The role of heparan sulphate proteoglycans in angiogenesis.

Abstract

The presence of HS (heparan sulphate) proteoglycans on the cell surface and in the extracellular environment is critical to many physiological processes including the growth of new blood vessels from pre-existing vasculature (angiogenesis). A plethora of growth factors and their receptors, extracellular matrix molecules and enzymes bind to specific sites on the HS sugar chain. For example, HS proteoglycans have profound effects on the bioactivity of the key angiogenic factor VEGF (vascular endothelial growth factor) (VEGF(165)), affecting its diffusion, half-life and interaction with its tyrosine kinase receptors. A number of HS structural features that mediate the specific binding of VEGF(165), including sulphation requirements, have been determined. In parallel, zebrafish embryos were used as a vertebrate model system to study the role in vascular development of the biosynthetic enzymes that create these specific binding sites on HS. It was discovered that knockdown of one of the HS 6-O-sulphotransferases in zebrafish with morpholino antisense oligonucleotides reduced vascular branching and corresponded to changes in the HS structure. The roles of the extracellular 6-O-sulphatase enzymes, the sulfs, in vascular development are now being investigated. Both oligosaccharides and small molecule biosynthetic enzyme inhibitors could be valuable HS-based strategies for controlling aberrant angiogenesis in diseases as diverse as cancer and heart disease.

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Cytokine–Proteoglycan Interactions451
The role of heparan sulphate proteoglycans
in angiogenesis
S.E. Stringer1
Division of Cardiovascular and Endocrine Sciences, School of Medicine, University of Manchester, Core Technologies Facility, Grafton Street,
Manchester M13 9NT, U.K.
Abstract
ThepresenceofHS(heparansulphate)proteoglycansonthecellsurfaceandintheextracellularenvironment
is critical to many physiological processes including the growth of new blood vessels from pre-existing
vasculature (angiogenesis). A plethora of growth factors and their receptors, extracellular matrix molecules
and enzymes bind to specific sites on the HS sugar chain. For example, HS proteoglycans have profound
effects on the bioactivity of the key angiogenic factor VEGF (vascular endothelial growth factor) (VEGF165),
affecting its diffusion, half-life and interaction with its tyrosine kinase receptors. A number of HS structural
features that mediate the specific binding of VEGF165, including sulphation requirements, have been deter-
mined. In parallel, zebrafish embryos were used as a vertebrate model system to study the role in vascular
development of the biosynthetic enzymes that create these specific binding sites on HS. It was discovered
that knockdown of one of the HS 6-O-sulphotransferases in zebrafish with morpholino antisense oligo-
nucleotides reduced vascular branching and corresponded to changes in the HS structure. The roles of the
extracellular 6-O-sulphatase enzymes, the sulfs, in vascular development are now being investigated. Both
oligosaccharides and small molecule biosynthetic enzyme inhibitors could be valuable HS-based strategies
for controlling aberrant angiogenesis in diseases as diverse as cancer and heart disease.
Angiogenesis
Angiogenesisisthetightlycontrolledfundamentalprocessby
which new blood vessels arise from pre-existing vasculature
[1,2]. It is of immense clinical interest as promoting angio-
genesis could bypass blocked coronary arteries or aid the
complications of diabetes, whereas inhibition of angiogenesis
starves tumours of their essential blood supply.
Angiogenesisisamulti-stepprocess.Localizedbreakdown
of the basement membrane and extracellular matrix precedes
the proliferation and migration of capillary endothelial cells
into the surrounding tissue, and formation of new vessels.
Angiogenesis contributes to development of the cardio-
vascular system in vertebrate embryos and is essential in
adults for wound healing and the female reproductive cycle.
Theswitchfromnormalquiescentvasculaturetoangiogenesis
is induced by a change in the balance of many pro- and anti-
angiogenic factors that are released predominantly by sur-
roundingpericytesandlymphocytes.Alargenumberofthese
factors have been found to bind to HS (heparan sulphate)
proteoglycans or the structurally related heparin, including
key angiogenic growth factors such as the FGFs (fibroblast
growthfactors)andVEGFs(vascularendothelialgrowthfac-
tors) [3]. However, the only growth factor observed almost
ubiquitously at sites of angiogenesis and whose levels cor-
Key words: angiogenesis, heparan sulphate, K5 lyase, proteoglycan, vascular endothelial growth
factor (VEGF), zebrafish.
Abbreviations used: FGF, fibroblast growth factor; GlcA, d-glucuronate; GlcNAc, N-acetyl-d-
glucosamine; HS, heparan sulphate; HS6ST, HS 6-O-sulphotransferase; IdoA, l-iduronate; S-
domain, N-sulphated domain; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.
1email sally.stringer@manchester.ac.uk
relate most closely with the spatial and temporal events of
blood vessel growth is VEGF A [4].
Roles of VEGF in angiogenesis
VEGFisapotentstimulatorofangiogenesisbothinvitroand
in vivo (for a review, see [4,5]). Inactivation of even a single
VEGF allele results in embryonic lethality due to abnormal
blood vessel development. VEGF is an almost ubiquitous
initiator of tumour angiogenesis. Its expression is potentiated
by hypoxia, by activated oncogenes, and by a variety of cyto-
kines. VEGF is able to regulate most, if not all, steps in the
angiogeniccascade;itregulatesproteinexpression,migration,
division and apoptosis of endothelial cells. VEGF interacts
with two type III receptor tyrosine kinases, VEGFR-1
(VEGF receptor-1; flt-1) and VEGFR-2 [flk-1/KDR (kinase
insert domain-containing receptor)], and with the isoform-
specific receptors, the neuropilins. In contrast with all other
known angiogenic factors, the proliferative action of VEGF
seems to be restricted almost entirely to endothelial cells,
making it an excellent agent or target for angiogenesis-based
therapies.
Interaction of VEGF with HS proteoglycans
HS proteoglycans are ubiquitous components of the cell sur-
face (syndecans and glypicans) and extracellular matrix (e.g.
Perlecan)[6].Theyconsistofaproteincorewithbetweenone
andfourpolysaccharidechains,whichcanbetransmembrane,
GPI (glycosylphosphatidylinositol)-anchored or embedded
in the matrix. It is mainly through the HS polysaccharide
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452Biochemical Society Transactions (2006) Volume 34, part 3
chains that they can bind to a variety of protein ligands, in-
cluding growth factors, growth factor receptors and serine
proteases. They have the ability to mediate angiogenesis
driven by a number of growth factors, including all but one
splicevariantofVEGF[7,8].Anumberofinvivoandinvitro
studies have examined the roles of HS proteoglycans in
VEGF activity: most importantly, HS seems to be essential to
spatially restrict VEGF to create the appropriate gradient
to allow blood vessel branching to occur [8]. HS proteo-
glycans can also re-activate oxidation-damaged forms of
VEGF,afunctionthatmaybeessentialinhypoxicsiteswhere
new vessels are required [9]. They potentiate VEGF binding
to its two specific signal-transducing receptors [10] and may
interact directly with the receptors [11–13] analogous to the
widely studied HS–FGF-2–FGF receptor signalling complex
[14,15].
Analysis of the binding site on HS for VEGF
The HS polysaccharide chain is initially synthesized as an
alternating polymer of GlcA (D-glucuronate) and GlcNAc
(N-acetyl-D-glucosamine).PartialN-sulphationcreatesthree
types of domains within the chain: blocks of N-sulphated
disaccharides[S-domains(N-sulphateddomains)]areflanked
by alternating regions of N-acetylated and N-sulphated
disaccharides and these sulphated areas are separated by N-
acetylatedstretches.Furtherstructuralvariabilityisgenerated
by O-sulphation of some of the disaccharides within the
S-domains and alternating regions. The fine structure and
spacing of these S-domains appears to be the major deter-
minant of specific binding to protein ligands (for reviews of
HS, see [6,16]).
The binding site on HS for the common, most potent
VEGF splice variant, VEGF165, has recently been analysed
[17]. The types of domains required for VEGF165 binding
wereassessedusingstructurallydistinctfragmentscreatedby
the enzymes heparanase I, heparinase III, platelet heparanase
and the phage-derived enzyme K5 lyase. Unlike members
of the FGF family [18], VEGF165did not bind strongly to
single S-domains isolated using heparinase III. In fact the
onlyfragmentswithstrongbindingwerethosecreatedbyK5
lyase. This enzyme cleaves within the N-acetylated stretches
of HS, leaving the S-domains and alternating regions intact.
The minimum size of K5 lyase fragment with good VEGF
binding was 22 saccharides. Modelling studies indicated that
a single heparin-binding site on VEGF165 could be filled
with a heptasaccharide. Therefore the 22+ oligosaccharides
could probably stretch across both of the heparin-binding
domains within the VEGF dimer and may consist of two
S-domains separated by an alternating region (Figure 1).
The sulphate dependency of HS binding to VEGF165was
also investigated [17]. Binding of specifically desulphated
HS to VEGF165suggested that N-, 2-O- and 6-O-sulphated
disaccharides all contributed to the interaction. However,
experiments where octasaccharide S-domains were bound to
a single VEGF site at sub-physiological salt concentrations
demonstrated that 6-O-sulphation was essential. This raised
Figure 1
oligosaccharide binding to the VEGF165dimer
Results from Robinson et al. [17] indicate that K5 lyase-released
oligosaccharides with strong binding to VEGF165are of sufficient length
to interact with both VEGF heparin-binding sites. These oligosaccharides
may contain two sulphated domains of at least seven sufficiently
sulphated saccharides to fill each binding site, appropriately spaced by a
short alternating region.
Hypothetical model of a K5 lyase excised
the question as to whether one or more of the enzymes that
6-O-sulphate HS may be critical for VEGF-mediated angio-
genesis.
Role of HS biosynthetic enzymes
in angiogenesis
Elevenfamiliesofenzymesarerequiredtocreatethecomplex
structure of HS (for a review, see [16]). Subsequent to chain
initiation on serine residues in the proteoglycan protein
core, the GlcNAc-GlcA bipolymer is synthesized. Partial N-
sulphationoccursbytheN-deacetylase/N-sulphotransferase
enzyme family followed by conversion of some of the GlcA
into IdoA (L-iduronate) within the S-domains. A single
enzyme adds 2-O-sulphate groups to some of the IdoAs
and GlcAs, whereas multigene families are responsible for 6-
O-sulphation and 3-O-sulphation of the glucosamines. The
incomplete action of these enzymes on the HS chain results
in its structural diversity.
Inrecentyears,reportsofmutationsintheHSbiosynthetic
enzymes in animal model systems have given important in-
sights into the roles of HS in vivo. Mutations that completely
preventedformationofHSaffectedmanydevelopmentalpro-
cesses. For example, mutation in the gene required for
production of the UDP-glucuronic acid precursor to HS
was found to prevent initiation of heart valve formation in
zebrafish [19], and was critical for patterning mediated by
Wingless (Wnt family member) and Decapentaplegic (related
to the bone morphogenetic proteins) in Drosophila [20–22].
Strikingly, it was found that fine modification of HS-
controlled specific physiological processes, for example 2-
O-sulphation, is important for kidney development [23], and
6-O-sulphationofHSisessentialforformationofthetracheal
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Cytokine–Proteoglycan Interactions453
system in Drosophila [24], which in many ways is analogous
to the vasculature in vertebrates. These findings suggested
the exciting possibility that fine-tuning of HS structure, by
influencing the activity of specific biosynthetic enzymes,
could be used to control individual physiological processes.
TheroleofHS6STs(HS6-O-sulphotransferases)inangio-
genesis was investigated in zebrafish embryos [25]. Zebrafish
are an excellent model system in which to study angiogenesis
due to their rapid development, ease of genetic manipulation
and comparability of vascular development to mammals
[26,27]. Morpholino antisense oligonucleotides were used
to individually block the translation of two of the four
HS6STs found in zebrafish and the decreased level of HS
6-O-sulphation was confirmed by a sensitive HPLC assay.
Reduction in expression of HS6ST-2 but not HS6ST-1 de-
creased vascular branching in the caudal vein plexus of the
zebrafish embryos, a paradigm for remodelling stages of
angiogenesis. The activity of the HS6ST-2 was thus shown
to be important for VEGF-mediated angiogenesis.
Role of sulfs in angiogenesis
In the last couple of years, a novel family of enzymes called
sulfs, which remove 6-O-sulphation from HS, were dis-
covered by Charles Emerson’s group [28]. These are thought
to fine-tune the structure of HS at the cell surface and were
initially found to promote Wnt activity in quail [29]. Sub-
sequent work using the chick chorioallantoic membrane
assay demonstrated that sulf1 activity could block FGF2-
mediated angiogenesis [30]. There are three sulf enzymes in
zebrafish. Their expression patterns and activities, especially
with respect to angiogenesis, are being investigated (S.E.
Stringer, unpublished work).
Discussion
The studies discussed here have demonstrated that HS 6-O-
sulphation is important both for VEGF binding and VEGF-
mediated angiogenesis in zebrafish. Inhibition of HS6STs or
the sulf enzymes could be a valuable way of preventing
orpromotingangiogenesisrespectively.Promisingresultsare
already emerging from groups screening for small molecules
that affect HS enzyme activity, while others are investigating
the use of specific oligosaccharides to control HS activity.
Such compounds may have clinical potential in diseases as
diverse as heart disease and cancer.
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