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5ß1 Integrin Expression and Luminal Edge Fibronectin Matrix Assembly by Smooth Muscle Cells after Arterial Injury
From the John P. Robarts Research Institute (Vascular Biology
Group), London Health Science Centre, Departments of Medicine
(Cardiology),*
Biochemistry,
Medical
Biophysics,
Anatomy and Cell
Biology,§
Surgery,¶
and Microbiology and
Immunology,||
University of Western Ontario,
London, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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5ß1 integrin. Whereas 5ß1 integrin was
not evident in the normal carotid artery, its expression was
induced after a vascular injury. By 14 days, the 5ß1
integrin was localized exclusively to the less differentiated smooth
muscle cells (SMCs) at the luminal surface of the neointima.
Platelet-derived growth factor-BB, dominant in neointimal
formation, selectively increased the expression of the 5ß1
integrin by human SMCs in culture. To track the assembly of fibronectin
fibers, fluorescence-labeled soluble fibronectin protomers were
added to cultured SMCs and to fresh segments of normal and
balloon-injured rat carotid arteries. Fibronectin fiber formation in
cultured SMCs could be detected within 10 minutes, and was
blocked by an RGD peptide, an anti-ß1 integrin
antibody, and an anti-5ß1 integrin antibody, but
not by an anti-ß3 integrin antibody. En face confocal microscopy of
arterial segments revealed that soluble fibronectin had polymerized on
the 5ß1 integrin-expressing SMCs of the luminal surface of the
injured arterial neointima, but not on the 5ß1
integrin-negative neointimal SMCs below this or on the
endothelial cells of uninjured arteries. Furthermore, in
situ fibronectin assembly by the neointimal SMCs was inhibited
by an RGD peptide and by an anti-ß1 integrin antibody. These studies
indicate that a subpopulation of SMCs in the repairing artery wall
orchestrates integrin-mediated fibronectin assembly.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After balloon-mediated arterial injury, there is rapid accumulation of fibronectin at the site of the injury, in association with neointimal formation.2,11-13 There are two potential sources for this newly deposited fibronectin. Some fibronectin may be derived from the circulatory system, where it exists as a plasma protein originally synthesized by the liver. In addition, fibronectin is synthesized locally, as a specific response to an injury, by resident vascular SMCs.2,13 Locally derived or cellular fibronectin is distinct from plasma fibronectin by virtue of the unique fibronectin domains that arise through alternative splicing. Cellular fibronectin contains the type III fibronectin modules ED-A and AD-B, whereas plasma fibronectin does not contain either of these splice variants.
Regardless of its origin, fibronectin is initially secreted from the cell as a soluble dimeric protein. Within the ECM, soluble fibronectin protomers polymerize to form insoluble, multimeric fibronectin. This assembly process is of paramount importance because only insoluble, fibrillar fibronectin can act as an adhesive ligand and regulate cell function. Moreover, fibronectin polymerization is not a spontaneous process, but requires specific cellular interaction.14 This contrasts with other ECM components, such as fibrillar collagen, basement membrane collagen, and laminin, which are capable of self-polymerization. Therefore, in the vessel wall, it is likely that a coordinated interplay between vascular cells and soluble fibronectin must occur to generate a fibronectin-rich ECM favorable to neointimal formation and growth.
The molecular basis of fibronectin assembly has been studied in culture
and found to involve cell surface fibronectin receptors, the actin
cytoskeleton, microtubule dynamics, and the Rho family of small
GTPases.14-16
Little is known however about fibronectin
matrix assembly in intact tissue, including the vessel wall. Based on
in vitro studies of nonvascular cells, the process can be
expected to depend on one or more members of the integrin superfamily
of heterodimeric adhesion receptors.15,17
Of these,
5ß1 integrin is a dominant fibronectin receptor, and we, as well
as others, have shown that the 5ß1 integrin is abundantly
expressed on the surface of human SMCs in culture.18,19
However, there is no information on the expression of this integrin in
the injured or diseased adult artery wall. This is noteworthy in light
of growing evidence that integrin expression in vitro may
not predict the same expression in vivo.20
This study elucidates the process by which soluble fibronectin is
converted to insoluble fibronectin fibers during neointimal formation.
We first established the 5ß1 integrin expression profile in normal
and balloon-injured rat carotid artery and related these findings to
the SMC phenotype, as indicated by the expression of smooth muscle
-actin. The effect of platelet-derived growth factor(PDGF)-BB on the
integrin expression in SMCs was also determined, recognizing the
central role of this growth factor in neointimal formation. We
subsequently tracked and characterized fibronectin assembly, using
fluorescently labeled fibronectin protomers, in cultured SMCs and in
fresh segments of normal and injured carotid arteries. Our findings
indicate that a specialized subpopulation of SMCs in the injured artery
wall orchestrates integrin-mediated fibronectin assembly during
vascular repair.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The monoclonal antibodies (mAbs) used included the human 1ß1
integrin-specific mAb Ts2/7,21
the human 2ß1
integrin-specific mAb BHA2.122
(Chemicon Inc., Temecula,
CA), the human 3ß1 integrin-specific mAb P1B5 (Life Technologies,
Inc., Gaithersburg, MD), a human 5ß1 integrin-specific mAb HA5
(Chemicon Inc., Temecula, CA), a function-blocking human 5ß1
integrin-specific mAb JBS5, a human ß1 integrin subunit-specific mAb
HB1.1 (Chemicon), and a human vß3 integrin-specific mAb 23C6
(Serotec). To study rat integrin expression, we used a rat 5ß1
integrin-specific mAb HM5–1(PharMingen), a rat ß1 integrin
subunit-specific mAb Ha2/5 (PharMingen), and a rat ß3
integrin subunit-specific mAb F11 (PharMingen). Control
antibodies for immunostaining, flow cytometry, and blocking experiments
included mouse immunoglobulin G (IgG; P3, ATCC), hamster IgG
(G235–2356, PharMingen), and hamster IgM (G235–1, PharMingen). The
peptides GRGDNP, RGES, and GPenGRGDSPCA were purchased from Life
Technologies.
SMC Culture
Primary cultures of rat and human arterial SMCs were initiated by
explant outgrowth from aortic segments and from segments of internal
thoracic artery retrieved at the time of coronary artery bypass
surgery, respectively.23,24
The identity of vascular SMCs
was confirmed morphologically and by positive immunostaining with a mAb
to smooth muscle -actin (1A4, DAKO). Cells were grown in M199 (Life
Technologies),and supplemented with the designated concentration of
fetal bovine serum. All experiments were performed using rat SMCs in
the fourth to sixth subculture and human SMCs in the third to sixth
subculture.
Balloon Injury to the Rat Carotid Artery
The left carotid artery of male Sprague-Dawley rats was injured using a 2F Fogarty catheter, as previously described.25,26 The uninjured right carotid artery was used as a control. At 0, 4, and 14 days after injury, rats were anesthetized, and the carotid arteries were perfused in situ with phosphate-buffered saline (PBS). Vessels were then harvested, embedded in OCT compound, frozen in liquid nitrogen, and cut into 6-µm cryostat sections. For some experiments, arteries were perfused in situ with methanol-Carnoy’s fixative (methanol:chloroform:glacial acetic acid, 6:3:1), immersed in the same fixative overnight, embedded in paraffin, and then sectioned at 6 µm thickness.
Immunostaining of Rat Tissues
Tissue sections of the skin, heart, large intestine, thoracic
aorta, uninjured carotid artery and injured carotid artery were
harvested from rats and examined for expression of the 5ß1
integrin. Frozen sections were dipped in acetone, pretreated with 10%
goat serum, and then incubated with the hamster anti-5ß1 integrin
antibody HMa5–1 (1:50 dilution) or isotype-matched control antibody
(G235–1) overnight at 4°C. The bound primary antibody was detected
with a biotinylated goat anti-hamster IgM antibody, an
avidin-biotin-peroxidase complex, and 3,3'-diaminobenzidine chromogen
(Sigma Chemical Co., St. Louis, MO). Staining for the smooth muscle
-actin was performed in paraffin-embedded sections of arteries fixed
in methanol-Carnoy’s solution. These sections were blocked with 10%
horse serum and endogenous peroxidase activity blocked with 3.0%
hydrogen peroxide in methanol. Sections were incubated for 2 hours at
room temperature with the anti-smooth muscle -actin mAb 1A4.The
bound primary antibody was detected with bio- tinylated horse
anti-mouse IgG antibody and color-developed as described above.
Sections were lightly counterstained with Harris’ hematoxylin. To
double-immunostain for the 5ß1 integrin and smooth muscle
-actin, frozen sections were first incubated with HM5–1, as
described above, and developed using a diaminobenzidine-nickel chloride
(0.4% in PBS) solution to yield a gray color. The same slide was then
blocked with 10% normal horse serum and immunostained for smooth
muscle -actin, using mAb 1A4. This reaction was developed using
diaminobenzidine alone to yield a brown color. Double-immunostained
sections were not counterstained. Immunostaining for leukocyte markers
was performed on frozen sections with mouse anti-rat CD4 mAb (MCA55,
Serotec; reactive against helper T-lymphocytes and cells of
monocyte/macrophage lineage), mouse anti-rat CD45RA (CLO33AP,
Cedarlane; reactive against B lymphocytes), and a mouse anti-rat
granulocyte mAb (HIS48; Pharmingen). The sections were color developed
and counterstained as for 5ß1 integrin staining.
Flow Cytometry
Flow cytometry for the detection of integrin expression was carried out by indirect immunofluorescence staining as described previously.22,27 Early confluent SMCs were trypsinized and washed in cold PBS with 1% bovine serum albumin. Cells were incubated for 30 minutes on ice with the control or integrin-specific mAbs at predetermined saturating concentrations. The washed cells were incubated with a fluorescein isothiocyanate (FITC)-labeled anti-mouse F(ab)'2 fragment (Becton-Dickinson, Ontario, Canada), and fluorescence staining was analyzed using a B.D. FacScan (Mountainview, CA).
Fibronectin Assembly by Cultured SMCs
Human plasma fibronectin was isolated by gelatin-sepharose chromatography as described.28 The purity of the isolate was verified by the presence of a single band on gel electrophoresis, which was confirmed to be fibronectin by Western blot analysis (data not shown). The fibronectin was then dialyzed against borate-buffered saline (170 mmol/L boric acid, 170 mmol/L sodium tetraborate, 75 mmol/L NaCl, pH 9.3) at 4°C overnight and then labeled with FITC by transferring the dialysate to a solution of borate-buffered saline containing 30 µg/ml FITC and mixing in the dark at room temperature for 1.5 hours. The physiological pH was restored, and unbound FITC was removed by dialysis against PBS at 4°C for 4 days. The concentration of FITC-labeled fibronectin was measured spectrophotometrically by measuring absorption at 280- and 493-nm wavelengths. The ratio of FITC to protein in the FITC-fibronectin conjugate was determined to be between 3.2 and 3.8, based on the approach of Mishell and Shiigi.29
Labeled soluble fibronectin was added to rat or human SMCs that were cultured on glass coverslips in M199 supplemented with fibronectin-free fetal bovine serum. The latter was obtained by passing fetal bovine serum through a gelatin-sepharose chromatography column and collecting the effluent. FITC-labeled fibronectin (100 nmol/L) was added to cultures for designated intervals, after which the cells were washed extensively with PBS and fixed with 4% paraformaldehyde. Cells were mounted in glycerol/PBS (9:1) containing Hoechst 33258 (2.5 µg/ml, Sigma) to identify the cell nuclei, and the presence of insoluble, polymerized fibronectin was evaluated by fluorescence microscopy. Blocking peptides or antibodies were added 16 hours after the SMCs were seeded onto coverslips and 30 minutes before the addition of FITC-labeled fibronectin protomers.
In Situ Fibronectin Assembly by Rat Carotid Artery
To study the assembly of fibronectin fibers by neointimal SMCs of the intact artery wall, we developed an in situ fibronectin assembly protocol. The left carotid artery of rats was balloon-injured and the middle one-third was harvested, unfixed, at 12 days after injury. Arterial segments were opened longitudinally and incubated with 2.5 mmol/L ethylenediaminetetraacetic acid in PBS for 15 minutes. After rinsing with Ca2+- and Mg2+-free PBS, the arterial segments were incubated for 36 hours with FITC-labeled soluble fibronectin (250 nmol/L) in the presence of 1.8 mmol/L Ca2+. After three washes with PBS, the artery fragments were pinned onto dental wax and then fixed with 3% formaldehyde. Segments were mounted whole in glycerol/PBS (9:1), containing Hoechst 33258, and imaged en face by confocal microscopy using a Zeiss LSM 410 microscope. An argon ion UV laser, emitting at 351 nm for the detection of Hoechst 33258, was used to establish nuclear morphology. These images could be used to distinguish endothelial cells, medial SMCs, and neointimal SMCs, based on their distinct nuclear morphology and alignment in situ. A krypton/argon laser emitting at 488 nm was used to detect FITC-fibronectin. Optical sectioning of samples was used to establish the relative position of cells and fibronectin fibers.
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Results |
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5ß1 Integrin Is Expressed in Injured, but Not Normal, Rat
Carotid Artery and Localizes to the Neointimal SMCs Subjacent to the
Lumen
To establish an expression profile of the 5ß1 integrin in
normal rat tissues, we immunostained frozen sections of the large
intestine, skin, thoracic aorta, and carotid artery. As shown in Figure 1, A and B
, SMCs in the muscle layers of
the large intestine were strongly positive, whereas the luminal
epithelial cells were negative. SMCs of the arrector pili muscle in the
skin were also positive (Figure 1C)
. In contrast to the SMCs in these
nonvascular tissues, SMCs in the media of the thoracic aorta and normal
carotid artery did not express immunodetectable 5ß1 integrin
(Figure 1D)
. Only rare adventitial cells, presumably fibroblasts, were
positive (Figure 1D)
.
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5ß1 integrin in SMCs forming the early
neointima, as well as those in the innermost layers of the media
(Figure 1E)
. By 14 days, the medial SMCs were no longer positive for
the 5ß1 integrin. Most of the neointimal SMCs were also negative,
with the notable exception of a rim at the luminal surface of one or
two cell layers (Figure 1F)
. This corresponds to the population of SMCs
well known to maintain proliferative activity during lesion
formation.25
5ß1 Integrin Expression in the Rat Carotid Artery Is Inversely
Correlated with That of Smooth Muscle -Actin
The temporal and spatial pattern of 5ß1-integrin expression
after an arterial injury suggested that this integrin was selectively
expressed in a subpopulation of SMCs that was actively contributing to
lesion formation. One of the hallmarks of SMC activation during lesion
formation is a fall in the expression of smooth muscle
-actin.30,31
To examine this, we immunostained sections
of normal and injured carotid arteries for smooth muscle -actin. As
illustrated in Figure 2
, the temporal and
spatial pattern of smooth muscle -actin expression was inversely
related to that of the 5ß1 integrin. Medial SMCs of the normal
carotid artery expressed smooth muscle -actin, but SMCs in the
neointima 4 days after injury were negative. At 14 days, most of the
neointimal SMCs had become positive for smooth muscle -actin, with
the important exception of those SMCs composing the luminal edge. None
of the cells at this edge expressed mononuclear inflammatory cell
markers, (CD4, CD45RA; Figure 2, D and E
), nor did they react with a
pan-granulocyte mAb (data not shown). These findings exclude the
possibility that the smooth muscle--actin-negative cells subjacent
to the lumen were colonized inflammatory cells. To verify the
reciprocal relationship between the 5ß1 integrin expression and
that of the smooth muscle -actin, we double-immunostained frozen
sections for both proteins. As shown in Figure 2F
, 5ß1
integrin expression was localized to the luminal-edge SMCs, whereas
smooth muscle -actin expression predominated below this.
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5ß1 Integrin on the Surface of Human SMCs Is
Increased by PDGF-BB
The finding of the 5ß1 integrin in the luminal cells of the
developing neointima prompted us to consider environmental factors that
might mediate localized induction of integrin expression. PDGF is an
established regulator of neointimal formation after a balloon injury of
the rat carotid artery32
and can be expected to be nearest
the luminal surface, owing to its release from adherent platelets and
expression by luminal SMCs.33
To determine the effect of
PDGF on the expression of the 5ß1 integrin, we
studied cultured SMCs by flow cytometry. Human SMCs were used for this
assessment for their relevance to human disease and to facilitate
comparison of 5ß1 integrin expression to that of several other
integrins. Monoclonal antibodies for human integrins are more readily
available than those for rat integrins. As shown in Figure 3
, stimulation with 10 ng/ml PDGF-BB
produced a notable increase in the expression of the cell surface
5ß1 integrin. In contrast, there was no detectable change in the
expression of the 2ß1 integrin, 3ß1 integrin, or vß3
integrin. There was a decline in the expression of the 1ß1
integrin. The expression of the ß1 integrin subunit was unchanged,
indicating an overall balanced effect of PDGF-BB on the total surface
pool of ß1 integrins.
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To test whether the 5ß1 integrin plays a role in the assembly
of a fibronectin matrix, the FITC-labeled fibronectin in its soluble,
protomeric form was added to both rat and human SMCs in culture. SMCs
were fixed after designated incubation periods, and fiber formation was
visualized by fluorescent microscopy. As shown in Figure 4
, for rat SMCs, fibronectin fibers were
clearly observed in association with cells, whereas noncellular areas
were devoid of fibers. The first evidence of fiber assembly was within
10 minutes, with short, streak-like fibers primarily localized at the
periphery of SMCs (Figure 4A)
. The fibers became brighter and longer
with an intricate network evident at 24 hours (Figure 4, B–D)
. The
pattern of the fibronectin matrix assembly by human SMCs was similar to
that of rat SMCs, although, qualitatively, the assembly process was
somewhat slower, and the fibers appeared finer (eg, Figure 5, G and I
). The addition of
cycloheximide to the cultures (50 µg/ml for 24 hours) did not inhibit
the assembly of exogenous fibronectin, indicating that the process was
not dependent on new protein synthesis or demonstrably influenced by
endogenously produced fibronectin (data not shown)
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5ß1 Integrin
To determine whether fibronectin assembly was integrin-mediated,
SMCs were pretreated with RGD peptide or function-blocking
anti-integrin antibodies for 30 minutes, followed by the addition of
FITC-labeled fibronectin in the presence of the respective blocking
reagent. After 2 hours, cells were washed, fixed, and examined by
fluorescence microscopy. The anti-integrin reagents were added 16
hours after the cells were seeded on coverslips and had no detectable
effect on cell morphology. Data from rat SMC cultures are illustrated
in Figure 5, A–F
. Compared with cultures incubated with a control
peptide (RGESP, 100 µg/ml), there was very little fibronectin in
cultures incubated with the RGD blocking peptide GRGDNP (100 µg/ml)
(Figure 5, A
versus B). Incubation with the anti-ß1
integrin antibody (Ha2/5) also substantially decreased fiber
polymerization compared with an isotype-matched control IgM (Figure 5, C
versus D). In comparison, the anti-ß3 antibody (F11) had
no demonstrable effect on fibronectin assembly compared with the
isotype matched control mAb (Figure 5, E
versus F). Cyclic
RGD (GPenGRGDSPCA), which preferentially inhibits the avß3 integrin,
also had no detectable effect (data not shown).
We performed the same assay in human SMC cultures, using a
function-blocking mAb that was specific to the human 5ß1 integrin
(JBS5). An analogous function-blocking antibody is not available for
rat SMCs, and we specifically verified that the anti-rat mAb used for
immunostaining (HM5–1) had no effect on the adhesion of rat SMCs to
fibronectin (data not shown). Incubation with mAb JBS5 (1:200 dilution
of ascites fluid) led to a striking inhibition of fiber formation,
compared with cultures incubated with the control IgG. Even in
confluent SMC cultures incubated with fibronectin protomers for 24
hours, fibronectin fibers were barely detectable (Figure 5, G
versus H). Incubation with mAb BHA2.1, an anti-2ß1
integrin mAb that we previously have shown inhibits SMC migration on
type I collagen,18
had no effect on fibronectin assembly
(Figure 5I)
.
Injured but Not Normal Rat Carotid Arteries Assemble a Fibronectin Matrix at Their Luminal Surface
We next determined whether the 5ß1 integrin-expressing cells
present on the surface of the injured rat carotid artery were capable
of polymerizing soluble fibronectin in situ. Arteries were
harvested 12 days after injury and subjected to a modification of the
fibronectin assembly assay, as described in Materials and Methods.
Figure 6A
depicts confocal microscopic
sections of the luminal surface of an uninjured rat carotid artery. The
characteristic morphology and alignment of endothelial cell nuclei can
be appreciated, particularly as the optical plane was lowered 1.2 µm
below the cell surface (more sharply focused nuclei in the middle panel
in Figure 6A
), confirming the artery to be intact and uninjured. There
was no evidence of de novo fibronectin fiber formation
associated with these cells. In contrast, optical sections through the
neointima of an injured carotid artery showed an extensive network of
polymerized fibronectin (Figure 6B)
. This was evident at the apical
surface of the innermost layer of neointimal cells, and it could also
be appreciated up to 5 µm into the tissue.
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5ß1 integrin-negative SMCs deeper within
the neointima were capable of mediating fibronectin assembly, arteries
were harvested 12 days after injury and longitudinally opened, and the
luminal-most neointimal cells were gently scraped away under
microscopic guidance, using a plastic coverslip. The artery was then
divided into two portions, one of which was frozen, sectioned, and
immunostained for the 5ß1 integrin. The other portion was
subjected to the fibronectin assembly protocol. In this way, the
soluble fibronectin protomers could be assured of access to SMCs that
were deeper in the neointima. As shown in Figure 6C
, arteries in which
the luminal surface was not removed displayed both the expression of
the 5ß1 integrin by the luminal SMCs and the capacity to assemble
a fibronectin matrix on this surface. In contrast, when the 5ß1
integrin-expressing lumenal SMCs were removed, in situ
fibronectin assembly was not detected. Fibronectin assembly was also
not evident on the adventitial side of arterial fragments, studied by
pinning arterial segments adventitia side up (data not shown).
To directly ascertain whether the assembly of fibronectin by arterial
neointima was integrin-mediated, arterial segments were incubated with
fluorescent fibronectin protomers and anti-rat integrin mAbs,
anti-integrin peptides, or their respective controls. As shown in
Figure 7
, fibronectin assembly in
situ by luminal SMCs was abrogated by the anti-ß1 integrin mAb
Ha2/5 and by the GRGDNP peptide. The anti-ß3 integrin mAb F11 had
little to no effect on fibronectin assembly.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5ß1 integrin-bearing SMCs orchestrates
integrin-mediated fibronectin polymerization in the repairing artery
wall.
The identification of 5ß1 integrin expression after arterial
injury augments the repertoire of integrins known to be expressed in
the remodeling adult artery wall. Despite the numerous integrins with
potential roles in vascular function,18,19,34
relatively
few have been identified in the intima of diseased or injured arteries.
The 1ß1 integrin is a collagen/laminin receptor identified in the
rat carotid artery neointima after balloon injury.35
The
vß3 integrin and vß5 integrin, which bind several ligands
including vitronectin, osteopontin, and fibronectin, have been
identified in human atherosclerotic plaque,36,37
although
the vß3 integrin was found to be more abundant in the arterial
media.36
Unlike the case for ß3 integrins, fibronectin
is the only ECM ligand for the 5ß1 integrin. Our observation that
the 5ß1 integrin was expressed in the injured but not normal rat
carotid artery suggests that 5ß1 integrin plays a role in artery
repair, but is not significantly involved in the integrity or function
of the normal rat carotid artery. This is consistent with the small
amount of fibronectin present in the media of the normal rat carotid
artery.31
Selective use of 5ß1 integrin-fibronectin
interactions during vascular remodeling has also been suggested during
the closure of the ductus arteriosus. The 5ß1 integrin is
expressed by ductus arteriosus SMCs only after birth, at which time
5ß1 integrin-fibronectin interactions are considered important in
mediating SMC motility and ductus closure.9,38,39
It is noteworthy that the expression of the 5ß1 integrin after
carotid artery injury was not distributed diffusely but was localized.
Four to five days after injury, the 5ß1 integrin was expressed in
SMCs of the medial layers adjacent to the internal elastic lamina and
in those of the primordial neointima. Subsequently, the expression
localized exclusively to the luminal edge SMCs. This temporal and
spatial pattern of expression parallels the well-established pattern
for SMC thymidine incorporation after balloon-injury,25
suggesting a linkage between the 5ß1 integrin and SMC replication.
An association between the 5ß1 integrin and those SMCs actively
contributing to lesion formation was further supported in the current
study by the inverse relationship between 5ß1 integrin expression
and smooth muscle -actin-containing stress fibers. The decline in
smooth muscle -actin in the developing rat carotid neointima is
consistent with the previous analyses of tissue lysate,30
and electron microscopy studies31
and reflects the
presence of less differentiated SMCs. Our findings indicate that the
5ß1 integrin expression, and fibronectin polymerization, are
specialized features of this population of less differentiated SMCs.
Twenty-eight days after injury, the luminal-edge cells expressed
immunodetectable smooth muscle -actin, in keeping with their SMC
identity, notwithstanding the lack of smooth muscle -actin staining
14 days earlier.
Factors that mediate the expression of the 5ß1 integrin by SMCs
in vivo remain to be elucidated. However, we previously
found that FGF2 modestly increased the expression of the 5ß1
integrin by human SMCs in culture.18
In the current study,
we observed that PDGF-BB also increased 5ß1 integrin expression in
cultured SMCs, and appeared to be selective in this response, because
there was no detectable increase in the expression of the 2ß1,
3ß1, or vß3 integrin and there was a decline in the
expression of the 1ß1 integrin. PDGF-BB is an especially
noteworthy candidate for mediating the localized expression of the
5ß1 integrin within the injured artery, because the tissue
concentration of PDGF-BB is likely highest near the luminal surface, by
virtue of its release from adherent platelets as well as local
expression by SMCs after injury, specifically at this
surface.33
By using fluorescence-labeled soluble fibronectin protomers, we tracked
the fibronectin assembly process, independently of fibronectin
synthesis by the cell. This enabled us to establish that fibronectin
polymerization by SMCs in vitro is a relatively rapid
process (evident within 10 minutes in vitro) that
progresses. The approach also allowed us to reliably probe for cell
surface receptors that mediate assembly, without any confounding
effects that the blocking reagents might have on fibronectin
production. This established that the 5ß1 integrin is essential
for fibronectin assembly by human SMCs in culture. The lack of potent
blocking antibodies for the rat 5ß1 integrin precluded absolute
confirmation of the specific integrin heterodimer involved in the
injured rat artery wall; however, both an RGD-containing peptide and an
anti-ß1 integrin mAb blocked in situ fibronectin assembly
by luminal neointimal SMCs. Neither the anti-ß3 integrin mAb nor a
cyclic RGD peptide that preferentially inhibits vß3 function had a
detectable effect on fibronectin assembly. Furthermore, after removal
of the 5ß1-integrin expressing cells subjacent to the lumen,
in situ fibronectin assembly was no longer detectable.
Together, these data strongly suggest that fibronectin assembly in the
injured rat carotid artery requires the 5ß1 integrin.
SMCs at the luminal surface of repairing arteries are potentially
vulnerable to the mechanical forces imposed by flowing blood,
particularly if there is little in the way of a supportive ECM. In the
injured rat carotid artery, this vulnerability may be especially
relevant for SMCs forming the primordial neointima (eg, 4–5 days after
injury), as well as SMCs at the luminal surface of the more developed
neointima that have newly arisen by mitosis. For these cells, there
must be a mechanism by which an insoluble ECM is rapidly formed. The
current findings indicate that this subpopulation of SMCs is
selectively equipped to rapidly generate an insoluble fibronectin
matrix from soluble precursors. The presence of polymerized fibronectin
would not only provide a structure for cell attachment but would
facilitate SMC survival by providing ligands for integrin-mediated
inhibition of apoptosis.40
At the same time, the insoluble
fibronectin would be conducive to SMC replication, migration, and
maintenance of a synthetic phenotype.6,41
Deposition of
fibrin on the injured lumen surface also likely occurs shortly after
injury,42
and we speculate that fibronectin assembly would
physically strengthen a provisional matrix initiated by fibrin, a
phenomenon suggested in other forms of wound healing.43
The 5ß1 integrin was not expressed in the deeper layers of the
neointima, and the SMCs in these layers were not observed to assemble a
fibronectin matrix. Presumably, the smooth muscle -actin-positive
SMCs in the deeper layers of the neointima interact with more permanent
ECM components such as collagen, which is consistent with a
heterogeneity of SMC-ECM interactions within the developing neointima.
We used plasma fibronectin as the source of soluble protomers for
integrin-mediated fiber assembly. This may be physiologically relevant
because plasma fibronectin is deposited within the vessel wall after
injury and is, theoretically, a ready source of substrate for matrix
assembly. On the other hand, the expression of alternatively spliced
cellular fibronectin is also a response to vessel injury.
Interestingly, cellular fibronectin has been localized specifically to
the SMCs at the luminal surface13
and, thus, would be
available for assembly by the 5ß1 integrin-expressing SMCs.
Although there are molecular differences between plasma and cellular
fibronectin, both are capable of binding the 5ß1 integrin, and it
is conceivable that both contribute to the fibronectin assembly
process. We do not know if other fibronectin-binding integrins such as
the 3ß1, 4ß1, or 8ß1 integrin play a role in fibronectin
matrix assembly in repairing arteries; these integrins have not,
however, been documented in intimal hyperplasia as yet. The ß3
integrin subunit has been identified in neointima after balloon
injury;44
however, the current study suggests that the
ß3 integrins do not have a dominant role in fibronectin assembly in
this context.
In summary, vascular injury induces expression of the 5ß1 integrin
by a subpopulation of SMCs that can orchestrate rapid,
integrin-mediated polymerization of soluble fibronectin. The efficiency
of this assembly process may be an important determinant of vascular
lesion growth and stability.
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Supported by a grant from the Heart and Stroke Foundation of Canada. J. G. P. was supported by a Career Investigator Award from the Heart and Stroke Foundation of Ontario (HSFO). B. M. C. C. was supported by a Research Scholarship from the Medical Research Council of Canada. E. R. received a studentship from the HSFO.
Accepted for publication October 14, 1999.
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5 ß 1 integrin fibronectin receptor, but not the 5 cytoplasmic domain, functions in an early and essential step in fibronectin matrix assembly. J Biol Chem 1993, 268:21883-218882ß1 integrin and disassembly of actin filaments. Circ Res 1997, 80:627-6372ß1) function in postextravasation movement of human rhabdomyosarcoma cells in the liver. Cancer Res 1996, 56:3142-3149 v ß 3 integrin mediates arteriolar vasodilation in response to RGD peptides. Circ Res 1996, 79:821-8261ß1 integrin is expressed during neointima formation in rat arteries and mediates collagen matrix reorganization. J Clin Invest 1996, 97:2469-2477[Medline]
vß3 integrin expression in normal and atherosclerotic artery. Circ Res 1995, 77:1129-1135 5 ß 1 integrin supports survival of cells on fibronectin and up-regulates Bcl-2 expression. Proc Natl Acad Sci USA 1995, 92:6161-6165This article has been cited by other articles:
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