Brain angiogenesis in developmental and pathologicalprocesses: therapeutic aspects of vascular endothelial growth factor Masabumi Shibuya1,2 1 Department of Molecular Oncology, Tokyo Med
Trang 1Brain angiogenesis in developmental and pathological
processes: therapeutic aspects of vascular endothelial
growth factor
Masabumi Shibuya1,2
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;
VEGF-E; VEGFR-1; VEGFR-2
Correspondence
M Shibuya, Department of Molecular
Oncology, Tokyo Medical and Dental
University, 1-5-45 Yushima, Bunkyo-ku,
Tokyo 113-8519, Japan
Fax: +81 3 5803 0125
Tel: +81 3 5803 5086
E-mail: shibuya@ims.u-tokyo.ac.jp
(Received 19 February 2009, revised 26
May 2009, accepted 15 June 2009)
doi:10.1111/j.1742-4658.2009.07175.x
The angiogenic process in the central nervous system (CNS) is basically regulated by typical angiogenic signaling systems such as vascular endothe-lial growth factor (VEGF)–VEGF receptors and angiopoietin–Tie recep-tors In addition to regular endothelial–pericyte interaction, the CNS vasculature has a unique system of cell to cell communication between endothelial cells and astrocytes which is known as the blood–brain barrier Among the pathological conditions of the CNS vascular network, stroke is
a major disease in which the supply of blood is decreased Pro-angiogenic therapy using natural VEGF-A has so far been unsuccessful, indicating the possible need for a new approach related to upstream or downstream regu-lators involved in the VEGF-signaling pathway, or alternate VEGF family members By contrast, a pathological increase in the blood supply in the CNS is seen in brain tumors, in particular malignant gliomas In phase II clinical trials, anti-VEGF therapies have been shown to suppress tumor growth and improve survival rates to some extent However, tumor inva-sion and the distant metastasis of gliomas can occur following anti-angio-genic therapy Further studies are needed to obtain safer clinical outcomes
by developing new strategies with combination therapy using known anti-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.
Trang 2[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 embryoangio-genesis and adulthood, and functions by binding with two tyrosine kinase recep-tors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR⁄ Flk-1),
Fig 1 The VEGF–VEGFR system and the
inhibitors of various signaling steps Five
VEGF family members and three VEGFRs
are encoded in the human genome In
addi-tion, VEGF-E encoded in the Orf virus binds
and activates only VEGFR-2 Anti-(VEGF-A)
IgG and aptamer, as well as VEGFR tyrosine
kinase inhibitors (multikinase inhibitors) have
been approved for the treatment of various
solid tumors Anti-(VEGF-A) IgG was
effec-tive in phase II clinical trials for glioblastoma
multiforme Other inhibitors or methods to
suppress VEGF–VEGFR system such as
VEGF–Trap, anti-VEGFR neutralizing IgG and
immunotherapy against VEGFRs are
under-going clinical trials.
Table 1 Activities of vascular endothelial growth factor (VEGF) family members PlGF, placenta growth factor; T.F svVEGF, Trimeresurus flavoviridis, snake-venom vascular endothelial growth factor.
a Vascular permeability activity detected by Miles assay (at acute phase: within 15 min).
Trang 3and 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
genome-encoded VEGF-like protein, activates only
VEGFR-2, resulting in angiogenesis without severe
vascular hyperpermeability or tissue edema [7,8] By
contrast, T flavoviridis, snake-venom VEGF, encoded
in the genome of the venomous ‘Habu’ snake, activates
VEGFR-1 strongly and VEGFR-2 weakly, resulting in
high vascular permeability with only minor angiogenic
activity [9]
Expression of VEGF and VEGFRs
VEGF-A, as well as VEGFR-1 and VEGFR-2, is
highly expressed in brain tissue in the early to middle
stages of embryogenesis, and gradually decreases at
the perinatal to postnatal stages However, expression
of VEGF-A and its two receptors is upregulated in
brain tumor tissue [10,11] The VEGF–VEGFR system
is generally used as a paracrine system in vivo
Neuro-nal cells, astrocytes, tumor cells and
bone-marrow-derived cells secrete VEGF-A, whereas VEGFRs are
specifically expressed in vascular ECs VEGFR-1 is
also expressed in macrophages Surprisingly however,
Lee et al.[12] recently reported that ECs do express a low level of VEGF-A, which contributes in part to EC survival in an autocrine manner Neuronal cells also express a low level of VEGFR under certain condi-tions, such as post injury, as discussed later
VEGFR-3 is expressed in lymph endothelial cells VEGF-C, as well as VEGF-D, binds and activates this receptor, resulting in the proliferation and migration
of lymph ECs and lymphangiogenesis [13]
Angiogenesis in brain diseases
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 fluid and edema in the brain may be dangerous because the volume of the brain tissue is tightly limited
by the cranial bone This limitation is unique among tissues in the body, and the risk of edema to brain functions should be carefully considered Brain edema may increase pressure in the cranial cavity and brain tissue, resulting in suppression of blood flow in the vessel network Therefore, it is not easy to control the administration of VEGF-A, i.e timing, dosage, dura-tion and combinadura-tion with other factors⁄ medicines
Trang 4In terms of vascular permeability, a VEGF family
member, VEGF-E, appears to have the attractive
char-acteristic of binding and activating only VEGFR-2,
inducing a relatively strong pro-angiogenic signal
Sev-eral reports have indicated that VEGF-E, but not
VEGF-A, has marked angiogenic activity without
causing severe edema or an inflammatory response
in vivo in transgenic mouse models as well as a
hind-limb ischemia model [7,8,16]
This seems reasonable because vascular permeability
is known to be induced after the simultaneous
activa-tion of VEGFR-1 and VEGFR-2 Furthermore,
inflammation after VEGF-A therapy could be
explained by a strong recruitment of macrophage
line-age cells via VEGF-A because macrophline-ages express
VEGFR-1, and VEGFR-1-dependent signaling
pro-motes the migration of macrophages Thus, VEGF-E
might be safer than VEGF-A in terms of dosage and
duration of administration (Fig 2) Because the
VEGF-Egene 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
Poesen et al [20] have studied this theme exten-sively, and found that: (a) VEGF-B is dispensable for the survival of motor neurons in healthy mice; (b) however, among mutant SOD1-overexpressing trans-genic mice, a model for amyotrophic lateral sclerosis, VEGF-B ) ⁄ ) mice showed faster motor neuron degeneration than VEGF-B +⁄) or + ⁄ + mice; (c) the VEGF-B receptor, VEGFR-1 (Flt-1), is expressed
in astrocytes and motor neurons after injury, and using VEGFR-1 (flt-1) TK) ⁄ ) mice, which are defi-cient in VEGFR-1 signaling [21], they showed that the VEGFR-1 expressed in motor neurons mediates the neuroprotective effect of VEGF-B In addition, the administration of VEGF-B was reported to increase the survival rate of amyotrophic lateral scle-rosis rats (Fig 3) Taken together, these results sug-gest that the VEGF-B–VEGFR-1 system (and maybe also the PlGF–VEGFR-1 system) is motor neuron protective in vivo, and may be a therapeutic target for diseases involving the degeneration of motor neurons
VEGF-A also binds and activates VEGFR-1, and so could be another candidate for the treatment of motor neuron degeneration However, as discussed previ-ously, VEGF-A simultaneously activates VEGFR-1 and VEGFR-2 on vascular endothelial cells, resulting
in strong hyperpermeability and brain edema In this regard, VEGF-B or a molecule with similar activity like PlGF is expected to be a potential therapeutic tool for this disease
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.
Trang 5tumors 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 maligmalig-nant 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
Rasis less frequent Gene amplification and activation
of EGFR occur in more than one third of cases, the
highest incidence among human tumors
Malignant glioma cells secrete a variety of angiogenic
factors such as VEGF and basic FGF [24] VEGF is
considered to have a major role in angiogenesis, as
suggested in other solid tumors like colon carcinoma
and breast carcinoma The molecular basis for the
upregulation of VEGF gene expression in gliomas has
at least four mechanisms (a) A hypoxia⁄ HIF-related
mechanism because of a low oxygen concentration in
growing malignant glioma tissues (b) Another involves
oncogenes, particularly the EGFR signaling pathway,
which stimulates VEGF gene expression via a
HIF-independent mechanism (c) It has been reported that
the FoxM1B transcription factor is upregulated in glioblastoma multiforme, but not in low-grade astro-cytoma, and stimulates VEGF expression independent
of HIF [25] (d) In addition to these mechanisms, Ido
et al.recently reported that HuR protein is upregulated
in glioblastoma multiforme under hypoxia [26] HuR functions to suppress the post-transcriptional degrada-tion of VEGF-A mRNA under hypoxia, contributing to
a further increase in VEGF levels
Accumulating evidence indicates that the VEGF and VEGFR system plays a major role in tumor angiogen-esis in malignant glioma, similar to most other solid tumors VEGF-A activates both VEGFR-1 and VEG-FR-2, but these two receptors differ biochemically The affinity of VEGFR-1 for VEGF-A is extremely high (Kd= 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 mice carrying wild-type cells In parallel with the decrease in tumor volume, the total number of tumor vessels, vessel density and number of infiltrating mac-rophage lineage cells were significantly reduced in mice carrying VEGFR-1 TK) ⁄ ) bone marrow cells These results strongly suggest that, in this model of cranial glioma, nearly half of all tumor growth is dependent
on VEGFR-1 signaling, possibly on bone marrow-derived VEGFR-1-expressing macrophages These macrophages may act as pro-angiogenic and
Fig 3 The VEGF-B–VEGFR-1 system expressed on motor neurons
acts to stop degeneration VEGFR-1 is expressed on motor
neu-rons, and its ligand VEGF-B activates the receptor to generate a
survival signal In VEGF-B ) ⁄ ) or VEGFR-1 TK() ⁄ )) condition, motor
neuron showed severe degeneration [20] Thus, the VEGFR-1
path-way activated either by VEGF-B or by PlGF may be useful for
pro-tecting motor neurons under certain conditions such as
amyotrophic lateral sclerosis.
Trang 6pro-tumorigenic cells similar to the tumor-associated
macrophages reported by several groups [28]
Glioma metastasis and VEGF
Intracranial invasion and metastasis are major
prob-lems in the prognosis of malignant glioma However,
their molecular basis is largely unknown Several
possi-bilities can be considered, including: (a) the
intravascu-lar migration of glioma cells into blood vessels and
their transfer to distant areas in the brain via blood
flow; (b) the rapid migration of tumor cells outside
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 sunitisolafe-nib, 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-nant glioma patients in phase II clinical trials [31–33]
However, a few reports suggest that glioma cells might
have acquired invasive and metastatic phenotypes via
the co-option of tumor cells with the vascular network
and via other mechanisms [30,34]
Under hypoxic conditions or at poor nutrition, some
tumors have been reported to become resistant, being
less apoptotic, and more invasive Our recent studies
also indicate that, in the malignant melanoma model,
tumor cells show a spheroid-like structure and become
more resistant to hypoxia–low nutrition double stress,
resulting in an aggressive phenotype in terms of inva-sion and metastasis in vivo [35]
Late in its clinical course, malignant glioma is known to show extensive cell migration, and become more invasive and metastatic Such metastasis within the brain is inoperable, and thus lethal In this regard, anti-angiogenic therapy should also be studied exten-sively in animal models to optimize the suppression of tumor growth as well as block the nerve dysfunction caused by the tumor mass without making the tumor more invasive or aggressive To this end, cellular responses of malignant glioma to anti-angiogenic stress (hypoxia and low nutrition) should be clarified, and combinations of anti-angiogenic drugs and inhibitors
to suppress aggressiveness induced by anti-angiogenic stress need to be considered
Other brain tumors
In other brain tumors, hemangioblastoma is relatively rare ( 3% of all tumors in the CNS), but occurs in 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
Trang 7which to rapidly and efficiently suppress vascular leaks
during the clinical course of brain diseases or during
the treatment of brain ischemia using pro-angiogenic
medicine
Acknowledgements
This work was supported by Grant-in-Aid Special
Pro-ject Research on Cancer-Bioscience 17014020 from the
Ministry of Education, Culture, Sports, Science and
Technology of Japan, a grant from the program
‘Research for the Future’ of the Japan Society for the
Promotion of Science, and the program ‘Promotion of
Fundamental Research in Health Science’ of the
Organi-zation for Pharmaceutical Safety and Research In May
2009, the FDA in the USA approved bevacizumab for
the treatment of patients with relapsed glioblastoma
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