Desenvolvimento da Vasculatura Retiniana

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ORIGINAL PAPER
Development of the retinal vasculature
Marcus Fruttiger
Received: 15 December 2006 / Accepted: 17 January 2007 / Published online: 24 February 2007
� Springer Science+Business Media B.V. 2007
Abstract Blood vessels that supply the inner portion
of the retina are extensively reorganized during
development. The vessel regression, sprouting angio-
genesis, vascular remodelling and vessel differentiation
events involved critically depend on cell–-cell signal-
ling between different cellular components such as
neurons, glia, endothelial cells, pericytes and immune
cells. Studies in mice using transgenic and gene dele-
tion approaches have started to unravel the genetic
basis of some of these signalling pathways and have
lead to a much improved understanding of the molec-
ular mechanisms controlling retinal blood vessel
behaviour both during development and under patho-
logical conditions. Such insight will provide the basis of
future therapeutic approaches aimed at manipulating
retinal blood vessels.
Keywords Retinal astrocytes � Endothelial cells �
Angiogenesis � Vasculogenesis � Vascular remodelling �
Vessel regression � Vaso-obliteration � Hyaloid
vasculature � Oxygen induced retinopathy �
Neovascularization
Introduction
The vascular network that supplies the inner portion
of the retina with oxygen and nutrients undergoes
dramatic changes and reorganization during devel-
opment (Fig. 1). Initially, the inner part of the eye is
metabolically supported by the hyaloid vasculature,
an arterial network in the vitreous. Blood enters
through the central hyaloid artery in the optic nerve,
runs through hyaloid vessels in the vitreous and then
exits through an annular collection vessel at the front
of the eye. In the latter stages of development the
hyaloid vasculature is replaced by the retinal
vasculature. This switch occurs in humans around
mid-gestation and in mice around birth. As hyaloid
vessels regress, a vascular plexus emerges from the
optic nerve head giving rise to the retinal vascula-
ture. The new vascular network spreads in the nerve
fibre layer across the inner surface of the retina. In
contrast to the hyaloid vasculature, it contains both
arteries and veins that both enter and exit through
the optic nerve. This primary vessel plexus at the
inner retinal surface subsequently remodels into
three parallel but inter-connected networks, located
in the nerve fibre layer and the plexiform layers. In
mice, the primary plexus reaches the periphery of the
retina within about 8 days after birth. Vessels at the
growing edge of the vascular network are less mature
than more central vessels. It is therefore possible to
observe different stages of vascular differentiation in
a peripheral to central gradient. Because of this
spatial separation of different aspects of vascular
development, and because it is easy to image the
virtually flat primary plexus, this vessel network has
become a popular model system for general studies
on vascular development. With the introduction of
novel and powerful tools in mouse genetics we have
now entered a new era of studying the genetic
components that control mouse retinal vasculature
development.
M. Fruttiger (&)
UCL Institute of Ophthalmology, 11-43 Bath Street,
London EC1V 9EL, UK
e-mail: m.fruttiger@ucl.ac.uk
123
Angiogenesis (2007) 10:77–88
DOI 10.1007/s10456-007-9065-1
Retinal astrocytes
Development of the retinal vasculature is preceded by
an invasion of migrating astrocytes into the retina
[1, 2]. They emerge from the optic nerve head and
spread as a proliferating cell population in a centrifugal
fashion across the inner surface of the retina, forming a
cellular network that provides a template for the blood
vessels in their wake (Fig. 2) [3, 4]. There is a strict
correlation between the presence of astrocytes and
blood vessels in the retina: No retinal astrocytes can be
found in the avascular retina of the possum [2]. In the
horse, retinal astrocytes are present only in a small
region around the optic nerve head, which is also the
only region that is vascularized [5]. In the rabbit retina,
vessels and astrocytes are confined to the region that
contains a broad horizontal band of myelinated nerve
fibres [2]. In primates and mice retinal astrocytes and
blood vessels cover the entire retina, with the excep-
tion of the primate fovea from which retinal astrocytes
and blood vessels are excluded [5–7]. Taken together
these observations suggest that the retinal astrocyte
network and the retinal vasculature are evolutionary
and developmentally linked.
Retinal astrocytes develop from an astrocyte pre-
cursor lineage in the optic nerve that expresses the
transcription factor Pax2 [8, 9]. These precursors give
rise to two astrocyte lineages, optic nerve astrocytes
Fig. 1 The vascular network inside the eye is remodelled during
development. (A) The hyaloid vasculature (hv) is supplied by the
hyaloid artery (ha) and drained into the venous, choroidal net
(ch) on the outside of the eye (the choroidal net is not shown in
B and C). (B) The hyaloid vasculature regresses as the primary
plexus (pp) of the retinal vasculature grows into the retina. The
primary plexus consists of arteries and veins. (C) The deeper
plexus (dp) of the retinal vasculature develops from veins in the
primary plexus
Fig. 2 Retinal astrocytes in whole mount preparations.
(A) Antibody labelling visualizes retinal astrocytes expressing
PDGFRa (green) and GFAP (red), and Collagen IV positive
blood vessels (blue). Retinal astrocytes, not yet covered by blood
vessels, express GFAP only weakly (red in A and B) but can be
identified by Pax2 expression (green in B). Once covered by
blood vessels, retinal astrocytes upregulate GFAP (bottom left in
A). (C) In situ hybridization shows high levels of VEGF mRNA
in the not yet vascularized part of the retina and lower levels in
the vascularized centre. Retinal astrocytes in the vicinity of
arteries (a) display less VEGF mRNA expression than astrocytes
associated with veins (v). Scale bars are 50 lm
78 Angiogenesis (2007) 10:77–88
123
and retinal astrocytes. The factors controlling the split
between the two lineages are as yet unknown, but the
earliest marker that distinguishes retinal astrocytes
from optic nerve astrocytes (and other astrocyte lin-
eages in the brain) is platelet derived growth factor
receptor alpha (PDGFRa). It is expressed by retinal
astrocytes in the optic nerve head several days before
they start to invade the retina [10]. The ligand for
PDGFRa is platelet derived growth factor A
(PDGFA) which is secreted by retinal ganglion cells
[11] and is a mitogen for retinal astrocytes. In trans-
genic mice that express increased levels of PDGFA in
retinal neurons, the size of the retinal astrocyte popu-
lation increases accordingly, suggesting that PDGFA
production limits retinal astrocyte proliferation. Thus
PDGFA matches retinal astrocyte numbers to neuro-
nal cell numbers [3]. Immature retinal astrocytes have
an elongated, bipolar morphology (spindle shaped)
[12] while they migrate as a proliferating cell popula-
tion across the retina. They continue to proliferate
when they reach the retinal periphery and establish a
mesh-like network (Fig. 2A, B), expressing low levels
of glial fibrillary acidic protein (GFAP) and high levels
of vimentin [8]. Before they are covered by blood
vessels, they experience hypoxia and strongly express
vascular endothelial growth factor (VEGF) (Fig. 2C)
[13, 14], which is a key stimulus for angiogenesis [15].
As the expanding vessel network grows over the retinal
astrocytes, they differentiate into a more mature phe-
notype [12]. Proliferation ceases, GFAP is upregulated,
vimentin and VEGF are downregulated and a stellate
morphology emerges [8, 14]. Oxygen provided by the
developing retinal vasculature can have a differentiat-
ing influence on retinal astrocytes, halting proliferationand limiting cell numbers. Since astrocytes induce
vessel formation, this creates a negative-feedback loop
that limits and stabilizes astrocyte numbers and the
density of blood vessels in the retina [14].
The orphan nuclear receptor Tlx (also known as
Nr2e1) also contributes to the regulation of retinal
astrocyte numbers [16]. It is expressed by immature
retinal astrocytes and downregulated as they mature
[17]. In Tlx knock-out mice, retinal astrocytes fail to
acquire a fully mature stellate morphology and remain
highly elongated [16]. But paradoxically, they prolifer-
ate only slowly and strongly express GFAP, which in
normal retinal astrocytes is associated with a mature
phenotype. Furthermore, retinal vascularization is dra-
matically delayed and abnormal in Tlx knock-out mice
[17]. Interestingly, this is not caused by abnormal VEGF
expression (which is not changed in these mice), but has
been attributed to impaired extracellular assembly of
fibronectin matrices by retinal astrocytes [17].
Vascular growth in the retina
The brain is predominantly vascularized by angiogen-
esis, a process in which proliferating endothelial cells
form new vessel sprouts and extend the vascular net-
work from pre-existing vessels [18]. Similarly, the
deeper networks of the retinal vasculature are also
believed to form by sprouting angiogenesis. In contrast,
it has been suggested by several authors that the pri-
mary inner vascular plexus in the retina is formed by
vasculogenesis [19–23], a process defined as the de
novo formation of blood vessels from isolated vascular
endothelial precursor cells that coalesce into cords and
then form a lumen [18]. This theory depends on the
identification of free-standing precursor cells distal to
the advancing primary vascular network, and in the
mouse retina no such precursors have been found to
date [12, 24]. It has been suggested that vascular pre-
cursor cells are present in the human retina, based on
the identification of ADPase/CD39 and CXCR4 posi-
tive cells [20, 25]. However, the identification and
lineage of endothelial precursor cells is still contro-
versial [26, 27], and further markers will be required to
identify endothelial precursor cells in the retina with
certainty. VEGF receptor 2 (VEGFR2, also known as
Krd or Flk1) is widely regarded as the earliest marker
of developing endothelial cells [28–31], and would
provide an alternative way to detect endothelial pre-
cursor in the retina. However, VEGFR2 positive
endothelial precursor cells have yet to be demon-
strated in the human retina. Nevertheless, it cannot be
excluded that endothelial precursor cells are involved
in the formation of the retinal vasculature [32]. For
example, it is possible that circulating endothelial
precursors are built into the growing network from
within blood vessels.
The notion that the primary plexus in the mouse
forms by sprouting angiogenesis [12] is supported by
the identification of specialized ‘‘endothelial tip cells’’
at the leading edge of the growing vascular network
[33]. Endothelial tip cells are a specialized subclass of
endothelial cells at the very tip of vascular sprouts that
form by angiogenesis. They extend numerous long
filopodia [34–36]—similar to axonal growth
cones—suggesting that they might steer the growth of
vascular sprouts by responding to attractant and
repellent guidance cues [37]. The gene expression
profile of endothelial tip cells differs from endothelial
cells in other positions in the vascular network. For
example, delta like 4 (Dll4), PDGFB and apelin
mRNA are upregulated at the leading front of the
vascular plexus [33, 38, 39]. Similarly, netrin receptor
Unc5b and VEGFR2 are also particularly strongly
Angiogenesis (2007) 10:77–88 79
123
expressed by endothelial tip cells [33, 40]. This clearly
demonstrates that endothelial cells are not a uniform
cell population and that they differentiate into various
subclasses depending on their position and function
within the vascular network. Experiments using a
three-dimensional cell culture system have suggested
that Notch signalling plays a role in establishing and
maintaining the different phenotypes of endothelial tip
cells and more proximal endothelial cells (stalk cells) in
angiogenic sprouts [41].
Guidance of sprouting angiogenesis
Having distinct identities allows endothelial cells to
respond to their environment differentially. This is
illustrated by the different behaviour of tip and stalk
cells. Cell proliferation is rare in tip cells whereas it is
extensive in stalk cells [33], despite the fact that tip
cells and the first few stalk cells are likely to be
exposed to similar concentrations of environmental
mitogens, such as VEGF. On the other hand, tip cells
form extensive bundles of filopodia in the direction of
vascular growth. These filopodia depend on signalling
via VEGFR2 and mediate migration towards higher
concentrations of VEGF [42]. Due to its regulation by
oxygen tension, VEGF mRNA is expressed in a gra-
dient in the presence of an oxygen-delivering vascular
plexus. This happens because VEGF mRNA is down-
regulated in the vascularized, normoxic centre of the
retina whereas in the not yet vascularized (hypoxic)
periphery of the retina VEGF mRNA is expressed at
high levels (Fig. 2C). Strong VEGF production in the
retinal periphery and low production in the centre are
likely to produce a gradient distribution of VEGF
protein. Although such a protein gradient has not yet
been directly demonstrated there is circumstantial
evidence that supports such a view. For example, dis-
turbances to the distribution of VEGF directly affect
the speed at which the vascular plexus spreads across
the retina. Intraocular injection of VEGF ‘‘floods’’ the
gradient and slows vascular growth [33]. This concept is
further supported by three mutant mouse strains in
which the affinity of VEGF to the extracellular matrix
(ECM) has been altered. Normally, alternative tran-
scription of VEGF yields VEGF120, VEGF164 and
VEGF188 isoforms, which differ in their ability to bind
ECM. The mutated strains selectively express only one
of these isoforms [43]. In mice that posses only the
short VEGF120 isoform (with a very weak affinity for
ECM), VEGF diffuses more freely, the VEGF protein
gradient is flattened [34], and vascular growth in the
retina is slowed [33, 44].
Despite the central role of VEGF in retinal vascular
development it is difficult to explain the intricate pat-
terning of the retinal vasculature on the basis of dif-
ferential VEGF expression alone, and other factors
that guide endothelial tip cell migration in the retina
are likely to exist. For example, intraocular injection of
anti-R-cadherin antibodies in two-day-old (P2) mouse
pups can perturb formation of the primary plexus [45].
The authors of this study have suggested that
R-cadherin normally mediates interactions between
the astrocyte template and growing blood vessels.
However, R-cadherin deficient mice show no defects in
retinal vascular development (personal communica-
tion, Holger Gerhardt, Cancer Research UK, London,
UK). Furthermore, there are a number of receptors for
axon guidance molecules that are expressed by endo-
thelial cells and have been shown to be involved in
vascular patterning elsewhere in the body, such as
PlexinD1, Robo1/4 and Unc5b [37, 46]. The ligands of
these receptors (Sema3E, Slit1/2 and Netrin1, respec-
tively) are all expressed in the retina [47–49], but so far
none of them have directly been implicated in retinal
vascular development. EphB4 and EphrinB2 is a fur-
ther receptor-ligand pair with a double role in axon
guidance and vascular patterning. Because EphB4 is
most strongly expressed in veins and EphrinB2 in
arteries, this signalling pathway has been implicated in
the demarcation of venous versus arterial territories
[50, 51]. However, EphB4 and EphrinB2 activity has
alsobeen linked to endothelial cell proliferation and
migration [52–55] and might play a role in retinal
angiogenesis.
Vascular remodelling and maturation
In the region behind the tip cells a relatively dense and
uniform capillary plexus is laid down. Over time, as
more vessels are added at the periphery, this primitive
plexus is remodelled and starts to mature into a hier-
archical vascular tree (Fig. 3). Differences in vessel
diameter emerge and arteries and veins can be distin-
guished. Some of the capillaries are pruned whilst
others are strengthened. Pruning is particularly evident
in the vicinity of arteries where capillary free zones
emerge (Fig. 3B). This can occur via migration and re-
localization of endothelial cells [56] or via selective
endothelial cell apoptosis [57]. Leukocytes adhere to
the vascular network through CD18 and induce selec-
tive endothelial cell death via Fas ligand (FasL).
Blockade of FasL or CD18 with antibodies increases
vascular density [57]. Interestingly, depletion of
macrophages via intraocular injection of clodronate
80 Angiogenesis (2007) 10:77–88
123
liposomes has the opposite effect and reduces vascular
density [58]. This demonstrates that immune cells can
have diverse roles during normal vascular develop-
ment. One possible molecular mediator is VEGF, be-
cause capillary pruning is defective in VEGF120 mice
[44]. The same mouse strain also shows changes in
vessel calibre [34, 44], suggesting VEGF signalling
could also influence this aspect of vascular network
maturation. This theory is supported by an in vitro
study showing that VEGF concentration in the culture
medium can directly influence the diameter of newly
forming vascular sprouts [59]. Transforming growth
factor beta (TGFb) superfamily signalling has also
been implicated in the control of vessel diameter in the
developing limb vasculature [60]. Furthermore, exper-
iments on the yolk sac of chick embryos have also
shown that blood flow can have a profound influence
on the patterning of the vascular tree [61].
Although the genetic components that control
remodelling and maturation of the retinal vasculature
are not well characterized, maturity of retinal vessels
can be tested by their susceptibility to hyperoxia [62,
63]. During the first two postnatal weeks it is possible
to ablate retinal vessels in mouse pups by increased
atmospheric oxygen; after that, retinal vessels are
resistant to hyperoxia exposure. Obliteration primarily
affects capillaries in the centre of the retina and around
arteries, and may be caused by direct oxidative damage
to endothelial cells by reactive oxygen species (ROS)
[64] and/or oxygen mediated downregulation of
VEGF, which acts as a survival factor for endothelial
cells [13, 65]. Capillaries near arteries are most af-
fected, because these areas contain the highest oxygen
concentrations [66] and the lowest VEGF levels [67].
The effects caused by hyperoxia may be an exaggeration
of the process that leads to capillary free zones around
arteries during normal development. Accordingly,
leukocytes, which are involved in normal remodelling
(see above), also participate in hyperoxia-induced
vaso-obliteration [57].
On a cellular level, resistance to hyperoxia in older
animals is based on maturation of the relationship
between endothelial and mural cells (pericytes and
smooth muscle cells). In kitten, maturity of the retinal
vasculature can be assessed via the percentage of des-
min expressing mural cells, which increases over time
and above a certain threshold value correlates with
resistance to hyperoxia induced vaso-obliteration [68].
Reciprocal cell–cell signalling between endothelial and
mural cells is at the heart of this maturation process.
Endothelial cells recruit mural cells via the secretion of
PDGFB, which acts through the PDGF receptor beta
(PDGFRb) on mural cells [69]. Signalling in the
reverse direction can occur via mural cell derived an-
giopoietin1 (Ang1) acting on endothelial cells through
the Tie2 receptor [70–72]. A pioneering study has
shown that intraocular injection of soluble PDGFB
causes mural cell detachment and can aggravate hy-
peroxia-mediated vaso-obliteration [73]. Similarly,
administration of a blocking antibody against
PDGFRb inhibits mural cell recruitment and leads to
severe remodelling defects in the retinal vasculature.
Simultaneous injections of recombinant Ang1 can
rescue this defect, at least in part [74]. In addition to
cell–cell signalling via PDGFB and angiopoietins,
sphingosine-1-phosphate-1 (S1P1) and TGFb1 also
play a role in vessel maturation [75], but their role in
the retinal vasculature is less well established.
Vessel maturation in the central nervous system also
includes the formation of permeability barriers by
Fig. 3 Developing mouse retinal vasculature stained with an
antibody against Collagen IV in retinal whole mount prepara-
tions. The relatively uniform plexus visible in 3-day-old (P3)
mouse pups (A) expands and remodels by P6 (B) into a network
with clearly distinguishable arteries (a) and veins (v). Arteries
can be identified by their capillary free zones (arrowheads in B).
At P9 (C) the primary plexus has reached the retinal periphery,
vessels start to sprout into the retina and are visible as white dots
(small arrowheads) establishing the deeper network of the
retinal vasculature. Some of the veins disappear from the
primary plexus (arrow) and relocate by a process of remodelling
to the deeper plexus (not visible). Scale bar is 200 lm
Angiogenesis (2007) 10:77–88 81
123
endothelial cells, resulting in the blood-brain barrier
(BBB) and the blood–retina barrier (BRB). In the
developing brain a barrier to protein and macromole-
cules exists at the earliest stages of brain vasculariza-
tion [76–78]. Later in development, astrocytes are
thought to drive further maturation of the BBB to also
exclude lower molecular weight compounds in adults
[79, 80]. Development of the BRB is less well under-
stood but it has been shown that the retinal vasculature
becomes impermeable to 4kD FITC-dextran by P10
(Poor SH et al. Invest Ophthalmol Vis Sci 2006;47:
ARVO E-Abstract 502). Several tight junction mole-
cules are expressed in adult retinal blood vessels, such
as occludin, zonula occludens-1 (ZO-1) and claudin-5
[81–83]. Occludin is more strongly expressed in arte-
rioles and capillaries in the deeper plexus than by
venules [84]. Whether this correlates with differential
permeability of retinal blood vessels is not known but
in culture systems occludin expression levels have been
linked to tightness of endothelial monolayers [85, 86].
Furthermore, in a diabetic rat model, protein extrava-
sation into the retina correlates with reduced occludin
content in the retinal vasculature [83, 84, 87]. This may
be mediated by astrocytes because they can increase
barrier properties of endothelial cells in vitro and
might play a role in the regulation of vascular perme-
ability in the retina [84, 88]. However, retinal astro-
cytes are limited to the primary plexus and cannot
influence the deeper vascular network in the retina. It
is possible that Müller cells induce barrier properties in
the deeper plexus [89].
Deeper plexus development
The deeper plexus of the retinal vasculature (also
known as outer plexus) develops by sprouting from the
primary plexus (Fig. 1) [90, 91]. In mice and rats this
occurs about one week after birth when the expanding
primary plexus reaches the margins of the retina [92].
Angiogenic sprouts emerge from veins, venules and
capillaries near veins, and start to penetrate the retina
perpendicular to the primary plexus (Figs. 3C, 4A).
This process commences in the centre of the retina and
expands towards the periphery. It is preceded by
transient expression of VEGF mRNA in somatas
located in the inner nuclear layer (INL) [15]. Vascular
sprouts grow along Müller cell processes into theretina
and turn ‘‘sideways’’ when they reach the inner and
outer boundary of the INL to establish two further
networks parallel to the primary plexus (Fig. 4B, C).
In contrast to primary plexus formation, the deeper
plexus develops independently of retinal astrocytes.
However, beyond this only little is known about the
cellular and molecular mechanisms that induce and
guide deeper plexus angiogenesis. The strict limitation
of these vessels to the INL boundaries can be disturbed
with intraocular injection of antibodies against
R-cadherin in mouse pups, which causes angiogenesis
through the normally avascular photoreceptor layer into
the subretinal space [45]. A very similar phenotype has
also been found in mice that lack the very low density
lipoprotein receptor (VLDLR) gene [93], but so far no
mechanism explaining this phenomenon has been
established. In the brain, signalling involving VLDLR,
reelin (RELN), apolipoprotein-E (ApoE) and disabled
homolog 1 (Dab1) guides neuronal migration along ra-
dial glial cells [94], but deeper plexus development in the
retina is normal in RELN, ApoE and Dab1 knock-out
mice (MF unpublished observation).
Further insight into deeper plexus development can
be gained from Ang2-deficient mice [95]. In these mice
the primary vascular network is slightly affected (it
develops late and incompletely) but the deeper plexus
never forms. Ang2 is normally expressed by horizontal
cells in the INL [96] and might promote angiogenesis in
the retina by antagonizing Ang1/Tie2 signalling [97].
There are two additional mutants in which deeper
plexus formation fails despite the presence of a rela-
tively normal primary plexus [98, 99]. The affected
genes in these mutants are the Norrie disease gene
(Ndph) and frizzled-4 (Fzd4). They encode a ligand
receptor pair which mediates activation of the classical
Wnt pathway [99]. Intriguingly, transgenic expression
of ectopic Ndph in the lens can completely rescue
deeper plexus formation in Ndph mutant mice [100].
However, so far it has not been established which cell
types in the retina interact through Ndph/Fzd4. In
humans, mutations in the homologous genes, NDPH
and FZD4, cause familial exudative vitreoretinopathy
(FEVR), an inherited disorder characterized by
incomplete vascularization of the peripheral retina
[101, 102]. Deeper plexus formation is not well char-
acterized in FEVR, but there is evidence that it may be
absent [103]. The low-density lipoprotein receptor re-
lated protein 5 (Lrp5) is a Wnt co-receptor and has also
been linked to FEVR [104]. Lrp5-deficient mice dis-
play persistent hyaloid vessels [105], but an analysis of
their retinal vasculature has so far not been published.
Regression of the hyaloid vasculature
Persistent foetal vasculature (PFV) is an ocular
pathology in which the hyaloid vasculature fails to
completely regress; this can lead to severe intraocular
82 Angiogenesis (2007) 10:77–88
123
haemorrhage [106]. In mice, regression of the hyaloid
vasculature can be inhibited by ablating macrophages.
This can be achieved in transgenic mice which express
diphtheria toxin specifically in macrophages [107] or by
injecting clodronate liposomes into the anterior
chamber of the eye [108]. In both cases capillaries in
the eye fail to regress, demonstrating a crucial
involvement of macrophages in vessel regression. More
recently this interaction between macrophages and
blood vessels has been shown to involve signalling of
Wnt7b derived from macrophages via Fzd4 receptor
and Lrp5 co-receptor on endothelial cells [109].
Accordingly, mice with a hypomorphic Wnt7b allele or
with Fzd4 or Lrp5 null mutations all display disturbed
hyaloid vessel regression [99, 105, 109]. Wnt7b-inde-
pendent Fzd4 activation might play a role as well, be-
cause Ndph-deficient mice (see above) also show
persistent hyaloid vessels [110]. The same phenotype is
also found in Ang2 knock-out mice implicating sig-
nalling via Tie2 receptor in hyaloid vessel regression
[95]. It is remarkable that in all mutants with disturbed
deeper plexus formation described above (Ndph-,
Fzd4- and Ang2-deficient mice) hyaloid vessel regres-
sion is also affected. This raises the possibilities that
deeper plexus formation and hyaloid vessel regression
are either functionally linked or share common sig-
nalling pathways. In heterozygous BMP4 knock-out
mice macrophages are absent in the vitreous and the
hyaloid vasculature fails to regress, but the underlying
signalling mechanisms and effects on inner plexus
development have not been elucidated yet [111].
There are other mutants with persistent hyaloid
vessels in which the molecular signalling mechanisms
are less well understood. In transgenic mice that over-
express a dominant negative fibroblast growth factor
receptor in the retina, the primary plexus never
develops, hyaloid vessels persist, and the retina is
eventually vascularized by sprouting hyaloid vessels
[112]. Similarly, VEGF188 mice (containing only the
VEGF188 isoform) [44] and Collagen XVIII knock-
out mice [113] have defects in primary plexus devel-
opment and subsequent participation of persistent
hyaloid vessels in retinal vascularization. It is feasible
that this phenomenon is part of a compensatory
mechanism.
Pathological vessel growth in the retina
The formation of new blood vessels, so-called neo-
vascularization, in the adult retina is a major compli-
cation in diseases such as diabetic retinopathy or age
related macular degeneration and is the leading cause
of blindness in the Western world. Understanding the
biological principles that govern blood vessel growth in
the retina thus has important clinical implications. The
most widely used mouse model for retinal vasculature
pathology recreates elements of retinal neovascular-
ization. The system is based on hyperoxia-induced
vaso-obliteration of capillaries in mouse pups (see
above) and their subsequent return to room air. This
triggers oxygen-induced retinopathy (OIR), a condi-
tion characterized by the growth of tortuous and leaky
vessels that form tuft-like structures towards the
vitreous [13]. The pathology is seen as part of a
vascular repair response, driven by the pronounced
Fig. 4 Vascular networks in the retina are depicted by fluores-
cein labelled lectin staining in retinal whole mount preparations
(A, C) and in a schematic of a retinal cross-section (B). Images in
A and C were taken at different focal planes and coloured red
(primary plexus), green (inner deeper plexus), blue (outer
deeper plexus) and superimposed using a computer. At P8 (A)
sprouts (green) are emerging from veins (v) and capillaries but
not arteries (a). At P14 (C) all three networks are established.
Arrowheads indicate connections between the primary and the
inner deeper plexus. Arrows indicate connections between the
inner and outer deeper plexus (RGC, retinal ganglion cells; IPL,
inner plexiform layer; INL, inner nuclear layer; OPL, outer
plexiform layer; ONL, outer nuclear layer; RPE, retinal pigment
epithelium). Scale bars are 100 lm
Angiogenesis (2007) 10:77–88 83
123
hypoxia and upregulation of VEGF in capillary-de-
pleted areas of the retina. However, it is less clear why
the remaining vessels are developing neovascular tufts
instead of re-vascularizing the capillary-depleted areas
through normal sprouting, as happens during devel-
opment.
One important difference between physiological and
pathological neovascularization might arise from the
way leukocytes and macrophages modulate vascular
growth. Blocking leukocyte activity with an anti-CD2
antibody exacerbates vascular pathology in OIR,
whereas inactivation of macrophages with clodronate
liposomes suppresses pathological neovascularization
[114]. The authors of this study also show that a
VEGF164-specific neutralizing aptamer reduces path-
ological neovascularization (with no effects on physi-
ological vascularization)and suggest that the
VEGF164 isoform might influence the inflammatory
component of neovascularization [114]. A further clue
implicating macrophages as a pathology-causing cell
type in OIR comes from experiments where antibody-
blocking of monocyte chemotactic protein-1 (also
known as CCL2) and macrophage inflammatory
protein-1alpha (also known as CCL3) reduced retinal
neovascularization [115]. However, the precise
mechanism through which macrophages exert their
pathology-causing influence in OIR remains to be
established. More importantly, it is also not yet known
whether macrophages are involved in neovascular tuft
formation in human diseases such as diabetic retinop-
athy or whether this is a peculiarity of the OIR model.
In recent years, numerous genetic studies in mice
have identified signalling molecules that have a pivotal
involvement in vascular growth, such as VEGF,
PDGFB, Angiopoietins, EphrinB2, Dll4 and others (for
recent reviews see [37, 46, 116–119]). It has often been
argued that these molecules, or the signalling pathways
through which they act, might represent useful thera-
peutic targets in pathologies of the retinal vasculature.
For example, inhibiting the action of VEGF can reduce
the occurrence of neovascular tufts in the vitreous dur-
ing OIR [120, 121]. However, hypoxia induced VEGF
expression is part of a natural repair response that
should, at least in theory, lead to re-vascularization and
re-oxygenation of ischemic tissue. Thus, a more desir-
able long term therapeutic aim should involve regener-
ation of the remaining vascular network, rather than
indefinite inhibition of factors that normally mediate
re-vascularization of hypoxic tissue.
In the OIR model one possible strategy to achieve
healthy vascular regeneration might come from the
manipulation of macrophage behaviour, but in human
disease, such as diabetic retinopathy, it is not yet clear
why the retinal vasculature fails to replace damaged
blood vessels. Attempts aimed at suppressing abnor-
mal blood vessels and regenerating healthy vessels in
the retina will have to take into account that growing
vessels encounter a different environment during nor-
mal development and in disease. Moreover, the retina
has unique properties and insights into angiogenesis
gained in non-ocular tissue will have to be interpreted
with caution [122]. For example, the entire retinal
vasculature possesses barrier properties, like vessels in
the brain, but, unlike brain vessels, the deeper plexus in
the retina is associated with Müller cells rather than
astrocytes. The unique character of ocular vasculature
is highlighted by Ndph knock-out mice, which display
their most dramatic vascular phenotype in the retina.
Thus the Ndph signalling pathway might provide a very
eye-specific drug target. This suggests that studying
retinal vasculature development could be useful in the
development of future therapeutic approaches. In fact,
it was a developmental study that first identified VEGF
as a key factor in retinal vessel growth [15], and it is
very likely that this research field will uncover further
factors with important roles in human retinal vascula-
ture pathologies.
However, it should be borne in mind that develop-
mental vascular biology has limited use in providing
models for retinal vascular diseases. Such pathologies
typically afflict adult individuals with a fully matured
vascular system. In proliferative diabetic retinopathy,
for example, capillaries are damaged and the normally
very quiescent, adult vasculature is re-activated and
starts to grow excessively in the retina. Knock-out mice,
so useful in the study of developmental angiogenesis, are
of limited value to investigate postnatal and adult blood
vessels. This is because genetic mutations affecting the
vasculature are often embryonically lethal and mutant
mice never reach adulthood. Techniques that allow the
targeting of specific genes in adult retina such as RNAi
[123] or inducible transgenic systems [124] have there-
fore great potential in this field. Such approaches could
be used to investigate the mechanisms that control vessel
maturation and drive vessels into quiescence in the ret-
ina. The reversal of this transition, from a quiescent to an
active vascular phenotype, occurs in retinal neovascular
diseases and the molecular mechanisms that control this
switch are clinically highly relevant. The development of
new models that allow the study of particular compo-
nents of human retinal disease, other than ‘‘neovascu-
larization’’, will be an important basis for future medical
applications. However, a thorough understanding of the
molecular and cellular processes that occur in such
models will be an essential prerequisite for any valid
clinical interpretation.
84 Angiogenesis (2007) 10:77–88
123
Acknowledgements The author thanks Joanne Taylor and
Christiana Ruhrberg for critical reading of the manuscript. The
work was made possible by funding from the Lowy Medical
Research Institute LTD and the Medical Research Council.
References
1. Watanabe T, Raff MC (1988) Retinal astrocytes are immi-
grants from the optic nerve. Nature 332:834–837
2. Stone J, Dreher Z (1987) Relationship between astrocytes,
ganglion cells and vasculature of the retina. J Comp Neurol
255:35–49
3. Fruttiger M, Calver AR, Kruger WH, Mudhar HS, Mic-
halovich D, Takakura N, Nishikawa S, Richardson WD
(1996) PDGF mediates a neuron-astrocyte interaction in
the developing retina. Neuron 17:1117–1131
4. Ling TL, Stone J (1988) The development of astrocytes in
the cat retina: evidence of migration from the optic nerve.
Brain Res 44:73–85
5. Schnitzer J (1987) Retinal astrocytes: their restriction to
vascularized parts of the mammalian retina. Neurosci Lett
78:29–34
6. Huxlin KR, Sefton AJ, Furby JH (1992) The origin and
development of retinal astrocytes in the mouse. J Neuro-
cytol 21:530–544
7. Engerman RL (1976) Development of the macular circu-
lation. Invest Ophthalmol 15:835–840
8. Chu Y, Hughes S, Chan-Ling T (2001) Differentiation and
migration of astrocyte precursor cells and astrocytes in
human fetal retina: relevance to optic nerve coloboma.
FASEB J 15:2013–2015
9. Mi H, Barres BA (1999) Purification and characterization of
astrocyte precursor cells in the developing rat optic nerve.
J Neurosci 19:1049–1061
10. Mudhar HS, Pollock RA, Wang C, Stiles CD, Richardson
WD (1993) PDGF and its receptors in the developing ro-
dent retina and optic nerve. Development 118:539–552
11. Fruttiger M, Calver AR, Richardson WD (2000) Platelet-
derived growth factor is constitutively secreted from neu-
ronal cell bodies but not from axons. Curr Biol
10:1283–1286
12. Fruttiger M (2002) Development of the mouse retinal vas-
culature: angiogenesis versus vasculogenesis. Invest Oph-
thalmol Vis Sci 43:522–527
13. Pierce EA, Foley ED, Smith LE (1996) Regulation of vas-
cular endothelial growth factor by oxygen in a model of
retinopathy of prematurity. Arch Ophthalmol 114:1219–
1218
14. West H, Richardson WD, Fruttiger M (2005) Stabilization
of the retinal vascular network by reciprocal feedback be-
tween blood vessels and astrocytes. Development 132:1855–
1862
15. Stone J, Itin A, Alon T, Pe’er J, Gnessin H, Chan-Ling T,
Keshet E (1995) Development of retinal vasculature is
mediated by hypoxia-induced vascular endothelial growth
factor (VEGF) expression by neuroglia. J Neurosci
15:4738–4747
16. Miyawaki T, Uemura A, Dezawa M, Yu RT, Ide C, Nis-
hikawa S, Honda Y, Tanabe Y, Tanabe T (2004) Tlx, an
orphan nuclear receptor, regulates cell numbers and astro-
cyte development in the developing retina. J Neurosci
24:8124–8134
17. Uemura A, Kusuhara S, Wiegand SJ, Yu RT, Nishikawa S
(2006) Tlx acts as a proangiogenic switch by regulating
extracellular assembly of fibronectin matrices in retinal as-
trocytes. J Clin Invest 116:369–377
18. Risau W (1997) Mechanisms of angiogenesis. Nature
386:671–674
19. Ashton N (1970)Retinal angiogenesis in the human em-
bryo. Br Med Bull 26:103–106
20. Chan-Ling T, McLeod DS, Hughes S, Baxter L, Chu Y,
Hasegawa T, Lutty GA (2004) Astrocyte-endothelial cell
relationships during human retinal vascular development.
Invest Ophthalmol Vis Sci 45:2020–2032
21. Chan-Ling TL, Halasz P, Stone J (1990) Development of
retinal vasculature in the cat: processes and mechanisms.
Curr Eye Res 9:459–478
22. Flower RW, McLeod DS, Lutty GA, Goldberg B, Wajer
SD (1985) Postnatal retinal vascular development of the
puppy. Invest Ophthalmol Vis Sci 26:957–968
23. Hughes S, Yang H, Chan-Ling T (2000) Vascularization of
the human fetal retina: roles of vasculogenesis and angio-
genesis. Invest Ophthalmol Vis Sci 41:1217–1218
24. Gariano RF (2003) Cellular mechanisms in retinal vascular
development. Prog Retin Eye Res 22:295–306
25. McLeod DS, Hasegawa T, Prow T, Merges C, Lutty G
(2006) The initial fetal human retinal vasculature develops
by vasculogenesis. Dev Dyn 235(12):3336–3347
26. Urbich C, Dimmeler S (2004) Endothelial progenitor cells:
characterization and role in vascular biology. Circ Res
95:343–353
27. Rehman J, Li J, Orschell CM, March KL (2003) Peripheral
blood ‘‘endothelial progenitor cells’’ are derived from
monocyte/macrophages and secrete angiogenic growth fac-
tors. Circulation 107:1164–1169
28. Yamashita J, Itoh H, Hirashima M, Ogawa M, Nishikawa S,
Yurugi T, Naito M, Nakao K, Nishikawa S (2000) Flk1-
positive cells derived from embryonic stem cells serve as
vascular progenitors. Nature 408:92–96
29. Shalaby F, Rossant J, Yamaguchi TP, Gertsenstein M, Wu
XF, Breitman ML, Schuh AC (1995) Failure of blood-island
formation and vasculogenesis in Flk-1-deficient mice. Nat-
ure 376:62–66
30. Dumont DJ, Fong GH, Puri MC, Gradwohl G, Alitalo K,
Breitman ML (1995) Vascularization of the mouse embryo:
a study of flk-1, tek, tie, and vascular endothelial growth
factor expression during development. Dev Dyn 203:80–92
31. Yamaguchi TP, Dumont DJ, Conlon RA, Breitman ML,
Rossant J (1993) flk-1, an flt-related receptor tyrosine ki-
nase is an early marker for endothelial cell precursors.
Development 118:489–498
32. Otani A, Kinder K, Ewalt K, Otero FJ, Schimmel P,
Friedlander M (2002) Bone marrow-derived stem cells
target retinal astrocytes and can promote or inhibit retinal
angiogenesis. Nat Med 8:1004–1010
33. Gerhardt H, Golding M, Fruttiger M, Ruhrberg C,
Lundkvist A, Abramsson A, Jeltsch M, Mitchell C, Alitalo
K, Shima D, Betsholtz C (2003) VEGF guides angiogenic
sprouting utilizing endothelial tip cell filopodia. J Cell Biol
161:1163–1177
34. Ruhrberg C, Gerhardt H, Golding M, Watson R, Ioannidou
S, Fujisawa H, Betsholtz C, Shima DT (2002) Spatially re-
stricted patterning cues provided by heparin-binding
VEGF-A control blood vessel branching morphogenesis.
Genes Dev 16:2684–2698
35. Marin-Padilla M (1985) Early vascularization of the
embryonic cerebral cortex: golgi and electron microscopic
studies. J Comp Neurol 241:237–249
36. Kurz H, Gartner T, Eggli PS, Christ B (1996) First blood
vessels in the avian neural tube are formed by a combina-
Angiogenesis (2007) 10:77–88 85
123
tion of dorsal angioblast immigration and ventral sprouting
of endothelial cells. Dev Biol 173:133–147
37. Eichmann A, le Noble F, Autiero M, Carmeliet P (2005)
Guidance of vascular and neural network formation. Curr
Opin Neurobiol 15:108–115
38. Claxton S, Fruttiger M (2004) Periodic Delta-like 4
expression in developing retinal arteries. Gene Expr Pat-
terns 5:123–127
39. Saint-Geniez M, Masri B, Malecaze F, Knibiehler B, Au-
digier Y (2002) Expression of the murine msr/apj receptor
and its ligand apelin is upregulated during formation of the
retinal vessels. Mech Dev 110:183–186
40. Lu X, le Noble F, Yuan L, Jiang Q, De Lafarge B, Sugiyama
D, Breant C, Claes F, De Smet F, Thomas JL, Autiero M,
Carmeliet P, Tessier-Lavigne M, Eichmann A (2004) The
netrin receptor UNC5B mediates guidance events control-
ling morphogenesis of the vascular system. Nature
432:179–186
41. Sainson RC, Aoto J, Nakatsu MN, Holderfield M, Conn E,
Koller E, Hughes CC (2005) Cell-autonomous notch sig-
naling regulates endothelial cell branching and proliferation
during vascular tubulogenesis. FASEB J 19:1027–1029
42. Gerhardt H, Betsholtz C (2005) How do endothelial cells
orientate? EXS 94:3–15
43. Carmeliet P, Ng YS, Nuyens D, Theilmeier G, Brusselmans
K, Cornelissen I, Ehler E, Kakkar VV, Stalmans I, Mattot
V, Perriard JC, Dewerchin M, Flameng W, Nagy A, Lupu F,
Moons L, Collen D, D’Amore PA, Shima DT (1999) Im-
paired myocardial angiogenesis and ischemic cardiomyop-
athy in mice lacking the vascular endothelial growth factor
isoforms VEGF164 and VEGF188. Nat Med 5:495–502
44. Stalmans I, Ng YS, Rohan R, Fruttiger M, Bouche A, Yuce
A, Fujisawa H, Hermans B, Shani M, Jansen S, Hicklin D,
Anderson DJ, Gardiner T, Hammes HP, Moons L, De-
werchin M, Collen D, Carmeliet P, D’Amore PA (2002)
Arteriolar and venular patterning in retinas of mice selec-
tively expressing VEGF isoforms. J Clin Invest 109:327–336
45. Dorrell MI, Aguilar E, Friedlander M (2002) Retinal vas-
cular development is mediated by endothelial filopodia, a
preexisting astrocytic template and specific R-cadherin
adhesion. Invest Ophthalmol Vis Sci 43:3500–3510
46. Klagsbrun M, Eichmann A (2005) A role for axon guidance
receptors and ligands in blood vessel development and tu-
mor angiogenesis. Cytokine Growth Factor Rev 16:535–548
47. Steinbach K, Volkmer H, Schlosshauer B (2002) Semaph-
orin 3E/collapsin-5 inhibits growing retinal axons. Exp Cell
Res 279:52–61
48. Livesey FJ, Hunt SP (1997) Netrin and netrin receptor
expression in the embryonic mammalian nervous system
suggests roles in retinal, striatal, nigral, and cerebellar
development. Mol Cell Neurosci 8:417–429
49. Erskine L, Williams SE, Brose K, Kidd T, Rachel RA,
Goodman CS, Tessier-Lavigne M, Mason CA (2000) Reti-
nal ganglion cell axon guidance in the mouse optic chiasm:
expression and function of robos and slits. J Neurosci
20:4975–4982
50. Adams RH, Wilkinson GA, Weiss C, Diella F, Gale NW,
Deutsch U, Risau W, Klein R (1999) Roles of ephrinB li-
gands and EphB receptors in cardiovascular development:
demarcation of arterial/venous domains, vascular morpho-
genesis, and sprouting angiogenesis. Genes Dev 13:295–306
51. Wang HU, Chen ZF, Anderson DJ (1998) Molecular dis-
tinction and angiogenic interaction between embryonic
arteries and veins revealed by ephrin-B2 and its receptor
Eph-B4. Cell 93:741–753
52. Kertesz N, Krasnoperov V, Reddy R, Leshanski L, Kumar
SR, Zozulya S, Gill PS (2006) The soluble extracellular
domain of EphB4 (sEphB4) antagonizes EphB4-EphrinB2
interaction, modulates angiogenesis, and inhibits tumor
growth. Blood 107:2330–2380
53. Steinle JJ, Meininger CJ, Chowdhury U, Wu G, Granger HJ
(2003) Role of ephrin B2 in human retinal endothelial cell
proliferation and migration. Cell Signal 15:1011–1017
54. Steinle JJ, Meininger CJ, Forough R, Wu G, Wu MH,
Granger HJ (2002) Eph B4 receptor signaling mediates
endothelial cell migration and proliferation via the phos-
phatidylinositol 3-kinase pathway. J Biol Chem 277:43830–
43835
55. Kim I, Ryu YS, Kwak HJ, Ahn SY, Oh JL, Yancopoulos
GD, Gale NW, Koh GY (2002) EphB ligand, ephrinB2,
suppresses the VEGF– and angiopoietin 1-induced Ras/
mitogen–activated protein kinase pathway in venous
endothelial cells. FASEB J 16:1126–1128
56. Hughes S, Chang-Ling T (2000) Roles of endothelial cell
migration and apoptosis in vascular remodeling during
development of the central nervous system. Microcircula-
tion 7:317–333
57. Ishida S, Yamashiro K, Usui T, Kaji Y, Ogura Y, Hida T,
Honda Y, Oguchi Y, Adamis AP (2003) Leukocytes
mediate retinal vascular remodeling during development
and vaso-obliteration in disease. Nat Med 9:781–788
58. Checchin D, Sennlaub F, Levavasseur E, Leduc M, Chem-
tob S (2006) Potential role of microglia in retinal blood
vessel formation. Invest Ophthalmol Vis Sci 47:3595–3602
59. Nakatsu MN, SainsonRC, Perez-del-Pulgar S, Aoto JN,
Aitkenhead M, Taylor KL, Carpenter PM, Hughes CC
(2003) VEGF(121) and VEGF(165) regulate blood vessel
diameter through vascular endothelial growth factor
receptor 2 in an in vitro angiogenesis model. Lab Invest
83:1873–1885
60. Vargesson N, Laufer E (2001) Smad7 misexpression during
embryonic angiogenesis causes vascular dilation and mal-
formations independently of vascular smooth muscle cell
function. Dev Biol 240:499–516
61. le Noble F, Moyon D, Pardanaud L, Yuan L, Djonov V,
Matthijsen R, Breant C, Fleury V, Eichmann A (2004) Flow
regulates arterial-venous differentiation in the chick em-
bryo yolk sac. Development 131:361–375
62. Smith LE, Wesolowski E, McLellan A, Kostyk SK,
D’Amato R, Sullivan R, D’Amore PA (1994) Oxygen-in-
duced retinopathy in the mouse. Invest Ophthalmol Vis Sci
35:101–111
63. Gu X, Samuel S, El Shabrawey M, Caldwell RB, Bartoli M,
Marcus DM, Brooks SE (2002) Effects of sustained hyper-
oxia on revascularization in experimental retinopathy of
prematurity. Invest Ophthalmol Vis Sci 43:496–502
64. Gu X, El Remessy AB, Brooks SE, Al Shabrawey M, Tsai
NT, Caldwell RB (2003) Hyperoxia induces retinal vascular
endothelial cell apoptosis through formation of peroxyni-
trite. Am J Physiol Cell Physiol 285:C546–C554
65. Alon T, Hemo I, Itin A, Pe’er J, Stone J, Keshet E (1995)
Vascular endothelial growth factor acts as a survival factor
for newly formed retinal vessels and has implications for
retinopathy of prematurity. Nat Med 1:1024–1028
66. Riva CE, Pournaras CJ, Tsacopoulos M (1986) Regulation
of local oxygen tension and blood flow in the inner retina
during hyperoxia. J Appl Physiol 61:592–598
67. Claxton S, Fruttiger M (2003) Role of arteries in oxygen
induced vaso-obliteration. Exp Eye Res 77:305–311
68. Chan-Ling T, Page MP, Gardiner T, Baxter L, Rosinova E,
Hughes S (2004) Desmin ensheathment ratio as an indicator
86 Angiogenesis (2007) 10:77–88
123
of vessel stability: evidence in normal development and in
retinopathy of prematurity. Am J Pathol 165:1301–1313
69. Hellstrom M, Kaln M, Lindahl P, Abramsson A, Betsholtz
C (1999) Role of PDGF-B and PDGFR-beta in recruitment
of vascular smooth muscle cells and pericytes during
embryonic blood vessel formation in the mouse. Develop-
ment 126:3047–3055
70. Nishishita T, Lin PC (2004) Angiopoietin 1, PDGF-B, and
TGF-beta gene regulation in endothelial cell and smooth
muscle cell interaction. J Cell Biochem 91:584–593
71. Satchell SC, Harper SJ, Mathieson PW (2001) Angiopoie-
tin-1 is normally expressed by periendothelial cells. Thromb
Haemost 86:1597–1598
72. Suri C, Jones PF, Patan S, Bartunkova S, Maisonpierre PC,
Davis S, Sato TN, Yancopoulos GD (1996) Requisite role
of angiopoietin-1, a ligand for the TIE2 receptor, during
embryonic angiogenesis. Cell 87:1171–1180
73. Benjamin LE, Hemo I, Keshet E (1998) A plasticity win-
dow for blood vessel remodelling is defined by pericyte
coverage of the preformed endothelial network and is reg-
ulated by PDGF-B and VEGF. Development 125:1591–
1598
74. Uemura A, Ogawa M, Hirashima M, Fujiwara T, Koyama
S, Takagi H, Honda Y, Wiegand SJ, Yancopoulos GD,
Nishikawa S (2002) Recombinant angiopoietin-1 restores
higher-order architecture of growing blood vessels in mice
in the absence of mural cells. J Clin Invest 110:1619–1628
75. Jain RK (2003) Molecular regulation of vessel maturation.
Nat Med 9:685–693
76. Risau W, Hallmann R, Albrecht U (1986) Differentiation-
dependent expression of proteins in brain endothelium
during development of the blood-brain barrier. Dev Biol
117:537–545
77. Bauer H, Sonnleitner U, Lametschwandtner A, Steiner M,
Adam H, Bauer HC (1995) Ontogenic expression of the
erythroid-type glucose transporter (Glut 1) in the telen-
cephalon of the mouse: correlation to the tightening of the
blood–brain barrier. Brain Res Dev Brain Res 86:317–325
78. Saunders NR, Knott GW, Dziegielewska KM (2000) Bar-
riers in the immature brain. Cell Mol Neurobiol 20:29–40
79. Arthur FE, Shivers RR, Bowman PD (1987) Astrocyte-
mediated induction of tight junctions in brain capillary
endothelium: an efficient in vitro model. Brain Res 433:155–
159
80. Risau W (1991) Induction of blood–brain barrier endothe-
lial cell differentiation. Ann NY Acad Sci 633:405–419
81. Tserentsoodol N, Shin BC, Suzuki T, Takata K (1998)
Colocalization of tight junction proteins, occludin and ZO-
1, and glucose transporter GLUT1 in cells of the blood–
ocular barrier in the mouse eye. Histochem Cell Biol
110:543–551
82. Russ PK, Davidson MK, Hoffman LH, Haselton FR (1998)
Partial characterization of the human retinal endothelial
cell tight and adherens junction complexes. Invest Oph-
thalmol Vis Sci 39:2479–2485
83. Barber AJ, Antonetti DA (2003) Mapping the blood vessels
with paracellular permeability in the retinas of diabetic rats.
Invest Ophthalmol Vis Sci 44:5410–5416
84. Barber AJ, Antonetti DA, Gardner TW (2000) Altered
expression of retinal occludin and glial fibrillary acidic
protein in experimental diabetes. The Penn State Retina
Research Group. Invest Ophthalmol Vis Sci 41:3561–3568
85. Kevil CG, Okayama N, Trocha SD, Kalogeris TJ, Coe LL,
Specian RD, Davis CP, Alexander JS (1998) Expression of
zonula occludens and adherens junctional proteins in hu-
man venous and arterial endothelial cells: role of occludin in
endothelial solute barriers. Microcirculation 5:197–210
86. Wong V, Gumbiner BM (1997) A synthetic peptide corre-
sponding to the extracellular domain of occludin perturbs
the tight junction permeability barrier. J Cell Biol 136:399–
409
87. Antonetti DA, Barber AJ, Khin S, Lieth E, Tarbell JM,
Gardner TW (1998) Vascular permeability in experimental
diabetes is associated with reduced endothelial occludin
content: vascular endothelial growth factor decreases oc-
cludin in retinal endothelial cells. Penn State Retina Re-
search Group. Diabetes 47:1953–1959
88. Gardner TW, Lieth E, Khin SA, Barber AJ, Bonsall DJ,
Lesher T, Rice K, Brennan WA Jr (1997) Astrocytes in-
crease barrier properties and ZO-1 expression in retinal
vascular endothelial cells. Invest Ophthalmol Vis Sci
38:2423–2427
89. Tout S, Chan-Ling T, Hollander H, Stone J (1993) The role
of Muller cells in the formation of the blood-retinal barrier.
Neuroscience 55:291–301
90. Gariano RF, Iruela-Arispe ML, Hendrickson AE (1994)
Vascular development in primate retina: comparison of
laminar plexus formation in monkey and human. Invest
Ophthalmol Vis Sci 35:3442–3445
91. Provis JM (2001) Development of the primate retinal vas-
culature. Prog Retin Eye Res 20:799–821
92. Engerman RL, Meyer RK (1965) Development of retinal
vasculature in rats. Am J Ophthalmol 60:628–641
93. Heckenlively JR, Hawes NL, Friedlander M, Nusinowitz S,
Hurd R, Davisson M, Chang B (2003) Mouse model of
subretinal neovascularization with choroidal anastomosis.
Retina 23:518–522
94. Trommsdorff M, Gotthardt M, Hiesberger T, Shelton J,
Stockinger W, Nimpf J, Hammer RE, Richardson JA, Herz
J (1999) Reeler/Disabled-like disruption of neuronal
migration in knockout mice lacking the VLDL receptor and
ApoE receptor 2. Cell 97:689–701
95. Hackett SF, Wiegand S, Yancopoulos G, Campochiaro PA
(2002) Angiopoietin-2 plays an important role in retinal
angiogenesis. J Cell Physiol 192:182–187
96. Hackett SF, Ozaki H, Strauss RW, Wahlin K, Suri C,
Maisonpierre P, Yancopoulos G, Campochiaro PA (2000)
Angiopoietin 2 expression in the retina: upregulation during
physiologic and pathologic neovascularization. J Cell
Physiol 184:275–284
97. Maisonpierre PC, Suri C, Jones PF, Bartunkova S, Wiegand
SJ, Radziejewski C, Compton D, McClain J, Aldrich TH,
Papadopoulos N, Daly TJ, Davis S, Sato TN, Yancopoulos
GD (1997) Angiopoietin-2, a natural antagonist for Tie2
that disrupts in vivo angiogenesis. Science 277:55–60
98. Luhmann UF, Lin J, Acar N, Lammel S, Feil S, Grimm C,
Seeliger MW, Hammes HP, Berger W (2005) Role of the
Norrie disease pseudoglioma gene in sprouting angiogenesis
during development of the retinalvasculature. Invest
Ophthalmol Vis Sci 46:3372–3382
99. Xu Q, Wang Y, Dabdoub A, Smallwood PM, Williams J,
Woods C, Kelley MW, Jiang L, Tasman W, Zhang K, Na-
thans J (2004) Vascular development in the retina and inner
ear: control by Norrin and Frizzled-4, a high-affinity ligand-
receptor pair. Cell 116:883–895
100. Ohlmann A, Scholz M, Goldwich A, Chauhan BK, Hudl K,
Ohlmann AV, Zrenner E, Berger W, Cvekl A, Seeliger
MW, Tamm ER (2005) Ectopic norrin induces growth of
ocular capillaries and restores normal retinal angiogenesis
in Norrie disease mutant mice. J Neurosci 25:1701–1710
Angiogenesis (2007) 10:77–88 87
123
101. Chen ZY, Battinelli EM, Fielder A, Bundey S, Sims K,
Breakefield XO, Craig IW (1993) A mutation in the Norrie
disease gene (NDP) associated with X-linked familial exu-
dative vitreoretinopathy. Nat Genet 5:180–183
102. Toomes C, Downey LM, Bottomley HM, Scott S, Woodruff
G, Trembath RC, Inglehearn CF (2004) Identification of a
fourth locus (EVR4) for familial exudative vitreoretinopa-
thy (FEVR). Mol Vis 10:37–42
103. Enyedi LB, de Juan E Jr, Gaitan A (1991) Ultrastructural
study of Norrie’s disease. Am J Ophthalmol 111:439–445
104. Toomes C, Bottomley HM, Jackson RM, Towns KV, Scott
S, Mackey DA, Craig JE, Jiang L, Yang Z, Trembath R,
Woodruff G, Gregory-Evans CY, Gregory-Evans K, Parker
MJ, Black GC, Downey LM, Zhang K, Inglehearn CF
(2004) Mutations in LRP5 or FZD4 underlie the common
familial exudative vitreoretinopathy locus on chromosome
11q. Am J Hum Genet 74:721–730
105. Kato M, Patel MS, Levasseur R, Lobov I, Chang BH, Glass
DA, Hartmann C, Li L, Hwang TH, Brayton CF, Lang RA,
Karsenty G, Chan L (2002) Cbfa1-independent decrease in
osteoblast proliferation, osteopenia, and persistent embry-
onic eye vascularization in mice deficient in Lrp5, a Wnt
coreceptor. J Cell Biol 157:303–314
106. Goldberg MF (1997) Persistent fetal vasculature (PFV): an
integrated interpretation of signs and symptoms associated
with persistent hyperplastic primary vitreous (PHPV). LIV
Edward Jackson Memorial Lecture. Am J Ophthalmol
124:587–626
107. Lang RA, Bishop JM (1993) Macrophages are required for
cell death and tissue remodeling in the developing mouse
eye. Cell 74:453–462
108. Diez-Roux G, Lang RA (1997) Macrophages induce
apoptosis in normal cells in vivo. Development 124:3633–
3638
109. Lobov IB, Rao S, Carroll TJ, Vallance JE, Ito M, Ondr JK,
Kurup S, Glass DA, Patel MS, Shu W, Morrisey EE,
McMahon AP, Karsenty G, Lang RA (2005) WNT7b
mediates macrophage-induced programmed cell death in
patterning of the vasculature. Nature 437:417–421
110. Richter M, Gottanka J, May CA, Welge-Lussen U, Berger
W, Lutjen-Drecoll E (1998) Retinal vasculature changes in
Norrie disease mice. Invest Ophthalmol Vis Sci 39:2450–
2457
111. Chang B, Smith RS, Peters M, Savinova OV, Hawes NL,
Zabaleta A, Nusinowitz S, Martin JE, Davisson ML, Cepko
CL, Hogan BL, John SW (2001) Haploinsufficient Bmp4
ocular phenotypes include anterior segment dysgenesis with
elevated intraocular pressure. BMC Genet 2:18
112. Rousseau B, Larrieu-Lahargue F, Bikfalvi A, Javerzat S
(2003) Involvement of fibroblast growth factors in choroidal
angiogenesis and retinal vascularization. Exp Eye Res
77:147–156
113. Fukai N, Eklund L, Marneros AG, Oh SP, Keene DR,
Tamarkin L, Niemela M, Ilves M, Li E, Pihlajaniemi T,
Olsen BR (2002) Lack of collagen XVIII/endostatin results
in eye abnormalities. EMBO J 21:1535–1544
114. Ishida S, Usui T, Yamashiro K, Kaji Y, Amano S, Ogura Y,
Hida T, Oguchi Y, Ambati J, Miller JW, Gragoudas ES, Ng
YS, D’Amore PA, Shima DT, Adamis AP (2003)
VEGF164-mediated inflammation is required for patho-
logical, but not physiological, ischemia-induced retinal
neovascularization. J Exp Med 198:483–489
115. Yoshida S, Yoshida A, Ishibashi T, Elner SG, Elner VM
(2003) Role of MCP-1 and MIP-1alpha in retinal neovas-
cularization during postischemic inflammation in a mouse
model of retinal neovascularization. J Leukoc Biol
73:137–144
116. Alva JA, Iruela-Arispe ML (2004) Notch signaling in vas-
cular morphogenesis. Curr Opin Hematol 11:278–283
117. Bicknell R, Harris AL (2004) Novel angiogenic signaling
pathways and vascular targets. Annu Rev Pharmacol Tox-
icol 44:219–238
118. Shibuya M, Claesson-Welsh L (2006) Signal transduction by
VEGF receptors in regulation of angiogenesis and lym-
phangiogenesis. Exp Cell Res 312:549–560
119. Thurston G (2003) Role of angiopoietins and Tie receptor
tyrosine kinases in angiogenesis and lymphangiogenesis.
Cell Tissue Res 314:61–68
120. Bainbridge JW, Mistry A, De Alwis M, Paleolog E, Baker
A, Thrasher AJ, Ali RR (2002) Inhibition of retinal neo-
vascularisation by gene transfer of soluble VEGF receptor
sFlt-1. Gene Ther 9:320–326
121. McLeod DS, Taomoto M, Cao J, Zhu Z, Witte L, Lutty GA
(2002) Localization of VEGF receptor-2 (KDR/Flk-1) and
effects of blocking it in oxygen-induced retinopathy. Invest
Ophthalmol Vis Sci 43:474–482
122. Campochiaro PA (2006) Ocular versus extraocular neo-
vascularization: mirror images or vague resemblances. In-
vest Ophthalmol Vis Sci 47:462–474
123. Shen J, Yang X, Xiao WH, Hackett SF, Sato Y, Campo-
chiaro PA (2006) Vasohibin is up-regulated by VEGF in the
retina and suppresses VEGF receptor 2 and retinal neo-
vascularization. FASEB J 20:723–725
124. Aronoff R, Petersen CC (2006) Controlled and localized
genetic manipulation in the brain. J Cell Mol Med 10:333–
352
88 Angiogenesis (2007) 10:77–88
123
	Development of the retinal vasculature
	Abstract
	Introduction
	Retinal astrocytes
	Vascular growth in the retina
	Guidance of sprouting angiogenesis
	Vascular remodelling and maturation
	Deeper plexus development
	Regression of the hyaloid vasculature
	Pathological vessel growth in the retina
	Acknowledgements
	References
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