(Received for publication, February 26, 1996)
From the
Although the importance of the vascular endothelial growth
factor (VEGF)/VEGF tyrosine kinase receptor (VEGFR) system in
angiogenesis is well established, very little is known about the
regulation of VEGFR expression in vascular endothelial cells. We have
cloned partial cDNAs encoding bovine VEGFR-1 (flt)
and -2 (flk-1) and used them to study VEGFR
expression by bovine microvascular- and large vessel-derived
endothelial cells. Both cell lines express flk-1, but not flt. Transforming growth factor 1 (TGF-
1) reduced
the high affinity
I-VEGF binding capacity of both cell
types in a dose-dependent manner, with a 2.0-2.7-fold decrease at
1-10 ng/ml. Cross-linking experiments revealed a decrease in
I-VEGF binding to a cell surface monomeric protein
corresponding to Flk-1 on the basis of its affinity for VEGF, molecular
mass (185-190 kDa), and apparent internalization after VEGF
binding. Immunoprecipitation and Western blot experiments demonstrated
a decrease in Flk-1 protein expression, and TGF-
1 reduced flk-1 mRNA levels in a dose-dependent manner. These results
imply that TGF-
1 is a major regulator of the VEGF/Flk-1 signal
transduction pathway in endothelial cells.
The formation of new blood vessels is essential for the development and maintenance of all organ systems. During embryogenesis, endothelial cells of primordial vessels differentiate from mesodermal precursors called angioblasts (vasculogenesis). During subsequent organogenesis, new capillary blood vessels are formed either by vasculogenesis or originate as endothelial sprouts from pre-existing vessels (angiogenesis). These two processes lead to the formation and maintenance of the vascular tree. Angiogenesis is also responsible for physiological and pathological neovascularization which occurs in the adult organism. Tumor angiogenesis is the most extensively studied example. It is permissive for tumor growth, and following tumor cell intravasation into newly formed vessels is responsible for the formation of metastases (reviewed by Folkman(1995) and Risau and Flamme(1995)).
Endothelial cell tyrosine kinase receptors are of
fundamental importance in the transmission of both differentiation and
angiogenic signals from the extracellular environment to the
endothelium. Five endothelial cell-specific tyrosine kinase receptors,
each of which has a specific role in blood vessel formation, have been
identified. These include Tie-1, Tie-2 (also known as Tek), Flt-1,
Flt-4, and Flk-1/KDR (reviewed by Mustonen and Alitalo(1995)). While
the ligands for Tie-1 and Tie-2 have not yet been identified, Flk-1/KDR
and Flt-1 (which in this paper are referred to as Flk-1 and Flt,
respectively) are receptors for vascular endothelial growth factor
(VEGF) ()(de Vries et al., 1992; Terman et
al., 1992). Flk-1 has been shown recently to be a receptor for
VEGF-C, a novel VEGF-related endothelial cell growth factor which also
binds to and induces autophosphorylation of Flt-4 (Joukov et
al., 1995). The importance of the VEGF/VEGF receptor (VEGFR)
system in blood vessel formation can be summarized as follows: (i)
spatiotemporal expression of both VEGF and VEGFRs correlates well with
phases of vasculogenesis and angiogenesis in the embryo, and with
phases of neovascularization in the adult (reviewed by Mustonen and
Alitalo(1995)); (ii) mice lacking Flk-1 or Flt die at early stages of
development, the former being essential for endothelial cell
differentiation, the latter for correct vascular assembly (Fong et
al., 1995; Shalaby et al., 1995); (iii) VEGF is the major
angiogenic factor in an animal model of ischemia-stimulated retinal
neovascularization (Aiello et al., 1995); (iv) antibodies to
VEGF, as well as a dominant-negative form of Flk-1, are able to block
tumor angiogenesis and tumor growth in vivo (Kim et
al., 1993; Millauer et al., 1994).
Angiogenesis is believed to proceed in at least two phases: (i) the activation phase, in which endothelial cells degrade their basement membrane, and form capillary ``sprouts'' by migrating and proliferating in the surrounding stroma; and (ii) the resolution phase, in which migration and proliferation cease and a new basement membrane is deposited (Pepper et al., 1996a). Both phases appear to be determined by a balance between positive and negative regulators. One of the elements likely to play a key role in this equilibrium is the regulation of expression of endothelial cell tyrosine kinase receptors. Consistent with this hypothesis, flk-1, flt, flt-4, tie-1, and tie-2 expression correlates well with phases of capillary growth, although in a few settings high expression of tyrosine kinase receptors persists after endothelial cell proliferation and migration have ceased (Mustonen and Alitalo, 1995; Pepper et al., 1996a). Although VEGF receptors (Matthews et al., 1991; Shibuya et al., 1990) have been characterized extensively at the level of expression (Mustonen and Alitalo, 1995), high affinity VEGF binding (de Vries et al., 1992; Terman et al., 1992), phosphorylation, and other signal transduction properties (Waltenberger et al., 1994), very little is known about factors which regulate expression in vascular endothelial cells. Hypoxia, which up-regulates VEGF expression in a variety of cell types (Brogi et al., 1994; Namiki et al., 1995; Shweiki et al., 1992), has been reported recently to induce the release of a factor from myoblasts and smooth muscle cells which increases expression of flk-1 (Brogi et al., 1996). The identity of this factor remains to be determined.
We have cloned partial cDNAs encoding bovine flk-1 and flt and used them as probes to study VEGFR expression in
bovine microvascular and large vessel (aortic) endothelial cells. Both
cell lines express flk-1, but not flt. Incubation of
the cells with the multifunctional angiogenic cytokine transforming
growth factor 1 (TGF-
1) (Pepper et al., 1996a)
results in a rapid and marked decrease in flk-1 expression at
levels of mRNA, total protein, and cell surface
I-VEGF
binding capacity.
To identify VEGF receptor(s) expressed by BME and BAE cells,
partial cDNAs for bovine flk-1 and flt were cloned by
RT-PCR using degenerate primers. 400-bp (flk-1) and
500-bp (flt) fragments were cloned and sequenced. As
expected on the basis of the position of the primers chosen, the cloned
bovine cDNA fragments showed strong sequence similarity with the first
two IgG-like domains of human and mouse flk-1 (Matthews et
al., 1991; Terman et al., 1991) and with the region
extending between the presumptive autophosphorylation site and the
translational end of human, mouse, and rat flt (Finnerty et al., 1993; Shibuya et al., 1990; Yamane et
al., 1994). Identities were: (i) for bovine flk-1: 86%
and 78% (nucleotides), 85% and 75% (amino acids) with human and mouse flk-1, respectively; (ii) for bovine flt: 87%, 82%.
and 83% (nucleotides); 92%, 87%, and 88% (amino acids) with human,
mouse, and rat flt (Fig. 1, A and B).
Complementary RNA probes transcribed from both fragments were protected
by adult bovine heart and kidney RNAs in an RNase protection assay,
thereby confirming their complementarity to RNAs of bovine origin (Fig. 2). BME and BAE cell RNA analysis revealed that both cell
lines express flk-1 but not flt (Fig. 2). flt expression was also undetectable in both BME and BAE cells
by RT-PCR using the same primers described for cDNA cloning (data not
shown).
Figure 1: Partial nucleic acid sequences of bovine RT-PCR-derived flk-1 (A) and flt (B) cDNAs and comparison with amino acid sequences from other species. flk-1 identity: bovine-human, 85%; bovine-mouse, 75%. A higher degree of conservation is observed between bovine, human, mouse, and rat flt proteins (bovine-human, 92%; bovine-mouse, 87%; bovine-rat, 88%) (EMBL accession numbers X94298 (bflk-1) and X94263 (bflt)).
Figure 2:
Ribonuclease protection assay of flk-1 and flt mRNAs in BME and BAE cells. Purified P-labeled 381-bp bovine flk-1 or 459-bp bovine flt cRNAs (probe) were hybridized to buffer (probe + h.m.), yeast tRNA (tRNA), or to 10
µg of total RNA from bovine adult heart, kidney, BME, or BAE cells. SP6, control template marker (New England Biolabs). To confirm
RNA integrity, 2 µg of total RNA from the samples used in the
ribonuclease protection assay were denatured by glyoxal,
electrophoresed in a 1.0% agarose gel, and stained with ethidium
bromide (bottom panel).
We have previously reported that TGF-1 acts in a
biphasic manner on VEGF-induced in vitro angiogenesis. At
picogram/ml concentrations, TGF-
1 enhanced the effect of VEGF,
whereas at nanogram/ml concentrations it had an inhibitory effect
(Pepper et al., 1993b). To explain this phenomenon, we
hypothesized that TGF-
1 might modulate flk-1 expression
in vascular endothelial cells. To explore this possibility, confluent
cultures of BME or BAE cells were incubated overnight (15 h) in the
presence of increasing concentrations (from 1 pg/ml to 10 ng/ml) of
TGF-
1. Cells were acid-treated (conditions known to detach growth
factors from their high affinity cell surface receptors (Haigler et
al., 1980)) and assayed for their capacity to bind
I-VEGF with high affinity. TGF-
1 decreased the
capacity of BME and BAE cells to bind
I-VEGF in a
dose-dependent manner, with a 2.0- to 2.7-fold decrease at 1-10
ng/ml TGF-
1 (Fig. 3). In parallel experiments performed
under identical conditions, treatment of BME and BAE cells with
2.2-100 ng/ml VEGF resulted in a 2.2- to 10-fold reduction in
their capacity to bind
I-VEGF (data not shown).
Figure 3:
TGF-1 decreases
I-VEGF
binding capacity of BME and BAE cells. Confluent cultures of BME or BAE
cells were incubated with the indicated concentrations of TGF-
1 or
with cytokine-free medium (C) for 15 h. At the end of the
incubation,
I-VEGF (150 pM) was bound to
acid-treated cells. Radioactivity of cell lysates from duplicate wells
was measured in a
-counter. A 2.0- to 2.7-fold decrease was
detected in BME and BAE cells incubated with 1-10 ng/ml
TGF-
1. Nonspecific binding was determined by adding a 50-fold
molar excess of cold VEGF in parallel cultures and was subtracted from
all samples. No significant differences in cell numbers were observed
between control and TGF-
1-treated cells (data not shown). Typical
results from a representative experiment are
shown.
To
further characterize the binding of I-VEGF to vascular
endothelial cells, confluent cultures of BAE cells were incubated for 8
h or 15 h in the presence of 1 ng/ml TGF-
1 or 30 ng/ml VEGF. At
the end of the incubation, the cells were acid-treated and chloramine
T-labeled
I-VEGF was cross-linked to the cell surface by
DSS. Cell lysates were electrophoresed under denaturing conditions.
Following autoradiography, a prominent band corresponding in size to
the expected molecular mass of the VEGF
-Flk-1 complex
(about 230-235 kDa) was detected (Fig. 4A). The
intensity of the band was markedly reduced (3.5-fold; 8 h) or almost
undetectable (15 h) in TGF-
1-treated cells when compared to
controls (Fig. 4A). While VEGF had no effect on flk-1 expression at both mRNA and total protein levels (data
not shown), the 230-235-kDa band was undetectable in VEGF-treated
cells (Fig. 4A), which is consistent with the cell
binding results. Similar results were obtained with different ligand
and DSS concentrations (100 pM
I-VEGF, 0.15
mM DSS; 100 pM
I-VEGF, 0.5 mM DSS; 500 pM
I-VEGF, 0.5 mM DSS),
as well as under nondenaturing conditions (data not shown). No bands
were detectable if IODOGEN-labeled VEGF was cross-linked to the cells
(data not shown), probably because of its low specific activity (see
``Experimental Procedures''). As the VEGF used in binding and
cross-linking assays had a M
of 45,000, and BAE
cells express flk-1 but not flt mRNA (see above), we
concluded that TGF-
1 down-regulates VEGF binding to a cell surface
monomeric molecule indistinguishable from Flk-1 on the basis of its
affinity for VEGF, molecular size (185-190 kDa), and apparent
internalization after VEGF binding (Matthews et al., 1991;
Millauer et al., 1993; Terman et al., 1992; Ullrich
and Schlessinger, 1990; Waltenberger et al., 1994).
Figure 4:
TGF-1 decreases cell surface and
total Flk-1 protein expression in BME and BAE cells. A,
confluent cultures of BAE cells were incubated with 1 ng/ml TGF-
1 (T), 30 ng/ml VEGF (V), or cytokine-free medium (C) for the times indicated. At the end of the incubation,
cells were processed as described in the legend to Fig. 3,
except that
I-VEGF (iodinated by chloramine T) was
cross-linked to the cell surface by DSS. Cell lysates (150
µg/sample) were electrophoresed in an SDS-5% polyacrylamide gel
under reducing conditions. B, confluent cultures of BME cells
were labeled by
S-Met/Cys and incubated for 8 h with (T) or without (C) 1 ng/ml TGF-
1. Cell lysates
(1.5
10
cpm/sample) were immunoprecipitated with a
polyclonal antibody (C-1158) recognizing a peptide (amino acids
1158-1345) in the mouse Flk-1 carboxyl terminus.
Immunoprecipitates were run in an SDS-5% polyacrylamide gel under
reducing conditions. C, confluent cultures of BAE cells were
incubated with 1 ng/ml TGF-
1 (T) or with cytokine-free
medium (C) for the times indicated. At the end of the
incubation, cell lysates (3 mg/sample) were immunoprecipitated as
described in B. After electrophoresis, immunoprecipitates were
transferred to a PVDF membrane. Membranes were incubated with a second
antibody to Flk-1 (C-20), recognizing a peptide in the mouse Flk-1
carboxyl terminus (amino acids 1348-1367) which is different from
that recognized by the antibody C-1158 (used for immunoprecipitation).
Flk-1/C-20 complexes were detected by the ECL immunodetection
system.
The
effect of TGF-1 on flk-1 expression was investigated in
confluent cultures of BME cells radiolabeled with
[
S]Met/Cys and incubated for 8 h in the presence
of 1 ng/ml TGF-
1. Cell lysates were immunoprecipitated with an
antibody (C-1158) recognizing a peptide mapping to the carboxyl
terminus of mouse Flk-1. Analysis of immunoprecipitates by SDS-PAGE
electrophoresis and autoradiography revealed a single band with an
apparent molecular mass of about 200 kDa (Fig. 4B),
consistent with our cross-linking results and with the published
molecular mass of Flk-1 (Matthews et al., 1991; Millauer et al., 1993; Terman et al., 1992; Waltenberger et al., 1994). The intensity of the band was decreased
2.5-fold in TGF-
1-treated cells when compared to controls (Fig. 4B), which is consistent with the cell binding
results (Fig. 3). To confirm the specificity of the antibody,
confluent cultures of BAE cells were incubated from 4 to 15 h with 1
ng/ml TGF-
1. Cell lysates were immunoprecipitated using the C-1158
antibody. Immunoprecipitates were electrophoresed, blotted onto a PVDF
membrane, and incubated with a second antibody (C-20) which recognizes
a peptide in the carboxyl terminus of Flk-1 (amino acids
1348-1367) which is different from that recognized by the
antibody C-1158 (amino acids 1158-1345). A major band of about
200 kDa and a faint band of about 170 kDa, possibly corresponding to
differentially glycosylated forms of Flk-1, were detected (Fig. 4C). At all time points tested, the intensity of
both bands decreased in cells treated with TGF-
1 when compared to
controls (Fig. 4C). By scanning densitometry, a
4.5-fold decrease of Flk-1 expression was detected after 15 h. No
signal was detectable if the antibody used for Western blotting was
preadsorbed with the corresponding peptide, thereby demonstrating the
specificity of both bands (data not shown).
Finally, we determined
the effect of TGF-1 on BME and BAE cell flk-1 mRNA
expression. Confluent cultures of BME or BAE cells were incubated
overnight (15 h) in the presence of increasing concentrations of
TGF-
1 (from 1 pg/ml to 10 ng/ml). Total cellular RNA was analyzed
by Northern blot (BME cells, Fig. 5A) or by RNase
protection (BAE cells, Fig. 5B). TGF-
1 decreased flk-1 mRNA levels in both cell lines in a dose-dependent
manner (Fig. 5, A and B). By scanning
densitometry, a 2.5-fold or a 9-fold decrease were detected in BME or
BAE cells treated with 1 or 3 ng/ml TGF-
1, respectively. In
contrast, TGF-
1 had no effect on FGFR-1/flg expression by
BME cells (Fig. 5A).
Figure 5:
TGF-1 decreases flk-1 mRNA
levels in BME and BAE cells. BME and BAE cells were incubated with the
indicated concentrations of TGF-
1 or with cytokine-free medium (C) for 15 h. A, replicate Northern blot filters
containing 5 µg of total BME cell RNA per lane were hybridized to a
P-labeled bovine flk-1 or human FGFR-1/flg cRNA probe. Methylene blue staining (bottom panel)
reveals 28 S and 18 S rRNAs and demonstrates uniformity of loading and
RNA integrity. B, purified
P-labeled 381-bp
bovine flk-1 cRNA (probe) was hybridized to buffer (probe
+ h.m.), yeast tRNA (tRNA), or 10 µg of total
RNA from BAE cells incubated for 15 h with cytokine-free medium (C) or with the indicated concentrations of TGF-
1. SP6, control template marker (New England Biolabs). To confirm
RNA integrity, 2 µg of total RNA from the samples used in the
ribonuclease protection assay were denatured by glyoxal,
electrophoresed in a 1.0% agarose gel, and stained with ethidium
bromide (bottom panel).
Endothelial cell activation status is determined by an equilibrium between positive and negative regulators of endothelial cell proliferation, migration, and proteinase production (Pepper et al., 1996a, 1996b). One element likely to play a central role in this equilibrium is the regulation of endothelial cell tyrosine kinase receptor expression by environmental factors. Among the well characterized endothelial cell tyrosine kinase receptors, Flt and Flk-1 are receptors for VEGF, an endothelial cell-specific mitogen whose importance in both physiological and pathological angiogenesis is well established (Mustonen and Alitalo, 1995; Pepper et al., 1996a). However, very little is known about factors which regulate VEGFR expression in vascular endothelial cells.
In the present study
we report that TGF-1 down-regulates the expression of bovine
endothelial cell Flk-1, the major signal-transducing tyrosine kinase
receptor for VEGF (Waltenberger et al., 1994). We have
characterized this effect at levels of (i) mRNA (by means of Northern
blotting and RNase protection assay), (ii) total protein (by
immunoprecipitation, alone or followed by Western blotting with an
antibody directed against a different Flk-1 peptide), (iii) cell
surface (by high affinity binding and cross-linking of iodinated VEGF
to the cells). The down-regulation of flk-1 expression by
TGF-
1 may be responsible for the inhibitory effect of this
cytokine (at nanogram/ml concentrations) on VEGF-induced in vitro angiogenesis (Pepper et al., 1993b).
The findings that
VEGF increases plasminogen activator (PA) activity in vascular
endothelial cells (Mandriota et al., 1995; Pepper et
al., 1991) and that plasmin is able to activate latent TGF-1
(Flaumenhaft et al., 1992; Lyons et al., 1990; Sato et al., 1990; Sato and Rifkin, 1989) raise the possibility
that a complex self-regulating mechanism of VEGF signal transduction
may exist during angiogenesis. Thus, in the initial phases of
angiogenesis, endothelial cells respond to VEGF by increasing PA
activity, migration, and proliferation, which results in the formation
of new capillary sprouts. As a consequence of increased plasmin
formation, latent TGF-
1 is activated. This in turn might decrease flk-1 expression and thereby reduce the effects of VEGF in
vascular endothelial cells. This would result in endothelial cell
quiescence, reduced levels of plasmin activity, and deposition of a new
basement membrane, all of which are characteristic of the later stages
of new vessel formation (Ausprunk and Folkman, 1977; Paku and Paweletz,
1991). Finally, as a consequence of decreased plasmin activity,
TGF-
1 would no longer be activated, flk-1 expression
would increase and endothelial cells of newly formed vessels could
enter another cycle of invasion in response to VEGF.
In conclusion,
our results, taken together with the recent finding that Flk-1 is a
receptor for VEGF-C, a novel VEGF-related endothelial growth factor
which also binds to and induces autophosphorylation of Flt-4 (Joukov et al., 1995), implicate TGF-1 as a major regulator of
VEGF/Flk-1-mediated signal transduction in vascular endothelial cells.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X94298 [GenBank](bflk-1) and X94263 [GenBank](bflt).