From the
Vascular endothelial growth factor (VEGF) is a potent angiogenic
factor and endothelial cell-specific mitogen that stimulates
urokinase-type plasminogen activator (uPA) activity in vascular
endothelial cells. Here, we report that VEGF increases the high
affinity binding of uPA to the same cells and that this binding is
prevented by a peptide corresponding to the uPA receptor (uPAR) binding
growth factor-like domain of uPA. Ligand cross-linking, ligand
blotting, and uPA-Sepharose affinity chromatography revealed an
increase in a cell surface uPA binding protein that corresponds to the
uPAR on the basis of its affinity for uPA, M
During angiogenesis, quiescent endothelial cells are induced to
locally degrade their basement membrane and to form new capillary blood
vessels by sprouting into the surrounding stroma (Ausprunk and Folkman,
1977). This phenomenon, which in the adult organism occurs in a number
of physiological and pathological settings (Folkman and Klagsbrun,
1987), requires tightly regulated extracellular proteolytic activity
(Pepper and Montesano, 1990). A similar process of sprouting occurs in
the formation of lymphatic capillaries during lymphangiogenesis (Clark
and Clark, 1932).
Angiogenesis is regulated by a number of cytokines
including basic fibroblast growth factor (bFGF)
Urokinase- and tissue-type plasminogen
activators (uPA, tPA) are serine proteinases involved in extracellular
proteolytic processes. PAs convert plasminogen, a zymogen present in
body fluids, to plasmin, a serine proteinase capable of degrading
directly or indirectly (through the activation of other zymogens
including latent metalloproteinases) most of the major protein
components of the extracellular matrix (ECM). Because of its strong
affinity for fibrin, tPA is primarily implicated in thrombolysis, while
uPA is associated with tissue remodeling and cell invasion (Vassalli
et al., 1991). Although the proteolytic cascade of zymogen
activation triggered by plasminogen activation appears to be a
fundamental component in many situations of cellular invasion including
angiogenesis (Mignatti and Rifkin, 1993), Carmeliet and co-workers
(1994) have recently reported that transgenic mice lacking uPA and/or
tPA develop and reproduce normally. However, the observation that tumor
growth and metastasis are blocked by anti-uPA antibodies in vivo (Ossowski and Reich, 1983; Ossowski et al., 1991b), may
indicate differences in the requirement for PA activity in
physiological versus pathological settings.
uPA expression
is regulated by hormones and growth factors (Vassalli et al.,
1991), and in the extracellular microenvironement, uPA activity is
modulated by several mechanisms. It is secreted as an inactive zymogen
(pro-uPA) that can be activated by a single proteolytic cleavage
(Petersen et al., 1988). Plasmin has been proposed as a
physiologic activator of pro-uPA, although other enzymes, including
cathepsin B, have also been implicated (Kobayashi et al.,
1991). Specific PA inhibitors (PAIs), often synthesized by the same
cells that produce uPA, are of importance in the maintenance of ECM
integrity, which is an essential requirement for cell invasion and
tissue remodeling processes (Pepper and Montesano, 1990).
An
important step in the regulation of uPA activity involves binding to a
high affinity cell surface receptor (uPAR) (Vassalli et al.,
1985; Stoppelli et al., 1985; Vassalli, 1994). uPAR is a
highly glycosylated, 55-60-kDa protein (Estreicher et
al., 1989; Roldan et al., 1990) linked to the plasma
membrane by a glycosylphosphatidylinositol anchor (Ploug et
al., 1991). Pro-uPA binds to uPAR via its non-catalytic
amino-terminal region (Vassalli et al., 1985; Appella et
al., 1987). The bound zymogen is activated and remains active on
the cell surface for several hours; binding of PAI-1 or PAI-2 to
uPAR-bound uPA results in rapid internalization and degradation of the
PAI
uPAR expression is associated with cell migration
(Pepper et al., 1993) and is localized to the leading front of
migrating monocytes and invading tumor cells (Estreicher et
al., 1990; Pyke et al., 1991). Binding of uPA to uPAR has
at least two major consequences: (i) localization of uPA activity on
the cell surface; (ii) an increase in the rate of plasminogen
activation (Ellis et al., 1989). Binding of uPA to uPAR
greatly enhances ECM degradation and cell invasion in vitro and in vivo (Hollas et al., 1991; Ossowski
et al., 1991a; Quax et al., 1991; Crowley et
al., 1993). Binding of uPA to uPAR could therefore be a critical
event in cell invasion.
Elevated levels of uPA expression are
typical of migrating endothelial cells in vitro (Pepper et
al., 1987). While quiescent endothelial cells in vivo do
not produce uPA (Larsson et al., 1984; Kristensen et
al., 1984), uPA is transiently induced in these cells at the time
of capillary growth (Bacharach et al., 1992). In
vitro, uPA activity is increased in blood and lymphatic vascular
endothelial cells in response to bFGF and VEGF (Saksela et
al., 1987; Pepper et al., 1990, 1991, 1994). Both
cytokines also stimulate the concomitant production of PAI-1, which is
consistent with the hypothesis that balanced proteolytic activity is
required for normal capillary morphogenesis (Pepper and Montesano,
1990). The objective of the present studies was to determine whether
VEGF increases uPAR expression in vascular endothelial cells in a
manner similar to that previously described for bFGF (Mignatti et
al., 1991).
For
Scatchard analysis, BAE or HUVE cells were grown to confluence in 23-mm
culture wells. HUVE cells were further grown for 4 days in the absence
of heparin and endothelial cell growth supplement. VEGF (30 or 100
ng/ml in the case of HUVE and BAE cells, respectively) was added for 15
h as described above. The cells were acid treated and incubated with
250 µl/well of binding medium containing increasing concentrations
of
VEGF increased the uPA binding capacity of BME, BAE, BLE,
and HUVE cells in a dose-dependent manner (Fig. 2). The most
prominent effect was seen with BAE cells, where 100 ng/ml VEGF
increased uPA binding capacity by 2.6-fold, and the smallest increase
was seen with BME and HUVE cells, where the same concentration of VEGF
increased uPA binding capacity by approximately 30%. Binding of human
uPA to BAE, BME, and BLE cells was inhibited in a dose-dependent manner
by the mouse uPA 13-33 peptide (Appella et al., 1987)
(Fig. 2), which inhibits human uPA binding to bovine uPAR
(Mignatti et al., 1991; Pepper et al., 1993). The
peptide was not used with HUVE cells because it has been previously
demonstrated that the mouse peptide is a poor inhibitor of human uPA
binding to human cells (Estreicher et al., 1989). These
findings further indicate that uPA binding to endothelial cells is
mediated by uPAR.
For cross-linking, confluent BAE cell monolayers were
incubated for 15 h with 500 pM bFGF (9 ng/ml) or VEGF (22.5
ng/ml) or with increasing concentrations of VEGF alone (1, 10, or 100
ng/ml). Acid-treated cells were exposed to 1 nM
The PA-plasmin system, and in particular the interaction of
uPA with uPAR, is an important element in the cohort of cellular
processes that mediate cellular invasion and tissue remodeling
(Mignatti and Rifkin, 1993; Vassalli, 1994). Although soluble uPA
efficiently converts plasminogen to plasmin, the uPAR-mediated binding
of uPA to the cell surface increases the efficiency of plasmin
formation severalfold (Ellis et al., 1989) and localizes
plasmin formation to cell-cell/cell-ECM contact sites. In the studies
reported in this paper, we have investigated whether VEGF, an
endothelial cell-specific mitogen that plays a major role in
neovascularization (Ferrara et al., 1992) and stimulates uPA
expression in vascular endothelial cells (Pepper et al., 1991,
1994), also increases uPAR expression in the same cells.
In
vitro, vascular endothelial cells constitutively express both uPA
and uPAR, and uPAR is occupied by endogenous uPA through an autocrine
mechanism. A similar phenomenon has been described in other cell types
(Stoppelli et al., 1986). By zymography, we have previously
found that VEGF increases cell-bound uPA activity in blood and
lymphatic vascular endothelial cells (Pepper et al., 1991,
1994, and data not shown). In this paper, we demonstrate that exposure
of both blood and lymphatic vascular endothelial cells to picomolar
concentrations of VEGF results in a dose-dependent increase in their
capacity to bind uPA with high affinity. Binding did not involve the
catalytic site of the enzyme, since diisopropylfluorophosphate-treated
uPA was efficiently bound and was prevented by the amino-terminal
growth factor-like domain of uPA, which is known to mediate binding of
uPA to uPAR (Appella et al., 1987). Scatchard analysis
revealed a single class of high affinity uPA binding sites on both BAE
and HUVE cells, with features similar to the previously characterized
endothelial cell uPAR (Barnathan et al., 1990; Haddock et
al., 1991; Mignatti et al., 1991). Cross-linking, ligand
blotting, and uPAR purification experiments confirmed that VEGF
increases the uPA binding capacity of vascular endothelial cells by an
increase in a uPA binding cell surface protein that corresponds by
M
Although the magnitude of the VEGF-induced increase in
uPAR mRNA levels was similar in all cell types, the VEGF-induced
increase in uPA binding capacity differed depending upon the
endothelial cell type assessed. Thus, 100 ng/ml VEGF increased the
binding of
The high number of uPAR molecules in HUVE cells is
probably a consequence of culture conditions, as these cells are
routinely cultured in the presence of a growth supplement (endothelial
cell growth supplement) containing endothelial cell mitogens (including
bFGF) capable of up-regulating uPAR. In addition, the
K
VEGF also increases tPA and PAI-1 in blood and lymphatic
vascular endothelial cells (Pepper et al., 1991, 1994, and
this paper). Although tPA has been implicated primarily in clot lysis,
it may participate in the proteolysis that occurs during tissue
remodeling (Tsafriri et al., 1989). This hypothesis is
consistent with the finding that anti-tPA antibodies inhibit
endothelial cell invasion of the human amnion basement membrane
(Mignatti et al., 1989). The VEGF-mediated induction of PAI-1
expression in endothelial cells is in agreement with our previous
hypothesis that finely tuned proteolytic activity is required for
normal capillary morphogenesis (Pepper and Montesano, 1990).
Originally proposed as a means of confining uPA activity to the
pericellular space, binding of uPA to uPAR has recently been shown to
regulate a number of cell functions, including c- fos expression, proliferation, migration, differentiation, and uPA
production (Fibbi et al. 1990; Nusrat and Chapman, 1991;
Rabbani et al., 1992; Busso et al., 1994; Dumler
et al., 1994). These effects occur independently of
uPA-mediated hydrolysis and appear to be mediated, at least in part, by
tyrosine kinase activities (Dumler et al., 1994; Busso et
al., 1994). Binding of uPA to uPAR also enhances cell adhesion
(Nusrat and Chapman, 1991; Waltz et al. 1993). The molecular
mechanisms underlying this phenomenon have been partially elucidated
and appear to be integrin-independent and to have vitronectin as a
primary target (Waltz and Chapman, 1994; Wei et al., 1994).
uPA-uPAR-mediated cell adhesion is decreased by PAI-1 (Waltz and
Chapman, 1993). In addition to its protease inhibitory activity, PAI-1
down-regulates cell surface uPA activity by promoting internalization
of PAI-1
We are grateful to Drs. M. B. Furie and S. C.
Silverstein for the BME cells, Dr. N. Maggiano for the HUVE cells, Dr.
J. Henkin for human tcuPA, Dr. P. Sarmientos for recombinant human
bFGF, Dr. F. Blasi for the human uPAR cDNA, Dr. W.-D. Schleuning for
bovine uPA, uPAR, and human tPA cDNAs, and Drs. A. Estreicher, P. Meda,
and A. Wohlwend for assistance during various phases of this work. We
are also grateful to Drs. R. Montesano and L. Orci for continued
support. Technical assistance was provided by C. Di Sanza, M. Quayzin,
and F. Tredici, and photographic work was done by B. Favri.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
of
50,000-55,000, and phosphatidylinositol-specific phospholipase C
sensitivity. By Scatchard analysis, VEGF increased the number of uPAR
molecules by 2.8-3.5-fold and concomitantly decreased their
affinity for uPA. By northern blotting uPAR mRNA was increased in a
dose- and time-dependent manner in response to VEGF. Taken together,
these findings demonstrate that VEGF-induced angiogenesis is
accompanied by increased uPAR expression and uPA activity on the
endothelial cell surface. These observations are consistent with the
notion that the uPA-uPAR interaction facilitates cellular invasion.
(
)
and vascular endothelial growth factor (VEGF, also known as
vascular permeability factor). Unlike bFGF that is not exported via the
classical secretory pathway, which acts on a broad spectrum of cell
types and whose receptor expression by endothelial cells in vivo is still controversial, VEGF is likely to be a major regulator of
physiological and pathological angiogenesis, since it is a secreted
endothelial cell-specific mitogen whose receptors are expressed almost
exclusively on vascular endothelial cells (Klagsbrun and D'Amore,
1991; Ferrara et al., 1992; Klagsbrun and Soker, 1993; Neufeld
et al., 1994).
uPA complex (Cubellis et al., 1990; Estreicher et
al., 1990).
Cell Culture
Adrenal cortex-derived
bovine microvascular endothelial (BME) cells, kindly provided by Drs.
M. B. Furie and S. C. Silverstein (Columbia University, NY) (Furie
et al., 1984), were grown in -modified minimum essential
medium (Life Technologies, Inc.) supplemented with 15% heat-inactivated
donor calf serum (Flow Laboratories, Baar, Switzerland). The cells were
used between passages 17 and 24. Bovine aortic endothelial (BAE) cells
(Pepper et al., 1993) were grown in Dulbecco's modified
Eagle's medium (Life Technologies, Inc.) containing 10% donor
calf serum and were used between passages 8 and 13. Human umbilical
vein endothelial (HUVE) cells were grown in medium 199 (Life
Technologies, Inc.), supplemented with 20% donor calf serum, 10
µg/ml heparin, and 30 µg/ml endothelial cell growth supplement
(Becton Dickinson Labware, Bedford, MA), and were used between passages
6 and 13. Bovine lymphatic endothelial (BLE) cells isolated from
mesenteric lymphatic vessels (clone A9, Pepper et al., 1994)
were grown in DMEM containing 20% fetal calf serum (Hyclone
Laboratories Inc., Logan, UT) and 1 mM sodium pyruvate and
used between passages 21 and 23. Confluent cultures of all cell types
were routinely split 1:4 and grown in gelatin-coated culture dishes or
flasks (Falcon Labware, Becton Dickinson Co., Lincoln Park, NJ) in the
presence of streptomycin (100 µg/ml) and penicillin (500 units/ml).
VEGF
Recombinant human VEGF (165-amino
acid species) was purified from transfected Chinese hamster ovary cells
as previously described (Ferrara et al., 1991). The purity of
the material was assessed by SDS-polyacrylamide gel electrophoresis and
by the presence of a single NH-terminal amino acid
sequence.
PA Activity Assay
Confluent BAE or BME
cell monolayers in 15-mm culture wells (Nunc Intermed, Roskilde,
Denmark) were incubated at 37 °C for 15 h with the indicated
concentrations of VEGF or bFGF as described below. The cells were
washed twice with phosphate-buffered saline and incubated for 1 h at 37
°C in the presence of 1 ml/well of DMEM containing 0.25 units/ml of
phosphatidylinositol-specific phospholipase C (PI-PLC; Boehringer
Mannheim). 10 µl of culture supernatant was tested for PA activity
by the I-fibrin plate assay (Gross et al.,
1982).
Radioiodination of uPA
Human single-chain
and two-chain uPA (scuPA, tcuPA) obtained from Serono (Serono
Laboratories, Aubonne, Switzerland) and from Dr. Jack Henkin (Abbott
Laboratories, Abbott Park, IL) were radiolabeled using
Na-I as described (Vassalli et al. 1984;
Mignatti et al., 1991).
I-uPA had a specific
activity of 4.5-8.0
10
cpm/µg.
VEGF and/or bFGF were added to the culture medium of
confluent endothelial cell cultures in 23-mm wells 36 h after the last
medium change. After 15 h of incubation, the cells were acid treated
and assayed for I-tcuPA Binding to
Cells
I-tcuPA binding capacity as described
(Mignatti et al., 1991), except that 100 pM (or 70
pM in the case of BME cells)
diisopropylfluorophosphate-treated
I-tcuPA was used.
Where mentioned, the indicated amounts of the mouse uPA 13-33
peptide, which corresponds to the uPAR binding region of mouse uPA
(Appella et al., 1987), were added to the binding medium (DMEM
containing 20 mM Hepes, pH 7.2, 200 Kunitz-inhibitory units/ml
of Trasylol, and 1 mg/ml bovine serum albumin). The peptide was
synthesized according to the sequence of mouse uPA as described by
Belin et al. (1985). Binding to cell-free gelatin-coated wells
was measured in parallel and subtracted from all samples. Cells were
washed four times with phosphate-buffered saline containing 1 mg/ml
bovine serum albumin to remove unbound ligand, lysed in 0.1 M Tris-HCl, pH 8.1, containing 0.2% Triton X-100, and centrifuged at
500
g for 15 min at 4 °C. The radioactivity
present in the supernatants was measured in a
-counter.
I-tcuPA (from 160 pM to 20 nM or
from 26 pM to 80 nM in the case of BAE or HUVE cells,
respectively). In parallel cultures, nonspecific binding was determined
by adding a 100-fold molar excess of diisopropylfluorophosphate-treated
unlabeled uPA to each concentration of
I-tcuPA.
Cross-linking of
Confluent BAE cell monolayers in 35-mm
culture dishes were exposed to VEGF or bFGF and processed as described
for the binding assay, except that 1 nM I-uPA to
Endothelial Cells
I-tcuPA
was added in 500 µl of binding medium/dish. After 1 h at 4 °C,
10 µl of 100 mM disuccinimidyl suberate (Pierce) in
Me
SO was added, and the cells were incubated at room
temperature for 20 min. The cross-linking reaction was blocked by
adding 0.7 µl of 7.5 M ammonium acetate. The cells were
washed four times with phosphate-buffered saline, lysed in 0.1 M Tris-HCl, pH 8.1, containing 0.2% Triton X-100, 200
Kunitz-inhibitory units/ml of Trasylol, 100 µg/ml
phenylmethylsulfonylfluoride, and sonicated. Protein concentration in
the cell lysates was determined by the BCA protein assay reagent
(Pierce) using bovine serum albumin as a standard. 25 µg of cell
extract protein were run in a SDS/6% polyacrylamide gel under
non-reducing conditions. The gel was dried and exposed to Kodak XAR-5
films (Eastman Kodak Co.) at
80 °C.
Ligand Blotting
Confluent BAE cell
monolayers in 10-cm culture dishes were incubated at 37 °C for 15 h
with the indicated concentrations of VEGF or bFGF and treated with
PI-PLC as described above. The PI-PLC washings were concentrated to 40
µl in 10-kDa cut-off Centricon tubes (Amicon GmbH, Witten,
Germany), electrophoresed in a SDS/10% polyacrylamide gel under
non-reducing conditions, and electroblotted onto a nitrocellulose
membrane (Hybond C-Extra, Amersham). The membrane was saturated for 1 h
at room temperature with 10 mM Tris-HCl, 150 mM NaCl,
0.1% Tween 20, pH 8.0 (TBS/Tween), containing 5% (w/v) skimmed milk and
hybridized for 1 h at room temperature to 2.5 nM I-scuPA in the same buffer. The membrane was
repeatedly washed in TBS/Tween and exposed to Kodak XAR-5 films at
80 °C.
Purification of
Confluent BAE cell monolayers in 10-cm culture dishes
were incubated with the indicated concentrations of VEGF or bFGF as
described above. The cells were incubated at 37 °C for 2.5 h in
methionine- and cysteine-free DMEM (Life Technologies, Inc.). The
medium was replaced with medium containing 300 µCi/ml
[S-Labeled
uPAR
S]Met/Cys (specific activity, 1173 Ci/mmol, ICN
ImmunoBiologicals, Costa Mesa, CA). After 3 h at 37 °C, the cells
were acid treated and incubated with PI-PLC as described above. The
PI-PLC washings were loaded onto a uPA-Sepharose column equilibrated
with 0.1 M Tris-HCl, 0.5 M NaCl, pH 8.0. After
several washings with the same buffer, bound protein was eluted with 50
mM glycine, 0.1 M NaCl, pH 3.0, and harvested in
tubes containing 0.5 M Hepes, 0.1 M NaCl, pH 7.5. The
eluates were concentrated to a final volume of 40 µl in 10-kDa
cut-off Centricon tubes and electrophoresed in a SDS/10% polyacrylamide
gel. The gel was fixed in 30% ethanol, 10% acetic acid for 1 h at room
temperature, washed with H
O, and incubated in
autoradiography enhancer buffer (DuPont NEN) for 1 h. The dried gel was
exposed to Kodak XAR-5 films at
80 °C.
RNA Purification and Northern Blot
Analysis
VEGF and/or bFGF were added to the culture medium
of confluent endothelial cell monolayers 36 h after the last medium
change. After the indicated times, total cellular RNA was purified as
described (Chomczynski and Sacchi, 1987). Northern blot analysis was
performed as described (Pepper et al. 1990). A 585-base pair
BamHI fragment of human uPAR cDNA (clone p-uPAR-1, position
501-1086) (Roldan et al., 1990) was subcloned into the
BamHI site of pBluescript KS (Stratagene). The plasmid was
linearized with EcoRI and transcribed with bacteriophage T7
RNA polymerase as described (Busso et al., 1986).
P-Labeled cRNA probes were prepared from bovine uPAR
(Kratzschmar et al., 1993), bovine uPA (Kratzschmar et
al., 1993), human tPA (Fischer et al., 1985), and bovine
PAI-1 (Pepper et al., 1990) cDNAs as described (Pepper et
al., 1990, 1993). Autoradiograms were scanned with a GenoScan
laser scanner (Genofit, Geneva, Switzerland).
VEGF Increases PI-PLC-sensitive uPA Activity in
Vascular Endothelial Cells
We have previously reported that
VEGF increases uPA activity in BME cells (Pepper et al.,
1991). Here, we show that treatment with PI-PLC releases PA activity
from cultured endothelial cells (Fig. 1). When cultured in the
presence of 100 ng/ml VEGF, PI-PLC-releasable PA activity was increased
5.7- and 2.0-fold in BAE and BME cells, respectively. Enzyme activity
released from VEGF-treated cells in the presence of PI-PLC was
inhibited by amiloride, which inhibits uPA but not tPA (Vassalli and
Belin, 1987) (Fig. 1). The increase in uPA activity in
VEGF-treated endothelial cells is unlikely to be due to a decrease in
PAI expression, since VEGF also increases PAI-1 expression by these
cells (Pepper et al., 1991). In addition, pro-uPA is rapidly
converted to active uPA in the fibrin plate assay; this rules out the
possibility that pro-uPA activation could account for the increase in
response to VEGF. These data demonstrate that the VEGF-induced uPA
activity in vascular endothelial cells is PI-PLC-releasable, a
characteristic feature of uPAR-bound enzyme.
Figure 1:
VEGF
increases PI-PLC-releasable cell-bound uPA activity in BME and BAE
cells. Cells were incubated for 15 h in the presence of the indicated
concentrations of VEGF, of 10 ng/ml bFGF ( F10), or of
cytokine-free culture medium as control ( C) and treated with
PI-PLC. Culture supernatants were assayed for their capacity to
solubilize I-fibrin; mean values of soluble
I-fibrin degradation products (c.p.m.) from duplicate
wells from a single representative experiment are shown. Where
indicated, PI-PLC was omitted (
PI-PLC), or 1 mM amiloride was added. This experiment has been repeated at least
twice for each condition in both cell lines.
VEGF Increases uPA Binding Capacity in Blood and
Lymphatic Vascular Endothelial Cells
To further explore the
possibility that the VEGF-mediated increase of cell-bound uPA activity
was due to an increase in uPAR, we studied the uPA binding capacity of
both human and bovine endothelial cells treated with VEGF. For this
purpose, we used I-labeled human uPA, which binds to the
bovine uPAR with high affinity (Mignatti et al., 1991). In
addition, cells were exposed to mild acidic conditions to remove
cell-bound endogenous uPA before assessing binding capacity (data not
shown).
Figure 2:
VEGF increases uPA binding capacity of
BME, BAE, BLE, and HUVE cells. Cells were exposed to the indicated
concentrations of VEGF for 15 h or to cytokine-free culture medium as
control ( C). Following acid treatment, cells were incubated
for 1 h at 4 °C in the presence of 100 pM I-tcuPA (70 pM in the case of BME cells).
The peptide competitor, which corresponds to the uPAR binding region of
mouse uPA, was added to the binding medium at molar excess over uPA, as
indicated. Radioactivity of the cell lysates of duplicate wells was
measured in a
-counter. Values are expressed relative to controls
and are the means ± S.E. from at least two experiments per
condition for each cell line. No significant differences in cell
numbers were observed between control and VEGF-treated cells (data not
shown).
Basic FGF and VEGF have a synergistic effect on
in vitro angiogenesis, and when tested separately at equimolar
concentrations, bFGF is a stronger angiogenesis inducer than VEGF
(Pepper et al., 1992). To compare the effect of bFGF and VEGF,
alone or in combination, on uPA binding by endothelial cells, confluent
BAE cells were treated with equimolar concentrations (50 or 500
pM) of bFGF or VEGF or with both cytokines together. Under
these conditions, uPA binding was greater in bFGF- than in VEGF-treated
cells; when the two cytokines were co-added, the resulting increase in
uPA binding was additive (Fig. 3). Similar results were obtained
with BME and BLE cells (data not shown). These findings demonstrate
that the synergistic effect on in vitro angiogenesis is not
mediated by synergism at the level of uPAR.
Figure 3:
Additive effect of VEGF and bFGF on the
uPA binding capacity of BAE cells. Cells were exposed to the indicated
concentrations of VEGF ( V), bFGF ( F), or both
cytokines ( F/V) for 15 h. I-tcuPA binding was
measured in duplicate samples as described in the legend to Fig. 2.
Values are expressed relative to controls and are the means ±
S.E. from two experiments per condition.
To determine the number
and affinity of uPA binding sites on endothelial cells, we performed a
Scatchard analysis on cells incubated overnight with a saturating
concentration of VEGF (100 or 30 ng/ml for BAE or HUVE cells,
respectively) or with culture medium alone (Fig. 4). A single
class of high affinity uPA binding sites was revealed in both cell
types, with features that are characteristic of uPAR (Barnathan et
al., 1990; Estreicher et al., 1989; Mignatti et
al., 1991). In control BAE cells, the number of uPAR
molecules/cell was 1.5 10
, with a
K
of 2.7 nM. VEGF increased the
number of uPAR molecules/cell by 3.5-fold (5.2
10
uPAR/cell) and slightly decreased their affinity for uPA
( K
= 4.5 nM). In HUVE
cells, VEGF increased the number of uPAR molecules/cell by 2.8-fold
(from 6.4
10
to 1.8
10
) and
also decreased their affinity for uPA ( K
increasing from 1.4 to 2.0 nM).
Figure 4:
Scatchard analysis of
I-tcuPA binding to VEGF-treated BAE or HUVE cells. Cells
were exposed overnight to 100 ng/ml or 30 ng/ml VEGF (BAE and HUVE
cells, respectively). Following acid treatment, cells were incubated in
the presence of increasing concentrations of
I-tcuPA (160
pM to 20 nM BAE and 26 pM to 80 nM HUVE) at 4 °C for 1 h. Nonspecific binding was determined by
adding a 100-fold molar excess of cold uPA to each concentration of
I-tcuPA in parallel cultures.
Characterization of uPA Binding Sites on VEGF-treated
Endothelial Cells
Cross-linking, ligand blotting, and uPA
affinity purification experiments were performed to further
characterize the binding of I-uPA to the endothelial cell
surface.
I-tcuPA, and bound uPA was cross-linked to the cell
surface by disuccinimidyl suberate. Cell extracts were analyzed by
SDS-polyacrylamide gel electrophoresis and autoradiography. BAE cell
extracts showed a prominent band corresponding to the expected position
of the uPA
uPAR complex (105 kDa, Fig. 5). Since human tcuPA
has an apparent M
of 55,000, this indicates an
apparent M
of approximately 50,000 for uPAR. Both
bFGF and VEGF increased the intensity of the uPA
uPAR complex, with
bFGF having a stronger effect. The effect of VEGF was dose dependent,
and
I-tcuPA binding was efficiently competed by a
1000-fold molar excess of the mouse uPA peptide (Fig. 5).
Figure 5:
Cross-linking of I-tcuPA to
BAE cells. Cells were incubated for 15 h in the presence of 500 pM bFGF ( F, 9 ng/ml), VEGF ( V, 22.5 ng/ml), of
increasing concentrations of VEGF, or of cytokine-free culture medium
as control ( C). The cells were acid treated and incubated for
1 h at 4 °C in the presence of 1 nM
I-tcuPA.
Where indicated, a 1000-fold molar excess of the uPA peptide competitor
was added to the binding medium.
I-tcuPA was cross-linked
to the cell surface by disuccinimidyl suberate. Cell lysates (25
µg/sample) were electrophoresed in a SDS/10% polyacrylamide gel
under non-reducing conditions.
uPAR
is linked to the plasma membrane by a glycosylphosphatidylinositol
anchor and can be detached from the cell surface by PI-PLC (Ploug
et al., 1991). Confluent BAE cells treated with increasing
concentrations of VEGF were incubated at 37 °C for 1 h in the
presence of 0.25 units/ml of PI-PLC. Concentrated supernatants were
characterized by ligand blotting with I-scuPA. A single
band corresponding to the expected position of uPAR (about 55 kDa) was
present in all samples (Fig. 6). The intensity of this band
increased in response to VEGF in a dose-dependent manner. By scanning
densitometry, the intensity of the band increased 3.5-fold in BAE cells
treated with 100 ng/ml VEGF (not shown), consistent with cell binding
data (see Fig. 4). The band was undetectable in samples incubated
in the absence of PI-PLC, indicating that it corresponds to a
glycosylphosphatidylinositol-anchored cell surface protein.
Figure 6:
Ligand blotting of I-scuPA
to PI-PLC-released BAE cell surface molecules. Cells were exposed for
15 h to the indicated concentrations of bFGF ( F), VEGF
( V), or to control medium ( C). After treatment with
PI-PLC, the PI-PLC washings were run in a SDS/10% polyacrylamide gel
and transferred to a nitrocellulose membrane. The membrane was
hybridized to
I-scuPA. A and B are
different radiographic exposure times of the same
membrane.
Finally,
PI-PLC-released uPAR was purified from S-labeled BAE cells
by uPA-Sepharose affinity chromatography. As shown in Fig. 7, a
single band corresponding in size to the expected position of uPAR
(approximately 55 kDa) was detected both in VEGF-treated and untreated
cells. The intensity of this band increased in VEGF-treated cells in a
dose-dependent manner. The 30-35-kDa band shown in
Fig. 7
most likely represents an incompletely glycosylated form of
uPAR or a uPAR degradation product. 10 ng/ml bFGF had an effect
comparable with that of 100 ng/ml VEGF.
Figure 7:
Purification of S-labeled
uPAR from BAE cells. Cells were exposed for 15 h to the indicated
concentrations of bFGF ( F), VEGF, or to cytokine-free culture
medium as control ( C) and labeled with
[
S]methionine/cysteine. Following PI-PLC
treatment, the PI-PLC washings were subjected to uPA affinity
chromatography and run in a SDS/10% polyacrylamide gel under
non-reducing conditions.
Taken together, these
findings demonstrate that VEGF increases the expression of a uPA cell
surface binding protein, which corresponds to uPAR on the basis of its
affinity for uPA, Mof 50,000-55,000, and
PI-PLC sensitivity.
uPAR mRNA Levels in VEGF-treated Endothelial
Cells
When confluent cultures of BAE cells were incubated
for 15 h in the presence of increasing concentrations of VEGF, uPAR
mRNA levels were increased in a dose-dependent manner, with a maximal
8.5-fold increase at 100 ng/ml (Fig. 8). Similar results were
obtained with BME cells (data not shown). VEGF also increased uPA, tPA,
and PAI-1 expression in BAE cells in a dose-dependent manner
(Fig. 8), confirming and extending our previous observations with
BME cells (Pepper et al., 1991 and data not shown). A kinetic
analysis revealed that in the presence of 100 ng/ml VEGF, uPAR
induction in BME cells began between 1 and 4 h of incubation and was
maximal (6.2-fold increase) after 24 h (Fig. 9). Similar results
were obtained with BAE cells (data not shown). The kinetics of uPA and
tPA induction were very similar to those seen for uPAR. In contrast,
PAI-1 induction was ephemeral, being maximal after 4 h and returning to
base-line levels after 24 h (Fig. 9).
Figure 8:
VEGF increases uPA, uPAR, tPA, and PAI-1
mRNAs in BAE cells. Cells were incubated for 15 h in the presence of
the indicated concentrations of VEGF or bFGF ( F). Replicate
filters containing total cellular RNA (5 µg/lane) were hybridized
with P-labeled bovine uPA, bovine uPAR, human tPA, and
bovine PAI-1 cRNA probes. Methylene blue staining ( bottom panel) reveals 28 and 18 S rRNAs and demonstrates
uniformity of loading and RNA integrity.
Figure 9:
Kinetics of uPA, uPAR, tPA, and PAI-1 mRNA
expression in BME cells. Cells were incubated for the indicated times
in the presence of 100 ng/ml VEGF. Replicate filters containing total
cellular RNA (5 µg/lane) were hybridized with
P-labeled bovine uPA, bovine uPAR, human tPA, and bovine
PAI-1 cRNA probes. Methylene blue staining ( bottom panel) reveals 28 and 18 S rRNAs and demonstrates
uniformity of loading and RNA integrity.
When bFGF and VEGF were
tested at equimolar concentrations, uPAR mRNA induction was greater in
bFGF- than in VEGF-treated BAE cells (data not shown). When tested
together, the effect of VEGF and bFGF on the expression of uPAR mRNA
was additive (data not shown), which is consistent with cell binding
data (Fig. 3). An additive effect of VEGF and bFGF on uPA, uPAR,
tPA, and PAI-1 mRNA levels was also observed in BME cells (data not
shown). uPAR mRNA levels were also increased in HUVE cells in a
dose-dependent manner. A maximal 8.4-fold increase was observed with 30
ng/ml VEGF (Fig. 10).
Figure 10:
VEGF increases uPAR mRNA levels in HUVE
cells. Cells were incubated for 15 h in the presence of the indicated
concentrations of VEGF. Total cellular RNA (5 µg/lane) was
hybridized with a P-labeled human uPAR cRNA probe.
Methylene blue staining ( bottom panel) reveals the 18
S rRNA and demonstrates uniformity of loading and RNA
integrity.
, PI-PLC sensitivity, and affinity for uPA to the
previously characterized uPAR. Moreover, the VEGF-induced PA activity
could be released from the endothelial cell surface by PI-PLC, and was
inhibited by amiloride, which is characteristic of uPAR-bound enzyme.
VEGF also increased uPA and uPAR mRNA levels in blood and lymphatic
vascular endothelial cells (Pepper et al., 1991, 1994, and
this paper). These data demonstrate that the VEGF-mediated increase in
cell-bound uPA activity in vascular endothelial cells is mediated by an
increase in uPA that binds to uPAR, which is also up-regulated in the
same cells.
I-human tcuPA (70 or 100 pM) by 2.6-,
1.3-, or 1.3-fold in BAE, HUVE, and BME cells, respectively. By
Scatchard analysis, we found different levels of basal uPAR expression
in different endothelial cell types: 1.5
10
and 6.5
10
uPAR/cell in BAE and HUVE cells, respectively.
Saturating concentrations of
I-uPA revealed that VEGF
increased the number of uPAR/cell by 3.5- and 2.8-fold in BAE and HUVE
cells, respectively. These data demonstrate that the amplitude of the
VEGF-induced uPAR increase is inversely related to the basal level of
uPAR expression. As previously reported for other cytokines and cell
types, the increase in receptor number in VEGF-treated cells is
associated with a decrease in receptor affinity (Estreicher et
al., 1989; Mignatti et al., 1991). Taken together, these
findings suggest that different post-transcriptional mechanisms,
including uPAR mRNA translation efficiency and changes in receptor
affinity and/or receptor half-life, may account for the differences in
binding capacity.
we measured (1.4 nM for
control HUVE) is consistent with K
values
reported for many tumor promoter- or growth factor-stimulated cell
types, whose unstimulated counterparts display a
K
of about 0.2-0.5 nM (Vassalli, 1994). Consistent with our findings, other authors have
reported numbers of uPA binding sites/cell varying from 1.3 to 6.5
10
and K
values
ranging from 0.5 to 5 nM in different strains of umbilical
vein-derived endothelial cells (Barnathan et al., 1990;
Haddock et al., 1991). It should be noted, however, that the
number of uPA binding sites/cell may not reflect the real number of
uPAR molecules per cell, as it is known that uPAR can be cleaved by uPA
and/or plasmin to yield a molecule incapable of binding uPA
(H-Hansen et al., 1992). This mechanism may in part
affect the determination of the number of uPAR molecules/cell both
between different cell types and between different samples of the same
cell line, particularly in those cases in which uPA expression is
increased.
uPA
uPAR complex (Cubellis et al., 1990;
Estreicher et al., 1990), and it has been proposed that this
may also be the mechanism by which PAI-1 decreases uPA-uPAR
vitronectin-mediated cell adhesion (Waltz et al., 1993; Wei
et al., 1994). Thus, uPA, uPAR, and PAI-1 may be key mediators
both in regulating pericellular proteolytic activity and in modulating
cellular adhesion. The increased expression of both uPA and uPAR in
endothelial cells in response to the angiogenic factor VEGF suggests
that the autocrine interaction between uPA and uPAR on the endothelial
cell surface is important for ECM degradation and endothelial cell
migration during capillary morphogenesis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.