MINIREVIEW
Brain angiogenesis in developmental and pathological
processes: therapeutic aspects of vascular endothelial
growth factor
Masabumi Shibuya
1,2
1 Department of Molecular Oncology, Tokyo Medical and Dental University, Japan
2 Jobu University, Takasaki, Japan
Introduction
The central nervous system (CNS) is a complex of
well-vascularized tissues through which oxygen and
nutrition are supplied to the brain via the carotid
artery. Actually, cells such as neurons and glial cells in
the CNS require a fresh supply of blood to function.
In embryogenesis, the formation of primitive blood
vessels from progenitors, hemangioblasts ⁄ angioblasts,
is dependent on the vascular endothelial growth fac-
tor ⁄ vascular endothelial growth factor receptor
(VEGF ⁄ VEGFR) system [1,2], and the further devel-
opment of blood vessels in various tissues and organs,
including the brain, is regulated by the VEGF system
in combination with other signaling systems such as
the angiopoietin–Tie, ephrin–Eph, Delta–Notch sys-
tems, and the Wnt pathway.
Furthermore, the blood vessel network in the CNS
has a unique stabilizing system at the postnatal to
adult stages known as the blood–brain barrier (BBB)
Keywords
macrophage; malignant glioma; motor
neuron; tumor angiogenesis; vascular
hyperpermeability; VEGF-A; VEGF-B;

angiogenic drugs or by developing unique medicines specifically targeting
the blood vessels in brain tumors.
Abbreviations
BBB, blood–brain barrier; CNS, central nervous system; EC, endothelial cell; FGF, fibroblast growth factor; HIF, hypoxia-inducible factor;
PlGF, placenta growth factor; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; VHL, von
Hippel–Lindau.
4636 FEBS Journal 276 (2009) 4636–4643 ª 2009 The Author Journal compilation ª 2009 FEBS
[3]. The BBB mainly consists of a strong interaction
between vascular endothelial cells and astrocytes, and
the tight junctions of vascular endothelial cells (ECs)
in the BBB are well organized with claudins and ZO-
proteins through a decrease in angiogenesis signaling
and an increase in the stability of ECs by the
AKAP12 ⁄ SSeCKS ⁄ Gravin and other systems [4].
Because the VEGF–VEGFR system is central to
angiogenesis in almost all stages of life, we briefly
introduce it here (Fig. 1), followed by discussion of
two pathological conditions of angiogenesis in the
brain, stroke and brain tumors, along with possible
therapeutic strategies.
VEGF and VEGFRs
The VEGF family
The VEGF family includes VEGF-A (also called
VEGF), placenta growth factor (PlGF), VEGF-B, -C,
-D and -E, and Trimeresurus flavoviridis, snake-venom
VEGF, although the latter two proteins are not
encoded in the human genome (Table 1) [1]. VEGF-A
is most important for vasculogenesis as well as angio-
genesis in both embryogenesis and adulthood, and
functions by binding with two tyrosine kinase recep-

Vascular permeability activity detected by Miles assay (at acute phase: within 15 min).
M. Shibuya Therapeutic aspects of VEGF in brain diseases
FEBS Journal 276 (2009) 4636–4643 ª 2009 The Author Journal compilation ª 2009 FEBS 4637
and neuropilin-1 [2]. VEGF-A has two major biologi-
cal roles, in angiogenesis and vascular permeability.
Angiogenesis
VEGF-A stimulates endothelial proliferation directly
via the activation of VEGFR-2 tyrosine kinase. VEG-
FR-2 tyrosine kinase has a strong kinase activity simi-
lar to epidermal growth factor receptor (EGFR), but
the signaling pathway towards proliferation differs
from that in EGFR. VEGFR-2 activates the phospho-
lipase Cc–protein kinase C–Raf–mitogen-activated
protein kinase pathway via a phosphorylated tyrosine
residue at position 1175 of the receptor, and stimulates
EC proliferation [5]. In adults, particularly under path-
ological conditions, VEGFR-1 also contributes to
angiogenesis indirectly via the recruitment of mono-
cyte ⁄ macrophage lineage cells which secrete various
angiogenic factors [6]. In addition, VEGFR-1
expressed in ECs generates mitotic and survival sig-
nals, although much less intensely than VEGFR-2.
Vascular permeability
VEGF-A stimulates the vascular leakage of fluids from
blood vessels in both an acute and a chronic manner.
Although the molecular basis of the signaling pathway
for vascular hyperpermeability within the cell is not
fully understood, both types of hyperpermeability
depend strongly on the simultaneous activation of two
receptors, VEGFR-1 and VEGFR-2. VEGF-E, a viral

Brain stroke
Stroke is induced through: (a) the obstruction of mid-
sized to large blood vessels, or (b) massive bleeding
from mid-sized to large vessels in the brain. These
lesions result in severe ischemia of neurons and astro-
cytes around and downstream of the lesions, eventually
inducing necrotic cell death. Several risk factors
including aging, hypertension, diabetes and atheroscle-
rosis have been described, but explain only about half
of the causes of stroke, suggesting that unknown
mechanisms are also involved in the onset [3].
Increased vascular density surrounding the ‘stroke’
area has been observed after stroke, and such an
increase in blood flow may rescue the ischemic and still
viable region of the brain called the ‘penumbra’ [14].
Therefore, degree of angiogenesis appears to correlate
with rate of recovery from stroke.
A variety of angiogenic factors such as VEGF,
fibroblast growth factor (FGF) and platelet-derived
growth factor are secreted from neuronal cells, astro-
cytes and inflammatory cells, including macrophages
that have infiltrated the stroke area. Zhang et al. [15]
have shown that the intravenous administration of
VEGF-A within 2 days after stroke induces angiogene-
sis in the penumbra, and contributes to a recovery in
neuron function from the ischemic events.
Administration of VEGF-A into the brain after
stroke may be effective for recovery. However, VEGF-
A not only has pro-angiogenic activity, but also
increases vascular permeability, and increases in tissue

duration of administration (Fig. 2). Because the
VEGF-E gene was originally found in a proangiogenic
sheep ⁄ goat (sometimes human)-oriented parapox virus,
‘Orf virus’, and does not exist in the human genome,
‘humanization’ of this protein to decrease its possible
antigenicity is needed. Such a trial has been already
carried out successfully [8].
Other factors unrelated to VEGF, including angio-
poietin or its modified molecule Comp-Ang1, FGF
and hepatocyte growth factor may also improve the
supply of blood into ischemic areas after stroke. In
addition, the transcription factor PGC-1a was recently
reported to have angiogenic activity via upregulation
of VEGF gene expression independent of the
hypoxia-inducible factor (HIF) system [17]. Further
study is needed to clarify which factor is most benefi-
cial for the recovery from brain ischemia.
Motor neuron degeneration
In 2001, Oosthuyse et al. [18] reported that deletion of
the hypoxia-response element of the VEGF-A gene
promoter and a reduction in VEGF-A expression
cause the degeneration of motor neurons. This study
raised the possibility that the VEGF and motor neuron
systems interact closely. Furthermore, Sun et al. [19]
found that VEGF-B, a member of the VEGF family,
has neuroprotective activity. VEGF-B knockout mice
showed increased severity after cerebral ischemic
injury. However, it was not clear whether the effect of
VEGF-B is direct or indirect, for example, via the
promotion of pericyte activity.

Brain tumors – malignant glioma
Major malignant tumors in the brain include high-
grade astrocytoma and glioblastoma multiforme. These
Fig. 2. The VEGFR-2-specific ligand VEGF-E may have a broad
range of therapeutic uses with less edema. VEGF-A activates both
VEGFR-1 and VEGFR-2, resulting in angiogenesis and vascular per-
meability. Therefore, the transfer of VEGF-A to ischemic tissue
such as brain stroke areas can easily induce tissue edema. An
inflammatory response may also be elevated via recruitment of
VEGFR-1-expressing macrophages. By contrast, VEGF-E and its
humanized version efficiently induced angiogenesis without severe
edema or inflammation. The safety of VEGF-E appears greater than
that of VEGF-A.
M. Shibuya Therapeutic aspects of VEGF in brain diseases
FEBS Journal 276 (2009) 4636–4643 ª 2009 The Author Journal compilation ª 2009 FEBS 4639
tumors have a relatively high incidence and are signifi-
cantly invasive and metastatic within the CNS in the
late stages. The origins of both tumors are thought to
be glial cells, thus, these tumors appear to be very sim-
ilar, and have been designated as a single entity, malig-
nant glioma. Because malignant glioma is a highly
vascularized tumor and its vascular density has been
reported to correlate with a poor clinical prognosis, it
is focused on here.
Malignant glioma cells show a loss of function in
tumor suppressor genes such as PTEN and p53, and
the activation of oncogenes such as gene amplification
of EGFR in either the wild-type or dominant active
form [22,23]. Some malignant gliomas also show c-myc
activation, but the dominant active mutant form of

high (K
d
= 1–10 pm), 10-fold that of VEGFR-2.
However, the tyrosine kinase activity of VEGFR-1 is
one order of magnitude lower than that of VEGFR-2
which is as strong as other typical tyrosine kinase
receptors like EGFR.
An important question is how tightly the signaling
from each receptor is linked to tumor angiogenesis and
the growth of malignant glioma in vivo. VEGFR-2 is
specifically expressed in vascular endothelial cells, and
directly transduces most of the mitotic signal towards
ECs, resulting in angiogenesis. However, VEGFR-1 is
expressed not only in vascular endothelial cells, but
also in monocyte ⁄ macrophage lineage cells. To clarify
the role of VEGFR-1 signaling in angiogenesis and
tumor growth in glioma, Kerber et al. [27] recently
studied the growth rate of intracranially transplanted
glioma cells in bone marrow-transplanted mice. They
used two systems, irradiated wild-type mice carrying
wild-type bone marrow cells, and irradiated wild-type
mice carrying VEGFR-1 (Flt-1) TK) ⁄ ) bone marrow
cells. VEGFR-1 TK) ⁄ ) mouse cells are deficient in sig-
naling from VEGFR-1 because of a lack of the tyro-
sine kinase domain [21]. They used three cell types, the
original glioma cells, VEGF-A-overexpressing glioma
cells and PlGF-overexpressing glioma cells. Remark-
ably, all three gliomas showed a significant decrease in
growth in vivo ( 30–50% decrease) in mice carrying
VEGFR-1 TK) ⁄ ) bone marrow cells compared with

blood vessels; and (c) the migration of tumor cells
independent of the vascular network, but via other
brain-specific structures such as neuronal fibers ⁄ axon
bundles. Under physiological conditions, glial cells and
vascular endothelial cells have cross-contacts, and
establish the BBB. It is of interest whether such a glial
cell–EC contact system is partly used for the rapid
migration of tumor cells through the vessel network.
The VEGF–VEGFR system is now widely accepted
as a major factor in a variety of solid tumors, as
strongly suggested to be the case in malignant glioma
also. Based on the results of phase III studies [29], bev-
acizumab, a humanized monoclonal anti-VEGF-A
neutralizing IgG, has been approved in many countries
for the treatment of colorectal cancer, lung cancer
(non-small cell, nonepithelial type) and breast cancer.
Furthermore, orally available small molecules, solafe-
nib and sunitinib, which inhibit a variety of tyrosine
kinases including VEGFRs, have been approved for
the treatment of renal cell cancer and liver cancer.
These anti-angiogenic drugs have significantly
improved the disease-free survival rate and total sur-
vival rate of cancer patients via at least two mecha-
nisms, (a) blocking of tumor angiogenesis and (b)
normalization of tumor vessels, although some adverse
effects have been observed [30]. Bevacizumab in com-
bination with cytotoxic agents such as irinotecan, and
other anti-angiogenic drugs such as VEGF-Trap have
recently been reported to be beneficial for the suppres-
sion of tumor growth and for longer survival in malig-

von Hippel–Lindau (VHL) patients. This tumor occurs
not in the cerebrum, but in limited areas such as the
retina, cerebellum, brainstem and spinal cord. Heman-
gioblastoma in VHL patients might be sensitive to
anti-VEGF–VEGFR therapy because VEGF-A is
thought to be abnormally upregulated because of con-
stitutive activation of the HIF pathway, similar to
VHL-deficient renal cell cancer. Treatment of a retinal
hemangioblastoma patient with SU5416, a VEGFR-
specific inhibitor, was effective in recovering visual
functions [36], suggesting that the above strategy may
work. Comparative studies with tumors in the brain
and other organs in terms of the molecular mechanism
for tumor angiogenesis are also important to obtain a
strategy to block the angiogenic pathway.
Conclusion and perspectives
Major brain diseases, i.e. ischemic diseases and brain
tumors such as malignant glioma, are closely linked to
the blood vessel system in the CNS. Therefore, thera-
peutic strategies in the near future will be directly
related to the artificial manipulation of vessel struc-
tures and functions via pro- or anti-angiogenic agents.
The basic regulators of blood vessels in the CNS
appear to be VEGF–VEGFR, angiopoietin–Tie and
BBB-related factors, but the molecular basis of these
signaling pathways is not fully understood. More stud-
ies on these pathways are needed in a CNS-specific
manner. In addition, the mechanism behind vascular
permeability and the formation of edema in brain
tissue needs to be clarified to obtain a strategy with

regulates angiogenesis and tight junction formation in
blood–brain barrier. Nat Med 9, 900–906.
5 Sakurai Y, Ohgimoto K, Kataoka Y, Yoshida N &
Shibuya M (2005) Essential role of Flk-1 (vascular
endothelial growth factor receptor-2) tyrosine residue-
1173 in vasculogenesis in mice. Proc Natl Acad Sci
USA 102, 1076–1081.
6 Sawano A, Iwai S, Sakurai Y, Ito M, Shitara K,
Nakahata T & Shibuya M (2001) Vascular endothelial
growth factor receptor-1 (Flt-1) is a novel cell surface
marker for the lineage of monocytes–macrophages in
humans. Blood 97, 785–791.
7 Kiba A, Sagara H, Hara T & Shibuya M (2003) VEG-
FR-2-specific ligand VEGF-E induces non-edematous
hyper-vascularization in mice. Biochem Biophys Res
Commun 301, 371–377.
8 Zheng Y, Murakami M, Takahashi H, Yamauchi M,
Kiba A, Yamaguchi S, Yabana N, Alitalo K & Shibuya
M (2006) Chimeric VEGF-E
NZ7
⁄ PlGF promotes angio-
genesis via VEGFR-2 without significant enhancement
of vascular permeability and inflammation. Arterioscler
Thromb Vasc Biol 26, 2019–2026.
9 Takahashi H, Hattori S, Iwamatsu A, Takizawa H &
Shibuya M (2004) A novel snake venom vascular endo-
thelial growth factor (VEGF) predominantly induces
vascular permeability through preferential signaling via
VEGF receptor-1. J Biol Chem 279, 46304–46314.
10 Plate KH, Breier G, Weich HA, Mennel HD & Risau

tivator PGC-1alpha. Nature 451, 1008–1012.
18 Oosthuyse B, Moons L, Storkebaum E, Beck H,
Nuyens D, Brusselmans K, Van Dorpe J, Hellings P,
Gorselink M, Heymans S et al. (2001) Deletion of the
hypoxia-response element in the vascular endothelial
growth factor promoter causes motor neuron degenera-
tion. Nat Genet 28, 131–138.
19 Sun Y, Jin K, Childs JT, Xie L, Mao XO & Greenberg
DA (2006) Vascular endothelial growth factor-B (VEG-
FB) stimulates neurogenesis: evidence from knockout
mice and growth factor administration. Dev Biol 289,
329–335.
20 Poesen K, Lambrechts D, Van Damme P, Dhondt J,
Bender F, Frank N, Bogaert E, Claes B, Heylen L,
Verheyen A et al. (2008) Novel role for VEGF-recep-
tor-1 and its ligand VEGF-B in motor neuron degenera-
tion. J Neurosci 28, 10451–10459.
21 Hiratsuka S, Minowa O, Kuno J, Noda T & Shibuya
M (1998) Flt-1 lacking the tyrosine kinase domain is
Therapeutic aspects of VEGF in brain diseases M. Shibuya
4642 FEBS Journal 276 (2009) 4636–4643 ª 2009 The Author Journal compilation ª 2009 FEBS
sufficient for normal development and angiogenesis in
mice. Proc Natl Acad Sci USA 95, 9349–9354.
22 Fueyo J, Gomez-Manzano C, Yung WKA & Kyritsis
AP (1998) The functional role of tumor suppressor
genes in gliomas: clues for future therapeutic strategies.
Neurology 51, 1250–1255.
23 Prigent SA, Nagane M, Lin H, Huvar I, Boss GR,
Feramisco JR, Cavenee WK & Huang HS (1996)
Enhanced tumorigenic behaviour of glioblastoma cells

30 Jain RK, Tong RT & Munn LL (2007) Effect of vascu-
lar normalization by antiangiogenic therapy on intersti-
tial hypertension, peritumor edema, and lymphatic
metastasis: insights from a mathematical model. Cancer
Res 67, 2729.
31 Vredenburgh JJ, Desjardins A, Herndon JE 2nd,
Dowell JM, Reardon DA, Quinn JA, Rich JN,
Sathornsumetee S, Gururangan S, Wagner M et al.
(2007) Phase II trial of bevacizumab and irinotecan in
recurrent malignant glioma. Clin Cancer Res 13, 1253.
32 Norden AD, Young GS, Setayesh K, Muzikansky A,
Klufas R, Ross GL, Ciampa AS, Ebbeling LG, Levy B,
Drappatz J et al. (2008) Bevacizumab for recurrent
malignant gliomas: efficacy, toxicity, and patterns of
recurrence. Neurology 70, 779–787.
33 Zuniga RM, Torcuator R, Jain R, Anderson J, Doyle
T, Ellika S, Schultz L & Mikkelsen T (2009) Efficacy,
safety and patterns of response and recurrence in
patients with recurrent high-grade gliomas treated with
bevacizumab plus irinotecan. J Neurooncol 91, 329–336.
34 Rubenstein JL, Kim J, Ozawa T, Zhang M, Westphal
M, Deen DF & Shuman MA (2000) Anti-VEGF anti-
body treatment of glioblastoma prolongs survival but
results in increased vascular cooption. Neoplasia 2,
306–314.
35 Aiello LP, George DJ, Cahill MT, Wong JS, Cavaller-
ano J, Hannah AL & Kaelin WG Jr (2002) Rapid and
durable recovery of visual function in a patient with
von Hippel–Lindau syndrome after systemic therapy
with vascular endothelial growth factor receptor inhibi-