© 2001 American Heart Association, Inc.
Integrative Physiology |
Regional Differences in Integrin Expression
Role of 
5ß1 in Regulating Smooth Muscle Cell Functions
From the Cardiovascular Research Center (K.L.D., D.W., R.N., Y.S., A.Z.), Department of Medicine (Cardiology), Thomas Jefferson University, Philadelphia; Department of Physiology (C.M.), Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia; and John S. Sharpe Research Foundation of Bryn Mawr Hospital (J.L.M.), Bryn Mawr, Pa.
Correspondence to Andrew Zalewski, MD, Division of Cardiology, Thomas Jefferson University, 1025 Walnut St, RM 410, Philadelphia, PA 19107. E-mail andrew.zalewski{at}mail.tju.edu
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Abstract |
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Abstract—There is increasing evidence to suggest that coronary smooth muscle cells (SMCs) differ from noncoronary SMCs. As integrin adhesion molecules regulate many SMC functions, we hypothesized that differences in integrin expression on coronary and noncoronary SMCs may account for cellular differences. Analysis of integrin expression on freshly isolated porcine coronary and noncoronary SMCs revealed that coronary SMCs express significantly less
5ß1 than noncoronary
SMCs, whereas the expression of total ß1 and
that of vß3 are
similar. Consistent with these findings, coronary SMCs demonstrated
significantly less adhesion to fibronectin, compared with carotid
artery SMCs. As
5ß1-mediated
signaling has been associated with cellular proliferation, the effects
of differential 5ß1
expression on cell proliferation were examined by comparing primary
coronary and carotid artery SMC proliferation. Coronary SMC growth was
significantly lower than that of carotid artery SMCs when plated on
fibronectin or type I collagen. Blocking
5ß1 function on
carotid artery SMCs produced a significant decrease in cellular
proliferation, resulting in growth similar to that of coronary SMCs.
Furthermore, blocking
5ß1, but not
vß3, inhibited loss
of -smooth muscle actin in proliferating SMCs. Proliferating
coronary SMCs were found to upregulate
5ß1 expression,
further indicating a role for
5ß1 in SMC growth.
These results suggest that dissimilar
5ß1 integrin
expression may mediate regional differences in phenotype of vascular
SMCs.
Key Words: integrin adhesion molecules • coronary smooth muscle cells • dedifferentiation • proliferation
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The integrin adhesion molecules, which are heterodimers consisting of noncovalently linked
and ß subunits,1
mediate many of the smooth muscle cell (SMC) functions associated with
atherosclerotic lesion formation, including SMC dedifferentiation,
migration, and
proliferation.2 At least 16
and 8 ß subunits have been identified, and members of both the
ß1 and ß3 integrin
families have been found to modulate vascular cell
functions.1 2 3
Although SMCs express several heterodimers containing the
ß1 subunit,
5ß1 has emerged as a
potentially important mediator of SMC responses.
5ß1, which is the
primary vascular fibronectin
receptor,4 plays a role in
SMC modulation from a contractile to a synthetic
phenotype,5 and
5ß1-mediated
fibronectin matrix assembly appears to be necessary for SMC
proliferation.6 Increased
5ß1 expression has
been demonstrated on dedifferentiated intimal SMCs after balloon
injury,7 and
5ß1 has been
implicated in constrictive vascular
remodeling.8 9 The
ß3 integrin
vß3 which is a
vitronectin receptor, although it can also bind thrombospondin,
fibronectin, von Willebrand factor, and
osteopontin,3 10 11
has also been demonstrated to participate in fibronectin matrix
assembly, cellular migration, and SMC
proliferation.12 13 14
Similar to 5ß1,
vß3 expression has
been shown to be upregulated in the media and adventitia during
hyperplastic responses in injured
arteries.15 16 In
addition, the blockade of
vß3 has been
demonstrated to decrease intimal thickening after arterial
injury.16 Despite the clinical relevance of coronary lesion formation, a limited number of studies have directly examined integrin expression and coronary SMC functions. Recent studies by our group and others have demonstrated that, unlike noncoronary vasculature, coronary arterial SMCs have a distinct phenotype and respond differently to in vivo and in vitro stimulation.17 18 19 20 For example, porcine coronary arteries lack a significant SMC response after endoluminal injury. Instead, activated adventitial fibroblasts proliferate and migrate, resulting in neointimal formation.17 18 21 Consistent with these in vivo findings, porcine and human coronary SMCs have been shown to behave differently in culture, demonstrating advanced differentiation and low proliferation when compared with SMCs from other vascular beds.19 20
In light of these findings, we hypothesized that differences
in integrin expression, in particular expression of
5ß1 and
vß3, on coronary
SMCs may account for the differential responses of these cells. Thus,
the objectives of this study were (1) to determine whether integrin
profile distinguishes porcine coronary SMCs from other vascular SMCs
and (2) to determine whether integrin profile plays a role in the
phenotype and proliferative functions observed in these
cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SMC Isolation
Porcine hearts and carotid arteries were obtained from the local abattoir. SMCs were isolated as described previously.19 Briefly, coronary and carotid artery media was incubated with collagenase type II (0.75 mg/mL, Worthington) and elastase (0.75 mg/mL, Sigma), and cells were collected after terminating digestion by addition of DMEM (Gibco BRL) with 10% FBS. The cell viability was consistently between 85% and 92% as determined by erythrosine B dye (Sigma) exclusion. In some experiments, iliac artery SMCs were used and were obtained as described above.
Indirect Immunofluorescence and Flow
Cytometry
Indirect immunofluorescence staining and flow
cytometry were performed as previously
described.22 Monoclonal
antibodies (mAbs) cross-reactive with the following porcine cell
surface markers were used:
5ß1 (CD49e/CD29, mAb
HA5 [10 µg/mL], mouse anti-human IgG2b, Chemicon International,
Inc), ß1 (CD29, mAb HB1.1 [10 µg/mL],
mouse anti-human IgG1, Chemicon), and
vß3 (CD41/CD61,
JM2E5, 1/5 dilution of mouse anti-porcine IgG1, Serotec). Mouse
IgG1 or IgG2b (Coulter-Immunotech) at 10 µg/mL was used as irrelevant
isotype control. SMCs in PBS with 0.2% BSA were incubated with
saturating concentrations of primary antibody or IgG control in the
presence of excess porcine 
-globulin (4 mg/mL, Sigma) and then
washed and incubated with excess goat anti-mouse FITC–conjugated
secondary antibody (1/50 dilution, BioSource International). Cells were
analyzed using a FACStarPlus flow cytometer (Becton-Dickinson), and the
data analyzed using LYSIS II software. Mean fluorescence intensity
(MFI) for IgG (ie, IgG1 or IgG2b) control was subtracted from the MFI
measured with the integrin antibodies to derive net
MFI.
Cell Adhesion Assay
Isolated SMCs were plated on 48-well plates (Falcon,
Becton Dickinson, 10 000 cells per well) coated with either bovine
plasma fibronectin (10 µg/mL, Sigma), human foreskin
fibroblast–derived cellular fibronectin (10 µg/mL, Sigma), or bovine
type I collagen (10 µg/mL, Becton
Dickinson).19 Cells were
allowed to adhere for 4 hours in DMEM with 0.1% BSA, and then the
wells were washed free of nonadherent cells. Adherent cells were lifted
with trypsin (Sigma) and counted in a Coulter counter
(Coulter-Immunotech). Percentage adhesion was calculated by dividing
the number of adherent cells by the total count of plated cells. Each
experiment was carried out in triplicate and repeated at least 3 times
on different occasions using cells isolated from multiple animals. In
additional experiments, cells were plated as above and allowed to
adhere for 48 hours in DMEM with 10% FBS. In these experiments,
disintegrin antagonists were used to determine the role of
5ß1 and
vß3 in adhesion of
carotid artery SMCs to plasma fibronectin and type I collagen.
Antagonists included the
5ß1 antagonist
EMF-10, the vß3
antagonist kistrin, and the
IIß3 antagonist
eristostatin (all 1
µg/mL).23 24
Cell Proliferation Assays
SMCs were plated as described for 48 hours of
adhesion. Proliferation was determined by counting cell numbers at 2,
4, 6, 8, and 10 days after plating, as described
previously.19 Each
experiment was carried out in triplicate and repeated 3 times on
different occasions using cells isolated from multiple animals. To
determine the role of
5ß1 and
vß3 in
proliferation, the disintegrins EMF-10
(anti-5ß1, 15 to 150
µmol/L), echistatin
(anti-5ß1, 150
µmol/L) kistrin
(anti-vß3, 150
µmol/L), and eristostatin
(anti-IIß3, 150
µmol/L) were added to the wells 48 hours after cell plating so as not
to interfere with initial cell
attachment.23 24
Antagonist was added every 48 hours when the growth medium was
changed.
To verify SMC proliferation data obtained using cell-counting methods, additional experiments were performed in which a cell proliferation was analyzed via 5-bromo-2'-deoxyuridine (BrdU) incorporation (Cell Proliferation ELISA, BrdU Colorimetric, Boehringer Mannheim). In these experiments, coronary and carotid artery SMCs were plated in 96-well plates (3000 cells per well) and allowed to grow for 6 to 8 days (70% confluence). Cells then underwent 3 hours of incubation with BrdU, and then the ELISA assay was performed. In the case of treated cells (ie, EMF-10, eristostatin), cells were treated for 24 hours and then the ELISA assay was performed.
In some experiments, passage 3 coronary and carotid artery SMCs were plated in 48-well plates at a density of 5000 cells per well and proliferation was examined over 10 days. Cells were passaged by plating 0.5 to 1x106 freshly isolated cells in T75 flasks. At 15 to 25 days after plating, proliferating cells were lifted with trypsin and subcultured. Coronary SMCs routinely took longer to reach numbers sufficient for subculture as a result of their decreased rate of proliferation, and primary cells did not reach confluence before subculture.
Histochemical Detection of SMC
Apoptosis
To determine whether disintegrin treatment of carotid
artery SMCs results in cell apoptosis, carotid artery SMCs were plated
on fibronectin-coated chamber slides and allowed to grow for 4
to 6 days. Cells were left either untreated or treated for either 2, 4,
6, 24, or 48 hours with EMF-10, kistrin, or eristostatin (150 µmol/L
for each) and then fixed and apoptosis examined via DNA fragmentation
methods (TdT-FragEl DNA Fragmentation Detection Kit, Oncogene Research
Products).
Immunostaining for -SM Actin
SMCs were plated on chamber slides (10 000 cells per
well) coated with fibronectin (10 µg/mL) and allowed to grow for 8
days. Cells were either untreated or treated with EMF-10 or kistrin as
described above. The Vectastain Elite ABC system was used (Vector
Laboratories) to stain for -SM actin (clone 1A4, IgG1, 1:50, Sigma)
as previously
described.19
Statistical Analysis
All data are presented as mean±SEM. Data were
compared by ANOVA using post hoc analysis with the Fisher corrected
t test. Probabilities of 0.05
or less were considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Regional Differences in Integrin Expression
Freshly Isolated Coronary SMCs Express Low Levels of Integrin
5ß1 Compared With
Other Vascular SMCsSurface expression of
5ß1, total
ß1, and
vß3 was compared on
freshly isolated coronary, carotid, and iliac artery SMCs by indirect
immunofluorescence and flow cytometry. As seen in the
Table
,
coronary SMC 5ß1
expression was significantly lower than that of carotid and iliac
artery SMCs. The difference in
5ß1 expression was
not due to lower total ß1 integrin, as total
ß1 expression was comparable among SMCs. In
addition, expression of the other integrin of interest,
vß3, was not
significantly different among the cell types
(Table).
Figure 1 shows representative histograms for coronary and
carotid artery SMC
5ß1 and total
ß1 expression. Freshly isolated coronary
artery SMCs showed a uniformly low level of
5ß1 expression.
Carotid artery SMC expression of
5ß1 was less
uniform; however, the vast majority of carotid artery cells show strong
positive staining for
5ß1. To assure that
the lack of 5ß1
surface expression was not a function of the isolation procedure,
coronary SMCs were placed in culture for 24 or 48 hours and then
examined. Expression of
5ß1 remained low
(data not shown), thus indicating that the lack of
5ß1 was not likely
the result of cell isolation procedures.
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Coronary SMCs Demonstrate Decreased
Adhesion to Fibronectin
To determine whether the low level of
5ß1 on coronary SMCs
affected their ability to adhere to fibronectin, the primary ligand for
5ß1, adhesion (4
hours in DMEM+0.1% BSA) of coronary and carotid artery SMCs to plasma
fibronectin, cellular fibronectin, and type I collagen was compared.
Consistent with the low level of
5ß1 expression,
coronary SMCs demonstrated significantly less adhesion (34.6±3.5%,
P<0.01, n=3) to plasma
fibronectin than carotid artery cells (56.3±6.9%, n=3)
(Figure 2). There was no significant difference between
adhesion of either SMC type to plasma or cellular fibronectin (data not
shown); thus, the remainder of the experiments (ie, proliferation,
differentiation, and apoptosis) were performed using only plasma
fibronectin coating. The low level of adhesion to fibronectin was
specific, as coronary and carotid artery SMCs adhered similarly to type
I collagen
(Figure 2). When adhesion was examined over 48 hours,
percentage adhesion to plasma fibronectin and type I collagen was very
similar to values observed at 4 hours for both cell types (data not
shown). At 48 hours, carotid artery SMC adhesion to fibronectin was
significantly inhibited by the
anti-5ß1 disintegrin
EMF-10 (51.4±6.1% inhibition, n=4,
P<0.01) but not by the
anti-vß3 disintegrin
kistrin (8.3±6.0% inhibition, n=4, NS) or the
anti-IIß3
disintegrin eristostatin (-10.6±3.2% inhibition, n=4, NS). Carotid
artery SMC adhesion to type I collagen was not significantly inhibited
by any of the disintegrins used (n=3 for all,
NS).
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Role of
5ß1 in Regulating
SMC Functions
Coronary SMCs Demonstrate Decreased
Proliferation Compared With Carotid Artery SMCs, a Function Associated
With 5ß1
Expression
As
5ß1 has been
associated with cell proliferation, experiments were performed to
determine whether the low level of
5ß1 expression on
coronary SMCs affected their ability to proliferate. As seen in
Figure 3, coronary SMC numbers at 10 days after plating on
fibronectin (22 578±6531 cells per well) and type I collagen
(22 455±5852 cells per well) were significantly lower than carotid
artery SMC numbers plated on the same matrix coatings (72 291±3766
and 55 013±2343 cells per well, respectively,
P<0.05). When cell
proliferation was examined using BrdU incorporation, very similar
results were observed; BrdU incorporation in coronary SMCs was 
45%
of that measured in carotid artery SMCs (data not shown). Differences
in cell proliferation could not be attributed solely to cell adhesion,
as there was no difference in coronary and carotid artery adhesion to
type I collagen
(Figure 2
).
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As seen in
Figure 4, blocking
5ß1 function on
carotid artery SMCs with the disintegrin EMF-10 (15 to 150 µmol/L) or
echistatin (150 µmol/L) resulted in a concentration-dependent
decrease in cellular proliferation, thus confirming a role for
5ß1in SMC
proliferation. As expected, the effects of
5ß1 antagonism were
greater when cells were plated on fibronectin (70% inhibition)
versus type I collagen (40% inhibition). Although the
vß3 antagonist
kistrin (150 µmol/L) also significantly inhibited cell proliferation
(Figure 4), for cells plated on fibronectin,
5ß1 blockade was
more effective than
vß3 blockade in
inhibiting cell proliferation. The effects of EMF-10 on SMC
proliferation were also verified using a BrdU incorporation
assay (data not shown). Similar inhibition of proliferation was
observed with both assays. The effects of EMF-10 and kistrin on
proliferation cannot be attributed to adhesion blockade, as disintegrin
treatment was initiated 48 hours after plating and none of the
disintegrins inhibited SMC adhesion to type I collagen (see adhesion
results, above). As expected, the
anti-IIß3 antagonist
eristostatin had no effect on SMC proliferation regardless of matrix
coating. These data indicate that in the presence of fibronectin
matrix, 5ß1, and not
vß3, may provide the
primary integrin signal for cell proliferation.
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As shown in
Figure 5, 5ß1 blockade on
carotid artery SMCs resulted in cell numbers similar to those of slowly
growing coronary SMCs at day 10 (25 505±2688 and 29 563±2428,
respectively, n=3). Thus, the low proliferative rate of coronary SMCs
was mimicked by blocking
5ß1 function on
carotid artery SMCs. Interestingly, blocking
5ß1 function on
coronary SMCs resulted in a significant reduction in cell numbers, when
cells were plated on fibronectin, suggesting that either the low level
of 5ß1 present at
the time of isolation is sufficient to signal for cell proliferation or
that 5ß1 may be
upregulated on coronary SMCs in response to serum
stimulation.
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Antagonism of
5ß1 Does Not Result
in SMC Apoptosis
To determine whether the decreased cell numbers
observed with treatment of carotid artery SMCs with either of the
peptide antagonists, EMF-10 or kistrin, was in part the result of cell
apoptosis, cell apoptosis was examined via DNA fragmentation methods.
Carotid artery SMCs were plated on fibronectin-coated chamber slides
and allowed to grow for 4 to 6 days. Cells were then either left
untreated or were treated for either 2, 4, 6, 24, or 48 hours with
EMF-10, kistrin, or eristostatin (150 µmol/L for each) and then
fixed, and apoptosis was examined via DNA fragmentation methods
(TdT-FragEl DNA Fragmentation Detection Kit, Oncogene Research
Products). Although even short-term treatment of carotid artery SMCs
with EMF-10 resulted in significant shape change (ie, rounding) of
cells, the cells remained firmly attached and did not undergo
apoptosis. For untreated cells and all treatments examined, <1% of
cells were found to be apoptotic at the times observed. These results
in conjunction with BrdU incorporation studies appear to indicate that
inhibition of proliferation is the primary means by which
5ß1 antagonism
results in decreased cell numbers.
Antagonism of
5ß1 Results in
Decreased SMC Dedifferentiation
As fibronectin binding through
5ß1 has been
hypothesized to be the primary signal for SMC dedifferentiation, the
effect of 5ß1
function blocking on -SM actin immunostaining was examined. Carotid
artery SMCs were plated on fibronectin-coated slides in the absence of
disintegrin or in the presence of EMF-10 or kistrin. As shown in
Figure 6, under normal growth conditions, only 48.7±3.5%
of carotid artery SMCs continue to express -SM actin by day 8. In
contrast, carotid artery SMCs grown in the presence of the
5ß1 disintegrin
EMF-10 continue to express high levels (>80% positive) of -SM
actin, similar to coronary SMCs, which have low baseline levels of
5ß1. This is in
contrast to carotid artery SMCs grown in the presence of kistrin, which
did not prevent SMC dedifferentiation
(Figures 6 and 7). As seen in
Figure 7, blockade of
5ß1 also altered
cell morphology. Untreated carotid artery SMCs
(Figure 7A) plated on fibronectin show a spindle-shaped
morphology, with cells generally possessing long, thin projections.
However, when cells were grown in the presence of EMF-10, the cells
were larger
(Figure 7B), with dense cell bodies lacking spindle
projections. These cells look very similar to coronary SMCs
(Figure 7D) both in their morphology and in their expression
of -SM actin.
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Proliferating Coronary SMCs Upregulate
5ß1 and Demonstrate
an Increased Rate of Proliferation
Despite the slower growth of coronary SMCs, a subset of
these cells proliferate in culture, and proliferation was inhibited by
blocking 5ß1
(Figure 5). To determine whether one of the mechanisms by
which coronary SMC begin to grow is through upregulation of
5ß1, coronary SMC
integrin phenotype was examined on primary (nonpassaged, 14 days after
culture) and passaged (passages 1 to 3) coronary SMCs. Primary coronary
SMCs significantly upregulate
5ß1. Freshly
isolated coronary SMCs had net MFI of 5.5±3.4 for
5ß1 expression,
whereas net MFI for
5ß1 expression at
day 14 after plating was 121.5±32.3 (n=3,
P<0.01). This value was not
significantly different from
5ß1 expression on
primary carotid artery SMCs at day 14 (net MFI 110.0±12.5, n=3). In
addition, passaged coronary SMCs continue to express
5ß1 at levels
similar to carotid artery SMCs (data not shown). To determine whether
the upregulation of
5ß1 on coronary SMCs
conferred an increased rate of proliferation and thus a rate similar to
carotid artery SMCs, passage 3 coronary and carotid artery SMCs were
plated in 48-well plates at a density of 5000 cells per well and
proliferation was examined over 10 days. Passage 3 coronary and carotid
artery SMCs demonstrated similar levels of proliferation (9.4±1.7-
versus 10.7±0.4-fold increase in cell number, respectively), further
indicating a role for
5ß1 in SMC
proliferation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is a growing body of evidence to suggest that coronary SMCs differ from noncoronary SMCs.17 18 19 20 The data presented herein suggest an explanation for these differences by demonstrating low basal expression of
5ß1 on coronary
SMCs, compared with SMCs from other vascular beds, which may account
for the highly differentiated phenotype of coronary SMCs (retention of
-SM actin and decreased proliferation). Our finding that
5ß1 expression is
not uniform among vascular beds may be of importance to the
understanding of vascular lesion formation, for two main reasons.
First, 5ß1 has been
linked to phenotypic changes in SMC function associated with lesion
development, including SMC
dedifferentiation5 7
and proliferation.6 Second,
our data imply that unlike SMCs from other vascular beds, upregulation
of 5ß1 by coronary
SMCs may be a prerequisite to coronary SMC proliferation and thus
intimal lesion formation.
The first evidence that
5ß1 may play a
significant role in vascular lesion formation was demonstrated by Hedin
and Thyberg,5 who showed that
fibronectin was the primary serum factor mediating modulation of SMCs
from a contractile to a synthetic phenotype. The fibronectin receptor
5ß1 or very
late–appearing antigen (VLA)–5 was soon identified as a member of the
integrin family of adhesion
molecules.3 25
Herein we show data that support the role of
5ß1 in mediating SMC
dedifferentiation by demonstrating that blocking
5ß1, but not
vß3, inhibits loss
of -SM actin in proliferating SMCs
(Figures 6 and 7). This is consistent with the finding of
Hedin and Thyberg,5 as they
demonstrated that rat aortic SMCs plated on fibronectin reduced their
myofilament bundles, even in the absence of serum. Most recently,
Pickering et al7 showed that
5ß1 expression was
strongly associated with dedifferentiated cells (ie, reduced -SM
actin) and fibronectin in the injury-induced intima. In contrast,
coronary SMCs, which express low levels of
5ß1, maintained
expression of SMC markers in
culture.19 20
In addition to its role in SMC phenotype switching,
5ß1 has been
demonstrated to play a role in SMC
proliferation6 as well as
proliferation of other cell
types.26 The role of
5ß1 as the primary
mediator of fibronectin matrix assembly has been demonstrated by
several
studies,13 27 28 29
which has led to the hypothesis that signaling through
5ß1 binding to
fibronectin stimulates cell
growth.6 13 27 28 29
Mercurius and Morla6 recently
demonstrated that peptide antagonists that block
5ß1-mediated
fibronectin matrix assembly, but not
5ß1 function,
effectively prevent SMC proliferation. The data presented herein show
for the first time that directly blocking SMC
5ß1 function is also
very effective in inhibiting SMC proliferation. Importantly,
vß3 has also been
shown to mediate fibronectin matrix assembly in the absence of
5ß1.13
These findings may explain the inhibition of cell growth observed with
the vß3-antagonist
kistrin and the ability of
anti-vß3 antagonists
to partially inhibit intimal formation in injured porcine coronary
arteries.16 Together, these
data suggest that multiple integrins are likely involved in the cell
proliferation associated with intimal formation. However, a significant
difference between
5ß1 and
vß3 may be their
impact on constrictive remodeling. Yee et
al8 have recently
demonstrated that
5ß1, but not
vß3, plays a crucial
role in SMC migration into and contraction of the fibrin clot, possibly
contributing to geometric remodeling of the vessel.
Given the importance of
5ß1 to SMC
functions, our observation that
5ß1 is expressed at
very low levels on quiescent coronary SMCs may provide new insight into
why these cells respond differently to both in vivo and in vitro growth
stimulation.17 18 19 20
Data presented herein suggest that the failure of coronary SMCs to
respond to in vivo stimulation may be due in large part to their
low-level expression of
5ß1. It remains to
be determined whether the differences in integrin expression arise from
the heterogenous lineages of vascular cells, with coronary vessels
originating from proepicardial cells in situ rather than from
ectodermal or mesodermal parts of the
aorta.30 The differential
integrin phenotype of coronary SMCs may confer a protective effect,
preventing rapid formation of occlusive coronary lesions in response to
noxious stimuli, and further supports a role of "invasive"
fibroblast in coronary vascular
remodeling.17 21
The roles of 5ß1 and
vß3 in coronary
fibroblast functions remain to be determined.
As cultured coronary SMCs did upregulate
5ß1, phenotypic
switching of coronary SMCs from low to high
5ß1 expression may
play a role in the loss of protective homeostasis in these cells. It is
likely that only a subset of coronary SMCs are capable of upregulating
5ß1 and thus
proliferating, given that the majority of coronary SMCs that adhere to
fibronectin and type I collagen fail to spread or take on a
dedifferentiated phenotype. Clonal analysis of coronary cells will be
necessary to more fully elucidate this phenomenon. Clonal heterogeneity
of medial SMCs has been observed in noncoronary vascular
beds31 ; however, this has
not been examined in the coronary media.
In conclusion, we have demonstrated that freshly isolated
porcine coronary SMCs express low levels of
5ß1 compared with
SMCs from other vascular beds, and this difference in
5ß1 may contribute
to the observed differences in coronary SMC responses to in vivo and in
vitro stimulation. Furthermore, unlike other SMCs, coronary SMCs appear
to have to upregulate
5ß1to proliferate.
Increasing our understanding of the importance of
5ß1 to coronary SMCs
may provide important new insight into our understanding of coronary
vascular
disease.
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Acknowledgments |
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This study was supported by NIH Grants HL-44150 and HL-60672 and the John S. Sharpe Foundation. We acknowledge Dr Rosario Scalia, Department of Physiology, Thomas Jefferson University, for providing generous use of flow cytometry facilities.
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Footnotes |
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Original received July 28, 2000; revision received November 8, 2000; accepted December 18, 2000.
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