Institute of Signaling, Developmental Biology
and Cancer Research, CNRS UMR 6543-Centre Antoine Lacassagne, 06189 Nice Cedex 2, France
Received 2 March 2001/Returned for modification 19 April
2001/Accepted 6 August 2001
Mouse capillary endothelial cells (1G11 cell line) embedded in type
I collagen gels undergo in vitro angiogenesis. Cells rapidly reorganize
and form capillary-like structures when stimulated with serum.
Transforming growth factor 
1 (TGF-1) alone can substitute for
serum and induce cell survival and tubular network formation. This
TGF-1-mediated angiogenic activity depends on phosphatidylinositol 3-kinase (PI3K) and p42/p44 mitogen-activated protein kinase
(MAPK) signaling. We showed that specific inhibitors of either pathway (wortmannin, LY-294002, and PD-98059) all suppressed TGF-1-induced angiogenesis mainly by compromising cell survival. We established that
TGF-1 stimulated the expression of TGF-
mRNA and protein, the
tyrosine phosphorylation of a 170-kDa membrane protein representing the
epidermal growth factor (EGF) receptor, and the delayed
activation of PI3K/Akt and p42/p44 MAPK. Moreover, we showed that all
these TGF-1-mediated signaling events, including tubular network
formation, were suppressed by incubating TGF-1-stimulated
endothelial cells with a soluble form of an EGF receptor (ErbB-1) or
tyrphostin AG1478, a specific blocker of EGF receptor tyrosine kinase.
Finally, addition of TGF- alone poorly stimulated angiogenesis;
however, by reducing cell death, it strongly potentiated the
action of TGF-1. We therefore propose that TGF-1 promotes
angiogenesis at least in part via the autocrine secretion of TGF-, a
cell survival growth factor, activating PI3K/Akt and p42/p44 MAPK.
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INTRODUCTION |
Angiogenesis or the formation of new
blood vessels from preexisting vasculature occurs in normal situations
such as embryonic development, wound healing, and during the female
reproductive cycle. However, activated blood vessel growth is also
found in many diseases, such as tumor progression, diabetic
retinopathy, and arthritis (24, 33). In the last few
years, several studies have led to the discovery of inducers and
inhibitors of the angiogenic process (6, 9, 12). Among the
inducers are factors such as vascular endothelial growth factor (VEGF)
and fibroblast growth factor 1 (FGF-1) and -2, which induce
angiogenesis in vivo and in vitro. They can also induce the
proliferation and migration of endothelial cells in two-dimensional
cultures. In contrast, other factors such as transforming growth factor
(TGF-) and tumor necrosis factor alpha induce angiogenesis in
vivo and in vitro but inhibit endothelial cell proliferation in vitro
(6, 9, 12).
TGF-1 is a 25-kDa peptide belonging to a family of multifunctional
cytokines that control the development and homeostasis of most tissues
by regulating diverse cellular functions, such as proliferation and
differentiation (49, 72). The receptors for this family
are basically two transmembrane serine/threonine kinases, termed
receptor type I and type II. The binding of the ligand causes the
heterodimerization of receptors I and II followed by the activation by
phosphorylation of receptor I. This receptor then phosphorylates and
activates the Smad family of proteins, which transduce the signal to
the nucleus (19, 36, 49, 72). The role of TGF- in
angiogenesis was first shown by new capillary formation after injection
of the factor into mice (23, 65) and by application of the
factor to the chicken chorioallantoic membrane
(80). Moreover, TGF-1 and TGF-2 are expressed during the development of angiogenically active tissues (35, 60). This proangiogenic activity of TGF- has been confirmed by
experiments using knockout mice. The knock out of TGF-1
(20), the type II receptor (59), and type I
receptor activin receptor-like kinase 1 (ALK1) (57, 74) is
lethal at 10.5 days of gestation due to defective vasculogenesis (the
initial formation of the primitive vasculature in the embryo), along
with defective endothelial cell differentiation and inadequate
capillary tube formation. Moreover, Smad5 knockout mice also die due to
defects in vasculogenesis and angiogenesis (14, 81).
Finally, mutations in the human ALK1 gene and in the endoglin gene,
which encodes a TGF-1-binding protein that presents TGF-1
to the type I and II receptors, all cause hereditary hemorrhagic
telangiectasia, a disease characterized by vascular malformations
(39, 50). Endoglin knockout mice also show a defective
angiogenesis and die at embryonic day 11.5 (44). In vitro,
TGF- inhibits endothelial cell proliferation in two-dimensional
cultures (3, 26, 34, 56) but induces tube formation when
endothelial cells are cultured inside three-dimensional collagen gels
(45, 53, 73). The differences between these studies have
been attributed to changes in type I and II receptor expression
(66). Finally, TGF-1 promotes the in vitro
differentiation of embryonic stem cells into endothelium cells
as well as the formation of cord-like structures (32).
However, the basic mechanisms underlying the proangiogenic action of
TGF- are largely unknown. With a mouse vascular endothelial cell
model (1G11 cell line) which rapidly forms capillary-like structures in
collagen and responds nicely to TGF-1, we have investigated the
basic signaling action of this angiogenic cytokine. We show that,
surprisingly, TGF-1 can stimulate the phosphatidylinositol 3-kinase
(PI3K)/Akt and the p42/p44 mitogen-activated protein kinase (MAPK)
pathways; however, this action is mediated by an autocrine mechanism,
implicating at least the production of TGF-. Moreover,
TGF-1-mediated stimulation of PI3K and p42/p44 MAPK signaling
cascades is essential for cell survival and formation of capillary-like structures.
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MATERIALS AND METHODS |
Materials.
Human TGF-1 was obtained from R&D Systems,
FGF-2 and platelet-derived growth factor BB (PDGF-BB) were obtained
from Pepro Tech, epidermal growth factor (EGF) and insulin were
obtained from Sigma, and TGF- was obtained from Boehringer.
LY-294002 was obtained from Alexis; wortmannin and rapamycin were
obtained from BioMol; PD-98059 was obtained from New England Biolabs;
cycloheximide, actinomycin D, brefeldin A, and tyrphostin AG1478 were
also obtained from Sigma. Genistein and SB202190 were obtained from
Calbiochem. Cell culture media, fetal calf serum (FCS), glutamine, and
antibiotics were obtained from Gibco-BRL. The most commonly used
chemicals were purchased from Sigma.
Cell culture.
Murine lung capillary endothelial cells (1G11
cell line) were obtained from Alberto Mantovani and Annunciata Vecchi
(Instituto Ricerche Farmacologiche Mario Negri, Milan, Italy)
(21). They were cultured in Dulbecco's modified Eagle's
medium (DMEM) containing 20% inactivated FCS; 50 U of penicillin, 50 µg of streptomycin sulfate, 150 µg of endothelial cell growth
supplement (Becton Dickinson), and 100 µg of heparin (Sigma)/ml; 1%
nonessential amino acids; and 2 mM sodium pyruvate. Before the
incubation with growth factors, cells were depleted for 24 h in a
1:1 mixture of DMEM and Ham's F-12 medium.
Murine heart capillary endothelial cells (H5V cell line) transformed by
polyoma middle T antigen (27) were obtained from P. Huber
(Grenoble, France) and cultured in DMEM containing 20% FCS.
Human umbilical vein endothelial cells (HUVEC) were obtained as
previously described (5) and were cultivated in SFM
(Gibco-BRL) supplemented with 20% FCS and 100 µg of heparin, 20 ng
of FGF-2, and 10 ng of EGF (Sigma)/ml.
HEK293 cells were cultured in DMEM containing 7.5% inactivated FCS, 50 U of penicillin/ml, and 50 µg of streptomycin sulfate/ml. Cells were
transiently transfected by the calcium phosphate method. HEK293 cells
were seeded at a density of 700,000 cells per well in six-well plates
and transfected with 0.25 µg of the green fluorescent protein
(GFP) plasmid only or cotransfected with 0.25 µg of GFP plasmid
together with 5 µg of expression vector CDM7-IgB-1, which codes for
the extracellular portion of EGF receptor (ErbB-1) fused to an Fc
portion of human immunoglobulin G1 (15). Forty-eight hours
after transfection, the culture medium from transfected HEK293 cells
was used to treat 1G11 cells.
Tubulogenesis and cell death counting assay.
To induce
capillary tube formation, 1G11 cells grown to confluence were
trypsinized and resuspended in 2× DMEM. Cells were added to a type I
collagen solution (Becton Dickinson; 4 mg/ml) to achieve a cell
concentration of 3 × 106 cells/ml and a
final collagen concentration of 2 mg/ml. Sixty microliters of this
preparation was placed in 24-well plates and incubated for 45 min at
37°C in a humidified incubator to allow polymerization, and complete
1G11 cell culture medium, DMEM alone, or DMEM containing 10 ng
of TGF-1/ml was added where indicated in the legends for Fig. 1, 2,
11, and 12.
To extract proteins from the collagen gels, cultures were lysed in
radioimmunoprecipitation assay buffer (phosphate-buffered saline with
1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate
[SDS], 40 mM -glycerophosphate, 200 µM sodium orthovanadate, 100 µM phenylmethylsulfonyl fluoride, 1 µM pepstatin A, 1 µg of leupeptin/ml and 4 µg of aprotinin/ml), homogenized with a minipotter homogenizer, and extracted for 15 min at 4°C. Insoluble
material was removed by centrifugation at 12,000 × g
for 10 min at 4°C.
To measure the numbers of live and dead cells after 24 h in
collagen gels, Hoechst stain (50-µg/ml bis-benzimide; Sigma) or propidium iodide (1 µg/ml; Sigma) was added and cells were incubated for 30 min at 37°C. The live (nuclei Hoechst stained) and dead cells
(nuclei with propidium iodide stain and condensed chromatin) were counted using an immunofluorescence microscope.
Western blot analysis.
Cells were washed twice with cold
phosphate-buffered saline and lysed in Triton X-100 lysis buffer (50 mM
Tris-HCl [pH 7.5], 100 mM NaCl, 50 mM NaF, 5 mM EDTA, 40 mM
-glycerophosphate, 200 µM sodium orthovanadate, 100 µM
phenylmethylsulfonyl fluoride, 1 µM pepstatin A, 1 µg of
leupeptin/ml, 4 µg of aprotinin/ml, 1% Triton X-100) for 15 min at
4°C. Insoluble material was removed by centrifugation at 12,000 × g for 5 min at 4°C. For conditioned medium from
1G11 cells, proteins from 10 ml were precipitated with acetone for
1 h at 
20°C and centrifuged at 5,000 × g for 5 min. The final pellet was resuspended in 50 µl of Laemmli sample buffer (40), followed by protein separation on an
SDS-15% polyacrylamide gel. Western blotting was performed as
described previously (76). The blots were incubated with a
polyclonal anti-Ets-1 antibody (Santa Cruz Biotechnology), polyclonal
antiphospho-(Ser473)-Akt (New England Biolabs), a polyclonal anti-Akt
antibody (New England Biolabs), a anti-poly(ADP-ribose) polymerase
(anti-PARP) monoclonal antibody (BioMol), a monoclonal
antiphospho-Erk1/2 antibody (Sigma), anti-p42/p44 MAPK antiserum EIB
(51), a polyclonal anti-TGF- antibody (R&D), and a
polyclonal antiphospho-Smad2 antibody (Upstate Biotechnology) in
blocking solution overnight at 4°C.
Where indicated (see Fig. 4B), the p70 S6K activity was
determined by a mobility shift assay as described previously
(76).
WGL and EGF receptor immunoprecipitation.
Quiescent cells
stimulated with or without the different agonists were lysed with
Triton X-100 lysis buffer for 15 min at 4°C. Insoluble material was
removed by centrifugation at 12,000 × g for 5 min at
4°C. Protein lysates (500 µg) were incubated for 1 h at 4°C
with wheat germ lectin (WGL) preadsorbed to Sepharose beads (Pharmacia)
or for 3 h (1 mg of lysate) with a specific polyclonal anti-EGF
receptor (Santa Cruz Biotechnology) preadsorbed to protein A-Sepharose
beads (Pharmacia). Precipitates were then washed four times with Triton
X-100 buffer. The final pellet was resuspended in 50 µl of Laemmli
sample buffer (40), followed by protein separation on an
SDS-7.5% polyacrylamide gel and Western blotting with an
antiphosphotyrosine monoclonal antibody or an anti-EGF receptor
antibody (Santa Cruz Biotechnology).
Northern blotting.
Poly(A)+ RNA was
isolated from confluent 1G11 cells treated for different times with 10 ng of TGF-1/ml or from H5V cells treated for 3 h with TGF-1
using a Micro-FastTack 2.0 kit (Invitrogen). A Northern blot assay was
performed as described previously (77). The mouse cDNA
probe for TGF- was a 190-bp PCR-amplified band obtained from 1G11
RNA using specific oligonucleotide primers for mouse TGF-
(5'-ATGGTCCCCGCGACCGGACAGCTC-3'; reverse oligonucleotide: 5'-ACATGCTGGCTTCTCTTCCTGCAC-3'). The identity of the
amplified TGF- band was confirmed by sequence analysis (Eurogentec).
Reverse transcription-PCR (RT-PCR).
Poly(A)+ RNA was isolated from confluent HUVEC
treated for 2 h with 10 ng of TGF-1/ml using a Micro-FastTack
2.0 kit (Invitrogen) or not treated. After reverse transcription using
an oligo(dT) primer (Amersham), cDNAs were amplified by PCR (Amersham)
using specific oligonucleotide primers for human TGF-
(5'-TTCTGGAGCTTCTCAAGGGAT-3'; reverse oligonucleotide:
5'-CCTGGTAAATCAATGGCTAGA-3') (35 cycles) and mouse actin
(5'-ATGGATGACGATATCGCTG-3'; reverse oligonucleotide: 5'-ATGAGGTAGTCTGTCAGGT-3') (33 cycles). The mouse cDNA probe
for TGF- was a 330-bp PCR-amplified band, and that for actin was a
500-bp band.
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RESULTS |
Inhibition of PI3K or p42/p44 MAPK blocks TGF-1-induced
endothelial tube formation and cell survival.
Like other capillary
endothelial cell models, as described previously (45, 55,
67), 1G11 lung mouse capillary endothelial cells completely
reorganize when cultured with complete medium in three-dimensional type
I collagen gels. The final result was a network of tubular structures
with multiple cell-cell contacts (Fig.
1), which caused the retraction of the
collagen gel (data not shown). A lack of this organization could be
seen when cells are cultured in the presence of growth factor-free DMEM
(Fig. 1). The same effect obtained in the presence of complete medium was observed when only TGF-1 was added. This TGF-1-induced tube formation was also observed in primary endothelial cell cultures from
mouse lung cultured inside collagen gels (D. Grall and J. C. Chambard, unpublished observations) and has already been described for
other microvascular endothelial cells (45, 53). The
tubular-growth remodeling induced by TGF-1 or complete medium was
dependent on mRNA and protein synthesis, since preincubation in the
presence of their respective inhibitors, actinomycin D and
cycloheximide, completely blocked tube formation (data not
shown). In contrast, when added to two-dimensional 1G11 cell cultures,
TGF-1 behaved as a growth inhibitor, as judged by the severe block
in thymidine incorporation stimulated by EGF or FGF-2 (data not shown).
This effect has also been described for other endothelial cell types (3, 26, 34, 56).
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FIG. 1.
TGF-1 and complete medium stimulate 1G11 capillary
endothelial cells to form a tubular network in type I collagen gels.
1G11 cells were mixed with type I collagen and placed on culture plates
to polymerize. After gel formation, DMEM alone (basal), complete medium
(20% FCS and 150 µg of endothelial cell growth supplement/ml), or
TGF-1 (10 ng/ml) was added for 48 h and gels were
examined by phase-contrast microscopy.
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To determine the signal transduction pathways important for
TGF-1-induced tube formation, we preincubated 1G11 cells grown in
collagen gels with specific inhibitors before addition of TGF-1. Rapamycin, a specific inhibitor of p70 S6K activation that blocks 1G11
vascular endothelial cell proliferation (76), did not have any effect on tube formation in collagen gels (Fig.
2A). In contrast, preincubation in the
presence of PI3K inhibitors LY-294002 (15 µM) or wortmannin (300 nM)
blocked tube formation induced by TGF-1 (Fig. 2A and data not
shown). In the presence of PI3K inhibitors cells were not organized and
appeared as isolated and refractile, similar to control cells cultured
in DMEM only (Fig. 1 and 2A, basal). A similar result was obtained when
p42/p44 MAPK activation was blocked by preincubating cells in the
presence of 30 µM PD-98059, an inhibitor of p42/p44 MAPK kinase 1 (MEK1) (Fig. 2A). In contrast, inhibition of p38 MAPK by SB202190 did
not have any effect. This blocking effect of PI3K and p42/p44 MAPK
inhibitors on TGF-1-induced tube formation was confirmed by the lack
of induction of a typical angiogenic marker, transcription factor Ets-1
(73, 75) (Fig. 2B).
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FIG. 2.
LY-294002 and PD-98059 prevent TGF-1-induced tube
formation and Ets-1 induction. (A) 1G11 endothelial cells immersed in
collagen gels were cultured for 24 h in the presence of DMEM alone
(basal) or 10 ng of TGF-1/ml either alone or with 10 µM SB202190,
15 µM LY-294002 (Ly), 10 nM rapamycin (rapa), or 30 µM PD-98059
(PD). Cells were examined by phase-contrast microscopy. Magnification,
×128. (B) 1G11 cells grown in collagen gels for 24 h in
the presence of 10 ng of TGF-1/ml either alone (TGF-1 lane ) or
with 15 µM LY-294002 (lane +Ly) or 30 µM PD-98059 (lane
+PD) or in the absence of TGF- (B lane ) were lysed, and Ets-1 was
detected by immunoblotting with a specific antibody. Identical amounts
of protein were loaded on the gel. The decrease in p42 MAPK content in
lanes +Ly and +PD reflects partial cell death. A representative Western
blot of three different experiments is shown.
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As observed in Fig. 1 and 2A, 1G11 cells cultured in collagen gels for
24 or 48 h in the absence of exogenous growth factors presented a
rounded aspect, with few cells presenting extensions. To a large
extent, cells presented a refractile aspect and condensed chromatin,
typical of dying cells. The number of cells presenting this morphology
dropped when growth factors or TGF-1 was added to the culture medium
but reappeared when cells were pretreated with LY-294002, wortmannin,
or PD-98059 before addition of TGF-1 (Fig. 2A). This result
suggested that one of the effects of TGF-1 on three-dimensional
cultures of microvascular endothelial cells was to provide survival
signals. To confirm this, we counted the live and dead cells (Hoechst
stain- or propidium iodide-positive nuclei, respectively) in the
presence and absence of TGF-1. As shown in Fig.
3A, when cells were incubated for 24 h in DMEM only, 64% of the cells were dead (live cell/dead cell ratio
of 0.6). Dead cells were round and refractile. In contrast, the
addition of TGF-1 to the cultures doubled the number of live cells
(live cell/dead cell ratio of 1.85). This cell survival effect of
TGF-1 was abolished by preincubation in the presence of LY-294002
(live cell/dead cell ratio, 0.54) or in the presence of PD-98059 (live cell/dead cell ratio, 0.7). Preincubation with LY-294002 or PD-98059 alone had no effect on the basal live cell/dead cell ratio. These results were confirmed by measuring apoptosis in collagen gels. Apoptosis was measured by evaluating cleavage of PARP, a
well-established substrate of caspase 3 and a marker of caspase
cascade activation during apoptosis. As observed in Fig. 3B, culture of
1G11 cells in collagen gels in the presence of growth factor-free DMEM
caused PARP cleavage. Treatment with TGF-1 completely precluded the apoptotic effect of collagen immersion, and this effect was reversed by
LY-294002 or PD-98059 pretreatment.
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FIG. 3.
TGF-1 stimulates endothelial cell survival in
collagen gels. (A) 1G11 cells immersed in collagen gels were cultured
for 24 h in the presence of DMEM alone (B), 15 µM LY-294002
alone (B+Ly), 30 µM PD-98059 alone (B+PD), 10 ng of TGF-1/ml
(TGF1), and TGF-1 in the presence of 15 µM LY-294002
(TGF1+Ly) or 30 µM PD-98059 (TGF1+PD). After this time, live
and dead cells were counted as described in Materials and Methods.
Results are expressed as the ratios of live cells/dead cells and are
averages of eight different experiments. (B) Proliferative 1G11 cells
(0) or cells immersed in collagen gels for 8 h in the absence or
presence of TGF-1 (10 ng/ml), LY-294002 (15 µM), or PD-98059 (30 µM) were lysed, and PARP was detected by immunoblotting with a
specific antibody. A representative Western blot is shown. The observed
difference in the mobility of PARP between proliferative cells and
cells immersed in collagen gels is due to the presence of collagen in
the SDS-polyacrylamide gel electrophoresis.
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TGF-1 stimulates PI3K and p42/p44 MAPK in collagen gels and in
two-dimensional cultures by an autocrine mechanism.
The
sensitivity of tube formation to PI3K and p42/p44 MAPK inhibitors
prompted us to investigate the action of TGF-1 on these two
signaling cascades, in particular during tubular network formation in
collagen gels. When cells were grown in collagen gels and in the
absence of growth factors, we observed a persistent activation of
p42/p44 MAPK (measured with an antibody against the phosphorylated active form of the kinases) (Fig. 4A).
This stimulation was probably due to integrin activation (8, 37,
69). Interestingly, under the same conditions of culture no
activation of PI3K, as indicated by the phosphorylation of its
downstream effector Akt, could be detected at short times; only after
8 h of culture on collagen gels was a weak increase in phospho-Akt
detected (Fig. 4A). In contrast, the addition of TGF-1 to
cells grown in collagen gels led to a marked activation of Akt (Fig.
4A). Akt activation was observable at 2 h, peaked at 8 h, and
persisted at least until 24 h (data not shown). For p42/p44 MAPK,
increased activation could also be seen after 1 h of stimulation
with TGF-1, with a maximal difference obtained at 4 h. These
results indicate that TGF-1 stimulated PI3K and enhanced p42/p44
MAPK in collagen gels. With the objective to better characterize the
observed PI3K and p42/p44 MAPK stimulation by TGF-1, we evaluated
whether the effect of TGF-1 on p42/p44 MAPK and PI3K activity could
also be observed on two- dimensional 1G11 cell cultures. Therefore,
cells were cultured on plates precoated with type I collagen or gelatin
and were stimulated for different periods of time with TGF-1. The activities of Akt, p70 S6K, which also depends on the activity of PI3K,
and p42/p44 MAPK were analyzed. As shown in Fig. 4B, TGF-1
stimulated all these signaling pathways as it did in cells cultured in
a three-dimensional collagen gel, but in a more transient manner. As
was observed in collagen gels, the effect of TGF-1 was only observed
after stimulation periods of over 1 h. Akt and p70 S6K activation
started at 2 h (data not shown), peaked at 4 h, and started
to decrease at 8 h. A weak and transient stimulation of p42/p44
MAPK, starting at 1 h, reaching maximum effect at 4 h, and
returning to basal levels at 8 h, was also observed.
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FIG. 4.
TGF-1 stimulates PI3K, p70 S6K, and p42/p44 MAPK
activities in endothelial cells grown in collagen gels and in
two-dimensional cultures. (A) 1G11 cells were grown in collagen gels
for the periods of time indicated. Cells were lysed, and phospho-Akt,
phospho-p42/p44 MAPK (pp42/pp44 MAPK), and p42 MAPK were detected by
immunoblotting with specific antibodies. A representative Western blot
is shown. (B) 1G11 cells were grown on collagen- or gelatin-coated
plates until confluence. After depletion of growth factors, cells were
stimulated with 10 ng of TGF-1/ml for the periods of time indicated
or with 20 ng of EGF for 30 min. Cells were lysed, and Western blotting
was performed using anti-phospho-Akt, anti-phospho-p42/p44 MAPK, or
p42/p44 MAPK. The same extracts were loaded on an SDS-9%
polyacrylamide gel (shift-up) and blotted with an anti-p70 S6K
antibody. Hyperphosphorylated and active forms of p70 S6K (arrows)
migrated more slowly than hypophosphorylated forms. The Western blots
are representative of three independent experiments.
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Given the latency observed for TGF-1-stimulated PI3K and p42/p44
MAPK activities, we hypothesized that TGF-1 could be having an
indirect effect on these pathways. In view of the signaling action of
TGF-1, it was possible that an autocrine factor released by 1G11
cells after TGF-1 stimulation was activating PI3K and p42/p44 MAPK.
To validate this hypothesis, we preincubated cells in the presence of
different inhibitors of mRNA synthesis (actinomycin D), protein
synthesis (cycloheximide), or vesicular traffic (brefeldin A, a
compound that disturbs the trans-Golgi apparatus) before addition of
TGF-1. All these inhibitors completely blocked the effect of
TGF-1 on Akt and p42/p44 MAPK stimulation (Fig.
5 and data not shown). This result
suggested that the effect of TGF-1 was caused by the synthesis and
secretion of an autocrine factor. This factor would then activate PI3K
and p42/p44 MAPK signaling pathways.
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FIG. 5.
Effect of TGF-1 on Akt and p42/p44 MAPK activation
depends on mRNA and protein synthesis and vesicular secretion.
Quiescent 1G11 cells were preincubated for 15 min in the presence of 5 µg of actinomycin D/ml, 10 µg of cycloheximide/ml, or 1 µg of
brefeldin A/ml or in the absence of inhibitors (), followed by a 4-h
stimulation with 10 ng of TGF-1/ml, a 30-min stimulation with 10 ng
of PDGF-BB/ml, or no stimulation (basal). Cells were lysed, and
phospho-Akt and Akt were immunodetected as previously (76)
described. A Western blot representative of three different
experiments is shown.
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TGF-1 stimulates the synthesis of TGF- and EGF receptor
phosphorylation.
Since this autocrine factor activates the PI3K
and p42/p44 MAPK pathways through cell surface receptors, we wanted to
evaluate the possible activation of the cell surface tyrosine kinase
receptor following TGF-1 stimulation. Therefore, we stimulated cells
for different periods of time with TGF-1 and isolated membrane
proteins by using Sepharose-coupled WGL, which binds glycoproteins.
After extensive washing, purified glycoproteins were separated on a polyacrylamide gel and phosphotyrosine-containing proteins were immunodetected with a specific antibody. Incubation with TGF-1 for 2 or 4 h induced the tyrosine phosphorylation of a number of
proteins on total lysates, some of which were also detected after
stimulation with FGF-2, EGF, and PDGF-BB (data not shown). Purification
of glycoproteins with lectin elicited the enrichment of cell surface
proteins such as growth factor receptors. The PDGF receptor and EGF
receptor, which were nearly undetectable in total lysates, could
clearly be seen after treatment with WGL (Fig.
6A). More interestingly, extracts from
cells stimulated for 2 or 4 h with TGF-1 increased tyrosine
phosphorylation on a 160- to 170-kDa protein. This molecular mass was
identical to that of the EGF receptor. This result suggested that
TGF-1 induces the phosphorylation of a member of the EGF receptor
family. To confirm this hypothesis, we immunoprecipitated the EGF
receptor with a specific antibody and evaluated its activity status by phosphotyrosine immunoblotting. As observed in Fig. 6B, incubation for
2 h with TGF-1 increased the tyrosine phosphorylation of the
EGF receptor, confirming the implication of a member of the EGF family
of growth factors in the TGF-1-mediated effect.
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FIG. 6.
Stimulation with TGF-1 causes EGF receptor
activation. (A) Quiescent 1G11 cells were stimulated or not (lane B)
for the indicated times with 10 ng of TGF-1/ml and for 10 min with
25 ng of FGF-2/ml, 10 ng of EGF/ml, or 10 ng of PDGF-BB/ml. Cells were
lysed, and proteins were incubated with Sepharose-WGL for 1 h.
After being washed, the final pellet was resuspended in Laemmli sample
buffer and loaded on a SDS-7.5% polyacrylamide gel.
Phosphotyrosine-containing proteins were immunodetected by using a
specific antibody. Arrow, phosphotyrosine-containing protein that
appeared after TGF-1 treatment. A representative Western blot is
shown. (B) Cells were treated as for panel A and lysed, and the EGF
receptor (EGFR) was immunoprecipitated (IP) by incubation with a
specific anti-EGF receptor antibody preadsorbed to protein A-Sepharose
beads. After being washed the pellet was treated as for panel A. WB,
Western blot.
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The EGF family is composed of at least six members that can activate
ErbB-1 or the EGF receptor (1). Moreover, only TGF- and
EGF have been implicated in the angiogenesis process (58, 68). To identify the factor implicated in the TGF-1-mediated effect, we performed RT-PCR experiments with mRNAs obtained from 1G11 cells treated for 2 h with TGF-1 or not treated. A
fragment corresponding to TGF- could be amplified in these cells; in
contrast there was no transcript corresponding to EGF (data not shown). We confirmed these results with Northern blot analysis using
poly(A)+ RNA from 1G11 cells and the PCR fragment
amplified from 1G11 cells as a specific TGF- probe. As shown in Fig.
7A, we detected a 4.5-kb band
corresponding to the TGF- mRNA. Treatment with TGF-1 caused a
transient increase in the TGF- transcript, which peaked at 2 h
and returned to nearly basal levels after 4 h. This result
indicates that TGF-1 stimulates the synthesis of TGF- in 1G11
capillary endothelial cells. Next, we investigated whether this effect
of TGF-1 on TGF- synthesis is also observed in other vascular
endothelial cells. We performed a Northern blot assay using
poly(A)+ RNA from H5V cells (a mouse heart
endothelial cell line transformed by polyoma middle T antigen
[27]) treated with TGF-1 or not treated. As observed
in Fig. 7A, treatment with TGF-1 also caused an increase in TGF-
mRNA in this endothelial cell model. Finally we performed RT-PCR
experiments with mRNAs obtained from primary cultures of HUVEC treated
for 2 h with TGF-1 or not treated. As shown in Fig. 7B,
stimulation with TGF-1 for 2 h also increased TGF- cDNA in
HUVEC.
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FIG. 7.
TGF-1 induces TGF- mRNA expression in endothelial
cells. (A) 1G11 and H5V cells stimulated with 10 ng of TGF-1/ml for
the indicated times or not stimulated were lysed, and
poly(A)+ mRNA was isolated. Gels were loaded with 2 µg of
mRNA, and Northern blotting was performed using a 190-bp
PCR-amplified fragment as a TGF--specific probe. Rat GAPDH
(glyceraldehyde-3-phosphate dehydrogenase) was used as a control. A
representative result is shown. (B) HUVEC stimulated with 10 ng of
TGF-1/ml for 2 h or not stimulated were lysed, and
poly(A)+ mRNA was isolated. After cDNA was obtained, PCR
was performed using specific primers for human TGF- or actin (as a
control). Samples were loaded on a 2% agarose gel. 1 and 2, two
independent preparations of HUVEC poly(A)+ for unstimulated
and stimulated cells.
|
|
Next, we performed Western blot experiments to detect the TGF-
protein. As is observed in Fig. 8, the
antibody used clearly detected commercial human TGF- as a 6-kDa band
in Western blot assays. However, we have never detected the mature and
secreted form of TGF- on conditioned medium from 1G11 cells treated
for 2 (Fig. 8) or 4 h (data not shown) with TGF-1 or not
treated. In contrast, in lysates from 1G11 cells we detected a 19-kDa
band that probably corresponded to an unprocessed form of the TGF- protein. This form increased after treatment with TGF-1 for 2 h
(Fig. 8). This result indicates that TGF-1 stimulates the synthesis of the TGF- protein in 1G11 capillary endothelial cells; this form
is then translocated to the membrane where it probably acts in
an autocrine or paracrine (cell-to-cell) manner, stimulating the ErbB-1
receptor.
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FIG. 8.
TGF-1 induces TGF- protein expression in 1G11
cells. Quiescent 1G11 cells were stimulated for 2 h with 10 ng of
TGF-1/ml or were not stimulated (lane B). After this time, medium
was collected and proteins were precipitated and loaded on a 15% gel.
As a control, 300 ng of human TGF- was also loaded. In parallel,
cells were lysed and 100 µg was loaded on the gel. TGF- was
immunodetected by using a specific antibody. A Western blot
representative of three different experiments is shown.
|
|
EGF receptor mediates TGF-1 stimulation of PI3K and p42/p44 MAPK
and cell survival.
We next wanted to determine whether EGF
receptor stimulation was responsible for PI3K and p42/p44 MAPK
stimulation after treatment with TGF-1. First, we incubated 1G11
cells in the presence of recombinant TGF- to examine if this growth
factor stimulated the same signaling pathways as TGF-1. As shown in
Fig. 9A, incubation for 1 h in the
presence of 10 ng of TGF-/ml stimulated both Akt and p42/p44 MAPK
phosphorylation. Second, prior to treatment with TGF-1, we
preincubated cells with a selective inhibitor of EGF receptor
tyrphostin AG1478 (43). This compound specifically inhibited TGF- and EGF signaling in 1G11 cells, while having no
effect on PDGF-BB-, insulin-, or FCS-induced PI3K or p42/p44 MAPK activities (Fig. 9A) or on the normal TGF-1 signaling,
indicated by Smad2 phosphorylation (Fig. 9B). Even more importantly,
preincubation with tyrphostin AG1478 precluded the activation of
p42/p44 MAPK by TGF-1 and decreased Akt phosphorylation by 50%
(Fig. 9). Moreover, the effect of AG1478 was also observed in
three-dimensional collagen gels, where it blocked TGF-1-stimulated
p42/p44 MAPK and Akt activities (data not shown). Third, to sequester
the possible TGF- or other EGF-like members secreted after TGF-1
stimulation, we used a soluble and extracellularly secreted form of the
extracellular portion of the EGF receptor fused in frame to an Fc
portion of human immunoglobulin G1, denoted CDM7-IgB-1
(15). This construction was expressed in HEK293 cells, and
the conditioned medium of these cells was added to 1G11 cells before
TGF-1 treatment. Thus, when 1G11 cells were pretreated for 1 h
in the presence of the medium from control transfected HEK293 cells, an
incubation for 4 h with TGF-1 stimulated Akt and p42/p44 MAPK
(Fig. 10). 1G11 cells pretreated with
CDM7-IgB-1 supernatants demonstrated a higher basal activation of Akt
and p42/p44 MAPK. However TGF-1 was no longer capable of stimulating
these two signaling pathways (Fig. 10). These results reinforce the
notion that TGF-1-stimulated Akt and p42/p44 MAPK signaling in 1G11
cells is mediated mainly through the EGF receptor.
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FIG. 9.
Tyrphostin AG1478, a specific inhibitor of the EGF
receptor, blocks TGF-1 stimulation of p42/p44 MAPK and Akt in the
absence of changes in TGF- signaling. (A) Quiescent 1G11 cells were
preincubated for 15 min in the presence (+) or the absence () of
tyrphostin AG1478 (1 µM). After this time, cells were stimulated for
4 h with 10 ng of TGF-1/ml (in duplicate in the presence of
tyrphostin AG1478) or for 1 h with 50 ng of TGF-/ml, 10 ng of
PDGF-BB/ml, 1 µM insulin, or 10% FCS or were left unstimulated (lane
B). Cells were lysed, and phospho-Akt, Akt, and phospho-p42/p44 MAPK
were immunodetected as described in Materials and Methods. A Western
blot representative of four different experiments is shown. (B)
Quiescent 1G11 cells were preincubated for 15 min in the presence (+)
or the absence () of tyrphostin AG1478 (1 µM). After this time,
cells were stimulated for the times indicated with 10 ng of TGF-1/ml
or for 15 min with 50 ng of TGF-/ml. After lysis, phospho-Smad2,
phospho-Akt, Akt, and phospho-p42/p44 MAPK were immunodetected as
described in Materials and Methods. A representative Western blot is
shown.
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|
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FIG. 10.
A soluble EGF receptor (IgB-1) blocks TGF-1
stimulation of Akt and p42/p44 MAPK. Quiescent 1G11 cells were
preincubated for 1 h with medium from control or soluble
extracellular EGF receptor IgB-1-transfected HEK293 cells. After this
period, cells were stimulated with TGF-1 for 4 h. Cells were
then lysed, and phospho-Akt, phospho p42/p44 MAPK, and p42/p44 MAPK
were immunodetected as described in Materials and Methods. A
representative Western blot from three different experiments is
shown.
|
|
We then wanted to evaluate whether inhibition of the EGF receptor could
affect tubular morphogenesis and cell survival in collagen gels. As
shown in Fig. 11, top, preincubation of
cells in the presence of tyrphostin AG1478 prior to the addition of TGF-1 inhibited capillary-like formation in collagen gels. Moreover, preincubation with tyrphostin AG1478 inhibited the protective effect of
TGF-1 on cell survival (Fig. 11, bottom). Finally, it was possible
that the in vitro angiogenic effect of TGF-1 was wholly mediated by
TGF-. To evaluate this possibility, 1G11 cells were incubated in the
presence or absence of TGF- alone. In this condition TGF-
increased cell survival (Fig. 11, bottom). After a 24-h treatment
TGF- did not stimulate tubular network formation; however, long-term
treatment (5 days) elicited tube formation although at a lesser level
than that elicited by TGF-1 (Fig. 12). Moreover, when TGF-1 was added
in the presence of TGF-, we noticed a very marked synergistic effect
on cell reorganization at concentrations of TGF-1 that alone
produced a limited effect (compare 2 and 5 ng of TGF-1/ml in
the presence or absence of TGF-). These results indicate that
TGF- is necessary but not sufficient for TGF-1-induced cell
reorganization in collagen gels. Taken together, all these results
suggest that TGF- secretion induced by TGF-1 is essential for
maintaining cell survival in three-dimensional cell cultures and plays
a role in the TGF-1-induced capillary-like formation in endothelial
cells.
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FIG. 11.
Inhibition of TGF- signaling blocks tube formation
and cell survival induced by TGF-1 in collagen gels. (Top) 1G11
endothelial cells grown in collagen gels were cultured for 24 h in
the presence of DMEM alone (basal) or 10 ng of TGF-1/ml either alone
or supplemented with 1 µM tyrphostin AG1478. After this time, cells
were examined by phase-contrast microscopy. Magnifications: left, ×90;
right, ×180. (Bottom) 1G11 endothelial cells grown in collagen
gels were cultured for 24 h in the presence of DMEM either alone
(lane B) or with 1 µM tyrphostin AG1478 (B+AG), in 10 ng of
TGF-1/ml either alone (TGF) or with 1 µM tyrphostin AG1478
(TGF+AG), in 50 ng of TGF-/ml either alone (TGF) or with
tyrphostin AG1478 (TGF+AG), or in 100 ng of FGF-2/ml either alone
(FGF) or with 1 µM tyrphostin AG1478 (FGF+AG). Dead and living cells
were counted as described in Materials and Methods. Results are
averages of three different experiments.
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|
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FIG. 12.
TGF- stimulates tube formation in collagen gels in
the long term and potentiates TGF-1 action in the short term. 1G11
cells grown in collagen gels were cultured for 24 h in the
presence of DMEM alone or different concentrations of TGF-1 (2 and 5 ng/ml) in the presence or the absence of TGF- (50 ng/ml). In
parallel, 1G11 cells were grown in collagen gels for 5 days in the
presence or the absence of TGF-1 (10 ng/ml) or TGF- (50 ng/ml).
Cells were examined by phase-contrast microscopy.
|
|
| |
DISCUSSION |
In this study we demonstrate that, besides its effects on cell
reorganization, TGF-1 promotes endothelial cell survival during in
vitro angiogenesis in collagen gels. This protective effect is mediated
by the activation of the PI3K and p42/p44 MAPK signaling pathways and
is obtained through an autocrine mechanism, which implicates the
synthesis and secretion of TGF- and the activation of the EGF receptor.
We show that TGF-1 has a clear effect on 1G11 endothelial cell
reorganization into capillary-like structures when cells are cultured
in collagen gels. However, this effect appears to depend on an
indirect antiapoptotic action of TGF-1. If this prosurvival effect
is inhibited by PI3K or p42/p44 MAPK inhibitors, cells do not organize
but rather die. The proangiogenic effect of TGF-1 reflects its role
in the later stages of angiogenesis, where endothelial cells pass from
a migratory and proliferative state to a more quiescent, mature, and
differentiated state (6, 64). This is characterized by
downregulation of endothelial cell proliferation, increased basement
membrane deposition, and final morphological organization of cells into
capillary tubes. These processes have been shown to be induced by
TGF- (45, 53, 61, 80), a key role confirmed by TGF-1
and TGF- receptor type II knockout mice. Indeed in both cases these
animals die early in embryogenesis due to defects in vasculogenesis.
Endothelial cell proliferation in wild-type animals was similar to that
in knockout animals; however, a problem in the terminal differentiation
and maintenance of the tube integrity was observed (20,
59). Differentiating cells entering growth-arrested
G0 need to maintain a good balance of cell
survival signals. Thus, it is not surprising that TGF-1, which alone
induces in vitro angiogenesis, can promote endothelial cell survival.
Two pathways, PI3K/Akt and p42/p44 MAPK, capable of inducing survival
signals have been well documented (10, 25, 41, 47). In
endothelial cells these signaling cascades provide a strong
antiapoptotic action. VEGF has a clear cell survival effect through the
activation of PI3K/Akt and stimulation of Bcl2 (28-30,
52). Moreover, endothelial cell-specific vascular
endothelial-cadherin forms a macrocomplex with the type II VEGF
receptor and PI3K, and this interaction is essential for VEGF
antiapoptotic signaling (13). Phorbol myristate acetate
induces tube formation and survival of HUVEC on collagen gels through
activation of p42/p44 MAPK and PI3K (38). In this context,
it is interesting that TGF-1 stimulates PI3K and p42/p44 MAPK (this
study) and induces cell survival. A similar effect of TGF-1 was
observed in macrophages, which TGF-1 protects from apoptosis
induced by serum deprivation (16).
In the majority of cells, stimulation by TGF-1 does not activate
PI3K or p42/p44 MAPK. In 1G11 endothelial cells we do not detect any
rapid activation of p42/p44 MAPK. The same results can be seen in HUVEC
(data not shown). In contrast, we observe a clear stimulation of PI3K
and p42/p44 MAPK by TGF-1 in 1G11 cells, but only 2 h after
TGF-1 addition. We show that this activation is mediated by EGF
receptor stimulation, probably through an autocrine loop involving
TGF- synthesis and secretion. This type of autocrine system has also
been implicated in TGF-1 induction of connective tissue cell
proliferation, mediated by an autocrine PDGF loop (7, 71).
This mechanism of PDGF production also exists in mouse embryo AKR-2B
cells (42) but is not present in endothelial cells
(6). Moreover, TGF-1 has been shown to induce FGF-2 production in bovine corneal endothelial cells (63).
Interestingly, TGF-1 induces VEGF in different cellular types but
not in HUVEC (62). Moreover, it causes the downregulation
of VEGF receptor flk-1 (46). All these results reflect the
role of TGF-1 in the later stages of angiogenesis, where endothelial
cells pass from a proliferative state (stimulated by VEGF) to a more
quiescent and differentiated state (less sensitive to VEGF). In our
study, TGF- seems to be the major factor responsible for TGF-1
stimulation of p42/p44 MAPK and PI3K, leading to endothelial cell
survival. However, we cannot discard the synthesis of other autocrine
factors that could also contribute to part of the response.
We have shown that TGF-1 stimulates TGF- mRNA and protein
synthesis. We have only detected synthesis of a high-molecular-mass form of the TGF- protein in 1G11 cell lysates. TGF- is derived from a larger 20- to 22-kDa transmembrane precursor that, after shedding, generates the soluble and mature 6-kDa form
(18). Higher-molecular-mass forms of biologically active
TGF- are not unusual and have been previously reported
(4). Moreover, transmembrane TGF- can activate the EGF
receptor but only at neighboring cells (11, 79). We have
never detected TGF- processing and release to the medium in our
experimental conditions. Moreover, Akt and p42/p44 MAPK stimulation by
TGF-1 increases with cellular confluence (data not shown). All these
results indicate that probably TGF- arrives at the cellular membrane
as a high-molecular-weight precursor and there exerts its effects in an
autocrine or paracrine (from a cell to its neighboring cell)
manner. Further experiments are required to validate or disprove this
hypothesis. TGF- is an important angiogenic factor, more
effective than EGF, and is highly expressed in neovascularized tumors
(68). Thus, it is possible that the effect of TGF-
production by TGF-1 is not restricted to the prosurvival effect and
also may contribute to tube formation. In fact, different studies
support a role for p42/p44 MAPK and PI3K in the angiogenic process.
Sustained integrin-induced p42/p44 MAPK activity is required for
FGF-2-induced angiogenesis in the chick chorioallantoic membrane
(22). Virally activated Ras cooperates with integrins to
induce tubulogenesis in sinusoidal endothelial cells, an effect that is
blocked by PD-98059 (48). Moreover, B-Raf and MEK-1
knockout mice die during embryonic development due to defects in
vascular endothelial cell differentiation and survival (B-Raf knockout)
(78) or in placental angiogenesis (MEK-1 knockout)
(31). The block of PI3K by wortmannin results in partial
inhibition of tumor-induced angiogenesis (2). However, TGF--stimulated signals, such as p42/p44 MAPK and PI3K, seem to be
necessary but not sufficient for tube formation in collagen gels.
TGF- alone is only partially able to reorganize endothelial cells
and form tubes, and only in the long term. This demonstrates that
TGF-1 unchains other signals that are important for the reorganization of cells on collagen gels. This could be the secretion of compounds of the extracellular matrix (such as fibronectin, collagens, etc.), the stimulation of integrins (8, 17), or the release of metalloproteinases (54, 70). Future studies will help us to identify these signals more precisely and understand the mechanisms of TGF-1-induced angiogenesis.
We thank A. Vecchi for 1G11 cells, J. Madri (Yale University) for
protocols for protein analysis in collagen gels; Y. Yarden (Weizmann
Institute, Israel) for CDM7-IgB-1 construction; S. Pagnotta, J. P. Laugier, and G. Nicaise (Centre de Microscopie Appliquée, Université de Nice-Sophia Antipolis) for microscopic studies; and
P. Huber for H5V cells. We particularly thank D. E. Richard and E. Berra for editorial help and L. Sevilla, R. Buscà, and F. Ventura
and all laboratory members for discussions and technical support.
This work was supported by research grants from CNRS, University of
Nice-Sophia Antipolis, INSERM, Association pour la Recherche contre le
Cancer, Ligue Nationale contre le Cancer, and the European Community
(EC contract B104-CT97-2071). F.V. is the recipient of a Marie Curie
Research Training Grant (EC contract FMBI-CT97-2706).
| 1.
|
Alroy, I., and Y. Yarden.
1997.
The ErbB signaling network in embryogenesis and oncogenesis: signal diversification through combinatorial ligand-receptor interactions.
FEBS Lett.
410:83-86[CrossRef][Medline].
|
| 2.
|
Arbiser, J. L.,
M. A. Moses,
C. A. Fernandez,
N. Ghiso,
Y. Cao,
N. Klauber,
D. Frank,
M. Brownlee,
E. Flynn,
S. Parangi,
H. R. Byers, and J. Folkman.
1997.
Oncogenic H-ras stimulates tumor angiogenesis by two distinct pathways.
Proc. Natl. Acad. Sci. USA
94:861-866[Abstract/Free Full Text].
|
| 3.
|
Baird, A., and T. Durkin.
1986.
Inhibition of endothelial cell proliferation by type beta-transforming growth factor: interactions with acidic and basic fibroblast growth factors.
Biochem. Biophys. Res. Commun.
138:476-482[CrossRef][Medline].
|
| 4.
|
Banerjee, S.,
P. P. Banerjee,
B. R. Zirkin, and T. R. Brown.
1998.
Regional expression of transforming growth factor-alpha in rat ventral prostate during postnatal development, after androgen ablation, and after androgen replacement.
Endocrinology
139:3005-3013[Abstract/Free Full Text].
|
| 5.
|
Barbieri, B.,
G. Balconi,
E. Dejana, and M. B. Donati.
1981.
Evidence that vascular endothelial cells can induce the retraction of fibrin clots.
Proc. Soc. Exp. Biol. Med.
168:204-207[Medline].
|
| 6.
|
Battegay, E. J.
1995.
Angiogenesis: mechanistic insights, neovascular diseases, and therapeutic prospects.
J. Mol. Med.
73:333-346[Medline].
|
| 7.
|
Battegay, E. J.,
E. W. Raines,
R. A. Seifert,
D. F. Bowen-Pope, and R. Ross.
1990.
TGF-beta induces bimodal proliferation of connective tissue cells via complex control of an autocrine PDGF loop.
Cell
63:515-524[CrossRef][Medline].
|
| 8.
|
Bazzoni, G.,
E. Dejana, and M. G. Lampugnani.
1999.
Endothelial adhesion molecules in the development of the vascular tree: the garden of forking paths.
Curr. Opin. Cell Biol.
11:573-581[CrossRef][Medline].
|
| 9.
|
Beck, L., Jr., and P. A. D'Amore.
1997.
Vascular development: cellular and molecular regulation.
FASEB J.
11:365-373[Abstract].
|
| 10.
|
Bonni, A.,
A. Brunet,
A. E. West,
S. R. Datta,
M. A. Takasu, and M. E. Greenberg.
1999.
Cell survival promoted by the ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms.
Science
286:1358-1362[Abstract/Free Full Text].
|
| 11.
|
Brachmann, R.,
P. B. Lindquist,
M. Nagashima,
W. Kohr,
T. Lipari,
M. Napier, and R. Derynck.
1989.
Transmembrane TGF-alpha precursors activate EGF/TGF-alpha receptors.
Cell
56:691-700[CrossRef][Medline].
|
| 12.
|
Bussolino, F.,
A. Mantovani, and G. Persico.
1997.
Molecular mechanisms of blood vessel formation.
Trends Biochem. Sci.
22:251-256[CrossRef][Medline].
|
| 13.
|
Carmeliet, P.,
M. G. Lampugnani,
L. Moons,
F. Breviario,
V. Compernolle,
F. Bono,
G. Balconi,
R. Spagnuolo,
B. Oostuyse,
M. Dewerchin,
A. Zanetti,
A. Angellilo,
V. Mattot,
D. Nuyens,
E. Lutgens,
F. Clotman,
M. C. de Ruiter,
A. Gittenberger-de Groot,
R. Poelmann,
F. Lupu,
J. M. Herbert,
D. Collen, and E. Dejana.
1999.
Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis.
Cell
98:147-157[CrossRef][Medline].
|
| 14.
|
Chang, H.,
D. Huylebroeck,
K. Verschueren,
Q. Guo,
M. M. Matzuk, and A. Zwijsen.
1999.
Smad5 knockout mice die at mid-gestation due to multiple embryonic and extraembryonic defects.
Development
126:1631-1642[Abstract].
|
| 15.
|
Chen, X.,
G. Levkowitz,
E. Tzahar,
D. Karunagaran,
S. Lavi,
N. Ben-Baruch,
O. Leitner,
B. J. Ratzkin,
S. S. Bacus, and Y. Yarden.
1996.
An immunological approach reveals biological differences between the two NDF/heregulin receptors, ErbB-3 and ErbB-4.
J. Biol. Chem.
271:7620-7629[Abstract/Free Full Text].
|
| 16.
|
Chin, B. Y.,
I. Petrache,
A. M. Choi, and M. E. Choi.
1999.
Transforming growth factor beta1 rescues serum deprivation-induced apoptosis via the mitogen-activated protein kinase (MAPK) pathway in macrophages.
J. Biol. Chem.
274:11362-11368[Abstract/Free Full Text].
|
| 17.
|
Collo, G., and M. S. Pepper.
1999.
Endothelial cell integrin alpha5beta1 expression is modulated by cytokines and during migration in vitro.
J. Cell Sci.
112:569-578[Abstract].
|
| 18.
|
Derynck, R.
1992.
The physiology of transforming growth factor-alpha.
Adv. Cancer Res.
58:27-52[Medline].
|
| 19.
|
Derynck, R.,
Y. Zhang, and X. H. Feng.
1998.
Smads: transcriptional activators of TGF-beta responses.
Cell
95:737-740[CrossRef][Medline].
|
| 20.
|
Dickson, M. C.,
J. S. Martin,
F. M. Cousins,
A. B. Kulkarni,
S. Karlsson, and R. J. Akhurst.
1995.
Defective haematopoiesis and vasculogenesis in transforming growth factor-beta 1 knock out mice.
Development
121:1845-1854[Abstract].
|
| 21.
|
Dong, Q. G.,
S. Bernasconi,
S. Lostaglio,
C. R. De,
P. I. Martin,
F. Breviario,
C. Garlanda,
S. Ramponi,
A. Mantovani, and A. Vecchi.
1997.
A general strategy for isolation of endothelial cells from murine tissues. Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants.
Arterioscler. Thromb. Vasc. Biol.
17:1599-1604[Abstract/Free Full Text].
|
| 22.
|
Eliceiri, B. P.,
R. Klemke,
S. Stromblad, and D. A. Cheresh.
1998.
Integrin alphavbeta3 requirement for sustained mitogen-activated protein kinase activity during angiogenesis.
J. Cell Biol.
140:1255-1263[Abstract/Free Full Text].
|
| 23.
|
Folkman, J., and M. Klagsburn.
1987.
Angiogenic factors.
Science
235:442-447[Abstract/Free Full Text].
|
| 24.
|
Folkman, J., and Y. Shing.
1992.
Angiogenesis.
J. Biol. Chem.
267:10931-10934[Free Full Text].
|
| 25.
|
Franke, T. F.,
D. R. Kaplan, and L. C. Cantley.
1997.
PI3K: downstream AKTion blocks apoptosis.
Cell
88:435-437[CrossRef][Medline].
|
| 26.
|
Frater-Schroder, M.,
G. Muller,
W. Birchmeier, and P. Bohlen.
1986.
Transforming growth factor-beta inhibits endothelial cell proliferation.
Biochem. Biophys. Res. Commun.
137:295-302[CrossRef][Medline].
|
| 27.
|
Garlanda, C.,
C. Parravicini,
M. Sironi,
M. De Rossi,
R. Wainstok de Calmanovici,
F. Corozzi,
F. Bussolino,
F. Colotta,
A. Mantovani, and A. Vecchi.
1994.
Progressive growth in immunodeficient mice and host cell recruitment by mouse epithelial cells transformed by polyoma middle-sized T antigen: implications for the pathogenesis of opportunistic vascular tumors.
Proc. Natl. Acad. Sci. USA
91:7291-7295[Abstract/Free Full Text].
|
| 28.
|
Gerber, H. P.,
V. Dixit, and N. Ferrara.
1998.
Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells.
J. Biol. Chem.
273:13313-13316[Abstract/Free Full Text].
|
| 29.
|
Gerber, H. P.,
K. J. Hillan,
A. M. Ryan,
J. Kowalski,
G. A. Keller,
L. Rangell,
B. D. Wright,
F. Radtke,
M. Aguet, and N. Ferrara.
1999.
VEGF is required for growth and survival in neonatal mice.
Development
126:1149-1159[Abstract].
|
| 30.
|
Gerber, H. P.,
A. McMurtrey,
J. Kowalski,
M. Yan,
B. A. Keyt,
V. Dixit, and N. Ferrara.
1998.
Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation.
J. Biol. Chem.
273:30336-30343[Abstract/Free Full Text].
|
| 31.
|
Giroux, S.,
M. Tremblay,
D. Bernard,
J. F. Cardin-Girard,
S. Aubry,
L. Larouche,
S. Rousseau,
J. Huot,
J. Landry,
L. Jeannotte, and J. Charron.
1999.
Embryonic death of Mek1-deficient mice reveals a role for this kinase in angiogenesis in the labyrinthine region of the placenta.
Curr. Biol.
9:369-372[CrossRef][Medline].
|
| 32.
|
Gualandris, A.,
J. P. Annes,
M. Arese,
I. Noguera,
V. Jurukowski, and D. B. Rifkin.
2000.
The latent transforming growth factor--binding protein-1 promotes in vitro differentiation of embryonic stem cells i |
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1 (TGF-
Signaling
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Abstract
Introduction
