From the Department of Medicine, University of North
Carolina, Chapel Hill, North Carolina 27599-7170 and the
§ Department of Biology, University of Michigan,
Ann Arbor, Michigan 48109-1048
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ABSTRACT |
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The aim of this study was to determine if
cultured porcine vascular smooth muscle cells (pSMCs) that had been
maintained on different extracellular matrix proteins had an alteration
in their expression of insulin-like growth factor binding protein-5
(IGFBP-5) and their responsiveness to insulin-like growth factor-I
(IGF-I). When pSMCs were plated on fibronectin, they synthesized
6.0 ± 1.2-fold more IGFBP-5 than did cells maintained on laminin
and type IV collagen. IGF-I increased IGFBP-5 gene expression 3-fold in
the cells plated on fibronectin. The addition of an RGD peptide and
echistatin to pSMC cultures that had been plated on fibronectin inhibited IGFBP-5 mRNA expression. The addition of an antibody against 2
1 integrin partially
reversed the inhibitory effects of laminin and type IV collagen on
IGFBP-5 expression. Cells maintained on fibronectin had a 5.0 ± 1.1-fold greater DNA synthesis response to IGF-I compared with those
maintained on laminin/type IV collagen, and echistatin significantly
inhibited the DNA synthesis response of the fibronectin-maintained
cells to IGF-I. The anti-
2
1 antibody partially reversed the inhibitory effect of laminin and type IV collagen on IGF-I-stimulated DNA synthesis. The addition of IGFBP-5 to
cultures plated on laminin and type IV collagen significantly increased
their response to IGF-I. Atherosclerotic plaques from pig aorta
contained abundant fibronectin and had increased IGFBP-5 mRNA
(4.5 ± 1.5-fold) compared with tissue from the normal vessel wall
that had a low fibronectin content. These results indicate that
fibronectin, laminin, and type IV collagen have major effects on
IGFBP-5 expression and on IGF-I-stimulated pSMC responses and that
these effects are mediated by their respective integrins.
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INTRODUCTION |
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Vascular smooth muscle cells
(SMCs)1 in the media of
normal blood vessels are surrounded by a basement membrane that
contains type IV collagen and laminin and maintain a differentiated,
contractile state characterized by abundant myofilaments and expression
of contractile protein isoforms such as -actin (1). They are quiescent, and the proliferative index is low (1). In contrast, SMCs in
the neointima seem to have dedifferentiated. This dedifferentiation includes a marked structural reorganization of the cells, with loss of
myofilaments and the formation of an extensive rough endoplasmic reticulum and large Golgi complex (2-4). Their abundance of smooth muscle
-actin decreases, whereas the expression of nonmuscle
-
and
-actin increases (2-4). They synthesize increased amounts of
fibronectin and deposit it into their extracellular matrix (ECM). This
is termed the synthetic phenotype, and these cells have acquired the
ability to proliferate, migrate, and synthesize ECM in response to
stimulation by growth factors and cytokines (5). Additionally, cells in
the synthetic phenotype constitutively synthesize peptide growth
factors, and they have the ability to replicate in the absence of
exogenously added mitogens (1).
Extracellular matrix has been shown to play an important role in the
modulation of the SMC phenotype in culture (6). Aortic SMCs assume the
synthetic phenotype when they are cultured on a fibronectin substratum
and will proliferate in response to stimulation by appropriate growth
factors. In contrast, if the cells are cultured on laminin and type IV
collagen, they will remain in the contractile phenotype. These ECM
proteins function by binding to their integrin receptors on the cell
surface. Cultured SMCs express the 5
1 and
V
3 integrins, which bind fibronectin, and
the
2
1 integrin, which binds to laminin
and several forms of collagen (7).
The growth and differentiation of SMCs are modulated by a number of
peptide growth factors, including the IGFs. SMCs in culture have been
shown to synthesize IGF-I, and IGF-I stimulates SMC proliferation in an
autocrine fashion (8-10). In vivo, IGF-I mRNA and
immunoreactive IGF-I are detected in atherosclerotic lesions that
develop after injury (11), and the increase in IGF-I mRNA and
immunoreactive peptide after balloon denudation injury precedes an
associated increase in SMC proliferation (12, 13). IGF-I has also been
shown to stimulate SMC migration (14), and this response is mediated by
the IGF-I receptors but also requires ligand occupancy of the
V
3 integrin. This ligand occupancy of integrins may play a role in controlling IGF-I responsiveness.
The bioactivities of IGFs are modulated by a group of specific, high affinity IGF binding proteins (IGFBPs) (15). Previous studies have shown that porcine aortic SMCs secrete IGFBP-2, -4, and -5 (16, 17) and that IGFBP-5 can modulate IGF-I-stimulated cell proliferation and migration (18). Because modulation of their phenotypic state alters pSMC responses to peptide growth factors, we determined the ability of specific EMC components to regulate IGFBP-5 expression and the responsiveness of these cells to IGF-I.
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EXPERIMENTAL PROCEDURES |
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Materials--
Human fibronectin, type IV collagen, laminin, and
echistatin were purchased from Sigma. The peptides GRGESP and GRGDSP
were purchased from Telios Pharmaceuticals Inc. (San Diego, CA).
Recombinant human IGF-I was obtained from Bachem Inc. (Torrance, CA).
Polyvinylidene difluoride filters were supplied by Millipore Corp.
Autoradiographic film was purchased from Eastman Kodak (Rochester, NY).
Fetal bovine serum, Dulbecco's modified Eagle's medium, and
penicillin-streptomycin were purchased from Life Technologies, Inc.
P1E6, a monoclonal antibody against the
2
1 integrin, was a gift from Dr. Leslie Parise (Department of Pharmacology at UNC-Chapel Hill). Trypsin was
obtained from Boehringer Mannheim. [3H]Thymidine was
purchased from ICN Biomedicals, Inc. (Costa Mesa, CA).
[32P]dCTP was purchased from Amersham Corp. Polyclonal
antisera against IGFBP-2 and -5 were prepared as described previously
(19, 20, 39). A monoclonal antibody that was specific for smooth muscle
-actin and rhodamine-labeled phalloidin were purchased from
Sigma.
Maintenance of Porcine Aortic Smooth Muscle Cells (pSMCs) in
Culture--
pSMCs were obtained from thoracic aortas of pigs using a
previously described method (21). They were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5/liter glucose, 100 units/ml penicillin, 100 µg/ml streptomycin, 4 mM
glutamine, and 10% fetal bovine serum (complete medium) in 10-cm
tissue culture plates (Falcon 3001). The cells were used between
passages 2 and 5. To culture the cells on specific ECM proteins, the
cells were detached with trypsin/EDTA, trypsin was neutralized with
10% fetal bovine serum, and the cells were rinsed twice with
serum-free DMEM and suspended in the same medium. They were then plated
on tissue culture plates or glass coverslips that had been exposed to
specific ECM proteins at a density of 2 × 104
cell/cm2. The surfaces of culture dishes or glass
coverslips were precoated with fibronectin (1 µg/cm2) or
type IV collagen (2 µg/cm2) plus laminin (1 µg/cm2) by incubating the mixture at room temperature for
4-5 h as recommended by the manufacturer. The liquid was then
aspirated, and the dishes were rinsed twice with PBS and maintained in
DMEM-H/0.01% bovine serum albumin for 15 min before use. To attach
IGFBP-5 to matrix proteins, the plates that contained the ECM proteins
were rinsed twice with binding buffer (Eagle's minimal essential
medium without bicarbonate, supplemented with 20 mM Hepes,
pH 7.4, and 0.01% bovine serum albumin) and incubated overnight at
4 °C with the same buffer and 100 ng/ml of IGFBP-5 or with buffer
alone. The wells were then rinsed twice with PBS before use. In some
experiments, the cells were incubated with an RGD peptide, echistatin,
or anti-2
1 antibody (P1E6) at the
indicated concentration for 15 min before the cells were plated on
culture dishes.
Immunocytochemistry--
The cells were plated on glass
coverslips precoated with ECM substrates for 2 days before they were
fixed in 2% formaldehyde in PBS for 20 min and then permeabilized with
0.2% Triton X-100 in PBS for 3 min. To determine the expression of
smooth muscle -actin, the cells were exposed to a 1:200 dilution of
a monoclonal antibody prepared against smooth muscle
-actin for
2 h followed by 1:500 dilution of fluorescein
isothiocyanate-labeled goat anti-mouse IgG for 30 min. To determine the
expression of total actin, the cells were exposed to 1:500 dilution of
rhodamine-labeled phalloidin. They were rinsed with PBS, mounted in
90% glycerol with 0.1% paraphenylenediamine, and then analyzed using
a Nikon Labophot microscope with epifluorescence optics.
RNA Isolation and Northern Blotting--
RNA was isolated from
cell cultures by Tri-Reagent following the manufacturer's instructions
(Molecular Research Center, Inc., Cincinnati, OH) and then quantified
by measuring UV absorbance at 260 nm. To isolate RNA from pig aortas,
the tissue specimens were obtained from atherosclerotic lesions and
from normal aortic subendothelium from six pigs who had been fed a high
fat diet for 4 months (22). These are early proliferative phase lesions that contain abundant monocytes, macrophages, and smooth muscle cells.
The lesion tissue was immediately frozen in liquid nitrogen and
pulverized at 80 °C using pre-cooled mortar and pestle. The pulverized tissues were then homogenized for 1 min with Polytron homogenizer in the presence of Tri-Reagent. RNA was isolated as described previously (17). The RNA samples were size fractionated on a
1.2% agarose/formaldehyde gel, blotted, fixed onto nylon membranes
(Biotrans, ICN Biochemical Inc.), and then hybridized with either a
[32P]dCTP-labeled human 627-base pair IGFBP-5 cDNA
probe (17) or a 542-base pair IGFBP-2 cDNA probe (15). The probes
were labeled to specific activities of 200 µCi/µg by random priming
(15). The relative abundance of the radiolabeled bands was determined by PhosphorImage analysis using Image Quant SF (Molecular
Dynamics).
Western Immunoblotting Analysis--
The pSMC cultures were
rinsed with PBS and scraped into SDS sample buffer containing 100 mM dithiothreitol. The protein concentration of each sample
was determined by the BCA protein assay (Pierce), and equal amounts of
protein (5 µg) per lane were loaded onto 12.5% SDS-polyacrylamide
gels. The separated proteins were then transferred to filters
(Immobilon PSQ, 0.45-mm pore size, Millipore). The filters were exposed
to a monoclonal antibody against smooth muscle -actin (diluted at
1:1,000 in TBS containing 3% bovine serum albumin) for 14 h at
4 °C. The immunoblots were developed with the enhanced
chemiluminescence detection system according to the manufacturer's
recommendation (Amersham). To determine the fibronectin content in the
aortic tissue specimens, the tissue was immediately frozen in liquid
nitrogen and then pulverized at
80 °C. The pulverized tissues were
transferred into tubes to which lysis buffer containing 20 mM Tris-HCl, pH 7.4, 2% Triton X-100, 10 mM
EDTA had been added. The suspension was homogenized for 1 min at
4 °C followed by centrifugation for 5 min at 12,000 × g at 4 °C. The protein concentration in the supernatant
was measured and adjusted to 1 mg/ml by adding 2 × Laemmli sample buffer with 0.1 M dithiothreitol. 30 µg of protein was
loaded per gel lane, and the proteins in the mixture were separated by 7.5% SDS-polyacrylamide gel electrophoresis (PAGE) in the presence of
0.1 M dithiothreitol. The separated proteins were
transferred to Immobilon PSQ filters. Fibronectin was detected with
anti-fibronectin polyclonal antibody using a 1:1000 dilution, and the
immune complexes were detected by chemiluminescence according to the
procedure cited above.
Immunoprecipitation of IGFBPs--
To analyze IGFBP synthesis
and secretion into conditioned medium, pSMC monolayers were exposed to
50 µCi/ml [35S]methionine (56 Ci/mmol) for 8 h in
methionine-deficient (106 M) DMEM. The medium
was incubated with a 1:1000 dilution of anti-IGFBP-5 or 1:1000 dilution
of anti-IGFBP-2 antisera, and the immune complexes were precipitated
with protein A-Sepharose as described previously (17). The precipitates
were analyzed by SDS-PAGE with fluorography and autoradiography
(17).
Cell Replication--
To measure [3H]thymidine
incorporation into porcine smooth muscle cells, pSMCs were plated at a
density of 2 × 104/cm2 in 48-well culture
plates (Costar 3548) that had been precoated with fibronectin or type
IV collagen and laminin with or without IGFBP-5, as described
previously. In some experiments, pSMCs were preincubated with
echistatin 107 M or the P1E6 antibody at a
dilution of 1:500 for 15 min before they were plated on the fibronectin
or laminin plus type IV collagen. The cultures were maintained for
24 h in serum-free DMEM (with or without the various treatments);
then, fresh DMEM (0.1 ml) supplemented with 0.2% human platelet-poor
plasma (21), 1 µCi/well [3H]thymidine (specific
activity, 35 mCi/mmol), and IGF-I (0-20 ng/ml) were added. After
24 h, the plates were placed on ice, washed with ice-cold
phosphate-buffered saline twice, and incubated with ice-cold 5%
trichloroacetic acid for 10 min. The trichloroacetic acid precipitates
were solubilized by adding 0.1 ml of 1% SDS, 0.1 N NaOH
overnight, and radioactivity was quantified using a Beckman
scintillation counter using ScintiSafe Econo 2 (Fisher Scientific) as a
scintillant. In some experiments, the cell number was determined in a
particle data counter (model ZBI, Coulter Electronics, Hialeah, FL).
For these experiments, the cells were plated at a density of 2 × 104 cells/cm2 in 24-well plates (Costar 3424).
After 24 h, the treatments were added, and the incubation
continued for 36 h. The cells were detached with 0.2% trypsin,
and the cell number was determined.
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RESULTS |
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Expression of Smooth Muscle (SM) -Actin--
SM
-actin was
used as a marker for SMC differentiation. Indirect immunofluorescence
microscopy showed that the percentage of cells with microfilament
bundles that stained intensely for smooth muscle
-actin was greater
when the cultures were plated on laminin plus type IV collagen compared
with cultures plated on fibronectin (Fig.
1A). Immunoblotting analysis
with a monoclonal antibody against smooth muscle
-actin was also
used to examine the effect of the different substrata on the level of
smooth muscle
-actin expression. A single band of 42 kDa was
detected. A direct comparison by immunoblotting showed that pSMCs
cultured on fibronectin contain 4.7-fold less smooth muscle
-actin
than the cells plated on laminin plus type IV collagen (Fig.
1B). Thus, the cells that were plated on laminin/type IV
collagen maintained this important property of differentiated pSMC. In
contrast, the expression of total actin was not decreased in pSMCs
plated on fibronectin, as demonstrated using rhodamine-labeled
phalloidin (Fig. 1C).
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Extracellular Matrix Proteins Regulate IGFBP-5 Synthesis and Secretion-- Cultures that were plated on fibronectin or laminin/type IV collagen had significant differences in the amount of IGFBP-5 that was synthesized and secreted. As shown in Fig. 2A, media obtained from the cells grown on fibronectin had a 1.7-fold ± 0.3 (p < 0.05) increase in intact IGFBP-5 compared with cultures maintained on plastic. More important, the amount of IGFBP-5 was 4.0 ± 1.0-fold (p < 0.01) greater than the cells that were plated on laminin/type IV collagen. In contrast, the synthesis of IGFBP-2 was unchanged when cells that were cultured using these conditions were compared (Fig. 2B).
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Influence of ECM on the Ability of IGF-I to Regulate IGFBP-5 mRNA Abundance-- As IGFBP-5 gene expression has been shown to be stimulated by IGF-I (17), we examined whether maintaining the cultures on different ECM proteins would alter this cellular response. As shown in Fig. 4, IGF-I increased IGFBP-5 mRNA abundance in the pSMCs that had been plated on either fibronectin or laminin plus type IV collagen. Although cells maintained in laminin/type IV collagen responded to IGF-I with an increase in IGFBP-5 expression (6.1 ± 1.0 to 41 ± 5.0, arbitrary units p < 0.01), the absolute level of IGFBP-5 mRNA was substantially below that detected in cultures that had been maintained on fibronectin (54 ± 4.0 to 140 ± 15, arbitrary units p < 0.01).
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Cell Replication Assays of pSMCs Cultured on Different ECM Substrata-- pSMCs cultured on different substrata were also analyzed for their ability to synthesize DNA in response to IGF-I. The results showed that the response to IGF-I was significantly greater (560 ± 84% compared with 112 ± 12%, p < 0.01) for cells plated on fibronectin compared with cells plated on laminin plus type IV collagen (Fig. 5). These data are consistent with the responses in Fig. 4 showing that cells plated on type IV collagen retain some capacity to respond to IGF-I, but the absolute level that can be attained is decreased significantly. The addition of IGFBP-5 to cultures that were plated on laminin and type IV collagen resulted in marked potentiation of the cellular response to IGF-I. In contrast, the addition of IGFBP-5 to cultures plated on fibronectin did not alter the cellular response to IGF-I significantly.
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The Effect of Different ECM Proteins Is Mediated by Their
Respective Integrins--
As pSMCs have been reported to express
5
1,
v
3, and
2
1 integrins, we examined whether
blocking ligand binding to these integrins would influence IGFBP-5
expression. Following a 30-min exposure to synthetic peptides or to
anti-integrin antibodies, the cells were plated on fibronectin or
laminin/type IV collagen dishes. The adherence of pSMC to the
substratum was unaffected by preincubating with the these reagents at
the given concentrations. After a 24-h incubation, IGFBP-5 mRNA
abundance was quantified. The synthetic RGD peptide that binds
5
1 and echistatin blocked the
fibronectin-induced change in IGFBP-5 expression (Fig.
6). A control RGE peptide had no effect.
The inhibition of IGFBP-5 expression that was induced by laminin and
type IV collagen was partially reversed by the antibody against
2
1 integrin. These data indicate that
binding of these matrix proteins to their respective integrins is an
important step in their ability to modulate IGFBP-5 expression. To
determine if these integrins were also involved in modulating the DNA
synthesis response to IGF-I, a similar experiment was conducted using
[3H]thymidine incorporation as an index of IGF-I action.
As shown in Fig. 7, exposure of pSMC
plated on fibronectin to echistatin attenuated the cellular DNA
synthesis response to IGF-I. In contrast, the P1E6 antibody partially
reversed the inhibitory effect of ligand occupancy of
2
1 on IGF-I-stimulated replication. To
determine if altering ligand occupancy of these integrins would alter
the cellular growth response to IGF-I, cell number was determined using
a similar experimental design. Quiescent pSMC cultures that were plated
on fibronectin increased their final cell number by 61 ± 7% in
response to 20 ng/ml of IGF-I. The addition of echistatin (10
7 M) decreased this response to 32 ± 4%. The cells plated on laminin/type IV collagen increased 11 ± 2.1%, and those that were exposed to the P1E6 antibody increased
18 ± 2.1%.
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Atherosclerotic Lesions Contain Abundant Fibronectin and Have Greater IGFBP-5 mRNA Expression Compared with Normal Aorta-- To determine if the SMCs in lesions express more IGFBP-5 than cells in the normal arterial wall, atherosclerotic lesions that were diet induced were extracted, and their RNA was analyzed by Northern blotting. The results showed that lesions contain 4.5 ± 1.5-fold (p < 0.05) (n = 4 separate animals) more IGFBP-5 mRNA compared with normal subendothelial tissue. A representative experiment is shown in Fig. 8A. The fibronectin content in these tissue specimens was also determined. The results show that a 5.9-fold greater amount of fibronectin was detected in the lesion tissue compared with normal aorta (Fig. 8B).
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DISCUSSION |
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In normal blood vessels, SMCs are attached to a basement membrane
that contains type IV collagen and laminin. Within the arterial wall,
most SMCs are maintained in a quiescent state and express contractile
protein isoforms. However, in response to vascular injury, SMCs alter
their phenotype and are stimulated to proliferate and migrate. This
phenotypic transition from the contractile to the synthetic state is
accompanied by enhanced expression of specific matrix proteins such as
fibronectin (9). The phenotypic transition can be modulated in
vitro by changing the composition of ECM substratum (6). Aortic
SMCs have been reported to convert to the synthetic phenotype when
cultured on a fibronectin substratum, and, if maintained on
fibronectin, they will proliferate if they are also exposed to
appropriate growth factors. In contrast, if the cells are cultured on
laminin plus type IV collagen, early passage (e.g. <p5)
cells can be maintained in a nonproliferative state for 1 week (6). This was confirmed in our study by showing abundant expression of
smooth muscle -actin and an increased number of microfilament bundles that stained for
-actin. In contrast, cells that were plated
and maintained on fibronectin had much lower
-actin content. Based
on these results, we believe that the in vitro modulation of
the pSMC phenotype was similar to that previously reported by others
(6).
A major finding in the present study is that, compared with cells cultured on laminin and type IV collagen, pSMCs cultured on fibronectin synthesize and release more IGFBP-5. The increase in IGFBP-5 synthesis is associated with increased IGFBP-5 mRNA expression. The effect becomes manifest between the 2nd and 4th days after changing the ECM substratum. The effect is specific for IGFBP-5 as IGFBP-2 protein and mRNA levels were unaltered by the ECM proteins. This suggests that expression of this gene may be an important component of phenotype transition. Because IGFBP-5 has been shown to potentiate the cellular growth-promoting effect of IGF-I (23), this may be relevant to the observation noted by several groups that synthetic phenotype cells are more responsive to growth factors (9, 24-26). This conclusion is strengthened by our observation that the addition of IGFBP-5 to cultures plated on laminin/type IV collagen enhanced their DNA synthesis response to IGF-I, suggesting that the enhanced expression of IGFBP-5 in the fibronectin-exposed cultures may be an important component of this enhanced replication response to IGF-I.
These changes were also reflected in the IGFBP-5 synthesis response to
IGF-I. We had previously shown that IGF-I induced a 6-fold increase in
IGFBP-5 mRNA (17); therefore, we analyzed the responses of pSMC
that expressed each phenotype. Although pSMC plated on fibronectin had
a greater increase in IGFBP-5 expression in response to IGF-I, cultures
maintained in the nonproliferative phenotype still showed some IGF-I
responsiveness. There are two possible interpretations of this result.
First, fibronectin is acting to directly stimulate basal (non-IGF-I
dependent) IGFBP-5 expression and to enhance the response to IGF-I.
This conclusion is supported by our data showing that peptide
antagonists of ligand binding to V
3 and
5
1 can inhibit IGFBP-5 expression.
Second, it is also possible that a low percentage of cells have not
been maintained in the nonproliferative phenotype and, therefore, have acquired IGF-I responsiveness. In either case, it seems that the synthetic phenotype cells are more responsive to IGF-I. In addition to
modulating the IGFBP-5 synthesis response to IGF-I, plating pSMC on
different substrata modulated the DNA synthesis response. This effect
also seemed to be ECM protein-mediated as cells that were plated on
laminin/type IV collagen had a reduced DNA synthesis response to IGF-I
compared with the response of cells plated on fibronectin.
To test the hypothesis that the effects of these ECM molecules were
mediated by their respective integrins, pSMCs were incubated with an
antibody or peptides that bind to and block integrin ligand occupancy.
The adherence of pSMCs to matrix-coated plates was unaffected by
preincubating the cells with either RGD peptide or the antibody at the
indicated concentrations. These results indicate that the effect of
fibronectin on IGFBP-5 expression is mediated by
5
1 and
V
3
integrins, as both the RGD peptide and echistatin, a disintegrin that
has been shown to block fibronectin binding to
V
3 integrin, inhibited the induction of
IGFBP-5 by fibronectin. Because we have previously shown that
vitronectin binding to
V
3 is required for
pSMC to migrate in response to IGF-I and because echistatin also
blocked the IGFBP-5 response to IGF-I, it is likely that
V
3 occupancy by fibronectin is also augmenting this response. In contrast to fibronectin, exposure to type
IV collagen/laminin resulted in maintenance of IGFBP-5 expression at a
low level. When occupancy of the
2
1
integrin was blocked with the P1E6 antibody, this inhibition of IGFBP-5 expression could be partially reversed. This strongly suggests that
2
1 integrin ligand occupancy
is acting to inhibit IGFBP-5 expression. Additionally, the
effects of these ECM proteins on IGF-I-stimulated DNA synthesis seem to
be integrin mediated as blocking binding to
2
1 or
V
3
partially reversed the effects of these ligands on IGF-I-stimulated
replication. Taken together, these results support the conclusion that
the effects of the ECM proteins used in this study are integrin
mediated and that integrin ligand occupancy modulates IGF-I
responsiveness. Therefore, the capacity of this growth factor to
stimulate pSMC replication is directly related to the context in which
the growth factor signal is presented to its cognate receptor.
IGFs exert multiple effects on target cells that are mediated by the IGF-I receptor on the cell surface. IGF-I has been shown to stimulate proliferation as well as migration of pSMC (14). In addition to the direct influences on the cells, IGF-I exposure is also required for the full activity of several other growth factors or cytokines. IGF-I has been shown to enhance the effects of platelet-derived growth factor (27), thrombin (28), and angiotensin II (29) on cultured SMCs. Selective inhibition of IGF-I binding to its receptor with anti-IGF-I antibody results in marked reduction in the mitogenic response to thrombin (28) and angiotensin II (29). In vivo, IGF-I mRNA and immunoreactive IGF-I are detected in intimal lesions that develop in humans (11). IGF-I mRNA and immunoreactive IGF-I levels both increase severalfold after balloon denudation injury in rats, and these increases are temporally preceded by an increase in SMC proliferation (12, 13). Thus, IGF-I may be important for SMC proliferation and migration in vivo and may play an important role in the development of atherosclerotic lesions.
Our findings suggest that variables such as enhanced binding of
fibronectin to V
3 or increased binding of
type IV collagen to
2
1 may also modulate
responsiveness of pSMC to other growth factors. Recent studies have
shown that ligand occupancy of the
V
3
integrin functions to regulate growth factor actions. Platelet-derived growth factor (30) and IGF-I (14) have a diminished capacity to
stimulate cell migration if ligand occupancy of this integrin is
attenuated. Similarly blocking integrin or growth factor
receptor-associated signaling pathways, such as induction of
phosphatidylinositol-3 kinase and inositol phosphate-3 formation,
results in inhibition of migration (30). The mechanistic explanations
that have been proposed to explain these observations include
coactivation of signal transduction elements by growth factor tyrosine
kinase receptors and integrins such as insulin receptor substrate-1
(31) or calmodulin-II kinase (32, 33) and, in the case of fibroblast growth factor, direct activation of the receptor kinase activity by
integrin occupancy (34). Our findings suggest that the
V
3 occupancy is activating IGF-I
signaling, resulting in not only enhanced migration but also in
enhanced replication. In contrast, no studies have been reported to
date that blocking ligand occupancy of
2
1
will enhance growth factor actions. In the case of platelet-derived growth factor, occupancy of this integrin was stated to enhance the SMC
migration response (35). Therefore, further studies that are directed
toward elucidating the mechanism by which
2
1 occupancy is inhibiting IGF-I actions
are warranted.
IGF bioactivities are modified by a family of six high affinity IGFBPs.
Among them, IGFBP-5 has the unique property of adhering tightly to ECM
(23). When associated with ECM, the affinity of IGFBP-5 is lowered
8-fold. This affinity shift results in a more favorable equilibrium
between IGF-I bound to ECM-associated IGFBP-5 and to the IGF-I
receptor, and this is associated with an enhanced cellular DNA
synthesis response to IGF-I. Additionally, the amount of ECM-associated
IGFBP-5 has been shown to be related to the degree of enhancement of
the cellular growth response (23). This hypothesis was confirmed by our
results showing that the addition of IGFBP-5 to cultures that were
plated on laminin and type IV collagen significantly increased the
cellular response to IGF-I. Therefore, factors such as the induction of
IGFBP-5 synthesis by fibronectin could result in further enhancement of the cellular responses to IGF-I. Similarly, an increase in the affinity
of V
3 for its ligands, which occurs
following IGF-I binding to its receptor (14), may contribute to this
response.
These observations may have some relevance to the development of
atherosclerotic lesions in vivo. This study demonstrated that atherosclerotic lesions contain more IGFBP-5 mRNA compared with comparable amounts of normal vessel wall. Although several cell
types exist in atherosclerotic plaques, SMCs are the predominant cell
type (5). Similarly, as the SMCs within lesions have assumed the
synthetic phenotype and because fibronectin is much more abundant, it
is possible that the increase that we observed in IGFBP-5 mRNA content in lesions reflects the differences that we observed in pSMC
cultures in vitro and that this increase could be related to
lesion enlargement. Because the V
3
integrin is not expressed in normal vessel wall but is abundant in
atherosclerotic lesions (36), it is possible that this change also
contributes to enhanced IGFBP-5 expression. Furthermore, because other
ECM proteins that bind to
V
3, such as
thrombospondin and osteopontin, are abundant in lesions and have the
potential to act through
V
3 to alter the
smooth muscle cell phenotype and responsiveness (37, 38), it will be of
interest to determine their effects on IGF-I and IGFBP-5 actions.
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ACKNOWLEDGEMENT |
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We thank George Mosley for help in preparing this manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HL-56580.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: Division of Endocrinology, CB 7170, 6111 Thurston Bowles Bldg., The University of North Carolina, Chapel Hill, NC 27599-7170.
1 The abbreviations used are: SMC, smooth muscle cell; IGFBP-5, insulin-like growth factor binding protein-5; IGF-I, insulin-like growth factor I; pSMC, porcine vascular smooth muscle cells; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; SM, smooth muscle.
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