Angiogenesis Activators and Inhibitors Differentially Regulate
Caveolin-1 Expression and Caveolae Formation in Vascular Endothelial
Cells
ANGIOGENESIS INHIBITORS BLOCK VASCULAR ENDOTHELIAL GROWTH
FACTOR-INDUCED DOWN-REGULATION OF CAVEOLIN-1*
Jun
Liu
,
Babak
Razani
§,
Shaoqing
Tang¶
,
Bruce
I.
Terman¶,
J. Anthony
Ware
¶
, and
Michael P.
Lisanti
**
From the Departments of
Molecular Pharmacology and
¶ Medicine, Albert Einstein College of Medicine, and ** The
Albert Einstein Cancer Center, Bronx, New York 10461
 |
ABSTRACT |
Angiogenesis is the process by which new blood
vessels are formed via proliferation of vascular endothelial cells. A
variety of angiogenesis inhibitors that antagonize the effects of
vascular endothelial growth factor (VEGF) and basic fibroblast growth
factor (bFGF) have recently been identified. However, the mechanism by which these diverse angiogenesis inhibitors exert their common effects
remains largely unknown. Caveolin-1 and -2 are known to be highly
expressed in vascular endothelial cells both in vitro and
in vivo. Here, we examine the potential role of caveolins in the angiogenic response. For this purpose, we used the well established human umbilical vein endothelial cell line, ECV 304. Treatment of ECV 304 cells with known angiogenic growth factors (VEGF,
bFGF, or hepatocyte growth factor/scatter factor), resulted in a
dramatic reduction in the expression of caveolin-1. This down-regulation event was selective for caveolin-1, as caveolin-2 levels remained constant under these conditions of growth factor stimulation. VEGF-induced down-regulation of caveolin-1 expression also
resulted in the morphological loss of cell surface caveolae organelles
as seen by transmission electron microscopy. A variety of well
characterized angiogenesis inhibitors (including angiostatin, fumagillin, 2-methoxy estradiol, transforming growth factor-
, and
thalidomide) effectively blocked VEGF-induced down-regulation of
caveolin-1 as seen by immunoblotting and immunofluorescence microscopy.
However, treatment with angiogenesis inhibitors alone did not
significantly affect the expression of caveolin-1. PD98059, a specific
inhibitor of mitogen-activated protein kinase and a known angiogenesis
inhibitor, also blocked the observed VEGF-induced down-regulation of
caveolin-1. Furthermore, we show that caveolin-1 can function as a
negative regulator of VEGF-R (KDR) signal transduction in
vivo. Thus, down-regulation of caveolin-1 may be an important step along the pathway toward endothelial cell proliferation.
 |
INTRODUCTION |
Angiogenesis is the development of new capillaries from
pre-existing blood vessels (1). In the adult, angiogenesis is primarily associated with pathological conditions such as tumor formation and
wound healing (2-4). With regard to tumor formation, angiogenesis is
essential for the rapid and continued growth of a variety of tumors
(5-7). This makes anti-angiogenic therapy an attractive and promising
treatment for cancer (8).
Several different angiogenic activators have been described thus far
(2-4). These include, but are not limited to, fibroblast growth factor
(FGF),1 vascular endothelial
growth factor (VEGF), and hepatocyte growth factor (HGF; also known as
scatter factor) (9). These growth factors bind and activate specific
receptor tyrosine kinases within endothelial cells that are coupled to
a variety of signal transduction pathways, most notably the Ras-p42/44
MAP kinase pathway (10, 11).
A number of endogenous angiogenesis inhibitors have been identified
that antagonize the effects of VEGF and FGF. Angiogenesis inhibitors
may be stored as inactive precursors within larger proteins and
released upon appropriate proteolytic processing (12-16). A notable
one is angiostatin, an ~38-40-kDa fragment of plasminogen (12, 13,
17). Others include endostatin (an ~18-kDa fragment of collagen
XVIII), TGF-
and thrombospondin-1 (14-16, 18-21).
Similarly, angiogenesis inhibitors derived from microbes (fumagillin,
from Aspergillus fumigatus; minocycline, a tetracycline derivative) have been identified by a variety of screening approaches (22-26). Others are chemically synthesized compounds, such as
2-methoxyestradiol (an endogenous estrogen metabolite) and thalidomide
(27-30). However, the mechanism of action of these anti-angiogenic
agents remains largely unknown.
Here, we examine the effects of both angiogenesis activators and
inhibitors on the expression of caveolin-1, a caveolae marker protein
that is known to be abundantly expressed in normal endothelial cells in
the adult (31). We find that angiogenesis activators and inhibitors
differentially regulate the expression of caveolin-1. As caveolin-1 is
thought to play a role as a negative regulator of signal transduction
(31), down-regulation of caveolin-1 by angiogenic growth factors may be
important for endothelial cell proliferation and subsequent angiogenesis.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Monoclonal antibodies directed against caveolin-1
(clone 2297) and caveolin-2 (clone 65) were the gift of Drs. John R. Glenney, Jr. and Roberto Campos-Gonzalez (Transduction Laboratories,
Lexington, KY) (32, 33). Reagents and other supplies were obtained from the following sources: polyclonal anti-caveolin-1 IgG from Santa Cruz
Biotechnologies, Inc. (Santa Cruz, CA); rhodamine-goat anti-rabbit IgG
from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA);
Slow-Fade anti-fade reagent from Molecular Probes, Inc. (Eugene, OR);
HGF from Sigma; fumagillin, 2-methoxyestradiol, thalidomide, and
PD98059 from Calbiochem; angiostatin from Angiogenesis Research Industries, Inc. (Chicago, IL); bFGF and TGF-
from Upstate
Biotechnology (Lake Placid, NY); VEGF from PeproTech EC Ltd. (Rocky
Hill, NJ), and the bicinchoninic acid protein assay kit from Pierce.
The cDNA for the human VEGF-R (KDR) was as described previously
(34). The cDNA for mutationally activated MEK-1 is included as a
positive control in the PathDetect Elk-1 trans-reporting system
(Stratagene, Inc.).
Cell Culture and Treatment with Angiogenesis Activators and
Inhibitors--
Human endothelial cells (ECV 304; CRL-1998) were grown
in medium-199 (Life Technologies, Inc.) with 10% heat-inactivated
fetal bovine serum (normal growth medium). Endothelial cells were
seeded at a density of ~1.0 × 104 cells/ml in
24-well plates. After incubation in normal growth medium overnight, the
medium was replaced by medium-199 containing 5% fetal bovine serum.
Protein Analysis--
Expression of caveolin-1 and -2 was
examined by Western blot analysis. Cells were solubilized with sample
buffer containing 0.125 M Tris-HCl (pH 6.8), 5% (w/v) SDS,
2.5% (v/v)
-mercaptoethanol, 5% glycerol in double distilled
water. After boiling for 4 min, proteins were separated by
SDS-polyacrylamide gel electrophoresis (5-15% gradient gels),
transferred to nitrocellulose, and subjected to Western blot analysis
using enhanced chemiluminescence. Prior to loading, the protein
concentration of the samples was measured with the bicinchoninic acid
method using bovine serum albumin as a standard.
Immunofluorescence Microscopy--
Briefly, cells were fixed
with methanol at
20 °C for 10 min, blocked with 2% bovine serum
albumin and stained with specific anti-peptide IgG directed against the
unique N terminus of caveolin-1 (pAb N-20, directed against human
caveolin-1 residues 2-21; Santa Cruz Biotechnologies, Inc.) (35).
Bound primary antibodies were detected using rhodamine-conjugated goat
anti-rabbit IgG. The immunostained cells were mounted in the presence
of Slow-Fade anti-fade reagent. Immunostaining was visualized using a
Zeiss Axio-photofluorescence microscope.
Electron Microscopy--
Transmission electron microscopy was
performed as described previously by our laboratory (36, 37).
In Vivo Assay for VEGF-R Signaling--
Coupling of the VEGF
receptor tyrosine kinase (KDR) to the MAP kinase cascade was assessed
using the PathDetect Elk trans-reporting system (Stratagene, Inc.).
Briefly, confluent ECV 304 or Chinese hamster ovary cells were
trypsinized and seeded at a density of ~8 × 104
cells/well for ECV 304 or 3 × 105 cells/well for
Chinese hamster ovary in 6-well plates. After incubation overnight in
growth medium, cells were transiently co-transfected with the following
plasmids in various combinations, as indicated in a given experiment:
pFR-luc (1 µg/well), pFA-Elk (0.1 µg/well), KDR (1 µg/well),
caveolin-1 (1 µg/well), MEK-1 (0.1 µg/well), or pCB7 (1 µg/well)
using the calcium/phosphate precipitation method (38, 39). 24 h
post-transfection, cells were treated with or without angiogenesis
inhibitors for an additional 20 h, as indicated in a given
experiment. Finally, cells were lysed, and luciferase activity was
measured as we described previously (38, 39).
 |
RESULTS AND DISCUSSION |
Angiogenesis Activators (VEGF, bFGF, and HGF) Down-regulate
Caveolin-1 Expression--
ECV 304 cells are a well characterized
human umbilical endothelial cell line (40-42). These cells express
both caveolin-1 and -2, but fail to express caveolin-3, a
muscle-specific caveolin protein (data not shown).
To evaluate the potential role of caveolins in angiogenesis, we studied
the effect of angiogenesis activators (VEGF and bFGF) on caveolin
expression in ECV 304 cells. In addition, we evaluated the effect of
another growth factor (HGF, hepatocyte growth factor; scatter factor)
that is known to activate the MET receptor tyrosine kinase and promote
angiogenesis (9).
Fig. 1 shows the effects of these growth
factors on caveolin expression. Treatment with VEGF, bFGF, or HGF all
dramatically down-regulated the expression of the caveolin-1 protein
(top panels). In striking contrast, the levels of the
caveolin-2 protein remained essentially unaffected (bottom
panels).

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Fig. 1.
Angiogenesis activators down-regulate
caveolin-1 expression. ECV 304 cells (seeded at a density of
1 × 104 cells/ml) were treated with or without 10 ng/ml VEGF, bFGF, and HGF in medium-199 containing 5% fetal bovine
serum for 24 h. The expression of caveolin-1 and -2 in these
endothelial cells was determined by Western blot analysis using a panel
of isoform-specific antibody probes that selectively recognize either
caveolin-1 or caveolin-2. Each lane contains an equal amount of total
protein.
|
|
As all three growth factors yielded similar results, we decide to focus
our efforts on the effects of VEGF, a well characterized stimulator of
angiogenesis. We next determined the time and concentration dependence
of the effects of VEGF. Interestingly, VEGF-induced down-regulation of
caveolin-1 appeared after only 8 h of treatment, exerting its
maximal effects at 24 h (Fig.
2A). VEGF induced the down-regulation of caveolin-1 at a minimal concentration of 3 ng/ml,
and the effect became maximal at 10 ng/ml (Fig. 2B). In contrast, caveolin-2 levels remained unaffected at every time and dose
investigated.

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Fig. 2.
VEGF-induced down-regulation of caveolin-1
expression is time- and dose-dependent. ECV 304 cells
were treated with 10 ng/ml VEGF for a period of up to 24 h (time
course, panel A) or with 0-20 ng/ml VEGF for 24 h
(concentration dependence, panel B). After the treatment,
cells lysates were subjected to immunoblot analysis with
isoform-specific antibodies that detect either caveolin-1 or
caveolin-2. Each lane contains an equal amount of protein.
|
|
Angiogenesis Inhibitors Block VEGF-induced Down-regulation of
Caveolin-1: Involvement of the p42/44 MAP Kinase
Cascade--
Angiogenesis inhibitors exert their effects by
antagonizing the effects of endothelial growth factors, such as VEGF.
However, their mechanism of action remains largely unknown.
Fig. 3A shows the effects of a
variety of well studied angiogenesis inhibitors (angiostatin,
fumagillin, 2-methoxyestradiol, TGF-
, and thalidomide) in
combination with VEGF. In all cases examined, these angiogenesis
inhibitors selectively blocked the ability of VEGF to induce the
down-regulation of caveolin-1. Caveolin-2 levels are shown for
comparison.

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Fig. 3.
Angiogenesis inhibitors abolish VEGF-induced
down-regulation of caveolin-1 expression. ECV 304 cells were
treated with 10 ng/ml VEGF for 24 h in the presence or absence of
angiostatin (5 µg/ml), fumagillin (0.2 µg/ml), 2-methoxyestradiol
(0.5 µg/ml), TGF- (3 ng/ml), thalidomide (1 µg/ml), or PD98059
(20 µM). After treatment, the expression of caveolin-1 in
these endothelial cells was monitored by immunoblot analysis
(panel A) and immunofluorescence microscopy (panel
B). The presence or absence of caveolae was monitored by
transmission electron microscopy (panel C). In panel
A, each lane contains an equal amount of protein.
Panel B: a, control; b, VEGF alone;
c, VEGF plus angiostatin; d, VEGF plus
thalidomide; e, VEGF plus PD98059. Panel C:
a, control (arrowheads point at caveolae);
b, VEGF alone; c, VEGF plus thalidomide.
Quantitation revealed that VEGF treatment reduced the number of
caveolae organelles by ~20-25-fold. Bar = 100 nm.
|
|
The effects of these inhibitors were also analyzed by
immunofluorescence microscopy (Fig. 3B). In untreated cells,
caveolin-1 staining was abundant and localized primarily to the
periphery of the cell. Growth factor stimulation using VEGF clearly
down-regulated caveolin-1 immunostaining; similarly, we find that VEGF
also induced the down-regulation of morphologically detectable caveolae
as seen by transmission electron microscopy (Fig. 3C).
Again, this down-regulation of caveolin-1 and caveolae organelles was
prevented by treatment with angiogenesis inhibitors, such as
angiostatin and thalidomide (Fig. 3, B and
C).
Because caveolin-1 levels are down-regulated in Ras-transformed NIH 3T3
cells and can be restored to normal levels by inhibiting the p42/44 MAP
kinase cascade, we next evaluated the effects of a well characterized
MEK inhibitor, PD 98059. PD 98059 also blocked the ability of VEGF to
down-regulate caveolin-1 expression, as seen by Western blotting (Fig.
3A) and immunofluorescence microscopy (Fig. 3B).
Thus, inhibition of the p42/44 MAP kinase cascade by PD 98059 is
sufficient to block growth factor-induced down-regulation of
caveolin-1. Interestingly, several recent reports indicate that PD
98059 can function as an angiogenesis inhibitor (10, 11, 43, 44).
Angiogenesis Inhibitors Alone Do Not Affect Caveolin-1
Expression--
Next we tested the effects of the angiogenesis
inhibitors alone on caveolin-1 expression (Fig.
4). Interestingly, in most cases, the
angiogenesis inhibitors alone had little or no effect on caveolin-1
levels, with one exception. In the case of fumagillin, caveolin-1
levels were slightly down-regulated. We do not yet know the
significance of this observation. Importantly, these results indicate
that the angiogenesis inhibitors exert their effects on caveolin-1
expression primarily in response to endothelial growth factors
stimulation via VEGF.

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Fig. 4.
Angiogenesis inhibitors alone do not affect
caveolin-1 expression. ECV 304 cells were treated with or without
angiostatin (5 µg/ml), fumagillin (0.2 µg/ml), 2-methoxyestradiol
(0.5 µg/ml), TGF- (3 ng/ml), thalidomide (1 µg/ml), or PD98059
(20 µM). After the treatment, cells lysates were
subjected to immunoblot analysis with isoform-specific antibodies that
detect either caveolin-1 or caveolin-2. Each lane contains an equal
amount of protein. Note that in most cases, the levels of caveolin-1
remain relatively constant with one exception (fumagillin).
|
|
Caveolin and VEGF-R Signal Transduction--
Caveolae have also
been implicated in signaling through the p42/44 MAP kinase pathway.
Morphological studies have directly shown that ERK-1/2 is concentrated
in plasma membrane caveolae in vivo using immunoelectron
microscopy (45). Evidence has been presented suggesting that other
components of the p42/44 MAP kinase cascade are localized within
caveolae membranes. These include receptor tyrosine kinases (EGF-R;
PDGF-R; Ins-R) (35, 46-48), H-Ras (47, 49), Raf kinase (47), 14-3-3 proteins (48), ERK (37, 48), Shc (48), Grb-2 (48), mSos-1 (48), and Nck
(48).
Recently, we examined the functional role of caveolins in regulating
signaling along the MAP kinase cascade (39). Co-expression with
caveolin-1 dramatically inhibited signaling from EGF-R, Raf, MEK-1, and ERK-2 to the nucleus in vivo (39). Using a
variety of caveolin-1 deletion mutants, we mapped this in
vivo inhibitory activity to caveolin-1 residues 32-95. In
addition, peptides derived from this region of caveolin-1
(i.e. the caveolin-scaffolding domain) also inhibited the
in vitro kinase activity of purified MEK-1 and ERK-2 (39).
Thus, caveolin-1 can inhibit signal transduction from the p42/44 MAP
kinase cascade both in vitro and in vivo by acting as a natural endogenous inhibitor of both MEK and ERK.
To assess whether caveolin-1 expression or angiogenesis inhibitors can
negatively regulate signaling from the VEGF receptor tyrosine kinase
(VEGF-R) along the MAP kinase cascade, we next used an established
in vivo signal transduction assay that measures activation
of the nuclear transcription factor, Elk-1. It is well known that
transient overexpression of receptor tyrosine kinases is sufficient to
cause receptor dimerization and signal transduction. Fig.
5, A and B, shows
that co-expression of VEGF-R with caveolin-1 in either Chinese hamster
ovary cells or ECV 304 cells significantly inhibits VEGF-R-induced
signaling, as compared with empty vector alone (pCB7). Similarly, we
find that co-expression of activated MEK-1 with caveolin-1 in ECV 304 cells dramatically inhibits MEK-induced activation of Elk-1 (Fig.
5C).

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Fig. 5.
Effects of caveolin-1 and angiogenesis
inhibitors on VEGF-R-induced signaling to the nuclear transcription
factor, Elk-1. VEGF-R signaling causes activation of p42/44 MAP
kinases (ERK 1/2), which translocate to the nucleus and activate the
transcription factor Elk. We measured VEGF-R (KDR) induced signaling
using a luciferase-based reporter system that reflects the activation
state of Elk-1 (see "Experimental Procedures"). Chinese hamster
ovary cells (panel A) or ECV 304 cells (panels B,
C, and D) were transiently transfected with a
plasmid encoding either VEGF-R (KDR) (panels A and
B) or mutationally activated MEK-1 (panel C). In
addition, these cells were co-transfected with a plasmid encoding
wild-type full-length caveolin-1 (pCB7-Cav-1) or with empty vector
alone (pCB7) as a negative control. Note that co-transfection with
caveolin-1 significantly inhibited VEGF-R-induced and MEK-1-induced
signaling. In panel D, ECV 304 cells were transfected with
VEGF-R (KDR) and incubated in the presence or absence of a variety of
angiogenesis inhibitors for an additional 20 h (detailed in the
legend of Fig. 4). Note that treatment of ECV 304 cells with either
PD98059 or thalidomide substantially reduced VEGF-R-induced signaling,
whereas treatment with angiostatin or fumagillin had little or no
effect. Data are the mean ± S.D.
|
|
Treatment of ECV 304 cells with either the well characterized MEK
inhibitor, PD98059, or thalidomide substantially reduced VEGF-R induced
signaling, suggesting that thalidomide affects VEGF-R induced
angiogenesis, at least in part, through inhibiting the p42/44 MAP
kinase pathway (Fig. 5D). In contrast, treatment with
angiostatin or fumagillin had little or no effect on VEGF-R-induced activation of Elk-1, suggesting that their effects are independent of
the p42/44 MAP kinase cascade. These observations are consistent with a
recent report that demonstrated that the actions of angiostatin are
independent of the p42/44 MAP kinase cascade (50).
Our current observations with angiogenesis activators and inhibitors
may be related to the ability of caveolin-1 to positively regulate
contact inhibition and growth arrest in normal cells (51). We have
previously shown (51) that caveolin-1 expression levels are
down-regulated in rapidly dividing NIH 3T3 cells and are dramatically
up-regulated at confluency. These results suggest that up-regulation of
caveolin-1 expression levels may be important to mediate normal contact
inhibition and to negatively regulate the activation state of the
p42/44 MAP kinase cascade and other signaling pathways (51). In support
of these findings, it has been shown that caveolins are most abundantly
expressed in terminally differentiated cells such as endothelial cells,
adipocytes, and muscle cells and are dramatically up-regulated during
adipogenesis and myotube formation (33, 52-54). Thus, down-regulation
of caveolin-1 expression and caveolae organelles may be a prerequisite
for endothelial cell proliferation and, therefore, angiogenesis.
 |
FOOTNOTES |
*
This work was supported by a National Institutes of Health
Grant R01-CA-80250 from the NCI, grants from the Charles E. Culpeper Foundation, the G. Harold and Leila Y. Mathers Charitable Foundation, and the Sidney Kimmel Foundation for Cancer Research (to M.P.L.).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 National Institutes of Health Medical Scientist
Training Program Grant T32-GM07288.
Supported by National Institutes of Health Grants R01-HL-47032
and HL51043.

To whom correspondence should be addressed: Dept. of Molecular
Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park
Ave., Bronx, NY 10461. Tel.: 718-430-8828; Fax: 718-430-8830; E-mail:
lisanti{at}aecom.yu.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
FGF, fibroblast
growth factor;
bFGF, basic FGF;
VEGF, vascular endothelial growth
factor;
VEGF-R, VEGF receptor;
MAP kinase, mitogen-activated protein
kinase;
TGF-
, transforming growth factor-
;
HGF, hepatocyte growth
factor;
ERK, extracellular signal-regulated kinase, MEK,
mitogen-activated protein kinase kinase.
 |
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