(Hypertension. 1999;34:1141-1146.)
© 1999 American Heart Association, Inc.
Scientific Contributions |
Matrix-Dependent Gene Expression of Egr-1 and PDGF A Regulate Angiotensin II–Induced Proliferation in Human Vascular Smooth Muscle Cells
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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Abstract—We 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 II–induced 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 II–induced 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 II–induced 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 II–induced 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 II–induced 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 factor–ß1 (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) proteins16 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 II–induced 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 II–induced 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 II–induced proliferation in cultured human VSM cells. We hypothesized that ECM proteins, such as collagen and fibronectin, that stimulate Ang II–induced growth, work by inducing differential activation of early growth response genes and subsequently of PDGF A. We found that Ang II–induced 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 II–induced 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 hundred–millimeter 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 anti–human 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 (AT1) receptor antagonist losartan was supplied by Merck Research Laboratories, and the selective angiotensin type 2 (AT2) 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 anti–PDGF 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]-dCTP–labeled 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 Student’s 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 II–Induced DNA Synthesis
To determine the effect of various ECM proteins on Ang II–induced 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 II–induced 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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Effect of ECM Proteins on Ang II–Induced 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 II–induced
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
II–induced increase in cell proliferation is mediated via the Ang II
receptor subtype AT1 (Figure 2).
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Effect of Anti-PDGF AA Antibodies on Ang II–Induced Increases in
DNA Synthesis, and Effect of Exogenous PDGF AA
To determine the contribution of PDGF AA to Ang II–induced
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 II–induced increase in DNA synthesis
was inhibited significantly by anti-PDGF AA antibodies (60% to 64%
inhibition), which suggested that PDGF AA participates in Ang
II–induced mitogenesis (Figure 3).
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If ECM-associated differences in Ang II–induced 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 II–induced 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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Effect of ECM Proteins on Ang II–Induced Increase in Expression of
PDGF A
Because Ang II–induced 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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Effect of ECM Proteins on Ang II–Induced 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 II–induced increase in Egr-1 expression
was blocked by losartan but not by PD 123319 (Figure 7).
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Discussion |
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In the present study, we have demonstrated that Ang II–induced mitogenesis in human VSM cells is mediated via the AT1 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 II–induced 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 II–induced 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 II–induced proliferation, it is unlikely that this receptor plays an important role in Ang II–induced proliferation in these cells, at least under the conditions we studied.
Ang II–induced mitogenesis is reportedly mediated, in part, via induction of PDGF A.6 7 8 Consistent with these reports is our observation that Ang II–induced increase in DNA synthesis was inhibited significantly by neutralizing antibodies to PDGF AA. In addition, we examined Ang II–induced 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 II–induced 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 II–induced mitogenesis, suggesting that when the decreased production of PDGF A on plastic and laminin is compensated for in this manner, equivalent Ang II–induced 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 II–associated VSM cell growth6 24 ; in the current study, however, we did not examine Ang II–induced changes in TGF ß1 expression, and hence it is unclear whether matrix-dependent changes in the production of this growth factor influence Ang II–induced 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 II–induced 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 II–induced 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 II–induced 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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