JOURNAL OF CELLULAR PHYSIOLOGY 177:439±452 (1998)
Vascular Endothelial Growth Factor (VEGF)-
C Synergizes With Basic Fibroblast Growth
Factor and VEGF in the Induction of
Angiogenesis In Vitro and Alters Endothelial
Cell Extracellular Proteolytic Activity
MICHAEL S. PEPPER,1* STEFANO J. MANDRIOTA,1 MICHAEL JELTSCH,2
VIJAY KUMAR,2 AND KARI ALITALO2
1Department of Morphology, University Medical Center, Geneva, Switzerland
2Molecular/Cancer Biology Laboratory, Haartman Institute,
University of Helsinki, Helsinki, Finland
Vascular endothelial growth factor-C (VEGF-C) is a recently characterized mem-
ber of the VEGF family of angiogenic polypeptides. We demonstrate here that
VEGF-C is angiogenic in vitro when added to bovine aortic or lymphatic endothe-
lial (BAE and BLE) cells but has little or no effect on bovine microvascular endothe-
lial (BME) cells. As reported previously for VEGF, VEGF-C and basic ®broblast
growth factor (bFGF) induced a synergistic in vitro angiogenic response in all
three cells lines. Unexpectedly, VEGF and VEGF-C also synergized in the in
vitro angiogenic response when assessed on BAE cells. Characterization of VEGF
receptor (VEGFR) expression revealed that BME, BAE, and BLE cell lines express
VEGFR-1 and -2, whereas of the three cell lines assessed, only BAE cells express
VEGFR-3. We also demonstrate that VEGF-C increases plasminogen activator
(PA) activity in the three bovine endothelial cell lines and that this is accompanied
by a concomitant increase in PA inhibitor-1. Addition of a2-antiplasmin to BME
cells co-treated with bFGF and VEGF-C partially inhibited collagen gel invasion.
These results demonstrate, ®rst, that by acting in concert with bFGF or VEGF,
VEGF-C has a potent synergistic effect on the induction of angiogenesis in vitro
and, second, that like VEGF and bFGF, VEGF-C is capable of altering endothelial
cell extracellular proteolytic activity. These observations also highlight the notion
of context, i.e., that the activity of an angiogenesis-regulating cytokine depends
on the presence and concentration of other cytokines in the pericellular environ-
ment of the responding endothelial cell. J. Cell. Physiol. 177:439±452, 1998.
q 1998 Wiley-Liss, Inc.
blast growth factors (FGF) families. However, althoughAngiogenesis is the formation of new capillary blood
a role for VEGF in the development of the embryonicvessels by a process of sprouting from preexisting ves-
vasculature and in tumor angiogenesis has been un-sels and occurs during development as well as in a
equivocally established (reviewed by Dvorak et al.,number of physiologic and pathologic settings. Angio-
1995; Ferrara and Davis-Smyth, 1997), the precise rolegenesis is necessary for tissue growth, wound healing,
of the FGFs in the endogenous regulation of angiogene-and female reproductive function, and is also a compo-
sis remains to be established (reviewed by Pepper etnent of pathologic processes such as tumor growth,
al., 1996a; Christofori, 1997). Two observations pointhemangioma formation, and ocular neovascularization
to the potential importance of interactions between(reviewed by Folkman, 1995; Pepper, 1997). A similar,
although far less well studied process also occurs in
the lymphatic system and is sometimes referred to as
lymphangiogenesis. Contract grant sponsor: Swiss National Science Foundation; Con-
tract grant number: 0031-43364.95.A number of polypeptide growth factors or cytokines
*Correspondence to: Dr. Michael S. Pepper, DeÂpartement de Mor-have been demonstrated to be angiogenic in vivo (re-
phologie, Centre MeÂdical Universitaire, 1 rue Michel Servet, 1211viewed by Klagsbrun and D'Amore, 1991; Leek et al.,
GeneÁve 4, Switzerland. E-mail: michael.pepper@medecine.unige.ch1994; Pepper et al., 1996a). These factors include the
vascular endothelial growth factor (VEGF) and ®bro- Received 14 January 1998; Accepted 11 May 1998
q 1998 WILEY-LISS, INC.
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PEPPER ET AL.440
these two cytokine families in the regulation of angio- scripts are detectable in many adult and fetal human
tissues and in a number of cell lines (Joukov et al.,genesis. First, VEGF and basic FGF (bFGF) have been
demonstrated to synergize in the induction of angiogen- 1996; Lee et al., 1996). Human VEGF-C has been
mapped to chromosome 4q34 (Paavonen et al., 1996).esis in vitro (Pepper et al., 1992a; Goto et al., 1993),
and this observation has been con®rmed in vivo in a One of the striking features of VEGF-C is that its
mRNA is ®rst translated into a precursor from whichrabbit model of hindlimb ischemia (Asahara et al.,
1995) and in the rat sponge implant model (Hu and the mature ligand is derived by cell-associated proteo-
lytic processing (Joukov et al., 1997). After biosynthe-Fan, 1995). Second, the in vitro angiogenic effect of
VEGF as well as its capacity to induce plasminogen sis, VEGF-C rapidly associates into a 58-kDa anti-par-
allel homodimer linked both by disul®de and noncova-activator (PA) activity are both dependent on endoge-
nous bFGF produced by endothelial cells (Mandriota lent bonds. This process is followed by proteolytic
processing of both N- and C-terminal propeptides inand Pepper, 1997).
Alterations in endothelial cell function induced by the terminal portion of the secretory pathway and at
the cell membrane, giving rise to a number of incom-members of the VEGF family are mediated by means
of transmembrane tyrosine kinase receptors, which at pletely processed intermediates. Mature VEGF-C is
then released from cells as a 21-kDa homodimer con-present include VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-
1), and VEGFR-3 (Flt-4). Despite the apparent lack taining two VEGF-homology domains linked by nonco-
valent interactions (Joukov et al., 1997). As a conse-of constitutive angiogenesis, VEGFs and VEGFRs are
expressed, sometimes at relatively high levels, in many quence of this processing, VEGF-C acquires the ability
to bind to and activate VEGFR-2 and also increasesadult tissues. However, both the ligands and receptors
are up-regulated during development and in certain its af®nity for and activating properties of VEGFR-3.
Based on these observations, it has been suggested thatangiogenesis-associated/dependent pathologic situa-
tions, including tumor growth (reviewed by Dvorak et the synthesis of VEGF-C as a precursor allows it to
signal preferentially through VEGFR-3 (Joukov et al.,al., 1995; Mustonen and Alitalo, 1995; Ferrara and
Davis-Smyth, 1997). The phenotypes of VEGFR-1- 1997), which is restricted to venous endothelium during
early stages of development and which becomes re-and -2-de®cient mice reveal an essential role for these
receptors in hematopoiesis and blood vessel formation stricted to lymphatic endothelium later in development
and in postnatal life (Kukk et al., 1996). Under circum-during development (Fong et al., 1995; Shalaby et al.,
1995), and the importance of VEGFR-2 in tumor angio- stances in which the processing mechanism becomes
activated, VEGF-C acquires the additional capacity togenesis has been demonstrated using a dominant-nega-
tive approach (Millauer et al., 1994, 1996). Ligands that signal through VEGFR-2, thereby providing an addi-
tional level of regulation of VEGF-C bioactivity. Proteo-have been identi®ed for VEGFR-1 include VEGF,
VEGF-B, and placenta growth factor (PlGF); ligands lytic processing might also promote the formation of
VEGFR-2/VEGFR-3 heterodimers.for VEGFR-2 include VEGF, VEGF-C, and VEGF-D;
whereas the ligands for VEGFR-3 include VEGF-C and One of the hallmarks of angiogenesis, both in vivo
and in vitro, is the induction of extracellular proteolyticVEGF-D (reviewed by Mustonen and Alitalo, 1995;
Thomas, 1996; Achen et al., 1998; Olofsson et al., 1998; activity (Pepper et al., 1996b). With respect to angio-
genic cytokines, we have previously reported thatfor references on VEGF-C, see next paragraph).
VEGF-C is a protein with structural homology to VEGF increases PA activity [both urokinase-type PA
(uPA) and tissue-type PA (tPA)] and uPA receptor ex-VEGF, which was isolated from the human prostatic
adenocarcinoma cell line PC-3 during a search for a pression, as well as synthesis of PA inhibitor-1 (PAI-1)
in endothelial cells (Pepper et al., 1991, 1994; Mandri-ligand for VEGFR-3 (Joukov et al., 1996). In an inde-
pendent search for a ligand for VEGFR-3, VEGF-re- ota et al., 1995). The co-induction of the PAs and PAI-1
is consistent with the notion of the ``proteolytic balance''lated protein (VRP, Lee et al., 1996) was isolated from
a human G61 glioma cell cDNA library screened with whereas proteases are necessary for cell migration and
morphogenesis, protease inhibitors play an equally im-probes based on an expressed sequence tag (EST) en-
coding an amino acid sequence with a high degree of portant permissive role by protecting the extracellular
matrix from inappropriate destruction (Pepper andsimilarity to VEGF (Lee et al., 1996). VEGF-C and VRP
are the same protein and will be referred to as VEGF- Montesano, 1991).
By using an in vitro model of angiogenesis thatC from hereon. VEGF-C displays a high degree of simi-
larity with VEGF, including conservation of the eight assays for extracellular matrix invasion and tube for-
mation (Montesano and Orci, 1985), we have previouslycysteine residues involved in intra- and intermolecular
disul®de bonding. The cysteine-rich C-terminal half, reported that bFGF and VEGF induce bovine microvas-
cular, lymphatic, and aortic endothelial cells grown onwhich increases the length of the VEGF-C polypeptide
relative to other ligands of this family, shows a pattern three-dimensional collagen or ®brin gels to invade the
underlying matrix within which they form capillary-of spacing of cysteine residues reminiscent of the Balbi-
ani ring 3 protein repeat. The C-terminal propeptide like tubular structures (Montesano et al., 1986; Pepper
et al., 1990, 1992a, 1993b, 1994, 1995a), demonstratingalso contains short motifs of EGF-like domains, which
may promote the interaction of secreted VEGF-C with that the angiogenesis-inducing properties of bFGF and
VEGF can be mediated by means of a direct effect onthe extracellular matrix. VEGF-C promotes the growth
of human and bovine endothelial cells, although it is endothelial cells. However, there is increasing evidence
that the nature of the response elicited by a speci®cless active than VEGF in this assay (Lee et al., 1996;
Joukov, 1997). VEGF-C has been reported to induce cytokine is contextual, i.e., that this response depends
on the presence or absence of other regulatory mole-endothelial cell migration in three-dimensional colla-
gen gels (Joukov et al., 1996, 1997). VEGF-C tran- cules present in the pericellular environment of the
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