© 2001 American Heart Association, Inc.
Vascular Biology |
Retroviral Overexpression of Decorin Differentially Affects the Response of Arterial Smooth Muscle Cells to Growth Factors
From the Department of Pharmacology (J.W.F.), Christian Albrechts University, Kiel, Germany; The Hope Heart Institute (M.G.K., T.N.W.) and the Departments of Pathology (M.G.K., T.N.W.) and Surgery (A.W.C.), University of Washington, Seattle; and the Department of Cardiology (B.L.), University of Münster, Münster, Germany.
Correspondence to Thomas N. Wight, PhD, The Hope Heart Institute, 1124 Columbia St, Suite 783, Seattle, WA 98104. E-mail twight{at}hopeheart.org
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Abstract—Decorin is a member of the family of small leucine-rich proteoglycans that are present in blood vessels and synthesized by arterial smooth muscle cells (ASMCs). This proteoglycan accumulates in topographically defined regions of atherosclerotic lesions and may play a role in the development of this disease. However, little is known about whether decorin has specific effects on the cellular events that contribute to atherosclerotic lesion formation. In the present study, rat ASMCs were transduced with a retroviral vector (LDSN) that carries the bovine decorin gene. Compared with vector control cells (LXSN), these cells constitutively overexpress decorin, as verified by Northern and Western analysis and by metabolic labeling. Experiments were performed to examine the responsiveness of decorin-overexpressing rat ASMCs to platelet-derived growth factor (PDGF) and transforming growth factor-ß1 (TGF-ß1), 2 growth factors that affect cell proliferation and extracellular matrix production in atherosclerosis. Decorin-overexpressing cells had decreased [3H]thymidine incorporation into DNA and increased the levels of the cyclin-dependent kinase inhibitors p21 and p27 in the first 24 hours of response to serum and PDGF-BB. However, these effects of decorin were not apparent at 48 or 72 hours after plating and did not result in reduced growth of decorin-overexpressing cells in response to serum and PDGF-BB. In contrast, the growth response of decorin-overexpressing ASMCs to TGF-ß1, as well as the expression of TGF-ß1–responsive genes, such as plasminogen activator inhibitor-1 and versican (an extracellular matrix proteoglycan), was diminished. These results indicate that decorin selectively inhibits the responsiveness of rat ASMCs to TGF-ß1 and suggests that the induction of constitutive decorin overexpression by ASMCs in vivo may have therapeutic value in the inhibition of TGF-ß1–mediated effects on the development of atherosclerotic lesions.
Key Words: transforming growth factor-ß1 • cell proliferation • extracellular matrix decorin • decorin • arterial smooth muscle
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Decorin is a family member of the small leucine-rich chondroitin/dermatan sulfate proteoglycans and is present in the extracellular matrix (ECM) of a variety of tissues and cell types.1 2 3 4 In blood vessels, decorin is confined mainly to the adventitia but is also present in lesser amounts in the smooth muscle–rich media.5 6 7 However, in atherosclerosis, decorin accumulates in defined locations throughout the lesions in association with deposited lipoproteins,8 9 collagen fibrils,6 8 9 10 and the plaque neovasculature.7 Such findings suggest that decorin might play a role in lipid retention,11 12 as well as in fibrosis and neovessel growth during the development of the atherosclerotic lesion.
Decorin has been shown to influence the proliferative capacity of cells. For example, decorin inhibits the growth of Chinese hamster ovary (CHO) cells13 14 and various cancer cell lines.15 16 The growth-inhibitory effect of decorin in malignant cell lines involves an increase in the cyclin kinase inhibitor p21.15 17 Moreover, the upregulation of decorin expression in nongrowing confluent arterial smooth muscle cells (ASMCs)18 suggests a relationship between the expression of decorin and growth quiescence in ASMCs. Decorin also influences ECM production and organization. Decorin binds to several ECM proteins, such as collagen,19 20 fibronectin,21 and thrombospondin,22 and mediates aspects of matrix protein fibrillogenesis and fibril packing.23 24 25 26 In decorin-null mice, the regulation of collagen fibril formation is obviously disturbed, and fibrils with irregular size and shape are deposited in collagenous tissues.23 Overexpression of decorin in in vivo disease models also alters ECM deposition. For example, an antifibrotic effect of decorin in vivo has been demonstrated in the bleomycin-induced hamster model of lung fibrosis27 and in an experimental animal model of glomerulonephritis.28 29 In addition, local overexpression of decorin in balloon-injured rat carotid arteries causes an increase in the density of collagen fibril packing within the neointima and decreases neointimal accumulation of versican and fibronectin, thereby reducing intimal volume.30
In addition to a direct effect on the assembly of the ECM proteins, decorin may mediate cellular and ECM changes by an influence on the activity of cytokines and growth factors that are involved in the regulation of cell proliferation and ECM production. For example, some studies have shown that decorin binds and inactivates transforming growth factor-ß1 (TGF-ß1)31 32 33 and reverses the effects of this cytokine on cells. The administration of purified decorin or gene therapeutic delivery of decorin reduces fibrosis in an experimental animal model of glomerulonephritis,28 29 in which fibrosis is dependent on TGF-ß1.34 35 Recent studies have demonstrated that decorin overexpression can also block the TGF-ß1–dependent suppression of immune surveillance of gliomas36 37 and the inhibition of lung epithelium morphogenesis by TGF-ß1.38
We have recently found that the accumulation of ECM in the intima of balloon-injured carotids that were seeded with decorin-overexpressing cells is decreased.30 The decreased volume of the lesion involves a decreased matrix volume with no change in cell number. To explore the mechanism by which decorin modulates this response, we have examined whether ASMCs that overexpress decorin have altered growth and ECM production in response to TGF-ß1, which is a cytokine that influences cell proliferation and ECM accumulation during atherosclerotic lesion development.39 40 41
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Materials
HEPES, NaCl, EGTA, dithiothreitol, sodium fluoride, Na3VO4, ß-glycerophosphate, leupeptin, aprotinin, urea, phenylmethylsulfonyl fluoride, Tween 20, chondroitin sulfate (type C), and TGF-ß1 (No. T7039) were from Sigma Chemical Co; 6-aminohexanoic acid and benzamidine were from Eastman Kodak Co; chondroitin ABC lyase was from ICN Pharmaceuticals; and DEAE-Sephacel was from Pharmacia Biotech, Inc. Prestained and 14C-labeled protein standards, glycine, SDS, N,N,N',N'-tetramethylethylenediamine, ammonium persulfate, and all cell culture supplies were from Life Technologies, Inc. Triton X-100 was from Boehringer-Mannheim Corp. Na2-[35S]sulfate (carrier free) and [3H]glucosamine were from ICN Radiochemicals, and 40% acrylamide solution was from Bio-Rad. Rabbit antibody (LF-94) against bovine decorin was generously provided by Dr L. Fisher, National Institute of Dental Research, National Institutes of Health, Bethesda, Md. Antibodies to TGF-ß1 receptor type I (No. sc398) and type II (No. sc400) and to p27 (No. sc528) were from Santa Cruz Biotech, Inc. The polyclonal antibody to p21 (No. PC55) was from Oncogene Research Products.
Recombinant human platelet-derived growth factor (PDGF)-BB was kindly supplied by Dr Charles Hart (Zymogenetics Inc, Seattle, Wash). Purified decorin, which was dissociatively extracted from bovine tendon and is active in a collagen fibrillogenesis assay, was kindly provided by Dr Kathryn Vogel, University of New Mexico, Albuquerque.
Construction of the Bovine Decorin Retrovirus
(LDSN) and Stable Transduction of Fischer 344 Rat Smooth Muscle
Cells
The cDNA of bovine decorin (PG28, courtesy of Dr
Marian Young, National Institute of Dental Research, National
Institutes of Health, Bethesda, Md) was inserted into the
EcoRI site of the
replication-defective retroviral vector LXSN (courtesy of Dr A.D.
Miller, Fred Hutchinson Cancer Research Center, Seattle,
Wash)42 to prepare the
retroviral vector for the expression of bovine decorin (LDSN).
Subsequently, the packaging cell lines were established, and Fischer
344 rat ASMCs were transduced as described
previously.26 30 42
Briefly, the retroviral vector containing the bovine decorin gene and
the control vector (LXSN) were transfected into the ecotropic packaging
cell line PE501 by use of the calcium phosphate precipitation
method.43 Transiently
produced virus was harvested and used to infect the second amphotropic
packaging cell line (PA317). After titering the virus
production in NIH 3T3 TK- cells,
the virus was harvested from clones producing between
5x105 and 5x106
virus/mL and used for stable transduction of Fischer 344 rat ASMCs in
the fourth passage after primary culture. Selection media for the PA317
cells and the ASMCs contained the neomycin analogue G418 at 600 µg/mL
and 800 µg/mL, respectively.
Cell Culture
ASMCs from male Fischer 344 rats were obtained as
described previously.44
Transduced cells were used for experiments between 4 and 8 passages
after the initial transduction. After selection by means of the
neomycin analogue G418 (800 µg/mL), ASMCs were maintained on tissue
culture plastic in DMEM supplemented with 10% calf serum.
Purified collagen (Vitrogen-100, Collagen Corp) was used according to the directions of the manufacturer to coat tissue culture plastic with monomeric collagen and polymeric collagen films. Briefly, for the monomeric coating, 100 µL of Vitrogen-100 solution was spread per well (24-well plates). The polymeric collagen coats were prepared by covering the tissue culture surface with a neutralized solution of Vitrogen-100 that was allowed to polymerize at 37°C for 1 hour. The polymeric and the monomeric collagen preparations were then air-dried overnight in the tissue culture hood. Before use, the wells were rinsed and rehydrated with serum-free tissue culture medium.
Growth Assays
To perform proliferation studies, cells were seeded
at 2x105 cells per well into 24-well
plates, grown for the indicated time periods, harvested by means of
trypsinization, and fixed in 3.7% formaldehyde. Cell number was
determined in a Coulter particle counter after dilution (1:10) with
PBS. The growth curves were established either directly without
preceding serum deprivation or after stimulation following
serum withdrawal (48 hours). DNA synthesis was measured by
incorporation of [3H]thymidine (10
µCi/mL), which was added 4 hours before the cells were harvested.
Subsequently, cells were washed twice with ice-cold PBS and then
incubated with ice-cold 10% trichloroacetic acid overnight at 4°C.
After 2 additional washes with 10% trichloroacetic acid, the
precipitated material was dissolved in 0.1N NaOH and analyzed
in a liquid scintillation counter, after an aliquot had been stored for
protein measurement (BCA, Pierce).
Metabolic Labeling and Proteoglycan
Analysis
For metabolic labeling of proteoglycans,
100 µCi/mL carrier-free
Na2-[35S]sulfate or
10 µCi/mL [3H]glucosamine was added to
the culture medium. Incorporation of radiosulfate and
[3H]glucosamine into total secreted
glycosaminoglycans was determined from duplicate
aliquots of culture medium by cetylpyridinium chloride
precipitation.45
For separation of radiolabeled proteoglycans by SDS-PAGE,
samples of proteoglycans in conditioned medium were partially purified
and concentrated on 0.5 mL DEAE-Sephacel columns in 8 mol/L urea with
0.5% Triton X-100, 0.01 Tris-HCl, pH 7.5, and 0.25 mol/L NaCl (urea
buffer), washed with 
10 vol urea buffer, and eluted by 3 mol/L NaCl
in urea buffer. After the addition of 30 µg chondroitin sulfate
carrier, the eluted material was precipitated at -20°C (2 hours) by
the addition of 3.5 vol of 95% ethanol containing 1.3% potassium
acetate. The pellet was dissolved in distilled water, and ethanol
precipitation was repeated without the addition of carrier. After the
final centrifugation, the supernatants were discarded,
and the pellet was air-dried. Samples were resuspended in 8 mol/L urea,
either with or without prior digestion by chondroitin ABC lyase (0.02
U) in enriched Tris buffer,46
pH 8, for 3 hours at 37°C. Subsequently, samples were boiled (3
minutes) in SDS-containing sample buffer with ß-mercaptoethanol.
Undigested radiolabeled samples were applied to a 4% to 12% gradient
SDS-polyacrylamide gel and detected by
autoradiography of the dried gels.
14C-labeled protein standards were used to
estimate the size of proteoglycan core proteins. In addition, digested
and undigested samples were run by SDS-PAGE and were blotted for
Western analysis (see below).
Western Analysis
Analysis of cyclin-dependent kinase
inhibitors was performed as described
previously.47 Briefly, cells
grown on tissue culture plastic were rinsed with PBS and harvested in
lysis buffer (50 mmol/L HEPES [pH 7.5], 150 mmol/L NaCl,
5 mmol/L EDTA, 2.5 mmol/L EGTA, 1 mmol/L dithiothreitol,
1 mmol/L NaF, 0.1 mmol/L
Na3VO4, 10 mmol/L
ß-glycerol phosphate, 0.5 mmol/L phenylmethylsulfonyl
fluoride, 10 µg/mL leupeptin, and 10 µg/mL aprotinin). Cell
extracts were incubated 10 minutes on ice and centrifuged at
27 000g for 30 minutes.
Subsequently, supernatants were analyzed for protein content
(BCA, Pierce). Equal aliquots of protein (20 to 50 µg) were separated
by 15% SDS-PAGE. After transfer to Immobilon membranes (Millipore
Corp), the proteins were immunoblotted with antibodies
against p21 and p27.47 For
detection of primary antibodies, a horseradish peroxidase–conjugated
second antibody was applied and developed by use of the enhanced
chemiluminescence method (Amersham-Pharmacia Biotech, Inc).
To detect decorin core protein before and after chondroitin ABC lyase digestion, Western analysis was performed as described previously.26 48 Briefly, samples were run on 10% SDS-polyacrylamide gels and were transferred to a nitrocellulose membrane (BA83, Schleicher and Schuell, Inc). Blots were blocked with 2% BSA in Tris-buffered saline with 0.1% Tween 20 and exposed to a primary antibody directed against bovine decorin core protein (LF-94). After incubation with an alkaline phosphatase–conjugated secondary antibody, the bound primary antibody was detected by use of an enzyme-linked chemiluminescence procedure (Tropix). To quantify total bovine decorin synthesis, secreted protein was collected 24 hours after medium change (complete medium with 0.2% FBS) from confluent LXSN- and LDSN-transduced cells, and the amount was determined by a blotting assay as described before,26 with purified bovine decorin from tendon used as a standard (provided by Dr Kathryn Vogel, University of New Mexico, Albuquerque). Expression of bovine decorin core protein was also verified by Western blots from passages 4 to 12 after the initial transfection to exclude the possibility that lack or loss of expression accounts for any negative results (not shown).
Northern Blot Analysis
Total RNA was isolated from cultured ASMCs by the
method of Chomczynski and
Sacchi.49 Fifteen micrograms
of total RNA was separated on 0.8% agarose gels containing
formaldehyde.50 Subsequently,
RNA was subjected to limited alkaline hydrolysis, transferred to
Zetaprobe (Bio-Rad), and cross-linked by UV light. Membranes were
prehybridized for 2 hours at 42°C in 50% (vol/vol) formamide (Life
Technologies, Inc), 6x
SSPE,50 5x Denhardt’s
solution,50 0.5% SDS, 5%
dextran sulfate, and 100 µg/mL salmon sperm DNA (Sigma). Probes were
32P-labeled by random priming, with the use
of 5'-[
-32P]dCTP (Amersham-Pharmacia
Biotech, Inc) as described
previously.51 Hybridization
with 32P-labeled cDNA probes (see below) was
carried out at 42°C in the same solution for 16 hours, followed by 3
washes with 2x SSPE/0.1% SDS at 42°C and 2 washes with 0.3x
SSPE/0.1% SDS at 65°C.
cDNA Probes
The same full-length bovine decorin cDNA (Pg28) used
for construction of the LDSN vector was used to detect bovine decorin
mRNA in the transduced ASMCs by Northern analysis. The rat
versican cDNA probe against the V3 form of
versican52 was used to
determine versican mRNA levels by Northern blotting. The rat
plasminogen activator inhibitor-1
(PAI-1) cDNA probe used in the present study for Northern
analysis was generously provided by Dr T. Gelehrter
(Albany Medical College, Albany,
NY).53
Statistical Analysis
Unpaired t
tests were performed where appropriate, and a 2-tailed value of
P<0.05 was considered
significant.
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Expression of Bovine Decorin Proteoglycan in Cultured Rat Vascular ASMCs
After transfection, the ASMCs were analyzed for expression of bovine decorin. Northern blot analysis (Figure 1A
) demonstrated bovine decorin mRNA expression in 4
ASMC lines transduced with the LDSN retrovirus compared with ASMCs
transduced with the "empty" LXSN retrovirus and the parental cell
line. The bovine decorin cDNA (Pg28) was used for hybridization. The
expression of bovine decorin core protein was demonstrated by use of a
species-specific antibody to bovine decorin (LF-94,
Figure 1B).
Figure 1 demonstrates that the expression levels in LDSN-1,
LDSN-2, and LDSN-4 cells were similar. Thus, these cell lines were used
for the subsequent experiments designed to study the function of
decorin. The amount of bovine decorin secreted by the confluent
transduced cells was 30 µg per 107
cells for 24 hours, as determined by comparison with a purified tendon
decorin standard in a quantitative blotting assay (see
Methods).
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To determine whether the retrovirally expressed decorin
contained a chondroitin sulfate/dermatan sulfate (CS/DS)
glycosaminoglycan chain, Western blot
analysis was performed before and after chondroitin ABC lyase
digestion of samples derived from conditioned tissue culture medium. As
shown in
Figure 2A, a strong band at 90 kDa was detected in ASMCs
transduced with LDSN, compatible with
Mr
estimates for
decorin.1 4 25 54 55 56
Digestion of the sample with chondroitin ABC lyase shifted the band to
40 kDa, which indicates the presence of CS/DS
glycosaminoglycan. To confirm the presence of newly
synthesized CS/DS glycosaminoglycan chains, cells
were metabolically labeled with
[3H]glucosamine and
[35S]sulfate. As shown in
Figure 2B, incorporation of
[3H]glucosamine and
[35S]sulfate into total secreted
proteoglycans is increased in the decorin-overexpressing cells. In
addition, electrophoretic separation of secreted proteoglycans on
SDS-PAGE showed markedly increased levels of a
35S-labeled proteoglycan at 90 kDa
(Figure 2C) in ASMCs transduced with LDSN. No changes in cell
morphology were observed on overexpression of decorin (not
shown).
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Effects of Decorin Overexpression on Levels of
Cdk Inhibitors and Proliferation
DNA synthesis, measured by
[3H]thymidine incorporation, was reduced
in decorin-overexpressing cells 24 hours after plating in 10% serum
(Figure 3A). This reduction of DNA synthesis was transient,
inasmuch as no difference was observed at 48 hours and 72 hours. To
determine whether changes occurred in the levels of cell
cycle–regulatory proteins, such as the cdk inhibitors p21
and p27, Western analysis was performed. Twenty-four hours
after plating, p21 and p27 levels were increased over the levels in
LXSN cells
(Figure 3B). If LXSN cells were plated (24 hours) in
conditioned medium from LDSN cells, the levels of p21 and p27 were
increased as well
(Figure 3C), suggesting that secreted bovine decorin is
responsible for the increase in cdk inhibitor levels. The
effect of decorin overexpression on cdk inhibitors was also
transient (72 hours,
Figure 3B), in agreement with the transient decrease in DNA
synthesis. The transient effect of decorin on
[3H]thymidine incorporation and cdk
inhibitors is not due to decreased decorin synthesis after
24 hours, because comparable levels of decorin are present in
medium collected from 0 to 24 hours and from 24 to 48 hours after
plating
(Figure 3A, right).
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To determine whether the initial inhibition of DNA synthesis
in LDSN cells has an effect on cell proliferation, the growth of ASMCs
was determined over the course of 6 days. For this purpose, ASMCs were
grown for 2 days after plating, synchronized by serum withdrawal for 48
hours, and stimulated by serum or PDGF-BB. No difference in growth rate
between LXSN and LDSN cells was found in response to 10% serum
(Figure 4A) or 2% serum (not shown). In addition,
proliferation in response to PDGF-BB (10 ng/mL) was identical in ASMCs
transduced with either LXSN or LDSN
(Figure 4B). Nor did retroviral overexpression of bovine
decorin in bovine aortic ASMCs affect proliferation (data not shown),
indicating that the failure of decorin overexpression to inhibit
proliferation is not species specific.
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Because a fibrillar collagen substratum alters the proliferative response of ASMCs to PDGF,47 additional growth assays were performed on monomeric and polymeric collagen films, with or without the addition of 5 and 25 µg/mL purified decorin, to determine whether the effect of decorin on cell growth was dependent on the nature of the substrate. The addition of purified decorin had no effect on the growth of ASMCs transduced with LXSN or LDSN on either substrate (data not shown). The same decorin preparation used in these assays inhibited the migration of endothelial cells without affecting cell proliferation,26 indicating that purified decorin is biologically active. These data suggest that retroviral overexpression of decorin does not affect ASMC growth in response to serum and PDGF in vitro.
Response of ASMCs That Overexpress Bovine
Decorin to Exogenous TGF-ß1 Is Reduced
In confluent rat ASMCs, TGF-ß1 stimulates DNA
synthesis, whereas in subconfluent ASMC cultures, TGF-ß1 inhibits
ASMC proliferation.39 The
stimulation of DNA synthesis in confluent ASMCs is mediated by the
induction of the autocrine PDGF
production.39 57
TGF-ß1, in the presence of 5% serum, also stimulates DNA synthesis
of confluent LXSN-transduced ASMCs and inhibits DNA synthesis in sparse
ASMCs
(Figure 5). Both of these density-dependent effects of
TGF-ß1 on [3H]thymidine incorporation by
ASMCs were inhibited in decorin-overexpressing cells
(Figure 5).
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The induction of 2 TGF-ß1–responsive genes,
PAI-158 59 and the
proteoglycan versican,60 was
assayed in LXSN and LDSN ASMCs to confirm directly the neutralization
of TGF-ß1 activity by decorin. The addition of TGF-ß1 to LXSN cells
caused a pronounced increase in PAI-1 and versican mRNA levels, but a
markedly lesser response was observed when LDSN cells were exposed to
the growth factor
(Figure 6). TGF-ß1 induced the accumulation of PAI-1 mRNA
in LXSN cells in a dose-dependent manner, with the strongest induction
at 5 ng/mL, whereas the dose-response curve for versican mRNA is
bell-shaped, with a peak of expression level at 0.1 ng TGF-ß1 per
milliliter
(Figure 6B). In LDSN cells, the dose-response curves for the
induction of PAI-1 and versican mRNA were shifted to higher TGF-ß1
concentrations. Notably, the effects of low concentrations (0.01 and
0.1 ng/mL) of TGF-ß1 were blocked completely in
decorin-overexpressing cells, whereas the effect of 5 ng/mL was only
partially inhibited.
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Medium-switching experiments were performed to test whether
decorin secreted by LDSN cells could inhibit the induction of PAI-1 and
versican expression by TGF-ß1 in LXSN cells
(Figure 6B
). Thus, LDSN-conditioned medium was applied to
LXSN cells before stimulation with 2 ng/mL TGF-ß1. In the presence of
LDSN-conditioned medium, TGF-ß1–mediated induction of PAI-1 and
versican mRNA was reduced in LXSN cells compared with the induction
observed in LXSN cells covered with LXSN-conditioned medium
(Figure 6B). These findings suggest that secreted decorin in
the medium of LDSN cells inhibits the activity of TGF-ß1 in rat
aortic ASMCs. No differences were observed between LXSN and LDSN
cultures when PDGF-BB (10 ng/mL) was used to stimulate PAI-1 mRNA
expression (data not shown). Western blot analysis indicated
that ASMCs transduced with LXSN or LDSN expressed similar levels of the
receptors I and II
(Figure 6C). This observation indicates that differences in
TGF-ß receptor expression cannot explain the decreased response to
TGF-ß1 in decorin-overexpressing
cells.
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ECM accumulation and ASMC proliferation are hallmarks in the development of intimal thickenings associated with the development of atherosclerotic lesions.61 Our recent work has shown that insertion of decorin-overexpressing cells into injured arteries causes a decrease in intimal volume.30 This decrease is due primarily to a decrease in ECM volume rather than a change in cell number within the intima. The results of the present in vitro study support these in vivo observations and, furthermore, suggest that the effects of decorin overexpression may involve the modulation of TGF-ß activity.
An initial decrease in DNA synthesis by
decorin-overexpressing cells was observed during the first 24 hours
after plating, but this relative decrease disappeared at later time
points. The relative decrease in LDSN cells of
[3H]thymidine incorporation and
increase of cyclin-dependent kinase inhibitors p21 and p27
at 24 hours after plating are similar to the effects that were reported
in studies of human cancer cell lines in which decorin inhibited growth
by the upregulation of
p21.15 17 However,
in contrast to those studies, the inhibitory effect of
decorin on DNA synthesis in ASMCs is transient; thus, no difference was
seen between LDSN and LXSN cells at 48 hours and 72 hours after
plating. Because the decorin content in the medium of LDSN cells at 48
hours is as high or higher than that in 24-hour conditioned medium, as
shown in
Figure 3A, decreased decorin concentration in the media at
later times after plating cannot explain the transient effect on DNA
synthesis. The transient nature of the decorin effect on cell
cycle–regulatory proteins may be due instead to the neutralization of
decorin by binding to other matrix components, such as fibronectin and
collagen,25 62
because these ECM components are produced in large amounts in
ASMCs.63 64 65
Alternatively, the deposition of ECM molecules, such as fibronectin and
collagen, may influence the proliferative response of ASMCs to growth
factors47 66 and
override the effects of decorin on cell proliferation. For example,
Koyama et al47 demonstrated
that ASMCs grown on fibrillar collagen had increased levels of
cyclin-dependent kinase inhibitors and a decreased growth
response to PDGF compared with cells cultured on monomeric collagen.
Thus, although decreased collagen synthesis or deposition by ASMCs that
overexpress decorin might be expected to allow continued proliferation
in response to PDGF and serum, the growth of rat ASMCs on fibrillar
collagen did not result in significant differences in the response of
LXSN or LDSN cells. Clearly, no difference in growth kinetics was
detected between LXSN and LDSN cells at later times in culture,
confirming the transient effect of decorin on DNA synthesis and p21/p27
levels in ASMCs.
Although the growth response of decorin-overexpressing cells
to serum and/or PDGF is not affected, TGF-ß1 responsiveness is
dramatically impaired. TGF-ß1 induces proliferation in dense cultures
of ASMCs that is due to the induction of
PDGF-AA39 57 and
inhibits growth in sparse cultures. The stimulation of DNA synthesis in
dense cultures and the inhibition in sparse cultures by exogenous
TGF-ß1 were blocked in decorin-overexpressing ASMCs
(Figure 5). These results are similar to those of previous
studies in which decorin inhibited the proliferation of CHO cells
because of an inhibition of TGF-ß1
utilization.14 It is thought
that the interaction between TGF-ß1 and decorin is mediated through a
binding sequence in the decorin core protein and does not involve the
glycosaminoglycan
chain.4 31 33
Several reports have demonstrated an inhibition of TGF-ß1 activity by
decorin,14 28 29
although enhancement of TGF-ß1 activity by decorin has also been
described.67 Other work
indicates that TGF-ß1/decorin complexes may be selectively
inactive,32 and thus, only
some TGF-ß1–dependent cellular responses are affected. The
variability in these observations may be due to the use of different
cell systems or differences in decorin preparations that were used,
because the folding and glycosylation of the decorin core protein are
dependent on the source and isolation procedure
used.56
The observation that decorin decreases the effect of TGF-ß1 on TGF-ß1–responsive genes provides further evidence that decorin influences TGF-ß1 activity. The effect of decorin on the responsiveness of ASMCs to TGF-ß1 does not appear to be mediated by changes in TGF-ß1 receptor expression, inasmuch as the TGF-ß receptor I and II expression levels were slightly upregulated in the decorin-transduced cells. Therefore, these experiments indicate that decorin is a functional antagonist of TGF-ß1 in rat ASMCs. In these studies, the PAI-1 mRNA level was used as a well-established reporter for TGF-ß1 activity.59 Earlier studies have also shown that TGF-ß1 induces the expression of versican by ASMCs.68 Therefore, the current observation that the induction of versican mRNA by decorin-overexpressing ASMCs is altered in response to TGF-ß1 is significant, inasmuch as versican is an important constituent of the ECM and an early response element in restenotic and atherosclerotic lesions.7 9 69 Moreover, decorin overexpression in vivo appears to significantly decrease the immunostaining for versican in rat carotid intimal lesions that develop in response to balloon cathetertization,30 although we have not attempted to determine whether PAI-1 expression is altered in that model. Therefore, some of the effects of decorin overexpression during atherosclerotic lesion development in vivo may involve the antagonism of endogenous TGF-ß activity.
In conclusion, the present study demonstrates that decorin overexpression clearly inhibits the response of ASMCs to TGF-ß1. Moreover, despite transient effects on cell cycle–regulatory proteins and DNA synthesis, decorin overexpression by ASMCs had little effect on long-term growth in response to serum or PDGF stimulation. These data establish that decorin overexpression modifies cellular processes that are fundamental to the development of vascular fibrosis.
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Acknowledgments |
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This study was supported by National Institutes of Health grants (HL-18645 and HL-52459), and Dr Jens W. Fischer was supported by a postdoctoral fellowship of Ernst Schering Research Foundation (Berlin, Germany). We thank Kathy Braun and Christina Tsoi for their excellent technical assistance.
Received November 22, 2000; accepted January 19, 2001.
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References |
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