528
Endothelial growth factors and their receptors may provide
important therapeutic tools for the treatment of pathological
conditions characterised by defective or aberrant
angiogenesis. Vascular endothelial growth factor (VEGF) is
pivotal for vasculogenesis and for angiogenesis in normal and
pathological conditions. VEGF-B and VEGF-C provide this
gene family with additional functions, for example, VEGF-C
also regulates lymphangiogenesis.
Addresses
*Ludwig Institute for Cancer Research, Box 240, SE-171 77
Stockholm, Sweden
†
Molecular/Cancer Biology Laboratory, Haartman Institute, PO Box 21
(Haartmaninkatu 3), University of Helsinki, SF-00014 Helsinki, Finland
‡
e-mail: Kari.Alitalo@helsinki.fi
Current Opinion in Biotechnology 1999, 10:528–535
0958-1669/99/$ — see front matter © 1999 Elsevier Science Ltd.
All rights reserved.
Abbreviations
IL interleukin
PDGF platelet-derived growth factor
PlGF placenta growth factor
TNF-α tumour necrosis factor α
VEGF vascular endothelial growth factor
VEGFR VEGF receptor
Introduction
The inner lining of blood and lymphatic vessels, as well as
the endocardium, consists of endothelial cells. The blood
vasculature forms by two processes: vasculogenesis, the
de novo formation of endothelial channels from differenti-
ating angioblasts; and angiogenesis, the sprouting or
splitting of capillaries from pre-existing vessels (reviewed
in [1]). Polypeptide growth factors and their receptors are
major components of the regulatory machinery that gov-
erns these processes. Two receptor tyrosine kinase
families, the vascular endothelial growth factor (VEGF)
receptors (VEGFR-1/Flt-1, VEGFR-2/KDR and VEGFR-
3/Flt4) and the angiopoietin receptors (Tie-1 and
Tie-2/Tek) are the key players, being largely specific for
endothelial cells. Other receptor families, such as the Eph
family, also provide major contributions to vessel differen-
tiation [2
•
,3]. Targeted gene disruptions in mice have
verified their central importance in vessel growth, remod-
elling and maturation ([4–8,9
••
], reviewed in [10]).
Although the adult vasculature is normally quiescent, it can
become activated to form new capillaries, for example, in
wound healing and tumourigenesis. There is convincing evi-
dence that tumours are angiogenesis dependent [11]. In the
prevascular phase, a tumour’s volume rarely exceeds a few
cubic millimetres and vessel density in invasive cancers (e.g.
in prostate cancer) positively correlates with metastatic
potential and prognosis [12]. During the so-called angiogenic
switch in tumourigenesis, the balance between angiogenesis
inhibitors (e.g. endostatin and thrombospondin-1) and
angiogenesis inducers (e.g. VEGF) is shifted and rapid ves-
sel ingrowth follows, supporting tumour expansion [11]. By
default, endothelial cell turnover rates are low in resting ves-
sels, whereas they are high in tumour vasculature.
Angiogenesis is suggested to be a rate-limiting step in
tumour development and angiogenesis inhibitors are thus
attractive drugs for anticancer therapy. There are several
benefits of directing drugs to the endothelium, including its
general accessibility through the blood circulation and the
absence of drug resistance in normal diploid and genetically
stable endothelial cells, as opposed to the frequent develop-
ment of resistance to cytotoxic therapy in genetically
heterogeneous and unstable cancer cells [13,14].
VEGF is a hypoxia-inducible endothelial cell mitogen. It
stimulates endothelial cell migration and vessel perme-
ability [15], and promotes survival of the newly formed
vessels (reviewed in [16]). VEGF is crucial for embryonic
development as targeted inactivation of even a single
VEGF allele results in embryonic lethality [17,18], and it
is also required for survival in early postnatal life when the
endothelium is still proliferating [19
•
]. Although VEGF is
highly specific for endothelial cells, it has become increas-
ingly clear that it also elicits responses in non-endothelial
cell types. For example, it is chemotactic for monocytes
[20,21] and can inhibit the maturation of dendritic cells
[22]. VEGF receptors are also expressed in certain non-
endothelial cell types in the testis and epididymis where
overexpression of VEGF caused spermatogenic arrest,
epithelial hyperplasia and infertility [23
•
]. VEGF is also
thought to be a regulator of bone formation via its effects
on the osteoblasts and osteoclasts of growth plates [24,25].
The different splice variants of VEGF seem to differ in
their function: in contrast to VEGF165, VEGF121 is
unable to bind to the non-tyrosine kinase receptor neu-
ropilin-1 [26], and in new-born gene-targeted mice,
VEGF120 cannot compensate for the loss of the longer
isoforms, leading to ischemic cardiomyopathy and
death [27
•
].
The family of VEGF-related molecules has recently
grown and contains presently five mammalian members:
VEGF; placenta growth factor (PlGF); VEGF-B;
VEGF-C; and VEGF-D. The viral homologues, collec-
tively called VEGF-E, are encoded by different strains of
the Orf virus [28].
VEGF-B, a protein that comes in two flavours
Two mRNA splice variants are generated from the
VEGF-B gene, which is located on human chromosome
11q13 [29–31]. The gene contains seven exons. The
coding sequence of the first five exons is incorporated
Current biology of VEGF-B and VEGF-C
Birgitta Olofsson*, Michael Jeltsch
†
, Ulf Eriksson* and Kari Alitalo
†‡
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Current biology of VEGF-B and VEGF-C Olofsson et al. 529
into both splice forms. Alternative splicing results in the
use of different, but overlapping reading frames in exon
6 (Figure 1). Consequently the two isoforms of the
polypeptide share the same 115 amino-terminal amino
acid residues, but have distinct carboxy termini [31].
After the 21 amino acid signal sequence has been
cleaved off, the two polypeptides are 167 (VEGF-B167)
and 186 (VEGF-B186) amino acids in length [30–32].
The apparent molecular masses of the secreted homod-
imers of VEGF-B167 and VEGF-B186 are 42 kDa and
60 kDa, respectively.
The amino acid sequences of VEGF-B167 and VEGF165
are ~44% identical and their intermolecular disulfide
bridging patterns are similar. The two subunits are joined
by disulfide bridges between the second and fourth cys-
teine residues of the platelet-derived growth factor
(PDGF) subtype cystine knot consensus sequence [33
•
].
Exon 6B of VEGF-B167 is homologous to exon 7 of
VEGF165; both encode protein sequences rich in basic
amino acid residues, which after secretion bind the growth
factor to cell-surface heparan sulphate proteoglycans [31].
In contrast, the carboxy-terminal domain of VEGF-B186 is
hydrophobic and contains many serine, threonine and pro-
line residues. VEGF-B167 and VEGF-B186 also differ in
their glycosylation pattern; whereas VEGF-B167 is not
glycosylated, VEGF-B186 contains O-linked glycans [31].
Furthermore, VEGF-B186 is proteolytically processed at
Arg127, giving rise to a 34 kDa dimer [33
•
,34
•
].
VEGF-C defines a subfamily within the
VEGF family
Within the VEGF family of growth factors, VEGF-C and
its closest relative, VEGF-D, constitute a subgroup, which
is characterised by the presence of unique amino- and car-
boxy-terminal extensions flanking the common
VEGF-homology domain [35–37,38
•
,39]. The carboxy-ter-
minal domain contains a repetitive pattern of cysteine
residues, Cys–X
10
–Cys–X–Cys–X–Cys, resembling a motif
characteristic of the Balbiani ring 3 protein, a secretory pro-
tein and a component of silk produced in larval salivary
glands of the midge Chironomus tentans. The central core
(the VEGF homology domain) exhibits ~30% identity to
VEGF [35] and is encoded by exons 3 and 4 of the seven
exons [40] (Figure 1), which is a feature conserved in other
members of the VEGF family [31,40,41]. The VEGF
homology domains of VEGF-C and VEGF-D are 61%
identical. The human VEGF-C gene has been localised to
chromosome 4q34 [29]. VEGF-C is synthesised as a pre-
cursor protein, which undergoes subsequent proteolytic
processing reminiscent of the PDGF-A and -B chain pro-
cessing, suggesting an evolutionary relationship
[35,42–44]. The carboxy-terminal domain is cleaved upon
secretion, but remains bound to the amino-terminal
domain by disulphide bonds giving rise to a disulfide-
linked tetramer composed of 29 and 31 kDa polypeptides.
Proteolytic processing of the amino-terminal propeptide
releases the mature form, which consists of two 21 kDa
polypeptide chains corresponding to the VEGF homology
domain [43]. The 29/31 kDa form seems to be the most
prevalent form of VEGF-C in various biological systems.
Dissimilar regulation of VEGF-B and VEGF-C
The promoters of the genes of VEGF family typically lack
a TATA-box and so transciption is initiated at more het-
erogeneous sites [40,41,45,46]. As for the VEGF-B
promoter, the VEGF-C promoter sequences also lack
putative binding sites for hypoxia-regulated factors [40]
and consequently neither VEGF-B nor VEGF-C mRNA
levels are regulated by hypoxia [47]. The VEGF-B pro-
moter contains binding sites for the Egr-1 transcription
factor, but lacks AP-1 sites that are present in the VEGF
promoter [45]. Several growth factors, including PDGF,
epidermal growth factor (EGF), transforming growth fac-
tor (TGF)-β and cytokines TNF-α and IL-1 (α and β), as
well as the diacylglycerol analogue phorbol myristate
acetate (PMA) increased the steady-state levels of VEGF-
C but not VEGF-B mRNA in human lung fibroblasts
[47,48]. The VEGF-C mRNA induction by IL-1 and
TNF-α might be mediated by the transcription factor
NF-κB binding sites in the VEGF-C promoter [40]. In
general, VEGF-C mRNA levels are downregulated by
steroid hormones [49,50]. In contrast, the VEGF-B
mRNA levels seem more or less invariable and show only
tissue-type-specific regulation.
VEGF-B is expressed early during fetal development and
is widely distributed, being prominently expressed in the
cardiac myocytes, in skeletal muscle and smooth muscle
cells of large vessels [51,52]. Interestingly, VEGF-B is also
Figure 1
Comparison of exon organisation and major
mRNA splice isoforms of VEGF, VEGF-B and
VEGF-C. The VEGF homology domain
containing the conserved cysteine residues of
the PDGF-subtype cysteine knot is shown in
light grey, the heparin-binding domain in black,
and the ‘silk’ homology domain in dark grey.
1 234576VEGF-C
145VEGF
1 23 4 5VEGF-B
2 3
6A
6B 7
86A 6B 7
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