Hypertension. 1999;34:1141-1146
(Hypertension. 1999;34:1141-1146.)
© 1999 American Heart Association, Inc.
Matrix-Dependent Gene Expression of Egr-1 and PDGF A Regulate Angiotensin IIInduced Proliferation in Human Vascular Smooth Muscle Cells
Shanhong Ling;
Aozhi Dai;
Yunn-Hwa Ma;
Emily Wilson;
Kanu Chatterjee;
Harlan E. Ives;
Krishnankutty Sudhir
From the Divisions of Nephrology and Cardiology, Cardiovascular Research
Institute, University of California (Y-H.M., E.W., K.S., H.E.I.), San
Francisco; and the Hormones and Vasculature Laboratory, Baker Medical Research
Institute and Alfred and Baker Medical Unit, Alfred Hospital (S.L., A.D.,
K.S.), Melbourne, Australia.
Correspondence to K. Sudhir, MD, PhD, Alfred and Baker Medical Unit, 3rd Floor, Alfred Hospital, Commercial Road, Prahran, VIC 3181, Australia. E-mail: krishna.sudhir{at}baker.edu.au
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Abstract
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AbstractWe have previously
shown, in a neonatal rat cell
line, that angiotensin II
(Ang II)induced proliferation
in vascular smooth muscle cells is
extracellular matrix (ECM)
dependent. We hypothesized that such an
effect might be mediated
via differences in Ang IIinduced increases
in the transcriptional
factor early growth response-1 (Egr-1) gene and,
consequently,
in platelet-derived growth factor (PDGF). Cultured
human newborn
aortic smooth muscle cells were studied on 4 different
surfaces:
(1) plastic, (2) laminin, (3) collagen, and (4) fibronectin.
Ang
IIinduced increases in DNA synthesis were significantly
greater
on collagen (2.0±0.3-fold) and fibronectin (1.9±0.3-fold)
than on
laminin (1.0±0.2-fold) or plastic (1.4±0.2-fold).
As with DNA
synthesis, at 48 and 72 hours, Ang IIinduced
increases in cell
numbers occurred only in cells grown on collagen
and fibronectin
culture plates and were blocked by an antagonist
to the
angiotensin type 1 (losartan, 10 µmol/L) but
not
the angiotensin type 2 (PD 123319, 10 µmol/L)
receptor.
Anti-PDGF AA antibody (6 µg/mL) blocked the increase in
DNA
synthesis by 60% to 64% in cells on collagen or fibronectin
cultures
but not on plastic cultures. When PDGF-AA (10 ng/mL)
and Ang II were
added together, DNA synthesis increased 2-fold
and did not differ on
the various ECM proteins. Increases in
PDGF A-chain mRNA were observed
only in cells grown on collagen
(3.21±0.65-fold) and fibronectin
(2.86±0.49-fold)
plates 2 to 8 hours after the addition of Ang II and
were blocked
by losartan but not PD 123319. Expression of
Egr-1, an early
growth response gene, increased at 15 minutes, peaked
at 30
minutes, and returned to normal after 2 hours with Ang II
treatment.
Ang IIinduced increases in Egr-1 mRNA were greater on
collagen
(4.82±0.66-fold at maximum) and fibronectin (4.01±0.56-fold)
than
on laminin (2.74±0.45-fold) or plastic (2.53±0.40-fold)
and were
blocked by losartan but not PD 123319. Thus, in human
vascular
smooth muscle cells in culture, Ang IIinduced
proliferation is
mediated via the angiotensin type 1 receptor,
dependent on
ECM proteins, and regulated by differential gene
expression of Egr-1
and PDGF-1.
Key Words: angiotensin II matrix early growth response-1 gene platelet-derived growth factor
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Introduction
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In some forms of hypertension, there is an increase in
smooth
muscle mass in the vascular wall.
1 2 This change is
the result
of smooth muscle hypertrophy or
hyperplasia.
3 4 It has been
suggested that
angiotensin II (Ang II) might participate in
this vascular
hypertrophy.
5 The effects of Ang II on
vascular
smooth muscle (VSM) cell growth are mediated in part via
enhanced
expression of endogenous growth factors, including
transforming
growth factorß1 (TGF-ß1) and platelet-derived
growth
factor (PDGF) A-chain.
6 In particular, exposure of
rat aortic
VSM cells to Ang II results in increased expression of PDGF
A,
7 8 which appears to mediate the increase in cell
size.
9 PDGF
A gene expression is also upregulated in the
vascular wall of
rats infused with Ang II.
10 In rat VSM
cells, the degree of
mitogenesis induced by Ang II can vary from 0- to
4-fold.
11 12 13 14 15 We have previously shown, in a neonatal rat
cell
line, that VSM proliferation induced by Ang II and mechanical
strain
is modified by extracellular matrix (ECM)
proteins
16 17 and
that PDGF plays a key role in the
interaction between Ang II
and strain.
16 However, the
mechanisms by which ECM proteins
influence Ang IIinduced growth have
not been defined.
In human VSM cells, Ang II reportedly induces a modest increase in DNA
synthesis,18 but its effect on cell proliferation is not
entirely clear. Although an increase in cell number has been reported
with Ang II,19 a recent study showed no effect of Ang II
on cell proliferation.20 However, the effects of different
matrices on Ang IIinduced growth have never been examined in human
VSM cells. The objectives of the present study were to determine
the effect of ECM proteins on Ang IIinduced proliferation in cultured
human VSM cells. We hypothesized that ECM proteins, such as collagen
and fibronectin, that stimulate Ang IIinduced growth, work by
inducing differential activation of early growth response genes and
subsequently of PDGF A. We found that Ang IIinduced DNA synthesis and
cell proliferation in human VSM cells were greater on collagen and
fibronectin, compared with either laminin or plastic, and that
ECM-associated differences in Ang IIinduced growth were closely
related to variable expression of early growth response-1 gene
(Egr-1) and PDGF A.
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Methods
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Materials
All materials were purchased from Sigma Immunochemicals , unless
otherwise
specified. One hundredmillimeter cell culture dishes
and
24- or 96-well cell culture plates were obtained from Collaborative
Biomedical
Products. The dishes and plates were coated with rat
tail collagen
1, human fibronectin, mouse laminin, or left uncoated
(plastic).
[
3H]-thymidine was purchased from
Amersham Corp. Recombinant
human PDGF AA (rhPDGF AA) and goat
antihuman PDGF AA
neutralizing polyclonal antibody were purchased
from R and D
Systems. This antibody neutralizes the biological activity
of
rhPDGF AA, rhPDGF AB, and natural human PDGF AA but not rhPDGF
BB or
natural human PDGF AB. The selective angiotensin type
1
(AT
1) receptor antagonist
losartan was supplied by Merck Research
Laboratories, and the
selective angiotensin type 2 (AT
2)
receptor
antagonist PD 123319 was supplied by Research
Biochemicals International.
Cell Culture
Primary cultures of human VSM cells harvested from the aorta of
a newborn autopsy patient were established at the University of
Washington, Seattle. From these cultures, a human newborn (HNB
18) cell line was established and was infected with the human
papilloma virus type E6E7 protein to immortalize the cells (named
HNB18E6E). Morphological, electron microscopic,
immunofluorescent, and biochemical analyses
demonstrated that these cells retain much of the phenotype of
normal aortic smooth muscle cells, including the expression of smooth
muscle markers and appropriate growth responses to PDGF and
heparin.21 The cells were generously supplied to us by Dr
Karen Yee and Dr Stephen Schwartz (University of Washington,
Seattle) at passage 3 after infection and were maintained in Waymouth
medium with 10% fetal bovine serum, 100 U/mL of penicillin, and 100
mg/mL of streptomycin (growth medium) in a humidified atmosphere of 5%
CO2/95% air at 37°C. Culture medium was
changed every 4 days until cells were confluent; they were subcultured
with 0.05% trypsin-versene and 0.2% pancreatin. Cells from passage 4
to 8 (after infection) were used for the present study.
Measurement of DNA Synthesis
Cells were plated on 24-well plates at a density of 10 000
cells per well in growth medium, incubated for 24 hours, growth
arrested in quiescence medium (Waymouth medium with 0.5% fetal bovine
serum and the same antibiotics as in the growth medium) for 24
hours, and then treated with Ang II (1 µmol/L), PDGF AA (10
ng/mL), or antiPDGF AA antibody (6 µg/mL) for 24 hours (4 wells for
each treatment). (The dose of Ang II used in this study was selected on
the basis of preliminary experiments in which the best increase in DNA
synthesis was obtained at 1 µmol/L.) During the final 6 hours, 1
µCi/mL of [3H]-thymidine was added to the
medium of each well and incubated at 37°C. Cells were then washed 3
times with PBS and extracted with 15% trichloroacetic acid at 4°C
for 30 minutes, and 0.5 mL/well of 1 mol/L NaOH was added for 20
minutes and neutralized with 0.5 mL/well of 1 mol/L HCl. The contents
of the wells were placed in scintillation vials for counting.
Determination of Cell Numbers
Cells were plated on 96-well plates (2500 cells per well) in
growth medium for 2 days and growth arrested in quiescence medium for
24 hours. Cells were then treated with Ang II (1 µmol/L) alone
or in the presence of losartan (10 µmol/L) or PD 123319
(10 µmol/L) for 24, 48, and 72 hours (8 wells for each treatment
at each time point). Cells were washed with PBS, recovered with 0.05%
trypsin (30 µL/well) for 5 minutes and mixed with PBS (70 µL/well),
and then counted with a hemocytometer.
RNA Isolation and Northern Blot Analysis
Cells on 100-mm dishes were grown nearly to confluence and were
growth arrested in quiescence medium for 24 hours and then treated with
Ang II (1 µmol/L) and/or losartan (10 µmol/L) or
PD 123319 (10 µmol/L) for 0 to 8 hours. Losartan and PD
123319 were added 30 minutes before Ang II. Total cellular RNA was
isolated with STAT-60 reagent (Tel-Test Inc) and quantified by
measuring the absorbance at 260 nm. RNA (10 µg per lane) was
electrophoresed on 1% agarose gels in the presence of glyoxal and
MOPS, transferred to Hybond nylon membranes (Amersham Corp) overnight,
and then fixed to the blots in a UV Statalinker (Stratagene) for
the optimal time period (auto mode). Blots were hybridized to
[
-32P]-dCTP (3000 Ci/mmol; Amersham Corp)
labeled cDNA probe for PDGF A-chain at 65°C overnight. After the
membranes were washed with SSC/0.1% SDS buffers, they were exposed to
x-ray films for 8 to 72 hours at -70°C until optimal signals were
obtained. The same blot was rehybridized to
[
-32P]-dCTPlabeled cDNA probes for Egr-1
and GAPDH, respectively, with a similar protocol to that used for PDGF
A, after stripping in 0.1% SDS solution at 95°C for 5 minutes. The
autoradiographic signals were scanned with a PowerLook II
Scanner (UMAX Data System, Inc) and relative levels of PDGF A and Egr-1
mRNA were normalized by comparison to the GAPDH mRNA signal.
Data Presentation and Statistics
All data are presented as mean±SEM. Comparisons between
2 means were made with Students t test. Multiple
comparisons were made by ANOVA. Values of P<0.05 were
considered significant.
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Results
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Effect of ECM Proteins on Ang IIInduced DNA Synthesis
To determine the effect of various ECM proteins on Ang IIinduced
increases
in DNA synthesis, [
3H]-thymidine
incorporation was measured
in response to Ang II (1 µmol/L) on
plastic (uncoated),
laminin, collagen, or fibronectin. Ang IIinduced
increases
in DNA synthesis were greater on collagen (2.0±0.3-fold)
and
fibronectin (1.9±0.3-fold) compared to laminin (1.0±0.2-fold)
and
plastic (1.4±0.2-fold) (Figure 1
).

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Figure 1. Human VSM cells grown in 24-well plates coated
with collagen, fibronectin, or laminin or without any ECM (plastic)
were treated with Ang II (1 µmol/L) for 24 hours (4 wells for
each treatment), and [3H]-thymidine incorporation was
measured. Data are expressed as fold increase over control (without Ang
II) on the same matrix and are the mean±SEM of 4 similar experiments.
*P<0.05, compared with plastic.
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Effect of ECM Proteins on Ang IIInduced Increases in Cell Number,
and Role of the AT1 Versus the AT2
Receptor
Similar to DNA synthesis, no increase in cell number was observed
in response to Ang II on either plastic or laminin. Ang IIinduced
increases in cell number occurred significantly only in cells grown on
collagen and fibronectin culture plates. This cell proliferation was
blocked by losartan but not PD 123319, which indicated that Ang
IIinduced increase in cell proliferation is mediated via the Ang II
receptor subtype AT1 (Figure 2).

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Figure 2. Human VSM cells were plated into 96-well plates
either uncoated with any ECM proteins (plastic) or coated with laminin,
collagen, or fibronectin. Cells were treated with Ang II (1
µmol/L) alone or in the presence of losartan (10
µmol/L) or PD 123319 (10 µmol/L) for 24, 48, and 72 hours, and
then cell number was counted. Data are mean±SEM of 8 wells for each
treatment at each time point. *P<0.05, vs control;
#P<0.05, vs Ang II treatment.
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Effect of Anti-PDGF AA Antibodies on Ang IIInduced Increases in
DNA Synthesis, and Effect of Exogenous PDGF AA
To determine the contribution of PDGF AA to Ang IIinduced
increase in DNA synthesis, the effect of Ang II on
[3H]-thymidine incorporation was measured in
the presence and absence of neutralizing antibodies to PDGF AA. On
collagen and fibronectin, the Ang IIinduced increase in DNA synthesis
was inhibited significantly by anti-PDGF AA antibodies (60% to 64%
inhibition), which suggested that PDGF AA participates in Ang
IIinduced mitogenesis (Figure 3).

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Figure 3. Human VSM cells were treated with Ang II (1
µmol/L) alone or in the presence of anti-human PDGF AA antibody (6
µg/mL) or nonspecific antibody for 24 hours, and then
[3H]-thymidine incorporation was measured. Data are
mean±SEM of 4 wells for each treatment. *P<0.05 vs Ang
II+ nonspecific antibody. Basal [3H]-thymidine
incorporation (cpm/well) was 3337±346 in plastic, 5898±518 in
collagen, 5791±560 in fibronectin, and 4060±439 in laminin.
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If ECM-associated differences in Ang IIinduced mitogenesis are
related to differential production of PDGF AA on various
matrices, then the addition of exogenous PDGF AA should abolish such a
difference. Consistent with this hypothesis, in the presence of
exogenous PDGF AA, Ang IIinduced DNA synthesis did not differ
substantially on different ECM proteins (Figure 4). PDGF AA, on its own, was a weak
mitogen, but again it induced greater increases in DNA synthesis on
collagen and fibronectin than on plastic or laminin (for data, see
legend to Figure 4).

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Figure 4. Human VSM cells were treated with Ang II (1
µmol/L) in the absence and presence of PDGF AA (10 ng/mL) for 24
hours and [3H]-thymidine incorporation was measured. Data
are mean±SEM of 4 wells for each treatment. *P<0.05,
vs Ang II treatment. Basal [3H]-thymidine incorporation
(cpm/well) was 9259±587 on plastic, 14 503±1298 on collagen,
13 810±394 on fibronectin, and 9663±557 on laminin. PDGF AA alone
induced increases in DNA synthesis of 1.2±0.15- and 1.15±0.2-fold on
plastic and laminin and 1.55±0.15- and 1.40±0.2-fold on collagen and
fibronectin.
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Effect of ECM Proteins on Ang IIInduced Increase in Expression of
PDGF A
Because Ang IIinduced mitogenesis is mediated in part via PDGF
A, we examined the effect of Ang II on PDGF A expression in the
presence of different ECM proteins. In response to Ang II, PDGF A-chain
mRNA increased only in the cells grown on collagen (3.21±0.65-fold
at maximum in 3 similar experiments) and fibronectin (2.86±0.49-fold)
plates, with Ang II treatment for 2 to 8 hours (Figure 5). PDGF A gene expression was inhibited
by losartan but not by PD123319 (Figure 6).

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Figure 5. Top, Photographs of Northern blots from 3 similar
experiments for Egr-1, PDGF A-chain, and GAPDH mRNA detection in human
VSM cells grown on plastic- (A), laminin- (B), collagen- (C), or
fibronectin-coated (D) culture plates and treated with Ang II (1
µmol/L) for 0 to 8 hours. Total RNA was isolated and Northern blot
analysis was performed to detect the mRNA of Egr-1, PDGF A, and
GAPDH. Bottom, Northern blot analysis of PDGF A-chain and Egr-1
expressions in human VSM cells grown on ECM-coated or -uncoated culture
plates and treated with Ang II (1 µmol/L) for 0 to 8 hours.
Relative PDGF A-chain and Egr-1 mRNA levels were normalized to GAPDH
mRNA signals. Fold induction of the mRNA relative to its basal level
(time 0) is shown as mean±SEM (n=3). *P<0.05, vs
plastic at the same time point.
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Figure 6. Top, Human VSM cells were grown on ECM-coated or
uncoated culture plates and treated with Ang II (1 µmol/L) alone
or in the presence of losartan (10 µmol/L) or PD 123319
(10 µmol/L) for 4 hours. Northern blot analysis was
performed to analyze PDGF A-chain mRNA. Photographs show 1
result from 3 similar experiments in cells grown on plastic- (A),
laminin- (B), collagen- (C), or fibronectin-coated (D) culture plates.
Bottom, Bar graphs show fold increase in PDGF A-chain mRNA (mean±SEM),
relative to control levels in response to Ang II, in the presence of
losartan (10 µmol/L) or PD 123319 (10 µmol/L).
*P<0.05, vs Ang II alone.
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Effect of ECM Proteins on Ang IIInduced Increase in the
Expression of Egr-1
Because transcriptional activation by Egr-1 appears to be a key to
the inducible expression of PDGF, the effect of Ang II on the
expression of Egr-1 was assessed on collagen, fibronectin, laminin, and
plastic. Steady-state Egr-1 mRNA increased in response to Ang II in
human VSM cells at 15 minutes; peak expression occurred at 30 minutes
and levels returned to normal after 2 hours (Figure 5). Egr-1
expression was also modified by ECM (Figure 5, bottom); Egr-1
was greater on collagen (4.82±0.66-fold at maximum) and fibronectin
(4.01±0.56-fold) than on laminin (2.74±0.45-fold) or plastic
(2.53±0.40-fold). Again, Ang IIinduced increase in Egr-1 expression
was blocked by losartan but not by PD 123319 (Figure 7).

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Figure 7. Top, Human VSM cells were grown on ECM-coated or
-uncoated culture plates and treated with Ang II (1 µmol/L)
alone or in the presence of losartan (10 µmol/L) or PD
123319 (10 µmol/L) for 30 minutes. Northern blot
analysis was performed to analyze Egr-1 mRNA.
Photographs show 1 result from 3 similar experiments in cells grown on
plastic- (A), laminin- (B), collagen- (C), or fibronectin-coated (D)
culture plates. Bottom, Bar graphs show fold increase in Egr-1 mRNA
(mean±SEM), relative to control levels in response to Ang II, in the
presence of losartan (10 µmol/L) or PD 123319 (10
µmol/L). *P<0.05, vs Ang II alone.
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Discussion
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In the present study, we have demonstrated that Ang
IIinduced
mitogenesis in human VSM cells is mediated via the
AT
1 receptor
and is ECM dependent, with it
greater on collagen and fibronectin
than on either laminin or plastic.
The differential mitogenesis
appears to be explained by a variable,
matrix-dependent expression
of Egr-1 and subsequently of PDGF A-chain,
with a resulting
increase in secretion or activity of PDGF.
We have confirmed in human VSM cells our previous observations that in
a neonatal rat cell line certain ECM proteins, such as collagen and
fibronectin, potentiate Ang IIinduced increases in DNA
synthesis.16 In parallel, we have shown that Ang II
increases cell numbers significantly only in cells grown on collagen
and fibronectin culture plates but not on laminin or plastic. Because
this cell proliferation was blocked by losartan, our
observations suggest that Ang IIinduced mitogenesis in human VSM
cells is mediated predominantly via the AT1
receptor. An inhibitory effect of the
AT2 receptor has been proposed,22
but because the addition of the selective AT2
receptor antagonist PD 123319 had no effect on Ang
IIinduced proliferation, it is unlikely that this receptor plays an
important role in Ang IIinduced proliferation in these cells, at
least under the conditions we studied.
Ang IIinduced mitogenesis is reportedly mediated, in part, via
induction of PDGF A.6 7 8 Consistent with these
reports is our observation that Ang IIinduced increase in DNA
synthesis was inhibited significantly by neutralizing antibodies to
PDGF AA. In addition, we examined Ang IIinduced increases in PDGF A
expression on different ECM proteins and found that PDGF A-chain mRNA
increased only in the cells grown on collagen and fibronectin. This
increase was inhibited by losartan, again suggesting that the
effect is mediated via the AT1 receptor. Thus, it
appears that ECM-associated differences in Ang IIinduced mitogenesis
may be related to differential production of PDGF A on various
matrices. Consistent with this hypothesis, the addition of
exogenous PDGF AA abolished the differential effect of ECM proteins on
Ang IIinduced mitogenesis, suggesting that when the decreased
production of PDGF A on plastic and laminin is compensated for
in this manner, equivalent Ang IIinduced mitogenesis is achieved,
irrespective of matrix. Despite a greater mitogenic effect
on collagen and fibronectin, exogenous PDGF AA was, in general, a weak
mitogen in these cells. Thus, in addition to an increase in PDGF
A-chain gene expression by Ang II on collagen and fibronectin, a
synergy between Ang II and PDGF AA on these matrices cannot be
excluded. We have previously reported a synergistic response between
Ang II and PDGF AB, a possible mechanism that underlies the
potentiation of the mitogenic activity of Ang II by
mechanical strain.16 Extracellular matrix is reportedly a
source of mitogenically active PDGF, which is readily
accessible to VSM cells by contact23 ; differential
interactions between Ang II and matrix-derived PDGF may also play a
role in ECM-dependent mitogenesis induced by Ang II. TGF-ß1 is also
reportedly an important growth factor in Ang IIassociated VSM cell
growth6 24 ; in the current study, however, we did not
examine Ang IIinduced changes in TGF ß1 expression, and hence it is
unclear whether matrix-dependent changes in the production of
this growth factor influence Ang IIinduced growth in human VSM
cells.
Egr-1 is a transcription factor25 that is
activated by diverse biochemical and mechanical stimuli, via
phosphorylation-dependent signaling pathways, which
converge at the Egr-1 promoter.26 Studies in
endothelial cells suggest that Egr-1 binds to the
proximal PDGF A promoter, prior to the inducible expression and
secretion of PDGF A.27 Recent work from our laboratory has
suggested a role for Egr-1 in the induction of PDGF-A by continuous
cyclic mechanical strain in neonatal rat VSM cells.28 We
hypothesized that the Ang IIinduced increase in PDGF A expression and
release observed in the present study in human VSM cells might also
be related to activation of Egr-1. We showed that steady-state mRNA of
Egr-1 increased in response to Ang II in human VSM cells at 15 minutes;
peak expression occurred at 30 minutes and levels returned to normal
after 2 hours, at which time an increase in PDGF A expression was first
observed. Egr-1 expression was also AT1 receptor
mediated and was modified by ECM proteins, being greater on collagen
and fibronectin compared with either laminin or plastic. Overall, our
observations are thus consistent with the induction of Egr-1 by
Ang II, with subsequent activation of the PDGF A gene, and release of
PDGF A. These processes are facilitated by collagen and fibronectin and
are attenuated on laminin or plastic, which results in ECM-dependent
effects of Ang II on DNA synthesis and cell proliferation.
In conclusion, in human newborn aortic smooth muscle cells, collagen
and fibronectin, but not laminin, favor Ang IIinduced increases in
DNA synthesis and cell proliferation. On collagen and fibronectin, Ang
II causes an increase in the expression of Egr-1 and subsequently that
of PDGF A, which presumably results in an increase in the secretion of
PDGF. Thus, matrix-dependent variations in PDGF A-chain expression
probably explain the effect of extracellular matrix on Ang IIinduced
proliferation. This interaction between Ang II and matrix proteins
might be of importance in pathological states, such as hypertension,
known to be associated with an increase in ECM proteins in the vascular
wall.29 30 Finally, the effects of Ang II on Egr-1 and
PDGF A expression and on cell proliferation are all mediated largely
via the AT1 receptor, with all the effects
substantially attenuated by a selective AT1
receptor antagonist.
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Acknowledgments
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This project was funded through a medical school grant from
Merck
Co and funds from the Foundation for Cardiac Research, University
of
California, San Francisco.
Received March 21, 1999;
first decision May 10, 1999;
accepted July 1, 1999.
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