac1 activation.50
Genetic studies in the Drosophila airway system reveal
that fibroblast growth factors (FGFs) determine the specifi-
cation of an epithelial tip cell.3 Indeed, the FGF homologue
Branchless, which binds to its receptor Breathless on tracheal
cells promotes outgrowth of terminal branches. Airway tip
cells respond directly to Branchless and lead branch out-
growth, whereas trailing stalk cells receive a secondary signal
to follow the lead cells and form a tube. These roles are not
genetically prespecified; rather, there is competition between
cells such that those with the highest FGFR activity take the
tip position, whereas those with less FGFR activity assume
subsidiary positions and form the branch stalk. Competition
appears to involve Notch-mediated lateral inhibition that
prevents extra cells from assuming the lead,3 analogous to the
endothelial system.1 Whether FGFR levels also determine the
specification of an endothelial tip versus stalk cell remains to
be determined.1 Interestingly, in a mixed EC population, in
which FGF receptor-1 (FGFR1) was silenced in half of the
cells, receptor-expressing cells pioneered migration in re-
sponse to FGF2, whereas silenced cells were unable to take
the lead but, nonetheless, were still capable of trailing behind
the leader.51 These findings suggest that FGFRs may regulate
endothelial tip cell migration as well (Figure 2b).
Angiomotin (Amot), a membrane-associated protein that
binds angiostatin, is expressed by activated endothelia, and
controls EC migration via its PDZ binding motif.52 Trans-
genic mice expressing a PDZ-deficient Amot in ECs lose
their response to growth factors, which leads to vessel
defects.52 Amot deficiency in mouse and zebrafish also
impairs vessel branching and integrity, with defective filop-
odia formation, and stalling of sprouting intersegmental
vessels (ISVs; Figure 2b).52 Although Amot does not affect
VEGF-induced EC survival or proliferation, it renders ECs
unresponsive to chemotactic stimuli. The subcellular local-
ization of Amot in lamellipodia and its ability to bind cell
polarity proteins indicate that it is required for cytoskeletal
reorganization during migration.52 Moreover, Syx, a RhoA
GEF, was recently identified as an important PDZ interacting
partner of Amot, regulating the activity of RhoA in the
leading front of the migrating cell.53
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Mechanisms of Vessel Branching 643
Another novel player is the Serum Response Factor (SRF)
transcription factor, which interacts with other transcription
factors including members of the ETS and GATA families.54
Late in development, SRF expression becomes confined to
ECs in small vessels, more precisely in tip and stalk ECs.54
Conditional endothelial SRF-deficient embryos succumb be-
cause of reduced vessel branching. This is not attributable to
a reduced number of tip cells or to abnormal expression of
Notch1 or Dll4 but to the presence of thinner and fewer
filopodia per tip cell, with a disorganized actin structure at the
base of each filopodium. This abnormal actin organization
also occurs in stalk cells, suggesting a fundamental role in
cytoskeletal rearrangements. In vitro, loss of SRF-function
results in aberrant EC migration and tube-formation (Figure
2b) attrituable to decreased F-actin formation. SRF has been
linked to RhoA signaling, with which it cooperates in
regulating transcription of ß-actin. Moreover, loss of SRF
reduces VE-Cadherin expression, which could explain the
loss of vessel integrity and stability.54 Another regulator of
EC filopodia formation is the Melanoma-associated antigen
(MAGE) D1, which was initially identified as a cell surface
antigen expressed by tumor cells. Overexpression of
MAGE-D1 inhibits EC migration and adhesion, and disrupts
cytoskeletal rearrangements and lamellipodia formation.55
Not surprisingly, some classical axon guidance molecules
also regulate tip cell navigation. The Netrin guidance receptor
Unc5B is also expressed by endothelial tip cells.17 Binding of
netrin-1 to Unc5B induces collapse of filopodia in the
developing retina (Figure 2b), whereas knockdown of Unc5B
induces ectopic filopodial extensions.17 In Dll4 heterozygous-
deficient mice, an increase in Unc5B-positive tip cells was
also observed.18 The bidirectional signaling system of trans-
membrane Eph receptors and ephrin ligands is involved in
arterial/venous boundary formation in the embryo.1 Ephrin/
Eph family members are also reciprocally expressed between
blood vessels and their surrounding tissues,1 and complemen-
tary ligand/receptor expression patterns provide guidance
cues during vascular development, similar to the mechanism
described for neuronal development.2 In the process of
spinogenesis, motile dendritic filopodia explore their envi-
ronment to contact with the appropriate presynaptic partner,
while EphB forward signaling is required for filopodia
motility and synaptogenesis.56 All these observations raise the
possibility that Ephrin/Eph signaling or other molecules
involved in this neuronal process might also regulate endo-
thelial filopodia formation.
The Invasiveness of the Tip Cell
Sprouting angiogenesis is an invasive process that requires
proteolytic degradation of the extracellular matrix (ECM).
Tip ECs express matrix metalloproteinases (MMPs), and their
expression is regulated by the composition of the surrounding
ECM they have to invade (Figure 2b). Membrane type-1
MMP (MT1-MMP; also known as MMP14) regulates angio-
genesis12 and appears indispensable for ECs to form invading
channels.33,57 There is ample evidence that MT1-MMP is
present at the leading tip of invading ECs,12,57 while its
expression is downregulated in stalk cells during vessel
stabilization and maturation as a result of an endothelial-
644 Arterioscler Thromb Vasc Biol May 2009
a Notch signaling induces b Mechanisms of lumen formation
a stalk cell phenotype
?
Dll4 pinocytosis
coalescence
Dll1Jagged1
Notch1
NICD
Intra-cellular
c “Push” model of vessel sprouting
Flt1 VEGFR2/3
Nrarp NRP1 EGFL7+/+
Wnt
Cell-cell
contact
pericyte crosstalk.12 The role and need for MMPs in vessel
branching may vary. Indeed, vessels in the adult are embed-
ded in a thick basement membrane (rich in type IV collagen
and laminin), whereas the basement membrane is merely a
thin layer or even absent during development, which might
explain why loss of MT1-MMP does not impair vascular
development in the embryo.12 These findings thus suggest
that degradation of the basement membrane is not limiting
vessel branching in the embryo, but a more critical hurdle in
the adult.
ECM components are often used by tip cells as scaffold to
navigate.58 Also, on arrest of VEGF-targeted therapy, vessels
regrow alongside ghost tracks of basement membrane.59
Integrin receptors induce cytoskeletal rearrangements, each
with specific effects; for instance, ␣5ß1 and ␣vß3 (both
receptors of fibronectin) differentially regulate activation of
Cofilin and thereby also EC migration (Figure 2b).60 Lami-
nins are heterotrimeric protein complexes, which are pro-
duced by both tip and stalk cells and deposited as pericellular
matrix.61 In the absence of laminin ß1 or ␥1 subunits, vessel
sprouts fail to form normally.61 When vessels branch, EC tip
cells are exposed to different ECM components than those
present in the basement membrane; some of these matrix
molecules also stimulate sprout formation.58
Endothelial Stalk Cells
A second endothelial subtype, called “stalk cell,” trails
behind the leading tip cell. Their task is to proliferate,
elongate the stalk, form a lumen, and connect to the circula-
tion.7,11 In contrast to tip cells, stalk cells do not extend
filopodia.7
Induction of Stalk Cells by Notch and Wnt
As explained above, lateral inhibition via Dll4/Notch under-
lies the selection of a tip cell at the expense of its neighbors,
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Figure 4. Molecular pathways of stalk
cell signaling. a, A stalk cell (purple) is
induced by lateral inhibition through Dll4/
Notch signaling, which reduces the
expression levels of VEGFRs and NRP
and, via Nrarp, stabilizes Wnt signaling.
Inter-cellular Other Notch ligands (Dll1 and Jagged1)
are also present. b, Schematic repre-
sentation of intracellular and intercellular
models of lumen formation. c, Perivas-
EGFL7-/- cular EGFL7 deposition (blue) in the
angiogenic sprout regulates vascular
morphogenesis.
Tip cell
Stalk cell
Phalanx cell
VEGF
EGFL7
which are instructed to become stalk cells (Figure 4a).1,19
Notch signaling in stalk cells dampens the migratory response
to VEGF, impairs filopodia extension, and, overall, inhibits
vessel branching by lowering the expression of VEGFR2,
VEGFR3, NRP1, and CXCR4.16,62 Hence, inhibition of
Notch signaling increases vessel branching, by promoting a
switch from stalk to tip cell differentiation and stimulating
migration and proliferation of tip cells, in the mouse retina
and hindbrain,18,19,63 zebrafish embryo,14 and tumor mod-
els.20,64,65 Notch-regulated ankyrin-repeat protein (Nrarp) is a
downstream target of Notch that counteracts Notch signaling
by destabilizing NICD. Recent studies reveal that Nrarp is
expressed in stalk cells at branch points, where it overcomes
the activity of Notch to induce cell cycle arrest and quies-
cence.66 At the same time, Nrarp stimulates Wnt signaling in
stalk cells, which stabilizes the stalk and prevents EC retrac-
tion in part via tightening EC junctions.66
Specification of the tip/stalk cell phenotype by Notch is
likely more complex. Indeed, Dll4 is not the only Notch
ligand expressed in sprouting vessels. Notably, loss of Dll4,18
Jagged1, or Dll121 each results in distinct vascular defects,
indicating that these 3 ligands are not functionally redundant.
Also, expression of these components is nonoverlapping in
postnatal retinal vessel development: Dll4 is the only
ligand expressed in tip cells, whereas Jagged1 and Dll1 are
present in stalk cells.21 Soluble Jagged1 reduces tip cell
number, filopodia, and vessels density.19,67 An outstanding
question is whether Jagged1 instructs cells at the vascular
front to become or remain tip cells, possibly via regulating
Notch signaling in this cell. Differences in Notch signaling
by distinct ligands may fine-tune vessel branching, as
occurs in other cellular systems, such as in lymphocyte
development.68
Dll4/Notch signaling upregulates the expression of genes
that influence EC migration and adhesion, such as VCAM1,
 De Smet et al
the RhoGTPase RND1, or the RasGEF RAPGEF5.69 Dll4/
Notch also promotes cell adhesion by activating ß1-integrins
through NICD transcription-independent mechanisms.70
Further, Notch1 stimulation by Jagged1 induces microtu-
bule stabilization in neurons,71 while promoting cell– cell
over cell–ECM interactions in fibroblasts72; if this would
be also the case in ECs, Notch would hereby promote stalk
stabilization and prevent stalk cell retraction. Interestingly,
the ECM protein Microfibril-Associated Glycoprotein-2
(MAGP-2) also induces actin rearrangements and vessel
branching through antagonizing Notch signaling in ECs.73
Fine Tuning of Stalk Morphogenesis by sFlt1
VEGFR1 (Flt1) and its soluble form, sFlt1, act as a trap for
VEGF, VEGF-B, and PlGF.74 Both endothelial tip and stalk
cells express VEGFR1 during retinal vascular development.7
VEGFR1 and its soluble form are upregulated on Dll4-acti-
vated Notch-signaling in stalk cells,69 yet neutralization of
VEGFR1 does not affect vessel branching in vivo.7 Also,
␥-secretase, which proteolytically activates Notch in stalk
cells, cleaves membrane-anchored VEGFR1: release of its
intracellular domain has been proposed to inhibit VEGFR2
signaling, while shedding of its extracellular sFlt1 domain
may further dampen VEGF by acting as a trap.75 All these
effects may potentially reduce or spatially restrict the overall
response of ECs to VEGF through sequestration of extracel-
lular VEGF. Although PlGF may activate ECs via signaling
through membrane-anchored VEGFR1 in pathological con-
ditions,74 it remains to be defined whether this involves an
effect on tip and/or stalk cells.
Stalk Cells Maintain Vessel Integrity
To elongate the stalk, stalk cells must divide, maintain
contact with the leading tip cell, and form a lumen. Similar to
tip cells, maintenance of a stalk cell phenotype requires
cytoskeletal restructuring. However, the molecular (down-
stream) mechanisms of how trailing cells remain in contact
with the leading front remain mostly unexplored. VE-
Cadherin is important to maintain cell– cell contacts, as its
absence induces random nondirectional migration of discon-
nected cells.51 Cadherins do not form rigid and fixed struc-
tures, but rather exhibit a flow-like movement on reorgani-
zation of the cytoskeleton.76 Indeed, when epithelial cells
move, cadherin clusters rapidly form at the leading edge,
while they are absorbed at the rear end. As mentioned above,
Nrarp-induced Wnt signaling is also important for stabiliza-
tion of the stalk and preventing EC retraction by improving
intercellular junctions.66 FGF signaling has also been impli-
cated in vascular homeostasis and integrity through tighten-
ing of EC junctions.77 Besides the above described “pull”
model, whereby the tip cell pulls stalk cells in the sprout,
recent data also provide evidence for a “push” model (Figure
4c), whereby dividing stalk cells push the sprout forward.
Indeed, in the absence of the vascular-specific secreted factor
EGFL7, an ECM protein mainly secreted by stalk cells,
newly formed ECs at the base of the stalk accumulate in
enlarged, but nonelongating sprouts.78
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Mechanisms of Vessel Branching 645
Robo4 Is Necessary for Stalk Cell Stabilization
Originally discovered in Drosophila neurons as receptors for
axon guidance cues, members of the Roundabout (Robo)
receptor family are also expressed by ECs. The Slit/Robo
system in vertebrates consists of three Slit proteins (Slit1–3),
which bind to Robo receptors (Robo1– 4). Robo4 is structur-
ally divergent from other Robo receptors and primarily
expressed by ECs, in particular in microvessels.79 Robo4
activates pathways involved in cell migration,79 including
WASP and Ena/Vasp family members in ECs.80 The role of
Robo4 in EC migration is, however, debated. Some reports
describe a role in repulsion,81,82 whereas others suggest a
promigratory effect.83,84 Genetic studies further show that
Robo4 stabilizes nascent vessels during postnatal retinal
angiogenesis.85 Robo4 is absent from most tip cells, but
expressed by stalk cells, where activation by Slit2 would
downregulate VEGF signaling and inhibit activation of Src-
kinase and Rac1 in this model. Also, Slit2 inhibits EC
migration, tube formation, and permeability, whereas vessels
in the ischemic retina are more leaky and unstable in Robo4
mutant mice.85 However, it remains debated whether Slit2 is
the ligand of Robo4. Indeed, overexpression studies show
that Slit2 can be immunoprecipitated with Robo4,81 but others
did not observe binding of Slit2 to Robo4.83 Recently, Robo4
was reported to form heterodimers with Robo1, which in-
creases the formation of filopodia in ECs.80 Thus, unraveling
the role of Robo-signaling in tip/stalk cells requires further
investigation.
Stalk Cells and Lumen Formation
After tip cell induction and stalk elongation, a vessel branch
needs to generate a lumen and initiate blood flow. Most of our
conceptual advances in lumen-formation have been generated
in vitro, by studying the behavior of ECs in two- or three-
dimensional gels.33 In these models, lumenogenesis relies on
the formation and coalescence of intracellular pinocytic
vacuoles. In zebrafish embryos, intra- and intercellular fusion
of vacuoles generates a lumen (Figure 4b).86 However, recent
findings suggest that the lumen in zebrafish vessels is formed
by the arrangement of ECs around an intercellular lumen.13
Vacuole formation depends on an interaction of integrins
with the ECM, whereas fusion of vacuoles requires rearrange-
ment of the actin and microtubule cytoskeleton.33 EC vacu-
oles accumulate in a polarized pattern, adjacent to the
centrosome.87 Both Rac1 and Cdc42, but not RhoA, localize
to vacuole membranes during formation of a lumen.33,87
Downstream signaling includes members of the WASP,
PAK, polarity proteins, and protein kinase C (PKC).33,87
However, an inhibitor of ROCK activity (which is down-
stream of RhoA) impairs vacuole formation,88 suggesting that
in some conditions, RhoA may regulate lumen formation.
Additionally, activation of endothelial RhoA-GTP on loss of
Cerebral Cavernous Malformations 2 (CCM2) causes cy-
toskeletal changes and impairs lumen formation.89 Once the
lumen has formed, additional signals drive EC proliferation,
growth, or enlargement, modifying the lumen size. Such
effects have been reported for different isoforms of VEGF,11
Notch1,67 and laminin.61
646 Arterioscler Thromb Vasc Biol May 2009

a behavior.5 Hence, by lowering the activity of an oxygen

Phd2+/+ b Phd2+/-
sensor, ECs readjust their shape and phenotype to improve
oxygen delivery in case of its shortage.
The molecular mechanisms underlying the endothelial
phalanx phenotype remain to be largely explored. Unlike tip
cells, phalanx cells extend few filopodia and migrate poorly
in response to VEGF, but form a tight barrier. They resemble
stalk cells by depositing a basement membrane and establish-
c Signaling pathways to maintain ing junctions, but differ from these cells by their increased
a phalanx cell phenotype quiescence, and reduced mitogenic response to VEGF. The
tip/stalk cell model of vessel branching suggests that high
levels of VEGF stimulate the induction of a migratory EC tip
FGFR cell phenotype, while intermediate levels promote the prolif-
VE-Cadherin erative stalk cell phenotype. Genetic studies show that loss of
FGF2 VEGF in ECs causes widespread EC dysfunction and desin-
VEGFR1 tegration, indicating that low levels of VEGF are critical for
migration, quiescent ECs to survive.90 It is also known that the junctional
proliferation molecule VE-Cadherin shifts the EC response to VEGF from
VEGF
proliferation and migration to survival and quiescence.91
Interestingly, phalanx cells in PHD2 haplodeficient mice are
more quiescent by expressing elevated levels of VE-Cadherin
VEGFR2
Quiescence and sFlt1, which acts as a VEGF trap to counteract the tip
Survival VE-Cadherin cell-inducing activity of VEGF (Figure 5c).5
Blood flow

Additional molecular pathways have been impli-

PHD2+/-
low O2
Shear sFlt
stress
PEDF
TIE2
BMP9
Ang TSP
TIE2
Figure 5
Figure 5. Molecular pathways of phalanx cell signaling. a and b,
Scanning electron microscopic images of the inner wall of tumor
vessels grown in wild-type (a) or PHD2⫹/⫺ (b) mice show
“abnormalized” versus “normalized” phalanx ECs. c, Signaling
mechanisms regulating phalanx cells quiescence.
Endothelial Phalanx Cells
Once a vessel branch is formed, ECs become quiescent, from
which only 0.01% are dividing in the healthy adult. The key
function of vessels is then to supply blood and oxygen to
tissues. While blood flow itself is already an important factor
for keeping ECs quiescent,34 some of the molecules, deter-
mining this endothelial phenotype were recently identified by
genetic studies. Indeed, endothelial-specific heterozygous
deletion of prolyl-hydroxylase domain-2 (PHD2), an oxygen
sensor, leads to “endothelial normalization” of tumor vessels.
In contrast to wild-type tumor vessels (Figure 5a), where ECs
are hyper-activated, extend multiple filopodia and loose tight
cell-cell contacts, ECs in PHD2 haplodeficient mice were
aligned in a smooth tight cobblestone monolayer, resembling
the “phalanx formation” of ancient Greek soldiers, hence
their name “phalanx cells” (Figure 5b).5 A shift of tumor ECs
to phalanx cells improves cancer perfusion and oxygenation,
thereby almost completely preventing metastasis and induc-
ing a shift from a malignant to a more benign tumor
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cated in EC quiescence and survival. Activation of
phosphatidylinositol-3-kinase (PI3K) and protein kinase B
(PKB)/Akt signaling promotes EC survival in response to
various signals, including VEGF, FGFs, Ang1 (Figure 5c),
and insulin-like growth factor (IGF).77,92–94 Furthermore, in
confluent (quiescent) ECs, Ang1 induces Tie2 translocation
to cell– cell contacts and the formation of homotypic Tie2–
Tie2 transassociated complexes that include the vascular
endothelial phosphotyrosine phosphatase, leading to inhibi-
tion of paracellular permeability (Figure 5c).95 Moreover,
FGF receptor inhibition disrupts endothelial integrity and
dissolves interendothelial junctions. 77 Also, bone-
morphogenic protein (BMP)-9, and its ALK1 receptor, and
pigment epithelium derived factor (PEDF) were recently
identified to promote EC quiescence.96,97 Interaction of
thrombospondin (TSP)-1 and -2 with their CD36 receptor
also diverge the proliferative and migratory stimuli of angio-
genic factors such as VEGF into survival signals (Figure
5c).98 Additionally, a number of molecular pathways have
been recently shown to induce tumor vessel normalization,
such as the formation of perivascular nitric oxide gradients,
but whether this involves a shift to a phalanx EC phenotype,
remains to be explored.99
Conclusion
The above proposed model of vessel branching is largely
based on seminal insights on tip, stalk, and phalanx cells from
the last 5 years. These studies have, however, also raised a
number of outstanding questions. For instance, are these
distinct EC phenotypes interchangeable, and what are the key
molecular determinants of this combinatorial code? Are
relative rather than absolute expression levels, or activation
status, of membrane receptors more relevant to specify EC
phenotypes? How do stalk cells maintain contact with the
leading tip cell and trail behind without dissolving the
 De Smet et al
elongating stalk? What is the molecular basis of lumen
formation? How do tip cells recognize their counterparts and
fuse with each other? What is the molecular code of the
phalanx phenotype? Is hypoxia codetermining the specifica-
tion of EC fates? What is the full implication of Wnt-
signaling? How do endothelial filopodia sense their environ-
ment, are guidance signals involved? Future explorations of
tip, stalk, and phalanx cell behavior will benefit from im-
proved in vivo imaging such as by fluorescence nanos-
copy.100 Finally, providing an answer to those questions will
be useful to translate these concepts into the development of
novel pro- and antiangiogenic therapies.
Sources of Funding
P.C. is supported by long-term structural funding (Methusalem
funding by the Flemish Government), Interuniversity Attraction Pole
(grant P60/30, funded by Belgian Government, BELSPO), FWO
G.0692.09 (Flemish Government), research grant by the Belgian
“Foundation against Cancer,” GOA 2006/11 - KU Leuven. P.J.H. is
recipient of a FEBS long-term fellowship. I.S. is recipient of a Marie
Curie Fellowship.
Disclosures
None.
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 Mechanisms of Vessel Branching: Filopodia on Endothelial Tip Cells Lead the Way
Frederik De Smet, Inmaculada Segura, Katrien De Bock, Philipp J. Hohensinner and Peter
Carmeliet

Arterioscler Thromb Vasc Biol. 2009;29:639-649; originally published online March 5, 2009;
doi: 10.1161/ATVBAHA.109.185165
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