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The Rockefeller University Press, 0021-9525/2003/06/1163/15 $8.00
The Journal of Cell Biology, Volume 161, Number 6, June 23, 2003 1163–1177
http://www.jcb.org/cgi/doi/10.1083/jcb.200302047
JCB

Article

1163

VEGF guides angiogenic sprouting utilizing
endothelial tip cell filopodia

Holger Gerhardt,

1

Matthew Golding,

2

Marcus Fruttiger,

3

Christiana Ruhrberg,

2

Andrea Lundkvist,

1


Alexandra Abramsson,

1

Michael Jeltsch,

4

Christopher Mitchell,

5

Kari Alitalo,

4

David Shima,

2


and Christer Betsholtz

1

1

Department of Medical Biochemistry, University of Göteborg, SE 405 30 Göteborg, Sweden

2

Endothelial Cell Biology Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, UK

3

Wolfson Institute for Biomedical Research, University College London, London WC1E 6AU, UK

4

Molecular/Cancer Biology Laboratory, Haartman Institute and Ludwig Institute for Cancer Research, Biomedicum,
00014 Helsinki, Finland

5

Department of Obstetrics and Gynaecology, University of Nottingham, City Hospital, Nottingham, NG5 1PB, UK

ascular endothelial growth factor (VEGF-A) is a major
regulator of blood vessel formation and function. It
controls several processes in endothelial cells, such
as proliferation, survival, and migration, but it is not known
how these are coordinately regulated to result in more
complex morphogenetic events, such as tubular sprouting,
fusion, and network formation. We show here that VEGF-A
controls angiogenic sprouting in the early postnatal retina
by guiding filopodial extension from specialized endothelial
cells situated at the tips of the vascular sprouts. The tip cells
V

respond to VEGF-A only by guided migration; the proliferative
response to VEGF-A occurs in the sprout stalks. These two
cellular responses are both mediated by agonistic activity
of VEGF-A on VEGF receptor 2. Whereas tip cell migration
depends on a gradient of VEGF-A, proliferation is regulated
by its concentration. Thus, vessel patterning during retinal
angiogenesis depends on the balance between two different
qualities of the extracellular VEGF-A distribution, which
regulate distinct cellular responses in defined populations
of endothelial cells.

Introduction

The development of branched tubular organs like the vascular
system, lung, kidney, and many glandular tissues poses several
fundamental biological questions. What determines the cellular
architecture of tubes and how do new branches arise? What
controls the size of a new branch and the direction of its out-
growth? How do branches fuse to form a continuous network?
The most pervasive vertebrate tubular organ, the vasculature,
is first assembled from scattered precursor cells that shape
blood islands, which fuse to create the first primitive plexus
of vessels (Risau and Flamme, 1995). Subsequently, enlarge-
ment and remodeling of the plexus, involving sprouting,
splitting, and regression of branches, shape hierarchical vascular
patterns that allow directional blood flow. These patterns
become precisely adapted to organ anatomy and physiology,
hence they differ extensively between organs.
Principally, at least two different mechanisms may lead to
organ-specific vascular patterns. First, the formation of a
primary vascular network may be a random process followed
by specific branch regression. This “vascular pruning” repre-
sents a major mechanism of vascular remodeling and is likely
regulated at the level of endothelial cell survival, which depends
on vascular endothelial growth factor (VEGF-A)* and un-
identified signals from surrounding vascular smooth muscle
cells or pericytes (Benjamin et al., 1999). Second, angiogenic
sprouting and fusion may be a guided process, leading to
specific primary vascular patterns. Such angiogenic guidance
is mainly inferred by the seemingly nonrandom angiogenic
sprouting in the developing central nervous system (CNS),
for example, in the mammalian retina, where a vascular

The online version of this article includes supplemental material.
Address correspondence to Christer Betsholtz, Dept. of Medical Bio-
chemistry, University of Göteborg, Medicinaregatan 9A, Box 440, SE
405 30 Göteborg, Sweden. Tel.: 46-31-7733460. Fax: 46-31-416108.
E-mail: christer.betsholtz@medkem.gu.se
David Shima’s present address is Eyetech Research Center, Eyetech Pharma-
ceuticals Inc., 42 Cummings Park, Woburn, MA 01801.
Key words: VEGF; endothelial cell; filopodia; astrocyte; migration; pro-
liferation

*Abbreviations used in this paper: CNS, central nervous system; GFAP, glial
fibrillary acidic protein; ILM, inner limiting membrane; P, postnatal day;
PECAM, platelet–endothelial cell adhesion molecule; PlGF, placenta
growth factor; VEGFR, vascular endothelial growth factor receptor.

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1164 The Journal of Cell Biology

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Volume 161, Number 6, 2003

plexus initially forms superimposed on a preexisting astrocyte
plexus (Stone and Dreher, 1987; Fruttiger et al., 1996).
Precision guidance of specialized cells is involved in the
formation of other pervasive organ systems. Axonal guidance
by attractive and repulsive forces is well established, and also
the formation of the insect tracheal system, which is both
structurally and functionally analogous to the vertebrate vas-
culature, relies on guidance of cells and subcellular processes
along predefined tracks (for review see Zelzer and Shilo,
2000). Certain molecules with a role in axon and/or tracheal
guidance have also been implicated in vascular morphogene-
sis (for review see Shima and Mailhos, 2000). For angiogen-
esis, however, the functions of these and other angiogenic
modulators remain ill defined.
The concept of precision guidance requires a sensor that
relays external signals into specific cell behavior. In axonal
guidance, this is provided by a specialized tip structure, the
growth cone. Also, the guidance of

Drosophila

tracheal
branches depends on specialized sensor cells situated at the
sprouting tips. These tip cells are unique in morphology and
gene expression and appear to respond to guidance cues con-
ferring positional information (Samakovlis et al., 1996).
Both the growth cone and the tracheal tip cells use dynamic
filopodia to sense guidance cues in their surroundings and to
migrate (Kater and Rehder, 1995; Ribeiro et al., 2002).
There is evidence from several studies that endothelial
sprouts can also extend multiple filopodia at their distal tips
(Bär and Wolff, 1972; Marin-Padilla, 1985 and earlier litera-
ture cited therein), indicating that growing vascular sprouts
are endowed with specialized tip structures with potential
functions in guidance and migration. These descriptions
have received surprisingly little attention, and with few re-
cent exceptions (Dorrell et al., 2002; Ruhrberg et al., 2002)
they go unnoticed in today’s concepts of vascular develop-
ment. Importantly, the numerous pro- and antiangiogenic
factors discovered during the past 15 yr have not been stud-
ied in relation to endothelial tip cells and their filopodia, and
in particular, the possibility that endothelial tip cells may re-
spond specifically to such factors has not been explored.
By analyzing mice lacking heparin-binding VEGF-A iso-
forms, we have recently provided evidence that the spatial
distribution of secreted VEGF-A is critical for the balance
between capillary branching and growth in vessel size (Ruhr-
berg et al., 2002). Here, we have used several genetic and
pharmacological gain and loss of function approaches to
show that different modes of VEGF-A distribution in the
extracellular space independently guide tip cell migration
and control proliferation in stalk cells. Collectively, our data
explain how the pattern of cellular expression and extracellu-
lar distribution of a single growth factor shapes vascular pat-
terns during angiogenic sprouting by regulating different
events in defined subpopulations of endothelial cells.

Results

We focused our studies of developmental angiogenesis on the
early postnatal mouse retina, which develops a stereotypical
vascular pattern in a well-defined sequence of events (Fig. 1).
Simultaneous vascular sprouting at the periphery and remod-
eling at the center (observable, for example, at postnatal day
[P]5), allows the study of different aspects of vessel forma-
tion, maturation, and specialization in a single preparation.
Retinas are ideal structures to visualize using whole-mount
immunostaining and in-situ hybridization techniques, cou-
pled with high resolution three-dimensional imaging by con-
focal laser scanning microscopy. We studied retinas from var-
ious mice between birth (P0) and P14. During this time,
spreading of the inner vascular plexus proceeds from the op-
tic disc to the peripheral margin. From approximately P6,
vascular branches also extend from the inner plexus into the
retina to form the outer plexuses (Fig. 1, P8, arrows).

Characterization of the endothelial tip cell

High resolution imaging of isolectin B4–stained retinas re-
vealed that the endothelial cells at the tips of vascular sprouts
extended long filopodia (Fig. 2, a–d and f). In the retina,
this was most evident at the edge of the expanding inner vas-
cular plexus (Fig. 2, a and b), at sites of sprouting into and
within the deeper retinal layers (Fig. 2 c), and at prospective
fusion sites in the central, remodeling zone (Fig. 2 d, ar-
rows). Endothelial filopodia were uniform in thickness
(



100 nm) but of variable length, with the longest extend-
ing



100



m. Staining of nuclei in combination with
isolectin B4, vascular endothelial (VE) cadherin, and fi-
bronectin (Fig. 2, a and e) revealed that the sprouting tip
consisted of a single, highly polarized endothelial cell, here-
after referred to as the tip cell. The endothelial identity of
this cell was further confirmed by staining for platelet–
endothelial cell adhesion molecule (PECAM)-1, endomucin
(unpublished data), and VEGF receptor (VEGFR)2 (see
Figure 1. Schematic presentation of retina development as a
model system for investigation of angiogenic sprouting in the CNS.
Corresponding top view micrographs of whole mount isolectin-
labeled specimen are shown to the right. The top view displays the
primary plexus in the fiber layer of the retina. Sprouting occurs
toward the periphery in the primary plexus (P1 and P5, arrows) and
subsequently into deeper layers (P8, arrows), where again branching
and fusion leads to plexus formation.