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From the Terrence Donnelly Research Laboratories, Division of Cardiology, St. Michael’s Hospital, and the Departments of Medicine and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
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1 or
2 integrins reduced attachment of SMCs to type VIII collagen by 29%
and 77%, respectively. We found that SMCs grown from the rat
neointima, but not medial SMCs, increased their
production of MMP-2 and -9 on adherence to type VIII collagen. This
suggests that there is an important difference in phenotype between
intimal and medial SMCs and that intimal SMCs have distinct
matrix-dependent signaling mechanisms. Our findings suggest that type
VIII collagen deposited in vascular lesions functions to promote SMC
attachment and chemotaxis, and signals through integrin
receptors to stimulate MMP synthesis, all of which are
important mechanisms used in cell migration and invasion.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the current study, we investigate the role of type VIII collagen in SMC migration. Type VIII collagen is a short-chain collagen expressed during active tissue remodeling, including angiogenesis, embryonic development of the heart, and glomerulonephritis.9-11 We showed dramatic up-regulation of type VIII collagen expression by SMCs, using differential display screening to identify genes that were overexpressed in the rat carotid artery after injury.12 The protein was present only transiently, during the first 7 days after balloon catheter injury, in coincidence with early SMC migration. Furthermore, when we inhibited SMC migration by treating rats with antibodies against platelet-derived growth factor-BB and fibroblast growth factor-2, both strong chemotactic stimuli, messenger RNA (mRNA) expression for type VIII collagen was reduced in correlation with migration. Subsequent studies confirmed our observations of type VIII collagen expression after rat carotid injury13 and in the balloon-injured iliacs of cholesterol-fed rabbits.14 The potential relevance to human vascular disease was suggested by expression in atherosclerotic plaques, but not in normal media of coronary arteries.15 Taken together, these studies suggest an important role in cell migration during vascular remodeling; however, there is still little concrete evidence of type VIII collagen function.
Type VIII collagen expression in the injured rat carotid12 was coincident with early and dramatic increases in MMP-2 and MMP-9 expression and activity, which are critical for SMC migration from the media to the intima.16-19 Very recently, reports have been published colocalizing type VIII collagen and MMP-1 expression in SMCs in the intima of injured iliac arteries14 and in macrophages of the atherosclerotic plaque.20 Taken together, these data suggest important parallels in type VIII collagen and MMP expression; however, it is not clear whether the two are directly related. In several other cell types, matrix components can stimulate MMPs,21-29 but this has never been investigated in SMCs.
When an artery is injured, SMCs within the media of the vessel undergo a switch from a quiescent to an active phenotype, which is characterized by proliferation, migration, and synthesis of extracellular matrix. SMCs that have migrated to the intima of an injured vessel maintain many aspects of this active phenotype, even when the cells are isolated and grown in tissue culture.30 This indicates that phenotypic dedifferentiation is not solely a function of the local environment. Intimal SMCs grow faster in culture, are more responsive to growth factors, and express a wide array of genes that are not expressed or expressed at very low levels by SMCs derived from the quiescent media of an uninjured vessel.31 In the current study, we have taken advantage of the ability to isolate and grow in culture SMCs derived from the media of an uninjured, quiescent rat carotid (medial SMCs) and activated SMCs derived from the intima of the injured rat carotid (intimal SMCs). We have compared interactions of these two types of SMC with type VIII collagen, because the responses of the different cell phenotypes may be very important in the pathogenesis of diseases such as atherosclerosis.
In this study we investigate the hypothesis that type VIII collagen
promotes SMC migration, using in vitro models to mimic
critical steps of the migration process. Type VIII collagen stimulates
SMC attachment, focal adhesion formation, and chemotaxis of medial and
intimal SMCs. These effects are mediated via 2ß1 and 1ß1
integrin receptors. We also show that type VIII collagen stimulates
MMP-2 and MMP-9 expression and activity in intimal, but not medial,
SMCs. These studies suggest that type VIII collagen plays a critical
role in regulating SMC invasion and migration.
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Materials and Methods |
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Smooth Muscle Cell Culture
Male Sprague-Dawley rats were obtained from Charles River
(Montreal, PQ, Canada). Uninjured carotid arteries were harvested and
stripped of adventitia and the endothelium was scraped off, then medial
SMCs were dispersed by digestion for 1 hour in 0.3 mg/ml elastase type
III, 1.8 mg/ml collagenase type I (Worthington, Freehold, NJ),
0.44 mg/ml soybean trypsin inhibitor, 2 mg/ml bovine serum albumin
(BSA).1
To obtain intimal SMCs, left carotid arteries of
rats were injured with a balloon catheter, and, 2 weeks later, the
thickened neointima was stripped from the vessel with the aid of a
dissecting microscope.30
Intimal SMCs were dispersed by
digestion with elastase and collagenase as described above. Six
carotids were pooled for isolation of medial SMCs, and six intimas were
pooled for isolation of intimal SMCs. In addition, to ensure
consistency of the SMC phenotypes across various different rats, we
obtained and maintained several independent dispersions, which were
randomly selected for experiments. Intimal and medial SMCs
were routinely grown in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal calf serum and 2% penicillin-streptomycin
and used between passages 5 and 10. Immunostaining for smooth muscle
-actin confirmed that the harvested cells were SMCs. DMEM,
penicillin-streptomycin, trypsin, FCS, and fibroblast growth
factor-2 were from Life Technologies, Inc. (Gaithersburg,
MD).
Human newborn aortic smooth muscle cells (HF16) were generously provided by Dr. Cecilia Giachelli of the University of Washington (Seattle, WA).
SMC Attachment Assay
Type VIII collagen was purified from bovine Descement’s membrane as previously described.32 To summarize, bovine eyes were obtained from a local slaughterhouse and corneas were dissected from the eyeballs, then the inner Descement’s membranes were peeled off with forceps. The membranes were digested with 0.5 mg/ml pepsin in 0.5 mol/L acetic acid for 12 hours and centrifuged, and the supernatant was lyophilized then resolubilized in 1 mol/L NaCl, 50 mmol/L Tris, pH 7.5. The collagens were separated from other proteins by a series of precipitations in NaCl (4 mol/L, 0.7 mol/L and 1.5 mol/L), each followed by dialysis against 0.5 mol/L acetic acid. Final separation of type VIII collagen from contaminating type V collagen was achieved by chromatography on an agarose A1.5-m column (BioRad, Hercules, CA). Purity of the preparation was confirmed by analysis of Coomassie-blue stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of the protein (one band evident at 50 kd), and Western blots probed with an antibody against bovine type VIII collagen also showed a single band at 50 kd.
Pepsin-solubilized bovine dermal type I collagen Vitrogen 100 (Collagen Biomaterials) was used as a positive control substrate based on previous reports of its ability to promote SMC attachment.6,33,34 Type VIII or type I collagen was diluted in phosphate-buffered saline (PBS) to the stated concentrations, then 50 µl per well was added to 96-well tissue culture plates and incubated overnight at 4°C. Coating efficiency was determined by iodinating type I and type VIII collagen (Iodogen, Pierce, Rockford, IL) and measuring the amount of protein coated as a percentage of total counts applied. Coating efficiency did not vary over the range of substrate concentrations applied; it was similar for both 10 µg/ml (37.1%) and 100 µg/ml (33.5%) type I collagen applied to wells. Coating efficiency was also similar for 10 µg/ml (27.4%) and 100 µg/ml (29.9%) type VIII collagen applied to wells. For attachment assays, after coating overnight, the wells were rinsed with PBS, and nonspecific binding sites were blocked with 10 mg/ml BSA at 37°C for 1 hour. SMCs were detached from culture flasks by minimal trypsinization (1–2 minutes in 0.05% trypsin), placed immediately into an equal volume of 0.5 mg/ml soybean trypsin inhibitor and centrifuged. Cells were resuspended in DMEM with 1 mg/ml BSA and counted using a hemocytometer. Cells (30,000) were plated in each well and allowed to attach at 37°C for 60 to 90 minutes. Nonadherent cells were rinsed off with PBS, and the remaining attached cells were fixed with 4% paraformaldehyde for 5 minutes, then stained for 5 minutes with 0.5% toluidine blue dissolved in 4% paraformaldehyde and rinsed in water. Attached, stained cells were solubilized with the addition of 100 µl of 1% sodium dodecyl sulfate, and absorbance was measured in a microtiter plate reader (Molecular Devices) at 595 nm. The validity of this measure of attachment was assessed by plotting toluidine blue absorption against cell counts, which were derived by trypsinizing to release cells from the wells and counting them with a Coulter counter. An excellent correlation (r2 = 0.986) between the two techniques was achieved. Experiments were performed in triplicate and repeated a minimum of three times. Attachment assays were performed using rat medial or intimal SMCs or human HF16 SMCs.
Vinculin Immunostaining
Glass coverslips placed in 24-well culture plates (Costar, Corning, NY) were coated with 10 µg/ml type VIII collagen. Medial SMCs (80,000) suspended in 1 mg/ml BSA/DMEM were added to each well and allowed to adhere for 2 or 4 hours, after which the coverslips were washed with PBS to remove nonadherent cells, and remaining cells were fixed in 4% paraformaldehyde for 7 minutes. The cells were treated with 0.2% Triton X-100 to block nonspecific binding, then stained with anti-human vinculin antibody at 1:600 dilution for 30 minutes, and then Cy3-conjugated anti-mouse immunoglobulin G (IgG) at 1:200 dilution (Jackson Immunoresearch, West Grove, PA). Fixing, blocking, and staining steps were followed by three 5-minute washes in PBS. The coverslips were mounted on glass slides with a 1:1 solution of PBS:glycerol and observed under a fluorescent microscope.
Integrin Receptor Blocking
Receptors for type VIII collagen were determined with blocking antibodies against integrin receptors to inhibit attachment or focal adhesion formation. Rat medial or intimal SMCs were incubated with anti-rat integrin ß1 antibody (clone Ha2/5, Pharmingen, San Diego, CA) or anti-rat integrin ß3 antibody (clone F11, Pharmingen) at a concentration of 20 µg/ml for 30 minutes at room temperature before plating for attachment or focal adhesion assays on 10 µg/ml type VIII collagen in the presence of antibody.
Human SMCs were used to determine the integrin subunit for type
VIII collagen, because there are few blocking antibodies available for
rat integrin subunits. Experiments were performed as described
above for rat SMCs. Blocking antibodies against human integrins were
obtained from Chemicon and included 1 (FB12), 2 (P1E6), 3
(P1B5), 4 (P1H4), 5 (P1D6), 6 (CLB701), v (P3G8), ß1
(6S6), and vß3 (LM609).
SMC Migration Assay
Type VIII or type I collagen was diluted to indicated concentrations in DMEM containing 200 µg/ml BSA and placed in the bottom wells of 24-well transwell chemotaxis chambers (Costar). Freshly trypsinized rat SMCs were washed twice in soybean trypsin inhibitor, then resuspended in DMEM containing 200 µg/ml BSA. Cells (50,000) were plated on polycarbonate filters with 8-µm pores, inserted into the wells containing chemoattractants, and allowed to migrate for 4 hours at 37°C in a humidified chamber. After the incubation period, the tops of the filters were scrubbed with a cotton swab to remove cells, and the cells that had migrated to the bottom of the filter were fixed and stained with Diff-Quick fixative and staining solutions (Dade Diagnostics). The filters were cut from the wells and mounted under oil on glass slides. Migration was quantitated by counting the number of cells in five random 200x fields/filter and expressed as the average number of cells per field. Counts were performed using a digital image analysis system (Simple, C-Imaging Systems). Each experiment was performed in triplicate and repeated three times. To differentiate chemotaxis from chemokinesis, we performed checkerboard assays. Type VIII or type I collagen, at concentrations ranging from 0 to 100 µg/ml, was dissolved in media in both the top and bottom chambers of the well or in the top chamber only. Under these conditions, in the absence of a chemotactic gradient, SMC migration is due to random chemokinesis.
Blocking antibodies against integrin receptors were used to inhibit migration toward type VIII collagen. SMCs were incubated with anti-rat integrin ß1 antibody (clone HA2/5, Pharmingen) or anti-rat integrin ß3 antibody (clone F11, Pharmingen) at a concentration of 20 µg/ml for 30 minutes at room temperature before plating SMCs in wells and performing migration assays in the presence of the blocking antibodies.
MMP Activity Measurement
MMP activity was measured using gelatin zymography. Intimal or medial SMCs were suspended in serum-free DMEM containing 200 µg/ml BSA and plated in 96-well plates coated with type VIII collagen. After 24 hours, conditioned medium was collected from the wells and assayed for MMP activity as we have previously described.16 Briefly, conditioned medium samples were subject to electrophoresis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels that contained 0.1% gelatin as a substrate for MMP digestion. After electrophoresis, the gels were incubated 16 hours and then stained with Coomassie blue (BioRad), and MMP activity was evident as cleared bands of substrate lysis. The MMPs were identified by their molecular weights and inhibition by ethylenediaminetetraacetic acid or phenanthroline.
Northern Blots for MMP-2 and MMP-9 mRNA Expression
Rat MMP-2 and MMP-9 complementary DNA (cDNA) probes were cloned by reverse transcriptase-polymerase chain reaction of RNA extracted from rat intimal SMCs. The primers for MMP-2 were as follows: sense, 5'-TTT GAT GAC GAT GAG CTA-3'; antisense, 5'-GGG AGC TCA GGC CAG AA-3'. Total length of probe 932 bp.35 Primers for MMP-9 were as follows: sense, 5'-GAT GGT TAT CGC TGG TGC GCC-3'; antisense, 5'-GTG CAG TGG AAC ACA TAG TGG-3'. Total length of this probe was 496 bp.36 Reverse transcription was performed using the Advantage 1st-strand cDNA synthesis kit by the manufacturer’s instructions (Clontech, Palo Alto, CA). To summarize, 0.2 µg of mRNA was heat denatured in diethyl pyrocarbonate-treated water for 2 minutes at 70°C and then incubated at 42°C for 1 hour in a total reaction volume of 20 µl containing 20 pmol of primer (oligodeoxythymidylic acid18 ), 0.5 mmol/L of each deoxynucleoside triphosphate, 1 U/µl of RNase inhibitor, and 200 U of Moloney murine leukemia virus reverse transcriptase. The reaction was stopped by heating at 94°C for 5 minutes, the mixture was diluted to a final volume of 100 µl, and aliquots were stored at -80°C until further use. PCR amplification of specific cDNA was achieved using 2.5 U of Taq DNA polymerase and 0.2 µmol/L primers in 100 µl of reaction mixture. Polymerase chain reaction cycling was carried out as follows: denaturation at 94°C for 45 seconds, annealing at 50°C for 45 seconds, and extension at 72°C for 1 minute for 30 cycles.
Intimal SMCs were plated in wells precoated with type VIII collagen at concentrations ranging from 5 to 100 µg/ml, and incubated for 24 hours. Total cellular RNA was isolated, and Northern blots were prepared by our previously published methods.12 The cDNA probes for rat MMP-2 and MMP-9 were labeled with 32P-labeled deoxycytidine triphosphate, using a Multiprime kit (Amersham). The hybridized blots were used to expose Hyperfilm-MP (Amersham).
Statistical Analysis
Attachment and migration assay results were analyzed by analysis of variance followed by Fisher’s protected least significant difference.
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Results |
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Medial SMCs attached to wells coated with type VIII or type I
collagen in a dose-dependent manner (Figure 1A)
. Maximal attachment occurred at 25
µg/ml type VIII collagen, with an optical density of 0.189 ±
0.010, or 100 µg/ml type I collagen, with an optical density of
0.386 ± 0.018. Attachment to type VIII collagen was significantly
less than attachment to type I collagen at all concentrations tested.
The molecular mass of type I collagen is ~95 kd, and the molecular
mass of type VIII collagen isolated by pepsin digestion from
Descement’s membrane is ~50 kd. When compared on an equimolar basis,
medial SMC attachment to type VIII collagen was still significantly
less than attachment to type I collagen. For example, when plates were
coated with a 1 mmol/L concentration of collagen, attachment to type
VIII collagen was 0.192 ± 0.022, whereas attachment to type I
collagen was 0.386 ± 0.018.
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. Attachment of intimal SMCs at low
concentrations of type VIII collagen (5–20 µg/ml) was equivalent to
attachment to type I collagen at these concentrations. However, at
higher concentrations (25–100 µg/ml), attachment of intimal cells to
type VIII collagen was significantly less than attachment to type I
collagen. When compared on an equimolar basis, at higher
concentrations, intimal SMC attachment to type VIII collagen was
significantly less than attachment to type I collagen. For example,
when plates were coated with a 1 mmol/L concentration of collagen,
attachment to type VIII collagen was 0.252 ± 0.024, while
attachment to type I collagen was 0.395 ± 0.010. SMCs Form Focal Adhesions on Type VIII Collagen
Assembly of focal adhesion complexes occurs after cell surface
integrins bind to the extracellular matrix and aggregate, initiating
reorganization of the cytoskeleton and a cascade of cell-signaling
events. To determine whether SMC binding to type VIII collagen was
mediated through integrins and focal adhesions, medial SMCs plated on
type VIII collagen were stained with an antibody against vinculin,
which is a cytoskeletal component of the focal adhesion complex.
Vinculin staining was localized at discrete focal adhesions 2 hours
after plating on 10 µg/ml type VIII collagen (Figure 2A)
, and by 4 hours the cells were well
spread and had formed numerous distinct focal adhesions (Figure 2B)
.
Similar results were obtained with intimal SMCs (data not shown). No
cell adhesion or spreading was observed in control wells coated with
only BSA.
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, whereas normal mouse IgG
had no effect (Figure 2C)
.
2ß1 and 1ß1 Integrin Receptors Mediate SMC Binding to
Type VIII Collagen
Blocking antibodies to various integrin receptors were used to
determine which and ß subunit pair(s) bind to type VIII collagen.
Pretreatment of medial SMCs with 20 µg/ml of an antibody against rat
ß1 integrin reduced attachment to 10 µg/ml type VIII collagen by
92% in comparison with normal mouse IgG-treated controls (Figure 3A)
. By contrast, pretreatment with an
antibody against rat ß3 integrin (20 µg/ml) had no significant
effect (Figure 3A)
. The results were similar for intimal SMCs; the
anti-ß1 antibody reduced intimal SMC attachment to type VIII collagen
by 83%, whereas anti-ß3 had no effect (Figure 3B)
. For these
blocking assays, SMCs were plated in wells coated with 10 µg/ml type
VIII collagen, a concentration that gave half maximal attachment.
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integrin subunits were
not available, experiments were performed using a human SMC strain,
HF16, to identify the subunit(s) that binds to type VIII collagen.
HF16 SMC attachment to type VIII collagen was maximal at a coating
concentration of 20 µg/ml, with an optical density of 0.314 ±
0.032. Pretreatment with an antibody against human ß1 integrin (20
µg/ml) reduced attachment by 97% (Figure 4)
. Blocking antibodies against 2 or
1 integrins reduced adhesion to type VIII collagen by 77% and 29%,
respectively (Figure 4)
. By contrast, antibodies against human 3,
4, 5, 6, v, and vß3 did not affect attachment to type
VIII collagen (data not shown). Pretreatment with soluble GRGDSP
peptides (Life Technologies) did not affect adhesion to type VIII
collagen, which suggested that binding was not mediated through
RGD sites (data not shown).
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We tested for a chemotactic effect of type VIII collagen on SMC
migration, using modified Boyden chambers. Type VIII collagen
stimulated the dose-dependent, directed migration of rat medial SMCs,
with maximal migration of 126.6 ± 32.1 cells/field occurring at a
concentration of 100 µg/ml (Figure 5A)
.
The chemokinetic effect of type VIII collagen was assessed using a
checkerboard assay, in which the chemotactic gradient was abolished by
adding equal concentrations of protein to both top and bottom wells of
the chemotaxis chamber or to the top well of the chamber only. Cell
movement due to chemokinesis was very low; less than five cells per
field migrated through the filter when 100 µg/ml type VIII collagen
was added to both wells or to the top well only. Type I collagen was
used as a positive control for the assay, because it is a known SMC
chemotactic factor.7
At equivalent concentrations of
collagen, chemotaxis toward type VIII collagen was not significantly
different from chemotaxis toward type I collagen (Figure 5A)
. Also,
comparing equimolar concentrations of collagen, there were no
significant differences between migration toward either substrate.
Dose-dependent increases in intimal SMC migration toward type VIII
collagen were similar to medial SMCs (data not shown).
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. By
contrast, anti-ß3 integrin antibody did not inhibit type VIII
collagen-stimulated migration. Attachment to Type VIII Collagen Stimulates Intimal SMC, but Not Medial SMC Gelatinase Expression and Activity
Production of MMPs is another important step for SMC migration and
invasion through three-dimensional matrices, thus we tested the
hypotheses that type VIII or type I collagen stimulates MMP expression
and activity. SMCs were plated on collagen, and MMP activity released
into the culture media was assayed using gelatin zymograms. Medial SMCs
plated on type VIII collagen produced two prominent bands of
gelatinolytic activity with molecular masses of 72 and 70 kd (Figure 6A)
. There was no change in activity in
these bands with increasing concentration of type VIII collagen.
Intimal SMCs plated on 5 µg/ml type VIII collagen also produced two
bands with molecular masses of 72 and 70 kd. Activity in the 72- and
70-kd bands was increased in a dose-dependent manner with increasing
type VIII collagen concentration (Figure 6A)
. In addition, two new
bands with molecular masses of 62 and 88 kd were induced in a
dose-dependent manner in the intimal SMCs. Based on molecular masses
and comparison with previous reports including our own, these bands
likely represent the following MMPs: latent zymogen forms of MMP-2 (72-
and 70-kd bands; MMP zymogens appear active on the zymogram because SDS
in the gel partially denatures the enzyme, exposing the active site);
MMP-2 active enzyme (62 kd); and MMP-9 active enzyme (88
kd).16
Medial or intimal SMCs plated on type I collagen
produced only the latent MMP-2 bands, and there was no relationship
between MMP production and type I collagen concentration (data not
shown).
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. Both MMP-2 and MMP-9 mRNA expression were increased
after plating intimal SMCs on increasing concentrations of type VIII
collagen ranging from 5 to 100 µg/ml (Figure 6B)
. Bands of 3.1 kb and
3.6 kb were detected on the blots, corresponding to mRNA transcripts
for MMP-2 and MMP-9, respectively.16
Thus the change in
mRNA levels for the two MMPs paralleled the change in activity,
suggesting that increased production was mediated at least in part
through transcription. |
Discussion |
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Many matrix molecules that promote cell migration act as attachment factors, because it is necessary for a cell to adhere to a substrate to gain traction for migration.37,38 We found dose-dependent increases in both medial and intimal SMC attachment to increasing concentrations of type VIII collagen. However, in general, SMC attachment to type VIII collagen was less than attachment to type I collagen, even when substrate concentrations were normalized on a mole-to-mole basis. This suggests that type VIII collagen was a less adhesive substrate than type I collagen. Because type I collagen is very abundant in the vessel wall, one might predict that very high concentrations of type VIII collagen would have to be present to compete for cell binding. Although we do not know the exact concentration in vivo, morphological evidence does suggest that type VIII collagen is deposited in local areas of the vessel after injury. Sibinga et al, using immunohistochemical staining, showed copious deposition of type VIII collagen restricted to medial SMCs immediately subjacent to the lumen and to the developing neointima in the injured rat carotid.13 In addition, our results suggest that, at some concentrations, SMCs of the activated intimal phenotype adhere equally well to type VIII and type I collagens. Thus it is likely that local deposition of high concentrations of type VIII collagen and the presence of activated intimal SMCs combine to make a significant contribution to vascular pathogenesis in vivo.
Type VIII collagen acted directly as a chemotactic factor for both medial and intimal SMCs in transwell migration assays. Our checkerboard assay results indicated a true chemotactic effect and not merely a chemokinetic effect, because SMCs migrated along a gradient of type VIII collagen and migration was dramatically diminished when the gradient was abolished. Chemotaxis is an important mechanism stimulating cell migration, and many matrix molecules deposited after injury exert chemotactic effects.1,3-5,39-42 Our results can be distinguished from the results of Sibinga et al, in which rat aortic SMCs were shown to migrate across type VIII collagen-coated filters in response to the chemoattractant platelet-derived growth factor-BB suspended in the bottom of the chamber.13 We have demonstrated for the first time that type VIII collagen can stimulate chemotaxis directly and independently of other chemoattractant agents.
An early step of SMC migration in the vessel wall is degradation of the matrix by MMPs, which enables cells to break contacts and clears a path for SMC movement from the media into the intima.16-18,43,44 In this report, we have shown that type VIII collagen can stimulate MMP-2 and MMP-9 synthesis in intimal SMCs grown in tissue culture. In two very exciting recent studies, Plenz et al reported coexpression of type VIII collagen and MMP-1 by SMCs in the intima of balloon-injured iliac arteries in cholesterol-fed rabbits and by macrophages in human atherosclerotic lesions.14,20 These results are consistent with our work, and we have significantly extended this by demonstrating for the first time an important functional role for type VIII collagen in stimulating MMP synthesis in intimal SMCs.
To the best of our knowledge, this is the first report that a matrix molecule can directly stimulate MMP synthesis by SMCs. Furthermore, we found that type I collagen, which is a normal matrix component found in abundance in the uninjured vessel wall, does not stimulate MMP synthesis. We suggest that this feedback regulation of matrix degradation is a property unique to matrix molecules that are induced after injury. We have evidence that osteopontin, another injury-induced matrix molecule, stimulates MMP-1 synthesis (unpublished observation). Evidence for this matrix feedback paradigm is accumulating from studies of several other cell types. Pioneering studies in fibroblasts,21-24 keratinocytes,25,26 melanoma cells,27 macrophages,28 and osteogenic cell lines29 have demonstrated MMP production regulated by feedback from the extracellular matrix.
In the injured vessel wall, the up-regulation of MMP synthesis precedes the appearance of SMCs in the intima, suggesting that medial SMCs are triggered to produce MMPs, and this may be part of the phenotypic shift from a quiescent to active state. Indeed, several growth factors and cytokines that mediate the phenotypic shift are also capable of stimulating MMPs.45 We show here for the first time that type VIII collagen can stimulate MMP synthesis in SMCs that have adopted the activated intimal phenotype. In vitro, type VIII collagen did not stimulate MMP synthesis in SMCs derived from a quiescent media, despite similar levels of attachment to substrate and the fact that attachment was mediated by same ß1 integrin receptor as intimal cells, and this further underlines the possibility of distinct signaling mechanisms in the two phenotypes.
SMCs plated on type VIII collagen formed focal adhesion plaques during
cell spreading, suggesting the involvement of integrin receptors in
binding. Using three different types of SMCs and integrin blocking
antibodies, we determined that adhesion to type VIII collagen was
mediated via the ß1 integrin subunit in rat intimal and medial SMCs
and human newborn aortic SMCs. Because antibodies against rat
integrin subunits were not available, we used the human SMCs to
determine which integrins were paired with the ß1 integrin in
binding to type VIII collagen. The 2 integrin was the predominant
receptor mediating over 70% of attachment, whereas 1 mediated 30%
of the attachment to type VIII collagen. These in vitro
results should be interpreted with some caution, because a previous
report has suggested that SMCs in vivo in the rat carotid do
not express the 2ß1 integrin receptor.6
Further work
is necessary to determine whether this holds true for all species and
tissues.
Our hypothesis is that type VIII collagen functions as a provisional matrix substrate in the vessel wall after injury. During wound healing in the skin, provisional matrices are transiently synthesized and enable attachment, activation, and migration of stromal and inflammatory cells.26 Once tissue healing is complete, these matrices are cleared and replaced by a more permanent matrix. Type VIII collagen is ideally suited to fulfill the role of a provisional substrate, because, in the injured rat carotid, we saw an extraordinarily rapid induction (within 24 hours) and clearance of type VIII collagen from the vessel wall by 7 days after injury.12 We hypothesize that SMCs adjacent to the vessel lumen and in the neointima increase type VIII collagen synthesis in response to growth factors released at the denuded arterial surface, creating a deposit of provisional matrix that stimulates nearby SMCs and attracts cells from deeper in the media.12 This is consistent with the pattern of immunohistochemical staining for type VIII collagen in the rat carotid, which was reported by Sibinga et al.13 This deposit of activating matrix may then promote cell attachment and stimulate proteinase secretion, facilitating migration into the intima. The protein is easily digested within its globular domains by a variety of serine proteinases and in the collagenous domain by MMPs.46 Up-regulation of plasmin and MMPs occurs between 1 and 7 days after arterial injury,16,18,47,48 and may provide the mechanism for rapid clearance of type VIII collagen from the vessel.
In conclusion, we report that type VIII collagen is able to support attachment, focal adhesion formation, and spreading of vascular SMCs and acts as a strong chemotactic factor. Furthermore, type VIII collagen stimulates MMP production, suggesting regulation of SMC invasion. These studies suggest that type VIII collagen plays a critical role in regulating SMC invasion and migration after vascular injury.
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Acknowledgements |
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Footnotes |
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Supported by the Heart and Stroke Foundation of Ontario, grant 3315, and grants from the Connaught Foundation and St. Michael’s Hospital. M. P. B. is supported by a Research Scholarship from the Heart and Stroke Foundation of Canada.
Accepted for publication October 14, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3 integrin to promote epidermal growth factor receptor phosphorylation and growth. J Cell Biol 1997, 139:279-2931ß1 integrin is expressed during neointima formation in rat arteries, and mediates collagen matrix reorganization. J Clin Invest 1996, 97:2469-2477[Medline]
1ß1 and 2ß1 integrin receptors by human vascular smooth muscle cells: 2ß1 integrin is required for chemotaxis across type I collagen-coated membranes. Am J Pathol 1994, 145:1070-1081[Abstract]
1 type VIII collagen in injured, platelet-derived growth factor-BB stimulated rat carotid arteries. Circ Res 1996, 79:524-5311ß1 and 2ß1 integrins. J Cell Biol 1995, 131:1903-1915vß3, and 5ß1 integrins during human melanoma cell invasion. Cancer Res 1993, 53:3411-34152ß1 is a positive regulator of collagenase (MMP-1), and collagen 1(I) gene expression. J Biol Chem 1995, 270:13548-13552This article has been cited by other articles:
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 U. Hopfer, N. Fukai, H. Hopfer, G. Wolf, N. Joyce, E. Li, and B. R. Olsen Targeted disruption of Col8a1 and Col8a2 genes in mice leads to anterior segment abnormalities in the eye FASEB J, August 1, 2005; 19(10): 1232 - 1244. [Abstract] [Full Text] [PDF]
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A. P. Wong, N. Nili, and B. H. Strauss In vitro differences between venous and arterial-derived smooth muscle cells: potential modulatory role of decorin Cardiovasc Res, February 15, 2005; 65(3): 702 - 710. [Abstract] [Full Text] [PDF]
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 M. S. Aguzzi, C. Giampietri, F. De Marchis, F. Padula, R. Gaeta, G. Ragone, M. C. Capogrossi, and A. Facchiano RGDS peptide induces caspase 8 and caspase 9 activation in human endothelial cells Blood, June 1, 2004; 103(11): 4180 - 4187. [Abstract] [Full Text] [PDF]
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 S. Stephan, M. J. Sherratt, N. Hodson, C. A. Shuttleworth, and C. M. Kielty Expression and Supramolecular Assembly of Recombinant {alpha}1(VIII) and {alpha}2(VIII) Collagen Homotrimers J. Biol. Chem., May 14, 2004; 279(20): 21469 - 21477. [Abstract] [Full Text] [PDF]
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N. Nili, A. N. Cheema, F. J. Giordano, A. W. Barolet, S. Babaei, R. Hickey, M. R. Eskandarian, M. Smeets, J. Butany, G. Pasterkamp, et al. Decorin Inhibition of PDGF-Stimulated Vascular Smooth Muscle Cell Function: Potential Mechanism for Inhibition of Intimal Hyperplasia after Balloon Angioplasty Am. J. Pathol., September 1, 2003; 163(3): 869 - 878. [Abstract] [Full Text] [PDF]
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P. Spessotto, M. Cervi, M. T. Mucignat, G. Mungiguerra, I. Sartoretto, R. Doliana, and A. Colombatti beta 1 Integrin-dependent Cell Adhesion to EMILIN-1 Is Mediated by the gC1q Domain J. Biol. Chem., February 14, 2003; 278(8): 6160 - 6167. [Abstract] [Full Text] [PDF]
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 A. I. Gotlieb, A. Rosenthal, and P. Kazemian Fibroblast growth factor 2 regulation of mitral valve interstitial cell repair in vitro J. Thorac. Cardiovasc. Surg., September 1, 2002; 124(3): 591 - 597. [Abstract] [Full Text] [PDF]
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 M. Zatyka, N. F. da Silva, S. C. Clifford, M. R. Morris, M. S. Wiesener, K.-U. Eckardt, R. S. Houlston, F. M. Richards, F. Latif, and E. R. Maher Identification of Cyclin D1 and Other Novel Targets for the von Hippel-Lindau Tumor Suppressor Gene by Expression Array Analysis and Investigation of Cyclin D1 Genotype as a Modifier in von Hippel-Lindau Disease Cancer Res., July 1, 2002; 62(13): 3803 - 3811. [Abstract] [Full Text] [PDF]
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 G. Hou, W. F. Vogel, and M. P. Bendeck Tyrosine Kinase Activity of Discoidin |
).
Plates were coated with the indicated concentrations of substrates.
Adhesion was quantitated by toluidine blue staining; the absorbance of
toluidine blue at 595 nm is proportional to the number of adherent
cells. Values are mean ± SEM. *Attachment to type VIII collagen
was significantly less than attachment to type I collagen at equivalent
concentrations.
