(Received for publication, August 17, 1994; and in revised form, October 16, 1994)
From the
Vascular endothelial growth factor (VEGF) is a potent and
specific mitogen for vascular endothelial cells and promotes
neovascularization in vivo. To determine whether
interleukin-1 (IL-1
), which is present in atherosclerotic
lesions, induces VEGF gene expression in vascular smooth muscle cells,
we performed RNA blot analysis on rat aortic smooth muscle cells
(RASMC) with a rat VEGF cDNA probe. IL-1
increased VEGF mRNA
levels in RASMC in a time- and dose-dependent manner. As little as 0.1
ng/ml IL-1
increased VEGF mRNA levels by 2-fold and 10 ng/ml
IL-1
increased VEGF mRNA by 4-fold. We also measured the half-life
of VEGF mRNA and performed nuclear run-on experiments before and after
addition of IL-1
to see if IL-1
increased VEGF mRNA levels by
stabilizing the mRNA or by increasing its rate of transcription. The
normal, 2-h half-life of VEGF mRNA in RASMC was lengthened to 3.2 h
(60%) by IL-1
, and IL-1
increased the rate of VEGF gene
transcription by 2.1-fold. In immunoblot experiments with an antibody
specific for VEGF, we found that IL-1
increased VEGF protein
levels in RASMC by 3.3-fold. Together these data indicate that
IL-1
induces VEGF gene expression in smooth muscle cells. This
IL-1
-induced expression of VEGF may accelerate the progression of
atherosclerotic lesions by promoting the development of new blood
vessels.
Neovascularization is frequently observed in human atheromatous
plaques(1, 2, 3, 4, 5) ,
and it has been implicated in the progression of atherosclerotic
lesions(5, 6) . In addition, neovascularization has
been associated with hemorrhage into atheromatous plaques, which can
lead to unstable angina and myocardial infarction. The fact that human
atherosclerotic plaques stimulate angiogenesis (7) indicates
the presence of angiogenic factors in these plaques. Although several
molecules have been shown to be important in angiogenesis, vascular
endothelial growth factor (VEGF) ()stands out because it is
a direct and specific mitogen for vascular endothelial
cells(8, 9) . Studies indicate that the FLT-1 and the
KDR/FLK-1 receptor tyrosine kinases function as endothelial
cell-specific receptors for
VEGF(10, 11, 12) , and VEGF, together with
its receptors, appears to function as the key regulator of physiologic (11) as well as pathologic
angiogenesis(13, 14) . For example, inhibition of
angiogenesis by a monoclonal antibody to VEGF (15) or by a
dominant-negative FLK-1 (which antagonizes FLK-1) (16) suppresses tumor growth in vivo.
The mRNAs of
several isoforms of the VEGF family (VEGF,
VEGF
, VEGF
, and VEGF
,
containing 121, 165, 189, and 206 amino acids, respectively) are
generated by alternative splicing from the same
gene(17, 18, 19, 20) . VEGF
is the predominant isoform secreted by a variety of normal and
transformed cells(18, 21) . Aortic smooth muscle cells
express VEGF
(22) ; however, it has not been known
whether interleukin-1
(IL-1
), which is present in
atherosclerotic lesions, regulates VEGF gene expression in aortic
smooth muscle cells.
We designed the present study to test whether
IL-1 induces VEGF mRNA and protein in rat aortic smooth muscle
cells (RASMC). We found that IL-1
induced VEGF mRNA in a time- and
dose-dependent manner and that this induction was due to a 211%
increase in the rate of VEGF mRNA transcription and a 60% increase in
its half-life. Additionally, the induction of VEGF mRNA by IL-1
was associated with a 3.3-fold increase in the level of VEGF protein in
RASMC. Our data suggest that IL-1
present in atherosclerotic
lesions may promote formation of new blood vessels by inducing
expression of VEGF in smooth muscle cells.
To promote
differentiation of RASMC in vitro, we coated 10-cm cell
culture dishes with 2.5 ml of Matrigel (Collaborative Research)
according to the manufacturer's instruction. Cells (2
10
) were evenly plated on the dishes and examined for
morphological changes after 24 h(24) .
Figure 4:
Effect of IL-1 on VEGF mRNA half-life
and rate of transcription in RASMC. A, RASMC were exposed to
vehicle (control, open circles) or IL-1
(10 ng/ml, filled circles) for 48 h before the addition of actinomycin D (ACD, 5 µg/ml). Total RNA was extracted from the cells at
the indicated times after actinomycin D administration. Northern blot
analyses were performed with 10 µg of total RNA/lane. After
electrophoresis the RNA was transferred to nitrocellulose filters,
which were hybridized to
P-labeled rat VEGF and 18 S
probes. To correct for differences in loading, the signal density of
each RNA sample hybridized to the VEGF probe was divided by that
hybridized to the 18 S probe. The corrected density was then plotted as
a percentage of the 0-h value against time (in log scale). B,
confluent RASMC were not stimulated (control) or stimulated with
IL-1
for 48 h. Nuclei were isolated, and in vitro transcription was allowed to resume in the presence of
[
-
P]UTP. Equal amounts of
P-labeled, in vitro-transcribed RNA probes from
each group were hybridized to 1 µg of denatured VEGF and
-actin cDNA that had been immobilized on nitrocellulose
filters.
Figure 3:
Response of VEGF mRNA to multiple cytokine
stimulation and effect of IL-1 on differentiated RASMC. A, RASMC were treated with vehicle (control), IL-1
(10
ng/ml), TNF-
(100 ng/ml), TGF-
1 (10 ng/ml), PDGF AA (20
ng/ml), PDGF BB (20 ng/ml), EGF (10
M), AII
(10
M), and ET-1 (10
M). Total RNA was extracted from the cells after 24 h of
stimulation. Northern blot analyses were performed with 10 µg of
total RNA/lane. B, RASMC were treated with vehicle (control)
or IL-1
(10 ng/ml) in the presence of Matrigel. After 24 h of
stimulation, total RNA was extracted from the cells. Northern blot
analyses were performed with 5 µg of total RNA/lane. After
electrophoresis (A and B) the RNA was transferred to
nitrocellulose filters, which were hybridized to
P-labeled
rat VEGF. To assess for differences in loading, the filters were also
hybridized to an 18 S oligonucleotide
probe.
Figure 1:
Time
course of VEGF mRNA induction by IL-1 in RASMC. RASMC were treated
with IL-1
(10 ng/ml), and total RNA was extracted from the cells
at the indicated times. RNA was also extracted from control cells
(control), which received vehicle (Dulbecco's phosphate-buffered
saline) but not IL-1
. Northern blot analyses were performed with
10 µg of total RNA/lane. After electrophoresis the RNA was
transferred to nitrocellulose filters, which were hybridized to a
P-labeled RASMC VEGF probe. The filters were also
hybridized with a GAPDH probe to assess loading differences. The
corrected density was then plotted as a percentage of
control.
Figure 2:
Dose-response of VEGF mRNA induction by
IL-1 in RASMC. RASMC were incubated for 48 h with the indicated
concentrations of IL-1
, and total RNA was extracted from the cells
at the end of each incubation. See Fig. 1for
details.
Passaged RASMC represent a
synthetic or dedifferentiated population of cells. To determine if
induction of VEGF mRNA by IL-1 is also a property of contractile
or differentiated smooth muscle cells, we performed experiments on
RASMC cultured on Matrigel. Matrigel is a solubilized basement membrane
matrix which has recently been used to maintain cultured vascular
smooth muscle cells in a highly differentiated phenotype(24) .
In the presence of Matrigel, IL-1
also increases VEGF mRNA levels
(3.5-fold) in RASMC (Fig. 3B).
Figure 5:
Induction of VEGF protein by IL-1 in
RASMC. Cell lysates from control and IL-1
-treated RASMC (48 h)
were fractionated by 10% SDS-polyacrylamide gel electrophoresis.
Protein on the gel was transferred to an Immobilon-P membrane. VEGF was
subsequently detected by immunoblotting with a polyclonal anti-human
VEGF antibody (1:500 dilution).
Neovascularization within the neointima may play an important role in the progression of atherosclerotic plaques and may cause plaque hemorrhage. Additionally, because new blood vessels express vascular cell adhesion molecule-1 they become an important site of continuous inflammatory cell recruitment into plaques(6) . Thus, a thorough understanding of the mechanisms that regulate neovascularization in atherosclerotic plaques may allow us to design strategies to modify the progression of these lesions. Human atherosclerotic plaques have been shown to stimulate angiogenesis(7) ; however, the angiogenic factors operating in these plaques have not been fully elucidated.
In this report we show
that IL-1, which is readily detectable in atherosclerotic
lesions(30) , increases VEGF mRNA in RASMC in a time- and
dose-dependent manner ( Fig. 1and Fig. 2). The induction
of VEGF mRNA by IL-1
does not indicate that the VEGF gene can be
induced by all cytokines. Stimulation of RASMC for 24 h with other
cytokines and growth factors demonstrated a significant increase in
VEGF mRNA by TGF-
1, but not by TNF-
, PDGF AA, PDGF BB, EGF,
AII, or ET-1 (Fig. 3A). The IL-1
-induced increases
in the level of VEGF mRNA in RASMC were apparently due both to an
increase in the transcriptional rate of the gene and to an increase in
the stability of its mRNA (Fig. 4). Although increases in the
rate of VEGF gene transcription have been suggested as the mechanism by
which agents such as phorbol esters, cyclic AMP, prostaglandins E1 and
E2, and transforming growth factor-
increase VEGF mRNA levels in
osteoblasts, preadipocytes, fibroblasts, and epithelial
cells(27, 31, 32, 33) , in these
studies no nuclear run-on experiments were performed to assess the rate
of VEGF gene transcription. To our knowledge, Fig. 4B represents the first demonstration that IL-1
increases the
transcriptional rate of the VEGF gene in RASMC. Tischer et al.(17) have shown that the promoter region of the VEGF gene
contains four potential AP-1 sites. Since AP-1 sites mediate the
transcriptional activation of IL-2 by IL-1
(34) , it is
possible that the IL-1
-induced increase in VEGF gene transcription
is also mediated through AP-1 sites.
IL-1-induced increases in
VEGF mRNA are associated with similar increases in VEGF protein in
RASMC (Fig. 5). Although IL-1
has been shown to be
angiogenic in vivo(35, 36) , it is not
mitogenic for vascular endothelial cells in culture(37) . It is
likely that the angiogenic effect of IL-1
in vivo is
mediated by the induction of VEGF in vascular smooth muscle (or other)
cells; the VEGF would then act on neighboring endothelial cells to
induce angiogenesis.
Our data raise the possibility that IL-1
stimulates vascular smooth muscle cells to express VEGF. This potent
angiogenic factor could then stimulate the formation of new blood
vessels in atherosclerotic lesions. The new vessels, which would
express adhesion molecules(6) , would subsequently attract
inflammatory cells. IL-1
released by these inflammatory cells
could then induce more VEGF gene expression in the atherosclerotic
lesions and initiate a vicious circle.
In summary, we have shown
that IL-1 can induce VEGF mRNA and protein in RASMC. Induction of
VEGF mRNA by IL-1
is mediated by a combined increase in the
transcriptional rate of the gene and the stability of the mRNA.
IL-1
-induced VEGF gene expression in vascular smooth muscle cells
may have a role in promoting neovascularization and, hence, the
progression of atherosclerotic lesions.
Note Added in
Proof-While this work was in progress, Brogi et al. (Brogi, E., Wu, T., Namiki, A., and Isner, J. M.(1994) Circulation90, 649-652) reported the induction
of VEGF mRNA by TGF-1 in vascular smooth muscle cells.