Download The heparin-binding domain confers diverse functions of VEGF

Molecular and Cellular Mechanisms of Angiogenesis
The heparin-binding domain confers diverse
functions of VEGF-A in development and disease:
a structure–function study
Dominik Krilleke, Yin-Shan Eric Ng and David T. Shima1
Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, 11–43 Bath Street, London EC1V 9EL, U.K.
Abstract
The longer splice isoforms of VEGF (vascular endothelial growth factor)-A, including VEGF164(165) , contain a
highly basic HBD (heparin-binding domain). This domain allows these isoforms to interact with and localize
to the HS (heparan sulfate)-rich extracellular matrix, and bind to the co-receptor Nrp-1 (neuropilin-1).
Heparin-binding VEGF-A isoforms are critical for survival: mice engineered to express exclusively the
non-heparin-binding VEGF120 have diminished vascular branching during embryonic development and die
from postnatal angiogenesis defects shortly after birth. Although it is thought that the HBD contributes to
the diverse functions of VEGF-A in both physiological and pathological processes, little is known about the
molecular features within this domain that enable these functions. In the present paper, we discuss the roles
of the VEGF HBD in normal and disease conditions, with a particular focus on the VEGF164(165) isoform.
Introduction
VEGF (vascular endothelial growth factor)-A is the most
prominent member of the PDGF (platelet-derived growth
factor)/VEGF family of secreted dimeric growth factors, a
family that consists of PlGF (placental growth factor),
VEGF-B, VEGF-C, VEGF-D and the virus-derived VEGFE variants [1,2]. PDGF/VEGF growth factors are pivotal in
regulating vascular development in the embryo, as well as the
formation of new blood vessels and vessel homoeostasis in
the adult [3]. VEGF-A is a potent chemoattractant for monocytes, a cell type implicated in pathological angiogenesis,
and can activate and induce monocyte chemotaxis across endothelial cell monolayers [4]. Cellular responses to VEGF are
primarily mediated by binding to VEGFR (VEGF receptor)1 [Flt-1 (Fms-like tyrosine kinase 1)] and VEGFR-2 [KDR
(kinase insert domain receptor)/Flk-1 (fetal liver kinase 1)]
[5], two structurally similar receptor tyrosine kinases predominantly expressed on vascular endothelial cells [6,7]. There
is abundant evidence for unique receptor functions, and
co-receptors such as Nrps (neuropilins) and HSPGs (heparan
sulfate proteoglycans) can also modulate VEGF-A functions.
VEGF exists as multiple biochemically distinct protein
isoforms which differ mainly by their ability to interact
with Nrps and HSPGs. The isoforms are produced as a
result of alternative mRNA splicing of a common transcript from a single Vegf gene [8]. In humans, although
multiple spliced isoforms have been identified, the most
Key words: cancer, heparan sulfate proteoglycan, heparin-binding domain, leukostasis, retinal
disorder, vascular endothelial growth factor-A (VEGF-A), vascular endothelial growth factor
receptor (VEGFR).
Abbreviations used: DR, diabetic retinopathy; HBD, heparin-binding domain; HS, heparan
sulfate; HSPG, HS proteoglycan; ICAM-1, intercellular adhesion molecule-1; Nrp, neuropilin;
PDGF, platelet-derived growth factor; ROP, retinopathy of prematurity; VEGF, vascular endothelial
growth factor; VEGFR, VEGF receptor.
1
To whom correspondence should be addressed (email [email protected]).
Biochem. Soc. Trans. (2009) 37, 1201–1206; doi:10.1042/BST0371201
common and well-studied isoforms are composed of 121, 165
and 189 amino acids, and the murine homologues lack one
amino acid per isoform. All VEGF isoforms share the same
N-terminal amino acid sequence, which contains the binding
sites for VEGFR-1 and VEGFR-2, but they may or may
not contain sequences encoded by exons 6 and 7 in the
C-terminus (Figure 1). This C-terminal region encodes two
HBDs (heparin-binding domains), each of which confers
the ability to bind HS (heparan sulfate) and HSPGs on cell
surfaces and basement membranes, thus determining the
localization of the VEGF-A isoforms in the extracellular
space [9,10]. VEGF121 , the shortest isoform, lacks an HBD
and is freely diffusible upon secretion. In contrast, the longer
splice form VEGF189 has two HBDs encoded by exon 6
and exon 7, and is almost completely sequestered in the
extracellular matrix and on the cell surface [9,11].
VEGF165 , the prototypic and most abundant VEGF isoform, differs from VEGF121 by the inclusion of a basic peptide
encompassing 44 residues encoded by exon 7. The moderate
affinity for heparin enables VEGF165 to act as both a soluble
and a cell-bound factor. This region also contains binding
determinants for Nrp-1, a VEGF165 isoform-specific coreceptor that has been shown to be expressed on endothelial
cells and certain tumour cells [12]. Nrp-1 is thought to
associate with VEGFR-2 upon VEGF165 binding, enhancing
VEGFR-2 activity and signalling [13]. The exact roles of
Nrp-1 in VEGF-A function are poorly understood, but there
is a growing body of evidence that Nrp-1 may participate in
VEGF-A signalling independent of VEGFR-2 [14,15].
Modulation of VEGF-A function by HSPG
The mechanisms by which HSPGs and certain GAGs
(glycosaminoglycans) regulate the activity of heparin-binding
VEGF-A isoforms have proven to be complex, as these matrix
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Figure 1 Human VEGF-A isoforms
Schematic representation of the three major VEGF isoforms depicted as monomers. The eight exons of the human VEGF-A
are alternatively spliced to produce several distinct isoforms that differ in the presence or absence of exon 6 and 7, the
HBDs. Binding sites for heparin and the different receptors are indicated. Arrowhead, plasmin cleavage site. Exons are not
drawn to scale.
molecules can bind to both VEGF-A and its receptors [16].
In the presence of HSPGs, VEGF165 , VEGFR-2 and Nrp-1
are thought to form an as-yet undefined signalling complex
through intermediate HS molecules [13,17]. The observed
regulatory effects of heparin or HSPGs on receptor binding
and activation by VEGF are affected by various experimental
factors [18]. Whereas heparin strongly potentiates 125 Ilabelled VEGF165 binding to VEGFR-2 in a cell-free assay,
the equivalent amount of heparin inhibits the binding to
NIH 3T3 cells overexpressing VEGFR-2 [19]. Heparin
augments VEGF-A binding to cells in which VEGFR-2 is
endogenously expressed [20]. In the case of VEGFR-1, both
VEGF121 and VEGF165 binding are reduced by exogenous
heparin [21,22]. Furthermore, binding to heparin also
increases the stability of VEGF-A, thereby controlling
VEGF-A activities by modulating its bioavailability and
protein half-life [23]. Further study will be needed to clarify
the effects of HSPGs on VEGF-A signalling activity.
in vitro, is not sufficient to drive extension of endothelial tip
cell filopodia and proper vascular branching. Normal vessel
architecture is fully restored, however, in mice engineered to
solely express VEGF164 (Vegf 164/164 ) or a combination of both
VEGF120 and VEGF188 (Vegf 120/188 ) [26,27]. Thus VEGF164
appears to be sufficient for normal vascular development, but
VEGF120 , which lacks certain functional properties provided
by the HBD in the longer isoforms, is not. The Vegf 120/120
mice also exhibit specific defects in developmental bone vascularization [28] and in postnatal angiogenesis of the myocardium [25], embryonic retina [27] and kidney glomerulus [29].
VEGF164 has previously been shown to participate in
neuronal patterning in vivo. Using mouse genetics and
explant cultures, Schwarz et al. [30] showed that VEGF164
is both necessary and sufficient for the correct migration of
facial motor neuron somata by acting as a guidance cue for
Nrp-1-expressing motor neurons. This study confirms the
importance of the HBD in VEGF-A-mediated vascular and
neuronal development.
Role of heparin-binding VEGF isoforms
in normal development: lessons from
mouse studies
Role of heparin-binding VEGF-A isoforms
in cancer
Bioavailability and spatial distribution of VEGF-A can be
controlled via production of both soluble and matrix-bound
isoforms. A finely balanced VEGF-A concentration gradient
consisting of isoforms with differential heparin-binding
affinities is necessary for proper blood vessel branching morphogenesis in mice [24]. In the Vegf 120/120 mice, which were
generated by targeted deletion of exons 6 and 7 of the
vegf gene and consequently are devoid of heparin-binding VEGF-A variants [25], the naturally occurring chemotactic VEGF-A gradient and matrix-derived chemoattractive
signals of VEGF-A are lacking. As a consequence, migration
of sprouting vessel tips is defective, branching complexity is
decreased and capillaries are enlarged compared with wildtype animals [24]. VEGF120 , despite showing similar potency
to VEGF164 in promoting endothelial cell proliferation
Deregulated VEGF-A expression contributes to the development of solid tumours by promoting tumour angiogenesis
[31]. The pathophysiological roles of the different VEGF-A
isoforms appear to be affected significantly by the microenvironment, such as expression of VEGF-A receptors and
extracellular matrix components at different anatomical sites
of tumours [32]. Various studies have shown that soluble
VEGF121 is less effective than heparin-binding VEGF-A
isoforms in supporting the neovascularization needed for
tumour survival. For example, human glioma cells overexpressing VEGF121 fail to induce tumour progression when
implanted in the subcutaneous space in mice, whereas
overexpression of VEGF165 and VEGF189 strongly augments
neovascularization and tumour growth relative to parental
cells [32]. VEGF120 is also incapable of supporting maximal
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Authors Journal compilation Molecular and Cellular Mechanisms of Angiogenesis
Figure 2 Structural fold of the VEGF165 HBD
(A) Ribbon diagram with secondary structure elements of the 55-residue HBD (residues 111–165 of VEGF165 ). The cysteine
residues shown are all involved in the formation of disulfide bonds. Charged residues are colour-coded, with basic and acidic
amino acids depicted in blue and in red respectively. (B) Surface topology model showing the distribution of electrostatic
potential and potential heparin-binding sites on the surface. The structural model is shown at two angles rotated by 180◦
about the vertical axis. The diagram was produced using PyMOL software (DeLano Scientific; http://pymol.sourceforge.net/)
based on published NMR data [49] (Protein Data Bank code 1KMX).
growth of tumour cells in mice receiving allografts of VEGFA-null embryonic fibroblasts stably expressing individual
VEGF isoforms [33]. In this study, the diffusible VEGF120
recruited peripheral vessels, but was incapable of vascularizing the tumour itself. In contrast, VEGF164 induced both
internal and external tumour vascularization, fully rescuing
growth of the angiogenesis-deficient parental tumour cells.
Furthermore, heparin-binding isoforms may confer a growth
advantage in certain human tumours, as altered VEGF-A
expression patterns in favour of the longer isoforms have
been associated with tumorigenesis and poorer outcomes
in certain cancers [34,35]. These data suggest that heparinbinding VEGF-A isoforms may be particularly important in
tumour angiogenesis, and that isoform-specific antagonists
may specifically target tumour angiogenesis [33,36].
Intraocular disease and the predominant
role of the VEGF165(164) isoform
Many studies suggest a causal role of VEGF-A activity in
proliferative neovascular pathologies of the eye [37]. In DR
(diabetic retinopathy), ROP (retinopathy of prematurity) and
the wet form of AMD (age-related macular degeneration),
abnormal VEGF-A expression causes uncontrolled neovascular growth and promotes vascular haemorrhages and leakage, conditions that eventually lead to irreversible retinal
damage and blindness [37]. During pathological proliferative
retinopathy, hypoxia-induced VEGF-A production by
ischaemic retinal cells results in aberrant vessel growth that
can extend beyond the retinal surface into the vitreous
cavity. Among the various isoforms, VEGF164 appears to be
preferentially involved in pathological retinal neovasculariz-
ation. This isoform selectively accumulates in the ischaemic
retina in a commonly used animal model of ROP [26,38].
Indeed, VEGF164 isoform-specific inhibition in this model
has been shown to be as effective as pan-VEGF-A blockade
in preventing neovascularization [26,38]. Although VEGF120
and VEGF164 are equally potent at rescuing retinal neuronal
cell apoptosis when injected intraocularly after ischaemic
injury, VEGF164 causes oedema and haemorrhage that is
not detected in retinas treated with VEGF120 [39]. Taken
together, these studies strongly suggest a pathological role
for heparin-binding VEGF164 in retinal disorders.
The mechanism by which VEGF164 exerts its pathological
function in ischaemic retinal disease may be related to the proinflammatory actions of VEGF-A in the presence of the
HBD [26]. VEGF164 is likely to be the most pro-inflammatory
isoform in the eye, capable of inducing leucocyte recruitment
(leukostasis) to the limbal and retinal vascular endothelium
[26,40]. On an equimolar basis, VEGF164 more potently induces retinal leukostasis and corneal inflammation than does
VEGF120 [40,41]. A comparison of VEGF120 with VEGF164
demonstrated that VEGF164 more potently induces both expression of ICAM-1 (intercellular adhesion molecule-1) and
translocation of P-selectin to the surface of HUVECs (human
umbilical vein endothelial cells) [40]. Both ICAM-1 and
P-selectin are important mediators of vascular inflammation.
In the retina, increased levels of VEGF-A and ICAM-1 have
been shown to coincide with elevated leucocyte counts during
experimental diabetes, with the VEGF164 isoform accounting
for 80 % of total VEGF-A detected [42,43]. VEGF-A may
also act directly on certain cells of the immune system,
including monocytes, lymphocytes and dendritic cells, as
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these cells have been shown to express active VEGF-A receptors [44,45]. Retinal inflammation and vessel leakage in both
early and established diabetes can be significantly reduced
when VEGF164 is inhibited by an isoform-specific aptamer
antagonist [41]. This is not surprising, given that VEGF165(164)
expression predominates over other isoforms in patients with
DR and in an animal model of this disease [43,46]. Taken
together, these observations suggest that VEGF165(164) is proinflammatory and the major pathological isoform in models
of neovascular eye disease and in human DR, although the
exact mechanistic roles of the HBD remain to be determined.
Molecular characterization of the
VEGF164 HBD
The differential functions of the VEGF isoforms suggest
that the HBD plays a critical role in modulating VEGF-A
isoform activity. Neither the sequence nor the structure
of this domain (residues 111–165 of VEGF165 ) bears any
similarity to known heparin-binding proteins outside the
VEGF family of growth factors [47]. The HBD comprises
55 residues, with clearly defined N-terminal and C-terminal
subdomains (residues 1–29 and 29–55 respectively), each
containing a two-stranded, antiparallel, β-sheet and two
disulfide bonds which dominate the fold. In addition, a
single α-helix is located in the C-terminal subdomain,
where it is packed against the β-sheet. The charge of this
overall highly basic domain (pI ∼11) is distributed rather
unevenly across the surface, with a surplus of positively or
negatively charged amino acids on each side of the domain
(Figure 2). The loop region at the interface of the two
subdomains (residues 11–17) and the relative orientation
of the subdomains within the HBD remain poorly defined.
The increased flexibility of this domain compared with the
rest of the protein complicates accurate prediction of heparinbinding sites within the HBD. Using a mutagenesis approach
to identify residues that are critical for mediating heparin
binding, a principal heparin-binding site consisting of Arg13 ,
Arg14 and Arg49 (corresponding to Arg123 , Arg124 and Arg159
respectively of VEGF165 ) has recently been proposed [48].
The residues, although non-contiguous in sequence, are
located along the interface of the clearly defined N-terminal
and C-terminal subdomains, where they form a continuous
binding surface. This model is supported by a refined NMR
solution structure showing that the heparin-binding residues
are in close contact with each other [49]. Spatial proximity
of heparin-binding residues in a three-dimensional context
is characteristic for many heparin-binding proteins [50].
Mutant VEGF164 variants in which Arg13 , Arg14 or Arg49
have been replaced with alanine are severely compromised
in their ability to bind to heparin and HS/HSPGs, although
they retain wild type-like potency in inducing endothelial
gene expression in vitro and angiogenesis in an ex vivo
aortic ring assay (Figure 3) [48]. Binding to Nrp-1 was only
minimally reduced, so these mutants might provide a tool to
help differentiate between Nrp-1 and HS/HSPG effects on
VEGF-A signalling. The VEGF164 mutants exhibited similar
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Authors Journal compilation Figure 3 VEGF164 heparin-binding deficient mutants display
wild-type-like capacity in inducing angiogenesis ex vivo
(A) The VEGF164 HBD mutants do not bind heparin. [3 H]Heparin was
incubated with increasing concentrations of VEGF variants in the presence
of 0.15 M NaCl for 1 h, after which VEGF–heparin complexes were
recovered using a nitrocellulose-binding assay. VEGF-bound [3 H]heparin
was quantified in a liquid-scintillation counter. The GraphPad Prism
program was used to generate a logarithmic curve fit according to the
one-site binding model and to calculate the dissociation constants.
The mutants were generated by arginine-to-alanine mutations of the
indicated amino acids of the proposed heparin-binding site. (B) Left-hand
panel: freshly prepared rat aortic rings were incubated with serum-free
medium in the presence of equimolar concentrations of native VEGF
isoforms or selected VEGF164 heparin-binding-deficient mutants for
7 days, and microvessel sprouts were stained with Isolectin B4.
Right-hand panel: representative images of aorta explants captured using epifluorescence microscopy (magnification ×5). Blood vessels were
traced for quantification using Photoshop (Adobe). Adapted from [48].
affinity towards VEGFR-2 [48]. These results suggest that
activation of VEGFR-2 is responsible for the proliferative activities of VEGF-A on endothelial cells, and that the HBD
does not play a direct role in VEGFR-2 activation. However,
Molecular and Cellular Mechanisms of Angiogenesis
the mutants exhibited a reduced binding affinity for VEGFR1, which is also expressed by leucocytes and mediates VEGFinduced migration [44]. The increased affinity of VEGF164
for VEGFR-1 relative to VEGF120 has previously been linked
to the presence of the HBD [10], and the data from the HBD
mutants confirm such a functional connection. It is possible
that the HBD contributes to the binding energy of the
VEGFR-1–VEGF-A interaction. VEGF-A, in particular the
isoforms containing the HBD, may act as a chemoattractant
for inflammatory cells by ligating and stimulating VEGFR-1.
As a result, elevated local VEGF164(165) concentrations
may provide recruitment signals for VEGFR-1-expressing
inflammatory cells. It will be interesting to see whether
the non-heparin-binding VEGF164 HBD mutants, which
bind to VEGFR-1 with lower affinity, have reduced proinflammatory activities compared with wild-type VEGF164
and activities similar to VEGF120 in various disease models.
Conclusions
The studies described above highlight the importance of
understanding the specific contributions and interplay
of the different VEGF-A isoforms in normal physiology and
in vascular pathologies. The pathobiology of the VEGF-A
HBD remains a matter of considerable interest, and there
is growing evidence that the heparin-binding isoforms have
additional functions beyond the endothelium, influencing
neural and immune cells. The HBD may represent a suitable
therapeutic target for pathologies associated with VEGF165
overexpression. Future studies should address the mechanism
of action of the HBD in VEGF-A-induced pathologies, and
the feasibility of targeting the pathological functions of the
HBD for more effective and safer anti-VEGF-A therapy.
Acknowledgements
We thank Richard Foxton and Anne Goodwin for critical reading of
the manuscript.
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Received 7 July 2009
doi:10.1042/BST0371201