211
Smooth Muscle Cell Immediate-Early Gene and
Growth Factor Activation Follows
Vascular Injury
A Putative In Vivo Mechanism for Autocrine Growth
Joseph M. Miano, Niksa Vlasic, Robert R. Tota, and Michael B. Stemerman
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To understand the molecular events governing smooth muscle cell (SMC) proliferation in vivo,
immediate-early gene (IEG) expression was assessed and related to growth factor ligand and receptor
mRNA and SMC DNA synthesis after aortic injury. Balloon catheter injury evoked increases in SMC
c-myc and thrombospondin (tsp) within 2 hours. The induction of these IEGs was followed by elevated
transcripts to platelet-derived growth factor-A (PDGF-A), transforming growth factor-/31 (TGF-01) and
a basic fibroblast growth factor (bFGF) receptor. Whereas PDGF type-/? receptor mRNA was demonstrated in SMCs from control and balloon-injured aortas, no detectable signal was observed for the PDGF
type-a receptor. To explore the potential linkage between LEG products and growth factor mRNA
expression, cycloheximide was employed to block early protein synthesis after balloon injury. Induction of
PDGF-A and TGF-01 was attenuated by cycloheximide, but bFGF induction was unaffected. Moreover,
cycloheximide superinduced IEGs and revealed PDGF-B transcripts, which were otherwise undetected.
Seven days after aortic injury, a spontaneous increase in c-myc and tsp mRNA was noted. This IEG
reactivation was followed 12 hours later by a twofold increase in SMC DNA synthesis. These findings
corroborate an autocrine mode of SMC proliferation in vivo and suggest that IEG products may control
such growth by stimulating growth factor genes. (Arteriosclerosis and Thrombosis 1993;13:211-219)
KEY WORDS • immediate-early gene • growth factors • smooth muscle cell proliferation •
vascular injury • autocrine growth
S
mooth muscle cell (SMC) proliferation is a major
determinant of vascular stenosis,1 yet the molecular signals governing such growth in vivo remain
undefined. The mRNA expression of platelet-derived
growth factor A-chain (PDGF-A),2-3 transforming
growth factor-/^ (TGF-/31),4-5 insulin-like growth factor
I,6 and the PDGF type-)9 receptor37 in vascular tissue,
as well as the detection of mitogenic activity from
vessel-derived SMCs,89 indicates that SMC proliferation may be controlled in part by a local autocrine
loop.10 However, despite these advances, the nature of
the stimulus for autocrine growth factor expression in
SMCs is unclear. Moreover, although evidence for SMC
autocrine growth has been documented in vitro,8-9 the
existence of similar mechanisms in vivo remains
conjectural.
Immediate-early gene (IEG) activation is a fundamental response to growth-stimulatory agents.11 These
genes are defined by their rapid/transient expression
From the Departments of Experimental Pathology (J.M.M.,
N.V., M.B.S.) and Medicine (R.R.T.), New York Medical College,
Valhalla.
Supported by National Institutes of Health grant HL-33742,
Bethesda, Md.
Address for correspondence: Dr. Michael B. Stemerman, Department of Experimental Pathology, New York Medical College,
Valhalla, NY 10595.
Received February 21, 1992; revision accepted November 4,
1992.
and protein synthesis-independent mechanism(s) of
induction.12 IEGs encode numerous products, including
transcription factors,13 cytokines,14 structural and extracellular matrix (ECM) proteins,15-16 and enzymes.17
Such functional diversity likely affords requisite flexibility in a cell's response to growth signals. For example,
certain IEG products may participate directly in the
Stimulation of DNA synthesis,18 whereas others may
function in some capacity independent of direct growth
stimulation.12 IEG activation occurs in response to
many cellular events,19 and there is compelling evidence
supporting a role for immediate-early proteins in cellular growth control.20-23 Although the notion of IEG
products that stimulate growth factor genes has been
shown or speculated to occur in culture,24-25 whether
such a mechanism is operative in an in vivo setting is
unclear. Moreover, few studies have examined the expression of IEG in in vivo models of cell proliferation.26
Thus, an analysis of IEG expression in vivo is required
to validate findings from cell culture studies.
Recently, the expression of immediate-early protooncogenes was demonstrated following balloon deendothelialization (BDE) of the rat carotid artery and
aorta.3-27 In the present study, we have initiated studies
to analyze the in vivo relation between IEG and growth
factor mRNA expression and SMC DNA synthesis. The
results demonstrate sequential IEG and growth factor
mRNA induction before the initiation of SMC DNA
synthesis. Furthermore, they suggest a plausible link
212
Arteriosclerosis and Thrombosis
Vol 13, No 2 February 1993
between certain IEG products and subsequent growth
factor gene activation. Finally, a second peak in IEG
expression is demonstrated, which appears to be associated with a second burst in SMC DNA synthesis.
These results support a concept of in vivo SMC autocrine growth whereby IEG products participate in the
initiation of SMC DNA synthesis and maintain such cell
growth by stimulating growth factor genes.
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Methods
Reagents
Type I porcine elastase, soybean trypsin inhibitor,
lactate dehydrogenase (LDH), and phenylmethylsulfonyl fluoride were purchased from Sigma Chemical Co.,
St. Louis, Mo. Type I collagenase was obtained from
Worthington Biochemical Corp., Freehold, N.J. Medium 199 was purchased from GIBCO, Gaithersburg,
Md. The reagents for RNA isolation and Northern
blotting were molecular biology grade and were purchased from Bethesda Research Laboratories, Gaithersburg, Md. [Me//iy/-3H]Thymidine (20 Ci/mmol), cytidine 5'-[a- 32 P]triphosphate (3,000 Ci/mmol), and
[35S]methionine (1,100 Ci/mmol) were obtained from
New England Nuclear, Boston, Mass. Cycloheximide
(CHX) was purchased from Boehringer Mannheim,
Indianapolis, Ind.
Animals
Approximately 200 male Sprague-Dawley rats (350375 g) were used for these studies. Animals were
obtained from Charles River Laboratories, Wilmington,
Mass., and were used in accordance with National
Institutes of Health and institutional, approved animal
care regulations.
Balloon Deendothelialization
BDE was performed in anesthetized rats as described.28 A minimum of five animals were pooled per
time point, and at least two independent experiments
were performed for each study. The following time
points after BDE were studied: 2, 6, 12, and 24 hours
and 2, 7, and 14 days.
Cell Culture
Two cell types were used as a source of positive or
negative control RNA for the genes under study. Human umbilical vein endothelial cells (HUVECs) were
propagated as described.29 BALB/C-3T3 cells (clone
A-31) were purchased from American Type Culture
Collection and grown according to the manufacturer's
specifications.
RNA Isolation/Northern Blotting!Densitometry
Total RNA was isolated from cultured cells and
medial SMCs, which were separated from the aorta by
an enzymatic digestion method as described.27 Briefly,
pooled aortas (n=5) were rapidly excised from the
animal; rinsed in medium 199; and incubated for 25
minutes in medium 199 plus 1% collagenase, 0.25%
elastase, and 1% soybean trypsin inhibitor.27 After
incubation in the enzymes, the adventitia and endothelium were rapidly stripped away from the media, which
was immediately snap-frozen in liquid nitrogen and
homogenized in 4 M guanidinium isothiocyanate con-
taining 2% /3-mercaptoethanol. Homogenates of medial
SMCs were sheared three times through a 23-gauge
needle and clarified by spinning at 10,000g for 10
minutes. The supernatant was layered over 5.7 M CsCl
and the RNA pelleted by ultracentrifugation as
described.30
Cultured cell RNA was isolated in a similar manner.
Equivalent amounts of total RNA (15-20 ^g/lane) were
fractionated in a 1.2% agarose gel containing 0.66 M
formaldehyde and 0.2 M morpholinopropanesulfonic
acid (pH 7). After capillary blotting, RNA was crosslinked to a nylon membrane (Zeta Probe, Biorad,
Rockville Centre, N.Y.) by UV irradiation (Stratagene,
La Jolla, Calif.). cDNA probes were labeled to a specific
activity of 109 cpm//xg by a random hexamer protocol
(Boehringer Mannheim). Hybridizations and washes
were performed as described.27 Blots were exposed to
x-ray film (Kodak X-OMAT, Rochester, N.Y.) with
intensifying screens at -80°C. A Hoefer GS-300 densitometer was used to semiquantify the changes in steadystate transcript levels. Levels of IEG and growth factor
mRNA were normalized to 18S rRNA as previously
described.27 Where reported, results are expressed as
fold increases above normalized controls (non-BDE).
Cycloheximide Studies
Animals (n=5) were injected intraperitoneally with
7.5 mg/kg body wt CHX either immediately or 2 hours
after BDE. Control BDE animals received 0.9% saline
vehicle. Total SMC RNA was isolated 6 hours after
BDE as described above and analyzed for IEG and
growth factor mRNA expression. The toxicity of CHX
was determined by measuring serum LDH 6 hours after
the combined BDE/CHX treatment.
The effect of CHX on SMC de novo protein synthesis
was determined by measuring the incorporation of
[^SJmethionine into trichloroacetic acid (TCA)-precipitable SMC protein 6 hours after BDE. Briefly, animals
underwent BDE in the absence or presence of 7.5 mg/kg
body wt CHX as described above. After 5 hours, all
animals received an intravenous injection of [^SJmethionine (0.5 mCi/kg body wt) as described.31 The radioactive
methionine was allowed to circulate for 30 minutes, at
which time the animals were killed and SMC protein was
isolated. Medial SMCs were isolated by enzymatic dissociation and homogenized in a solution of phosphatebuffered saline containing 1 mM phenylmethylsulfonyl
fluoride and 2 mM EDTA. The homogenate was clarified
by spinning at ll,000g for 20 minutes and the pellet
solubilized with the aforementioned phosphate-buffered
saline solution containing 1% sodium dodecyl sulfate.
Samples were heated to 95°C and then spun at 14,000g
for 20 minutes. The supernatant was collected and stored
at -80°C. Samples of liver were collected and processed
similarly to further document the effect of CHX on
protein synthesis inhibition. Aliquots of stored samples
were precipitated with TCA, and the radioactivity was
measured in a scintillation counter. Protein concentration was determined by the^method of Lowry et al.32
Results are expressed as TCA-precipitable counts per
minute per microgram of total protein.
DNA Specific Activity
The measurement of SMC DNA synthesis was performed as described33 with minor modifications. One
Miano et al Vascular Injury-Induced EEG and GF Expression
213
FIGURE 1. Photomicrographs showing effect of enzyme digestion on aortic histology. Panel A: Undigested
rat thoracic aorta. Panel B: Rat thoracic aorta after
25-minute digestion in enzymes. Note complete removal
of the adventitia and endothelium. x320.
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hour before they were killed, the animals received an
intravenous injection of [mef/jy/-3H]thymidine (0.5
mCi/kg body wt). The animals were then killed and
vessels subjected to enzyme digestion to isolate medial
SMCs. At least five animals were pooled per time point
in two independent studies. The following time points
were examined: 0, 2, 7, 7.5, and 8 days. Results were
analyzed by one-way analysis of variance. Tukey's studentized range test was employed for post hoc multiple
comparisons.34 A statistical difference less than 0.05 was
considered significant.
cDNA Probes
The following cDNA probes were obtained from
individual investigators and prepared by established
methods35: a 0.8-kb EcoRl fragment of human basic
fibroblast growth factor (bFGF) in pBR322 (Judith
Abraham, CalBio Inc., Mountain View, Calif.); a 1.3-kb
EcoRl fragment of human PDGF-A in pUC13 (Christer
Betsholtz, Ludwig Institute for Cancer Research, Uppsala, Sweden); a 0.75-kb EcoRl-Acc I fragment of
human PDGF type-a receptor in pUC19 and a 0.75-kb
Pst I fragment of human PDGF type-/3 receptor in
pUC19 (Lena Claesson-Welsh, Ludwig Institute for
Cancer Research); a 1.0-kb EcoRl fragment of human
TGF-01 in pSP64 (Rik Derynck, University of California at San Francisco); a 4.5-kb Sal I fragment of human
thrombospondin in pUC18 (Vishva Dixit, University of
Michigan, Ann Arbor); a 1.4-kb EcoRI-Mndlll fragment of murine c-myc in pSP65 (William Lee, University of Pennsylvania, Philadelphia); an 18S rRNA fragment in pHRR118 (Ester Sabban, New York Medical
College, Valhalla); a 1.8-kb Sac I fragment of human
von Willebrand factor in pUC18 (J. Evan Sadler, Howard Hughes Medical Institute, Washington University
School of Medicine, St. Louis, Mo.); a 0.4-kb 5a/ 1-Ap<f
I fragment of chicken bFGF receptor in pBluescript
KS+ (Lewis T. Williams, Howard Hughes Medical
Institute, University of California at San Francisco);
and a 2.0-kb BamHl fragment of human PDGF-B in
pBR322 (Randy Zicht, National Cancer Institute, Bethesda, Md.). The c-myb, c-fms, glyceraldehyde phosphate dehydrogenase, and /3-actin probes were obtained
from American Type Culture Collection, Bethesda,
Md., and Oncor Probes, Gaithersburg, Md.
Results
SMC Growth Factor Ligand mRNA Expression After
Balloon Deendothelialization
Growth factor transcripts have been demonstrated in
rat aortic tissue after vascular manipulations.4-6 These
studies measured steady-state mRNA levels from a
mixed population of cells of the media and adventitia.
To ascertain whether growth factor ligand transcripts
are expressed specifically in aortic SMCs, we employed
a previously described enzymatic digestion procedure.27
Figure 1 documents the complete removal of the adventitia and endothelium from a normal aorta treated with
enzymes. Such treatment also resulted in partial digestion of the neointima from 7- and 14-day BDE vessels
(data not shown). The latter preparations of RNA,
therefore, represent both medial and neointimal SMCs.
The purity of our SMC preparations was further evaluated by probing the RNA pool with cell-specific markers. For example, von Willebrand factor mRNA could
easily be demonstrated in undigested aortas; however,
such expression was absent from enzyme-treated vessels
(data not shown).
Results in Figure 2 demonstrate BDE-induced expression of PDGF-A, bFGF, and TGF-/31 in aortic
SMCs over a 14-day period. Expression of PDGF-A was
barely detected in SMCs obtained from non-BDE aortas (time zero). However, 6 hours after BDE, the 2.3and 2.9-kb transcripts were elevated more than threefold. PDGF-A transcripts were seen to decrease after 6
hours but were discernible at 12 and 24 hours and 7 days
after BDE. In contrast to PDGF-A, steady-state expression of PDGF-B mRNA was not detected at any time
point after BDE, although a prominent signal was
obtained from HUVECs (Figure 2, lane C2).
A single 6.0-kb bFGF transcript was observed in
SMCs from non-BDE aortas (Figure 2). Levels of bFGF
mRNA increased approximately threefold 2 hours after
BDE and then gradually declined over the remaining
time period. The pattern of TGF-/31 mRNA expression
was distinct from that of PDGF-A and bFGF. This gene
was coinduced with PDGF-A and bFGF 6 hours after
BDE; however, steady-state levels of TGF-/31 mRNA
remained elevated above non-BDE controls at every
time point studied subsequently (Figure 2).
The induction of growth factor ligand transcripts in
SMCs after BDE was not associated with a generalized
214
Arteriosclerosis and Thrombosis
Vol 13, No 2 February 1993
0 2h 6h 12h 24h 2d 7d 14d C1 C2
{PDGF-A
PDGFAR
JPDGF-B
PDGF- BR
bFGF
TGF-H1
18S
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FIGURE 2. Growth factor ligand mRNA expression after
balloon deendothelialization (BDE), as shown by autoradiographs from three independent studies showing smooth muscle cell (SMC) growth factor ligand mRNA expression after
BDE. At the times indicated, total SMC RNA was isolated
and processed for Northern blotting. Note the BDE-induced
increase in steady-state transcripts to platelet-derived growth
factor-A (PDGF-A, 2.3 and 2.9 kb), basic fibroblast growth
factor (bFGF, 6.0 kb), and transforming growth factor-Bl
(TGF-B1, 2.5 kb). The 1.7-kb transcript to PDGF-A is not
shown because it was not easily distinguished from nonspecific
binding of the probe to 18S rRNA. Hybridization to an 18S
rRNA probe verified equal RNA loading. Lanes Cl and C2
correspond to control RNA from quiescent BALB/c-3T3 cells
and human umbilical vein endothelial cells, respectively. Lane
C2 in the PDGF-A panel represents RNA from serumstimulated BALB/C-3T3 cells, h, Hours; d, days.
increase in RNA, since levels of 18S rRNA (Figure 2) as
well as £-actin, c-Ha-ras, c-myb, glyceraldehyde phosphate
dehydrogenase, and c-fms mRNA (data not shown)
showed little or no change in expression between control
and BDE aortic SMCs subjected to BDE. Finally, the
effect of enzymes on growth factor ligand mRNA was
assessed by measuring steady-state transcript levels in
whole aortic tissue (i.e., vessels not subjected to enzyme
digestion). The induction of growth factor ligand transcripts in such tissue was comparable to that observed in
SMCs isolated by the enzyme cocktail (data not shown).
Thus, transcripts to at least three growth factors are
elevated specifically in aortic SMCs after BDE.
SMC Growth Factor Receptor mRNA Expression
After Balloon Deendothelialization
To determine whether growth factor receptor transcripts are coinduced with their ligands, steady-state
mRNA levels of PDGF type-a and -8 receptors and a
bFGF receptor were measured over the same 14-day
period. No signal corresponding to the PDGF type-a
receptor transcript was detected in SMCs obtained from
non-BDE aortas or at any time point after BDE (Figure
3). On the other hand, BALB/c-3T3 cells showed a
notable 6.5-kb transcript corresponding to the PDGF
bFGFR
FIGURE 3. Growth factor receptor mRNA expression after
balloon deendothelialization (BDE), as demonstrated by
steady-state mRNA transcripts of type-a and -B plateletderived growth factor (PDGF) receptors (designated
PDGF-AR and PDGF-BR, respecHvely) and a basic fibroblast
growth factor receptor (bFGFR) after BDE. No detectable
transcripts to the type-a PDGF receptor were observed in two
independent experiments, although the 6.5-kb transcript is
clearty present in BALB/c-3T3 cells. In contrast, both type-B
PDGF (5.7 kb) and a bFGF receptor mRNA (4.7 kb) were
readily observed in aortic smooth muscle cells after BDE. The
increase in type-B PDGF receptor expression at 7 days was
not a consistent finding. The increase in bFGF receptor
mRNA was approximately 10-fold, as determined by densitometry. Note the absence of PDGF type-a and -B receptors
in human umbilical vein endothelial cells, h, Hours; d, days.
type-a receptor (Figure 3). Since BALB/c-3T3 cells are
derived from a mouse and rats are close to mice on the
evolutionary ladder, it is unlikely that the absence of the
PDGF type-a receptor mRNA in rat aortic SMCs is due
to the heterologous probe employed. Moreover, the
PDGF type-a receptor probe hybridizes to the highly
conserved tyrosine kinase domain.36 The possibility
remains, however, that PDGF type-a receptor transcripts are present in aortic SMCs but are below the
level of sensitivity of Northern blotting.
In contrast to the PDGF type-a receptor, transcripts to
the PDGF type-/3 receptor were observed in SMCs from
both control and BDE vessels (Figure 3). Indeed, the
5.7-kb PDGF type-/? receptor transcript was detected at
every time point after BDE, indicating little or no modulation of this receptor mRNA. A consistent increase in
expression of a bFGF receptor transcript37 was noted
between 2 and 12 hours after BDE (Figure 3). Levels of
this bFGF receptor mRNA were detectable over the
remaining 14-day period with no further modulation.
Thus, at least two growth factor receptor transcripts are
expressed in aortic SMCs during arterial repair.
Effect of Protein Synthesis Inhibition on Balloon
Deendothelialization-Induced SMC Growth Factor
Ligand mRNA Expression
Previously, we demonstrated the rapid induction of
several IEGs in SMCs after BDE.27 To examine the
possibility that IEG products may regulate growth factor gene expression in vivo, CHX was used to strategi-
Miano et al Vascular Injury-Induced IEG and GF Expression
1 2
3
215
0 2h 6h 12h 24h 2d 7d 14d
4
PDGF-A
c-myc
TGF-P1
TSP
bFGF
18S
PDGFB
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18S
FIGURE 4. Effect of cycloheximide (CHX) on growth factor
ligand mRNA induction. This figure shows attenuation of
platelet-derived growth factor-A (PDGF-A) and transforming growth factor-Bl (TGF-B1) transcripts after combined
balloon deendothelialization (BDE)ICHX treatment. Animals underwent BDE and received either saline vehicle (lane
1), CHX (7.5 mglkg body wt, lane 2), or CHX 2 hours after
BDE (lane 3). All animals were killed 6 hours after BDE.
Note partial induction of PDGF-A and TGF-B1 mRNA levels
with delayed CHX delivery (lane 3). No changes in basic
fibroblast growth factor message levels were detected when
CHX was injected immediately or 2 hours after BDE. Levels
ofPDGF-B mRNA were detected only after CHX treatment.
Lane 4 represents equally loaded RNA from human umbilical
vein endothelial cells and serves as a positive control for
growth factor ligand mRNA expression. Results were confirmed in one additional experiment.
cally arrest protein synthesis, particularly immediateearly transcription factors. The toxicity of CHX was
evaluated by measuring serum LDH levels. The levels of
LDH in vehicle-treated control rats was 505 ±107
units/L. At 25 mg/kg body wt, CHX caused a profound
increase in serum LDH levels (3,050±501 units/L), and
the animals became visibly sick. However, at a dose of
7.5 mg/kg body wt, CHX elicited only modest increases
in serum LDH (l,016±222 units/L) with no evidence of
overt toxicity. In addition, such levels of CHX resulted
in an 80% inhibition of SMC and liver protein synthesis
(data not shown). Thus, all studies concerning CHX
were carried out at a dose of 7.5 mg/kg body wt.
Figure 4 shows steady-state expression of PDGF-A,
TGF-01, bFGF, and PDGF-B mRNA in aortic SMCs 6
hours after BDE in the presence or absence of CHX.
When CHX was administered immediately after BDE,
induction of PDGF-A and TGF-/91 was reduced by 50%
(Figure 4, lane 2). However, if CHX treatment was
delayed for 2 hours and SMC RNA was isolated 4 hours
later, PDGF-A and TGF-01 mRNA induction was
partially restored (Figure 4, lane 3), indicating that new
proteins that were synthesized during the initial 2 hours
FIGURE 5. Expression of c-myc and thrombospondin (tsp)
mRNA after balloon deendothelialization (BDE). This figure
demonstrates steady-state expression of smooth muscle cell
immediate-early gene over a 14-day period after BDE. Note
the biphasic increase in c-myc and tsp with peak early
expression occurring at 6 hours and later reactivation at 7
days. Message levels for each immediate-early gene were
depressed significantly after 14 days. These results were
confirmed in two additional experiments, h, Hours; d, days.
after BDE were necessary for full growth factor
expression.
In contrast to PDGF-A and TGF-/31, CHX treatment
had no effect on steady-state levels of bFGF transcripts.
Interestingly, CHX treatment either immediately or 2
hours after BDE revealed PDGF-B mRNA, which was
otherwise undetected by Northern blotting (compare
PDGF-B blots in Figures 2 and 4). Finally, several IEGs
were superinduced in aortic SMCs after the combined
BDE/CHX treatment (data not shown), further substantiating the specificity of CHX's effect on aortic SMC
gene expression.
Phasic Expression of Immediate-Early Genes in
SMCs After Balloon Deendothelialization
Growth factor stimulation represents a major route
for IEG induction.11 To determine whether SMC IEG
induction recurs after growth factor mRNA expression,
which would be consistent with SMC autocrine growth
stimulation, we measured steady-state transcript levels
of c-myc and (thrombospondin) tsp in aortic SMCs over
a 14-day time course after BDE. The results demonstrate a phasic pattern of IEG expression after a single
injury at time zero (Figure 5). Levels of SMC c-myc and
tsp mRNA showed early (2-6 hours) induction, with
levels returning toward baseline at 24 hours. After a
general diminution in mRNA expression, both IEG
transcripts showed a consistent increase at 7 days. By 14
days, steady-state IEG mRNA levels had returned
toward baseline (Figure 5). The increase in IEG expression observed at 7 days is unlikely to be due to regenerating endothelial cells or monocytes; Northern analysis showed few, if any, transcripts to von Willebrand
factor, c-fms, or c-myb (data not shown). Thus, two
IEGs, whose expression is classically induced by growth
factors such as PDGF,16'38-19 show reactivation at a time
coinciding with the most rapid phase of intimal
hyperplasia.33
216
Arteriosclerosis and Thrombosis
Vol 13, No 2 February 1993
FIGURE 6. Bar graph of smooth muscle cell (SMC)
DNA specific activity (SA) after balloon deendothelialization (BDE), showing spontaneous reactivation of
SMC DNA SA 7.5 days after BDE. This increase was
significantly different *p<0.05 from DNA specific activity measured at 7 and 8 days. Results are expressed as the
mean±SEM SA, which is defined as the trichloroacetic
acid-precipitable counts per minute per microgram of
DNA. Graph represents pooled data from two independent experiments.
Control
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Spontaneous SMC DNA Synthesis
To determine whether the reactivation of IEG at 7
days predicts SMC growth, SMC DNA specific activity
was evaluated at 0, 2, 7, 7.5, and 8 days after BDE. A
large increase in DNA specific activity was observed 2
days after BDE (Figure 6). Seven days after vascular
injury, DNA specific activity declined but remained
above control levels. A twofold increase in DNA specific
activity was observed 7.5 days after BDE (p<0.05).
This spontaneous increase in SMC tritiated-thymidine
incorporation was transient; DNA specific activity at 8
days was equivalent to 7-day measurements. Thus, at
least two waves of IEG expression precede elevated
levels of SMC thymidine incorporation.
Discussion
Autocrine growth stimulation is postulated to play an
important role in the proliferation of SMCs.40 However,
in vivo proof supporting this thesis is incomplete. In this
report we show that PDGF-A, bFGF, and TGF-/31
mRNA are induced specifically in aortic SMCs after
BDE. The elevation in two PDGF-A transcripts 6 hours
after injury is similar to that reported previously in the
rat carotid artery.3'41 Since we and others42 have been
unable to demonstrate PDGF type-a receptor transcripts in aortic SMCs, the function of PDGF-A during
arterial remodeling remains unclear. A recent report,
however, indicates that PDGF-AB binds to cells in the
absence of detectable type-a receptors.43 Although
PDGF-B mRNA is normally undetected in aortic
SMCs, CHX treatment reveals readily detected
PDGF-B transcripts. This in vivo finding is consistent
with cell culture studies that show superinduction of
PDGF-B after CHX treatment.44 Whether the presence
of PDGF-B mRNA after CHX administration reflects
stabilization of the message or the inhibition of a labile
repressor molecule is not known. Nevertheless, if
PDGF-B mRNA is present at low but rapidly turning
over levels, then it is conceivable that PDGF-AB heterodimers may form and bind the PDGF type-B receptor, whose mRNA is detected at every time point
studied. Such molecules may participate in stimulating
SMC proliferation and migration during arterial repair.
Levels of bFGF mRNA are elevated in SMCs early
after BDE and are coinduced with a receptor mRNA37
whose translated product recognizes this growth factor
ligand. These findings suggest that SMC-derived bFGF
may stimulate SMC growth in an autocrine manner. In
support of this concept are the findings of Reidy and
colleagues, who showed accelerated intimal thickening
after administration of bFGF 45 and a significant reduction in SMC thymidine index with antibodies to this
growth factor.46 Although the extrusion of stored bFGF
from SMCs after BDE may account for the subsequent
growth of these cells, it is equally plausible that newly
synthesized bFGF stimulates autocrine growth, perhaps
through an intracrine loop.47
Whereas BDE-induced mRNA levels of PDGF-A
and bFGF are transient, the expression of TGF-/31
mRNA remains elevated over the time course studied.
This finding is consistent with studies in the injured rat
carotid artery5 and suggests that this growth factor may
be functional over the entire 14-day period. In addition
to being a bifunctional regulator of SMC growth,48
TGF-/31 acts as a chemoattractant and stimulates ECM
protein production.49 Indeed, the production of ECM in
the growing intimal mass may result from autocrine
stimulation of SMCs by TGF-/31. Supporting the latter
concept is the recent demonstration of sequential
TGF-^1 and ECM gene expression in the carotid artery
after BDE.5
Importantly, mRNA induction of PDGF-A, bFGF,
and TGF-01 in aortic SMCs coincides with the immunodetection of several immediate-early transcription
factors.50 To examine the possibility that such growth
factor activation is controlled by IEG products, CHX
was administered immediately after BDE, and growth
factor mRNA levels were measured 6 hours later.
Consistent with cell culture studies,25 CHX blocked
BDE-induced SMC PDGF-A gene activation and superinduced IEGs such as c-myc. Protein synthesis inhibition also attenuated SMC TGF-/31 mRNA levels 6
hours after BDE. Levels of these growth factor mRNAs
can be partially restored by postponing CHX administration for 2 hours. Taken together, the results indicate
that PDGF-A and TGF-/31 are delayed-early genes
whose expression, unlike that of IEG, is dependent on
early protein synthesis.51 Such newly synthesized proteins likely include immediate-early transcription factors that may bind to growth factor genes and stimulate
their transcription. This notion is further supported by
the complete inhibition of immunoreactive IEG products after CHX treatment.50
There is precedence for the notion of IEG products'
stimulating growth factor gene induction because regulatory sequences to TGF-/31 and PDGF-A contain
canonical cis elements corresponding to AP-1 and
Egr-1, respectively.24-52 Indeed, the TGF-/31 gene ap-
Miano et al Vascular Injury-Induced IEG and GF Expression
TSP
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pears to be governed by Fos and Jun proteins, at least in
transient cotransfection assays.24 Taken together with
the results reported here, the existing data are consistent with the proposition that IEG products can influence SMC proliferation by directly activating autocrine
growth factors (see Figure 7). In this way, a short-term
signal (IEG activation) can result in a long-term response (autocrine growth factor activity), as has been
suggested for neuronal signaling.53
Unexpectedly, CHX had no effect on steady-state
bFGF mRNA expression. These in vivo results have
recently been demonstrated in vascular SMCs in vitro54
and raise the possibility that bFGF has unique control
mechanisms that regulate its transcription. Perhaps
bFGF gene expression, like that of IEG, requires only
posttranslational modifications of preexisting protein(s).55 Since bFGF is considered to be a primary
growth factor for SMC proliferation in vivo,46 knowledge pertaining to its transcriptional regulation may be
vital to a complete understanding of the molecular
mechanisms underlying SMC growth.
We have documented a biphasic expression pattern of
two functionally distinct IEGs. Expression of c-myc has
been reported previously27 and is now shown to be
reactivated 7 days after BDE. Similarly, results demonstrating tsp mRNA induction support the work of others56 and imply that the tsp signal detected at 7 days may
be the consequence of the same stimulus that evokes
c-myc reactivation. Such a stimulus may be the generation of locally derived growth factors. This notion is
consistent with the second peak in DNA specific activity
observed 12 hours after IEG reactivation (see below).
Monocytes and endothelial cells elaborate paracrine
growth factors such as PDGF, 5758 which may account
for the elevated IEG transcripts at 7 days and the
217
FIGURE 7. Schematic representing a hypothetical
model for immediate-early gene
(IEG)-mediated
smooth muscle cell (SMC) autocrine growth. Dotted
lines indicate pathways for which there is little or no
experimental evidence. One route of SMC growth
stimulation may be activation of membrane receptors*
(1) and transmission/amplification of growth signals to
the nucleus. Such signals activate rapid expression of
IEG (2) and may stimulate growth factor (GF) genes
such as basic fibroblast growth factor (2a), whose
expression appears to be independent ofde novo protein
synthesis. IEG products (3) diverge into at least two
pathways. Secretory IEG products include thrombospondin (TSP) (4); TSP may communicate extracellular matrix signals through the plasma membrane (5).
An alternative route for immediate-early protein transport is the nucleus, where such transcription factors (6)
directly or indirectly participate in DNA replication (7)
and transregulate GF genes such as platelet-derived
growth factor-A and transforming growth factor-pl
(8). Protein products of GF (9) stimulate SMC growth
through internal (10) or external (11) autocrine mechanisms. Paracrine growth stimulation is also possible
(lla). SMC autocrine growth stimulation may proceed
through (2) or (2a) and may account for the reactivation of IEG observed at 7 days. * This figure is not
meant to exclude other mechanisms of IEG induction.
subsequent rise in DNA specific activity. Such an effect
seems unlikely for several reasons. First, there are
virtually no monocytes present in the aortic wall after
BDE.59 The lack of monocytes in the vessel wall is
substantiated by the absence of c-fms expression, which
has been used as a marker for macrophages.60 Second,
endothelial cell regeneration is concentrated at the
dorsal surface of the rat aorta.61 In contrast, intimal
thickening is greatest at the ventral surface 7 days after
BDE.61 Thus, it is difficult to envisage an endothelial
cell-mediated paracrine effect on SMC hyperplasia,
although it is possible that endothelial cells release
mitogens that may act in an endocrine manner by
stimulating SMC growth distally.
The reinduction of SMC c-myc and tsp mRNA at 7
days is most consistent with autocrine growth factor
stimulation; these IEGs are induced by growth factors,
and their reactivation occurs subsequent to SMC growth
factor ligand and receptor mRNA expression. It must be
emphasized, however, that it is unclear at this time
whether IEG transcripts at 7 days accumulate in proliferating SMCs of the neointima. Nor is it known whether
IEG transcripts colocalize with growth factor ligand/
receptor proteins. Studies combining in situ hybridization with immunocytochemistry and tritiated-thymidine
uptake will clarify these points.
Previous work has established a second wave in SMC
DNA specific activity after reinjury to the rabbit abdominal aorta.62 In the present report, a similar increase
in SMC DNA synthetic activity occurs 7.5 days after
BDE- This increase arises spontaneously without further experimental manipulation beyond the initial BDE
event. We hypothesize that this increase in SMC DNA
specific activity represents a cyclic phenomenon of SMC
growth during arterial repair. Such growth likely pro-
218
Arteriosclerosis-and Thrombosis
Vol 13, No 2 February 1993
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ceeds through autocrine mechanisms, since the induction of IEG, whose expression is classically induced by
growth factors, ensues before heightened SMC DNA
specific activity. Future studies will examine additional
time points to further assess the relation between IEG
expression and DNA specific activity. In addition,
blocking studies will be performed to ascertain whether
IEG reactivation at 7 days is necessary for the subsequent rise in SMC DNA specific activity.
In summary, we have shown the mRNA induction of
functionally disparate IEGs and growth factor ligands
and receptors in aortic SMCs after BDE. Protein synthesis inhibition studies reveal a plausible link between
immediate-early transcription factors and subsequent
growth factor gene activation. The surge in IEG expression at 7 days followed by increased SMC DNA specific
activity is suggestive of an autocrine mode of SMC
growth (Figure 7). Accordingly, control of SMC autocrine growth factor expression may have consequences
for the progression of vascular stenosis associated with
atherosclerosis and restenosis after angioplasty. Our
study implies that such control may be achieved by
interrupting immediate-early transcription factor expression. Studies in this laboratory have been initiated
to address this potential mechanism of growth factor
gene activation.
Acknowledgments
We are grateful to the following individuals for providing us
with their cDNA clones: Judith Abraham, Christer Betsholtz,
Lena Claesson-Welsh, Rik Derynck, Vishva Dixit, William
Lee, Ester Sabban, J. Evan Sadler, L.T. Williams, and Randy
Zicht. We also acknowledge Umesh Bhatia, Jane Lin, and Eric
Olson for reading this manuscript and Dr. Pal Czobor, Nathan
Kline Institute, Orangeburg, N J., for performing the statistics
on DNA specific activity. Finally, the technical assistance of
Patrick) Villalon is much appreciated.
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Smooth muscle cell immediate-early gene and growth factor activation follows vascular
injury. A putative in vivo mechanism for autocrine growth.
J M Miano, N Vlasic, R R Tota and M B Stemerman
Arterioscler Thromb Vasc Biol. 1993;13:211-219
doi: 10.1161/01.ATV.13.2.211
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