MINIREVIEW
Brain angiogenesis in developmental and pathological
processes: regulation, molecular and cellular
communication at the neurovascular interface
Hye Shin Lee
1,2
, Jiyeon Han
1,2
, Hyun-Jeong Bai
1,2
and Kyu-Won Kim
1,2,3
1 Neurovascular Coordination Research Center, College of Pharmacy, Seoul National University, Korea
2 Research Institute of Pharmaceutical Science, Seoul National University, Korea
3 Department of Molecular Medicine and Biopharmaceutical Sciences, Seoul National University, Korea
Development of the brain vasculature
Blood vessels form via two distinct processes: vasculo-
genesis and angiogenesis. Vasculogenesis involves the
proliferation and differentiation of mesoderm-derived
angioblasts into endothelial cells [1]. Before the heart
even begins to beat, the primary vascular plexus is
formed throughout the body by vasculogenesis [2]. The
extracerebral vascular plexus is established by vasculo-
genesis within the brain vasculature [2]. Early in
embryogenesis, angioblasts invade the head region and
form the perineural vascular plexus, which ultimately
covers the entire neural tube [3]. After the primary vas-
cular plexus is formed by vasculogenesis, a more com-
plex vascular network is established via angiogenesis
(i.e. the production of vessel branches from pre-exist-
ing vessels). Indeed, the vascular network of the brain

vascular smooth muscle cells, neurons and brain macrophages. Each cell
type plays a unique role, and works with other types to maintain environ-
mental homeostasis and to respond to certain stimuli. Taken together, this
minireview emphasizes the importance of the coordinated action of mole-
cules and cells at the neurovascular interface, with regards to the regulation
of angiogenesis and barriergenesis.
Abbreviations
Ang-1, angiopoietin-1; AQP4, aquaporin4; BBB, blood–brain barrier; BDNF, brain-derived neurotrophic factor; CNS, central nervous system;
HIF, hypoxia-inducible factor; NGF, nerve growth factor; NT, neurotrophins; SEMA, semaphorin; SSeCKS, Src-suppressed C kinase
substrate; TGF, transforming growth factor; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor;
vSMC, vascular smooth muscle cell.
4622 FEBS Journal 276 (2009) 4622–4635 ª 2009 The Authors Journal compilation ª 2009 FEBS
process, vascular sprouts from the pia mater invade
the brain and extend toward the ventricles [4]. Like
other vascular networks, brain vessels undergo forma-
tion, stabilization, branching, pruning and specializa-
tion. In brief, the nascent vasculatures formed by
vasculogenesis and angiogenesis are stabilized via the
recruitment of mural cells and generation of the extra-
cellular matrix. The nascent vasculatures are then fine-
tuned in response to environmental cues from
neighboring cells [5]. Finally, vessels acquire features
suitable for the function of each respective organ.
Brain vessels have extremely specialized characteris-
tics that allow them to form the blood–brain barrier
(BBB). The concept of the BBB was first suggested
more than 100 years ago when Paul Ehrlich discovered
that dyes injected into the vascular system did not pen-
etrate brain tissues but were easily absorbed by periph-
eral tissues [3]. The BBB consists of interendothelial

tified as BBB components [7]; however, their role in
the function of the BBB remains unclear.
The physical barrier resulting from tight, adherens
and gap junctions enhances transcellular, rather than
paracellular, transport when the brain parenchyma
and blood exchange factors across the vessel wall.
Because the physical barrier primarily functions to
protect the brain from toxins in the blood, a special-
ized transport system is needed to absorb essential
molecules and release substances from the brain.
Nutrients are typically transported from the blood
to the brain via a carrier-mediated transport system.
Because glucose is one of the brain’s primary energy
sources, the Glut-1 transporter is of principal impor-
tance to the BBB [11]. The Glut-1 transporter is asym-
metrically distributed, with a greater abundance found
at the abluminal side than at the luminal membrane.
This distribution ensures that the proper level of glu-
cose is supplied to the brain by preventing the accumu-
lation of glucose in the interstitial fluid [12,13].
Essential amino acids, nucleosides and vitamins also
use carrier systems. For example, the L1 system trans-
ports large neutral amino acids, whereas the y+ sys-
tem transports cationic amino acids and the CNT2
adenosine transporter serves as a carrier for nucleo-
sides [13]. In addition to carrier-mediated transporters,
the BBB endothelium has a receptor-mediated trans-
porter system used by proteins, such as insulin, trans-
ferrin and leptin, to cross the BBB [13].
Molecular basis of brain angiogenesis

during barriergenesis
Hypoxic signals are no longer needed once they induce
new vessel formation in areas lacking oxygen and
nutrients. New vessels then undergo maturation steps
suitable for their environment. Vessel maturation in
the brain involves the acquisition of specialized fea-
tures, including the BBB.
In an attempt to connect the missing link between
angiogenesis and barriergenesis, Lee et al. [19] identified
the Src-suppressed C kinase substrate (SSeCKS) protein
(also known as AKAP12 or gravin in humans), which
is upregulated by changes in oxygen tension during
reoxygenation after hypoxic insult. In cultured primary
astrocytes, overexpression of SSeCKS reduced VEGF
expression and induced angiopoietin-1 (Ang-1), thereby
promoting the expression of tight junction proteins and
strengthening the bonds between brain endothelial cells
[19]. Recent studies indicate that SSeCKS ⁄ AKAP12
downregulates HIF-1a expression by enhancing interac-
tions with von Hippel-Lindau tumor suppressor protein
(pVHL) and prolyl hydroxylase domain 2 (PHD2) [20].
These findings strongly suggest that SSeCKS may trig-
ger the transition from angiogenesis to barriergenesis.
Extracellular factors regulating angiogenesis and
barriergenesis
VEGF
Within the brain, formation of the primary vascular
plexus is largely dependent on VEGF signaling. The
interaction between VEGF and the vascular endothe-
lial growth factor receptor (VEGFR) is thought to

Angiopoietin has also been identified as a potent
angiogenic factor during embryonic vessel develop-
ment. Ang-1 deficiency leads to embryonic vascular
defects in the central nervous system (CNS) and many
other parts of the body, because of an inappropriate
association of the extracellular matrix and supporting
cells [28]. Knockout mice deficient in Ang-1, tyrosine
kinase with immunoglobulin-like and EGF-like
domains (Tie)-1 and Tie-2 receptors experienced vascu-
lar defects at a relatively later stage than did VEGF
null mutant mice [29,30]. These findings indicate that
the Ang-1–Tie system may function during vessel mat-
uration and stabilization, rather than during vessel
sprouting. Although Ang-1-overexpressing transgenic
mice experienced increased vascularization, Ang-1 also
increases the tightness of BBB endothelial cells and
reduces vessel permeability [19]. Despite the contro-
versy surrounding the role of Ang-1 as either an angio-
genic factor or a maturation factor, recent studies
clearly show that Ang-1 is a prominent regulator of
vascular development. In addition to its role in vascu-
lar maturation, angiopoietin seems to play an impor-
tant role in the maintenance of BBB homeostasis.
Previous studies have shown that Ang-1 mRNA levels
decrease in conditions that induce BBB breakdown,
such as middle cerebral artery occlusion, whereas the
expression of Ang-2, an endogenous antagonist of
Ang-1, increases [31]. Moreover, when mice with ische-
mic lesions that had been induced by middle cerebral
artery occlusion or VEGF application were treated

In addition to the classical angiogenic regulators dis-
cussed above, Wnt family growth factors have
recently been highlighted as key molecules for CNS
angiogenesis and barriergenesis. Wnts are a large
family of growth factors crucial for a variety of bio-
logical processes; in particular, their functions are
well established in CNS development, for example,
they control dorsal–ventral, anterior–posterial pattern-
ing of CNS tissues, dendrite morphogenesis and
synaptogenesis (for a review, see reference [39]).
According to recent reports, b-catenin, an effecter
molecule of canonical Wnt pathway, is expressed in
the developing CNS vasculature and has critical roles
in embryonic vascular development [40–42]. Interest-
ingly, Wnt ⁄ b-catenin signaling is not only responsible
for angiogenesis, but also regulates barriergenesis.
Conditional knockout of b-catenin in endothelial cells
results in a reduction in CNS vessels, vascular hemor-
rhage and malformation [40]. At the same time, it
impairs Glut-1 expression and claudin-3-mediated
endothelial tightness, reflecting the importance of this
pathway for BBB induction [40,41]. Various types of
Wnt ligands exist in neural tissues to transmit signals
to the perineural endothelium. Wnt7a and Wnt7b are
expressed in ventral–lateral spinal cord, whereas
Wnt1, Wnt3 and Wnt3a are located in dorsal part of
the spinal cord [40,42].
Neurogenic factors involved in angiogenesis and
barriergenesis
Vessels and nerves are located in close proximity to

NT-3 TrkC Inhibit proliferation of cerebral endothelial cells [88]
H. S. Lee et al. Regulation of angiogenesis and barriergenesis
FEBS Journal 276 (2009) 4622–4635 ª 2009 The Authors Journal compilation ª 2009 FEBS 4625
Axon guidance cues
Recently, it has become widely accepted that four
major axon guidance cues (ephrins, semaphorins, slits
and netrins) are responsible for vascular patterning
[44]. In particular, ephrin B2 contributes to arterial–
venous specification, mural cell recruitment, lymphatic
vessel development and tumor angiogenesis via its
receptor, EphB4 [45]. Semaphorins may perform two
functions, with regards to angiogenesis. Class 3 sem-
aphorins (SEMA), such as SEMA3A and SEMA3F,
inhibit angiogenesis via competition with VEGF for
their common receptor, neuropilin. By contrast,
SEMA4D functions as a pro-angiogenic factor that
induces tumor angiogenesis [46]. Slit2 and Netrin 1
also participate in vessel development and tumor
angiogenesis via their receptor UNC5B, as well as the
ROBO4 receptor for slit and the NeogeninA2b recep-
tor for Netrin [47].
Neurotrophins
Neurotrophins (NTs) are well-known trophic factors
involved in neuronal proliferation, survival and path-
finding. The NT family consists of four members [i.e.
nerve growth factor (NGF), brain-derived neuro-
trophic factor (BDNF), NT-3 and NT-4]. Besides their
classical functions on neuronal cells, a growing body
of evidence suggests that NTs play other roles in
non-neuronal tissues, especially blood vessels.

in the vascular environment, including pericytes, astro-
cytes, vascular smooth muscle cells, neurons and brain
macrophages (Fig. 1).
Pericytes
Pericytes are vascular mural cells belonging to the vas-
cular smooth muscle cell (vSMC) lineage. Although
these cells were discovered more than 100 years ago,
pericytes seldom attracted interest because they were
merely considered mural cells that supported endothe-
lial cells. Recent studies have established that pericytes
not only provide physical support to endothelial cells,
but also play critical roles in vessel functioning. Most
importantly, pericytes and endothelial cells share a
basement membrane, enabling them to communicate
directly. In fact, pericytes form focal contacts with
endothelial cells at sites known as peg–socket contacts.
At these contacts, pericytes are connected to endothe-
lial cells through tight, gap and adherence junctions
(Fig. 1) [54]. Pericyte coverage varies among different
types of vessels. The pericyte ⁄ endothelial cell ratio
ranges from 1 : 100 in skeletal muscle to 1 : 1 in the
retina. In general, vessels in the CNS exhibit the high-
est pericyte coverage, highlighting the importance of
pericytes in the formation and maintenance of CNS
vasculature [13,54].
During embryonic angiogenesis, pericyte recruitment
is the first event to stabilize the primary vascular
Table 2. Angiogenic regulatory factors affecting nervous system.
Ang-1, angiopoietin-1; FGF, fibroblast growth factor; IGF, insulin-like
growth factor; VEGF, vascular endothelial growth factor.

consequently induces the differentiation of pericyte
precursors to mature pericytes [57]. The opposite situa-
tion also seems possible, in which pericytes induce and
guide vessel sprouting. In the developing human brain,
migrating pericytes are found in front of growing ves-
sels and pericyte-driven angiogenesis participates in the
organization of growing vessels [58].
Another question that arises is: why are pericytes
abundant in the brain vasculature? Brain pericytes
may perform specialized roles involved in the develop-
ment and maintenance of brain vessels. First and fore-
most, pericytes are thought to enhance BBB integrity.
Generally, in vitro models of the BBB involve the
co-culturing of endothelial cells and astrocytes. How-
ever, when pericytes are added to the co-culture, endo-
thelial cells reorganized into stable, capillary-like
structures [59]. Furthermore, pericytes play a protec-
tive role in hypoxia-induced disruption of the BBB
[60]. Ang-1, a key factor regulating barriergenesis, also
contributes to pericyte-induced BBB formation; in fact,
pericyte-derived Ang-1 induces occludin expression in
cultured brain endothelial cells [34].
Pericytes are sometimes confused with vSMCs,
because a specific marker capable of distinguishing
pericytes from vSMCs has not yet been developed.
However, it seems clear that the mural cells located in
the brain microvessels are pericytes. Like vSMCs in
other parts of the body, pericytes are able to regulate
vessel diameter and blood flow. One of the observa-
tions supporting this idea is that pericytes express a

e
x
PM
BM
AC
Endf eet
N
Blood vessel

c
u
lGe
so
A Adenosine
Amino acid
AQP4
Neurotransmitter
Adherence junction
Gap junction
Tight junction
AA
ulGt1
1L
2T
N
C
Fig. 1. Cellular communication at the neurovascular interface. The neurovascular unit consists of neurons (N), endothelial cells (EC) and other
types of cells located in the neurovascular unit, i.e. astrocytes (AC), pericytes (PC), vascular smooth muscle cells (vSMC), microglia (MG) and
perivascular macrophages (PM). Endothelial cells form a blood–brain barrier characterized by tight, adherence and gap junctions, as well as a
specialized transporter system (i.e. consisting of Glut-1, L1 and CNT2). Pericytes share basement membranes with blood vessels and directly

Astrocytes are also interesting with regards to brain vas-
culature, because they regulate the formation and main-
tenance of BBB, modulate neurovascular coupling and
maintain several parts of brain homeostasis. In this
minireview, we focus on the active functions of astro-
cytes in regards to brain vasculature. Anatomically,
most astrocytes have stellate shapes containing multiple
processes. These cells expand toward neurons and ves-
sels. The ends of the cells, so-called endfeet, contact the
vessel wall and form large compartments that enclose
most blood vessels of the brain (Figs 1, 2A). Thus, one
astrocyte can contact several synapses, in addition to
blood vessels, making it possible to integrate signals
generated from both neurons and vessels. Consequently,
astrocytes are believed to function as key mediators of
neurovascular coordination.
Roles in BBB formation and maintenance
Vessel sprouting is completed before birth, whereas
astrocyte differentiation occurs during the late embry-
onic and early postnatal periods. Because of the dis-
cordance in developmental timing, it seems difficult for
astrocytes to modulate developmental angiogenesis.
Rather, astrocytes may play a role in barriergenesis.
The period of astrocyte differentiation coincides with
that of BBB formation. Differentiating astrocytes may
extend their processes to the vessel wall, thereby send-
ing signals to acquire BBB properties. When cultured
astrocytes were transplanted into a rat anterior eye
chamber or a chick chorioallantoic membrane (where
vessels are leaky), the vessels acquired BBB properties

2+
ion. Vari-
ous neurotransmitters generated from synaptic activi-
ties increase intracellular Ca
2+
levels in astrocytes [66].
For example, glutamate, an excitatory neurotransmit-
ter, stimulates astrocytes via the metabotropic mGluR
receptor, and activated mGluR consequently triggers a
Ca
2+
increase [67]. Local increases in Ca
2+
concentra-
tion diffuse throughout the entire body of astrocytes,
including the endfeet. The Ca
2+
signals released by as-
trocytes subsequently alter vascular tone and promote
either vasoconstriction [68] or vasodilation [67]. In
astrocytes, vasoconstriction and vasodilation both
require arachidonic acid, but the next step is different.
The conversion of arachidonic acid to 20-hydroxyeico-
satetraenoic acid occurs during vasoconstriction, and
arachidonic acid is converted to prostaglandin E
2
or
Regulation of angiogenesis and barriergenesis H. S. Lee et al.
4628 FEBS Journal 276 (2009) 4622–4635 ª 2009 The Authors Journal compilation ª 2009 FEBS
epoxyeicosatrienoic acid during vasodilation [66]. In

into astrocytes.
The clearance of neurotransmitters and ions is accom-
panied by the movement of water, which is buffered by
astrocytes. The increased Na
+
concentration caused by
glutamate transport, and increased intracellular Ca
2+
levels from mGluR activation, lead to water uptake and
slight swelling of the astrocytes [10,67]. The astrocytic
foot processes that surround blood vessels have a high
density of aquaporin4 (AQP4), a water channel, which
transports water bidirectionally between the blood and
the brain. Astrocytes secrete water into the perivascular
space via AQP4, thereafter maintaining water homeo-
stasis in the brain environment (Fig. 1) [69]. During
pathogenesis, AQP4 is likely responsible for the forma-
tion and clearance of brain edema. Interestingly, AQP4
plays opposite roles in cytotoxic and vasogenic edema
(Fig. 2B). Deletion of AQP4 worsens vasogenic edema
and prevents water elimination, whereas AQP4 null
mutants protect against cytotoxic edema by reducing
the flow of water into the brain [71].
vSMCs
vSMCs are myocytes that mediate vasoconstriction
and vasodilation. The thickness of the vSMC layer dif-
fers according to the size of the vessels. In the brain,
pial arteries invade the brain parenchyma and reduce
the width of arterioles, then form deep branches that
become small capillaries. As vessels become smaller,

tact with specialized regions [74]. It is now widely
accepted that our brain contains neural stem cells
throughout our entire lifespan, and that these stem
cells are located at certain regions (i.e. the subgranu-
lar zone of the hippocampus and the subventricular
zone of the cerebral cortex) known as the stem cell
niche. Anatomical analyses of the stem cell niche
have suggested that the environment surrounding neu-
ral stem cells, especially the vascular environment, is
important for maintenance and differentiation. Inter-
estingly, the stem cell niche has high angiogenic
potential, with part of the proliferating cell popula-
tion composed of endothelial precursor cells [75].
These findings indicate that angiogenesis and neuro-
genesis share common signals and blood vessels con-
tribute to neural stem cell behavior by generating
environmental cues. The direct effects of endothelial
cells on neural stem cells were demonstrated via an
in vitro co-culture system. When neural stem cells
H. S. Lee et al. Regulation of angiogenesis and barriergenesis
FEBS Journal 276 (2009) 4622–4635 ª 2009 The Authors Journal compilation ª 2009 FEBS 4629
were co-cultured with endothelial cells, they exhibited
greater self-renewal activity followed by extensive neu-
rogenesis [76]. These findings suggest that the vascular
environment of the neural stem cell may contribute
to the maintenance and proper differentiation of the
stem cell population under certain conditions, with
the help of soluble factors.
Perivascular nerves participate in functional hyper-
emia. These nerves originate from the peripheral ner-


H
2
O
H
2
O
H
2
O
ursiDpnoitfo T JMB &
att
eDh
cm
etn
a

fos
ortc
y
t
e
A(C)e ndfeet
osaVegcinedeam
y
Cto
t
o
x
ic e

e( CL
)
i
n
ifltartino
E damenoitamrof
Blood vessel
Blood vessel
Blood vessel
Blood vessel
BM
RBC
RBC
IgG
MG
N
N
N
AC
AC
CK
EC
BM
TJ
AC
BM
MG
LC
Fig. 2. Neurovascular dysfunction. A number of brain disorders can disrupt homeostasis of the neurovascular unit. (A) Degradation of junc-
tions in the blood–brain barrier (BBB) disrupts neurovascular interactions. (B) Brain edema is a clinically important symptom induced by

from blood to the brain parenchyma (Fig. 2C) [13].
The transmigration of leukocytes through the BBB
occurs via both the paracellular and transcellular path-
ways. Leukocyte and endothelial cell interactions are
necessary for extravasation, a process during which
rolling leukocytes dock to the luminal membrane of
endothelial cells via interactions between selectins,
chemokines and integrins. After docking to the vessel
wall, leukocytes extend their processes toward interen-
dothelial junctions to search for abluminal chemokine
cues. Chemokine–chemokine receptor interactions
encourage leukocytes to migrate to the perivascular
space. Some leukocytes are retained at the perivascular
space, whereas others keep migrating toward brain
parenchyma across the glia limitance [82]. In this pro-
cess, leukocytes migrate through the extracellular
matrix with the help of matrix metalloproteinase [82].
The consequent event of brain macrophages is one
of the most important defense mechanisms used by the
brain. However, this phenomenon sometimes leads to
neuroinflammatory disorders, such as multiple sclerosis
[83] and neuro-AIDS [84]. The inflammatory response
resulting from the activation of microglia and leuko-
cyte infiltration affects normal cells, which, in turn,
causes neuronal dysfunction.
Cell–cell interaction in the
neurovascular unit
As discussed, all types of cells in the brain have
unique roles and coordinate with each other to main-
tain brain homeostasis and enable proper reactions to

precise mechanisms underlying cellular communication
within the neurovascular unit. However, the in vitro
systems used for studying cellular communication (e.g.
the co-culture system or treatment with conditioned
medium) have inevitable limitations and cannot
entirely reproduce the in vivo environment. For exam-
ple, co-cultures of astrocytes and endothelial cells elim-
inate the effects of the basement membrane and
differences in the luminal–abluminal polarity of the
endothelium. To bridge the gap between in vitro condi-
tions and the actual environment, experiments should
incorporate improved in vivo imaging techniques, con-
struct clear marker systems and develop proper animal
models for certain brain diseases.
Acknowledgements
This work was supported by the Korea Science and
Engineering Foundation (KOSEF) grant funded by the
Ministry of Education, Science & Technology (MEST)
through the Creative Research Initiatives Program
(Grant R16-2004-001-01001-0, 2008).
H. S. Lee et al. Regulation of angiogenesis and barriergenesis
FEBS Journal 276 (2009) 4622–4635 ª 2009 The Authors Journal compilation ª 2009 FEBS 4631
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