© 1995 American Heart Association, Inc.
Articles |
A Subpopulation of Smooth Muscle Cells in Injured Rat Arteries Expresses Platelet-Derived Growth Factor–B Chain mRNA
From the University of Washington, Department of Pathology, Seattle.
Correspondence to Volkhard Lindner, MD, PhD, University of Washington, Department of Pathology SJ-60, Seattle, WA 98195.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract Proliferation of smooth muscle cells (SMCs) and formation of a neointima are characteristics of the response of rat carotid arteries to balloon injury. Rat platelet-derived growth factor (PDGF)-B was cloned, thus allowing us to use species-specific probes to carry out in situ hybridization on the surface of injured arteries. A distinct population of luminal SMCs (7% to 10%) in the developing neointima expressed PDGF-B mRNA, but very few luminal SMCs still expressed PDGF-B (0.5%) when the lesion had stopped growing. Primary SMC cultures revealed expression of PDGF-B mRNA in 1.6% of SMCs derived from normal tunica media and in 11% of SMCs derived from the neointima. These data demonstrate that SMCs in the injured vessel wall are heterogeneous with regard to PDGF-B expression and that subculturing of these cells may give rise to cultures that are either positive or negative for PDGF-B expression. Furthermore, with abundant expression of the PDGF receptor ß-subunit expressed by intimal SMCs, our findings provide evidence that PDGF-B synthesized by these cells may be involved in intimal lesion formation via a paracrine or autocrine mechanism.
Key Words: platelet-derived growth factor • intima • migration • platelet-derived growth factor receptor • in situ hybridization
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The concept that platelet-derived growth factor (PDGF) plays a role in formation of the neointima after balloon catheter injury1 was derived originally from studies showing that PDGF is a mitogen and chemoattractant for smooth muscle cells (SMCs) in culture.2 This idea was modified with the discovery that PDGF-B is only a weak mitogen, at least in the rat, for SMCs in vivo.3 4 Nonetheless, in vivo studies did show that infused PDGF-BB can act as a chemotactic factor, stimulating the migration of medial cells across the internal elastic lamina and increasing the extent of neointimal formation after injury.3 4 These data, however, do not rule out a replicative role for PDGF-BB, which might be acting as a comitogen with other growth factors. A role for PDGF-B in intimal thickening was also demonstrated by Nabel et al,5 who transfected PDGF-B plasmid DNA into pig arteries and demonstrated intimal lesion formation.
In the rat, the process of neointimal formation in response to arterial balloon injury continues for several weeks,6 but adhesion of platelets to the denuded surface is seen only during the first few days after denudation.7 Thus, stimulatory effects on underlying SMCs due to PDGF released from adhering platelets may be limited to a few days after injury. Given the short half-life in circulation, any effects of PDGF on intimal lesion formation beyond this time may only be possible if stimulation of SMCs occurs via a paracrine or autocrine mechanism within the vessel wall. The possibility that such a pathway exists was initially provided by the finding of PDGF-A mRNA expression in the balloon-injured vessel wall.8 PDGF-A, however, is probably not a good candidate for an autocrine factor, since in vitro studies showed that PDGF-A is a poor mitogen9 and may even act as an inhibitor, rather than a stimulant, of migration.10 11 In contrast, PDGF–B chain is usually a good mitogen in vitro,2 and the PDGF receptor ß-subunit, which can only respond to binding of the PDGF–B chain ligand, was also expressed at high levels in the intima. The levels of PDGF-B mRNA are not modulated after injury, and only low expression was detectable by Northern analysis in RNA extracted from the vessel wall.8 In addition, attempts to localize the cells expressing PDGF-B mRNA by in situ hybridization of vessel cross sections with a probe for mouse PDGF-B were unsuccessful. In summary, infused PDGF-B appears to be chemotactic rather than mitogenic, and evidence for an autocrine/paracrine loop in neointimal formation was still missing, since we did not see local expression of PDGF-B.
Evidence for a possible autocrine role of PDGF-B, however, did exist in vitro when SMCs from the neointima were grown in culture in which PDGF–B chain mRNA levels increased with passage number.12 Expression of the same transcript in cultures derived from normal tunica media, however, was either absent or detectable only after many passages were performed.12 This observation suggests that SMCs expressing PDGF-B mRNA represent a subpopulation with differences in phenotype that could account for many of the unusual properties of the neointima.
The inability to identify cells expressing PDGF–B chain in vivo raises concerns that the unique properties found in cultured intimal cells12 13 could be reflective of culture conditions rather than the properties of these cells in their original tissue environment. Thus, the present report sought to reexamine cells expressing PDGF-B chain by a new method for in situ hybridization using an en face technique. This method allowed us to demonstrate that a small population of SMCs in the neointima of injured rat arteries expresses PDGF-B and that this proportion, though small, is elevated during the period of most marked formation of the neointima. We will discuss the possibility that these in vivo PDGF-B–producing cells could play a key role in forming the neointima and that the appearance of PDGF–B chain in cultured neointimal cells may represent an amplification of a preexisting subpopulation found in vivo.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Arterial Injury Model
Male Sprague-Dawley rats (400 g, 3 to 4 months old, Bantin & Kingman, Edmonds, Wash) were used in all the experiments. All surgical procedures were carried out with the animals under general anesthesia (2.2 mg/kg body wt IP xylazine [AnaSed, Lloyd Laboratories] and 50 mg/kg body wt IP ketamine [Ketaset, Aveco Co, Inc]). Right and left carotid arteries were denuded with a 2F Fogarty balloon catheter as previously described.6 These vessels were used to study SMCs that had migrated onto the denuded surface from the underlying tunica media
4 days after denudation, where they
formed an intimal lesion over the course of several weeks. This model
allowed us to study luminal SMCs at early times after injury, when
cells were replicating (up to 2 weeks after injury), and at late times,
when proliferation had stopped (6 weeks). Deendothelialized segments of
arteries were identified by intravenous injection of Evans blue (0.3 mL
of 5% solution in saline) 10 minutes before the animals were killed.
All animals were perfusion-fixed with phosphate (0.1 mol/L, pH
7.4)–buffered 4% paraformaldehyde. For in situ hybridization and
immunostaining, rats were killed at the indicated times after injury
(between 4 days and 6 weeks). Denuded carotid arteries were divided
into three segments, each measuring 8 to 10 mm in length. This
allowed tissue to be used from the same animal for in situ
hybridization with different probes as well as for immunostaining. For
each time point, segments were also obtained for embedding in paraffin
to carry out in situ hybridization on cross sections. For colocalization of replicating and PDGF-B–expressing SMCs on the luminal surface of an 8-day neointima, 3 rats were injected intraperitoneally with a single dose of 5-bromo-2'-deoxyuridine (BrdU, 25 mg/kg, Boehringer Mannheim) 1 hour before death. Immunostaining on en face preparations with an antibody against BrdU was carried out as previously described.14 Replication of luminal SMCs was expressed as a percentage of all luminal SMCs.
Cell Culture
Two independent isolations of neointimal SMCs from
carotid arteries injured 2 weeks earlier and medial SMCs from uninjured
carotid arteries were grown in Waymouth MB 752/1 medium (GIBCO/BRL)
supplemented with 10% bovine serum (Hyclone Laboratories Inc) as
previously described.15 Cells were grown in primary
cultures on chamber slides (Lab Tek, Nunc, Inc) until they were
confluent, 10 days after plating. The cells were then stored in
phosphate (0.1 mol/L, pH 7.4)–buffered 4% paraformaldehyde until they
were processed for in situ hybridization. Colocalization of PDGF-B mRNA
expression and DNA synthesis was carried out in previously
characterized rat SMC cell lines,16 of which one did
(WKY12-22) and one did not (WKY3M-22) express PDGF-B mRNA. These
cells were grown to confluence on chamber slides using Waymouth MB
752/1 medium and 10% bovine serum and were then switched to serum-free
medium. Twenty-four hours later, the cells were restimulated with 10%
bovine serum. One hour before fixing the cells with phosphate (0.1
mol/L, pH 7.4)–buffered 4% paraformaldehyde at 0, 6, 12, and
24 hours after serum stimulation, the cells were pulsed with BrdU (30
µmol/L). Immunostaining for BrdU was carried out as previously
described.14
DNA Probes and Cloning of Rat PDGF-B
A cDNA library from cultured rat newborn aortic
SMCs17 was screened for sequences related to the human
PDGF-B cDNA clone, pSM-1,18 by using the hybridization
conditions previously described.17 Twenty cDNA clones
remained positive after three rounds of selection, with the largest
clone (3-4a) containing a 3-kb insert representing a nearly
full-length clone. The putative translated region was sequenced by the
dideoxynucleotide chain termination method (Sequenase, USB), and each
sequence was confirmed by sequencing the complementary strand. Sequence
analysis was performed by using PCGENE sequencing
software (Intelligenetics). Multiple alignments were performed by using
the CLUSTAL program. Other DNA probes used for in situ
hybridization were a 0.6-kb Pst I fragment of the
extracellular domain of the rat PDGF receptor
ß-subunit.19
In Situ Hybridization and Immunostaining
In situ hybridization and immunostaining were carried out on
cross sections and on en face preparations of vessel segments as
recently described.14 After hybridization the slides were
coated with autoradiographic emulsion (Kodak, NTB2), exposed for 3
weeks, and then developed (Kodak, D-19). Preparations were observed
under the light microscope by using dark-field, bright-field, and a
combination of epiluminescence and bright-field illumination
(reflective light).
A monoclonal antibody recognizing rat macrophages and monocytes
(ED1, Bioproducts for Science Inc) and smooth muscle 
-actin (HHF-35,
a generous gift from Dr A. Gown) was used to identify macrophages and
SMCs on en face preparations. A staining protocol was followed as
previously described.20 21
Quantification of PDGF-B–Positive SMCs
The nonspecific background level of hybridization was determined
in two ways: (1) by hybridizing specimens with labeled sense probes and
(2) by hybridizing adult medial SMC cultures that were known not to
express PDGF-B mRNA by Northern analysis. In almost all cases these
negative controls showed fewer than five grains per cell.
PDGF-B–expressing cells were easily identified after hybridization
with the antisense probe by the presence of an abundance of silver
grains, which made grain counts almost impossible. The distribution of
silver grains was therefore considered to be bimodal. A total of 56
arterial segments derived from 28 different animals were studied, of
which 21 and 35 were hybridized with the sense and antisense probes,
respectively. Analysis of SMCs in primary cultures was carried out in
an identical fashion. Twenty-three fields for medial SMC cultures and
39 fields for intimal SMCs cultures were analyzed at x400
magnification. Statistical analysis on the in vivo data was
performed by using Fisher's test for multiple comparisons, and the
t test (unpaired, two tailed) was used to compare cultured
medial and intimal SMCs.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cloning of Rat PDGF-B cDNA
A neonatal rat SMC library was screened with a human cDNA clone for PDGF-B (pSM-1).18 Of the 20 clones identified in this manner, the largest one, 3-4a, was chosen for sequence analysis. As shown in Fig 1
, the translated region of clone 3-4a
showed high homology to the published amino acid sequences for porcine,
human, and feline PDGF-B.18 22 23 The degree of homology
with murine PDGF-B was 94% at the nucleic acid level.24
When used for Northern blot analysis, 3-4a detected an 3.5-kb
transcript in cultured rat pup SMCs that was indistinguishable from the
band detected by pSM-1 (not shown). Thus, 3-4a most likely
represents the rat homologue of PDGF-B.
|
Expression of PDGF-B mRNA in Injured Arteries
The normal rat carotid artery only rarely contains intimal SMCs.
Removal of the endothelium from the carotid artery induces
proliferation of the underlying medial SMCs, with migration of SMCs
into the intima a few days later.6 These
neointimal cells can remain uncovered by regenerating
endothelium for weeks and months after injury. Thus, after denudation
intimal SMCs can be studied on the luminal surface.
Previous studies using Northern analysis of total RNA extracted
from rat carotid arteries demonstrated that the transcript for PDGF-B
was expressed at low levels,8 with no change in response
to balloon injury. Moreover, in situ hybridization carried out on cross
sections of arteries with a murine probe8 failed to
localize PDGF-B mRNA to specific cells. Using the labeled rat probe on
cross sections, we could identify occasional cells expressing PDGF-B
mRNA, and these cells were only seen on the luminal surface of the
neointima (Fig 2a). To obtain more
convincing evidence for the expression of PDGF-B mRNA by luminal SMCs,
we used an en face approach that examined the entire cell population
present on the luminal surface. With this technique, denuded
carotid arteries were studied at different times after injury. A small
number of SMCs on the luminal surface at 5 days after injury were found
to express the mRNA for PDGF-B (Fig 2b). Occasionally, these cells were
arranged in clusters suggesting either expansion from a common
precursor or convergence of groups of migrating cells (Fig 2d).
|
The percentage of PDGF-B–positive cells on the luminal surface ranged
between 7.1% and 10.7% and did not change significantly up to 14 days
after injury (Fig 3). We also carried out in situ
hybridization on luminal SMCs of mature lesions (6 weeks after
denudation), in which cell replication is an infrequent
event.6 25 In these lesions only occasional
PDGF-B–positive SMCs were found (0.5%). This value was significantly
lower compared with all the other time points (P<.05).
|
We used an antibody (ED1) that recognizes rat macrophages to eliminate
the possibility that the PDGF-B–expressing cells on the luminal
surface were macrophages, since these cells have been reported to
express PDGF-B26 27 under certain conditions. Macrophages
were not detected on the luminal surface of injured vessels at the time
points up to 2 weeks (data not shown). The identity of the cells on the
luminal surface as SMCs was further verified by staining with an
antibody against SMC -actin (data not shown).
In agreement with previous studies, we found high expression levels of
PDGF receptor ß-subunit in all luminal SMCs at the time points
studied (Fig 2e
).8
The possibility that expression of PDGF-B mRNA is linked to cell
replication prompted us to determine whether there is a correlation
between PDGF-B expression and SMC replication. A single injection of
BrdU was given to rats 8 days after balloon injury of the carotid
artery, and the number of replicating SMCs on the denuded luminal
surface was quantified with an antibody against BrdU. Unfortunately, we
were unable to perform both immunostaining for BrdU and in situ
hybridization on the same specimens (Fig 2c). The replication index
(percentage) for luminal SMCs (Fig 3), however, was approximately four
times higher than the percentage of luminal SMCs expressing PDGF-B
mRNA.
Expression of PDGF-B mRNA in Primary Cultures
SMC cultures derived from normal adult media generally give rise
to cultures that do not express PDGF-B mRNA, whereas cultures derived
from the neointima or pup aorta do express this
mRNA,12 13 and expression levels increase with passage
number. These differences in PDGF-B mRNA expression may be the result
of different levels of expression by the entire cell population or may
reflect differences in the percentage of cells expressing PDGF-B mRNA
in the culture. To discriminate between these two possibilities, we
determined the percentage of PDGF-B–positive cells in cultures derived
from 2-week-old intima and from uninjured tunica media. Intimal SMCs
were isolated by enzymatic digestion from the entire intima and grown
until confluent for 10 days, and 10.9% of these cultured intimal SMCs
expressed PDGF-B (Fig 3). However, primary cultures obtained from the
tunica media of uninjured carotid arteries showed that only 1.6% of
these SMCs expressed PDGF-B, which was significantly lower (86%,
P
.01) than the value determined for intimal SMCs (Fig 3).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cultured SMCs can synthesize PDGF–B chain mRNA and may do so at a high level. However, the ability to make PDGF-B in vitro has consistently been seen only in cultured SMCs derived from 2-week-old rats, ie, rat pups, or from the neointima of balloon-injured carotid arteries (Fig 2g
and 2h).12 In
contrast to the in vitro data, however, PDGF–B chain mRNA has only
been seen in the vessel wall at low levels by Northern blotting, and
there was no evidence of its presence by in situ hybridization carried
out on arterial sections. Thus, it seemed that either the in vitro
phenomenon was a property only of cultured cells or that detection in
vivo might be limited with the previous techniques used. Using an en
face approach, we report that expression of PDGF-B mRNA by individual
SMCs can be detected in vivo. This finding shows that PDGF-B expression
by SMCs is not a phenomenon limited to SMCs in culture; instead, it
demonstrates that SMCs in the vessel wall are heterogeneous with regard
to PDGF-B expression. There are several explanations as to why these PDGF-B SMCs have not been previously detected by in situ hybridization on cross sections of arteries.8 With the cloning of rat PDGF-B cDNA, a species-specific probe was available that remained hybridized during the high-stringency wash of the specimens, which includes a ribonuclease digestion step. Using this probe for in situ hybridization, we did indeed find very few luminal SMCs on some cross sections that expressed PDGF-B. With the en face technique used in the present study, we were able to demonstrate more convincingly that PDGF-B–positive SMCs are present on the luminal surface. Because this technique allowed us to examine the entire luminal cell population, it was possible to quantify the frequency of PDGF-B–expressing cells. To analyze a similar number of cells on cross sections would require a large number of sections; this is especially true when only very few positive cells are present, eg, at 6 weeks after denudation.
PDGF-B is one of several mRNAs that showed constitutive differences in
expression, ie, higher in pup or intimal cells than in medial
unmanipulated cells.12 17 28 29 Both the
PDGF-B–expressing pup/intimal phenotype (Fig 2g) and the nonexpressing
adult medial SMC phenotype are stable in culture.16 The
presence of cells with these properties in vitro led us to propose that
vascular SMCs within injured vessels are a heterogeneous population,
containing at least two different subpopulations. PDGF-B mRNA, however,
has been localized only in cultured SMCs until now. Interestingly,
expression levels for PDGF-B in intimal SMCs in vitro increased with
passaging until steady levels were reached after approximately six
passages.12 Our data on the percentage of PDGF-B–positive
SMCs in primary cultures of medial and intimal SMCs relate to this
issue and demonstrate that the PDGF-B–expressing cells in the in vivo
intima give rise to the PDGF-B–producing population of cultured cells
derived from intimal cells. Serial passaging of the cultured cells may
change the ratio of PDGF-B–expressing cells. For medial SMC cultures
with a low frequency of PDGF-B–positive cells (1.6%), one possibility
is that subculturing may cause a further decrease in PDGF-B–expressing
cells and that they may eventually be lost. Alternatively, after
long-term subculture and expansion of the PDGF-B–expressing
population, significant levels of PDGF-B mRNA may be found only after
extensive subculturing. These two possibilities provide an explanation
for the observations made earlier12 : variable amounts of
PDGF-B mRNA were found in higher passage numbers of medial SMCs.
However, these PDGF-B–positive SMCs are less likely to be lost from
cultures of intimal SMCs in which the percentage of PDGF-B–positive
cells is sevenfold higher than in the corresponding medial SMC
cultures. The increase in PDGF-B expression with successive passage
numbers of these cells seems to suggest that the increase in the
PDGF-B–positive population of SMCs might possibly be the result of a
growth advantage under the culture conditions.
Another interesting question is whether the increased number of SMCs expressing PDGF-B mRNA in intimal cultures is due to a selective advantage of these cells in migrating from the media into the intima in response to balloon injury. Indeed, our recent data have shown that an infusion of PDGF-BB causes an increase in the accumulation of SMCs in the intima of injured rat carotid arteries,3 which occurs as a result of the increased migration of SMCs from the media into the intima. Thus, SMCs expressing their own PDGF-B and PDGF receptor ß-subunit may have an increased capacity to migrate, leading to a higher percentage of PDGF-B–positive SMCs in the intima. An alternative explanation is that PDGF-B–expressing SMCs have evolved as a consequence of vascular injury associated with balloon catheter denudation. The fact that we cannot detect PDGF-B mRNA expression in SMCs of the uninjured media lends support to this possibility. As a result of balloon injury, a situation is created in which SMCs in the neointima provide the luminal lining of the vessel wall. In that regard, it should be noted that PDGF-B–positive SMCs were found only on the luminal surface of denuded vessels. One difference between these luminal cells and the SMCs deeper in the intimal lesion is the fact that the luminal cells are exposed to shear stress. It is of interest that Resnick et al30 have identified a fluid shear stress–responsive element in the PDGF–B chain promoter. However, it remains unclear why shear stress would induce PDGF-B mRNA only in some luminal SMCs and not in the entire luminal SMC population. In any case, PDGF-B synthesized by SMCs on the luminal surface might be stimulating intimal lesion formation via its chemotactic properties by recruiting SMCs into the intima.
It is also of interest that the number of SMCs in the intimal lesion
expressing PDGF-B at 6 weeks after injury was significantly lower than
at the earlier time points. From previous studies we know that the
intimal lesion at these late time points is quiescent; ie, SMC
replication is a rare finding, and the size of the lesion is no longer
increasing.6 At the earlier time points (5 to 14 days),
however, migration of SMCs from the media into the intima occurs, and
intimal SMCs are dividing rapidly, causing the lesion to
grow.6 14 31 Expression of PDGF-B mRNA does not appear to
correlate with replication of intimal SMCs per se, since 40% of the
luminal SMCs at 8 days after injury are replicating at any given time
(Fig 3), yet no more than 10% of these cells express PDGF-B mRNA.
Furthermore, as recently shown by Lemire et al,16 some SMC
cell lines constitutively express PDGF-B mRNA (WKY12-22), whereas
others do not (WKY3M-22, Fig 2f). Labeling of replicating SMCs with a
1-hour pulse of BrdU at various times after serum stimulation revealed
that no more than 18% of the cells in the PDGF-B–positive cell line
were replicating at any of the time points (data not shown), whereas
all of the cells were expressing PDGF-B mRNA (Fig 2h). Together, these
findings argue that PDGF-B mRNA is expressed only by a subpopulation of
SMCs, and there appears to be a positive correlation between the time
when growth of the intimal lesion occurs and the time when PDGF-B is
expressed by SMCs.
The possible contribution of PDGF-B to the formation of the
neointima is not clear. The predominant receptor expressed
in the neointima is PDGF receptor
ß-subunit,8 a molecule that can only bind PDGF-B. The
present data demonstrate that a small portion of the cells in the
intima, 10% at the surface of the neointima, express
PDGF-B and could therefore serve as the source for an autocrine or
paracrine agonist long after platelet release is over. It should be
emphasized, however, that PDGF-BB is only a weak mitogen for SMCs in
vivo3 ; therefore, the mitogenic effect of the growth
factor may not be critical to the growth of the lesion. Furthermore,
injection of neutralizing antibodies to PDGF did not inhibit
replication of intimal SMCs, although they did inhibit lesion
formation.4 A possible mechanism for PDGF as a stimulant
of migration has recently been proposed by Yabkowitz et
al,32 who demonstrated that PDGF-B–induced migration of
SMCs is mediated via thrombospondin. In addition, PDGF-B has been found
to induce integrins in vascular SMCs that are thought to be important
in SMC migration,33 34 35 including ß1 and
ß3 integrins.
Our data demonstrate the heterogeneity of vascular SMCs in injured arteries in vivo with regard to PDGF-B mRNA expression. Furthermore, they provide the basis for a potential PDGF autocrine/paracrine loop in neointimal formation.
|
Acknowledgments |
|---|
This study was supported by National Institutes of Health grants HL-03174, HL-41103, and HL-42270.
Received March 8, 1994; accepted February 24, 1995.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Ross R. The pathogenesis of atherosclerosis: an update. N Engl J Med. 1986;314:488-500. [Medline] [Order article via Infotrieve]
2. Grotendorst GR, Chang T, Seppa HEJ, Kleinman HK, Martin GR. Platelet-derived growth factor is a chemoattractant for vascular smooth muscle cells. J Cell Physiol. 1982;113:261-266. [Medline] [Order article via Infotrieve]
3. Jawien A, Bowen-Pope DF, Lindner V, Schwartz SM, Clowes AW. Platelet-derived growth factor promotes smooth muscle migration and intimal thickening in a rat model of balloon angioplasty. J Clin Invest. 1992;89:507-511.
4.
Ferns GAA, Raines EW, Sprugel KH, Motani AS, Reidy MA,
Ross R. Inhibition of neointimal smooth muscle
accumulation after angioplasty by an antibody to PDGF.
Science. 1991;253:1129-1132.
5. Nabel EG, Yang Z, Liptay S, San H, Gordon D, Haudenschild CC, Nabel GJ. Recombinant platelet-derived growth factor B gene expression in porcine arteries induces intimal hyperplasia in vivo. J Clin Invest. 1993;91:1822-1829.
6. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury, I: smooth muscle growth in the absence of endothelium. Lab Invest. 1983;49:327-333. [Medline] [Order article via Infotrieve]
7. Jørgensen L, Grøthe AG, Groves HM, Kinlough-Rathbone RL, Richardson M, Mustard JF. Distribution of cellular responses in rabbit aortae following one and two injuries with a balloon catheter. Br J Exp Pathol. 1988;69:351-365. [Medline] [Order article via Infotrieve]
8.
Majesky MW, Reidy MA, Bowen-Pope DF, Hart CE, Wilcox
JN, Schwartz SM. PDGF ligand and receptor gene expression during
repair of arterial injury. J Cell Biol. 1990;111:2149-2158.
9.
Majack RA, Majesky MW, Goodman LV. Role of
PDGF-A expression in the control of vascular smooth muscle cell growth
by transforming growth factor-ß. J Cell Biol. 1990;111:239-247.
10.
Koyama N, Morisaki N, Saito Y, Yoshida S.
Regulatory effects of platelet-derived growth factor-AA
homodimer on migration of vascular smooth muscle cells. J
Biol Chem. 1992;267:22806-22812.
11.
Koyama N, Hart CE, Clowes AW. Different
functions of the platelet-derived growth factor- and -ß receptors
for the migration and proliferation of cultured baboon smooth muscle
cells. Circ Res. 1994;75:682-691.
12.
Majesky MW, Giachelli CM, Reidy MA, Schwartz SM.
Rat carotid neointimal smooth muscle cells reexpress
a developmentally regulated mRNA phenotype during repair of arterial
injury. Circ Res. 1992;71:759-768.
13.
Majesky MW, Benditt EP, Schwartz SM. Expression
and developmental control of platelet-derived growth factor A-chain and
B-chain/Sis genes in rat aortic smooth muscle cells. Proc
Natl Acad Sci U S A. 1988;85:1524-1528.
14.
Lindner V, Reidy MA. Expression of basic
fibroblast growth factor and its receptor by smooth muscle cells and
endothelium in injured rat arteries: an en face study.
Circ Res. 1993;73:589-595.
15.
Walker LN, Bowen Pope DF, Ross R, Reidy MA.
Production of platelet-derived growth factor-like molecules by
cultured arterial smooth muscle cells accompanies proliferation after
arterial injury. Proc Natl Acad Sci U S A. 1986;83:7311-7315.
16. Lemire JM, Covin CW, White S, Giachelli CM, Schwartz SM. Characterization of cloned aortic smooth muscle cells from young rats. Am J Pathol. 1994;144:1068-1081. [Abstract]
17. Giachelli CM, Bae N, Lombardi D, Majesky M, Schwartz SM. Molecular cloning and characterization of 2B7, a rat mRNA which distinguishes smooth muscle cell phenotypes in vitro and is identical to osteopontin (secreted phosphoprotein I, 2aR). Biochem Biophys Res Commun. 1991;177:867-873. [Medline] [Order article via Infotrieve]
18.
Ratner L, Josephs SF, Jarrett R, Reitz MSJ, Wong-Staal
F. Nucleotide sequence of transforming human c-sis
cDNA clones with homology to platelet-derived growth factor.
Nucleic Acids Res. 1985;13:5007-5018.
19. Herren B, Pech M. Expression of a rat PDGF receptor b ectodomain generates a low affinity ligand antagonist. J Recept Res. 1993;13:725-738. [Medline] [Order article via Infotrieve]
20. Ferns GA, Reidy MA, Ross R. Balloon catheter de-endothelialization of the nude rat carotid: response to injury in the absence of functional T lymphocytes. Am J Pathol. 1991;138:1045-1057. [Abstract]
21. Lindner V, Majack RA, Reidy MA. Basic fibroblast growth factor stimulates endothelial regrowth and proliferation in denuded arteries. J Clin Invest. 1990;85:2004-2008.
22. Stroobant P, Waterfield MD. Purification and properties of porcine platelet-derived growth factor. EMBO J. 1984;3:2963-2967. [Medline] [Order article via Infotrieve]
23.
Van den Ouweland AMW, Van Groningen JJM, Schalken JA,
Van Neck HW, Bloemers HPJ, Van de Ven WJM. Genetic organization
of the c-sis transcription unit. Nucleic Acids Res. 1987;15:959-971.
24. Bonthron DT, Sultan P, Collins T. Structure of the murine c-sis proto-oncogene (Sis, PDGFB) encoding the B chain of platelet-derived growth factor. Genomics. 1991;10:287-292. [Medline] [Order article via Infotrieve]
25.
Lindner V, Lappi DA, Baird A, Majack RA, Reidy MA.
Role of basic fibroblast growth factor in vascular lesion
formation. Circ Res. 1991;68:106-113.
26. Shimokado K, Raines EW, Madtes DK, Barrett TB, Benditt EP, Ross R. A significant part of macrophage-derived growth factor consists of at least two forms of PDGF. Cell. 1985;43:277-286. [Medline] [Order article via Infotrieve]
27. Wilcox JN, Smith KM, Williams LT, Schwartz SM, Gordon D. Platelet-derived growth factor mRNA detection in human atherosclerotic plaques by in situ hybridization. J Clin Invest. 1988;82:1134-1143.
28.
Giachelli CM, Majesky MW, Schwartz SM.
Developmentally regulated cytochrome P-450IA1 expression in
cultured rat vascular smooth muscle cells. J Biol
Chem. 1991;266:3981-3986.
29. Giachelli CM, Bae N, Almeida M, Denhardt DT, Alpers CE, Schwartz SM. Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques. J Clin Invest. 1993;92:1686-1696.
30.
Resnick N, Collins T, Atkinson W, Bonthron DT,
Dewey CF Jr, Gimbrone MA Jr. Platelet-derived growth factor B
chain promoter contains a cis-acting fluid shear-stress-responsive
element. Proc Natl Acad Sci U S A. 1993;90:4591-4595.
31. Jackson CL, Reidy MA. Basic fibroblast growth factor: its role in the control of smooth muscle cell migration. Am J Pathol.. 1993;143:1024-1031. [Abstract]
32. Yabkowitz R, Mansfield PJ, Ryan US, Suchard SJ. Thrombospondin mediates migration and potentiates platelet-derived growth factor-dependent migration of calf pulmonary artery smooth muscle cells. J Cell Physiol. 1993;157:24-32. [Medline] [Order article via Infotrieve]
33. Clyman RI, Mauray F, Kramer RH. b1 and b3 integrins have different roles in the adhesion and migration of vascular smooth muscle cells on extracellular matrix. Exp Cell Res. 1992;200:272-284. [Medline] [Order article via Infotrieve]
34.
Clyman RI, McDonald KA, Kramer RH.
Integrin receptors on aortic smooth muscle cells mediate
adhesion to fibronectin, laminin, and collagen.
Circ Res. 1990;67:175-186.
35. Janat MF, Argraves WS, Liau G. Regulation of vascular smooth muscle cell integrin expression by transforming growth factor b1 and by platelet-derived growth factor-BB. J Cell Physiol. 1992;151:588-595.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  R. C.M. Siow and A. T. Churchman Adventitial growth factor signalling and vascular remodelling: Potential of perivascular gene transfer from the outside-in Cardiovasc Res, September 1, 2007; 75(4): 659 - 668. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Mallawaarachchi, P. L. Weissberg, and R. C. M. Siow Antagonism of platelet-derived growth factor by perivascular gene transfer attenuates adventitial cell migration after vascular injury: new tricks for old dogs? FASEB J, August 1, 2006; 20(10): 1686 - 1688. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. Garanich, M. Pahakis, and J. M. Tarbell Shear stress inhibits smooth muscle cell migration via nitric oxide-mediated downregulation of matrix metalloproteinase-2 activity Am J Physiol Heart Circ Physiol, May 1, 2005; 288(5): H2244 - H2252. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. S. Schwartz, N. A. Chronos, and R. Virmani Preclinical restenosis models and drug-eluting stents: Still important, still much to learn J. Am. Coll. Cardiol., October 6, 2004; 44(7): 1373 - 1385. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. S. Santiago and L. M. Khachigian Ets-1 Stimulates Platelet-Derived Growth Factor A-Chain Gene Transcription and Vascular Smooth Muscle Cell Growth via Cooperative Interactions With Sp1 Circ. Res., September 3, 2004; 95(5): 479 - 487. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. C. Berk Vascular Smooth Muscle Growth: Autocrine Growth Mechanisms Physiol Rev, July 1, 2001; 81(3): 999 - 1030. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Leppanen, N. Janjic, M.-A. Carlsson, K. Pietras, M. Levin, C. Vargeese, L. S. Green, D. Bergqvist, A. Ostman, and C.-H. Heldin Intimal Hyperplasia Recurs After Removal of PDGF-AB and -BB Inhibition in the Rat Carotid Artery Injury Model Arterioscler. Thromb. Vasc. Biol., November 1, 2000; 20 (11): e89 - e95. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Palumbo, C. Gaetano, G. Melillo, E. Toschi, A. Remuzzi, and M. C. Capogrossi Shear Stress Downregulation of Platelet-Derived Growth Factor Receptor-{beta} and Matrix Metalloprotease-2 Is Associated With Inhibition of Smooth Muscle Cell Invasion and Migration Circulation, July 11, 2000; 102(2): 225 - 230. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. M. Taylor and L. M. Khachigian Induction of Platelet-derived Growth Factor B-chain Expression by Transforming Growth Factor-beta Involves Transactivation by Smads J. Biol. Chem., May 26, 2000; 275(22): 16709 - 16716. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. G. Pickering, L. H. Chow, S. Li, K. A. Rogers, E. F. Rocnik, R. Zhong, and B. M. C. Chan {alpha}5{beta}1 Integrin Expression and Luminal Edge Fibronectin Matrix Assembly by Smooth Muscle Cells after Arterial Injury Am. J. Pathol., February 1, 2000; 156(2): 453 - 465. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A Facchiano, F De Marchis, E Turchetti, F Facchiano, M Guglielmi, A Denaro, R Palumbo, M Scoccianti, and M. Capogrossi The chemotactic and mitogenic effects of platelet-derived growth factor-BB on rat aorta smooth muscle cells are inhibited by basic fibroblast growth factor J. Cell Sci., January 8, 2000; 113(16): 2855 - 2863. [Abstract] [PDF]
|
|
|
|
|
|
C. Mao, O. T.-B. Malek, M. E. Pueyo, P. G. Steg, and F. Soubrier Differential Expression of Rat Frizzled-Related frzb-1 and Frizzled Receptor fz1 and fz2 Genes in the Rat Aorta After Balloon Injury Arterioscler. Thromb. Vasc. Biol., January 1, 2000; 20(1): 43 - 51. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Schwartz The Intima : A New Soil Circ. Res., November 12, 1999; 85(10): 877 - 879. [Full Text] [PDF]
|
|
|
|
|
|
L. D. Adams, J. M. Lemire, and S. M. Schwartz A Systematic Analysis of 40 Random Genes in Cultured Vascular Smooth Muscle Subtypes Reveals a Heterogeneity of Gene Expression and Identifies the Tight Junction Gene Zonula Occludens 2 as a Marker of Epithelioid "Pup" Smooth Muscle Cells and a Participant in Carotid Neointimal Formation Arterioscler. Thromb. Vasc. Biol., November 1, 1999; 19(11): 2600 - 2608. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-o Deguchi, M. Makuuchi, T. Nakaoka, T. Collins, and Y. Takuwa Angiotensin II Stimulates Platelet-Derived Growth Factor-B Chain Expression in Newborn Rat Vascular Smooth Muscle Cells and Neointimal Cells Through Ras, Extracellular Signal-Regulated Protein Kinase, and c-Jun N-Terminal Protein Kinase Mechanisms Circ. Res., October 1, 1999; 85(7): 565 - 574. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. E. Vagnoni, N. D. Christiansen, G. R. Holyoak, M. A. Janowiak, and P. H. Martin Cellular Source in Ewes of Prostaglandin-Endoperoxide Synthase-2 in Uterine Arteries Following Stimulation with Lipopolysaccharide Biol Reprod, September 1, 1999; 61(3): 563 - 568. [Abstract] [Full Text]
|
|
|
|
|
|
G. Bilder, T. Wentz, R. Leadley, D. Amin, L. Byan, B. O'Conner, S. Needle, H. Galczenski, J. Bostwick, C. Kasiewski, et al. Restenosis Following Angioplasty in the Swine Coronary Artery Is Inhibited By an Orally Active PDGF-Receptor Tyrosine Kinase Inhibitor, RPR101511A Circulation, June 29, 1999; 99(25): 3292 - 3299. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. R. Bonin, K. Madden, K. Shera, J. Ihle, C. Matthews, S. Aziz, N. Perez-Reyes, J. K. McDougall, and S. C. Conroy Generation and Characterization of Human Smooth Muscle Cell Lines Derived From Atherosclerotic Plaque Arterioscler. Thromb. Vasc. Biol., March 1, 1999; 19(3): 575 - 587. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. S. Santiago, H. C. Lowe, F. L. Day, C. N. Chesterman, and L. M. Khachigian Early Growth Response Factor-1 Induction by Injury Is Triggered by Release and Paracrine Activation by Fibroblast Growth Factor-2 Am. J. Pathol., March 1, 1999; 154(3): 937 - 944. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Floege, T. Ostendorf, U. Janssen, M. Burg, H. H. Radeke, C. Vargeese, S. C. Gill, L. S. Green, and N. Janjic Novel Approach to Specific Growth Factor Inhibition in Vivo : Antagonism of Platelet-Derived Growth Factor inGlomerulonephritis by Aptamers Am. J. Pathol., January 1, 1999; 154(1): 169 - 179. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. W. Courtman, A. Cho, L. Langille, and G. J. Wilson Eliminating Arterial Pulsatile Strain by External Banding Induces Medial but Not Neointimal Atrophy and Apoptosis in the Rabbit Am. J. Pathol., December 1, 1998; 153(6): 1723 - 1729. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Rafty and L. M. Khachigian Zinc Finger Transcription Factors Mediate High Constitutive Platelet-derived Growth Factor-B Expression in Smooth Muscle Cells Derived from Aortae of Newborn Rats J. Biol. Chem., March 6, 1998; 273(10): 5758 - 5764. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. L. Couper, S. R. Bryant, J. Eldrup-Jorgensen, C. E. Bredenberg, and V. Lindner Vascular Endothelial Growth Factor Increases the Mitogenic Response to Fibroblast Growth Factor-2 in Vascular Smooth Muscle Cells In Vivo via Expression of fms-Like Tyrosine Kinase-1 Circ. Res., December 19, 1997; 81(6): 932 - 939. [Abstract] [Full Text]
|
|
|
|
|
|
R. D. Kenagy, C. E. Hart, W. G. Stetler-Stevenson, and A. W. Clowes Primate Smooth Muscle Cell Migration From Aortic Explants Is Mediated by Endogenous Platelet-Derived Growth Factor and Basic Fibroblast Growth Factor Acting Through Matrix Metalloproteinases 2 and 9 Circulation, November 18, 1997; 96(10): 3555 - 3560. [Abstract] [Full Text]
|
|
|
|
|
|
M. R. Ward, A. Agrotis, P. Kanellakis, R. Dilley, G. Jennings, and A. Bobik Inhibition of Protein Tyrosine Kinases Attenuates Increases in Expression of Transforming Growth Factor-ß Isoforms and Their Receptors Following Arterial Injury Arterioscler. Thromb. Vasc. Biol., November 1, 1997; 17(11): 2461 - 2470. [Abstract] [Full Text]
|
|
|
|
|
|
H.-I Yeh, F. Lupu, E. Dupont, and N. J. Severs Upregulation of Connexin43 Gap Junctions Between Smooth Muscle Cells After Balloon Catheter Injury in the Rat Carotid Artery Arterioscler. Thromb. Vasc. Biol., November 1, 1997; 17(11): 3174 - 3184. [Abstract] [Full Text]
|
|
|
|
|
|
A. Kumar and V. Lindner Remodeling With Neointima Formation in the Mouse Carotid Artery After Cessation of Blood Flow Arterioscler. Thromb. Vasc. Biol., October 1, 1997; 17(10): 2238 - 2244. [Abstract] [Full Text]
|
|
|
|
|
|
J. S. Mondy, V. Lindner, J. K. Miyashiro, B. C. Berk, R. H. Dean, and R. L. Geary Platelet-Derived Growth Factor Ligand and Receptor Expression in Response to Altered Blood Flow In Vivo Circ. Res., September 19, 1997; 81(3): 320 - 327. [Abstract] [Full Text]
|
|
|
|
|
|
J.-i. Abe, J.-o Deguchi, T. Matsumoto, N. Takuwa, M. Noda, M. Ohno, M. Makuuchi, K. Kurokawa, and Y. Takuwa Stimulated Activation of Platelet-Derived Growth Factor Receptor In Vivo in Balloon-Injured Arteries : A Link Between Angiotensin II and Intimal Thickening Circulation, September 16, 1997; 96(6): 1906 - 1913. [Abstract] [Full Text]
|
|
|
|
|
|
L. Capron, J. Jarnet, D. Heudes, D. Joseph-Monrose, and P. Bruneval Repeated Balloon Injury of Rat Aorta : A Model of Neointima With Attenuated Inhibition by Heparin Arterioscler. Thromb. Vasc. Biol., September 1, 1997; 17(9): 1649 - 1656. [Abstract] [Full Text]
|
|
|
|
|
|
H. Ueba, M. Kawakami, and T. Yaginuma Shear Stress as an Inhibitor of Vascular Smooth Muscle Cell Proliferation : Role of Transforming Growth Factor-ß1 and Tissue-Type Plasminogen Activator Arterioscler. Thromb. Vasc. Biol., August 1, 1997; 17(8): 1512 - 1516. [Abstract] [Full Text]
|
|
|
|
|
|
R. L. Panek, T. K. Dahring, B. J. Olszewski, and J. A. Keiser PDGF Receptor Protein Tyrosine Kinase Expression in the Balloon-Injured Rat Carotid Artery Arterioscler. Thromb. Vasc. Biol., July 1, 1997; 17(7): 1283 - 1288. [Abstract] [Full Text]
|
|
|
|
 |
|
 T. K. Dahring, G. H. Lu, J. M. Hamby, B. L. Batley, A. J. Kraker, and R. L. Panek J. Pharmacol. Exp. Ther., June 1, 1997; 281(3): 1446 - 1456. [Abstract]
|
|
|
|
 |
|
 E. Allaire and A. W. Clowes Endothelial Cell Injury in Cardiovascular Surgery: The Intimal Hyperplastic Response Ann. Thorac. Surg., February 1, 1997; 63(2): 582 - 591. [Abstract] [Full Text]
|
|
|
|
|
|
D. deBlois, M. Viswanathan, J.E. Su, A.W. Clowes, J.M. Saavedra, and S.M. Schwartz Smooth Muscle DNA Replication in Response to Angiotensin II Is Regulated Differently in the Neointima and Media at Different Times After Balloon Injury in the Rat Carotid Artery: Role of AT1 Receptor Expression Arterioscler. Thromb. Vasc. Biol., September 1, 1996; 16(9): 1130 - 1137. [Abstract] [Full Text]
|
|
|
|
|
|
J. M. Lemire, S. Potter-Perigo, K. L. Hall, T. N. Wight, and S. M. Schwartz Distinct Rat Aortic Smooth Muscle Cells Differ in Versican/PG-M Expression Arterioscler. Thromb. Vasc. Biol., June 1, 1996; 16(6): 821 - 829. [Abstract] [Full Text]
|
|
|
|
 |
|
 R. K. Dubey, D. G. Gillespie, Z. Mi, F. Suzuki, and E. K. Jackson Smooth Muscle Cell–Derived Adenosine Inhibits Cell Growth Hypertension, March 1, 1996; 27(3): 766 - 773. [Abstract] [Full Text]
|
|
|
|
|
|
A. B. Firulli, J. M. Miano, W. Bi, A. D. Johnson, W. Casscells, E. N. Olson, and J. J. Schwarz Myocyte Enhancer Binding Factor-2 Expression and Activity in Vascular Smooth Muscle Cells : Association With the Activated Phenotype Circ. Res., February 1, 1996; 78(2): 196 - 204. [Abstract] [Full Text]
|
|
|
|
|
|
S. M. Schwartz, D. deBlois, and E. R. M. O'Brien The Intima : Soil for Atherosclerosis and Restenosis Circ. Res., September 1, 1995; 77(3): 445 - 465. [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||