Cross talk between the cardiovascular and nervous systems: neurotrophic effects of vascular endothelial growth factor (VEGF) and angiogenic effects of nerve growth factor (NGF)-implications in drug development.

Abstract

Both blood vessels and nerves are guided to their tissue targets by "specific" growth factors such as vascular endothelial growth factor (VEGF) and nerve growth factor (NGF), originally discovered as growth factors specific for endothelial and neuronal cells, respectively. While the eminent role of VEGF in the formation of new blood vessels (angiogenesis) is unquestioned, recent studies indicate that VEGF also has direct effects on the nervous system in terms of neuronal growth, survival (neurotrophic), axonal outgrowth (neurotropic), and neuroprotection. Conversely, NGF, a neurotrophin that plays a crucial role in promoting neurotrophic and neurotropic effects in sympathetic neurons, has recently been identified as a novel angiogenic molecule exerting a variety of effects on endothelial cells and in the cardiovascular system in general. VEGF and NGF have also been implicated in both neurodegenerative and vascular diseases. The pleiotropic effects of these growth factors have raised interest in assessing their therapeutic potential. The challenge for the future is to unravel to what extent the effects of these growth factors are interrelated with regards to their angiogenic, and neurotrophic effects and how to design selective drugs interfering with their respective actions. Most biological actions of NGF and VEGF are mediated by their cognate receptor protein tyrosine kinases, tropomyosin related kinase (trkA for NGF) and kinase insert domain-containing receptor (KDR, VEGFR-2, flk-1 for VEGF), which activate a complex and integrated network of signaling pathways in neurons and endothelial cells. Two small molecules, K252a and SU-5416, which are antagonists of trkA and VEGFR-2, respectively, may serve as key tools in dissecting the role of NGF and VEGF in angiogenesis and neurogenesis. Development of selective drugs specific for the trkA and VEGFR-2 subtypes of receptors will provide new tools for the treatment of neurodegenerative diseases, such as Alzheimer's and Parkinson's, as well as of numerous angiogenesis-dependent diseases, such as cancer, diabetes, and arthritis.

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Current Pharmaceutical Design, 2006, 12, 2609-26222609
1381-6128/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd.
Cross Talk between the Cardiovascular and Nervous Systems:
Neurotrophic Effects of Vascular Endothelial Growth Factor (VEGF) and
Angiogenic Effects of Nerve Growth Factor (NGF)-Implications in Drug
Development
Philip Lazarovici1, Cezary Marcinkiewicz2 and Peter I. Lelkes3,*
1Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine, The Hebrew
University of Jerusalem, Jerusalem 91120, Israel; 2Department of Neuroscience, Center for Neurovirology, Temple
University, Philadelphia, PA 19122, USA and 3School of Biomedical Engineering, Science and Health Systems, Drexel
University, Philadelphia, PA 19104, USA
Abstract: Both blood vessels and nerves are guided to their tissue targets by “specific” growth factors such as vascular
endothelial growth factor (VEGF) and nerve growth factor (NGF), originally discovered as growth factors specific for en-
dothelial and neuronal cells, respectively. While the eminent role of VEGF in the formation of new blood vessels (angio-
genesis) is unquestioned, recent studies indicate that VEGF also has direct effects on the nervous system in terms of neu-
ronal growth, survival (neurotrophic), axonal outgrowth (neurotropic), and neuroprotection. Conversely, NGF, a neurotro-
phin that plays a crucial role in promoting neurotrophic and neurotropic effects in sympathetic neurons, has recently been
identified as a novel angiogenic molecule exerting a variety of effects on endothelial cells and in the cardiovascular sys-
tem in general. VEGF and NGF have also been implicated in both neurodegenerative and vascular diseases. The plei-
otropic effects of these growth factors have raised interest in assessing their therapeutic potential. The challenge for the
future is to unravel to what extent the effects of these growth factors are interrelated with regards to their angiogenic, and
neurotrophic effects and how to design selective drugs interfering with their respective actions. Most biological actions of
NGF and VEGF are mediated by their cognate receptor protein tyrosine kinases, tropomyosin related kinase (trkA for
NGF) and kinase insert domain-containing receptor (KDR, VEGFR-2, flk-1 for VEGF), which activate a complex and
integrated network of signaling pathways in neurons and endothelial cells. Two small molecules, K252a and SU-5416,
which are antagonists of trkA and VEGFR-2, respectively, may serve as key tools in dissecting the role of NGF and
VEGF in angiogenesis and neurogenesis. Development of selective drugs specific for the trkA and VEGFR-2 subtypes of
receptors will provide new tools for the treatment of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s, as
well as of numerous angiogenesis-dependent diseases, such as cancer, diabetes, and arthritis.
Key Words: NGF, VEGF, trkA, VEGFR-2, K252a, SU-5416, angiogenesis, neurotrophic, neuron, endothelial cell.
1. ANGIOGENESIS
Angiogenesis, viz, the generation of new capillaries, is a
complex, yet tightly regulated process involving multiple
cells, growth factors and extracellular matrix modeling. An-
giogenesis, in general, occurs by capillaries sprouting from
the existing microvasculature. Newly formed blood vessels
carry oxygen, hormones, nutrients and electrolytes and se-
crete a variety of growth factors with paracrine and jux-
tacrine activity on the endothelial, fibroblasts and pericytes
forming the neovessels [1]. Angiogenesis plays a critical role
in the morphogenesis of many normal tissues during embry-
onal life. Once these new capillaries have formed and ma-
tured, the endothelial cells lining the newly formed blood
vessels stop proliferating and become quiescent. Angiogene-
sis occurs under tight regulation also in the adult in the fe-
male reproductive system and during wound healing [2].
Unbalanced angiogenesis is a recognized pathology in
*Address correspondence to this author at the School of Biomedical Engi-
neering, Science and Health Systems, Drexel University, Philadelphia, PA
19104, USA; Tel: 215.895.2219/215.762.2071; Fax: 215.895.4983;
E-mail: pilelkes@drexel.edu
different disease states such as rheumatoid arthritis, diabetic
retinopathy, psoriasis and juvenile hemangiomas [3-6]. More
than 30 years ago Judah Folkman recognized that angiogene-
sis is essential for the sustained growth of solid tumors [7].
Solid tumors that lack adequate vascularization become ne-
crotic [8] and/or apoptotic [9] and fail to grow beyond a lim-
ited size. However, tumors that undergo neovascularization
enter a phase of rapid growth and exhibit increase metastatic
potential [10] since the leaky blood vessels invading and
surrounding the tumor provide a way for cancer cells to enter
the circulation and to metastasize to distinct organs. Thus,
many if not all organs may involve diseases in which angio-
genesis is an important component. Inhibition of the early
steps of angiogenesis has been identified as an attractive
approach for the treatment of human cancers [11, 12]. An-
gio-inhibition is a promising therapeutic approach, with very
mild side effects, because throughout the body the vascula-
ture is largely quiescent with very low turnover rates; normal
adult tissues are already vascularized and do not require new
blood vessels. A basic knowledge of the mechanism of ac-
tion of putative angiogenic and anti-angiogenic growth fac-
tors my lead to the discovery of the means how to control

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2610 Current Pharmaceutical Design, 2006, Vol. 12, No. 21Lazarovici et al.
their interplay. This in turn may allow the development of
specific anti-angiogenic drugs for the therapy of cancer and
other angiogenesis-dependent diseases.
2. VASCULAR ENDOTHELIAL GROWTH FACTOR
(VEGF)
VEGF is a member of a family of closely related growth
factors which include VEGFs A, B, C, D, E and PlGF, pla-
centa growth factor [13]. Alternative splicing of human
VEGF A mRNA from a single gene containing eight exons
gives rise to at least five different isoforms of 121, 145, 165,
189, and 206 amino acid residues [14]. Exon 6 is absent in
VEGF121 and VEGF165 and exon 7 is absent from VEGF121
and VEGF145. While all VEGFs are secreted, VEGFs 189
and 206 remain sequestered in the extracellular matrix upon
their secretion. By contrast, VEGFs 121, 145, and 165 form
dimeric proteins that can act in a paracrine fashion and are
also found in the circulation. All isoforms except VEGF121,
bind heparin to different degrees via a domain rich in
charged basic amino acids encoded by exon 6 and 7 [14].
Human VEGF165 is glycosylated at Asn75 [14] and is typi-
cally expressed as a 46 kDa homodimer of two 23 kDa
monomers. VEGF165 is the most abundant and most biologi-
cally active form in vitro [15]. The major role of VEGF A in
angiogenesis has been highlighted by the occurrence of ab-
normal blood vessel development and lethality in embryos
lacking a single VEGF allele [16]. Transgenic homozygous
mice expressing only VEGF120 and lacking the heparin-
binding isoforms VEGF165 and VEGF189 isoforms coded by
exons 6 and 7 die shortly after birth due to bleeding and
ischemic cardiomyopathy [17]. A hallmark of all isoforms of
VEGF A is that their expression is regulated by hypoxia due
to the binding of hypoxia inducible transcription factor (HIF)
to the VEGF gene promoter region [18].
The role of other VEGFs is not well understood at pre-
sent: VEGF C and D may be growth factors for lymphatic
vessels [19], VEGF B may regulate the expression and ac-
tivity of urokinase type plasminogen activator [20], and a
role of placenta growth factor in placental angiogenesis has
been proposed [21]. Among the different VEGFs, VEGF A
is the key molecular regulator of pathological angiogenesis
and will be mainly addressed in this review.
3. VEGF RECEPTOR STRUCTURE AND SIGNALING
IN ANGIOGENESIS
Two major receptor tyrosine kinases have been identified
for VEGF, the high affinity receptor VEGFR1 / Flt1 (Kd 16-
114 pM) and a second receptor with a somewhat lower af-
finity, VEGFR2 / KDR/ Flk1 (Kd 0.4-1 nM). Both receptors
share a 44% amino acid identity [15] and consist of a cyto-
plasmic region containing a tyrosine kinase domain divided
into two parts by an insert sequence, a single transmembrane
hydrophobic domain, and an extracellular region comprising
seven IgG-like domains [22]. Most of the biologically rele-
vant VEGF signaling in endothelial cells is mediated by
VEGFR2 [23, 24]. Similar to other tyrosine kinase receptors,
VEGFR2 is activated through ligand-stimulated receptor
dimerization and trans-autophosphorylation of tyrosine resi-
dues in the cytoplasmic kinase domain. To date, six major
autophosphorylation sites have been identified in VEGFR2:
Y951 and Y996 in the kinase insert region, Y1054 and
Y1059 in the kinase domain, and Y 1175 and Y 1214 in the
carboxy terminal tail [25, 26]. The functions of these sites
are being investigated: Y 951 was identified as the binding
site for the SH2 (Src homology 2) domain protein and
Y1175 is a binding site for PLC-gamma (phospholipase C-
gamma) [27, 28]. VEGFR2 has also been shown to associate
with other SH2 domain proteins, such as growth factor re-
ceptor-bound protein 2 (Grb2), SH2-containing protein (Shc)
and SH2 and SH3 containing protein (Nck) as well as protein
tyrosine phosphatases, such as suppressor of high-copy PP1,
2 (SHP1, 2) and human cellular protein tyrosine phosphatase
A (HCPTPA), which supposedly regulate VEGFR2 function
[29, 30]. The central role of VEGF in angiogenesis is de-
pendent upon its ability to coordinately regulate, through the
activation of signaling pathways downstream from VEGFR2,
multiple endothelial functions: survival, proliferation, differ-
entiation, adhesion and migration, vascular permeability and
others.
A major VEGFR2-mediated pathway, which promotes
endothelial cell survival, is the activation of PI3-kinase
(phosphoinositide 3-kinase) responsible for induction of the
anti-apoptotic kinase Akt/protein kinase B [31], which in
turn phosphorylates and inhibits the pro-apoptotic proteins
Bcl-2 homologue containing BH3 motif (Bad) and caspase-
9. VEGF increases tyrosine phosphorylation of FAK (focal
adhesion kinase) [32]. FAK is a major component of the
integrin β-3 and β-1 signaling pathways, an important sur-
vival system for nascent blood vessels during angiogenesis
[33]. Thus, activation of FAK may also contribute to
VEGFR2 survival signaling. Presently, other signaling path-
ways are also considered to play a role in VEGF stimulation
of endothelial cell survival. For example, phorbol myristate
acetate, a protein kinase C activator, promoted survival of
human umbilical vein endothelial cells and endothelial cell
tube formation in 3-D collagen gels [34].
VEGF stimulates VEGFR2-mediated DNA synthesis and
proliferation in vitro in a variety of endothelial cells [35].
Mitogenic signaling of VEGF plays a central role in angio-
genesis: mitogen activated protein kinases 1 and 2 (erk1 and
erk2) are strongly activated in VEGFR2 stimulation of en-
dothelial cell proliferation [36, 37]. However, in contrast to
other tyrosine kinase receptors such as trkA, the NGF re-
ceptor, VEGF stimulation of erks is ras-independent and may
involve activation of alternative signaling pathways, such as
src, phospholipase C gamma, increase in intracellular cal-
cium, activation of certain PKC isoforms, stimulation of NO
production and other cellular effects which indirectly stimu-
late activation of the raf-erk pathway. As yet, however, the
exact mechanisms involved are unresolved [35].
VEGFR2–induced FAK activation and the subsequent
phosphorylation of focal adhesion-associated protein sub-
strates, such as paxillin, is critical for regulating focal adhe-
sion turnover, actin filament organization and cell migration
[36]. VEGF increases FAK phosphorylation at both Y397
and 861, selectively stimulating Y861 phosphorylation via a
src-dependent pathway [37]. VEGF–induced cell migration
is also dependent on the activation of the p38 mitogen acti-
vated protein kinase [38], increased NO production [39] and
activation of RhoA and Rac [40], suggesting redundancy and

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Angiogenic and Neurotrophic Effects of NGF and VEGFCurrent Pharmaceutical Design, 2006, Vol. 12, No. 21 2611
compensatory mechanisms in VEGF-mediated chemotactic
signaling.
VEGF was originally identified as a brain glioma-derived
factor which increased vascular permeability (hence it was
originally termed Vascular Permeability Factor, VPF) [41].
The signaling mechanisms, which may explain this physio-
logical effect, are not clear. Mechanisms proposed in the past
include: VEGF-induced formation of abundant fenestrae
[42], increased formation of caveolae [35], increased phos-
phorylation of tight junction proteins [43, 44], VEGF-
induced NO and PGI2 production [45], and activation of
phospholipase C gamma [46].
Despite the significant progress in understanding signal
transduction of VEGFR2, important signaling aspects, spe-
cifically identification of unique down stream targets and of
the complex signaling network underlying the individual
physiological responses, remain to be elucidated.
4. ANGIOGENIC EFFECTS OF VEGF IN THE
NERVOUS SYSTEM
The vasculature of the central nervous system is derived
by angiogenesis from the perineural vascular plexus which
invades the neural tube early in development, actively con-
tinues during the growth of the brain in the postnatal period
and is significantly decreased in the adult. In the developing
brain, VEGF is expressed in the ventricular zone while the
VEGF receptors are expressed in endothelial cells of the
perineural capillary plexus infiltrating the neuroectoderm
[47]. This topographical selective expression strongly sug-
gests that VEGF is involved in the directional growth of
blood vessels in the developing brain [48]. A direct evidence
for such a role was provided by targeted inactivation of the
VEGF gene resulting in abnormal blood vessel development
in the brain [49, 50]. Cerebral angiogenesis and VEGF ex-
pression are also tightly associated in the postnatal develop-
ing brain [51]. VEGF expression is mainly restricted to cor-
tical neurons and mediates cortical angiogenesis by provid-
ing a gradient along which new blood vessels grow [51].
When the vascular bed begins to stabilize, predominant
VEGF expression is shifted to glial cells, which interact with
the blood vessels while neuronal VEGF expression is re-
duced to basal level. Brain angiogenesis is a strictly con-
trolled process that is down regulated after the first three
postnatal weeks. In the adult brain, angiogenesis can resume
under conditions of ischemia or tumor growth [52]; angio-
genesis and blood brain barrier dysfunction occur mainly
after injury to the brain. Administration of VEGF165 to fetal
and adult cortical tissue induced significant angiogenesis
[53, 54] suggesting that a targeted increase in the vascular
capillary density (and with that the oxygen supply in the
brain) may help to mediate orderly development of vascular
patterning and remodeling of the vasculature.
In the brain, most of the VEGF-induced effects, such as
proliferation, migration, and (anti-apoptotic) survival are
mediated through activation of VEGFR2 [55, 56]. Recent
studies indicate however, that VEGFR2 is also found ex-
pressed in neurons and, hence, that the action of VEGF in the
brain is much broader than the well known vascular effects.
Most recent studies demonstrate that VEGF causes a variety
of diverse neuronal effects, which suggests that this previ-
ously “vascular endothelial-specific growth factor” may be
intimately involved in neuronal repair and development by
mechanism(s) independent of its effects in the vasculature
[55].
5. NEURONAL EFFECTS OF VEGF IN THE NERV-
OUS SYSTEM
Apart from its central role in the cardiovascular system,
recent evidence indicates that VEGF also plays an important
role in the nervous system. As presented in Table 1, VEGF
exerts a large number of diverse neuronal effects in vitro and
in vivo, both in the central and peripheral nervous system.
VEGF stimulates neurogenesis (proliferation), increases sur-
vival and promotes growth (neurotrophic factor) for a variety
of neuronal and glial cell types of different species and nerv-
ous tissue origin. In addition, VEGF induces neurite out-
growth (neurotropic factor) in ganglia and neurons and also
protects neuronal tissues in vitro and in vivo (neuroprotec-
tive) from cell death induced by a variety of insults such as
ischemia, trauma, spinal cord injury, peripheral neuropathy,
typical for numerous neurodegenerative disorders, e.g.,
Amyotropic Lateral Sclerosis (ALS, or Lou Gehrig’s dis-
ease) and Parkinson’s disease. As an example, in the
ischemic brain increased VEGF levels play a neuroprotective
role in the pathophysiological processes that follow stroke.
VEGF administered exogenously or overexpressed by gene
delivery into rat brains, reduced the size and severity of
ischemic brain infarcts and the incidence of hypoxic neu-
ronal cell death. The neuroprotective mechanisms of
VEGFR2-mediated cellular signaling events is based mainly
on the activation of the PI3K/Akt pathway, inhibition of
caspase -3 activity, inhibition of specific potassium currents
(Kv1.2 channel) as well as enhanced proliferation, migration
and differentiation of neuronal progenitors [79]. Obviously,
an important issue in understanding VEGF actions in the
nervous system is whether an enhanced angiogenesis can be
correlated with or responsible for some or all neuronal ef-
fects of VEGF [80, 81] or whether VEGF effects are not
restricted to the vasculature. Selective VEGFR2 receptor
antagonists, such as SU-5416, will definitely provide impor-
tant tools to separate the individual actions of this important
growth factor. Presently, we are in earliest stages of under-
standing the cross-talk between the cardiovascular system
and nervous system mediated by VEGF. While inhibiting
VEGF-induced angiogenesis is an important approach for
cancer treatment, the emerging evidence for a neuroprotec-
tive role of VEGF in some neurodegenerative disorders pro-
vides a rationale for considering the therapeutic potential of
VEGF in the nervous system. Therefore both neuronal-
specific VEGF agonists and antagonists need to be studied
and might also have to be developed de novo.
6. SU-5416, A PROTOTYPIC SELECTIVE INHIBITOR
OF THE TYROSINE KINASE ACTIVITY OF VEGFR2
Selective protein–tyrosine kinase inhibitors (PTKIs)
comprise a new, rapidly evolving family of low molecular
weight anticancer drugs which block downstream signaling
of a variety of receptor tyrosine kinases, such as epidermal
growth factor receptor and related oncogenic forms or the
various forms of VEGF receptors, specifically of VEGFR2
[82]. The first inhibitor of VEGFR2 to undergo large scale

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2612 Current Pharmaceutical Design, 2006, Vol. 12, No. 21Lazarovici et al.
clinical trial as an anti-cancer drug, was the anti-angiogenic
compound, Semaxanib / SU-5416 developed by SUGEN
(now Pfizer) (Fig. 1) [83].
Fig. (1). Chemical structure of the VEGFR2 antagonist SU-5416.
SU-5416 is a potent, relative selective, ATP- competitive
inhibitor of the tyrosine kinase catalytic domain of VEGFR2
with a Ki of 0.16 µM [83] and long lasting (72hr) inhibitory
activity in vitro and in vivo [84]. SU-5416 is 20 fold less
potent inhibitor of platelet derived growth factor receptor
(PDGFR) and a weak inhibitor of fibroblast growth factor
receptor (FGFR, Ki of 20 µM). Moreover, SU-5416 does not
inhibit epidermal growth factor receptor (EGFR), insulin-like
growth factor receptor (IGFR) and other non receptor protein
kinases [83]. Regrettably, research on SU-5416 was stopped
in February 2002 after disappointing phase 3 results [84]. A
similar compound developed since then, SU-6668 (Fig. 1), is
a less selective, ATP-competitive inhibitor of VEGFR2,
PDGFR and FGFR, with IC50 values in the micromolar
range, as determined by using isolated receptors or cellular
assays [85]. Consistent with these properties, SU-6668 (Fig.
2) potently inhibits angiogenic signaling in cellular assays
and exhibits strong anti-angiogenic activity in mice tumors
[85, 86]. Pfizer has now a third generation VEGFR2 inhibi-
tor, SU-11248 (Fig. 2) which showed encouraging phase 1
results inducing partial remission in patients with acute
myeloid leukemia [87]. This inhibitor is less selective than
SU-6668 or SU-5416, blocking VEGFR2, PDGFR,
VEGFR1 and KIT kinase activity [88]. Other orally admin-
istered VEGFR2 inhibitors, of different potencies, selectivity
and chemical structures (see Figs. 2 and 4), such as CEP-
7055 [89], ZD-6474 [90], PTK 787/ZK222584 [91], BAY
43-9006 [92], and AZD 2171 [93] are under active research
and development as promising anti-angiogenic drugs for the
therapy of different cancers.
Similar to some other small anticancer drugs, the lipo-
philic VEGFR2 inhibitor SU-5416 caused neurotoxic side
effects, viz CNS hemorrhage, hypertension, headache and
fatigue [94-96]. Although there is minimal penetration of
SU-5416 into the central nervous system (cerebrospinal
fluid) after intravenous injection in nonhuman primates [94],
these toxic effects may be explained by neuro-inhibitory
and/or neuro-stimulatory effects of this compound in both
the central and peripheral nervous system The most plausible
pharmacological explanation is that SU-5416 acts as a mixed
agonist-antagonist drug: in the absence of VEGF, it binds to
the ATP binding domain of VEGFR2 and induces partial
receptor activation. In this case, like VEGF, it induces the
production of nitric oxide in vascular endothelial cells, de-
creasing the peripheral resistance followed by hypertension
due to compensatory mechanisms in the cardiovascular sys-
tem. Like VEGF, SU-5416 increases brain capillary perme-
ability inducing hemorrhage. In the presence of VEGF, SU-
5416 blocks receptor autophosphorylation resulting in anti-
angiogenic effects. Alternatively, the different pharmacol-
ogical side effects of SU-5416 may be attributed to the indis-
criminate inhibition of cellular kinases which posses a highly
conserved ATP binding domain [173]. We anticipate that
future generations of VEGFR2 inhibitors will a) address
other enzymatic mechanisms of inhibition, b) be less lipo-
philic to decrease neuronal permeability, c) behave as full
Table 1.Neuronal Effects of VEGF in the Nervous System
Cell TypeOrigin EffectReferences
Neuronal precursors, astrocytes, endothelium Mouse brain cortexProliferation [57]
Neurons and neuronal explants Rat brain cortexSurvival, neurite outgrowth [58]
Superior cervical and dorsal root ganglionMouse Survival, proliferation, axonal outgrowth[59-61]
Mesencephalic explantsRat fetus Survival, growth[62]
Cortical and retinal progenitorsRat Proliferation[63, 64]
Astrocytes RatProliferation [53, 62, 65]
Motor neurons from embryo and adultMouse, rabbitNeuroprotection [66-68]
Motor neuronsHuman Neuroprotection in amyotrophic lateral sclerosis (ALS) [68]
Cortical neuronsRatsNeuroprotection in ischemia[69-71]
Cortical neuronsRat Neuroprotection in brain trauma [72]
Spinal cord motor neurons and inter-neuronsRat, rabbitNeuroprotection in spinal cord injury [73, 74]
Peripheral neuronsRat, humanNeuroprotection in diabetic and ischemic neuropathy [75, 76]
Dopaminergic neurons Rat Neuroprotection in Parkinson’s model[77, 78]
H
N
O
H
N

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Angiogenic and Neurotrophic Effects of NGF and VEGFCurrent Pharmaceutical Design, 2006, Vol. 12, No. 21 2613
antagonists to increase efficacy and reduce side effects. In
our opinion, such compounds will be ideally suited for
treating peripheral solid tumors by an anti-angiogenic ap-
proach. In this aspect an important lesson about small mole-
cule VEGFR2 inhibitors, is provided by Avastin (Bevacizu-
mab), the humanized form of a mouse anti-VEGF antibody
[97] In February 2004, the FDA approved this anti-VEGF
drug for the treatment of metastatic colon cancer. Avastin
was the first anti-angiogenic compound to receive FDA ap-
proval, after one of the most successful phase 3 anticancer
drug trials in the last decades. Patients receiving Avastin had
a median end-point survival time of 5 months longer than
placebo or other control treatments [98]. Being a protein, this
drug is very hydrophilic and does not penetrate into the
nervous system, therefore lacking neurotoxic side effects.
The major problem with this immunotherapeutic approach is
the systemic shielding of all subtypes of VEGF receptors
from the ligand, which, as a natural compensatory mecha-
nism, might lead to the build-up of clinical resistance to
antiangiogenic cancer therapy as well as to the generation of
antibodies against the drug in chronically treated patients.
Taken together, we anticipate that the development of
novel, more-specific anti-angiogenic drugs in concert with
improved management protocols, e.g., carefully adjusting the
timing of anti-angiogenesis therapy, drug dosage, targeting
more angiogenic factors/receptors at the same time (such as
SU-11548), and/or combining angiogenic therapy with che-
motherapy, immunotherapy, and radiotherapy, will create a
more effective outcome for the therapy of a large range of
diverse cancers.
7. NERVE GROWTH FACTOR (NGF)
NGF is an evolutionary conserved, polypeptide neurotro-
phin, which plays a crucial role in the sympathetic and sen-
sory nervous systems [99]. A large number of NGFs have
been discovered from different species and tissues, charac-
terized by a known consensus sequence [100] which differ in
40 out of 118 amino acids of their β-chain, the chain that is
solely responsible for NGF’s neurotropic and neurotrophic
activities [101]. The majority of research on NGF has been
performed using NGF isolated from the male mouse sub-
maxillary gland (NCBI, NP38637). More recently human
recombinant NGF (NCBI, AAA59931) has also become
available. NGF is produced by the murine submaxillary
gland as a precursor complex of about 130 kD MW (also
named 7S). This complex is composed of three subunits: α,
β, and γ, which disassociate at a very acidic pH, enabling the
isolation of the active β-subunit [102]. Both the β and γ
subunits are members of the kallikrein family of serine pro-
teases and are involved in the cleavage of this 7S precursor
and generation of β-NGF. The β-subunit, known as β-NGF
(2.5S), is the active subunit of NGF and exhibits all of the
biological activities classically described for NGF, e.g., pro-
motion of neuronal survival, proliferation and differentiation
(morphologically expressed as neurite outgrowth) [103]. In
humans, the β-NGF gene is located on chromosome 1 within
band 1p13 [104]. The crystal structure of 7S-NGF shows that
the α subunit contacts the β and γ subunits while γ subunits
contact each other and make a zinc ion mediated contact
with the α subunit [105]. β-NGF contains a characteristic
structural motif, the cystein knot, denoted by a ring structure
formed by two disulfide bridges penetrated by a third disul-
fide bridge [106]. Recently, the 2.3-angstrom crystal struc-
ture of the NGF, complexed with its p75 receptor was deter-
mined, indicating an asymmetrically, non-covalent homodi-
mer binding [107]. The crystal structure of NGF bound to its
second, tyrosine kinase-trkA receptor was determined at a
resolution of 2-angstrom, identifying a receptor binding do-
main comprised of three leucine-rich motifs flanked by two
cysteine-clusters followed by two immunoglobulin-like do-
mains [108]. Although both the 7S-NGF precursor and 2.5S-
β-NGF, induce neurite outgrowth in PC12 pheochromocy-
toma cells, there are differences in the potencies of these two
related compounds, [109].
Over the last years the interest in NGF precursors was
renewed, when it was found that many tissues and cell-lines
synthesize proneurotrophins, such as pro-NGF, as immature,
7S-like precursors, which are proteolytically cleaved by furin
and other matrix metalloproteinases [110]. Unprocessed
proNGF can discriminate between the two types of NGF
receptors [111, 112], viz, p75 and trkA (see below). This
discovery may completely change our understanding of NGF
physiology. Interest in NGF and in its receptors has in-
creased in the last decade [113, 114], due to the discovery of
the therapeutic potential of human-NGF [116] in neurode-
generative disorders [115, 116]. Furthermore, new develop-
ments in biotechnology facilitate the large-scale production
of highly purified, human NGF according to GMP standards
for industrial use [117, 118] and clinical purposes [119].
8. NGF RECEPTOR STRUCTURE AND SIGNALING
The biological functions of NGF are mediated through
two classes of cell surface receptors: the p75 neurotrophin
receptor (p75 NTR), common to all members of the neu-
Fig. (2). The chemical structures of the VEGFR2 inhibitors A-SU 5416; B-SU 11248; C-SU 6668.
N
H
O
N
H
CH3
CH3
A
N
H
O
N
H
CH3
B
F
H3C
O
N
H
N
CH3
CH3
N
H
O
N
H
CH3
C
H3C
COOH

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2614 Current Pharmaceutical Design, 2006, Vol. 12, No. 21Lazarovici et al.
rotrophin family, and the tropomyosine kinase related re-
ceptor (trkA), belonging to the tyrosine kinase-neurotrophin
receptor family [120]. p75 NTR is a member of the family of
tumor necrosis factor/cell death receptors and will not be
discussed in the present review. For details see the recent
excellent review by Kaplan and Miller [174]. TrkA was
originally discovered as a colon carcinoma oncogene, re-
sulting from the gene fusion of tropomyosin and trkA genes
resulting in a constitutively active receptor, composed of the
extracellular tropomyosin domain and intracellular trkA ty-
rosine kinase domain [121]. Later, the trkA proto-oncogene
was identified as the major, biologically relevant, receptor of
NGF [122, 123]. The 25 kb human trkA gene, localized like
NGF on chromosome 1 in band 1q21-q22 [124], is organized
into 17 exons [125]. An alternative splicing of exon 9 pro-
duces two isoforms [126]. The trk A gene encodes a mem-
brane-bound receptor molecule comprised of extracellular,
transmembrane and intracellular domains. The extracellular
domain contains two cysteine-rich regions interrupted by a
leucine-rich domain and two immunoglobulin-like regions
[127] involved in generation of NGF binding domain. Both
the transmembrane and the juxtamembrane domains are
critical for receptor signaling as evident from site-directed
mutagenesis studies [128, 129]. Upon NGF binding to the
extracellular domain, the intracellular tyrosine kinase region
is autophosphorylated due to trkA receptor occupancy and
dimerization [130]. Similar to the VEGFR2 signaling, trkA
receptor-mediated signal transduction is initiated by the
autophosphorylation of several tyrosine residues, viz Y490,
Y674/675, Y751, and Y785 [130].
The Y674/675 phosphorylation sites lie within the cata-
lytic domain of trkA [130]. Phosphorylation of Y490 is re-
quired for the association of trkA with SHC or other adaptor
proteins, such as FRS-2, and the subsequent activation of the
ras-MAPK signaling pathway. This primary signaling cas-
cade leads to the phosphorylation and activation of a variety
of cellular substrates and transcription factors involved in
every facet of NGF action [130]. Phosphorylated Y751 pro-
vides the binding site for the p85 subunit of inositol phos-
phate 3 kinase (IP3K), resulting in IP3K activation, which in
turn activates Akt (PkB) kinase promoting a phosphoryla-
tion-based downstream signaling cascade, which is essential
for cell survival [130]. Phosphorylation at Y785 enables the
activation of phospholipase Cγ, which is responsible for the
generation of inositol 3-phosphate and increase in intracel-
lular calcium [130]. All these signaling pathways coordi-
nately constitute the complex phosphorylation cascade in-
duced by NGF and are responsible for the majority of NGF’s
“neuronal effects” such as survival, proliferation, differen-
tiation, growth, and maturation [131].
TrkA receptors are expressed in neuroblastomas and me-
dulloblastomas, two of the pediatric tumors of the neuroec-
toderm [132]. Overexpression of trkA, which has also been
demonstrated in many non-neuronal neoplasms, such as
prostate, pancreas, tyroid, melanocytic, breast, ovary and
lung cancer, uniformly serves as a poor prognosis marker for
these diverse types of tumors [133-139]. Recent studies on
trkA signaling in these tumors suggest that trkA engagement
leads to the activation of a variety of intracellular pathways,
which include proteins encoded by proto-oncogenes and
tumor suppressor genes, most of which are essential for both
neuronal development and tumorogenesis [140]. Therefore,
we believe, that selective inhibitors of trkA will contribute to
the development of new therapeutic strategies against some
of the most aggressive tumors [141, 142], using a concept
similar to that for anti angiogenic therapy.
9. NEURONAL EFFECTS OF NGF
As discussed above, β-NGF belongs to a family of neu-
rotrophic factors that regulate the survival and differentiation
of neurons in the peripheral and central nervous systems
[99]. NGF is crucial for the survival and maintenance of sen-
sory and sympathetic neurons, and also affects septal cho-
linergic neurons in the brain [143] and neurosecretory chro-
maffin cells in the adrenal medulla [144]. The neurological
losses observed in knockout mice lacking either NGF or its
trkA receptor provided compelling evidence in support of the
concept that NGF is responsible for the survival of nocicep-
tive and thermoceptive sensory neurons as well as ganglionic
and certain brain neurons [145]. The critical role of NGF in
neuronal development has been extensively characterized.
Recent findings point to an unexpected diversity of biologi-
cal actions of NGF throughout adulthood and aging, in addi-
tion to its role during embryonic development [131]. Over
the last decade, a growing body of evidence suggests that
NGF signaling provides neuroprotective and repair functions
in physiological and pathological conditions of the nervous
system [131]. The recently expanded role of NGF in the non-
neuronal, peripheral tissues such as the immune system [146]
indicates a further, unexpected diversity of this important
ubiquitous growth factor. Specifically, there is an emerging
body of data suggesting that neurotrophins may act as novel
mediators of angiogenesis [147]. Several recent publications
(Table 2), focus on dissecting the mode of action of neu-
rotrophins in blood vessel development and remodeling
[148].
10. ANGIOGENIC EFFECTS OF NGF
During embryonic and fetal development there is a cross-
talk and interdependence between the vascular and the neu-
ronal systems [175]. According to prior dogma, blood vessel
development guides the development of the (peripheral)
nervous system. For example, the migration of sympathetic
(and sensory) neurons will occur along preformed vascular
beds, such as the aorta [176]. Similarly, in vivo experiments
indicate that neovascularization along an FGF-1 (acidic
FGF) treated, collagen-coated
(PTFE) fiber is accompanied/ de novo innervation of this
synthetic conduit [177]. More recent studies, suggests that
the interactions between the vascular and the nervous system
are truly bidirectional: the nervous system plays an important
role in the development of the embryonic cardiovascular
system. For example, sensory nerves determine the pattern of
arterial differentiation and blood vessel branching [149].
This is also supported by findings that mutations that disrupt
peripheral sensory neurons or Schwann cells prevent arterio-
genesis. Furthermore, in neurogenin1/neurogenin2 double
homozygous mutants lacking peripheral sensory neurons and
phox2b homozygous mutants lacking peripheral autonomic
nerves, resulting in neuronal disorganization, the alignment
of arteries with misrouted axons is still maintained [150].
These findings suggest that the nervous system might con-
polytetrafluoroethylene

Page 7

Angiogenic and Neurotrophic Effects of NGF and VEGFCurrent Pharmaceutical Design, 2006, Vol. 12, No. 21 2615
tribute to the control of embryonic vascularization, most
probably by a paracrine route involving neurotrophins such
as NGF. Reparative neovascularization in the adult occurs
mainly through capillary endothelial cells sprouting, en-
largement of existing arterioles by proliferation of endothe-
lial cells and smooth muscle cells (arteriogenesis) [178] and
de novo vascularization from circulating endothelial cell
precursors [151]. Recent studies, mostly in vitro but also in
vivo, provide mounting evidence for a direct involvement of
NGF in angiogenic processes, in addition to the well-
established role of VEGF and basic FGF in neovasculariza-
tion (Table 2). For example, NGF induces in vitro prolifera-
tion of umbilical cord, brain capillary, choroidal and dermal
microvasculature endothelial cells. NGF increases the migra-
tion of endothelial cells and upregulates expression of adhe-
sion proteins such as ICAM. The few studies in existence to-
date in animal models, indicate that NGF upregulates VEGF
production and induces, reparative neoangiogenesis, arterio-
genesis and wound-healing Whether these processes occur
directly via NGF-induced angio/arteriogenesis or indirectly
via the induction of classical “angiogenic factors” such as
VEGF, remains to be clarified. In any case, the involvement
of NGF-trkA receptor in proliferation of cultured endothelial
cells was clearly demonstrated by measuring the inhibitory
effect of K252a, a well known relative selective antagonist
of NGF actions [142, 164] (Fig 1). Other in vitro studies
have implicated MAPK signaling in the NGF-induced vas-
cular effects [153] and suggested that that NGF may play an
autocrine role in the endothelium [159, 160]. Thus, NGF
expression in human umbilical vein endothelial cells was
increased by serum deprivation suggesting that NGF up-
regulation may be part of an endothelial cell response to nu-
trient/trophic depletion. It is very tempting to suggest that
nutrient/trophic deprivation together with hypoxia may acti-
vate a cross-talk between epigenetic, autocrine (NGF pro-
duction by endothelium) and paracrine (VEGF production by
stromal or tumor cells) signals leading to endothelial cells
survival and proliferation. Thus NGF could represent a novel
pro-angiogenic candidate growth factor in the cardiovascular
system, in the embryonal nervous system, as well as during
inflammation and tumor growth.
11. K252A, A PROTOTYPIC SELECTIVE INHIBITOR
OF TRKA RECEPTOR TYROSINE KINASE ACTIV-
ITY
K252a, an indolecarbazole microbial derivative (Fig. 3),
was isolated from the culture broth of Nocardiopsis sp. and
characterized as a potent (1-25 nM), nonselective, in vitro
protein-kinase inhibitor, which acts by competing with the
ATP binding site in the catalytic domains of the different
protein kinases [142].
Fig. (3). Chemical structure of the trkA antagonist K252a.
K252a was also established in cellular studies as a rela-
tive specific and potent NGF antagonist [142, 164]. Once the
trkA proto-oncogene product gp140trk was identified as the
high affinity NGF receptor, further studies on the mechanism
of action of K252a revealed that this compound is a potent
antagonist of trkA tyrosine kinase activity, resulting in inhi-
bition of the majority of the cellular effects of NGF [142].
For example, in PC12 cells, K252a efficiently inhibited the
increase in NGF-induced c-fos oncogene transcription, as
well as the increase in intracellular calcium and the stimula-
Table 2.Angiogenic Effects of NGF in the Cardiovascular System
Cell typeOrigin EffectReferences
Umbilical vein endothelial cellsHumanProliferation[152]
Chorioallantoic membrane ChickNeoangiogenesis [152]
Superior cervical ganglia RatNeoangiogenesis [153]
Cornea RatNeovascularization, healing [154-156]
Skin ulcer of diabetes type I MiceReparative capillarization and accelerated wound closure [157]
Limb ischemia Mice Reparative neovascularization and arteriogenesis[158]
Aortic endothelial cellsMice Survival and maintenance [159]
Immortalized brain endothelial cells RatProliferation [160]
Eye choroidal endothelial cellsHumanMigration and proliferation[161]
Dermal microvascullar endothelial cellsHuman Proliferation and expression of ICAM[162]
ArteriesRat Regulation of VEGF [163]
H
N
O
N
N
O
HO
H
O
O

Page 8

2616 Current Pharmaceutical Design, 2006, Vol. 12, No. 21Lazarovici et al.
tion of the phosphorylation cascade resulting in neuronal
differentiation [164]. The highly selective inhibition by
K252a of neurotrophin action in cellular and animal studies
is in striking contrast to the nonselective inhibitory action of
this drug on different protein kinases in cell-free systems
[142]. K252a, in addition to being a selective antagonist of
trkA, has significant NGF-like effects and potentiates NGF
action. Therefore, K252a could be considered as a mixed
agonist/antagonist of NGF. To date, K252a has become a
widely used tool in biological research on trkA signal trans-
duction, cellular functions and the pathophysiology of neu-
rologic and neoplastic disorders.
Since trkA receptors were found to be overexpressed in a
variety of human tumors (representing a marker for poor
prognosis), K252a was used as a lead compound by Cepha-
lon company for the development of the closely related, anti-
cancer derivatives CEP-701 and CEP-751 (Fig. 4). These
drugs inhibit autophosphorylation and signaling of receptors
of the trk family at nanomolar concentration in vivo [165-
167]. CEP-701, which potently inhibited RET tyrosine
kinase in tyroid carcinoma [168] and FLT3 tyrosine kinase in
myeloid leukemia [169], was well tolerated in carcinoma
patients in a phase 1 clinical trial [170]. CEP-751 may be a
useful therapeutic agent for neuroblastoma, meduloblastoma
[171] and prostatic cancers [166, 167] by inhibiting trkA and
inducing tumor apoptosis. Similarly, CEP-2563, a lysinyl-
beta-alanyl ester of CEP-751 (Fig. 4), with increased solu-
bility, was proven safe in a recent phase 1 clinical trial in
patients with refractory solid tumors [172]. Interestingly,
novel CEP-derivatives, such as CEP 5214 and its dimethyl-
glycine ester, the prodrug clinical candidate CEP 7055 (Fig.
4) were developed as potent inhibitors of VEGFR2. Both
these compound showed promising anti-angiogenic activity
and anti-tumor efficacies in preclinical studies [89, 179].
Taken together, CEP drugs, by antagonizing NGF and/or
VEGF represent a novel family of promising chemothera-
peutic agents.
12. ANGIOGENIC AND NEUROTROPHIC EFFECTS
OF VEGF AND NGF: IMPLICATIONS IN DRUG DE-
VELOPMENT
Angiogenesis and neurogenesis are distinct yet, as we
know now, related biological processes that primarily occur
during embryonal development but continue to play an im-
portant physiological role postnatal. Upon injury, the com-
bined and concerted action of angiogenic factors, such as
VEGF, and neurotrophins, such as NGF, contribute to the
healing process of both nerves and blood vessels. When en-
dothelial cells are injured, e.g., by hypoxia, ischemia, or se-
rum starvation, and loose their contact inhibition, they begin
to proliferate, migrate, and in turn, produce/release angio-
genic and/or neurotrophic factors. These factors, either in an
autocrine or paracrine fashion, are partially responsible for
the cross talk between the cardiovascular and nervous system
(Fig. 5).
We believe that this crosstalk is instrumental for proper
neovascularization as well as inervation/regeneration of the
damaged tissue. During the past decade increasing evidence
suggests that at least two such regulatory molecules, VEGF
and NGF, possess reciprocal angiogenic and neurotrophic
effects on neurons and blood vessels (Fig. 5).(For these ac-
tivities they both utilize their respective tyrosine kinase re-
ceptors, viz, Flk and trkA, which show a much broader tissue
Fig. (4). The chemical structures of the trkA antagonists: A-K252a; B-CEP 701; C-CEP 751; D-CEP 2563; E-CEP 7055.
H
N
NN
O
OH
H
O
A
H
N
N
N
O
B
O
H3C
HO
OH
H
N
NN
O
C
O
H3C
H3C-O
OH
H
N
N
N
O
D
O
H3C
H3C-O
ON
H
OO
NH2
NH2
H
N
N
O
E
O
N
CH3
O
CH3
O
CH3
H3C

Page 9

Angiogenic and Neurotrophic Effects of NGF and VEGFCurrent Pharmaceutical Design, 2006, Vol. 12, No. 21 2617
distribution than hitherto assumed. Thus, insufficient protec-
tion by VEGF and NGF may represent novel etiologic
mechanisms of neuronal and vascular degeneration. Simi-
larly, there is growing evidence in tumor biology to suggest
that both NGF and VEGF may contribute to tumor angio-
genesis and cancer cell growth. The increased knowledge
about the role of trkA and Flk in the etiology of and in the
pathogenic pathways involved in tumor development has
enabled the implementation of novel more specific therapeu-
tic approaches based on the inhibition of tyrosine kinase re-
ceptors, as opposed to the classical, systemic use of nonspe-
cific anticancer drugs. Lead compounds, such as SU-5416
and K252a, and their drug derivatives SU-11248 and CEP-
2563 (Figs. 2 and 4) that potently and relative selectively
inhibit VEGF and NGF, are currently a major focus in the
development of newer therapeutic modalities in oncology.
Besides their therapeutic utility, these compounds may be
also pan out as important pharmacological tools in investi-
gating the role of NGF in the cardiovascular system and that
of VEGF in the nervous system. The major pharmaceutical
challenges in this field are to elucidate the molecular mecha-
nisms of these promising drugs and to design new deriva-
tives which retain their anti-tumor selectivity but lack side
effects on the cardiovascular or neuronal systems. We fully
anticipate that the research on VEGF and NGF will continue
to contribute to our understanding of the role of these two
growth factors in both ontogenesis and oncogenesis and with
that further bridge the gap between cancer and neurology.
ACKNOWLEDGMENTS
PIL and PL gratefully acknowledge the generous support
by the Louis and Bessie Stein Foundation and by Drexel
University. Part of this work was also supported by a grants-
in aid from NASA (NNC9-130 and NNJ04HC81G to PIL).
We thank Mr. Shimon Lecht (Hebrew University, Jerusalem)
and Mr. Ilya Rakhman (Drexel University, Philadelphia) for
their help with preparing the figures
Fig. (5). Schematic of the cross talk between the cardiovascular (left) and nervous system (right). The mediators of this cross-talk are NGF
and VEGF that specifically interact with their receptors trkA and flk, respectively. Selective inhibitors od trkA and flk are indicated as
angiostatic and anti-tumoral compounds.

Page 10

2618 Current Pharmaceutical Design, 2006, Vol. 12, No. 21 Lazarovici et al.
ABBREVIATIONS
NGF
VEGF
VEGFR
TrkA
= Nerve growth factor
= Vascular endothelial growth factor
= VEGF receptor
= Tropomyosine related kinase type A -
NGF receptor
= VEGFR 2
= trkA antagonist
= flk1 antagonist
= Focal adhesion kinase
= Protein kinase C
= Phospholipase C
= Nitric oxide
= Prostaglandin I
= Molecular weight
= Kilodalton
Flk1
K252a
SU5416
FAK
PKC
PLC
NO
PGI
MW
kD
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