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1
activation and smooth muscle growth morphology
1 Section of Vascular Surgery and 2 Section of Surgical Gastroenterology, Yale University School of Medicine, New Haven, Connecticut 06520
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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We examined whether endothelial cells (ECs)
inhibit smooth muscle cell (SMC) transforming growth factor-
1
(TGF-1) activation in bilayer coculture. Western analysis showed
that SMCs cocultured with ECs as a bilayer had lower amounts of active
TGF-1 protein compared with SMCs cultured alone and SMCs cocultured
with ECs as a monolayer. EC inhibition of TGF-1 activation could be
blocked with plasminogen activator inhibitor-1 (PAI-1) antibody.
Similarly, SMC hill-and-valley growth, a marker for TGF-1 activity,
was present in SMCs cultured alone and SMCs cocultured with ECs as a
monolayer but was absent in SMCs cocultured as a bilayer. SMCs cocultured with ECs as a bilayer migrated at a greater rate than SMCs
cultured either alone or cocultured as a monolayer. The EC effect on
SMC migration was inhibited by the addition of 5 ng/ml TGF-1. ECs
had no effect on SMC RNA levels of TGF-1. PAI-1 levels were
increased in ECs and ECs cocultured with SMCs compared with SMCs
cultured alone. ECs inhibit TGF-1 activation in bilayer coculture.
This appears to be mediated through an increase in EC PAI-1 release.
Alterations in coculture conditions, in particular the degree of EC-SMC
cell contact, have profound effects on this process.
transforming growth factor; endothelial cells; smooth muscle cells; plasminogen activator inhibitor; migration
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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AFTER VASCULAR WALL injury smooth muscle cells (SMCs) dedifferentiate from a contractile phenotype characterized by a spindle-shaped morphology and a relative decrease in synthetic organelles to a synthetic phenotype that is found in developing vascular tissue and is characterized by increased synthetic organelles and a more hypertrophic appearance. After SMC dedifferentiation, cells migrate into the subintimal space, proliferate, and secrete extracellular matrix. These are central mechanisms in the development of intimal hyperplasia (7, 29). Proliferative vascular lesions are the major cause for the 50% incidence of early restenosis after coronary angioplasty as well as the majority of early failures after vascular surgical procedures (4).
Numerous growth factors have been identified that have varying degrees
of importance in the development of restenosis. Type I transforming
growth factor-1 (TGF-1), for example, has multiple effects on
SMCs, some of which vary depending on cell seeding density and culture
conditions. In general, TGF-1 is thought to be a potent stimulant of
extracellular matrix accumulation as a result of increased matrix
synthesis and decreased matrix degradation. This growth factor has been
identified both in animal models of restenosis as well as in human
intimal hyperplastic lesions. TGF-1 is secreted by SMCs and
platelets in a latent form and requires plasmin for activation (26).
Our laboratory has previously shown that endothelial cells (ECs) can
influence SMC phenotype expression, proliferation, migration, and
matrix synthesis in bilayer coculture (5, 21-25). We have previously shown that ECs maintain cultured vascular SMCs in a more
contractile phenotype compared with SMCs cultured alone (23, 24). ECs
also inhibit SMC type I collagen synthesis and stimulate SMC migration
and proliferation when cocultured as a bilayer (25). In addition, ECs
can prevent SMC hill-and-valley growth in culture. Majack (15) has
previously reported that SMC hill-and-valley growth in culture is due
to active TGF-1 (15). We have confirmed these findings and have
shown that TGF-1 antibody, the plasmin inhibitor aprotinin, and ECs
all block SMC hill-and-valley growth in culture (22). At least several
of the EC effects on SMC function in bilayer coculture, such as the EC
inhibition of SMC hill-and-valley growth, appear to be a result of EC
inhibition of TGF-1 activation.
ECs stimulate SMC migration by an as yet undetermined mechanism (21).
Numerous investigators have previously shown that TGF-1 inhibits SMC
migration in vitro (8, 17, 19). Koyama and co-workers (10) have shown
that TGF-1 inhibits platelet-derived growth
factor-induced migration in SMCs (10). EC inhibition of
TGF-1 may account for the differences in SMC migration in the
presence and absence of ECs we have previously reported.
The mechanism by which ECs may inhibit TGF-1 activation in bilayer
coculture is unclear. ECs have been shown to secrete plasminogen activator inhibitor 1 (PAI-1) in response to TGF-1 and as a result could prevent plasmin-mediated activation of this growth factor. Sato
and Rifkin (26) have reported that PAI-1 inhibition in monolayer
coculture prolongs the period in which active TGF-1 is detectable in
conditioned media. On the other hand, SMCs have been shown to increase
their secretion of plasminogen activators in response to TGF-1,
which could result in increased TGF-1 activity (14).
Coculture conditions also appear to have a profound effect on EC
regulation of SMC function and TGF-1 activation. Previous investigators have shown that when ECs are cocultured as a monolayer, with SMCs or pericytes, TGF-1 activation is increased. The mechanism for the activation of latent TGF-1 in monolayer
coculture appears to occur through enhanced plasmin-mediated cleavage
of the latency-associated peptide (LAP) from the larger inactive latent
TGF-1 complex (19). The large latent TGF-1 complex has been shown
to specifically bind only to SMCs in monolayer coculture with ECs and
activation of TGF-1 is enhanced as a result of the close
approximation of EC-bound plasminogen (5a).
In the present study, we have examined the effect of different
coculture conditions on the ability of ECs to inhibit or stimulate TGF-1 activation, SMC macroscopic growth features, and migration. We
also sought to determine if PAI-1 mediated this response. The hypothesis tested in this investigation was that coculture conditions determine the EC's ability to inhibit TGF-1 activation and control SMC macroscopic growth features.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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EC-SMC coculture.
Bovine aortic ECs and SMCs were harvested using the collagenase method
for ECs and the explant method for SMCs. Cells were grown 3-5
passages from primary cultures in Dulbecco's modified Eagle's medium
(DMEM)-10% calf serum (CS). ECs were identified using a monoclonal
mouse antibody to human von Willebrand factor (Dako-VWF, F8/86, Dako,
Carpinteria, CA), and SMCs were identified using an anti-
-actin
antibody (Sigma A-2547, Sigma Chemical, St. Louis, MO). SMC cultures
were established by plating cells on a 13-µm-thick polyethylene
terephthalate (PET) membrane at a subconfluent density of 5 × 104 cells/25
mm2 area circular well
(n = 6 wells/group). Each membrane
contains 1.6 × 106
pores/cm2; each pore is 0.45 µm
in diameter (Cyclopore membrane, Falcon cell culture insert, Becton
Dickinson, Bedford, MA). EC-SMC bilayer cocultures were established by
plating ECs (1 × 105
cells/well) on the outer side of the membrane and were grown to
confluence over 3-5 days. Once the ECs were confluent (as
determined by phase-contrast microscopy), SMCs were plated on the
opposite side of the PET membrane. EC-SMC monolayer cocultures were
established by plating SMCs on the PET membrane. Two hours later ECs (1 × 105 cells/well) were
plated directly on to the SMC cultures.
Western analysis for TGF-1 in SMC lysates.
Whole cell lysates were prepared by scraping SMCs with a rubber
policeman into 200 µl of suspension buffer in the presence of the
proteinase inhibitors aprotinin (4 µg/ml), leupeptin (4 µg/ml),
phenylmethylsulfonyl fluoride (100 µg/ml), and pepstatin (4 µg/ml).
The cell suspension was passed through a 25-g needle, centrifuged, and
pelleted. This step was repeated five times. Protein was measured using
a bicinchoninic acid spectrophotometric assay (Pierce,
Rockford, IL). Stacking gels (4% acrylamide) and resolving gels (8%
acrylamide) were loaded with 20 µg protein/lane and run at 20-mA
through the stacking gel and 40 mA through the resolving gel for 37 min. Proteins were transferred to nitrocellulose. Membranes were
primary stained with a monoclonal antibody to TGF-1 (R&D Systems,
Minneapolis, MN). This antibody is specific for the active form of
TGF-1 and does not cross react with latent TGF-1 or other
isoforms of TGF-. Filters containing the proteins were then
secondary stained and exposed against Kodak XAR film using the enhanced
chemiluminescence technique. Densitometry was performed in
the linear portion of development using a Visage 2000 densitometer.
Assays for TGF-1 in SMC conditioned media.
Conditioned media from SMCs cultured alone and SMCs cocultured as a
monolayer and bilayer were collected and centrifuged to remove cell
debris. Active and total TGF-1 levels were measured in full-strength
conditioned media using an enzyme-linked immunosorbent assay (ELISA;
Quantikine, R&D Systems) as described by the manufacturer after 24, 48, and 72 h in culture. Total TGF-1 was quantitated by activating
conditioned media with 1 N HCl to a pH < 2.0 for 20 min before assay.
Results were compared with a standard curve of known TGF-1
concentrations.
1 was also measured in the SMC conditioned media using a mink
lung epithelial cell (MV 1 Lu, American Type Culture Collection, Rockville, MD) bioassay as described by Tucker et al. (27). Briefly, MV
1 Lu cells were cultured in minimal essential medium supplemented with
5% fetal bovine serum. Cells were harvested for passage at
subconfluence with trypsin-EDTA. MV 1 Lu cells were plated in 24-well
plates at 1 × 104
cells/cm2 in minimal essential
medium with 0.5% fetal bovine serum. Twenty-four hours later
conditioned medium was added in a 1:1 dilution. Twenty hours later the
MV 1 Lu cells were pulsed for 4 h with 2 µCi/ml tritiated thymidine.
MV 1 Lu cells were harvested by dissolution with 1.5 ml of 0.3 N KOH.
SMC DNA was then precipitated with 0.5 ml of 0.9 N HCl-25%
trichloroacetic acid solution at 4°C over 24 h. The DNA precipitate
was pelleted and resuspended in 0.5 ml 0.1 N KOH, and MV 1 Lu cell
proliferation was measured as uptake of tritiated thymidine as measured
in a scintillation counter. Results were compared with a standard curve
of known TGF-1 concentrations. A neutralizing antibody to TGF-1
was used to block the growth-inhibitory effects of TGF-1 on a
control group of MV 1 Lu cells.
Light microscopy.
Previous reports have shown that hill-and-valley growth that occurs in
SMCs cultured alone is caused by TGF-1 (15, 22). We have thus used
the presence or absence of hill-and-valley growth as a qualitative
measure of TGF-1 activity. After 3 days, cocultured SMCs and SMCs
cultured alone were washed with phosphate-buffered saline (PBS), fixed
in 10% Formalin, and stained with toluidine blue. SMCs were examined
by light microscopy for hill-and-valley growth.
Sheet migration assay.
SMC migration was measured by release from contact inhibition using a
steel fence as described by Basson et al. (1, 2, 14). SMCs were seeded
(5 × 104 cells/well) in the
center portion of the coculture membrane by utilizing a stainless steel
fence (see Fig. 1). In the case of the
monolayer cocultures, 1 × 104 ECs were added directly onto
SMC cultures. After 6 h the fences were removed and the SMCs were
treated for 2 h with 20 µg/ml of mitomycin C (an inhibitor of SMC
proliferation but not migration). This dose of mitomycin C was chosen
because compared with control cells it continued to suppress SMC
tritiated thymidine uptake at 6 days without causing cell death. After
4 h media were removed and cells were washed with PBS and placed in
DMEM-2.5% CS for the remainder of each experiment. SMCs were either
cultured alone or cocultured with ECs as a bilayer or monolayer. SMCs
in each group were treated with either 25 µg/ml neutralizing antibody to TGF-1, 100 µg/ml of aprotinin (a plasmin inhibitor that
prevents the plasmin-mediated activation of TGF-1), or 5 ng/ml
TGF-1 at 1 and 3 days. The TGF-1 antibody does not cross react
with latent TGF-1 or other TGF- isotypes (R&D Systems). TGF-1
inhibition was confirmed by the absence of hill-and-valley growth in
SMCs. Cells were refed every 3 days and after 6 days in culture cells were washed with PBS, fixed in 10% Formalin, and stained with toluidine blue. Cell migration was measured by scanning the inserts on
a computer scanner (Sigma Scan; Jandel Scientific, Montgomeryville, PA,
and Macintosh Power PC, Apple, Cupertino, CA). The radial migration
rate was measured in square millimeters over 6 days. To account for
differences in seeding density between experiments, we normalized data
to the control group (SMCs cultured alone), pooled, and expressed as
percentage of control.
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Northern analysis.
Total RNA was extracted by a modification of the method of Chomczynski
and Sacchi (3) and measured by spectrophotometry. Fifteen micrograms of
total RNA per lane were fractionated on a 1% formaldehyde agarose gel
and transferred to a Genescreen nylon membrane (NEN Research Products,
Boston, MA). cDNA probes were labeled with
[32P]dCTP by random
primer labeling method. Labeled probes were purified by Sephadex G-50
spin columns. A human 1.6-kilobase cDNA probe for TGF-1 was
graciously supplied by Dr. Rik Derynck (University of California, San
Francisco, CA). Hybridization was performed at 65°C for 20 h in
hybridization buffer consisting of 1 M NaCl, 50 mM
tris(hydroxymethyl)aminomethane · HCl, 1% sodium
dodecyl sulfate (SDS), 10% dextran sulfate, and 100 µg/ml salmon
sperm. The filters were then washed in 2× SSPE (3.6 M NaCl, 0.2 M
NaH2PO4, 0.02 M EDTA, pH 7.7),
0.1% SDS at 65°C for 30 min and exposed to Kodak XAR film. For
assessment of lane loading, membranes were stripped and reprobed with
an 18S ribosomal cDNA probe. Densitometry was performed as previously
described.
Assay for PAI-1 in SMC conditioned media. Measurements of PAI-1 in full-strength conditioned media prepared as described above were performed using the Immubind ELISA kit (American Diagnostica, Greenwich, CT) in accordance with the manufacturer's recommendations.
Data and statistical analysis. All experiments were performed in triplicate, data were pooled, and results are expressed as means ± SE. Significant differences between groups were tested by analysis of variance and post hoc Tukey test using a Macintosh computer and commercial software (Systat, Evanston, IL.). P < 0.05 was considered significant.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Effect of ECs on active TGF-1 protein levels.
As shown in Fig. 2, after 48 h in culture,
SMCs cocultured as a monolayer (EC-SMC mix) had a greater than
threefold increase in active TGF-1 levels compared with SMCs
cocultured as a bilayer (EC-SMC) (EC-SMC mix 180 ± 7% vs. EC-SMC
51 ± 5%; P < 0.01) and an
almost twofold increase compared with SMCs cultured alone (EC-SMC mix
180 ± 7% vs. SMC 100%; P < 0.05).
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Effect of PAI-1 inhibition on active TGF-1 in
bilayer coculture.
As shown in Fig. 3, the addition of 250 µg/ml of PAI-1 antibody (American Diagnostica) increased active
TGF-1 levels in SMCs cocultured with ECs as a bilayer by more than
fivefold compared with SMCs cocultured as a bilayer in the absence of
PAI-1 antibody (EC-SMC PAI-1 antibody 306 ± 24% vs. EC-SMC 59 ± 14%, P < 0.05). The addition
of 200 µg/ml of the plasmin inhibitor aprotinin to SMCs cultured
alone (SMC aprotinin) resulted in a decrease in active TGF-1 levels
compared with SMCs cultured alone (SMC aprotinin 75 ± 16% vs. SMC
100%).
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Effect of ECs on TGF-1 protein in SMC conditioned
media.
There was no difference in total TGF-1 levels in the conditioned
media from SMCs cultured alone (2.46 ± 0.10 ng/ml) compared with
SMCs cocultured as a bilayer (2.44 ± 0.13 ng/ml). Total TGF-1 levels were increased in SMC cocultured with ECs as a monolayer (2.90 ± 0.19 ng/ ml) compared with SMCs cocultured as a bilayer or SMCs
cultured alone (P < 0.05)
1 in the conditioned
media of SMCs cultured alone or SMCs cocultured with ECs as a monolayer
or bilayer when measured by either ELISA or MV 1 Lu cell assay at 24, 48, and 72 h (data not shown).
Effect of ECs on SMC hill-and-valley growth.
As shown in Fig. 4, SMC hill-and-valley
growth, a marker for TGF-1 activity, was observed in SMCs cultured
alone (SMC) and in SMCs cocultured as a monolayer (EC-SMC mix group).
This distinct morphology was absent in the SMCs cocultured as a bilayer
(EC-SMC group). In addition, the hill-and-valley growth response seen in SMCs cultured either alone or with ECs as a monolayer was inhibited by aprotinin or TGF-1 antibody. The addition of TGF-1 to SMCs cocultured with ECs as a bilayer resulted in SMC hill-and-valley growth.
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Effect of ECs and TGF-1 on SMC migration.
As shown in Fig.
5A,
migration of SMCs cocultured as a monolayer was decreased by 25%
compared with SMCs cultured alone and decreased by 50% compared with
SMCs cocultured with ECs as a bilayer. As shown in Fig.
5B, the migration rate of SMCs
cultured alone was increased by 22 ± 3% after the addition of 25 µg/ml of a neutralizing antibody to TGF-1. The addition of
TGF-1 antibody to cocultured SMCs had no effect, whereas the
addition of TGF-1 inhibited the migration of SMCs cultured either
alone or in the presence of ECs.
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Effect of ECs on SMC TGF-1 gene expression.
Coculture with ECs as a bilayer did not affect SMC TGF-1 gene levels
after 2 days in culture as determined by Northern analysis (Fig.
6).
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Effect of ECs on PAI-1 levels in conditioned media. Compared with SMCs cultured alone, PAI-1 levels were increased in conditioned media from ECs cocultured as a bilayer with SMCs, as well as SMCs cocultured with ECs and ECs cultured alone (Fig. 7). This was true at all time points studied.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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TGF-1 is a 25-kDa heterodimer growth factor that is secreted as a
large latent TGF-1 complex by numerous cell types, including SMCs
and platelets (13). The large latent TGF-1 complex consists of the
LAP, the active TGF-1 molecule, and the latent TGF-1 binding
protein (LTBP) and can be activated by either acidification or
proteolytic cleavage of the LAP from the inactive latent TGF-1 (9).
The serine protease plasmin appears to be the physiological mechanism
of TGF-1 activation as demonstrated by Sato and Rifkin (26) and by
Lyons and co-workers (11). This growth factor's effects depend on the
exposed target cell. TGF-1 has been shown to increase SMC
extracellular matrix production, stimulate confluently plated SMCs to
proliferate while growth inhibiting sparsely plated SMCs, increase SMC
proliferation in vivo while inhibiting SMC proliferation in vitro,
inhibit SMC migration in vitro, and inhibit EC proliferation and
migration (8, 14).
Several recent in vivo studies provide evidence that TGF-1 is
involved in the development of intimal hyperplasia. Nikol and co-workers (18) have identified mRNA transcripts to TGF-1 in human
coronary restenosis specimens that have been retrieved during coronary
artery arthrectomy. Majesky et al. (16) have identified an upregulation
of TGF-1 transcripts after balloon catheter injury in rat carotid
arteries. In these same experiments infusion of TGF-1 after rat
carotid balloon injury resulted in increased intimal thickness and an
increase in SMC DNA synthesis. Others have similarly shown that
infusion of a neutralizing antibody to TGF-1 inhibits the
development of intimal hyperplasia in both rat and rabbit carotid
balloon injury models (6, 28). TGF-1 may contribute to the
development of restenosis by both increasing SMC matrix synthesis and
decreasing matrix degradation.
Previous studies using ECs and SMCs cocultured as a monolayer have
shown that TGF-1 activation is increased in conditioned media (12,
20). These cocultures were established by plating SMC or pericytes in
direct contact with ECs in the same dish. Monolayer cocultures differ
from bilayer coculture in several respects. The ECs are not confluent,
as SMCs or pericytes are interspersed between ECs. More importantly
there is a much greater degree of cell-to-cell contact in the monolayer
compared with bilayer cocultures. This cell-to-cell contact as
previously described is critical for the increased TGF-1 activation
observed in monolayer coculture. In the monolayer coculture system
TGF-1 activation is thought to occur through an enhancement of
plasmin-mediated activation of the latent TGF-1 molecule bound to
the SMC surface as a result of the close approximation to plasminogen
bound to the EC surface (5a).
Our studies confirmed that monolayer coculture results in increased
TGF-1 activation as measured by Western blot and the presence of SMC
hill-and-valley growth compared with bilayer cocultures and SMCs
cultured alone. The main difference in our study compared with previous
investigators is the undetectable levels of active TGF-1 in the
conditioned media in any of the culture conditions as measured by ELISA
or MV 1 Lu cell assay. One explanation for this difference is that we
measured the conditioned media at 24, 48, and 72 h. Previous
investigators have shown that active TGF-1 levels are present only
during the initial 8-12 h after coculture and rapidly thereafter
become undetectable.
Our study demonstrates that, as measured by Western blot, ECs inhibited
activation of SMC TGF-1 when cocultured as a bilayer. The primary
antibody used in these studies is specific for the active form of
TGF-1 and does not cross react with either latent TGF-1 or other
TGF-1 isoforms. Although Western blotting has not been as frequently
used to assay for active TGF-1 as bioassays, this technique has been
used by Flaumenhaft and co-workers (5a) to measure active TGF-1.
Because the latent growth factor has been shown to bind selectively to
the SMC before activation, we felt that measuring the cell
lysate-associated active TGF-1 in addition to measuring levels in
the conditioned media would reveal useful information.
The formation of SMC hill-and-valley growth in monolayer cocultures and
SMCs cultured alone but not in bilayer cocultures suggests decreased
active TGF-1 is present in bilayer coculture. These data also
suggest that while active TGF-1 levels in the conditioned media may
be short-lived, active TGF-1 could be detected in lysates from SMCs
cultured alone or cocultured as a monolayer for up to 48 h and that
hill-and-valley growth was sustained indefinitely in these same culture
groups.
The mechanism by which ECs inhibit TGF-1 activation in bilayer
coculture is unclear. Madri and co-workers (14) have shown that
exposure of ECs to TGF-1 results in EC secretion of PAI-1. PAI-1
inhibits the conversion of plasminogen to plasmin, which could then
inhibit the plasmin-mediated activation of TGF-1. In contrast, the
exposure of SMCs to TGF-1 has been shown to stimulate SMC secretion
of plasminogen activators, which is the opposite of the effect of
TGF-1 on ECs (14). Increased secretion of plasminogen activators by
SMCs in response to TGF-1 could result in increased TGF-1
activation and thereby generate a positive-feedback loop. This
differential response of ECs and SMCs to TGF-1 may be one mechanism
by which ECs regulate TGF-1 activation and as a result control SMC
phenotype expression and growth.
The results of the present study are consistent with the above
hypothesis. PAI-1 levels were increased in ECs cultured alone and in
the conditioned media obtained from bilayer-cocultured ECs and SMCs
compared with the conditioned media of SMCs cultured alone. In
addition, PAI-1 inhibition in SMCs cocultured with ECs as a bilayer
resulted in a marked increase in active TGF-1. This suggests that EC
release of PAI-1 may be a mechanism by which ECs control TGF-1
activation in bilayer coculture. The increased PAI-1 levels observed in
the conditioned media from SMCs cultured opposite ECs may be due to
diffusion of PAI-1 in the EC conditioned media across the semipermeable
membrane. However, the possibility that ECs stimulate SMC PAI-1 release
cannot be dismissed.
The effect of ECs on SMC migration has not been extensively studied. In
the present study we have shown that, compared with SMCs cultured alone
and SMCs cocultured as a monolayer, ECs stimulate SMC migration when
cocultured as a bilayer. This effect can be inhibited by TGF-1. EC
stimulation of SMC migration is at odds with what would be expected to
occur in vivo. However, the contribution of migration to the SMC
response to injury is likely not as significant as other SMC functions,
such as the secretion of extracellular matrix, which contributes up to
80% of the bulk of restenotic arterial lesions. As previously
described TGF-1 is increased in human and animal models of
restenosis and may be responsible for the matrix accumulation in these
lesions. In vitro coculture models in which TGF-1 activity is
inhibited (i.e., bilayer coculture) may be more physiologically
relevant than those coculture models in which TGF-1 activation is
increased, such as monolayer coculture.
In conclusion, our data suggest that ECs in the absence of direct SMC
contact inhibit SMC hill-and-valley growth and stimulate SMC migration
in vitro. This mechanism appears to involve EC regulation of TGF-1
activation, which may be mediated by EC PAI-I release. Finally,
coculture conditions, in particular the degree of EC-SMC cell contact,
are critical in this process.
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ACKNOWLEDGEMENTS |
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This work was supported by a Department of Veterans Affairs Merit Review Grant.
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FOOTNOTES |
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Address for reprint requests: R. J. Powell, Dartmouth Hitchcock Medical Center, Section of Vascular Surgery, One Medical Center Drive, Lebanon, NH 03756.
Received 22 August 1997; accepted in final form 10 October 1997.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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