Physiol Rev 89: 607– 648, 2009;
doi:10.1152/physrev.00031.2008.
Role and Therapeutic Potential of VEGF in the Nervous System
CARMEN RUIZ DE ALMODOVAR, DIETHER LAMBRECHTS, MASSIMILIANO MAZZONE,
AND PETER CARMELIET
The Vesalius Research Center, KU Leuven and VIB, Leuven, Belgium
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I. Introduction: the Neurovascular Link–Common Principles and Mechanisms
II. Biological Activities of Vascular Endothelial Growth Factor
A. VEGF family members
B. VEGF receptor tyrosine kinases
C. Neuropilin coreceptors
D. Regulation of VEGF expression
E. Hypoxia as a key regulator of VEGF expression
III. Role of Vascular Endothelial Growth Factor in the Developing Nervous System
A. VEGF regulates vessel and neuronal wiring in the CNS
B. VEGF at the crossroad of peripheral innervation and vascularization
IV. Role of Vascular Endothelial Growth Factor in the Nervous System in the Healthy Adult
A. Role of VEGF in neurogenesis
B. Role of VEGF in synaptic plasticity
C. Role of VEGF in neuroprotection and neuroregeneration
D. Intracellular signaling of VEGFRs in neural and vascular cells
V. Role and Therapeutic Potential of Vascular Endothelial Growth Factor in Neurological Disease
A. Reduced expression of VEGF triggers motor neuron degeneration
B. VEGF is a modifier of sporadic ALS in humans
C. Therapeutic potential of VEGF in motor neuron degeneration
D. Therapeutic window of VEGF delivery in the brain
E. VEGF in other motor neuron disorders
F. Role of VEGF in excitotoxicity of motor neurons
VI. Vascular Endothelial Growth Factor in Other Neurodegenerative Diseases
A. VEGF in Alzheimer’s disease
B. VEGF in Parkinson’s disease
VII. Vascular Endothelial Growth Factor in Peripheral Neuropathies
A. VEGF in diabetic and chemotherapy-induced neuropathies
B. VEGF and nerve repair
C. VEGF in retinal diseases
VIII. Vascular Endothelial Growth Factor in Acute Neurological Disorders
A. VEGF in ischemic brain disease
B. VEGF in status epilepticus
C. VEGF in multiple sclerosis
D. VEGF in spinal cord injury
IX. Vascular Endothelial Growth Factor-B: an Angiogenic or Neurotrophic Factor?
A. Angiogenic activities of VEGF-B
B. Novel role for VEGF-B in the nervous system
C. VEGFR-1 mediates neuroprotection and glial activation
X. Concluding Remarks
Ruiz de Almodovar C, Lambrechts D, Mazzone M, Carmeliet P. Role and Therapeutic Potential of VEGF in the
Nervous System. Physiol Rev 89: 607– 648, 2009; doi:10.1152/physrev.00031.2008.—The development of the nervous
and vascular systems constitutes primary events in the evolution of the animal kingdom; the former provides
electrical stimuli and coordination, while the latter supplies oxygen and nutrients. Both systems have more in
common than originally anticipated. Perhaps the most striking observation is that angiogenic factors, when
deregulated, contribute to various neurological disorders, such as neurodegeneration, and might be useful for the
treatment of some of these pathologies. The prototypic example of this cross-talk between nerves and vessels is the
vascular endothelial growth factor or VEGF. Although originally described as a key angiogenic factor, it is now well
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0031-9333/09 $18.00 Copyright © 2009 the American Physiological Society
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established that VEGF also plays a crucial role in the nervous system. We describe the molecular properties of VEGF
and its receptors and review the current knowledge of its different functions and therapeutic potential in the nervous
system during development, health, disease and in medicine.
I. INTRODUCTION: THE NEUROVASCULAR
LINK–COMMON PRINCIPLES
AND MECHANISMS
FIG. 1. Anatomical parallelisms between vessels and nerves patterning. A and B: drawing by A. Vesalius, illustrating the similarities in the
arborization of the vascular and nervous system. C: vessels (red) and nerves (green) running in parallel alongside nerves. [Adapted from Carmeliet
and Tessier-Lavigne (43).]
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Five centuries ago, the Belgian anatomist Andreas
Vesalius already documented the anatomical parallelisms
between vessel and nerve patterning (Fig. 1, A and B).
Despite their distinct function, the vascular and nervous
systems exhibit architectural similarities and are both
patterned in ramified and hierarchically ordered networks. Yet, it is only recently that the common molecular
basis of neuron and vessel specification, growth, navigation, and survival has started to become elucidated. Both
systems are composed of efferent and afferent networks,
such as motor and sensory nerves in the nervous system,
and arteries and veins in the vascular system (43) (Fig. 1,
A and B). Intriguingly, nerves and vessels regularly exhibit
comparable patterning, with vessels running in parallel
alongside nerves (Fig. 1C). The anatomical association
between both networks has raised intriguing questions as
to whether there are developmental links between both
networks and to what extent their development is controlled by similar (classes of) molecules. In the next paragraphs, we illustrate this neurovascular link with a few
examples.
Recent studies show that common genetic pathways
regulate, at least in part, the differentiation of the cellular
players and development of both networks. For instance,
neurogenesis and angiogenesis are closely intertwined,
with endothelial cells (ECs) in vascular niches releasing
cues for neural stem cells (NSCs) (see sect. IVA). Furthermore, the organization of vascular and nervous networks
relies, in part, on the segregation of distinct cell populations with establishment of tissue boundaries so that cells
with common functions group together, while cells with
different activities are excluded. Examples include the
segmentation of the hindbrain in rhombomeres (64, 187,
385) and the formation of boundaries between arterial
and venous ECs in the vasculature. In both cases, the
molecules, which help to establish these boundaries, belong to the Ephrin family of receptor tyrosine kinases and
their membrane-bound ephrin ligands (65, 255).
More similarities between both systems have been
identified when exploring the mechanisms of axon and
blood vessel navigation during development. Specialized
endothelial “tip” cells at the forefront of navigating vessels extend numerous filopodia that sense the surrounding tissue and respond to guidance cues, comparably to
axonal growth cones that sense and navigate to their
targets (107) (Fig. 2). Similar classes of cues, which control axon guidance (such as Slits, Netrins, Semaphorins,
and Ephrins), also regulate vessel navigation (43), while
vice versa angiogenic molecules also regulate neuronal
and axon patterning. These processes will be further described in section III, A and B.
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Another example of a close interaction between neuronal and vascular cells is the innervation of resistance
arteries by autonomic nerves (29, 35). With the development of a pressurized circulation, certain arteries became innervated to allow proper control of the distribution of flow to vital organs (35). These autonomic
nerves arise from neural crest cells that segregate from
the neural tube and migrate to peripheral regions in the
embryo (78, 184). Neural crest cells also differentiate to
smooth muscle cells (SMCs) and pericytes (PCs), that
cover some of the large thoracic arteries and vessels in
the forebrain (78, 85). When deregulated, this process
causes metameric appearance of vascular anomalies in
the central nervous system (CNS) of patients suffering
the PHACES syndrome (posterior fossa malformations,
hemangiomas, arterial anomalies, cardiac defects, eye
abnormalities and sternal or ventral defects). The neural crest thus contributes directly to the formation of
both the nervous system and the vascular system.
The cross-talk between vessels and nerves is also
important for proper functioning of neurons and blood
vessels in adults. Arterioles in the nervous system consist
of a single layer of ECs that are surrounded by PCs; these
vessels lay in intimate contact with foot processes of glial
cells and nerve terminals. This neurovascular unit allows
blood vessels to match oxygen and glucose delivery with
neuronal metabolic demands (223). Active neurons not
only induce vasodilation by releasing vasoactive substances (46, 272, 299, 312, 369, 448) but also regulate
neuronal perfusion indirectly through release of glutamate, which evokes secretion of vasoactive substances
from astrocytes (448).
Since VEGF is the prototypic example of the crosstalk between nerves and vessels, we focus the rest of this
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review primarily on VEGF. Because its role in angiogenesis has already extensively been reviewed (39, 92, 171),
we here describe the molecular properties of VEGF and
its receptors during angiogenesis more briefly, before
highlighting its role in the nervous system in more detail.
II. BIOLOGICAL ACTIVITIES OF VASCULAR
ENDOTHELIAL GROWTH FACTOR
A. VEGF Family Members
Given that excellent reviews on the molecular properties of the VEGF family and its receptors have been
published previously (92, 289, 343), we restrict our discussion here to those characteristics that are relevant for
understanding their role in neurobiology. VEGF-A (termed
VEGF from hereon) is the founding member of a family of
homodimeric glycoproteins that are structurally related
to the platelet-derived growth factors (PDGF). Initially
isolated as a factor increasing vascular permeability
(334), the VEGF gene was thereafter cloned from pituitary
cells (93). Exon 1 and part of exon 2 encode a hydrophobic signal sequence, exons 3 and 4 encode domains responsible for binding to its receptors, while exons 6
and 7 encode basic domains capable of binding to heparin
(Fig. 3).
Through alternative RNA splicing, several VEGF isoforms are generated. The VEGF121 isoform (121 amino
acids in humans and 120 in mice; VEGF isoforms have 1
less amino acid in mice) is freely diffusible, since it lacks
the basic amino acid residues responsible for heparin
binding and, therefore, does not, or only minimally, binds
to the extracellular matrix (ECM) (Fig. 3). The larger
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FIG. 2. Similarities between endothelial cell (EC) tip cells and axon growth cones. A: image of EC tip cells with filopodia extension
(arrowheads) in the developing retinal vasculature. (Image kindly provided by Li-Kun Phng and Dr. H. Gerhardt.) B: image of a cohort of growth
cones (arrowheads) with actin filaments and microtubules labeled, respectively, in pink and light blue. (Image kindly provided by Dylan Burnette
and Dr. Forscher.)
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forms, consisting of 145, 165, 183, 189, or 206 amino acids,
bind to heparin and heparan sulfate proteoglycans. Because VEGF165 contains some basic residues (encoded by
exon 6), it is partly diffusible and partly bound to the
pericellular matrix (Fig. 3). VEGF189 contains even more
basic residues, explaining why it remains spatially restricted to the matrix around the VEGF-producing cell
(Fig. 3). Matrix-bound VEGF isoforms are released by
proteinases, such as heparinases and matrix metalloproteinases (MMPs), and thereby regulate vessel shape and
number (216); plasmin generates an active VEGF fragment of 110 NH2-terminal residues, named VEGF110 (139).
Because of their different affinity for the ECM, VEGF
isoforms lay down a spatial VEGF gradient for vessel
patterning (42, 363, 364), with VEGF121 diffusing over long
distances, VEGF165 reaching distant and nearby target
cells, and ECM-bound VEGF189 providing guidance cues
over short ranges (Fig. 4). This gradient not only chemoatPhysiol Rev • VOL
tracts ECs from large distances, but also guides navigating ECs closer to the VEGF-producer cell. Hence, in mice
expressing exclusively VEGF120, vessels enlarge at the
expense of vessel branching, while in mice expressing
only VEGF188, vessels branch excessively but have a tiny
lumen (41, 43, 107, 323, 364) (Fig. 4). Other isoforms have
been identified, such as VEGF165b, similar in length as
VEGF165 but with a different COOH-terminal tail, that may
act as an inhibitor of angiogenesis (21, 185, 314, 421);
VEGF111 is absent in healthy tissues but induced in tumor
cells by irradiation and genotoxic drugs (265).
The other members of the VEGF family include placental growth factor (PlGF), VEGF-B, VEGF-C, and
VEGF-D (Fig. 5). The VEGF-E gene, which is encoded by
the parapoxvirus Orf virus (257) and svVEGF, which is a
snake venom VEGF (155), will not be discussed. Each of
these family members is characterized by the presence of
eight conserved cysteine residues, which form a typical
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FIG. 3. Transcriptional and posttranscriptional regulation of vascular endothelial growth factor (VEGF). Transcription of VEGF can start from
either the P1 TATA less promoter (at nucleotide position ⫹1) or the internal promoter (at nucleotide position ⫹633). Hypoxia and nutrient
deprivation enhances VEGF transcription through hypoxia-inducible transcription factor (HIF)-1␣ (by binding to the HRE, which is indicated with
an orange box in the P1 TATA less promoter) or alternatively through PGC-1␣ [by coactivating the orphan nuclear estrogen-related receptor-␣
(ERR␣) on conserved binding sites found within the promoter and the first intron (red boxes)]. VEGF mRNA stability is also regulated through
stress-induced factors, that bind to adenylate-uridylate-rich elements (AREs) in its 3⬘-untranslated (UTR) region (green stripes). The 5⬘-UTR contains
two IRES (A and B) (blue) that ensure efficient initiation of the translation under stress condition, thus bypassing the 5⬘ cap recognition required
for the assembly of the translation initiation complex. However, cap-dependent hypoxia-regulated translation mechanisms also exist. Three in-frame
CUGs translation initiation codons can also initiate the synthesis of a long VEGF (L-VEGF). Normally, translation starts however from the canonical
ATG. Maturation in the endoplasmic reticulum generates the different VEGF isoforms. Three coding exons are alternatively spliced and give rise
to, at least, six VEGF isoforms that possess different affinity to heparin sulfate proteoglycans (HSPG) depending on the absence or presence of one
or two HSPG binding sites. All VEGF isoforms can be cleaved by plasmin to generate a 110-amino acid-long VEGF. Exons 7 and 8 (indicated in
purple) code for the regions described to bind NRP1.
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cysteine-knot structure (341). These homologs have been
implicated in angiogenesis or lymphangiogenesis, although with distinct roles (Fig. 5). For instance, in addition to its key role in angiogenesis, VEGF also regulates
lymphangiogenesis (122, 132, 165, 277). VEGF-C regulates
lymphatic vessel growth, but also promotes angiogenesis
(156); the role of endogenous VEGF-D, even though capable of stimulating lymphangiogenesis when overexpressed, in lymphatic growth remains less clear in mice
(156), while it acts as a modifier of lymphangiogenesis in
tadpoles (284) (Fig. 5). PlGF stimulates angiogenesis selectively in pathological conditions (41, 94, 95), while
VEGF-B is a relatively weak angiogenic stimulator, with
apparently restricted activity in ischemic myocardium
(39, 95, 226) (Fig. 5). Of the VEGF family members,
VEGF-B, VEGF-C, and PlGF also have a role in the nervous system. Their functions will be discussed in this
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review as well (see sect. IIIA for VEGF-C and sect. IX for
VEGF-B and PlGF). No role for VEGF-D in the nervous
system is known thus far.
B. VEGF Receptor Tyrosine Kinases
Three VEGF receptors (VEGFRs) have been identified: VEGFR-1 (also known as fms-like tyrosine kinase 1
or Flt1), VEGFR-2 (termed kinase insert-domain containing receptor, KDR in humans or fetal liver kinase 1, Flk1
in mice), and VEGFR-3 (Flt4). The members of the VEGF
family bind to these distinct receptors, each with different
affinities and selectivities: VEGF binds to VEGFR-1 and
VEGFR-2, VEGF-B and PlGF bind to VEGFR-1, whereas
VEGF-C and -D bind to VEGFR-3 and (depending on the
species) with a lower affinity to VEGFR-2 (39, 92, 171)
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FIG. 4. VEGF gradient determines blood vessel branching. Top panels: images from neonatal murine retinal vascular networks in wild-type
(left), VEGF120 (middle), and VEGF188-expressing mice (right). Proper elongation and branching of vessels is determined by a spatial gradient of
VEGF isoforms (middle panels), with matrix-associated VEGF188 (red) closer to VEGF producer cells, soluble VEGF120 (green) diffusing most
distantly from the VEGF producer cells, and VEGF164 (black) exhibiting an intermediate spatial distribution. In wild-type mice (left panels), a proper
VEGF gradient controls the ramification of large vessels into smaller branches. In VEGF120 mice (middle panels), the normal VEGF isoform gradient
is replaced by a uniform field of ectopic VEGF120-expressing cells, causing vessel enlargement at the expense of vessel branching. In contrast, in
VEGF188 mice (right panels), the VEGF isoform gradient is replaced by local spots of restricted VEGF188 expression, inducing supernumerary
branching of smaller vessels. [Adapted from Carmeliet and Tessier-Lavigne (43).]
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(Fig. 6). In contrast to VEGF, proteolytic cleavage of
VEGF-C and -D by plasmin generates mature forms,
which bind VEGFR-3 and -2 with much higher affinity than
their full-length forms (252). VEGFRs contain seven immunoglobulin (Ig)-like loops (except for VEGFR-3 which
contains only 6) in their extracellular part and a split
tyrosine-kinase domain in their intracellular region (289).
The second and third Ig domain mediate ligand binding,
while the fourth and seventh domain mediate receptor
dimerization. VEGF receptors form homodimers, but also
heterodimerize, although their function remains more
enigmatic (15, 76) (Fig. 6).
VEGFR-1 and VEGFR-2 differ from each other in
several aspects. VEGFR-2 is the best characterized signaling receptor, driving angiogenesis in health and disease; it
stimulates EC proliferation, migration, navigation of tip
cells, survival and vascular permeability (289). VEGFR-2
has a strong tyrosine kinase (TK) activity (see below)
(289). In the nervous system, VEGFR-2 also stimulates
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migration, proliferation, and survival of various neural
cell types (151, 153, 286, 358, 359, 419). Recently, a soluble
form of VEGFR-2 has been discovered in human and
mouse plasma (79), which traps VEGF; its levels inversely
correlate with tumor progression (80). However, whether
this is a splice variant or possibly a cleaved form needs
further investigation.
Although VEGFR-1 was discovered before VEGFR-2
(344, 386), its role remains more enigmatic. Besides a
soluble VEGFR-1 (also termed sFlt1), which traps
VEGF, the transmembrane form of VEGFR-1 has weak
tyrosine kinase activity but high affinity for VEGF; deletion of this domain does not affect vascular development (134). Hence, VEGFR-1 may act as a “decoy”
receptor that prevents excessive VEGFR-2 activation
by trapping VEGF (289). This seems to be particularly
the case in development, as embryos lacking VEGFR-1
die due to disorganization of the vascular network (96).
In vitro, sFlt1 is more potent in rescuing abnormal
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FIG. 5. VEGF homologs and orthologs. Phenogram (built using the Neighbor Joining method with Poisson correction) of VEGF homologs
(VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF) and orthologs in human (Homo sapiens, hs), mouse (Mus musculus, mm), frog [Xenopus tropicalis
(xt) and Xenopus laevis (xl)], zebrafish (Danio rerio, dr), fly (Drosophila melanogaster, dm), and worm (Caenorhabditis elegans, ce). In flies
and worms, all the VEGF homologs belong to the ancestral family of PDGF/VEGF-like factors (PVF). Zebrafish lack VEGF-B but have a protein
similar to PlGF (drPlGF-like). Frogs (Xenopus tropicalis) contain a primitive form of VEGF-B (only 26.2% of homology to the hsVEGF-B). The
percentages in black indicate the conservation degree (similarity score) between a specific member of the VEGF family and the respective
human ortholog. The percentages in blue indicate the similarity score of each human homolog compared with hsVEGF-A. Boxes on the right
indicate the established functions as well as still-debated roles (as indicated with the question mark) of the different members of the VEGF
family.
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613
vessel morphogenesis of VEGFR-1-deficient ECs than
membrane-anchored VEGFR-1, presumably because it
is diffusible and thereby shapes a pericellular gradient
of VEGF (162, 168, 169). Other studies show, however,
that VEGFR-1 stimulates postnatal angiogenesis
through intracellular signaling. Indeed, mice expressing
a variant VEGFR-1 without tyrosine kinase domain exhibit impaired pathological angiogenesis (133, 273,
342). Also, PlGF promotes angiogenesis directly by activating specific VEGFR-1 signaling in ECs, as well as
indirectly via VEGFR-1 signaling in CD45⫹ vascularmodulatory inflammatory cells (15, 94, 95). Evidence is
now emerging that VEGFR-1 also exerts neuroprotective effects during pathological conditions (227) (see
sect. IX). VEGFR-3 is the primary receptor inducing
(lymph)angiogenesis (156), but also modulates angiogenesis, especially by transmitting sprouting signals in
endothelial tip cells (378); it also induces proliferation
of oligodendrocyte precursors and certain other neural
progenitors, as discussed below (213).
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C. Neuropilin Coreceptors
Another class of receptors for VEGF are the neuropilins (NRPs) (105). Neuropilin-1 (NRP1) and neuropilin-2
(NRP2) were originally discovered as receptors for semaphorins (51, 128), the latter belonging to a large family of
membrane-bound and secreted proteins. Although membrane-associated semaphorins interact directly with plexins, class 3 secreted semaphorins (Sema3A to F) bind to
the NRP receptors, which form complexes with the plexins to trigger signal transduction (186, 393, 443). Genetic
studies in fruit flies and mice show that semaphorins are
usually repellent cues for axons and neuronal cells (393),
although Sema3A can also function as a chemoattractant,
depending on intracellular levels of cyclic nucleotides
(361).
NRPs are single-spanning transmembrane glycoproteins, with extracellular domains A and B mediating semaphorin binding (105, 106, 118, 278) (Figs. 6 and 7A). Mutagenesis studies show that Sema3A requires an interac-
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FIG. 6. VEGF family members and receptors. A: the mammalian VEGF family members are VEGF-A, -B, -C, -D, and PlGF. VEGF binds to both
VEGFR-1 and VEGFR-2. VEGF-B and PlGF only bind to VEGFR-1. VEGF-C and -D bind to VEGFR-3 and VEGFR-2. B: VEGF and PlGF can also bind
to heterodimeric receptor complexes formed by VEGFR-1 and -2. Likewise, VEGF-C and -D are known to bind heterodimers formed by VEGFR-2
and -3. C: NRP1 and NRP2 can act as coreceptors for certain VEGF-VEGFR complexes and modulate VEGFR activation and signaling. VEGF-A and
all its family members bind to both NRP1 and NRP2, except for VEGF-B, which only binds to NRP1.
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tion with the A and B domain for its binding to NRP1,
while only domain B of NRP1 is necessary for VEGF
binding (118). However, the additional presence of the A
domain of NRP1 enhances VEGF binding considerably
(237). Domain B also binds heparin and thereby enhances
the interaction of VEGF165 with NRP1 more than 100-fold
(237, 400). Domain C in NRP1 is important for oligomerization with VEGFR-2 (105). Initial studies showed that
NRP1 binds VEGF165 and VEGF189 (297), but not VEGF121,
which lacks exon 7 (encoding for the NRP1 binding site)
(110, 130). A recent study shows, however, that VEGF120
is also capable of binding NRP1 in vitro (297), likely due
to the fact that the COOH-terminal tail of VEGF, encoded
by exon 8, may bind directly to NRP1 (13). NRP2 interacts
with VEGF145 and VEGF165 but not VEGF121 (110, 280).
The heparin-binding form of PlGF binds NRP1 and NRP2
(110, 260), and VEGF-B binds NRP1 only (235, 260). Finally, VEGF-C and VEGF-D have also been described to
bind to NRP1 and NRP2 in a heparin-independent and
-dependent manner, respectively (163).
Neuropilins enhance VEGF signaling by acting as
coreceptors for VEGF receptors. When coexpressed with
VEGFR-2, NRP1 associates with VEGFR-2 and amplifies
VEGFR-2 phosphorylation and signaling (105). As the cytoplasmic domain of NRPs only contains 40 amino acids,
it has been postulated that this domain does not transduce biological signals, but instead, requires an interaction with other signaling molecules, such as those interacting with VEGFR-2. Other studies have shown that
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NRPs contain an intracellular PDZ-binding domain (Fig.
7A), which interacts with intracellular proteins, such as
synectin (105). Deletion of this PDZ-binding domain decreases complex formation between NRP1 and VEGFR-2,
and diminishes the EC response to VEGF165 (304), suggesting that synectin acts as a bridge between NRPs and
intracellular signaling molecules (36, 410).
During development, NRP1 expression becomes restricted to ECs in arteries, whereas NRP2 labels ECs of
venous and lymphatic vessels (81). Murine embryos deficient for NRP1 exhibit defects in the vascular system,
including impaired neural vascularization and abnormalities in vascular remodeling (166). In contrast, mice lacking NRP2 are viable, with no evidence of cardiovascular
defects, although small lymphatic vessels and capillaries
fail to form (50). Accordingly, monoclonal antibodies
against NRP1 or NRP2 inhibit tumor angiogenesis and
lymphangiogenesis, respectively (47, 296). Loss of synectin in mice and zebrafish also causes selective arterial
defects (59).
D. Regulation of VEGF Expression
The expression of VEGF is tightly regulated; for instance, loss of even a single VEGF allele results in embryonic lethality because of defective vascular development
(40, 266). Furthermore, even a reduction of VEGF levels
by only 25–30% causes paralyzing motor neuron degener-
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FIG. 7. Cross-talk between VEGF and
Sema3A. A: NRP1 is composed of three extracellular domains, a transmembrane domain
(TM) and a very short cytoplasmic tail (CT),
containing a PDZ-binding domain. Domain A
(a1a2) and domain B (b1b2) are responsible
for Sema3A binding while domain B is necessary for VEGF binding. The A domain of NRP1
enhances VEGF binding considerably, whereas domain C is important for NRP1 dimerization and oligomerization with VEGFR-2.
B: published evidences regarding the role
of Sema3A in vascular development.
C: VEGF and Sema3A have been proposed
to compete with each other for the binding
site in domain B of NRP1. This table summarizes the evidences supporting or refuting this hypothesis.
VEGF IN THE NERVOUS SYSTEM
E. Hypoxia as a Key Regulator
of VEGF Expression
Hypoxia is a strong stimulus for angiogenesis: when
cells suffer hypoxia, they release, in a feedback loop,
angiogenic factors to reestablish oxygen supply through
vessel formation. Hypoxia activates hypoxia-inducible
transcription factors (HIFs), which function as master
switches to induce expression of angiogenic factors, including VEGF. At the transcriptional level, hypoxic induction of VEGF is conferred by binding of a HIF complex to
a core “hypoxia responsive element” (HRE) sequence in
the VEGF promoter (264) (Fig. 3). HIF complexes are
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heterodimeric transcription factors, consisting of ␣- and
␤-subunits, which belong to the basic helix-loop-helix
PAS family of transcription factors (408). The ␤-subunit,
also known as the aryl hydrocarbon receptor nuclear
translocator (ARNT), is constitutively expressed, while
activation of the ␣-subunit is tightly regulated by oxygen
tension. Several HIF␣ subunits exist, i.e., HIF-1␣, HIF-2␣
(also known as endothelial PAS domain, EPAS) and HIF3␣, each encoded by different genes (298, 417). HIF-1␣
and HIF-2␣ share conserved structural domains, dimerize
with HIF-␤, and induce gene expression in a hypoxiainducible manner (229); HIF-3␣ on the other hand is
poorly characterized.
HIFs execute the cellular response to hypoxia, but do
not sense changes in oxygen tension themselves. This is
performed, in part, by HIF-prolyl hydroxylase domain
(PHD) proteins, which are dioxygenases that use oxygen
to hydroxylate proline residues in HIF␣ subunits (97,
158). Under normoxic conditions, hydroxylated HIF␣ interacts with the von Hippel-Lindau (pVHL) protein, a
component of the E3 ubiquitin ligase complex, that triggers ubiquitinization and, thereby, marks HIF␣ for proteasome-dependent degradation (161, 250). In hypoxia, PHDs
are inactive, and HIF␣ subunits become stabilized and
activate transcription of target genes (141, 142). The transcriptional activity of HIFs is also regulated by another
type of sensor, i.e., the factor-inhibiting HIF-1 (FIH-1).
HIF␣ subunits contain two transactivation domains, an
oxygen-regulated COOH-terminal domain (CAD) and a
NH2-terminal domain (NAD) (229). The activity of CAD is
regulated by hydroxylation of an asparaginyl residue by
FIH-1 (209, 210, 229). In normoxia, this residue is hydroxylated and CAD activity is repressed, while in hypoxia, FIH-1 is inactive, hydroxylation of CAD does not
occur, and HIF␣ subunits bind then, through their CAD
domain, to the transcriptional coactivators CBP/p300 to
activate transcription of target genes (229). Intriguingly,
in conditions of low oxygen and nutrients, the transcriptional coactivator and key metabolic regulator PGC-1␣
also upregulates VEGF expression and angiogenesis, independently of the canonical HIF pathway (14).
Hypoxia also increases the half-life of the VEGF
mRNA by increasing its stabilization (219, 434) (Fig. 3).
Under low oxygen conditions, ARE elements in the 3⬘UTR bind mRNA hypoxia-induced stabilizing proteins,
such as HuR (an Elav-like protein), poly(A)-binding protein 2 (PAIP2), and heterogeneous nuclear ribonucleoproteins L (hnRNPL) or K (hnRNPK) (90, 219, 222, 290, 345,
434). As discussed above, translation of VEGF mRNAs in
hypoxic conditions occurs through IRES sequences (365)
(Fig. 3), although IRES-independent mechanisms also exist (435). The relevance and relative contribution of each
of these mechanisms regulating VEGF translation in hypoxic conditions require further investigation.
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ation after birth (see sect. VA) (292). Molecular studies
reveal that VEGF expression is regulated at the transcriptional and posttranscriptional levels (Fig. 3). Concerning
the first, two transcription initiation sites have been identified in the VEGF gene, with the most frequently used site
located in the TATA-less promoter, and a second one in
the 5⬘-untranslated region (5⬘-UTR) (6, 294) (Fig. 3). A
wide range of signals also stimulate VEGF transcription,
such as (inflammatory) cytokines and oncoproteins, including interleukin (IL)-1, IL-6, insulin-like growth factor
I, transforming growth factor (TGF)-␤1, c-Src, v-Raf, and
Ras among others (reviewed in Ref. 294); the translational
regulation of VEGF expression during hypoxic conditions
will be described separately (see sect. IIE).
VEGF expression is also regulated at the posttranscriptional level. Indeed, the VEGF mRNA contains an
unusually long 5⬘-UTR of 1,038 nucleotides, which harbors three in-frame CTG translation initiation start
codons and two internal ribosome entry sites (IRES) (6,
262, 365) (Fig. 3). Translation from the three in-frame
initiation codons can produce a long VEGF (L-VEGF),
which after being cleaved generates a secreted and intracellular VEGF (N-VEGF) of 206 amino acids (20). Normally, translation starts however from the canonical ATG
(Fig. 3). IRES sequences serve to position ribosomes in
the vicinity of the translation start codon, thereby bypassing the 5⬘-cap recognition required for the assembly of the
translation initiation complex. Both elements cooperate
to ensure initiation of translation in stress conditions
(such as hypoxia or starvation), when synthesis of most
proteins is shut down. The activity of IRES-A is also
regulated through interactions with mRNA sequences, encoded by VEGF exons 6 and 7 (32), while a small upstream open reading frame within IRES-A suppresses the
expression of VEGF121 (20), although the precise mechanisms and consequences remain to be elucidated. VEGF
mRNA stability is also regulated through binding of proteins, that bind to adenylate-uridylate-rich elements
(AREs) in its 1,892 nucleotide long 3⬘-UTR region (220,
221) (Fig. 3).
615
616
RUIZ DE ALMODOVAR ET AL.
Although initial studies indicated that VEGF is an
endothelial cell-specific factor, more recent findings revealed that VEGF also has important effects in the nervous system. We now describe the evidence on the role of
VEGF in the nervous system both in development and
health.
III. ROLE OF VASCULAR ENDOTHELIAL
GROWTH FACTOR IN THE DEVELOPING
NERVOUS SYSTEM
A. VEGF Regulates Vessel and Neuronal Wiring in
the CNS
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In higher organisms, the neural tube (NT), from
which the brain and spinal cord develop, becomes vascularized by a process involving VEGF. Indeed, current evidence indicates that vascularization of the NT occurs via
the formation of a perineural vascular plexus (PNVP)
surrounding the NT and by subsequent inward vessel
sprouting from the PNVP (136, 195, 196). In cocultures of
NT explants with presomitic mesoderm, which provides
the source of ECs, VEGF inhibitors inhibit PNVP formation (136). Likewise, formation of the PNVP is blocked
when presomitic mesoderm explants from VEGFR-2-deficient embryos are cocultured with wild-type NT explants.
At the time of vessel sprouting, VEGF is specifically expressed in the NT by the floor plate, the neural progenitors in the ventricular zone, and the motor neurons, suggesting that VEGF also plays a role in directing vessel
sprouting into the NT (136, 276, 311). One of the molecules responsible for the timely and highly specific expression of VEGF in the NT is Sonic hedgehog, an angiogenic factor which also participates in NT vascularization
in part by regulating VEGF expression (211, 276, 302, 322).
On the other hand, invading endothelial precursors derived from the lateral plate mesoderm and somites have
also been found in the NT from quail embryos (9, 10, 58,
136, 283), suggesting that vascularization of the NT might
also occur through other mechanisms apart of PNVP vessel sprouting.
The intracerebral vasculature develops through sprouting
of the pial vessels located on the brain surface. The vessel
sprouts grow into the brain parenchyma and converge
radially towards the ventricles (195). From there, they
extend branches that surround the ventricles and navigate in the reverse direction, radially towards the pial
surface again (116). A recent model proposes, however,
that vascular differentiation in specific brain regions may
be regulated in a similar way as neuronal specification.
Indeed, in the developing telencephalon, brain vascularization starts from periventricular vessels, which develop
independently, according to compartment-specific expression of homeobox transcription factors that also determine neuronal specification (402).
Vascularization of other brain regions is also regulated by VEGF. For instance, VEGF is expressed by Purkinje cells and astrocytes during cerebellar development,
while VEGFRs are expressed by ECs (5). Interestingly, a
spatial VEGF gradient, established by various VEGF isoforms, is necessary for proper vessel patterning in the
brain. Hence, in mice lacking the heparin-binding VEGF
isoforms, endothelial tip cells fail to extend filopodia to
the midline of the hindbrain, and vessels are disorganized
and misshaped (323). In addition, in mouse embryos, in
which VEGF is eliminated in neural progenitors, directed
ingrowth of capillaries in the developing brain is impaired
(121, 311). NRP1 also contributes to the VEGF signaling,
as vascularization of the nervous system is impaired in
NRP1-deficient mice (108, 166).
Blood vessels also cross-talk with neural cells during
CNS vascularization. For instance, in the developing retina, astrocytes grow centrifugally from the center to the
outside layers and thereby provide a template for growing
vessels. Indeed, initially, the developing retina is avascular and hypoxic, which upregulates VEGF expression in
astrocytes. Increased VEGF then triggers blood vessel
growth (308, 366), and supply of oxygen by the nascent
neovasculature will, in turn, downregulate VEGF expression in astrocytes and trigger astrocyte differentiation
(418).
In addition to effects on vascularization, VEGF also
regulates neuronal cell migration in the CNS. For instance, migration of facial motor neurons in the developing mouse hindbrain is regulated by VEGF and NRP1
(330) (Fig. 8A). Facial motor neurons are born in a ventral
position of the hindbrain segment (rhombomere 4) and
move their soma caudally to rhombomere 6. VEGF controls this migration process by interacting with NRP1.
However, VEGF is not required for their axon guidance;
instead, Sema3A and Sema3F cooperate to pattern facial
branchiomotor neuron axons by binding to NRP1/PlexinA4 and
NRP2/Plexin A3, respectively, without controlling the pathfinding of their somata (330, 331) (Fig. 8). VEGF also
stimulates axonal outgrowth of cortical neurons and retinal ganglion neurons (30, 54, 151, 358, 359, 445). Another
ligand of the VEGF family, VEGF-C, regulates expansion
of the population of oligodendrocyte precursor cells
(OPCs) and neural progenitors in vitro (213). VEGF-C acts
as a trophic factor for these cells in vivo, since VEGF-Cdeficient mouse embryos show a selective loss of OPCs in
their optic nerve (213).
The observation that VEGF is involved in wiring the
developing nervous system is perhaps not surprising,
given that VEGF and its receptors appeared first in the
nervous system of invertebrate species, such as worms
and flies, which lack a well-developed vascular network
(Fig. 5). In C. elegans, a family of four tyrosine kinase
receptors, structurally similar to VEGF receptors, has
been identified. These receptors (vascular endothelial
VEGF IN THE NERVOUS SYSTEM
617
FIG. 8. VEGF and neuronal migration. Proposed model for the
control of axon and soma guidance in facial branchiomotor neurons by NRP1 ligands. VEGF164 binds to NRP1-containing receptors on the cell body of neurons in the hindbrain and controls their
soma migration from rhombomere 4 (r4) to rhombomere (r6).
Sema3A and Sema3F bind to NRP1 and Plexin receptors in the
growth cone to control axon guidance in the periphery. NRP1
associates with Plexins to transmit semaphorin signals in the
growth cone, but the signaling coreceptor in the soma is not
known. [Adapted from Schwarz et al. (330), with permission from
Genes & Development.]
B. VEGF at the Crossroad of Peripheral
Innervation and Vascularization
In the peripheral nervous system, some vessels and
nerves migrate along the same path and track alongside
each other. For instance, in the skin of the embryonic
limb, arteries are intimately associated with sensory
nerves (Fig. 1C). In mouse mutants with disrupted axon
patterning, arterial patterning follows that of disorganized
nerves (271). Interestingly, these vessels express arterial
Physiol Rev • VOL
markers once they establish close contact with nerves,
indicating that nerve-vessel association regulates arterial
specification, at least, in this condition (271). Coculture
experiments of ECs with dorsal root ganglia (DRG) neurons and Schwann cells further demonstrate that both cell
types promote arterial differentiation of ECs through release of VEGF (271). A conditional gene inactivation strategy further reveals that both nerve- and Schwann cellderived VEGF is required to induce arterial differentiation
of these vessels (270). The coalignment of vessels and
nerves is, however, not dependent on the release of VEGF
(270). In other cases, the opposite also occurs: vessels
release guidance cues, such as VEGF, artemin, neurotrophin-3, or endothelin-3 to attract axons to track alongside
the vessels (22, 138, 194, 236, 242).
VEGF also affects neural cells in the PNS. It prolongs
the survival and stimulates proliferation of Schwann cells
in explants of superior cervical ganglia (SCG) and DRG
(328). Even though Schwann cells express VEGFR-1,
VEGFR-2, and NRP1, the effect of VEGF on their migration appears to be mediated predominantly by VEGFR-2
(328). Nonetheless, the precise role of VEGF in peripheral
innervation remains incompletely understood.
In mice, expressing a variant NRP1, that only binds
VEGF but not Sema3A, neural but not vascular morphogenesis is perturbed (119) (Fig. 7, B and C). Moreover,
genetic studies in zebrafish embryos show that VEGF
regulates axon outgrowth via NRP1; indeed, silencing of
NRP1 induces aberrant branching of motor axons and
migration defects of motor neurons (89). Coinjection of
Sema3A or VEGF morpholinos in combination with NRP1
morpholinos, each at a suboptimal concentration, induces
similar defects, suggesting that NRP1 integrates signals
from both Sema3A and VEGF during axonal outgrowth
(89) (Fig. 7C). Likewise, the role of Sema3A in peripheral
vascularization is not fully characterized: Sema3A deficiency did not impair the formation of the major axial
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growth factor receptors or ver genes) are expressed by
specialized cells of neural origin, such as glial cells, chemosensorial neurons, and neurons of the dorsal ganglia
(303). A PDGF/VEGF-like growth factor (pvf1) has been
characterized in the worm with biochemical properties
similar to vertebrate PDGF and VEGF (Fig. 5), which bind
to VEGFR-1 and VEGFR-2 and induce angiogenesis in
vertebrate models (384).
The fruit fly D. melanogaster expresses a receptor
tyrosine kinase related to mammalian PDGF and VEGF
receptors (pvr), as well as three VEGF orthologs (pvf1 to
pvf3); this receptor is involved in the tubular formation of
salivary glands (126) and in migration of various cell
types, including hemocytes, which are the primitive blood
cells in the fruit fly (60, 287). Loss of pvr also induces
defects in axon patterning and positioning of glial cells
(287, 333): midline neurons secrete pvf ligands, which
bind to pvr on glial cells and regulate their survival and
chemoattract them to the midline (214, 287), whereas
gain-of-function experiments show that pvf induces supernumerary glia at the midline (214). Since glial cells are
required for axon guidance, fasciculation, and axon ensheathment during development and after axon injury,
VEGF may affect wiring of the developing nervous system
indirectly by acting through glial cells in this invertebrate
species.
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RUIZ DE ALMODOVAR ET AL.
IV. ROLE OF VASCULAR ENDOTHELIAL
GROWTH FACTOR IN THE NERVOUS
SYSTEM IN THE HEALTHY ADULT
A. Role of VEGF in Neurogenesis
Neurons and glial cells arise from NSCs; ECs influence this process. In specific areas of the nervous system
(“vascular niches”), such as in the neurogenic subgranular
zone of the hippocampus and the subventricular zone,
NSCs proliferate in small clusters around dividing capillaries (295); ECs closely interact with NSCs and astroglial
cells in these niches (143, 438) (Fig. 9). Furthermore, ECs
release factors that induce differentiation of neuronal
precursors (258) (Fig. 9). For example, when subventricular zone explants are cocultured with ECs, the maturation, neurite outgrowth, and migration of neurons are
enhanced (218).
Emerging evidence indicates that VEGF participates
in this cross-talk between ECs and neural progenitors.
VEGF promotes neuronal cell proliferation indirectly by
stimulating ECs (Fig. 9). In adult birds, testosterone-induced neurogenesis is preceded by an upregulation of
VEGF, which stimulates expansion of vocal cord capillaries (231). ECs in turn secrete brain-derived neurotrophic
factor (BDNF), which supports the survival and integration of neurons that are newly recruited from the overlying ventricular zone (231). However, VEGF also exerts a
direct mitogenic effect on VEGFR-2 expressing neural
progenitors (249). Genetic studies with neurospheres
from mice, lacking VEGFR-2 in NSCs, show that VEGFR-2
signaling is essential for the survival of cultured NSCs
(407). Furthermore, intracerebroventricular delivery of
VEGF stimulates adult neurogenesis in the subventricular
FIG. 9. VEGF at the neurovascular
niche. Neural stem cells (NSCs) reside in
vascular niches in defined CNS regions,
such as in the subventricular zone (or SVZ).
Neurogenesis occurs in close spatiotemporal association with vessel growth in these
niches. VEGF, secreted by ECs, NSCs, and
astrocytes (together with other factors,
which have also been depicted), promotes
astrocyte differentiation and neurogenesis,
the latter by stimulating NSC proliferation,
migration, and differentiation. In addition,
VEGF secreted from NSCs also promotes EC
proliferation and angiogenesis. [Adapted from
Zacchigna et al. (436).]
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vessels, vessel branching, or vessel remodeling, neither in
an inbred or outbred genetic background (404) (Fig. 7, B
and C). In contrast, another study analyzing Sema3A-null
mouse embryos described vascular defects (335). The
reason for these contradicting findings is unclear. Silencing of PlexinD1 or Sema3A expression also induced vascular defects in zebrafish embryos (391). Indeed, Sema3A
morphants displayed intersomitic vessel-patterning defects, with vessels emerging from the aorta at irregular
positions, and elongating sprouts failing to track along the
intersegmental boundaries and connecting with each
other in an aberrant pattern (391) (Fig. 7, B and C).
Further studies are thus required to resolve the controversy about the role of Sema3A in vascular development
(Fig. 7B).
Since VEGF165 and Sema3A interact with the B domain
of NRP1, both molecules have been proposed to bind to
overlapping sites and functionally compete with each other
(Fig. 7C). Indeed, it has been reported that VEGF antagonizes the axon-collapsing effect of Sema3A in DRG neurons
(118) and inhibits its repellent effect on migrating neuroectodermal progenitors (17). In the vascular system, high concentrations of Sema3A inhibit EC migration, lamellipodia
formation, and microvessel assembly in vitro, and these
effects are reversed by VEGF (259, 279) (Fig. 7C). On the
other hand, recent crystallographic studies, providing the
first detailed picture of VEGF and Sema3A binding to NRP1,
indicate that VEGF and Sema3A do not directly compete for
binding to the same site in NRPs. In vitro data also reveal
that VEGF fails to inhibit Sema3A-induced growth cone
collapse of DRG neurons (13) (Fig. 7C), thereby favoring the
latter hypothesis that VEGF and Sema3A are not competing
for binding to NRP1.
VEGF IN THE NERVOUS SYSTEM
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FIG. 10. Different environmental stimuli regulate neurogenesis
through VEGF. A: in the hippocampus, VEGF becomes upregulated after
different environmental stimuli such as enriched environment, exercise,
or antidepressants. Upregulated VEGF in turn induces EC and NSC
proliferation, resulting in neurogenesis. B: however, certain conditions,
such as chronic stress or aging, downregulate VEGF and reduce proliferation of putative NSCs near blood vessels in the hippocampus. As a
mediator of the cross-talk between endothelial and neural stem cells,
VEGF is thus a crucial mediator of neurogenesis. [Adapted from WarnerSchmidt and Duman (414), with permission from Elsevier.]
learning (38), while silencing hippocampal VEGF expression blocks neurogenesis in response to environmental
enrichment (38). In behavioral models, infusion of VEGF
in the brain mimics the neurogenic effect of antidepressants (414). Electroconvulsive seizures, which increase
neurogenesis in adult animals, rescue defective hippocampal neurogenesis induced by low doses of irradiation. VEGF is a mediator of this effect, since inhibition of
VEGF signaling attenuates NSC proliferation, while VEGF
infusion partially restores proliferation in irradiated animals (415).
B. Role of VEGF in Synaptic Plasticity
Emerging evidence suggests that VEGF affects neuronal plasticity in the CNS in the healthy adult animal. In
cultured hippocampal neurons, it induces long-lasting increases in protein synthesis, in part by modulating the
expression of calcium/calmodulin protein kinase II (CaMKII),
cAMP-responsive element binding protein (CREB), and
mammalian target of rapamycin (mTOR), suggesting that
VEGF may participate in protracted changes of synaptic
efficacy (176). Indeed, field-recording studies in hippocampal slices revealed that VEGF application prior to
high-frequency stimulation of hippocampal neurons increases long-term potentiation (LTP), while a VEGFR-2
inhibitor reduces this effect (176). VEGF is also secreted
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and subgranular zone of the hippocampal dentate gyrus
(151), and promotes subsequent neurite outgrowth (172,
317).
Several factors mediate or codetermine the angiogenic and neurogenic activity of VEGF. For instance,
erythropoietin promotes angiogenesis by enhancing the
secretion of VEGF from neural progenitors and upregulating VEGFR-2 expression in ECs (409). Cocultures of
NSCs with brain-derived ECs elicit vascular tube formation and maintenance through the release of nitric oxide
(NO) by NSCs and the subsequent NO-mediated upregulation of VEGF and BDNF expression in ECs. VEGF and
BDNF then act in an autocrine manner to activate
VEGFR-2 and the BDNF receptor TrkB on ECs, and in a
paracrine manner to induce NSCs proliferation (225). In
vitro, VEGF-mediated proliferation of NSCs relies on the
presence of basic fibroblast growth factor (bFGF), which
upregulates VEGFR-2 expression in NSCs (423). Granulocyte colony-stimulating factor (G-CSF) also stimulates
neurogenesis in vitro by increasing the release of VEGF
from NSCs and the expression of VEGFR-2 in the same
NSCs. In agreement, VEGFR-2 tyrosine kinase inhibitors
block neurogenesis induced by G-CSF (154), thus indicating that G-CSF exerts its proliferative effect through
VEGF/VEGFR-2.
VEGF has been implicated in adult neurogenesis in
various conditions. For instance, VEGFR-2 signaling is
involved in the induction of neurogenesis by antidepressant treatments (414); the latter increase VEGF expression in the hippocampus (7), particularly in granule cells
(414) (Fig. 10A). Conversely, chronic stress conditions,
such as cold immobilization, forced cold swimming, or
isolation, downregulate the expression of VEGF in hippocampal astrocytes and VEGFR-2 in hippocampal granule cells and reduce proliferation of putative NSCs near
blood vessels in the hippocampus (129) (Fig. 10B). Hippocampal VEGF levels in rats decline from young to
middle-age, coincident with the age-associated decrease
in dentate neurogenesis (339) (Fig. 10B). Intriguingly, exercise-induced neurogenesis in part relies on circulating
VEGF levels, as peripheral blockade of VEGF abolishes
running-induced neurogenesis (Fig. 10A). Since VEGF
does not cross the blood-brain barrier, these observations
may suggest that VEGF controls neurogenesis through
effects on the brain vasculature (87).
Although the exact contribution of VEGF-induced
adult neurogenesis remains unclear, VEGF exerts beneficial effects in a number of assays, in which functional
outcome has been associated with neurogenesis. For instance, rats raised in a physically enriched environment
or trained in a Morris water maze task show increased
hippocampal expression of VEGF, coincident with neurogenesis and improved learning (38) (Fig. 10A). Intracerebral administration of a viral vector encoding VEGF also
improves hippocampus-dependent associative and spatial
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RUIZ DE ALMODOVAR ET AL.
by hippocampal neurons upon activation of NMDA receptors or L-type voltage-activated channels (176). In addition, VEGF can be released from cortical neurons and
astrocytes by shedding of extracellular vesicles (307,
327). Although the biological significance of VEGF secretion is not yet understood, these observations suggest that
secretion of VEGF could influence the effect of neurotransmitters at the postsynaptic level.
C. Role of VEGF in Neuroprotection
and Neuroregeneration
TABLE
1.
VEGF exerts direct effects on neural cells in the central nervous system
Neural Cells
Effect
Receptor/Pathway
Reference Nos.
Neurons
Embryonic cortical neurons
Retinal ganglion cells
Cerebellar granule cells
Hippocampal neurons
Cortical neuron precursors
Embryonic ventral mesencephalic neurons
Cortical neurons
Cortical neurons
Primary CNS neuronal cultures
Cholinergic neurons (in vivo)
SVZ neural progenitors
Cortical neurons
Retinal explant cultures
SVZ neural progenitor cells
Primary motor neurons
Primary hippocampal neurons
Survival
Neurite outgrowth
Survival after K⫹ deprivation, glutamate, or 3-NP
treatment
Survival after hypoxia or NMDA-induced
excitotoxicity
Proliferation
Survival and neurite outgrowth
Survival after hypoxia
Neurite outgrowth
Neuronal growth and maturation
Survival after NMDA stimuli
Proliferation and differentiation
Neurite outgrowth
Reduction of apoptosis of retinal neurons in an
ischemia/reperfusion model
Migration
Survival after hypoxia or hypoglycemia
Survival after glutamate-induced toxicity
VEGFR-2: MEK
VEGFR-2
VEGFR-2: Akt/PKB
286
30
419
Inhibition of excitotoxicity
374
MEK, PLC, PKC, and PI3K
ND
Inhibition of casp-3 activation
VEGFR-2: MEK, PI3K-Akt
VEGFR-2: MAPK
ND
ND
VEGFR-2: Rho/ROK
VEGFR-2
445
300
148
317
172
268
256
149
282
439
397
247
Motor neurons
Cerebral cortical cultures, SVZ and SCG
neuronal progenitors
Definite neural stem cells
Dopaminergic neurons
Dopaminergic neurons
Cortical neurons
Survival after AR polyglutamine-induced toxicity
Proliferation
ND
ND
VEGFR-2: PI3K/Akt and MEK/
ERK
ND
VEGFR-2
Survival
Survival
Survival after 6-hydroxy-dopamine treatment
Survival
VEGFR-2: NF␬B
ND
ND
TNFR-1 dependent
407
348
431
382
Astroglia
Proliferation
238
Astrocytes
Astrocytes
Primary microglial cultures
Proliferation
Astrocyte activation
Proliferation, chemotaxis
VEGFR-1: MAPK/ERK and
PI3K
ND
ND
VEGFR-1: Akt
362
151
Astrocytes and microglia
348
318
99
VEGF-mediated effects are summarized per cell type, receptor through which VEGF exerts its effect, as well as the described downstream
pathways that are stimulated. ND, not determined.
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VEGF also has neuroprotective effects on many postmitotic neuronal cell types in the CNS (cortical, hippocampal, dopaminergic, cerebellar, and retinal neurons)
and peripheral nervous system (sympathetic neurons)
(Tables 1 and 2). It protects these cells against death
induced by a wide variety of different noxious stimuli,
including hypoxia, serum withdrawal, or excitotoxic stimuli; VEGFR-2 predominantly mediates this neuroprotec-
tive effect, although VEGFR-1 may also transmit some of
these effects. The molecular cascades, underlying this
neuroprotection, are described in the next section.
Besides effects on neuroprotection, VEGF also stimulates neuroregeneration. In a model of axotomy, regenerating motor nerves (expressing VEGFR-2) align with
vasa nervorum, which release VEGF (22). A VEGF trap
impairs regeneration of these axotomized nerves; however, it still needs to be determined whether VEGF acts
directly on VEGFR-2 expressing nerves or indirectly
through effects on the vasculature (22). In another model,
VEGF enlarges the growth cone of sympathetic neurons
in vitro and promotes reinnervation of denervated arteries in vivo (242). Since SMCs express VEGF and sympathetic nerve fibers express VEGFRs, SMC-derived VEGF
might promote growth of sympathetic axons. VEGF also
affects glial cells in the adult nervous system. For instance, VEGF has mitogenic effects on astrocytes in mesencephalic explant cultures in vitro and following intracerebral VEGF delivery in vivo (348). Most astrocytes
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TABLE
2.
VEGF exerts direct effects on neural cells in the peripheral nervous system or on neural cell lines
Neural Cells
Effect
Receptor/Pathway
Reference Nos.
Neurons
SCG and DRG neurons
DRG neurons
Axonal outgrowth, survival
DRG growth cone formation
Major pelvic ganglia
Fiber outgrowth, induction of NOS, and TH
expression
VEGFR-2: MAPK
NRP1: prostacyclin and
prostaglandin formation
ND
358, 359
54
228
Schwann cells
Survival, migration in hypoxia and normoxia
conditions
ND
328
Schwann cells in SCGs
Survival, proliferation
ND
358
NSC34 motor neurons
Survival in normal conditions and after
hypoxia, H2O2 stimuli, serum deprivation,
TNF-␣ or in SOD1G93A
Survival after serum deprivation
Survival, proliferation, and migration after
Sema3A repellent action and Sema3Ainduced apoptosis
Survival
Survival after an ischemic insult (hypoxia
with glucose deprivation)
Proliferation, chemotaxis
Survival after serum deprivation
VEGFR-2 and NRP1: PI3K/Akt
224
VEGFR-2: PI3-K/Akt and NF␬B
NRP1/VEGFR-1
152
17
VEGFR-2
326
123
Cell lines
HN33 immortalized hippocampal neurons
Neuroectodermal progenitor cell line (Dev)
Clonally derived adult rat neural stem cells
A1 human hybrid clonal neurons
Microglial cell line BV-2
SK-N-SH neuroblastoma cells
ND
VEGFR-1
VEGFR-2: PKA, MEK/ERK1/2,
Akt, p38 MAPK
99
111, 112
VEGF-mediated effects are summarized per cell type, receptor through which VEGF exerts its effect, as well as the described downstream
pathways stimulated by VEGF. ND, not determined.
express VEGFR-1, but little or no VEGFR-2. VEGF increases astroglial proliferation and expression of glial fibrillary
acidic protein (GFAP) and nestin through VEGFR-1 signaling (238). VEGF also induces proliferation of microglia
through VEGFR-1 (99).
D. Intracellular Signaling of VEGFRs in Neural
and Vascular Cells
Although the intracellular signaling cascades of the
VEGF receptors have been studied in more detail in ECs
than in neurons, many similarities and parallelisms between both cell types exist. In general, binding of VEGF to
its receptor induces receptor dimerization, upon which
specific tyrosine residues become phosphorylated. Activation of VEGFR-2 results, for instance, in the phosphorylation of tyrosine residues at position 951 in the kinase
insert domain, 1054 and 1059 in the kinase domain (positive regulatory sites), and 1175 and 1214 in the COOHterminal tail (289). These phosphorylated tyrosines then
act as docking sites for intracellular signaling pathways.
We will briefly discuss some of the VEGFR-2 pathways
according to their biological effect. It needs to be taken
into account, however, that the cascades, mediating proliferation, survival, and migration are complex, interact
with each other, and partially overlap. For further details,
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the interested reader is referred to previous reviews (289,
340).
1. Regulation of proliferation
By signaling through VEGFR-2, VEGF acts as a potent inducer of proliferation in ECs. In particular, through
phosphorylation of tyrosine residue 1175 (i.e., Tyr1175),
VEGFR-2 serves as a docking site for phospholipase C-␥
(PLC␥), which activates protein kinase C (PKC) through
generation
of diacylglycerol and increased intracellular
2⫹
Ca concentrations. PKC in turn activates mitogen-activated protein kinase (MAPK) and extracellular signalregulated kinase 1/2 (ERK1/2) via Raf-1 (375, 376) or Ras
(253, 254, 347). Via a clathrin-dependent pathway,
VEGFR-2 is then internalized in endosomes (28, 86, 207).
The extent to which VEGFR-2 is endocytosed determines
the magnitude, duration, and qualitative nature of its signaling. Indeed, in confluent quiescent ECs, which form
tight contacts through adhesion with junctional VE-cadherin molecules, EC growth is inhibited. In these ECs,
VEGFR-2 forms a complex with VE-cadherin at the cell
surface, resulting in dephosphorylation of specific tyrosines in VEGFR-2 and the attenuation of MAPK signaling (115). However, when VE-cadherin is absent or not
clustered at intercellular contacts, as is for instance the
case in active ECs, that do not form intercellular contacts,
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Primary Schwann cells
622
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2. Regulation of survival
VEGF also promotes the survival of ECs through
VEGFR-2. One of the molecules involved in triggering this
effect is the adaptor molecule Shb, which binds to Tyr1175
and activates PI3K/Akt signaling pathways (102, 137); the
latter also inhibits the proapoptotic proteins Bad and
caspase-9 (234) and activates NF␬B, which in turn will
upregulate the expression of various survival factors, including Bcl-2, Blc-xL, Mcl1, c-IAPs, and c-FLIP (234).
VEGF also protects neuronal cells against cell death.
Indeed, VEGF promotes the survival of neural progenitors
and numerous postmitotic neurons of the CNS and peripheral nervous system. The molecular mechanisms
whereby VEGF regulates neuronal survival are incompletely understood, yet they seem to rely, at least partly,
Physiol Rev • VOL
on the activation of the PI3K/Akt and MAPK pathways
(63, 152, 224, 247, 286, 374, 419) (Tables 1 and 2). Interestingly, VEGF also promotes neuronal survival by inducing phosphorylation of the voltage-gated potassium channel Kv1.2, via a mechanism linked to the PI3K pathway
(309). In addition, VEGF protects hippocampal neurons
against ischemic cell death by inhibiting voltage-activated
channels and reducing Ca2⫹ influx and overload caused
by the ischemic insult (233).
3. Regulation of migration
Endothelial tip cells and axon growth cones use
filopodia at the leading edge of protruding lamellipodia to
sense guidance cues in their surroundings and to navigate
in response to these signals (103, 107). The initiation and
elongation of these filopodia require the precise regulation of polymerizing, converging, and cross-linking actin
filaments (248). Once formed, lamellipodia and filopodia
are then stabilized by adhering to the ECM or adjacent
cells with their transmembrane receptors, which connect
to the actin cytoskeleton. These newly formed adhesions
then act as traction sites when the cell moves forward
over them.
VEGF induces EC migration through VEGFR-2 by
activating Src kinases. Activated VEGFR-2 is also capable
of forming a multiprotein complex with VEGFR-associated protein (VARP), also known as T-cell specific adaptor (or TsAd), and Src kinases to trigger migration (246).
Indeed, phosphorylated Tyr951 in VEGFR-2 acts as a docking site for TsAd, which forms a complex with the Src
family kinases, whereas phosphorylated Tyr1214 recruits
the Src kinase Fyn (82, 201, 246, 319, 320, 437). Activation
of Src kinases by VEGFR-2 subsequently leads to phosphorylation of guanine nucleotide exchange factors, such
as C3G and Vav2, which then activates small GTPase
proteins from the Rho family, including Rap1, Rac1, and
Cdc42 localized in the migratory edge of endothelial cells
(44, 104, 202). While Rap1 and Rac1 promote formation of
lamellipodia and membrane ruffling (354, 355), Cdc42
induces filopodia formation (203). Conversely, the PI3K
catalytic subunit p110␣ activates the small GTPase RhoA,
which is required for cell detachment during endothelial
cell motility (114).
Numerous proteins bind Rho, Rap1, Rac, and Cdc42
in a GTP-dependent manner and mediate their effects on
the actin cytoskeleton. For example, serine/threonine kinases, such as Rho-kinase, ROK␣ (RhoA-binding kinase
␣), and its close relative p160 ROCK (ROK␤), phosphorylate the myosin binding subunit of myosin light chain
(MLC) through inactivation of MLC phosphatase and direct phosphorylation of MLC (reviewed in Ref. 315). This,
in turn, enhances binding of myosin to actin filaments and
promotes the formation of stress fibers, which are involved in cell contractility (8). VEGF also triggers the
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VEGFR-2 is more efficiently internalized and remains active as a signaling molecule for prolonged periods (207).
Dynamin-2, a signal-transducing GTPase involved in receptor endocytosis, has been suggested to bind VEGFR-2
and mediate VEGFR-2 endocytosis (28). However, further
studies are needed to shed more light on the molecular
mechanisms regulating VEGFR-2 internalization.
Because VEGFR-1 exhibits only weak tyrosine kinase
activities, its signaling pathway is less well understood.
Molecular studies identified, however, several signaling
molecules as possible interacting partners, including
PLC␥, the p85 regulatory subunit of PI3K, growth factor
receptor-bound-2 (Grb2) protein, the Src homology 2 domain-containing protein tyrosine phosphatase SHP2, and
Src homology 2-containing adapter molecule Nck (289).
This suggests that VEGFR-1 can trigger relevant biological effects. Deletion of VEGFR-1 also reduces EC proliferation and induces premature EC senescence due to
constitutive Akt activation (281). Consistent herewith,
PlGF amplifies VEGF-driven angiogenesis (41), in part
because activation of VEGFR-1 by PlGF transphosphorylates VEGFR-2 and thereby amplifies its signaling (15).
Intriguingly, VEGF and PlGF each induces a specific tyrosine phosphorylation pattern of VEGFR-1, i.e., Tyr1213 is
phosphorylated by VEGF and Tyr1309 by PlGF. In addition,
a distinct gene expression profile is triggered by both
molecules in ECs (15). VEGF and PlGF also activate
MAPKs differently in ECs: PlGF by acting through its
cognate receptor VEGFR-1 and VEGF by acting through
VEGFR-2, but not VEGFR-1 (208).
In neurons, VEGF also binds to VEGFR-2 and stimulates proliferation via similar pathways as in ECs, including the PLC␥ and the MAPK pathways (99, 445) (Tables 1
and 2). Although VEGF affects neurons and Schwann
cells predominantly via VEGFR-2, it also affects astrocytes and microglia by activating the MAPK/ERK and
phosphatidylinositol 3-kinase (PI3K) signaling pathways
through VEGFR-1 (238).
VEGF IN THE NERVOUS SYSTEM
V. ROLE AND THERAPEUTIC POTENTIAL OF
VASCULAR ENDOTHELIAL GROWTH FACTOR
IN NEUROLOGICAL DISEASE
A. Reduced Expression of VEGF Triggers Motor
Neuron Degeneration
Strong evidence for a role of VEGF in the nervous
system has come from studies in amyotrophic lateral
sclerosis (ALS) or Lou Gehrig’s disease, which is a progressive, adult-onset neurodegenerative disorder characterized by loss of motor neurons in the spinal cord, brain
stem, and cerebral cortex, leading to muscle atrophy,
paralysis, and death, usually within 5 yr after onset of
clinical symptoms (205). A role for VEGF in motor neuron
degeneration was discovered by generating transgenic
VEGF⭸/⭸ mice, in which the HRE in the VEGF promoter is
deleted (Fig. 3). Under baseline conditions, this results in
a 25– 40% reduction in VEGF expression levels in neural
tissue, but not in muscle, heart, and fibroblasts, and a
reduction of 60–75% under hypoxic conditions (291) (Fig. 11, A
and B). Unexpectedly, VEGF⭸/⭸ mice suffer from adultPhysiol Rev • VOL
onset and progressive motoneuron degeneration with
neuropathological and clinical features similar to those of
ALS. Beyond 6 mo of age, VEGF⭸/⭸ mice develop muscle
weakness resulting from the progressive degeneration of
lower motoneurons in the spinal cord and brain stem.
Several of the neuropathological hallmarks detected in
VEGF⭸/⭸ mice are strikingly similar to those found in
patients with ALS. They include muscle atrophy in the
absence of myopathic signs, collateral nerve-terminal
sprouting, specific loss of choline acetyltransferasepositive motor neurons, presence of axonal spheroids and
aberrant neurofilament inclusions, as well as selective
loss of large, myelinated axons. One phenotypic difference with ALS in humans is that VEGF⭸/⭸ mice have a
slower progression and normal longevity, unlike patients
with ALS.
Reduced levels of VEGF also increase the severity of
motor neuron degeneration in the standard model of ALS,
i.e., in mice overexpressing the G93A mutant SOD1 protein (i.e., SOD1G93A mice). Indeed, VEGF⭸/⭸/SOD1G93A
double-transgenic mice exhibit earlier onset of muscle
weakness due to motor neuron loss and reduced life span
(206). Although reduced VEGF levels in VEGF⭸/⭸ mice do
not result in an abnormal number or size of blood vessels
in the spinal cord, laser-Doppler and microsphere measurements in the brain and spinal cord reveal that baseline neural blood flow is reduced by 50% in paralyzed
VEGF⭸/⭸ mice (291). It is not entirely clear, however,
whether reduced neural perfusion is present before the
onset of motor neuron degeneration or is the consequence of neuronal loss, and whether reduced neural
blood flow leads to suboptimal delivery of oxygen to
neuronal tissue, thereby triggering the selective degeneration of motor neurons in VEGF⭸/⭸ mice.
A second mechanism whereby reduced VEGF levels in VEGF⭸/⭸ mice might contribute to motoneuron
degeneration is by reducing the direct neuroprotective
effects of VEGF. Indeed, VEGF exerts direct neuroprotective effects on many different types of neurons in
vitro, including primary motor neuron cultures and cell
lines (291, 397). When transgenic Thy:VEGFR-2 mice,
overexpressing VEGFR-2 under control of the Thy1.2
promoter which drives expression in postnatal neurons, were intercrossed with SOD1G93A mice, these
double-transgenic Thy:VEGFR-2 ⫻ SOD1G93A mice exhibit improved motor performance with deterioration
at a later age compared with single transgenic SOD1G93A mice
(367). Transgenic mice overexpressing VEGFR-2 also
live longer, confirming that VEGFR-2 in neurons delays
the degeneration of spinal motor neurons in SOD1G93A
mice by transmitting survival signals of endogenous
VEGF (367). Intriguingly, intrathecal infusion of VEGFR-2
antisense oligonucleotides in rats, followed by a daily
hypoxic challenge, also results in loss of half of the motor
neurons (346). The activation of Akt and ERK under
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activation of the focal adhesion kinase FAK in a RhoAROCK-dependent manner. Phosphorylation of FAK is required to recruit paxillin and vinculin to FAK, which,
together with the activation of IQGAP1 (IQ motif containing GTPase activating protein 1), ensure formation and
turnover of focal adhesions (3, 4, 212, 425). Simultaneously, VEGF-induced activation of stress-activated protein kinase 2 (SAPK2/p38) leads to the phosphorylation of
the small heat shock protein HSP27, which induces the
release of phosphorylated HSP27 from capped actin filaments, and thereby stimulates actin reorganization to promote EC migration (202, 319, 320). By acting through the
small GTPases of the Rho family, VEGF also activates Akt
signaling, which promotes phosphorylation of the actinbinding protein Girdin, thereby inducing EC migration
and sprouting (183).
VEGF regulates neuronal migration, but its underlying pathways remain incompletely understood. Interestingly, VEGF signaling through VEGFR-2 regulates neural
progenitor migration in vitro by activation of IQGAP1,
which recruits additional partners such as Cdc42, Rac1,
and the microtubule-binding protein Lis1 (18). The PI3K/
Akt and MAPK pathways have also been implicated in
VEGF-induced neurite outgrowth (317, 358). In addition,
neurite outgrowth after VEGFR-2 activation is blocked by
inhibitors of ROCK and Rho (149). Thus, although some
studies indicate that VEGF activates similar intracellular
pathways in neurons as in ECs, it remains to be determined how these signals and players are integrated to
mediate cytoskeleton rearrangements for neural migration and neurite outgrowth.
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hypoxic conditions is markedly inhibited in motor neurons of these rats (346), indicating that interference with
the direct neuroprotective activities of VEGFR-2 renders
motor neurons more susceptible to degeneration.
Expression of mutant SOD1 also destabilizes VEGF
mRNA and downregulates VEGF protein levels in the
spinal cord of SOD1G93A mice before the onset of weakness (60 days of age). This dysregulation is mediated
through binding of a complex containing mutant SOD1 to
the AREs in the VEGF 3⬘-UTR (232), indicating that posttranscriptional processing of VEGF mRNA is blocked by
mutant SOD1 and results in lower VEGF expression levels
(Fig. 11C). Other studies confirm that VEGF expression in
SOD1G93A motor neurons is minimally upregulated by
hypoxia, unlike in wild-type mice (275). Exposing mutant
SOD1G93A mice for prolonged periods to hypoxia does not
affect their life span (397). Moreover, VEGF levels are
reduced before disease onset and become progressively
Physiol Rev • VOL
lower with disease progression in SOD1G93A rats (424). In
summary, these data suggest that mutant SOD1 dysregulates posttranscriptional processing of VEGF, thereby reducing VEGF expression and accelerating neurodegeneration in ALS.
B. VEGF Is a Modifier of Sporadic ALS in Humans
The connection between VEGF and ALS in rodents was
confirmed in humans, as VEGF plasma levels in Swedish
ALS patients are reduced by ⬃50% compared with healthy
spouses (206). High levels of VEGF and erythropoietin are
also observed in the cerebrospinal fluid (CSF) from hypoxemic neurological controls, whereas only erythropoietin, but
not VEGF, is increased in the CSF from hypoxemic ALS
patients (157). Likewise, VEGF levels in the CSF from hypoxemic ALS patients are lower than in normoxemic ALS
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FIG. 11. VEGF is causally involved in neurodegeneration through various mechanisms. A: under normal conditions, HIF-1 in complex with
CBP/p300 binds to the hypoxia-response element (HRE) in the VEGF promoter to drive VEGF expression. B: deletion of the HRE element in the
VEGF promoter leads to impaired transcription and reduced expression of VEGF in VEGF⭸/⭸ mice, causing adult-onset progressive degeneration of
motor neurons. C: expression of mutant SOD1 (SOD1G93A) also destabilizes VEGF mRNA and downregulates VEGF protein levels in the spinal cord
of SOD1G93A mice before the onset of weakness. D: A C to A single nucleotide polymorphism 2578 bp upstream of the AUG start codon (i.e., the
⫺2578C/A SNP) downregulates VEGF expression levels and is more common in (male) amyotrophic lateral sclerosis (ALS) patients than healthy
individuals. This indicates that reduced VEGF expression is also a risk factor for ALS in humans. E: overexpression of the androgen receptor (AR)
interferes with transcription of the VEGF gene by squelching the transcriptional coactivator CREB-binding protein (CBP), which together with its
coactivator p300 is necessary to allow the HIF-1 complex to induce VEGF gene transcription. As a result, a reduction in VEGF expression, similar to the
one observed in VEGF⭸/⭸ mice, is noted long before AR mice develop a neurogenic muscle atrophy. F: VEGF also coaggregates with A␤ and is only slowly
released from the coaggregated complex. Continuous deposition of VEGF in the amyloid plaques could thus result in VEGF depletion and may contribute
to neurodegeneration and vascular dysfunction in the progression of AD. (Adapted from Lambrechts and Carmeliet. Biochim Biophys Acta, 2006.)
VEGF IN THE NERVOUS SYSTEM
Physiol Rev • VOL
small populations, originally claiming a positive association of candidate genes with ALS, were subsequently
challenged by reports lacking any association or even
contradicting it (336). One approach to determine whether a
gene contributes to ALS susceptibility is to combine all
published data in meta-analyses (146, 147). The strength of
this approach lies in its ability to identify subtle genetic
effects that none of the single studies would have the
power to detect. In addition, a meta-analysis is considered
to give a more robust estimate of the overall relevance
and risk in humans. A recent meta-analysis of all the
association studies on VEGF in ALS, combining all 8
European and 3 American populations and analyzing over
7,000 patients in total, revealed, however, that homozygous carriers of the ⫺2578A risk allele exhibit a significantly increased risk for ALS (204) (Fig. 11D). This effect
is specific for male ALS patients. The increased susceptibility to ALS in male patients thus reappraises the link
between reduced VEGF levels and ALS, as originally revealed by mouse genetic studies.
C. Therapeutic Potential of VEGF in Motor
Neuron Degeneration
Insights on the significance of reduced VEGF in the
pathogenesis of ALS also raised the question whether
VEGF could have therapeutic effects in ALS rodent models. To evaluate this possibility, a rabies-G pseudotyped
equine infectious anemia virus (EIAV) lentiviral vector
encoding the human VEGF gene, EIAV-VEGF, was constructed. Intramuscular administration of virus revealed
that it is retrogradely transported to the neuronal cell
body and efficiently transduces motor neurons (16). Indeed, 4 wk after the injection of an EIAV vector, carrying
the LacZ reporter gene into muscles of SOD1G93A mice,
reporter gene expression is observed in up to 60% of the
motor neurons. When EIAV-VEGF treatment is started
before onset of symptoms in SOD1G93A mice, at the age of
3 wk, VEGF remarkably improves motor performance and
prolongs survival by, respectively, 28 and 38 days, thereby
resembling the survival effect of gene therapy with insulin-like growth factor I, as one of the most effective therapeutic strategies in the field (164). When treatment is
started at disease onset, a smaller but still significant
effect is also observed.
Although clinical trials with viral vectors are being
considered, the clinical applicability of gene therapy for
ALS still remains to be established. Delivery of recombinant neurotrophic growth factors, on the other hand, is
clinically attractive, as it offers flexible control of the dose
and duration of the administered protein. Although systemic delivery of recombinant VEGF to SOD1G93A mice
prolonged the survival of SOD1G93A mice in one study
(441), this effect was not replicated in another study
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patients, whereas hypoxemic neurological controls displayed higher levels than normoxemic controls (267). VEGF
baseline levels in the CSF of ALS patients are lower than in
controls during the first year of the disease (73). Each of
these studies thus supports a causative role for reduced
VEGF expression in ALS patients. Other studies report, however, that VEGF is not reduced in the CSF or serum from
ALS patients (68, 144), or is even increased in serum
from ALS patients (285). These contradictory results
could be due to the small groups of patients and controls studied. Moreover, as platelets release VEGF quite
abundantly, serum levels of VEGF are difficult to interpret. Finally, impaired respiratory function leading to
hypoxia in ALS patients could also affect VEGF expression levels, warranting caution to correctly interpret
these data. Stratification according to the disease stage
of ALS patients in which VEGF levels are measured
might be advisable.
In ALS patients, fewer motor neurons express VEGF or
its receptor VEGFR-2 (33). As VEGF⭸/⭸ mice lacking the HRE
in the VEGF promoter developed an ALS-like phenotype,
ALS patients were screened for the presence of mutations in
the HRE of the VEGF gene, without, however, revealing any
mutations (206). Intriguingly, however, haplotypes of three
genetic variations, consisting of the at risk ⫺2578A, ⫺1154A,
and ⫺634G alleles, in the VEGF gene increased the risk of
sporadic ALS in four distinct European populations from
Sweden, England, and Belgium (206). When assessing the
influence of these haplotypes in vitro, they reduced VEGF
expression by ⬃40%. Individuals with these genotypes also
exhibit significantly lower VEGF in plasma. In a smaller
follow-up association study, these at-risk haplotypes exhibit
a threefold increased risk for ALS (387). On the contrary,
studies performed in British (34), Dutch (399), American
(52), and Italian populations (72) reported that there was no
association between VEGF haplotypes and sporadic ALS.
Yet another study reported that the role of VEGF in ALS in
the German population might be dependent on the gender of
the patients; the results of this study, however, did also not
entirely confirm findings of the initial study, as the common
⫺1154G allele, rather than the ⫺1154A risk allele, is associated with female ALS in this study (91). Thus, although
genetic studies in mice firmly establish a link between VEGF
and ALS, the identification of a genetic link in human ALS
has remained more challenging.
ALS is, however, a heterogeneous disease, in which
multiple different pathways contribute to motor neuron
death (205). A wide variety of genetic variations might
therefore increase the risk for ALS, but the size of each
risk effect would be expected to be modest. This implies
that genetic studies in ALS would require the inclusion of
several thousands of cases and controls to reliably identify real associations. Given the modest effect of risk
factors and the typically small sample sizes employed, it is
not surprising that several genetic reports in relatively
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Physiol Rev • VOL
ered VEGF approach may be attractive for other neurons
in CNS diseases (see below).
D. Therapeutic Window of VEGF Delivery
in the Brain
The angiogenic activity of VEGF may, however, limit
its therapeutic usefulness. Indeed, when different doses
of VEGF are infused into the right lateral ventricle in rats,
the vessel density increases dose-dependently in animals
receiving daily doses of 1 and 5 ␮g VEGF/kg body wt
(125). Significant enlargement of the lateral ventricles and
capillary permeability are also observed in the highestdose group (125). VEGF also induces an angiogenic response when infused directly into the cortex of the adult
rat brain, or when applied to the cerebellum by way of
injecting an adenoviral vector (306). Likewise, when administered to the brain surface or parenchyme, VEGF
increases vascular permeability (77). In another model,
delivery of VEGF in the frontoparietal cortex induces
extravasation of serum proteins and fluorescent microspheres (67). Remarkably, even a low dose of VEGF,
which produces no appreciable changes in vascular morphology, leads to extravasation of leukocytes, as determined with the pan-leukocyte marker OX1 (67). As another response to VEGF infusion, VEGFR-1 is upregulated
in reactive astroglia, while VEGFR-2 expression is increased in vascular endothelium and neuronal somata
(188).
These studies indicate that delivery of (high doses of)
VEGF directly into the brain parenchyme induces inflammatory and angiogenic responses with potential detrimental effects. However, when delivered intracerebroventricularly, 0.2 ␮g VEGF䡠kg⫺1 䡠day⫺1 is safe, does not
induce inflammation or angiogenesis, but exerts neuroprotective effects in ALS rats (367), thus confirming a
therapeutic window for the intracerebroventricular delivery of VEGF in the brain, in which VEGF is neuroprotective but not angiogenic.
E. VEGF in Other Motor Neuron Disorders
X-linked spinal and bulbar muscular atrophy
(SBMA; Kennedy’s disease) is a rare lower motor neuron disease (170) caused by an expansion of a polymorphic CAG repeat in the first exon of the androgen
receptor gene (197). Transgenic mice expressing an
expanded human androgen receptor develop a progressive disorder, characterized by muscle weakness, atrophy, and early death, and manifested histologically by
the loss of motor neurons from the anterior horns of
the spinal cord and neurogenic muscle fiber atrophy
(362). Motor neurons from these mice show reduced
viability in cell culture, which can be rescued by VEGF
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(442). Previous clinical trials with systemic delivery of a
neurotrophic factor have also failed to show therapeutic
effects, because these recombinant factors are insufficiently active as they are not capable of crossing the
blood-brain barrier, trigger an immunogenic reaction, or
are rapidly cleared from the circulation. As an alternative
method to deliver recombinant proteins to motor neurons, recombinant VEGF was therefore delivered into the
CSF from SOD1G93A rats (367). Distribution studies with
radiolabeled 125I-VEGF revealed that intracerebroventricularly administered 125I-VEGF diffused from the CSF
into the parenchyme of the brain and spinal cord, where
it reached motor neurons and remained intact for several
hours. Subsequent studies revealed that intracerebroventricular delivery of 0.2 ␮g VEGF䡠kg⫺1 䡠day⫺1 delays onset
and prolongs life expectancy in SOD1G93A rats. VEGF
treatment also changed the disease subtype from a severe
to a much milder form. Indeed, fewer SOD1G93A rats
suffered from severe forelimb onset type of disease after
VEGF than after artificial CSF delivery. Compared with
CSF-treated rats with hindlimb or forelimb onset, VEGFtreated rats, respectively, survived 17 or 27 days longer.
The more pronounced therapeutic effect of VEGF on
forelimb than hindlimb muscles is likely attributable to
the higher VEGF levels in the bulbar/cervical than in the
lumbar spinal cord upon intracerebroventricular delivery.
This is a relevant finding, as involvement of brain stem
and cervical disease results in a worse prognosis in both
rats and humans. Intriguingly, the levels of phospho-Akt
in motor neurons were also increased after intracerebroventricular administration of VEGF to mutant SOD1 rats
(74). Overall, these findings revive interest in delivering
recombinant neurotrophic factors to ALS patients.
SOD1G93A mice were also crossed with mice overexpressing VEGF in neurons and shown to exhibit delayed
motor neuron loss, motor impairment, and prolonged survival (413). Expression of the mutant human SOD1 protein in zebrafish embryos also induced a dose-dependent
motor axonopathy. Lowering VEGF in SOD1G93A-overexpressing embryos induced a more severe phenotype, whereas
upregulating VEGF rescued the mutant SOD1G93A axonopathy (217). The effect of VEGF on motor neurons is thus
also replicated in a small animal model for ALS. Overall,
these findings have primed interest in the use of VEGF as
a novel candidate for the possible treatment of ALS, and
clinical trials to deliver recombinant VEGF intracerebroventricularly to ALS patients are underway.
A recent study also assessed the potential of delivering VEGF directly into the CNS following intranasal administration (426). The highest CNS tissue concentration
following intranasal delivery was found in the trigeminal
nerve, followed by the optic nerve, olfactory bulbs and
tubercle, striatum, medulla, frontal cortex, midbrain, etc.
Although this delivery method may not be attractive to
target VEGF to the motor neurons, the intranasally deliv-
VEGF IN THE NERVOUS SYSTEM
(362). Intriguingly, in this form of muscular atrophy, the
overexpressed levels of the androgen receptor also
interfere with transcription of the VEGF gene by
squelching the transcriptional coactivator CREB-binding protein (CBP), which together with its coactivator
p300 is necessary to allow the HIF-1␣ complex to induce VEGF gene transcription (362). As a result, a
reduction in VEGF expression, approximating that observed in the HRE-deleted VEGF⭸/⭸ mice (291), is noted
long before androgen receptor mice develop a neurogenic muscle atrophy (Fig. 11E). Thus a disturbance in
the neuroprotective effects of VEGF is also observed in
an animal model of SBMA.
VEGF also protects motor neurons in a nontransgenic paradigm of motor neuron degeneration: in rat
spinal cord organotypic cultures challenged with a
model of chronic glutamate excitotoxicity, in which
glutamate transporters are inhibited by threohydroxyaspartate leading to sustained elevation of glutamate
levels, VEGF exerts neuroprotective effects (389). This
effect is mediated through the PI3K/Akt signal transduction pathway as it is blocked by the specific PI3K
inhibitor LY294002 (389). VEGF is also effective in an in
vivo model of excitotoxic motor neuron degeneration,
triggered by infusing the glutamate receptor agonist
␣-amino-3-OH-5-methyl-4-isoxazole propionate (AMPA)
in the lumbar spinal cord. AMPA infusion produced
dose-dependent progressive hindlimb motor deficits,
reaching complete bilateral paralysis in ⬃10 days,
which was correlated with the loss of spinal motoneurons. VEGF administration together with AMPA completely prevented the motor deficits in this model and
reduced motor neuron death by 75% (392).
VI. VASCULAR ENDOTHELIAL
GROWTH FACTOR IN OTHER
NEURODEGENERATIVE DISEASES
A. VEGF in Alzheimer’s Disease
Alzheimer’s disease (AD) is a chronic neurodegenerative disease characterized by a progressive impairment
of cognitive functions and memory loss. Neurofibrillary
tangles, ␤-amyloid plaques, neuron loss, and astrogliosis
are major hallmarks of the AD brain (124, 381). In contrast
to the traditional neurocentric view of AD, recent findings
indicate that neurovascular dysfunction contributes to
cognitive decline and neurodegeneration in AD. According to this hypothesis, faulty clearance of amyloid ␤-pepPhysiol Rev • VOL
tide (A␤) across the blood-brain barrier (BBB), aberrant
angiogenesis, and senescence of the cerebrovascular system could initiate neurovascular uncoupling, vessel regression, brain hypoperfusion, and neurovascular inflammation (109, 446, 447). As a factor exerting important
effects on ECs and neurons, VEGF is a likely candidate to
be involved in AD.
Indeed, in AD, increased VEGF levels in the CSF
(383) and plasma (57) have been reported. VEGF immunoreactivity is also enhanced in clusters of reactive astrocytes in the neocortex, in the walls of many large intraparenchymal vessels, and in different perivascular deposits (160). It has been proposed that the increase in VEGF
expression levels occur as a secondary response to hypoperfusion and hypoxia in the AD brain. On the other hand,
a reduction in VEGF production has also been demonstrated in peripheral immune cells of AD subjects (356)
and in serum from AD patients (244). This observation
has been explained by the toxic effects of A␤ on VEGF
expression. In the brains of patients with AD, VEGF is
colocalized with A␤ plaques. VEGF also coaggregates
with A␤ and is only slowly released from the coaggregated complex (427). Continuous deposition of VEGF in
the amyloid plaques could thus result in VEGF depletion
and may contribute to neurodegeneration and vascular
dysfunction in the progression of AD (Fig. 11F). Moreover, VEGF inhibits A␤-induced cytotoxicity on PC12
cells, possibly by exerting direct neuroprotective effects
on these cells (428).
A functional polymorphism within the promoter region (⫺2578C/A) of the VEGF gene that lowers VEGF
expression has also been associated with increased risk
of AD in an Italian population (71). However, in subsequent studies, this polymorphism did not confer a greater
risk for AD, nor did it modulate the extent of brain vascular lesions in AD patients from French or Spanish populations (48, 245). In a fourth study, the ⫺2578AA genotypes were associated with an increased risk of developing AD and with an accelerated cognitive decline in
APOE⑀4-positive patients with AD (57). Additional studies
are therefore warranted to either confirm or refute the
association of this low-VEGF allele with AD. Intriguingly,
low VEGF ⫺2578AA genotypes are also significantly overrepresented in long-lived subjects compared with a population of young and elderly subjects, suggesting that
VEGF variability can be considered as a genetic factor
influencing life span (70).
B. VEGF in Parkinson’s Disease
VEGF has a neuroprotective effect on dopaminergic
neurons, which are the main target cells of neurodegeneration in Parkinson’s disease (PD) (Table 1). Indeed,
VEGF protects cultured mesencephalic dopaminergic
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F. Role of VEGF in Excitotoxicity
of Motor Neurons
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VII. VASCULAR ENDOTHELIAL GROWTH
FACTOR IN PERIPHERAL NEUROPATHIES
Various studies provide evidence that administration
of VEGF might also be promising for the treatment of
peripheral neuropathies, such that clinical trials testing
this concept have either been proposed or are underway.
Here, we discuss findings that VEGF is involved in neuPhysiol Rev • VOL
ropathies, associated with diabetes, chemotherapy, ischemia, and nerve injury. An intriguing question is whether
the beneficial properties of VEGF in these conditions are
linked to its ability to promote vascular remodeling in the
compromised tissue, or whether VEGF also confers direct
neuroprotection when the blood supply is intact.
A. VEGF in Diabetic and
Chemotherapy-Induced Neuropathies
Although the exact mechanisms of diabetic microvascular complications, such as diabetic neuropathy, are
largely unknown, reduced vascular perfusion has been
causally linked with diabetic neuropathies. Indeed, the
number of vessels and the extent of nerve blood flow are
markedly attenuated in rats with streptozotocin (STZ)induced diabetes (329), suggesting that diabetic neuropathies may actually improve when angiogenesis is stimulated through increased expression of VEGF (394). In two
different animal models of diabetes, i.e., in STZ and alloxan-induced diabetic models, respectively, in rats and rabbits, VEGF gene transfer reverses the deficits in nerve
conduction velocities (329). Since VEGF also improves
the nerve blood flow in these models, the vessel perfusion-promoting effects of VEGF seem to be responsible
for these effects. Similar observations were obtained in
chemotherapy-induced neuropathies: in animal models of
cisplatin-, taxol-, and thalidomide-induced neuropathies,
nerve blood flow is attenuated and the number of vessels
in the vasa nervorum is reduced, suggesting that these
neuropathies might benefit from angiogenic cytokine
therapies as well. Indeed, intramuscular gene transfer of
plasmid DNA encoding VEGF results in recovery of vascularity and improved nerve electrophysiology (179, 180).
Several other studies have confirmed the therapeutic
potential of VEGF in diabetic neuropathies: subcutaneous
inoculation of herpes simplex virus (HSV) vector-mediated gene transfer, resulting in the expression of VEGF in
nerves and DRGs, prevents sensory nerve amplitudes,
preserves autonomic function measured by pilocarpineinduced sweating, reduces nerve fiber loss in the skin, and
improves neuropeptide calcitonin gene-related peptide
and substance P expression in DRG neurons in a mouse
model of diabetic neuropathy (49). Gene transfer of an
engineered zinc finger protein transcription factor, designed to upregulate expression of VEGF, also protects
against diabetic neuropathy in rats (305). In addition, in a
rat model of diabetes, VEGF gene transfer in the corpus
cavernosum also improves erectile function, a cause of
decreased quality of life in more than 70% of diabetic men
(69). Progressive endothelial dysfunction, a reduction in
VEGF expression and loss of intraepidermal nerve fibers
have also been observed in the foot skin of diabetic
patients with increasing neuropathic severity (310). Fre-
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neurons against 6-hydroxydopamine (6-OHDA)-induced
cell death. Immunohistochemical analysis of midbrain tissues from PD patients further reveal prominent VEGF
immunoreactivity in the substantia nigra of patients, in
particular in reactive astrocytes (406). VEGFR-1, but not
VEGFR-2, expression is also upregulated in ECs, astrocytes, and neurons (406), but VEGF serum levels are not
associated with PD (145). In vivo, transplantation of
VEGF-secreting baby hamster kidney (BHK) cells in the
striatum of adult rats also protects these animals against
6-OHDA, both at the behavioral and pathological level.
Interestingly, a protective effect is still evident when
transplantation is performed after induction of the
6-OHDA lesion (430, 432, 433). Transplantation of human
neural progenitor cells into the subthalamic nucleus also
induces significant functional recovery following amphetamine-induced rotations, possibly through VEGF, as the
transplanted neural progenitor cells secrete VEGF for up
to 5 mo after implantation in the host brain (11). VEGF
may thus have a therapeutic potential in PD. VEGF may
also be involved by acting through the vasculature, since
nonexercised rats display age-dependent decreases in the
density of nigral microvessels and VEGF mRNA expression, which were reversed by physical exercise (405).
When assessing the effect of VEGF on human embryonic stem cells, VEGF upregulates the expression of the
neuroectodermal genes Sox1 and Nestin during germ
layer formation in embryoid bodies and efficiently increases the number of neural rosettes expressing both
Pax6 and Nestin (175). The neural progenitors, generated
by VEGF treatment of embryonic bodies, further differentiate into cells that show a similar pattern of gene
expression as observed during dopaminergic neuronal
development (175). Intriguingly, conditional inactivation
of HIF-1␣ in murine neural progenitor cells, which reduces the expression of VEGF, also reduces expression of
these dopaminergic markers, including tyrosine hydroxylase and aldehyde dehydrogenase (263). The number of
tyrosine hydroxylase positive neurons in these mice is
also reduced by 31% compared with wild-type mice (263),
without, however, an effect on dopamine concentrations
or locomotor behavior in these mice. Overall this indicates that HIF-1␣, at least partially by acting through
VEGF, is a key transcription factor during development
and survival of substantia nigra dopaminergic neurons.
VEGF IN THE NERVOUS SYSTEM
B. VEGF and Nerve Repair
VEGF is also protective in various nerve injury models. When 2-cm gaps in rat peroneal nerves are repaired
with VEGF-treated acellular peripheral nerve isografts,
there is a significant increase in the total number of axons
and in the percentage of neural tissue in the VEGF-treated
group (321). Also, intracavernosal VEGF injection facilitates nerve regeneration and recovery of erectile function
after cavernosal nerve injury (140). Another study demonstrated that VEGF application via Matrigel in a silicone
sciatic nerve chamber enhances myelin axon counts by
78% and significantly improves motor performance (135).
Physiol Rev • VOL
In these experiments, VEGF increases angiogenesis in the
chamber, but also enhances Schwann cell proliferation
and migration. Schwann cells also upregulate VEGF expression secondary to chronic nerve compression injury
and can thus also be involved in mediating the effects of
VEGF after a nerve injury (120).
C. VEGF in Retinal Diseases
Diabetic retinopathy has traditionally been regarded
as a disease of the retinal microvasculature. Histologically, vascular lesions in the early stages of diabetic retinopathy are characterized by capillary basement membrane thickening and microaneurysms, pericyte-deficient
capillaries, blood-retinal barrier breakdown, and other
vascular cell changes (100). Damage to nonvascular cells
of the retina has also been documented: loss of retinal
ganglion cells (RGCs) has been detected in diabetic rats
and humans, whereas glial cells change from a quiescent
to an injury-associated phenotype, with high levels of
expressed GFAP (19, 230, 324). VEGF is highly expressed
during early stages of diabetic retinopathy (37, 360). Intravitreal injection of VEGF protein and delivery of slowVEGF release implants also cause vascular abnormalities
similar to those observed in diabetic retinopathy patients,
including hemorrhage, retinal detachment, microaneurysms, venous tortuosity, and capillary nonperfusion (200,
338, 398). Mice overexpressing modest to high levels of
VEGF under control of a truncated rhodopsin promoter
are characterized by similar abnormalities (Fig. 12A). The
degree of vascular abnormalities in these models depends
on the extent to which VEGF is overexpressed, with more
severe changes documented in mice overexpressing the
highest VEGF levels (200, 338, 398). This illustrates that
increased VEGF expression contributes to diabetic retinopathy by acting on the vasculature.
VEGF also affects other cell types in the retina.
Transgenic Nse:VEGF mice, which express VEGF under
control of the neuronal Nse promotor and only modestly
overexpress VEGF, do not exhibit vascular changes in
their retina (174). When Nse:VEGF mice are challenged in
a model of RGC axotomy, which normally induces a
stereotypic pattern of apoptotic death in 80% of ganglion
cells, RGC neurons are protected (174) (Fig. 12B). Western blots also revealed increased phosphorylated ERK1/2
and Akt, reduced phosphorylated p38, and reduced activated caspase-3 levels in axotomized VEGF-transgenic
retinas (173). In a model of ischemia/reperfusion injury,
VEGF also induces a dose-dependent reduction in retinal
neuron apoptosis. While mechanistic studies show that
VEGF elevates blood flow to the retina, which is partially
responsible for the neuroprotection, ex vivo studies on
retinal cultures also demonstrate a direct neuroprotective
effect for VEGF (282). Ischemic preconditioning 1 day
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quency-modulated electrical stimulation, which improves
pain control and nerve conduction velocity in diabetic
patients, also results in an increase in circulating VEGF
levels (26). Preliminary results from clinical studies further demonstrate that the sensory neuropathy in diabetic
patients improves after intramuscular injection of a plasmid DNA encoding VEGF (351).
One study reports that intramuscular VEGF gene
transfer promotes the recovery of sensory deficits without
inducing angiogenesis in the sciatic nerve, thereby suggesting that other mechanisms than angiogenesis in the
endoneurium of the peripheral nerve may be responsible
for the observed effects (274). A possible mechanism is
that VEGF exerts direct neuroprotective effects on sensory DRG neurons. An intense signal for VEGF is detectable in DRG cell bodies and sciatic nerve fibers of normal
and STZ-induced diabetic animals (325, 357, 358). In vitro,
supplementation of VEGF has also been shown to have
neuronal growth-promoting effects on sensory neurons
(359), indicating that VEGF is present on DRG neurons
and may indeed exert neuroprotective effects in neuropathies. It is, however, difficult to assess whether neural
repair or neuroprotection is the result of a direct neurotrophic effect in vivo, or, rather, are secondary to VEGFdriven angiogenesis and improved perfusion of the ischemic territory. Another mechanism whereby VEGF may
exert its therapeutic effects is by directly affecting
Schwann cells. Although a direct in vivo effect of VEGF
on Schwann cells during diabetic neuropathy has not yet
been reported, VEGF stimulates chemotaxis and proliferation of Schwann cells in vitro (328). VEGF stimulation of
Schwann cells also leads to phosphorylation of VEGFR-2,
indicating that VEGFR-2 binding sites are functionally
active in these cells. VEGF may thus also act directly on
Schwann cells during nerve recovery (328). High levels of
VEGF have also been implicated in the pathogenesis of
the POEMS (peripheral neuropathy, organomegaly, endocrinopathy, M-protein, and skin changes) syndrome, suggesting that VEGF is also involved in the pathogenesis of
this disorder (416).
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RUIZ DE ALMODOVAR ET AL.
before ischemia reperfusion injury also increases VEGF
levels in RGC neurons, and substantially decreases the
number of apoptotic retinal cells. The protective effect of
ischemic preconditioning is reversed after inhibition of
VEGF. Overall, these data indicate that modest increases
in VEGF expression seem to exert beneficial neuroprotective effects in ischemic and injury-induced retinopathies without affecting the vasculature. Higher levels of
VEGF, however, rather contribute to diabetic retinopathylike abnormalities.
Many pathological conditions in the eye are known to
result from the formation of abnormal neovessels, including age-related macular degeneration, which is the most
common cause of blindness in the elderly. Intriguingly,
these conditions are all characterized by overexpression
of VEGF (37). Recently, antagonists directed against
VEGF have successfully been developed for the treatment
of age-related macular degeneration (12, 159). Based on
the common pathological grounds of these ocular diseases, therapeutic strategies involving intravitreal injections of anti-VEGF agents are also being applied as new
therapies for progressive diabetic retinopathy and diabetic macular degeneration (350). However, the use of
intravitreous anti-VEGF treatment in diabetes patients
has yet to be evaluated in phase III trials, and some
caution is warranted. Indeed, one study reported that
chronic inhibition of VEGF in the adult mouse eye could
lead to a significant loss of neuronal RGCs (282) (Fig.
12C). Another concern is that prolonged VEGF inhibition
may damage fenestrated vascular beds, which are more
dependent on VEGF, such as for instance the choroicapillaris in the retina. Although a recent study failed to
Physiol Rev • VOL
report regression of established choroidal neovascularization as well as damage to RGC axons in the optic nerve
after transgenic overexpression of a VEGF antagonist
(395), caution is warranted. Anti-VEGF drugs delivered
within the vitreous can also pass into the systemic circulation, where neutralization of circulating VEGF levels
could produce systemic adverse effects, ranging from
teratogenicity, thrombosis, hypertension, bleeding, disrupted wound healing, hypothyroidism, fatigue, glomerular thrombotic microangiopathy, proteinuria and edema,
posterior leukoencephalopathy, leucopenia, and skin toxicity (like rash and hand-foot-syndrome), increased incidence of stroke, and myocardial infarction (84, 98, 403).
VIII. VASCULAR ENDOTHELIAL
GROWTH FACTOR IN ACUTE
NEUROLOGICAL DISORDERS
VEGF has also been implicated in several acute neurological disorders, in which it can have both positive and
negative effects, depending on the context. Indeed, in
addition to its protective effects on neurons and ECs,
VEGF has also negatively been implicated in BBB breakdown after ischemia and in mediating inflammatory responses (238, 348). These pleiotropic activities of VEGF
make it challenging to foresee whether VEGF will aggravate or improve clinical outcome of experimental paradigms, and whether these effects are due to neuro-, glio-,
or endotheliotrophic activities. Here, we first discuss the
role of VEGF in ischemic brain diseases, followed by a
description of the functions of VEGF in spinal cord injury,
status epilepticus, and multiple sclerosis.
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FIG. 12. Effects of VEGF expression in
the retina. A: intravitreal injection of VEGF protein and slow-VEGF release implants cause
vascular abnormalities, including hemorrhage,
retinal detachment, microaneurysms, venous
tortuosity, and capillary nonperfusion. B: in
contrast, normal or moderately increased levels of VEGF obtained by overexpressing VEGF
in neurons under the control of the neuronal
promoter Nse protect retinal ganglion cells
against cell death in a model of axotomy. C:
chronic inhibition of VEGF in the adult mouse
eye could also lead to a loss of retinal ganglion
cells, neurodegeneration, and vessel regression. [Retina image adapted with permission
from the Children’s Hospital Boston (www.
childrenshospital.org).]
VEGF IN THE NERVOUS SYSTEM
A. VEGF in Ischemic Brain Disease
Physiol Rev • VOL
The beneficial effects of VEGF in these studies are at
least partly related to its positive effects on the vasculature, as a denser vasculature in the ischemic penumbra
correlated with improved neurological recovery (373,
440). Moreover, exercise-induced expression of VEGF increased microvessel densities in the brain and reduced
neurological deficits and infarct volume after stroke (75).
In addition, various studies report that VEGF also protects neurons from ischemic death. Although these neuroprotective effects might occur indirectly, as a result of
improved perfusion of the ischemic penumbra, there is
evidence that VEGF also directly affects the neurons. For
instance, low doses of systemic VEGF promote neuroprotection of ischemic brains without inducing angiogenesis,
whereas a high dose of VEGF induces angiogenesis but
does not protect ischemic brains (239). VEGF overexpression, by the neuronal Nse promoter, in mice also significantly alleviates neurological deficits, infarct volume, and
reduces disseminated neuronal injury and caspase-3 activity, thereby confirming earlier observations that VEGF
has neuroprotective properties (440). The increased
VEGF levels in these mice also result in a faster and more
pronounced angiogenic response in areas exhibiting neuronal injury. However, cerebral blood flow studies suggest that VEGF overexpression induces a hemodynamic
steal phenomenon, in which the higher vessel density in
transgenic animals promotes a pronounced steal of blood
flow away from ischemic to nonischemic areas which
becomes more apparent towards the core of the ischemic
area (440). Caution is thus warranted to prevent that
inappropriate VEGF administration evokes such unfavorable hemodynamic effects. Intracerebroventricular infusion of VEGF also decreases infarct volume and brain
edema without affecting cerebral blood flow (125). A
study focusing on the association of VEGF expression to
remote cortical areas after an infarct in the primary motor
cortex also detects increased expression of VEGF in neurons of cortical areas functionally or behaviorally related
to the area of infarct, suggesting that enhanced expression of VEGF in related remote cortical areas may be
neuroprotective and stimulate recovery of function after
stroke (368). VEGF thus seems to confer direct neuroprotection in the context of cerebral ischemia. These neuroprotective effects of VEGF are mediated, at least partly,
by signaling through VEGFR-2 and its downstream PI3K/
Akt signaling pathways (173).
A third mechanism by which VEGF might exert beneficial effects after brain ischemia is by stimulating neurogenesis. Intracerebroventricular delivery of VEGF stimulates neurogenesis, as bromodeoxyuridine (BrdU) incorporation in the two principal neuroproliferative zones of
the mammalian brain, i.e., the subgranular zone (SGZ) of
the hippocampal dentate gyrus and the rostral subventricular zone (SVZ), colocalizes with the neuronal lineage
marker Dcx (151). It is thus likely that VEGF also con-
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VEGF has been implicated in brain ischemia, where it
can mediate recovery according to three different mechanisms: VEGF can stimulate angiogenesis and modulate
vascular permeability, exert direct neuroprotective effects, or promote neurogenesis. We will now discuss the
evidence supporting a role for VEGF in each of these
processes. In patients who have suffered a stroke, neuronal loss is proportional to the reduction in blood flow, and
early reperfusion significantly correlates with survival
(193, 241). Autopsy studies show that brain ischemia stimulates angiogenesis, especially in the ischemic penumbra,
where blood flow is reduced and small increases in perfusion can determine the difference between cell death or
survival (192). Histological studies have also demonstrated that HIF-1␣ triggers many downstream molecules
in the ischemic border, including the expression of VEGF
and its receptors, which can drive angiogenesis in the
ischemic border (243). Overall, these observations suggest that enhancing the VEGF/VEGFR signaling pathway
to stimulate angiogenesis after cerebral ischemia may
exert beneficial effects in the brain.
Numerous studies investigating the therapeutic potential of VEGF have been performed. The first studies
focusing on the role of VEGF in stroke did, however, not
indicate that VEGF has a beneficial effect in models of
brain ischemia. Indeed, intravenous delivery of VEGF
early after the insult (within 1 h) worsens stroke outcome
by increasing BBB leakage and hemorrhagic transformation of the ischemic lesions (396, 440). These detrimental
effects of VEGF are due to its potent vessel permeability
effects, which enhance acute brain edema immediately
after ischemic stroke. In addition, VEGF administration
enhances BBB disruption specifically in the ischemic penumbra through activation of the NO synthase pathway
(55, 56). Consistently, intravenous delivery of VEGF antagonists immediately after the ischemic insult also reduce brain edema and infarct size (178). However, when
systemically administered at later time points, i.e., after
48 h of the ischemic insult, VEGF exerts beneficial effects
(440). Topical or intracerebral delivery of VEGF early
after a stroke also exerts acute beneficial effects in various models of stroke (127, 167, 380). The continuous
intracerebral administration of VEGF through encapsulated grafts (215, 261, 430) and viral-mediated hypoxiainducible VEGF expression cassette has also been demonstrated to be beneficial (337). A study focusing on the
functional outcome of intracerebroventricular delivery of
VEGF further demonstrates that the sensorimotor and
cognitive deficiencies following focal cerebral ischemia
are improved after VEGF delivery (411). The delivery
route (systemic versus local) and the timing of VEGF
delivery (early versus late) thus seem to determine the
outcome of VEGF therapy after an ischemic insult.
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RUIZ DE ALMODOVAR ET AL.
B. VEGF in Status Epilepticus
One day after a status epilepticus, VEGF expression
is highly upregulated both in neurons and glial cells of the
hippocampus and limbic cortex (66). VEGF also protects
cultured hippocampal neurons against glutamate excitotoxicity (247). Intrahippocampal infusion of VEGF appears to protect hippocampal neurons from seizure-induced damage, as there is less neuronal loss with fewer
pyknotic cells, whereas a VEGF trap significantly worsens
cell loss relative to animals treated with the control (66).
Seizure scores are, however, not significantly different
from controls in these experiments. When VEGF is administered to adult rat hippocampal slices, it reduces the
Physiol Rev • VOL
amplitude of excitatory responses elicited after stimulation and is able to suppress epileptiform activity in epileptic rats, but not in normal rat slices (251). VEGF may
thus also play a role to decrease epileptiform activity in
the epileptic brain.
C. VEGF in Multiple Sclerosis
There is considerable evidence that VEGF is also
involved in various autoimmune disorders, as serum
VEGF levels correlate with disease activity in a large
number of autoimmune diseases (45). Very little is known,
however, about the role of VEGF in multiple sclerosis
(MS), the most common autoimmune disorder of the
nervous system. Already over 130 years ago, MS lesions
were found to be associated with abnormal blood vessels,
and fingerlike projections (Dawson’s fingers) of demyelination were shown to extend into the white matter alongside the course of blood vessels (379). Increased formation, permeability, and perfusion of vessels have also been
documented in MS lesions, whereas perfusion of grey
matter is reduced, possibly reflecting the decreased metabolism secondary to neuronal and axonal loss (181,
182). Evidence for the occurrence of neovascularization
in MS is observed with contrast-enhanced MRI in the
appearance of “ring enhancement” at the periphery, but
not at the center of chronic lesions (131). Another MRI
study shows a positive correlation between VEGF serum
levels and the length of spinal cord lesions, suggesting
that VEGF might be involved in the formation of spinal
cord lesions of MS (370). Increased levels of VEGF and its
receptor VEGFR-1 are found in astrocytes in MS plaques
during the inflammatory phase (113, 182, 306). In addition,
intrastriatal infusion of VEGF aggravates plaque inflammation at the site of VEGF injection (306). Moreover, as
VEGF expression is highly influenced by inflammatory
cytokines and ischemia (see further below), the accumulation of VEGF may be the result, not the cause, of MS.
When given intracerebrally to experimental autoimmune
encephalomyelitis (EAE) rats, recombinant sVEGFR-1 reduces disease severity compared with untreated rats and
decreases ED1 immunoreactivity, as a marker of inflammatory cells, in the CNS (444). While all these findings
may suggest that VEGF, as a factor affecting vessels or
inflammatory cells, aggravates MS, VEGF, as a neuroprotective factor, can also protect against axonal damage in
MS. Thus the precise role of VEGF in MS remains enigmatic. Possibly, VEGF exerts a dual role in MS lesions:
increased levels of VEGF can amplify vascular permeability in vessels and inflammation in glial cells during the
acute phase of the disease, but can also stimulate the proliferation of neurons and their axons during the chronic
phases of the disease.
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tributes to neurogenesis in a paradigm of cerebral ischemia.
However, as an ischemic insult also increases BrdU labeling
in the dentate gyrus and SVZ (150), the stimulatory effect of
VEGF on neurogenesis can be masked by the ischemic
insult. VEGF also exerts a survival effect on neuronal progenitor cells in the ischemic brain, as significantly more
BrdU-labeled cell from the neuronal lineage are present in
the ischemic zone after 4 wk in the VEGF-treated group,
suggesting that VEGF not only stimulates the proliferation
but also enhances the survival of neural progenitor cells. In
addition, neuronal overexpression of VEGF enhances migration of newborn neurons toward sites of ischemic injury to
replace neurons that undergo ischemic death (412). All these
findings thus suggest that VEGF enhances postischemic
neurogenesis. This also offers VEGF an additional therapeutic advantage for chronic brain repair.
Intriguingly, the therapeutic effects of VEGF in ischemic brain injury are also observed in models of spinal cord
injury: in a model of severe spinal cord ischemia induced by
clamping the thoracic vessels, most of the ventral horn
neurons degenerate leading to complete paralysis in mice
(206). VEGF treatment rescues some of the motor neurons
from death in this model and results in improved neurological outcome (206). In another study, intubation of spinal
subarachnoid space with particles releasing VEGF, also improves rehabilitation after a spinal cord ischemia, as evidenced by improved spinal-evoked and motor-evoked potentials after the VEGF treatment (53).
However, as mentioned previously, VEGF can also
exert toxic side effects when delivered in the nervous
system. This is further illustrated by the observation that
ischemic brains treated with low doses of VEGF, which
are insufficient to induce angiogenesis, exhibited reduced
numbers of macrophages, whereas ischemic brains
treated with an angiogenic dose of VEGF showed high
macrophage density (240). Thus, although VEGF is a therapeutic candidate for stroke and other ischemic disorders, therapeutic use of this molecule warrants caution
and careful consideration.
VEGF IN THE NERVOUS SYSTEM
D. VEGF in Spinal Cord Injury
Physiol Rev • VOL
IX. VASCULAR ENDOTHELIAL GROWTH
FACTOR-B: AN ANGIOGENIC OR
NEUROTROPHIC FACTOR?
A. Angiogenic Activities of VEGF-B
VEGF-B is a secreted growth factor with sequence
homology to VEGF-A. The VEGF-B gene, located on human chromosome 11q13, yields two polypeptide forms,
i.e., VEGF-B167 and VEGF-B186 through alternative splicing (117, 288, 293). The amino acid sequence of VEGF-B167
is 44% identical to that of VEGF165, and the intermolecular
disulfide bridging pattern of both molecules is similar.
VEGF-B is therefore predicted to form a cysteine-knotted,
disulfide-linked homodimeric protein, similar to VEGF
(332). Unlike VEGF-B186, VEGF-B167 has a COOH-terminal
heparin binding domain, allowing it to associate with
pericellular heparin-like glycosaminoglycans, that anchor
this growth factor in the extracellular matrix (288). The
predominant isoform in mice is VEGF-B167, which is expressed at approximately four times the level of VEGFB186. Interestingly, the VEGF-B promoter lacks putative
binding sites for hypoxia-regulated factors, and consequently, VEGF-B is not regulated by hypoxia (83).
VEGF-B is abundantly expressed in most tissues: during
fetal development, it is expressed in cardiomyocytes,
skeletal muscle, and SMCs of large vessels, while in adult
mice, VEGF-B is abundant in heart, kidney, and brain in
addition to other sites (1, 24, 198, 199). Mice lacking
VEGF-B are healthy and fertile, with apparently normal
developmental and physiological angiogenesis, indicating
that this molecule is redundant in healthy conditions.
Studies using VEGF-B knockout mice have yielded
conflicting results regarding the role of VEGF-B in angiogenesis and the development of the cardiovascular system. One study reported that VEGF-B knockout mice
have smaller hearts, dysfunctional coronary arteries, and
impaired recovery from experimentally induced myocardial ischemia (24), while another study documented that
these mice show a subtle cardiac phenotype and that
VEGF-B is not required for proper development of the
cardiovascular system either during development or for
angiogenesis in adults (2). Some reports also documented
an angiogenic effect of VEGF-B (269, 349, 422), whereas
others did not (2, 27, 313, 316, 377). More recently, reevaluation of the activity of VEGF-B in knockout mice on a
congenic background proposed that the angiogenic activities of VEGF-B are restricted to the infarcted area of the
myocardial heart, where it promotes angiogenesis and
myocardial function (226, 388). Overall, VEGF-B seems to
have a rather weak and restricted angiogenic activity,
raising the question whether it might have arisen in evolution as a molecule with other activities, perhaps in the
nervous system.
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VEGF has also been investigated for its ability to
promote nerve repair following spinal cord injury. Expression of VEGF and its receptors is increased in traumatic injury lesions within the spinal cord or brain (352,
401). In the injured brain, VEGFR-2 expression is mainly
upregulated in ECs (188), whereas the upregulation of
VEGF and VEGFR-1 is predominantly associated with
glial cells in the vicinity of the lesion (190). After a contusion injury, VEGF and VEGFR-1 expression is visible
within 1 day of the injury in microglia, macrophages, and
reactive astrocytes and persists for at least 14 days (61).
Expression of VEGF stimulates axonal regeneration in
preparations of sciatic nerves in vitro (135), suggesting
that VEGF could exert beneficial effects in nervous system injury models. Indeed, adenoviral VEGF gene transfer
promotes regeneration of corticospinal tract axons in rats
following transection of the spinal cord (88). After a
traumatic spinal cord injury, local delivery of VEGF also
improves the recovery, which appears to be associated
with increased vessel density and reduced neuronal apoptosis in the lesion area (420). Inhibition of VEGFR-2
immediately after brain injury enlarges the hemorrhagic
area, increases serum levels of the neural injury marker
neuron-specific enolase and the glial injury marker S100␤,
and leads to increased neuronal apoptosis (353), whereas
delivery of a hypoxia-inducible VEGF plasmid improves
the outcome in a rat spinal cord injury model (62). Although it is attractive to postulate a direct neuroregenerative role of VEGF in these studies, it is difficult to assess
whether neural repair or neuroprotection are the result of
a direct neural effect or are secondary to VEGF-driven
angiogenesis and hence improved perfusion of the injured
region. On the other hand, caution is warranted, since
VEGF therapy can also worsen spinal cord injury, possibly secondary to its effect on vascular permeability (25).
VEGFR-2 and VEGFR-1 seem to have a differential
role during the response to brain injury (189). Infusion of
anti-VEGFR-2 antibodies decreases vascular proliferation
and causes endothelial cell degeneration, however, without an effect on astrogliosis. In contrast, infusion of neutralizing antibodies to VEGFR-1 inhibits astroglial proliferation and increases endothelial degeneration slightly
(189). Neutralization of VEGFR-1, but not of VEGFR-2,
also reduces astroglial expression of the growth factors
CNTF and FGF-2. This suggests that after injury, VEGF
acts through VEGFR-1 on reactive astrocytes to increase
their proliferation and to facilitate expression of trophic
factors (189). Endogenous VEGF may thus also play an
important role in the formation and maintenance of the
astroglial scar, which is important to provide structural
support and isolate the injury by restricting the migration
of inflammatory cells into healthy tissue.
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B. Novel Role for VEGF-B in the Nervous System
C. VEGFR-1 Mediates Neuroprotection
and Glial Activation
In the nervous system, the VEGF-B receptor VEGFR-1 is
also detectable in cortical and hippocampal neurons of neonatal and adult rat brain (429), as well as in motor neurons from adult mice (301). In situ hybridization studies
further reveal that VEGFR-1 mRNA is detectable in the
proliferating stem cells of the ventricular and subventricular zones, as well as in postmitotic differentiating
cells of the embryonic cortex and hippocampus (177). In
addition, VEGFR-1 is expressed in proliferating progenitor cells of the anterior horn (SVZa) of the cerebral lateral
ventricle of adult macaques, as well as in cortical neurons
from humans (390). Intriguingly, VEGFR-1 expression is
strongly upregulated in reactive astrocytes in response to
injury, e.g., following permanent and transient occlusion
of the middle cerebral artery, neural grafting, tumor cell
implantation, and after mechanical spinal injury (190,
Physiol Rev • VOL
TABLE 3. Distinct angiogenic versus neural effects
of VEGF-A and VEGF-B
Cell Type
VEGF-A
VEGF-B
Neurons
Schwann cells
Neural progenitor cells
Microglia
Astrocytes
Endothelial cells
⫹⫹
⫹
⫹
⫹
⫹
⫹⫹⫹
⫹⫹
ND
⫹
ND
ND
⫾
VEGF, originally considered as an endothelial specific growth
factor, has recently been shown to have direct effects on different neural
cell types. These include the following: neuronal cells, on which VEGF
stimulates survival and axonal outgrowth; neural stem cells, where
VEGF activates proliferation, differentiation, and migration; Schwann
cells, astrocytes, and microglia, on which VEGF triggers proliferation,
survival, and migration. In contrast to VEGF, the effect of VEGF-B has
little or no effect on the vascular system. However, VEGF-B also exerts
potent effects on neural progenitor cells and neurons.
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In contrast to the minor effects of VEGF-B in the
vascular system, recent studies established a more convincing role for VEGF-B in the nervous system. First of all,
studies focusing on the role of VEGF-B in cortical neurons, spinal motor neurons, and RGCs revealed that
VEGF-B is expressed in these cell types (227, 301, 372).
Unlike VEGF, VEGF-B is not essential for development or
maintenance of these neurons under normal conditions,
suggesting that VEGF-B is less important than VEGF in
the nervous system (227, 301, 372). However, upon ligation of the middle cerebral artery, the infarct volume in
mice lacking VEGF-B increases by as much as 40% and
results in a more severe neurological impairment (371).
Since the cortical vasculature seemed unaffected in
VEGF-B-deficient mice undergoing a ligation and VEGF-B
protects cortical neurons in vitro against hypoxia, the
protective activity of VEGF-B in these mice is most likely
mediated through a direct neuroprotective effect of
VEGF-B on neurons. This hypothesis was indeed confirmed in another study, which demonstrated that intracerebroventricular delivery of VEGF-B increases BrdU
incorporation in cells of neuronal lineage both in vitro and
in vivo (372). VEGF-B-deficient mice also display impaired neurogenesis, whereas intraventricular delivery of
VEGF-B in VEGF-B-deficient mice restores neurogenesis
to wild-type levels (372), thus indicating that VEGF-B is
capable of regulating adult neurogenesis. Moreover, VEGF-B
treatment rescues neurons from apoptosis in the retina
and brain in mouse models of ocular neurodegenerative
disorders and stroke, respectively, without promoting
neovascularization, thus supporting the role of VEGF-B in
the nervous system (227).
191). Likewise, VEGFR-1 expression is strongly upregulated in activated astrocytes surrounding degenerating
motor neurons from paralyzed SOD1 mice (301). Application of VEGF to cortical explants also increases astroglial
proliferation, presumably by acting though VEGFR-1 expressed on reactive astrocytes, as silencing of VEGFR-1
further decreases astrogliosis (238). In focal cortical dysplasia patients, VEGFR-1 is expressed in reactive astrocytes, as well as in cells from the microglial/macrophage
lineage (31). Some studies also found that endothelial
cells upregulate VEGFR-1 (352, 420), although this was
not confirmed by others (189).
A recent study confirmed that VEGF-B, through
VEGFR-1 signaling, is critical for cell survival by downregulating genes involved in apoptosis pathways in models of optic nerve crush injury, NMDA-induced retinal
neuron apoptosis, or ischemic neuronal death induced by
middle cerebral artery occlusion (227). VEGF-B also increases BrdU incorporation into cells of neuronal lineage
(372) and protects motor neurons against degeneration.
Indeed, when primary motor neuron cultures are isolated
from normal mice, VEGF-B is neuroprotective (Table 3).
In contrast, when motor neurons are isolated from tyrosine kinase-deficient VEGFR-1 mice, VEGF-B fails to
exert any effect, thus confirming the role of VEGFR-1 as a
neuroprotective receptor. In vivo, mice lacking VEGF-B
or expressing a tyrosine kinase-deficient VEGFR-1, also
develop a more severe form of motor neuron degeneration when intercrossed with mutant SOD1 mice (301).
Intracerebroventricular delivery of VEGF-B186 also prolongs the survival of mutant SOD1 rats. Notably, at a dose
effective for neuron survival, VEGF-B treatment does not
seem to affect normal or pathological angiogenesis or
influence blood vessel permeability. Compared with a
similar dose of VEGF, VEGF-B is thus safer and does not
cause vessel growth or BBB leakiness. Rather than acting
as a molecule with combined angiogenic and neuropro-
635
VEGF IN THE NERVOUS SYSTEM
tective activities (such as VEGF), VEGF-B thus seems to
have a more restricted neuroprotective effect in the nervous system.
Upregulation of PlGF, which also binds selectively to
VEGFR-1, in the CNS has also been observed after focal
cerebral ischemia (23). Although the precise role of PlGF
in the CNS is not known, PlGF significantly reduces astrocyte cell death induced by oxygen and glucose deprivation (101), suggesting that it may also be protective
under pathological conditions. In addition, PlGF-2, a PlGF
isoform that binds to NRP1, was shown to chemoattract
DRG axons in vitro (54).
Since the discovery of the neuroprotective effects of
VEGF on DRGs in 1999 and the fortuitous discovery in
2001 that a reduction in VEGF causes motor neuron degeneration in mice, a formidable amount of research has
been performed to unravel the role of VEGF in the nervous system. By now, it is well-established that VEGF
exerts direct in vitro effects on a variety of neural cells
(Fig. 13). However, the molecular mechanisms activated
by VEGF in neurons are mostly unknown. The unexpected effect of VEGF on neurons raises numerous exciting questions. For instance, is VEGF also acting as an
axon guidance cue? Does VEGF regulate cytoskeleton
rearrangements in neurons? What are the intracellular
pathways activated by VEGF that lead to VEGF-induced
neuronal proliferation survival, migration, or differentiation? How is VEGF regulating synaptic plasticity?
VEGF also has in vivo effects in acute or chronic
models of neurodegeneration, including nerve and brain
injuries. VEGF can thus be considered as a modifier ca-
FIG. 13. Pleiotropic effects of VEGF in the CNS.
VEGF enhances vascular perfusion (resulting in enhanced O2 delivery) and ensures EC survival, stimulates the release of neurogenic signals from ECs,
promotes the survival of multiple types of adult neurons and protects them against a variety of stress
conditions, increases neurogenesis, regulates neuronal migration and improves cognitive performance
and memory, affects synaptic transmission, stimulates neurite extension, stimulates remyelination
through its effects on Schwann cells, and promotes
muscle regeneration. VEGF also acts on astrocytes
and microglial cells, which modulate neurodegeneration. These multiple actions of VEGF underlie its
important role during development and its therapeutic
potential, as demonstrated in preclinical rodent models
of ALS. NMJ, neuromuscular junction. [Adapted from
Zacchigna et al. (436).]
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X. CONCLUDING REMARKS
pable of aggravating or reducing neurodegeneration, depending on whether its expression is reduced or increased. Many other growth factors have been reported to
exert similar effects in these models. The effects of VEGF
are, however, of particular interest, since they may result
from stimulating both the nervous and vascular systems
(Fig. 15). The protective effects of VEGF may indeed
result from the combined effect on vessels, neuronal and
nonneuronal cells; in nerve injury models, for instance,
VEGF stimulates axonal outgrowth, Schwann cell proliferation, and nerve perfusion. In fact, these pleiotropic
“multitasking” effects make VEGF a particularly suitable
and promising candidate for therapeutic studies, especially since the treatment of neurodegeneration has so far
been largely neurocentric.
There is also clear evidence that reduced VEGF levels cause neurodegenerative diseases. A causative role for
VEGF has been documented in various motor neuron
degenerative diseases. Motor neurons thus seem to be
particularly dependent on sufficient VEGF for their survival. The observation that reduced levels of VEGF are
causally involved in neurodegeneration might have implications for antiangiogenic therapies targeting VEGF in
cancer or ocular diseases. Despite potential side effects,
the evidence provided in this review illustrates that VEGF
can be considered as an attractive therapeutic target.
Indeed, gene-based therapy using a VEGF-expressing lentiviral vector, MoNuDin, is being developed for human
use, and clinical studies delivering VEGF intracerebroventricularly for ALS patients have been initiated. Deciphering the therapeutic value of VEGF thus promises to
be an exciting journey, which will hopefully lead to the
discovery of an effective therapy for the neurodegenerative diseases.
636
RUIZ DE ALMODOVAR ET AL.
ACKNOWLEDGMENTS
We apologize for not being able to cite every study published in the VEGF field. We acknowledge all previous and
current collaborators who have contributed to the studies of the
role of VEGF in the neurovascular link and thank Dr. L. Claesson-Welsh and Dr. S. Plaisance for helpful discussions on the
manuscript, D. Brunette and Dr. Foscher for providing the
growth cone image, and Li-Kun Phng and Dr. H. Gerhardt for
providing the endothelial tip cell image.
Address for reprint requests and other correspondence: P.
Carmeliet, The Vesalius Research Center (VRC), Flanders Interuniversitary Institute for Biotechnology (VIB), KU Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium
(e-mail: [email protected]).
Some of the studies discussed in this overview were funded
with the help of Geneeskundige stichting Koningin Elisabeth
(GSKE), the GOA 2006/11, the FWO (G 0210.07), long-term
structural funding: Methusalem funding by the Flemish Government, the Muscular Dystrophy Association (MDA), the Amytrophic Lateral Sclerosis Association (ALSA), and the Motor Neuron Disease (MND) Association. During the process of writing
this manuscript, C. Ruiz de Almodovar was a recipient of a FEBS
postdoctoral fellowship and is currently supported by the Research Foundation Flanders (FWO), Belgium. D. Lambrechts is
supported by the Research Foundation Flanders (FWO), Belgium. M. Mazzone is a recipient of an EMBO postdoctoral fellowship.
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