Tumor Necrosis Factor-
Regulates Expression of Vascular
Endothelial Growth Factor Receptor-2 and of Its Co-receptor
Neuropilin-1 in Human Vascular Endothelial Cells*
Enrico
Giraudo
,
Luca
Primo,
Enrica
Audero,
Hans-Peter
Gerber§,
Pieter
Koolwijk¶,
Shay
Soker**,
Michael
Klagsbrun
,
Napoleone
Ferrara§, and
Federico
Bussolino
From the Vascular Biology Laboratory, Department of Genetics,
Biology and Biochemistry, Medical School, University of Torino,
Torino, 10126 Italy, the § Department of Cardiovascular
Research, Genentech, Inc., South San Francisco, California 94080, the ¶ Department of Vascular and Connective Tissue Research
Gaubius, Laboratory TNO-PG, 2333 CK Leiden, The Netherlands,
** Departments of Urology and Surgery, Children's Hospital,
Harvard Medical School, and the 
Departments of Surgery
and Pathology, Children's Hospital, Harvard Medical School, Boston,
Massachusetts 02115
 |
ABSTRACT |
Tumor necrosis factor-
(TNF-
) modulates
gene expression in endothelial cells and is angiogenic in
vivo. TNF-
does not activate in vitro migration
and proliferation of endothelium, and its angiogenic activity is
elicited by synthesis of direct angiogenic inducers or of proteases.
Here, we show that TNF-
up-regulates in a dose- and
time-dependent manner the expression and the function of
vascular endothelial growth factor receptor-2 (VEGFR-2) as well as the expression of its co-receptor neuropilin-1 in human endothelium. As
inferred by nuclear run-on assay and transient expression of VEGFR-2
promoter-based reporter gene construct, the cytokine increased the
transcription of the VEGFR-2 gene. Mithramycin, an inhibitor of binding
of nuclear transcription factor Sp1 to the promoter consensus sequence,
blocked activation of VEGFR-2, suggesting that the up-regulation of the
receptor required Sp1 binding sites. TNF-
increased the cellular
amounts of VEGFR-2 protein and tripled the high affinity
125I-VEGF-A165 capacity without affecting the
Kd of ligand-receptor interaction. As a
consequence, TNF-
enhanced the migration and the wound healing
triggered by VEGF-A165. Since VEGFR-2 mediates angiogenic
signals in endothelium, our data indicate that its up-regulation is
another mechanism by which TNF-
is angiogenic and may provide
insight into the mechanism of neovascularization as occurs in
TNF-
-mediated pathological settings.
 |
INTRODUCTION |
A well regulated angiogenesis is critical for embryonic growth,
bone remodeling, menstrual cycle, corpus luteum formation, and tissue
repair. The stable vascular bed occurring in these physiologic
conditions results from a balance of signals that favor angiogenesis
and those that promote vascular regression. In contrast, a deregulated
angiogenesis is pivotal in tumor progression and inflammatory and viral
diseases (1-3). A number of naturally occurring growth factors can
directly induce angiogenesis by stimulating endothelial cell
proliferation and migration or act indirectly by triggering endothelial
cells themselves or accessory cells (monocyte/macrophage, mastocytes, T
cells) to release direct angiogenic inducers (1-3).
Tumor necrosis factor-
(TNF-
)1 is a powerful
activator of angiogenesis in vivo in several animal models
when used at low doses (4-6) but is inhibitory at high doses (7).
However, the ability of TNF-
to induce in vitro
biological responses related to angiogenesis is weak. TNF-
stimulates in vitro chemotaxis of bovine adrenal capillary
endothelial cells (4) but inhibits wound repair (8) and is devoid of
mitogenic activity (5). Angiogenesis promoted by TNF-
seems
necessarily to be due to indirect effects. TNF-
activates in
endothelial cells the synthesis of B61 (9), basic fibroblast growth
factor (FGF) (10), and platelet-activating factor (11), all known to be
angiogenic (6, 12), and of tissue factor (13), which is a regulator of
vessel formation (14). In endothelial cells, TNF-
promotes the
synthesis of urokinase-type plasminogen activator (15), which is
involved in the progression phase of angiogenesis characterized by a
remodeling of extracellular matrix proteins by proteolytic enzymes
(Ref. 16; for reviews, see Refs. 1-3).
TNF-
cooperates with basic FGF, vascular endothelial growth factor-A
(VEGF-A), and interleukin-8 to induce capillary-like tubular structure
of human microvascular endothelial cell growth in a three-dimensional
gel of extracellular matrix proteins (17, 18). In these systems, the
type of extracellular matrix seems to address the features of the
angiogenic model. TNF-
does not induce angiogenesis in
vitro when the cells are plated on three-dimensional fibrin
matrix, but it is permissive for the activity of basic FGF and VEGF-A.
TNF-
up-regulates the activity of urokinase-type plasminogen
activator, which is required for the formation of capillary structure
in addition to the angiogenic molecules (17). Otherwise, TNF-
induces in vitro angiogenesis of endothelium plated on
collagen type I. This activity is mediated by the release of
VEGF-A, basic FGF, and interleukin-8 (18). Furthermore, TNF-
induces
mesenchymal or tumor cells to release angiogenic molecules, including
VEGF-A (7, 19). Finally, it has been reported that TNF-
regulates
the expression of integrins involved in adhesion of endothelial
cells to extracellular matrix and in angiogenesis (8, 20).
The puzzling effects of TNF-
on endothelial cells and new vessel
growth suggest the presence of more than one angiogenic signaling
pathway and that this cytokine may have different activities on
endothelial cells depending on the microenvironment. In light of the
relevance of the cooperation between TNF-
and VEGF-A (7, 17, 18) in
angiogenesis, we studied the effect of TNF-
on the expression and
function of VEGF receptors. Adult endothelial cells express on their
surface VEGF receptor (VEGFR)-1 encoded by Flt-1 (21) and
VEGFR-2 by KDR/Flk-1 (22, 23), but recent findings suggest
that the latter alone is able to mediate the mitogenic and chemotactic
effect of VEGF-A in endothelial cells (22, 24, 25). More recently, it
has been reported that neuropilin-1, a receptor that mediates neuronal
cell guidance (26), is expressed by endothelial cells and enhances the
binding of VEGF-A165 isoform to VEGFR-2 (27). Here, we
demonstrate that the pretreatment of endothelial cells with TNF-
is
followed by an increased migration and wound repair induced by
VEGF-A165. An augmented expression of VEGFR-2 and
neuropilin-1 genes causes this effect.
 |
EXPERIMENTAL PROCEDURES |
Cell Cultures--
Human umbilical vein endothelial cells,
prepared and characterized as described previously (28), were growth in
medium 199 (Life Technologies, Inc.) supplemented with 20% fetal calf
serum (FCS) (Irvine, Santa Ana, CA), endothelial cell growth supplement (100 µg/ml), porcine heparin (50 units/ml), 100 units/ml of
penicillin, and 100 µg/ml of streptomycin (all from Sigma), in
gelatin (Life Technologies, Inc.)-coated tissue culture plates (Falcon,
Becton Dickinson, Plymouth, UK). They were used at early passages
(I-III). Human fibrosarcoma 8378 cells, which respond to TNF-
(29), were maintained in Dulbecco's modified Eagle's medium containing 10%
FCS. Porcine aortic endothelial cells transfected with human VEGFR-2 (24) were cultured in Ham's F-12 (Sigma) supplemented with
10% FCS. Human foreskin microvascular endothelial cells isolated as
described previously (17), were cultured on fibronectin-coated dishes
in medium 199 buffered with 20 mM Hepes containing 10% human serum, 10% newborn calf serum, endothelial cell growth
supplement (150 µg/ml), and porcine heparin (5 units/ml).
Experimental Design--
To verify the effects of TNF-
on the
expression of VEGF receptors and on the biological activities elicited
by VEGF-A165, the following experimental conditions have
been used: confluent endothelial cell growth at a CO2 level
of 5% in atmospheric air was treated with TNF-
(1 × 107 units/mg of protein; Genentech, Inc., San Francisco,
CA) in medium 199 supplemented with 20, 5, and 1% FCS or 1% bovine
serum albumin (BSA) (lipopolysaccharide-free, Sigma), twice washed with
medium 199, and then used to extract RNA. Alternatively, cells were
stimulated with VEGF-A165 (a gift of Dr. H. A. Weich,
GBF, Braunschweig, Germany) (30) in medium 199 containing 1% FCS in
chemotaxis or 3% BSA in wound healing experiments. In some
experiments, endothelial cells were starved for 24 h in medium 199 containing 1% FCS and 1% BSA before adding TNF-
. The effect of
mithramycin (Sigma), which inhibits gene expression by blocking Sp1
binding to the CG box (7, 31), was studied by treating the cells for
12 h in medium 199 containing 5% FCS with or without TNF-
.
RNA Extraction and Northern Analysis--
Total cellular RNA was
isolated by guanidinium isothiocyanate extraction and centrifugation
through cesium chloride (32). Equal amounts of total RNA (15 µg/lane)
were electrophoresed in 1% agarose gels containing 6.3% formaldehyde
in MOPS buffer (Sigma) and blotted on a Nylon Duralon-UV membrane
(Stratagene) by the traditional capillary system in 10× SSC (1.5 M NaCl, 150 mM sodium citrate, pH 7) (32).
Filters were cross-linked with UV light (0.5 J/cm2) and
prehybridized for 4 h at 42 °C in 50% formamide deioinizate, 10% dextran sulfate, 1% SDS, 1 M NaCl, and 100 µg/ml
denatured salmon sperm DNA. Hybridization was carried out overnight at
42 °C with [
-32P]dCTP-labeled (3000 Ci/mmol,
Amersham, Buckinghamshire, United Kingdom, UK) human VEGFR-2 (a
0.729-kb HindIII-EcoRI of KDR cDNA) (23),
VEGFR-1 (a 1.347-kb HindIII-BglII fragment of
human FLT-1 cDNA) (21), neuropilin-1 (a 0.735-kb
PstI-PstI fragment of human neuropilin-1
cDNA) (27) and
-actin cDNAs (33). cDNAs were labeled
using Rediprime random primer labeling kit (Amersham) according to
manufacturer's instructions. Posthybridization washes were
performed at high stringency (once in 2× SSC, 0.1% SDS for 30 min,
once in 0.2× SSC, 0.1% SDS for 30 min and twice in 0.1× SSC, 0.1%
SDS for 30 min) at 57 °C, and the membranes were exposed on
autoradiography with Hyperfilm-MP (Amersham).
Nuclear Run-on--
Nuclei were isolated from cultured
endothelial cells essentially according to Ref. 34. Briefly, cells
(2 × 107 cells/assay) were washed twice with ice-cold
phosphate-buffered saline (PBS), scraped and collected in a 15-ml
centrifuge tube by centrifugation at 500 × g for 5 min
at 4 °C. Subsequent steps were performed at 4 °C. The cells were
resuspended in 4 ml of lysis buffer (10 mM Tris-HCl, pH
7.4, 10 mM NaCl, 3 mM MgCl2, 0.5%
Nonidet P-40) and allowed to stand on ice for 5 min. and then
centrifuged at 500 × g at 4 °C for 5 min. Nuclei
were resuspended in 200 µl of glycerol storage buffer (10 mM Tris-HCl, pH 8.3, 40% (v/v) glycerol, 5 mM
MgCl2, 0.1 mM EDTA) and frozen in liquid N2. In vitro transcription and isolation of the
resulting nuclear RNA were performed as described by Ikeda et
al. (35). Two-hundred µl of frozen nuclei were thawed and mixed
with 200 µl of 2× reaction buffer (10 mM Tris-HCl, pH
8.0, 5 mM MgCl2, 300 mM KCl 10 mM dithiothreitol, 400 units/ml placental ribonuclease
inhibitor (Stratagene), 20 mM creatine phosphate (Sigma),
200 µg/ml creatine phosphokinase (Sigma), a 1 mM
concentration each of ATP, CTP, and GTP (Stratagene), and 100 µCi of
[
-32P]UTP (3000 Ci/mmol, Amersham). Samples were
incubated at 30 °C for 30 min with shaking and for 5 min in the
presence of 20 units of DNase I (RNase-free, Life Technologies, Inc.).
After the addition of proteinase K (150 µg/ml, Sigma) and SDS (0.5%
final concentration), incubation was continued at 37 °C for 30 min.
Extracted RNA was resuspended in TES buffer (10 mM TES, pH
7.4, 10 mM EDTA, 0.2% SDS) at 5 × 106
cpm/ml. Linearized plasmids containing the target cDNAs (15 µg) were immobilized onto a nylon Duralon-UV membrane (Stratagene) using a
Bio-Dot SF microfiltration apparatus (Bio-Rad). The filters were
prehybridized overnight at 42 °C with hybridization buffer containing 20 mM PIPES (Sigma), pH 6.4, 50% formamide
(Sigma), 2 mM EDTA, 0.8 M NaCl, 0.2% SDS, 1×
Denhardt's solution (0.02% Ficoll, 0.02% BSA, 0.02%
polyvinylpyrrolidone), 200 µg/ml E. coli tRNA (RNase-free,
Stratagene). Hybridization was at 42 °C for 48 h in the same
solution supplemented with 15 × 106 total cpm of
labeled RNA. The filters were washed twice in 2× SSC, 0.5% SDS at
42 °C for 30 min, twice in 0.3× SSC, 0.5% SDS, at 42 °C for 30 min and then incubated with 10 µg/ml RNase A in 2× SSC at 37 °C
for 30 min. Further washed were done in 2× SSC at 37 °C for 30 min
and then in 0.3× SSC at 37 °C for 30 min. The filters were exposed
on autoradiography with Hyperfilm-MP and intensifying screens at
80 °C. The amount of VEGFR-2 mRNA was standardized by
comparison with the amount of
-actin mRNA. Densitometric
analysis was performed with a GS250 Molecular Imager (Bio-Rad).
Western Blot--
Endothelial cells were washed twice with PBS
and lysed on ice with 1 ml of 50 mM Tris buffer (pH 7.5)
containing 150 mM NaCl, 0.1% Triton X-100, 10% glycerol,
5 mM EDTA, 50 µg/ml pepstatin, 100 µg/ml leupeptin, 10 µg/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride, 500 µg/ml soybean trypsin inhibitor (all from Sigma). After
centrifugation (20 min at 4 °C at 13,000 × g),
protein were solubilized, separated by SDS-polyacrylamide gel
electrophoresis (7%), transferred onto polyvinylidene difluoride
membranes (Immobilon, Millipore Corp., Bedford, MA), probed with rabbit
anti-VEGFR-2 antibody (C-1158, Santa Cruz Biotechnology, Inc., Santa
Cruz, CA), and detected by ECL (Amersham).
Binding Assay and Analysis--
Recombinant
VEGF-A165 (2 µg) was dissolved in 200 µl of sodium
phosphate buffer 20 mM, pH 7.4, and transferred in
IODO-GEN-coated tubes (50 µg/ml) (Pierce), where
VEGF-A165 was iodinated (5 min, 4 °C) with 1 mCi of
125I (Amersham). Twenty µl of phosphate buffer 20 mM, pH 7.2, containing 1% BSA, 0.4 M NaCl,
0.1% CHAPS (Pierce) was added, and the reaction products were
separated on Sephadex-G10. The specific activity of the tracer was
90,000 cpm/ng. 125I-VEGF-A165 retained its
biological activity as measured by migration of endothelial cells (28).
For specific binding studies confluent cells plated in 24-well plates
were incubated an orbital shaker at 4 °C for 2 h in 200 µl/well of binding medium (medium 199 containing 20 mM
Hepes buffer, pH 7.4, 0.1% BSA, 100 µg/ml soybean trypsin inhibitor)
with increasing concentrations of
125I-VEGF-A165 in the presence of a 100-fold
excess of unlabeled ligand. Endothelial cells were washed three
times with ice-cold PBS containing 0.1% BSA and lysed in 200 µl/well
of SDS 2% in PBS. Lysates were counted using a Beckman
5500B
counter. Triplicate samples under each condition were obtained for each
experiment. Specific binding, calculated subtracting from the total cpm
bound after incubation with a 100-fold excess of unlabeled
ligand, was approximately 80%. The Kd was estimated
by Scatchard plot using the Ligand program (Elseviere-Biosoft,
Cambridge, UK).
Transient Transfection of pGL2basicFLK Plasmid--
The 2.0-kb
XhoI/SacI fragment of Flk-1 promoter
(36) was subcloned in pGL2basic plasmid (Promega, Madison, WI) to
generate the luciferase reporter vector
pGL2basicFlk-1.2 Human
fibrosarcoma 8387 cell line (2 × 105 cells/well) was
transfected with 3 µg of pGL2basicFlk-1 or pGL2basic using Superfect
Transfection Reagent according to the manufacturer's instructions
(Qiagen, Inc., Valencia, CA).The generated plasmid-liposome complex in
0.6 ml of Dulbecco's modified Eagle medium containing 10% FCS was
incubated with the cells for 3 h at 37 °C in 5%
CO2. The medium was then replaced with fresh medium, and
the cells were stimulated for 4 h with TNF-
(20 ng/ml) or left
untreated. pSVgal construct (2.5 µg) was co-transfected to correct
for the variability in transfection efficiency, and
-galactosidase
activity was assayed with chlorophenol red
-D-galactopyranoside (Boehringer Mannheim GmbH,
Mannheim, Germany) as a substrate. For final luciferase assay, cells
were lysed in 0.2 ml of passive lysis buffer (dual luciferase assay,
Promega) at 4 °C, and 20 µl of cleared (12,000 × g for 2 min at 4 °C) cell extract containing 50 µg of
protein were mixed with 0.1 ml of luciferase assay buffer. Light
production was measured for 5 s in a luminometer (Magic Lite
Analyzer, Ciba Corning, Milano, Italy), and results were normalized to
the
-galactosidase activity.
Migration Assay--
Migration assay was performed as described
previously with Boyden's chamber technique (28). Polycarbonate filters
(5-µm pore size polyvinylpyrrolidone-free; Neuroprobe, Pleasanton,
CA) were coated with 0.1% gelatin for 6 h at room temperature.
VEGF-A165 in medium 199 supplemented with 1% FCS was
seeded in the lower compartment of the chamber, and 1.25 × 105 resuspended cells in 50 µl of medium 199 containing
1% FCS were then seeded in the upper compartment. At the end of the
incubation (37 °C in air with 5% CO2 for 6 h),
filters were removed and stained with Diff-Quik (Baxter Spa, Rome,
Italy), and 10 high power oil immersion fields were counted. The
results obtained were analyzed by one-way analysis of variance and the
Student-Newman-Keuls test (Statistic Software; Bio-Soft).
Wound Healing--
Human endothelial cells were grown at
confluence on 24 wells and monolayers were treated for 24 h with
TNF-
(10 ng/ml) or vehicle alone in medium 199 containing 5% FCS.
After washes, the monolayer was wounded with a razor blade (lesion
surface: 20 mm2) as described (28) and incubated in medium
199 containing 3% BSA with or without VEGF-A165. After
24 h, the cells were fixed and stained as described (28). To
quantify the repair process, phase-contrast microscopic pictures of
wounded monolayer were recorded with a still video camera recorder
(R5000H; Fuji Photo Film Co., Tokyo, Japan), and cell number was
counted in 10 fields of 1 mm2 randomly selected, with a
Cosmozone image analyzer (Nikon, Tokyo, Japan).
 |
RESULTS |
Increase of VEGFR-2 and Neuropilin-1 mRNA in Endothelial Cells
Challenged with TNF-
--
Several studies have shown that 7.0-kb
VEGFR-2 (23, 37) and 7.5-kb VEGFR-1 mRNAs (21) are expressed
constitutively by endothelial cells in culture (37, 38). The second
band of 3.4 kb recognized by VEGFR-2 cDNA represents an alternative
transcript, as previously reported (37). Furthermore, neuropilin-1 has
been recently demonstrated to be a specific co-receptor of VEGFR-2 for
the binding of VEGF-A165 isoform (27). TNF-
stimulation of endothelial cells from human umbilical cord for 24 h induced an
increase in VEGFR-2 (Fig. 1) and
neuropilin-1 (Fig. 3B) mRNA levels. The effect on
VEGFR-2 transcript was observed in different conditions of culture:
medium supplemented with 5% FCS (Fig. 1B), 20% FCS (Fig.
1C), or 1% FCS associated with 1% BSA (Fig.
1A). In medium 199 containing 20% FCS, TNF-
was active
at 1 ng/ml. As shown in Fig. 2, the
levels of VEGFR-2 mRNA began to increase after 4 h of
incubation with TNF-
(20 ng/ml), reached a maximum level after
around 24 h and was gradually reduced after 48 h. TNF-
was
also active on foreskin microvascular endothelial cells. In the basal
condition, these cells did not express amount of mRNA detectable by
Northern technique. However, after treatment with TNF-
, the VEGFR-2
mRNA was evident in microvascular endothelial cells (Fig.
3A). The expression of
VEGFR-1, the second receptor expressed on endothelial cell membrane,
was not modified by TNF-
treatment (Fig. 3B).

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Fig. 1.
Effect of TNF- on VEGFR-2 mRNA
expression in human endothelial cells. Northern blot analysis of
total RNA extracted from confluent endothelial cells stimulated for
24 h with TNF- in medium 199 containing 1% FCS and 1% BSA
(A), 5% FCS (B), or 20% FCS (C).
Fifteen µg of total RNA were run in a formaldehyde-agarose gel and,
after blotting to Duralon membrane, hybridized to VEGFR-2 cDNA
labeled with [ -32P]dCTP. Transcripts have been
visualized by autoradiography. The lower panel
displays an image of the respective ethidium bromide-stained nylon
membranes to demonstrate even loading and transfer. This experiment is
representative of three performed with similar results.
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Fig. 2.
Time course of TNF- -induced up-regulation
of VEGFR-2 mRNA expression in human endothelial cells.
A, VEGFR-2 mRNA level from starved and confluent
endothelial cells stimulated with 20 ng/ml TNF- in medium 199 containing 20% FCS was determined by Northern blotting as detailed in
the legend to Fig. 1. B, an image of the respective ethidium
bromide-stained nylon membranes to demonstrate even loading and
transfer. This experiment is representative of two experiments
performed with similar results.
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Fig. 3.
Effect of TNF- on expression of VEGFR-2
mRNA in human foreskin endothelial cells (A), of
neuropilin-1 mRNA (B), and of VEGFR-1 mRNA in human
endothelial cells from umbilical cord (C).
A, confluent human endothelial cells from vein cord
(lanes 1 and 2) and from foreskin
endothelium (lanes 3 and 4) were
stimulated for 24 h with 20 ng/ml TNF- in medium 199 containing
10% (foreskin endothelium) or 20% FCS (umbilical endothelial cells)
(lanes 2 and 4) or vehicle alone
(lanes 1 and 3). VEGFR-2 mRNA was
determined by Northern blotting as detailed in the legend to Fig. 1.
B, confluent human endothelial cells from vein cord were
stimulated for 24 h with 20 ng/ml TNF- in medium 199 containing
5% FCS (lane 2) or vehicle alone
(lane 1). Neuropilin-1 mRNA was determined by
Northern blotting as detailed in the legend to Fig. 1. C,
confluent human endothelial cells from vein cord were stimulated for
24 h with 20 ng/ml TNF- in medium 199 containing 5% FCS
(lane 2) or vehicle alone (lane
1). VEGFR-1 mRNA was determined by Northern blotting as
detailed in the legend to Fig. 1. The lower
panels display an image of the respective ethidium
bromide-stained nylon membrane to demonstrate even loading and
transfer. These experiments are representative of two performed with
similar results.
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Since it has been reported that TNF-
reduces the VEGFR-2 mRNA
expression in human endothelial cells (39), two Northern blots have
been performed in two different laboratories (Dr. M. Introna, "Mario
Negri" Institute, Milano, Italy; Dr. V. van Hinsbergh, TNO Prevention
and Health, Leiden, The Netherlands) with results similar to those
shown in Fig. 1. Furthermore, a marked increase of VEGFR-2 mRNA was
also observed in porcine aortic endothelial cells transfected with the
VEGFR-2 gene and stimulated with TNF-
(20 ng/ml) for 12 h (data
not shown).
Induction of VEGFR-2 Gene Expression by TNF-
--
In order to
investigate whether TNF-
activates transcription of the VEGFR-2 gene
in endothelial cells, nuclear run-on assay was performed. Nuclei were
prepared from endothelial cells cultured with medium alone and with 20 ng/ml of TNF-
for 4, 24, and 48 h, and RNAs transcribed from
these nuclei were hybridized with VEGFR-2 cDNA. We observed that
TNF-
increased the transcription rate of VEGFR-2, without affecting
the transcription of
-actin gene and of pBluescript plasmid, used as
negative control (Fig. 4). The
densitometric analysis done on three independent run-on assays showed
that the transcriptional rate of the VEGFR-2 gene was respectively
elevated of 1.2 ± 0.6-, 6.0 ± 0.5-, and 3.5 ± 0.3-fold after 4, 24, and 48 h of TNF-
incubation.

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Fig. 4.
Nuclear run-on analysis after exposure of
endothelial cells to TNF- . Nuclei were prepared from cells
incubated in medium 199 containing 5% FCS with or without 20 ng/ml
TNF- . Transcription in the isolated nuclei was analyzed by
hybridization of 32P-labeled RNA to 15 µg of VEGFR-2,
-actin, and pBluescript cDNAs immobilized on nitrocellulose
membrane. This experiment is representative of two experiments
performed with similar results.
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|
To further confirm that TNF-
regulates the transcription of VEGFR-2
gene, the mouse VEGFR-2 promoter-based reporter gene construct (36) was
transiently transfected in 8378 human fibrosarcoma cells, which were
treated for 4 h with TNF-
(20 ng/ml). This cell line was
selected instead of endothelial cells, because it was more easily
transfected with the construct than endothelial cells and expresses a
functional active TNF receptor (29). Analysis of the respective
luciferase expressions in untreated and TNF-
-stimulated cells
revealed notable basal activity of the pGL2basicFlk-1 construct, which
was increased after treatment with the cytokine by a factor of 2 (Fig.
5).

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Fig. 5.
Activity of VEGFR-2 promoter-based luciferase
construct in human fibrosarcoma 8387 cell line. Cells were
transfected with 3 µg of pGL2basicFlk or pGL2basic and subsequently
treated for 4 h with 20 ng/ml TNF- or vehicle alone. Extracts
were analyzed for luciferase level. Transfection efficiency was
corrected by cotransfection with pSV gal. Mean ± S.D. of three
experiments performed in duplicate is shown.
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Human and mouse VEGFR-2 promoter contain five Sp1 elements (40), a
transcription factor involved in TNF-
-induced gene expression in
endothelial cells (41). We evaluated the role of Sp1 elements in the
regulation of VEGFR-2 transcription by incubating endothelial cells
with mithramycin, an inhibitor of the binding of the transcription factor to the CG box (7, 31). Fig. 6
shows that mithramycin at 1 and 10 nM inhibited the
TNF-
-induced expression of VEGFR-2, suggesting that Sp1 binding
sites in the promoter are involved in up-regulation of VEGFR-2
molecules.

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Fig. 6.
Effect of mithramycin on endothelial cell
increase in VEGFR-2 mRNA levels by TNF- . Cells were
stimulated in medium 199 supplemented with 5% FCS with 20 ng/ml
TNF- in the absence or presence of 1 and 10 nM
mithramycin. The cellular levels of VEGFR-2 transcript were determined
by Northern blotting as detailed in the legend to Fig. 1. The
lower panel displays the Northern blot performed
with -actin cDNA. This experiment is representative of two
experiments done with similar results.
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|
TNF-
Increased the Expression of VEGFR-2 on Endothelial Cell
Surface--
The effect of TNF-
on the up-regulation of VEGFR-2
expression was further investigated with the analysis of
125I-VEGF-A165-specific binding at equilibrium
on the endothelial cell surface. Since TNF-
does not up-regulate
VEGFR-1 (Fig. 3C), the binding studies were performed by
incubating endothelial cells with
125I-VEGF-A165 concentrated to 50 pM or higher. This experimental condition excluded the
analysis of the binding of VEGF-A165 to VEGFR-1 (42), which
has a Kd ranging from 9 to 16 pM (24,
43), and indicated a single high affinity VEGF-A165 binding site on endothelial cells (Fig.
7A). Cell treatment for
24 h with TNF-
(20 ng/ml) in medium 199 containing 5% FCS
produced a significant increase in 125I-VEGF binding to
cell surface. Nonlinear regression analysis (Fig. 7B) of the
data reported in Fig. 7A indicated a Kd = 137 ± 23 pM (n = 3) in untreated
cells and a Kd = 122 ± 21 pM
(n = 3) in TNF-
-treated cells. In contrast, TNF-
triplicates the number of binding sites expressed on cell membrane (in
untreated cells, Bmax = 79 ± 14 fmol; in
TNF-
-treated cells, Bmax = 243 ± 16 fmol, n = 3). Similar results have been obtained with
TNF-
-treated endothelial cells in presence of 20% FCS (data not
shown).

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Fig. 7.
Effect of TNF- on binding of radiolabeled
VEGF-A165 to human endothelial cells. A,
specific ligand binding curve. Monolayers were incubated with TNF-
(20 ng/ml) ( ) or vehicle alone ( ) for 24 h in medium 199 containing 5% FCS. After washes, cells were incubated with indicated
concentrations of 125I-VEGF-A165 for 2 h
at 4 °C in the presence of a 100-fold excess of cold ligand.
B, Scatchard plot of the data reported in A. The
data shown are representative of three experiments.
|
|
To analyze the expression of the protein encoded by VEGFR-2 gene,
endothelial cells were treated with TNF-
for 24 h, and then the
proteins from cell lysate were separated by SDS-polyacrylamide gel
electrophoresis and probed with anti-VEGFR-2 antibodies. Fig. 8 shows that TNF-
treatment increased
the amount of a 210-kDa protein recognized by an antibody anti-VEGFR-2.
TNF-
did not modify the expression of proteins recognized by
antibody anti-VEGFR-1 (data not shown).

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|
Fig. 8.
Western blot analysis of VEGFR-2 in human
endothelial cells stimulated with TNF- . Confluent endothelial
cell monolayers were treated for 24 h with TNF- in medium 199 containing 5% FCS. 200 µg of lysed proteins were separated by
SDS-polyacrylamide gel electrophoresis (7%), blotted onto
polyvinylidene difluoride membrane, probed with rabbit anti-VEGFR-2
antibody, and detected by enhanced chemiluminescence.
|
|
Effect of TNF-
on VEGF-A165-induced Endothelial Cell
Migration--
VEGF-A165 induced in a
dose-dependent manner the migration of endothelial cells as
evaluated by the Boyden chamber technique in agreement with previous
observations (24, 44). The maximal migration was obtained with a
concentration of VEGF-A165 of 10 ng/ml (3.7-fold the
control value, p < 0.05). TNF-
alone did not
influence endothelial cell migration. However, when endothelial cells
were stimulated for 24 h with TNF-
(20 ng/ml) in the presence of 20% FCS (Fig. 9) or lower FCS amounts
(1 and 5%; not shown), they showed an increased motility after
challenge with VEGF-A165. TNF-
treatment sensitized
endothelial cells to an ineffective dose of VEGF-A165 (1 ng/ml) and was able to double the number of migrating cells stimulated
with VEGF-A165 used at optimal concentration (10 ng/ml)
(Fig. 9). The migration of endothelial cells triggered by hepatocyte
growth factor (28) was not enhanced by cell treatment with TNF-
,
suggesting that the effect of this cytokine is specific for
VEGF-A165 (Table I).

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|
Fig. 9.
Effect of TNF- on migration of human
endothelial cells elicited by VEGF-A165. The migration
of cells was measured by the modified Boyden chamber technique, as
described under "Experimental Procedures." Human endothelial cells
were treated with 20 ng/ml TNF or vehicle alone for 24 h in medium
199 with 5% FCS. Suspended cells (1.25 × 105) in
medium 199 containing 1% FCS were seeded in the upper compartment of
the chamber, and VEGF-A165 suspended in the same medium
containing 1% FCS was placed in the lower compartment. Cells that
migrated after 6 h of incubation to the lower surface of the
filter were counted after coding samples. The numbers are
the mean ± S.D. of eight experiments performed in triplicate. *,
p > 0.05.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Effect of TNF- on endothelial cell migration stimulated by
VEGF-A165, and hepatocyte growth factor
Endothelial cells were pretreated with TNF- (20 ng/ml) or vehicle
alone for 24 h and then stimulated in Boyden's chamber with 10 ng/ml each molecule in medium 199 containing 1% FCS and processed as
detailed in the legend of Fig. 1. The values are the means ± S.D.
of three experiments done in triplicate.
|
|
Effect of TNF-
on VEGF-A165-induced Wound Repair in
Endothelial Cell Monolayers--
A wound healing assay in
vitro, i.e. the ability of filling artificial gaps
created in cell monolayers, requires both cell growth and activation of
cell movements. In preliminary experiments, we demonstrated that
VEGF-A165 used at 10 ng/ml induced repair of mechanical
wounds generated in human endothelial cell monolayers within 24 h
but was ineffective at 1 ng/ml. The experiment given in Fig.
10 shows that pretreatment of the cells
with TNF-
(20 ng/ml) for 24 h evidently enhances the
VEGF-A165-induced wound repair. Table
II provides a quantitative analysis of
cells migration into and across the wound, supporting the qualitative
experiment in Fig. 10.

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|
Fig. 10.
Effect of TNF- on wound healing of human
endothelial cell monolayer induced by VEGF-A165.
Confluent endothelial cell monolayers were treated for 24 h with
TNF- (20 ng/ml) in medium 199 containing 5% FCS and washed twice
with medium without FCS. The monolayer was wounded with a cross-shaped
scratch and stimulated with VEGF-A165 (1 ng/ml) for 24 h in medium 199 containing 3% BSA. At the end of incubation, cells
were fixed and stained by crystal violet. Magnification was ×2. The
picture is representative of five experiments with similar
results.
|
|
View this table:
[in this window]
[in a new window]
|
Table II
Effect of TNF- on endothelial cell wound repair stimulated by
VEGF-A165
Endothelial cell monolayer was treated with TNF- (10 ng/ml) or
vehicle alone for 24 h and then wounded with a razor blade. After
washes, cells were stimulated with VEGF-A165 (1 ng/ml) in
medium 199 containing 3% BSA and processed as described in the legend
of Fig. 2. At the end of stimulation, the wounded area was recorded
with a still video camera recorder. Cells in 10 fields of 1 mm2
were counted with an image analyzer. The values are the means ± S.D. of 10 fields in one experiment representative of three performed
with similar results.
|
|
 |
DISCUSSION |
TNF-
is an inflammatory cytokine with a wide spectrum of
biological activities including angiogenesis (1, 45, 46). TNF-
acts
particularly on the formation of new vessels by multiple indirect ways
instead of promoting directly the sprout of endothelial cells and their
growth, as the direct angiogenic inducers. The release of direct
angiogenic molecules and up-regulation of proteolytic systems seem to
be the biological events triggered by TNF-
to participate in
angiogenesis (6, 7, 10, 12, 13, 17-19). In this report, we add a new
piece to this mosaic, showing that TNF-
increases the transcription
rate of the VEGFR-2 gene in vascular endothelial cells, resulting in
augmented number of molecules expressed on cell surface and enhances
the biological response of endothelial cells to VEGF-A165.
This statement is based on five major observations: 1) in the binding
analysis performed with 125I-VEGF-A165
concentrated to 50 pM or higher in order to render negligible the contribution of VEGFR-1 (42), TNF-
triples the number
of high affinity binding sites for VEGF-A165 on endothelial membrane without affecting the affinity of the receptor for the ligand;
2) this effect is coupled to an early increase of mRNA expression
of VEGFR-2, whereas the VEGFR-1 transcript is unchanged; 3) the
up-regulation of VEGFR-2 mRNA results from an increase of the
transcription as demonstrated by nuclear run-on assay and by the mouse
VEGFR-2 promoter activation transfected in a human fibrosarcoma cell
line responsive to and challenged with TNF-
; 4) human endothelial
cells pretreated with TNF-
are more responsive to
VEGF-A165 than untreated cells in terms of migration and
ability to repair a wounded monolayer; 5) the enhancement effect of
TNF-
on endothelial cell migration is not observed when hepatocyte growth factor, an activator of endothelial motility (28), is used as
stimulus, suggesting a relative specificity of the system.
The effect of TNF-
on mRNA expression of VEGFR-2 is
dose-dependent and consistently detected with 1 ng/ml
cytokine. The mRNA expression appears within 4 h after TNF-
stimulation and persists up to 24 h and then declines to basal
level within 48 h. This time course is similar to that of other
genes activated by TNF-
to direct endothelial cells toward a
proinflammatory phenotype (47). Among these genes, E-selectin and
vascular cell adhesion molecule-1 regulate leukocyte transmigration,
but the soluble forms of the encoded proteins have been reported to be
angiogenic too (48).
The control of VEGFR-2 transcription is entrusted by a promoter
characterized by putative binding sites for AP-2, Sp1, and NF-
B
transcription factors (36, 40). The TNF-
-induced activation of
VEGFR-2 was inhibited by mithramycin, an inhibitor of Sp1 interaction with its consensus sequence (7, 31), suggesting that the rapid increase
of VEGFR-2 might thus be mainly mediated through the activation of Sp1
in endothelial cells. Recently, it has been reported in endothelial
cells that Sp1 is the major nuclear protein binding to VEGFR-2 promoter
(49) and that TNF-
up-regulates Sp1 transcription and expression
with a time course similar to that described in this study for VEGFR-2
(41). Sp1 oligonucleotide antisense inhibits the stimulating effect of
TNF-
on VEGF-A production by endothelium and on in vitro
angiogenesis (18). Since the VEGF-A promoter has Sp1 binding sites
(50), Yoshida and co-workers have hypothesized that inhibition of
VEGF-A synthesis may be the mechanism by which TNF-
-induced in
vitro angiogenesis is affected (18). Viewed in light of the
results shown here, it is also reasonable to explain the inhibition of
Sp1 oligonucleotide antisense on angiogenesis as an effect on the
mechanisms leading to the up-regulation of VEGFR-2 induced by
TNF-
.
TNF-
also increases the transcription of neuropilin-1,
which enhances the binding of VEGF-A165 to VEGFR-2 and
VEGF-A165-mediated chemotaxis (27). The binding of
VEGF-A165 to neuropilin-1 is mediated through the amino
acid sequence encoded by exon-7, absent in other VEGF-A isoforms (25),
suggesting the high specificity of this co-receptor for
VEGF-A165 (27). Further experiments could discriminate
whether the effect of TNF-
is restricted to VEGF-A165 or
also present in other isoforms.
The observed up-regulation of VEGFR-2 by TNF-
is in disagreement
with the results published by Patterson and co-workers (39), who
demonstrated that TNF-
down-regulates VEGFR-2 expression in human
endothelial cells from veins or arteries. This discrepancy could be due
to differences in experimental conditions. However, human microvascular
endothelial cells from omental tissue increase VEGFR-2 mRNA when
challenged with TNF-
(18).
Therefore, this study brings new insight into the conditions regulating
the endothelial cell response to VEGF-A165. Previous studies on the mechanism responsible for the regulated expression of
VEGFR-2 have focused on TGF-
1 (51) or hypoxia (42). Notably, neutralizing anti-TNF-
antibodies did not neutralize the
up-regulation of VEGFR-2 by conditioned media from hypoxic cells (42).
Hypoxia does not affect directly the VEGFR-2 promoter (36), but the up-regulation is mediated by an unknown factor present in ischemic tissues (42). TGF-
1 decreases the expression of VEGFR-2 after a
prolonged time of incubation, by a presently unknown molecular mechanism (51). Indeed, TNF-
is the first identified cytokine that
increases the endothelial cell response to VEGF-A165 by a direct effect on the VEGFR-2 transcription.
Our in vivo preliminary experiments agree with our in
vitro results. The treatment of DBA2 mice with TNF-
(750 ng
intraperitoneally followed by a second dose 3 days after) allows an
ineffective angiogenic concentration of VEGF-A165 to
promote vascularization in a Matrigel plug (52) injected subcutaneously
on the day of the second TNF-
injection (vehicle-treated mice were
injected with 5 ng of VEGF-A165/0.75 ml of Matrigel
(n = 5), and vascularized area was 6 ± 4% of
total Matrigel area; vehicle-treated mice were injected with 0.75 ml of
Matrigel (n = 3), and vascularized area was 3 ± 2% of total Matrigel area; TNF-
-treated mice were injected with 5 ng of VEGF-A165/0.75 ml of Matrigel (n = 5), and vascularized area was 34 ± 10% of total Matrigel area;
TNF-
-treated mice were injected with 0.75 ml of Matrigel
(n = 5), and vascularized area was 7 ± 3% of
total Matrigel area). Involvement of VEGFR-2/VEGF-A system has recently
been demonstrated in vivo in cancer disease and in chronic
inflammation (44, 53, 54), processes in which TNF-
is markedly
up-regulated (55-57). Furthermore, we have recently demonstrated that
VEGFR-2 is also the receptor of Tat (58), a protein of immunodeficiency
virus-1 involved in the angiogenesis associated with Kaposi's sarcoma
(59). Since our results demonstrate that TNF-
presents as a potent
inducer of VEGFR-2 synthesis, the TNF-
-mediated up-regulation of the
unique VEGF receptor capable to mediate mitogenic and motogenic signals
inside endothelial cells (22, 24) is likely to play an important role
in the initiation and maintenance of angiogenesis and increased
vascular permeability in these conditions.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Lena Claesson-Welsh (Uppsala
University, Uppsala, Sweden), Dr. Martino Introna (Istituto "Mario
Negri," Milano, Italy), and Dr. P. M. Comoglio (Institute for
Cancer Research, Torino, Italy), who provided porcine aortic
endothelial cells transfected with VEGFR-2, human fibrosarcoma 8378 cells, and human recombinant hepatocyte growth factor, respectively.
Dr. Martino Introna and Victor van Hinsbergh provided mRNA of
TNF-
-stimulated and control endothelial cells. VEGFR-2 and VEGFR-1
were kindly provided by Dr. Bruce Terman (Wyeth Ayerst Research, Pearle
River, NY), and recombinant VEGF-A165 was provided by Dr.
Herbert Weich (GBF, Braunschweig, Germany).
 |
FOOTNOTES |
*
This study was supported by grants from the European
Community (Biomed-2 Project: BMHL-CT96-0669), the Italian Association for Cancer Research, Istituto Superiore Ri Sanità (X AIDS
Project, Multiple-Sclerosis Project, Program on Tumor Therapy), the
Dutch Cancer Society (TNOP 97-1511), National Institutes of Health (CA 37392 and 45548), Centro Nazionale Ricerche (P. F. Biotecnologie), and
Ministero Dell' Universita e Della Ricerca Scientifica Technologica (60%).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Supported by a grant from the Italian Foundation for Cancer
Research.
To whom correspondence should be addressed: Dept. of Genetics,
Biology and Biochemistry, Via Santena 5bis., 10126 Torino, Italy. Tel.:
39-11-6706684; Fax: 39-11-6635663; E-mail:
Bussol{at}molinette.unito.it.
The abbreviations used are:
TNF-
, tumor
necrosis factor-
; BSA, bovine serum albumin; FCS, fetal calf serum; FGF, fibroblast growth factor; PBS, phosphate-buffered saline; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; kb, kilobase
pair(s); MOPS, 3-(N-morpholino)propanesulfonic acidTES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acidPIPES, piperazine-N,N'-bis(2-ethanesulfonic
acid)CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
2
H. Gerber and J. Park, personal
communication.
 |
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