Peroxisome Proliferator-activated Receptor
Ligands Are Potent
Inhibitors of Angiogenesis in Vitro and in
Vivo*
Xiaohua
Xin,
Suya
Yang,
Joe
Kowalski, and
Mary E.
Gerritsen
From the Department of Cardiovascular Research, Genentech, Inc.,
South San Francisco, California 94080
 |
ABSTRACT |
Peroxisome proliferator-activated receptor
(PPAR
) is a nuclear receptor that functions as a transcription
factor to mediate ligand-dependent transcriptional
regulation. Activation of PPAR
by the naturally occurring ligand,
15-deoxy-
12,14-prostaglandin J2
(15d-PGJ2), or members of a new class of oral antidiabetic agents, e.g. BRL49653 and ciglitizone, has been linked to
adipocyte differentiation, regulation of glucose homeostasis,
inhibition of macrophage and monocyte activation, and inhibition of
tumor cell proliferation. Here we report that human umbilical vein
endothelial cells (HUVEC) express PPAR
mRNA and protein.
Activation of PPAR
by the specific ligands 15d-PGJ2,
BRL49653, or ciglitizone, dose dependently suppresses HUVEC
differentiation into tube-like structures in three-dimensional collagen
gels. In contrast, specific PPAR
and -
ligands do not affect tube
formation although mRNA for these receptors are expressed in HUVEC.
PPAR
ligands also inhibit the proliferative response of HUVEC to
exogenous growth factors. Treatment of HUVEC with 15d-PGJ2
also reduced mRNA levels of vascular endothelial cell growth factor
receptors 1 (Flt-1) and 2 (Flk/KDR) and urokinase plasminogen activator
and increased plasminogen activator inhibitor-1 (PAI-1) mRNA.
Finally, administration of 15d-PGJ2 inhibited vascular
endothelial cell growth factor-induced angiogenesis in the rat cornea.
These observations demonstrate that PPAR
ligands are potent
inhibitors of angiogenesis in vitro and in
vivo, and suggest that PPAR
may be an important molecular target for the development of small-molecule inhibitors of angiogenesis.
 |
INTRODUCTION |
Angiogenesis, the formation of new blood vessels, is not only
critically involved in a number of normal physiological processes such
as embryonic development, ovulation, and wound healing, but is also a
critical step in many pathological conditions including solid tumor
growth, diabetic retinopathy, and age-related macular degeneration (1).
The complex steps involved in new vessel formation have been well
characterized in recent years and involve the degradation of the
basement membrane by cellular proteases, the penetration and migration
of endothelial cells into the extracellular matrix, endothelial
proliferation, and the resultant formation of patent, interconnected
vascular networks (2, 3). It is now well established that the
progression of solid tumor growth and metastasis is dependent on
angiogenesis, with the resultant global effort by many laboratories to
identify new angiostatic therapies for the treatment of cancer.
Peroxisome proliferator-activated receptors
(PPARs)1 are members of the
steroid receptor superfamily and, as such, are ligand-activated transcription factors (4, 5). Three subtypes of PPAR,
,
(also
known as
or NUCI), and
have been identified and cloned. Like
other members of this superfamily, PPARs mediate transcriptional regulation by their central DNA binding domain that recognizes response
elements in the promoters of specific target genes (6, 7). Activation
of PPAR
has been linked to adipocyte differentiation and regulation
of glucose homeostasis in rodents and humans (8). Recent evidence also
shows that the natural receptor ligand for PPAR
,
15d-PGJ2 (9, 10), and synthetic antidiabetic
thiazolidinedione drugs (e.g. BRL49653 and ciglitizone),
inhibit macrophage and monocyte activation (11, 12) and suppress tumor
cell growth (14-16). PPAR
can heterodimerize with at least one
other member of the steroid receptor superfamily, retinoid acid
receptor (RXR) (6, 17). Specific ligands for the PPAR
and RXR have
been shown to act synergistically to induce terminal differentiation of
human liposarcoma cells (18) in vitro and to enhance insulin sensitivity in diabetic animals (19). Activation of PPAR
has been
demonstrated to mediate lipid catabolism. Treatment of animals with
PPAR
activators results in the proliferation of peroxisomes and the
induction of hepatic genes involved in the
-oxidation of fatty acids
(20). Mice that lack functional PPAR
accumulate lipid droplets in
their livers (21). Several selective PPAR
activators have been
described including WY 14643 and clofibrate (22). Inhibition of human
aortic smooth muscle cell activation by PPAR
ligands has also been
reported recently (13). Although PPAR
has been found to be
ubiquitously expressed, specific functions for this receptor are not
known at the present time.
In this study we report on the potent and novel inhibitory activity of
PPAR
ligands on HUVEC differentiation into tube-like structures and
proliferation in vitro, and the inhibition of VEGF elicited
angiogenesis in vivo. These studies demonstrate that PPAR
is an important molecular target for the development of small molecule
inhibitors of angiogenesis, which may be useful therapeutic agents in
the treatment of cancer and other vasculoproliferative disorders.
 |
EXPERIMENTAL PROCEDURES |
Materials--
PGA1, PGA2,
PGB1, PGB2, PGD1, PGD2,
PGE1, PGE2, ciglitizone, WY 14643, clofibrate,
and 15d-PGJ2 were from Cayman Chemical (Ann Arbor, MI).
BRL49653 was kindly synthesized at Genentech by Dr. Jim Marsters (South
San Francisco, CA). HUVEC were purchased from Clonetics (San Diego, CA)
and maintained in Clonetics EGM medium supplemented with 10% fetal
bovine serum (FBS) and endothelial cell growth supplements provided by
the company. Type I rat tail collagen and recombinant basic fibroblast
growth factor (bFGF) were purchased from Collaborative Research (Becton
Dickinson Labware, Bedford, MA). Recombinant VEGF was from Genentech.
10× medium 199 (M199) and PMA were purchased from Sigma. FBS was from
Hyclone (Logan, Utah). ITS (insulin, transferrin, and selenium A),
trypsin, and versene were from Life Technologies, Inc. The mouse
monoclonal antibody to PPAR
(E-8, SC-7273) and goat polyclonal
antibodies to PPAR
(N-19, SC-1985) and PPAR
(C-20, SC-1983) were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Magnetic
protein A beads (Dynabeads) were from Dynal (Lake Success, NY).
Endothelial Tube Assay--
Collagen gels were formed by mixing
together ice-cold gelation solution (10× M199, H2O, 0.53 M NaHCO3, 200 mM
L-glutamine, type I collagen, 0.1 M NaOH,
100:27.2:50:10:750:62.5 by volume) and cells in 1× basal medium (see
below) at a concentration of 3 × 106 cells/ml at a
ratio of 4 volumes gelation solution:1 volume of cells. After gelation
at 37 °C for 30 min, the gels were overlaid with 1× basal medium
consisting of M199 supplemented with 1% FBS, 1× ITS, 2 mM
L-glutamine, 50 µg/ml ascorbic acid, 26.5 mM
NaHCO3, 100 units/ml penicillin, and 100 units/ml
streptomycin supplemented with 40 ng/ml bFGF, 40 ng/ml VEGF, and 80 nM PMA. All drugs were added to the 1× basal medium
immediately after gelation. To quantitate tube formation, the number of
tubes per high power (20X) field was determined at 48 h after
addition of the basal medium. A tube was defined as an elongated
structure comprised of one or more endothelial cells that exceeded 100 µm in length (long axis). Five independent fields separated by 100 µm optical sections were assessed for each well, and the average
number of tubes/20X field determined. Cytoxicity was assessed using
cell proliferation kit II from Boehringer Mannheim.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Assays--
Synthesis of complementary DNA (cDNA) was performed
using 6 µg of total RNA extracted from human liver (positive control) or HUVEC cultured on three-dimensional collagen gels for 24 h by
random primer according to the manufacturer's instructions (Stratagene
Co., San Diego, CA). Subsequent amplifications of the partial cDNA
encoding PPAR
,
, and
were performed using different amounts
of reverse transcribed mixture (4 µl for liver
,
, and
and
5 µl for HUVEC
and
and 6 µl for HUVEC
) as templates
with specific oligonucleotide primers as follows: PPAR
sense
5'-CCAGTATTTAGGACGCGGTCC-3' and antisense
5'-AAGTTCTTCAAGTAGGCCTGCG-3'; PPAR
sense 5'-AACTGCAGATGGGCTGTGAC-3'
and antisense 5'-GTCTCGATGTCGTGGATCAC-3' and PPAR
sense
5'-TCTCTCCGTAATGGAAGACC-3' and antisense 5'-GCATTATGAGACATCCCCAC-3'. The expected sizes of PCR products for PPAR
,
, and
were 492, 484, and 474 base pairs, respectively. Negative controls for
reverse transcription and PCR amplifications were included. The PCR
mixtures were subjected to 35 cycles of amplification by denaturation
(30 s at 94 °C), hybridization (30 s at 55 °C), and elongation
(45 s at 55 °C). The PCR products were analyzed by 1.5% agarose gel electrophoresis with ethidium bromide.
Real Time RT-PCR (Taqman) Assay--
This technique has been
used to quantitatively monitor mRNA expression and has been
described in detail previously (26, 27). Total RNA was extracted from
HUVEC cultured in three-dimensional collagen gels or on collagen-coated
flasks for various times in 1× basal medium consisting of M199
supplemented with 1% FBS, 1× ITS, 2 mM
L-glutamine, 50 µg/ml ascorbic acid, 26.5 mM
NaHCO3, 100 units/ml penicillin, and 100 units/ml
streptomycin supplemented with 40 ng/ml bFGF, 40 ng/ml VEGF, and 80 nM PMA. A gene-specific PCR oligonucleotide primer pair and
an oligonucleotide probe labeled with a reporter fluorescent dye at the
5'-end and a quencher fluorescent dye at the 3'-end were designed using
the Oligo 4.0 software (National Bioscience, Plymouth, MN) following
guidelines suggested in the Taqman Model 7700 sequence detection
instrument manual (PE Applied Biosystem). The primers and probes used
were as follows: human PPAR
gene forward primer
5'-GGACGTGCTTCCTGCTTCAT-3', reverse primer
5'-CACCATCGCGACCAGATG-3', and probe 5'-TTGGAGCTCGGCGCACAACCA-3'; human
PPAR
gene forward primer 5'-TGACCTGCGGCAACTGG-3', reverse primer
5'-TTCGGTCTTCTTGATCCGCT-3', and probe 5'-CACCGAGCACGCCCAGATGATG-3'; human PPAR
gene forward primer 5'-GCCAAGCTGCTCCAGAAAAT-3', reverse primer 5'-TGATCACCTGCAGTAGCTGCA-3', and probe
5'-ACAGACCTCAGACAGATTGTCACGGAACAC-3'; human Flk/KDR gene forward primer
5'-CACCACTCAAACGCTGACATGTA-3', reverse primer
5'-CCAACTGCCAATACCAGTGGA-3', and probe 5'-TGCCATTCCTCCCCCGCATC-3'; human Flt-1 gene forward primer 5'-ACCCAGATGAAGTTCCTTTGGA-3', reverse
primer 5'-CCCAGTTTAGTCTCTCCCGG-3', and probe
5'-CCTTATGATGCCAGCAAGTGGGAGTTTG-3'; human urokinase-type plasminogen
activator (uPA) gene forward primer 5'-ACGCTTGCTCACCAGAATGA-3', reverse
primer 5'-GCGCACACCTGCCCTC-3', and probe
5'-ATTGCCTTGCTGAAGATCCGTTCCAA-3'; human PAI-1 gene forward primer
5'-TCGTCCAGCGGGATCTGA-3', reverse primer 5'-GTGCTCCGGAACAGCCTG-3', and
probe 5'-CCAGGGCTTCATGCCCCACTTCT-3'; human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene forward 5'-GAAGGTGAAGGTCGGAGTC-3', reverse
5'-GAAGATGGTGATGGGATTTC-3', and probe 5'-CAAGCTTCCCGTTCTCAGCC-3'. Total RNA (100 ng) was added to a 50-µl RT-PCR reaction mixture according to the manufacturer's protocol (Roche Molecular Systems, Inc., Branchburg, NJ). The thermal cycling conditions included 1 cycle
at 48 °C for 30 min, 1 cycle at 95 °C for 10 min, 40 cycles at
95 °C for 15 s, at 60 °C for 1 min, and finally hold at
25 °C for 2 min. Standard curves for expression of each gene were generated by serial dilution of the total RNA isolated from HUVEC. Expression of GAPDH gene was not significantly altered during the times
(up to 48 h) of incubation with drugs and vehicle. Therefore, the
relative mRNA expression of each gene was normalized to the level
of GAPDH in the same RNA preparation.
Western Blot Assays--
Confluent HUVEC grown on type I
collagen-coated flasks were washed once with ice-cold
phosphate-buffered saline, then scraped and pelleted by centrifugation.
Pellets were lysed in ice-cold lysis buffer (20 mM
Tris/HCl, 150 mM NaCl, 2 mM EDTA, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 40 units/ml
aprotinin, 15 µg/ml leupeptin, 0.36 mM
1,10-phenanthroline, 1% Nonidet P-40, and 1% Triton X-100, pH 7.5).
An aliquot equivalent to 100 µg of protein was incubated for 16 h at 4 °C with 20 ng of anti-PPAR antibody in a total volume of 500 µl. Protein-A agarose beads (50 µl) were then added, and the
mixture incubated for 2 h at 4 °C. The beads were washed three
times with 500 µl of lysis buffer, and the proteins remaining on the
beads were solubilized by heating at 100 °C for 5 min in 2× SDS
sample buffer. The precipitated complexes were then analyzed by Western
blotting. Proteins were visualized using the ECL chemiluminescence kit
(Amersham Pharmacia Biotech).
Proliferation Assay--
HUVEC were seeded on collagen-coated
96-well plates at 6,000 cells/cm2 in Clonetics EGM
containing endothelial cell growth supplements and 10% FBS, 2 mM L-glutamine, 100 units/ml penicillin, and
100 units/ml streptomycin and allowed to attach for 4 h. Medium
was then replaced with 1× basal medium consisting of M199 supplemented with 1% FBS, 1× ITS, 2 mM L-glutamine, 50 µg/ml ascorbic acid, 26.5 mM NaHCO3, 100 units/ml penicillin, and 100 units/ml streptomycin supplemented with 40 ng/ml bFGF, 40 ng/ml VEGF, and 80 nM PMA. Cells were
cultured in the above medium in the presence of drugs or vehicle for
4 h. 5 µl (100 µM) of 5'-bromo-2'-deoxyuridine (BrdUrd) was added in a final volume of 100 µl/well, and cells were
incubated for another 20 h. BrdUrd incorporation was evaluated by
an enzyme-linked immunosorbent assay kit from Boehringer Mannheim.
Corneal Angiogenesis Assay--
A 1.5-mm incision was made
approximately 1 mm from the center of the cornea of isoflurane-ketamine
(60-80 mg/kg) xylazine (10-15 mg/kg) anesthetized Sprague-Dawley
rats. Using a curved spatula, the incision was bluntly dissected
through the stroma toward the outer canthus of the eye. A hydron pellet
(2 × 20 mm) containing VEGF (200 ng), sucralfrate (100 ng) with
or without (control) 15d-PGJ2 (10 µg/pellet) was inserted
into the base of the pocket. After surgery the eyes were coated with
gentamicin ointment. Animals were observed at 24-48 h for the
occurrence of nonspecific inflammation and then daily thereafter. At
day 6, the animals were euthanized and injected with fluorescein
isothiocyanate-dextran to allow for visualization of the vasculature.
Corneal whole mounts were made of the enucleated eyes and analyzed for
neovascular area using computer-assisted image analysis (Image Pro-Plus
2.0, Silver Spring, MD).
Statistics--
Data are expressed as the mean ± S.E.
Statistic analysis was performed using one-way analysis of variance
(INSTAT, Graph Pad Software, Sorrento Valley, CA). Multiple comparisons
against the control were analyzed using Bonferroni modification of
Student's t test to determine differences between groups. A
p value < 0.05 was accepted as significant.
 |
RESULTS AND DISCUSSION |
Endothelial Tube Formation in Three-dimensional Collagen
Gels--
In this study we used a model of in vitro
angiogenesis initially described by Davis et al. (23). In
this model, endothelial cells suspended in three-dimensional collagen
lattices comprised of type I collagen in the presence of VEGF, bFGF,
and PMA undergo rapid morphogenesis. Numerous vacuoles are observed in
the majority of endothelial cells within 4 h; the formation of
tube-like structures can be observed at 24 h, and an
interconnected network of tube-like structures is observed (Fig.
1A) at 48 h. Inhibitors
of mRNA synthesis (actinomycin D) and protein synthesis
(cycloheximide) completely blocked the formation of tube-like
structures (data not shown), demonstrating the requirement for new
mRNA and protein synthesis.

View larger version (63K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of eicosanoid derivatives on HUVEC
tube formation in three-dimensional collagen gels.
Panel A, representative photomicrograph
documenting the formation of network of tube-like structures containing
lumens in HUVEC grown in three-dimensional collagen gels for 48 h
in the presence of 40 ng/ml VEGF, 40 ng/ml bFGF, and 80 nM
PMA; panel B, inhibitory effects of
15d-PGJ2 on tube formation. HUVEC were grown in
three-dimensional collagen gels in the presence of VEGF, bFGF, and PMA
as well as 10 µM 15d-PGJ2 for 48 h. Note
that most cells in the gel remain rounded and devoid of vacuoles or
lumen-like structures. Parallel experiments using the vehicle and
growth factors were similar to panel A.
Panel C, effects of different eicosanoids on tube formation
of HUVEC in three-dimensional collagen gels. Drugs were tested at 1, 10, and 100 µM. Data are expressed as percent inhibition
compared with controls incubated with growth factors and the vehicle
and are shown as mean ± S.E. of n 3. *,
significantly different from vehicle treated control (Bonferroni
modified Student's t test for multiple comparisons).
|
|
Effects of Eicosanoids on Tube Formation by HUVEC--
It has
recently been shown that a number of eicosanoid metabolites can serve
as PPAR ligands (8-10, 22), and activation of PPARs has been linked to
numerous functions in a number of cell types (8, 11-16). To determine
whether PPARs could affect HUVEC differentiation, we investigated the
effect of 15d-PGJ2, a naturally occurring endogenous ligand
of PPAR
on HUVEC tube formation. HUVEC were grown in
three-dimensional collagen gels in medium containing VEGF, bFGF and PMA
with addition of 15d-PGJ2 (10 µM) for 48 h and were analyzed for tube formation. As shown in Fig. 1B,
15d-PGJ2 markedly reduced HUVEC tube formation. The majority of cells in the gels remained rounded and devoid of vacuoles or lumen-like structures. Vehicle-treated control groups did not show
any significant inhibitory effect (data not shown). We extended our
observations to a variety of metabolites of 20:4 and 20:3 fatty acids
for their ability to inhibit tube formation in three-dimensional collagen gels (Fig. 1C). PGJ2 and
PGD2 and their derivatives/analogs demonstrated
dose-dependent inhibition of tube formation over the
concentration range of 1 to 100 µM. PGB1 and
PGB2 possessed only weak inhibitory effects. In contrast,
two prostanoids known to activate cell surface G-protein-coupled
receptors, namely PGE1 and PGE2, had no
significant effect over this concentration range. Among the tested
drugs, 15d-PGJ2 was the most potent inhibitor of HUVEC tube
formation. Data from parallel cytotoxicity assays indicated that none
of the tested prostanoids exhibited significant toxicity over the
time course (48 h) of the experiment (data not shown). Previous studies
(24, 25) have demonstrated that PGD2 can spontaneously
convert to PGJ2, and in the presence of serum or albumin,
to 15d-PGJ2, a natural selective ligand for PPAR
. Therefore, activation of PPAR
may modulate endothelial morphogenesis.
PPAR
Is Expressed in HUVEC--
RT-PCR was used to demonstrate
the expression of all three PPAR (
,
, and
) mRNAs in HUVEC
cultured in the three-dimensional collagen gels. In two independent
experiments conducted with different HUVEC populations derived from
different donors, the mRNA expression of three isoforms of PPAR
were demonstrated (Fig. 2A).
By using real time quantitative RT-PCR (Taqman) (26, 27), the relative mRNA expression levels of each isoform were determined to be
PPAR
> PPAR
> PPAR
(Fig. 2B). Preliminary observations also
suggested that expression of PPAR
mRNA was not altered in medium
containing VEGF, bFGF and PMA at 4, 24 or 48 h of incubation with
15d-PGJ2 or vehicle in HUVEC cultured in three-dimensional
collagen gels or on collagen-coated surfaces (data not shown). The
expression of PPAR
protein in HUVEC was confirmed by Western blot
analysis (Fig. 2C), and PPAR
protein levels were not
significantly altered by the growth factor mixture when HUVEC were
cultured on collagen-coated surfaces (Fig. 2C). Using
commercially available antibodies, we were unable to demonstrate
protein expression of PPAR
and -
in HUVEC (data not shown). The
expression of PPAR
mRNA in HUVEC has been previously reported
(28); however, this study demonstrates for the first time the
expression of PPAR
mRNA and protein and PPAR
mRNA by
HUVEC.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 2.
Expression of PPAR in HUVEC.
A, RT-PCR analysis using specific primers for PPAR , - ,
or - as described under "Experimental Procedures." Specific
cDNA were synthesized from human liver RNA (positive control) and
from HUVEC RNA using random primer in the presence of 50 units of
Moloney murine leukemia virus reverse transcriptase. PCR products were
resolved on a 1.5% agarose gel. The DNA ladder was included as a
marker to indicate the sizes of the PCR products of PPAR
(lanes 1 and 4), - (lanes 2 and
5), and - (lanes 3 and 6).
B, quantitative analysis of mRNA expression of PPARs in
HUVEC by real time RT-PCR (Taqman). Total RNA (100 ng) were extracted
from HUVEC grown in three-dimensional collagen gels in medium
containing VEGF, bFGF and PMA. The relative expression of PPAR isoforms
were normalized to GAPDH levels measured in the same RNA preparation.
Data shown are from three independent experiments analyzed in duplicate
and are expressed as mean ± S.E. C, Western blot
analysis of PPAR expression in HUVEC. Tissue extract from mouse
adipose tissue (20 µg of protein, positive control) or HUVEC cell
lysates (100 µg of protein) prepared from cells cultured on
collagen-coated surfaces in 1× basal medium without (lane
1) or containing (lane 2) VEGF, bFGF, and PMA for
24 h were immunoprecipitated by PPAR monoclonal antibody.
Following SDS-polyacrylamide gel electrophoresis and transfer to
nitrocellulose, the proteins were immunoblotted with the same PPAR
monoclonal antibody. Similar results were obtained from two independent
experiments with different HUVEC cultures.
|
|
PPAR
Mediates the Inhibitory Effect on HUVEC Tube Formation and
Proliferation--
To determine which PPAR isoform(s) mediated the
inhibition of HUVEC tube formation, the effect of specific PPAR ligands
was assessed. As shown in Fig.
3A, treatment of HUVEC with
the specific PPAR
ligands, 15d-PGJ2, BRL49653, and
ciglitizone, dose dependently inhibited tube formation with
half-maximal inhibition concentrations (IC50) of 2.8, 6.2, and 2.7 µM, respectively. In contrast, the selective
agonists for PPAR
(WY 14643 and clofibrate) and PPAR
(erucic
acid) (29) did not significantly inhibit tube formation (Fig.
3B). These observations demonstrated that activation of PPAR
results in inhibition of endothelial cell differentiation into
tube-like structures in vitro. We further investigated
whether PPAR
ligands also regulate the proliferative response of
HUVEC to growth factors. BrdUrd incorporation assays were performed to
detect proliferation of HUVEC cultured on type I collagen-coated surface in medium containing VEGF, bFGF, and PMA in the presence of
vehicle or PPAR
ligands. As shown in Fig. 3C,
15d-PGJ2 and BRL49653 dose dependently inhibited HUVEC
proliferation with IC50 of 2.4 and 15.7 µM,
respectively. In contrast, PGE1 and PGE2 did not affect HUVEC proliferation (data not shown). These results demonstrate that PPAR
ligands also repress growth factor-induced HUVEC proliferation in vitro.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 3.
Effects of PPAR ligands on HUVEC tube
formation and proliferation. HUVEC were cultured in
three-dimensional collagen gels in medium containing VEGF, bFGF, and
PMA for 48 h with addition of either PPAR ligands or vehicle
(A and B). A,
dose-dependent inhibition of HUVEC tube formation by
PPAR ligands, 15d-PGJ2, BRL49653, and ciglitizone
(Cgt); B, selective ligands of PPAR (WY 14643, 100 µM and clofibrate, 100 µM) and PPAR
(erucic acid, 200 µM) do not inhibit tube formation;
C, PPAR activation inhibits HUVEC proliferation. HUVEC
cultured on type I collagen-coated surfaces in medium containing VEGF,
bFGF, and PMA were treated with various concentrations of
15d-PGJ2 or BRL49653. Cell proliferation was monitored as
BrdUrd incorporation. Data in A, B, and
C are the mean ± S.E. from three to four independent
experiments. Data are expressed as percent inhibition (A and
C) or percent of control (B) compared with
vehicle treated groups in the presence of growth factors.
|
|
15d-PGJ2 Regulates Gene Expression Events Associated
with Angiogenesis--
Flk/KDR and Flt-1 are two structurally related
endothelial cell tyrosine kinase receptors for VEGF. The importance of
these two receptors during angiogenesis has been clearly demonstrated by the findings that KDR functions as a transducer to signal
endothelial cell proliferation and differentiation and that Flt-1 is a
critical survival factor involved in endothelial cell morphogenesis
(30-32). We examined whether activation of PPAR
alters Flt-1 and
KDR gene expression using real time quantitative RT-PCR. Both KDR and
Flt-1 mRNA were up-regulated by the mixture of growth factors in
HUVEC grown in three-dimensional collagen gels (Fig.
4, A and B,
open bars). 15d-PGJ2 inhibited the induction of
both VEGF receptor mRNA in HUVEC cultured in three-dimensional
collagen gels (Fig. 4, A and B, hatched
bars). Similar data were obtained when HUVEC were grown on type I
collagen-coated surface with growth factor stimulation (not shown). It
is also well known that the production of proteases (e.g.
plasminogen activators) and their inhibitors (e.g.
plasminogen activator inhibitor I, PAI-1) is correlated with
endothelial cell degradation of extracellular matrix and migration, the
two critical steps of the angiogenic processes (33). We analyzed the
effects of 15d-PGJ2 on gene expression of uPA and PAI-1 in
three-dimensional collagen gels. Treatment of HUVEC with
15d-PGJ2 reduced uPA mRNA at 4 h and increased
PAI-1 gene expression at 24 h, respectively (Fig. 4, C
and D, hatched bars). Taken together, these
observations indicate some possible molecular mechanisms by which
PPAR
ligands mediate inhibition of angiogenesis.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
Regulation of angiogenesis associated gene
expression by 15d-PGJ2. Total RNA (100 ng) were
isolated from HUVEC grown in three-dimensional collagen gels for the
indicated times in medium containing VEGF, bFGF, and PMA in the
presence of 10 µM 15d-PGJ2 (hatched
bars) or vehicle (open bars). The mRNA levels of
the indicated genes were determined by real time quantitative RT-PCR
(Taqman) analysis. The relative expression levels of each gene were
normalized to the levels of GAPDH measured in the same RNA preparation.
#, significantly different from control groups at 4 h; *,
significantly different from time-matched control groups. Data are
shown as means ± S.E. from three to five independent experiments
and were analyzed by one-way analysis of variance.
|
|
15d-PGJ2 Demonstrates Anti-angiogenic Activity in
Vivo--
To determine the potential anti-angiogenic effect of PPAR
ligand 15d-PGJ2 in vivo, hydron pellets
containing 200 ng of recombinant VEGF with or without 10 µg of
15d-PGJ2 were implanted into the corneas of Sprague-Dawley
rats. Summary data from the experiment show that pellets containing the
combination of 15d-PGJ2 and VEGF demonstrated a significant
reduction in vessel area compared with the VEGF only positive controls
(Fig. 5, A, B, and
C). Results from this experiment were repeated in a
second independent experiment. These in vivo data strongly
support our in vitro observations that activation of PPAR
results in marked inhibition of angiogenesis.

View larger version (65K):
[in this window]
[in a new window]
|
Fig. 5.
Anti-angiogenic effect of
15d-PGJ2 in vivo. Representative
flatmount photographs of rat corneas 5 days after implantation of
hydron pellets containing 200 ng/pellet VEGF (A) or 200 ng/pellet VEGF and 10 µg/ml 15d-PGJ2 (B). Bar
graph (C) shows the summary data of the angiogenic response
induced by 200 ng/pellet VEGF alone or 200 ng/pellet VEGF and 10 µg/ml 15d-PGJ2. The area of new vessels was assessed on
day 6 postimplantation. Data are expressed as mean ± S.E. *,
significantly different from control (Ctr); +, significantly
different from VEGF alone groups (Mann-Whitney test for nonparametric
values).
|
|
Recent evidence has demonstrated that PPAR
is highly expressed in
most colon cancer cells and breast cancer cells (14, 16). Activation of
the PPAR
resulted in growth arrest and induction of differentiation
of cancer cells and reduction of tumor cell growth rate, suggesting
potential implications of PPAR
as a target for treatment of human
cancers. In this study we have documented, for the first time, the
expression of PPAR
in human endothelial cells. Activation of PPAR
with either naturally occurring ligand or synthetic selective ligands
results in potent inhibition of endothelial differentiation into
tube-like structures and proliferation in vitro and
suppression of VEGF-induced angiogenesis in vivo. PPAR
activation also inhibits the expression of at least three important
genes in the angiogenic process, the VEGF receptors Flk/KDR, Flt-1, and
the protease uPA. This study thus provides a new insight into the
mechanism of PPAR
ligands as inhibitors of solid tumor growth and
suggests that such drugs may also provide novel means to control other
angiogenic disorders.
 |
ACKNOWLEDGEMENTS |
We thank the DNA synthesis group at Genentech
for oligonucleotides and real time RT-PCR probes.
 |
FOOTNOTES |
*
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.
To whom correspondence should be addressed: Dept. of Cardiovascular
Research, MS 42, Genentech, Inc., 1 DNA Way, South San Francisco, CA
94080. Tel.: 650-225-6870; Fax: 650-225-6327; E-mail: meg{at}gene.com.
 |
ABBREVIATIONS |
The abbreviations used are:
PPARs, peroxisome
proliferator-activated receptors;
HUVEC, human umbilical vein
endothelial cells;
15d-PGJ2, 15-deoxy-
12,14-prostaglandin J2;
RXR, retinoid acid
receptor;
VEGF, vascular endothelial growth factor;
PMA, phorbol
myristate acetate;
RT-PCR, reverse transcription-polymerase chain
reaction;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
BrdUrd, 5'-bromo-2'-deoxyuridine;
FBS, fetal bovine serum;
bFGF, basic
fibroblast growth factor;
ITS, insulin, transferrin, and selenium A;
uPA, urokinase-type plasminogen activator.
 |
REFERENCES |
-
Folkman, J.
(1995)
Nat. Med.
1,
27-31[Medline]
[Order article via Infotrieve]
-
Folkman, J.
(1986)
Cancer Res.
46,
467-473[Medline]
[Order article via Infotrieve]
-
Liotta, L. A.,
Steeg, P. S.,
and Stetler-Stevenson, W. G.
(1991)
Cell
64,
327-336[Medline]
[Order article via Infotrieve]
-
Issemann, I.,
and Green, S.
(1990)
Nature
347,
645-650[CrossRef][Medline]
[Order article via Infotrieve]
-
Schoonjans, K.,
Martin, G.,
Staels, B.,
and Auwerx, J.
(1997)
Curr. Opin. Lipidol.
8,
159-166[Medline]
[Order article via Infotrieve]
-
Kliewer, S. A.,
Umesono, K.,
Noonan, D. J.,
Heyman, R. A.,
and Evans, R. M.
(1992)
Nature
358,
771-774[CrossRef][Medline]
[Order article via Infotrieve]
-
Palmer, C. N. A.,
Hsu, M. H.,
Griffin, K. J.,
and Johnson, E. F.
(1995)
J. Biol. Chem.
270,
16114-16121[Abstract/Free Full Text]
-
Spiegelman, B. M.
(1998)
Diabetes
47,
507-514[Abstract]
-
Kliewer, S. A.,
Lenhard, J. M.,
Willson, T. M.,
Patel, I.,
Morris, D. C.,
and Lehmann, J. M.
(1995)
Cell
83,
813-819[Medline]
[Order article via Infotrieve]
-
Forman, B. M.,
Tontonoz, P.,
Chen, J.,
Brun, R. P.,
Spiegelman, B. M.,
and Evans, R. M.
(1995)
Cell
83,
803-812[Medline]
[Order article via Infotrieve]
-
Ricote, M.,
Li, A. C.,
Willson, T. M.,
Kelly, C. J.,
and Glass, C. K.
(1998)
Nature
391,
79-82[CrossRef][Medline]
[Order article via Infotrieve]
-
Jiang, C.,
Ting, A. T.,
and Seed, B.
(1998)
Nature
391,
82-86[CrossRef][Medline]
[Order article via Infotrieve]
-
Staels, B.,
Koenig, W.,
Habib, A.,
Merval, R.,
Lebret, M.,
Torra, I. P.,
Delerive, P., A, F.,
Chinetti, G.,
Fruchart, J.,
Najib, J.,
Maclouf, J.,
and Tedgui, A.
(1998)
Nature
393,
790-793[CrossRef][Medline]
[Order article via Infotrieve]
-
Elstner, E.,
Muller, C.,
Koshizuka, K.,
Williamson, E. A.,
Park, D.,
Asou, H.,
Shintaku, P.,
Said, J. W.,
Heber, D.,
and Koeffler, H. P.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
8806-8811[Abstract/Free Full Text]
-
Kubota, T.,
Koshizuka, K.,
Williamson, E.,
Asou, H.,
Said, J.,
Holden, S.,
Miyoshi, I.,
and Koeffler, H. P.
(1998)
Cancer Res.
58,
3344-3352[Abstract]
-
Sarraf, P.,
Mueller, E.,
Jones, D.,
King, F. J.,
DeAngelo, D. J.,
Partridge, J. B.,
Holden, S. A.,
Chen, L. B.,
Singer, S.,
Fletcher, C.,
and Spiegelman, B. A.
(1998)
Nat. Med.
4,
1046-1052[CrossRef][Medline]
[Order article via Infotrieve]
-
Schulman, I. G.,
Shao, G.,
and Heyman, R. A.
(1998)
Mol. Cell. Biol.
18,
3483-3494[Abstract/Free Full Text]
-
Tontonoz, P.,
Singer, S.,
Forman, B. M.,
Sarraf, P.,
Fletcher, J. A.,
Fletcher, C. D. M.,
Brun, R. P.,
Mueller, E.,
Altiok, S.,
Oppenheim, H.,
Evans, R. M.,
and Spiegelman, B. M.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
237-241[Abstract/Free Full Text]
-
Mukherjee, R.,
Davies, P. J. A.,
Crombie, D. J.,
Bischoff, E. D.,
Cesario, R. M.,
Jow, L.,
Hamam, L. G.,
Boehm, M. F.,
Mondon, C. E.,
Nadzan, A. M.,
Paterniti, J. R., Jr.,
and Heyman, R. A.
(1997)
Nature
386,
407-410[CrossRef][Medline]
[Order article via Infotrieve]
-
Issemann, I.,
and Green, S.
(1990)
Nature
347,
645-650[CrossRef][Medline]
[Order article via Infotrieve]
-
Lee, S. S.,
Pineau, T.,
Drago, J.,
Lee, E. J.,
Owens, J. W.,
Kroetz, D. L.,
Fernandez-Salguero, P. M.,
Westphal, H.,
and Gonzalez, F. J.
(1995)
Mol. Cell. Biol.
15,
3012-3022[Abstract]
-
Forman, B. M.,
Chen, J.,
and Evans, R. M.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
4312-4317[Abstract/Free Full Text]
-
Davis, G. E.,
and Camarillo, C. W.
(1996)
Exp. Cell Res.
224,
39-51[CrossRef][Medline]
[Order article via Infotrieve]
-
Fitzpatrick, F. A.,
and Wynalda, M. A.
(1983)
J. Biol. Chem.
258,
11713-11718[Abstract/Free Full Text]
-
Giles, H.,
and Leff, P.
(1988)
Prostaglandins
35,
277-300[CrossRef][Medline]
[Order article via Infotrieve]
-
Heid, C. A.,
Stevens, J.,
Livak, K. J.,
and Williams, P. M.
(1996)
Genome Methods
6,
986-994
-
Gerber, H.,
Condorelli, F.,
Park, J.,
and Ferrara, N.
(1997)
J. Biol. Chem.
272,
23659-23667[Abstract/Free Full Text]
-
Inoue, I.,
Shino, K.,
Noji, S.,
Awata, T.,
and Katayama, S.
(1998)
Biochem. Biophys. Res. Commun.
246,
370-374[CrossRef][Medline]
[Order article via Infotrieve]
-
Johnson, T. E.,
Holloway, M. K.,
Vogel, R.,
Rutledge, S. J.,
Perkins, J. J.,
Rodan, G. A.,
and Schmidt, A.
(1997)
J. Steroid Biochem. Mol. Biol.
63,
1-8[CrossRef][Medline]
[Order article via Infotrieve]
-
Fong, G. H.,
Rossant, J.,
Gertsenstein, M.,
and Breitman, M. L.
(1995)
Nature
376,
66-70[CrossRef][Medline]
[Order article via Infotrieve]
-
Ferrara, N.,
and Davis-Smyth, T.
(1997)
Endocr. Rev.
18,
4-25[Abstract/Free Full Text]
-
Ilan, N.,
Mahooti, S.,
and Madri, J. A.
(1998)
J. Cell Sci.
111,
3621-3631[Abstract/Free Full Text]
-
Chapman, H. A.
(1997)
Curr. Opin. Cell Biol.
9,
714-724[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.