© 1998 American Heart Association, Inc.
Original Contributions |
Tetraspanin CD9 Is Associated With Very Late–Acting Integrins in Human Vascular Smooth Muscle Cells and Modulates Collagen Matrix Reorganization
From the Laboratoire de Pharmacologie et Physiologie Cellulaires, ERS CNRS 653, Faculté de Pharmacie, Université Louis Pasteur de Strasbourg, Illkirch (A.S., A.B.); INSERM U 311, Etablissement de Transfusion Sanguine, Strasbourg (S.M., D.O.A., F.L.); and Laboratoire de Pharmacognosie, Faculté de Pharmacie, Université Louis Pasteur de Strasbourg, Illkirch (G.H.-A.), France.
Correspondence to Alain Beretz, Faculté de Pharmacie, BP24, 67401 Illkirch, France. E-mail aberetz{at}pharma.u-strasbg.fr
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Abstract—CD9, a member of the tetraspanin family, and very late–acting (VLA) integrins are known to associate and form functional units on the surface of several cell types. We studied the changes in expression of CD9 and ß1-integrins (CD29, VLA) in human vascular smooth muscle cells (VSMCs) under in vitro culture conditions mimicking proliferative vascular diseases. We also investigated possible interactions between CD9 and VLA integrins in VSMCs. We found that CD9 is highly expressed in VSMCs and is subject to modulation, depending on the proliferative/contractile state of the cells. In the contractile phenotype, the levels of CD9, CD81, another tetraspanin, and CD29 are
50% of those found in the proliferative phenotype.
Coimmunoprecipitation experiments showed physical association between
CD9 and CD29. CD9 was mainly associated with 
2 and
3-integrins (CD49b and c) and also with
5-integrin to a weaker extent. Functionally, the
addition of anti-CD9 monoclonal antibodies (MoAbs) doubled the extent
of collagen gel contraction mediated by VSMCs, a model for the
reorganization of the extracellular collagen matrix occurring in the
vessel wall. Anti-CD29 MoAbs inhibited gel contraction, but anti-CD9
MoAbs counteracted this inhibitory effect of anti-CD29
MoAbs. Transfection of human CD9 into Chinese hamster ovary cells more
than doubled the extent of Chinese hamster ovary cell–mediated
collagen gel contraction (130% stimulation), confirming a role for CD9
in extracellular matrix reorganization. Thus, CD9 seems to be involved
in the modulation of VLA integrin–mediated collagen matrix
reorganization by VSMCs. These findings suggest that high CD9
expression is associated with a proliferative state of VSMCs. The role
of CD9 could be to modulate the function of VLA integrins on the
surface of VSMCs.
Key Words: CD9 • tetraspanins • collagen • integrins • smooth muscle
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Introduction |
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During atherosclerosis, vascular smooth muscle cells (VSMCs) display various functional changes such as hypertrophy, hyperplasia, and cell migration.1 2 Three main processes are involved in the formation of atherosclerotic lesions: (1) proliferation of VSMCs, as well as macrophages and possibly lymphocytes; (2) formation by VSMCs of the fibrous plaque, a dense, connective-tissue matrix comprising collagen fibers,3 elastic fibrillary proteins, and proteoglycans; and (3) accumulation of lipids enriched in cholesterol in the matrix and the associated cells.4
VSMCs can occur in 2 main distinct states, referred to as a synthetic, or proliferative, phenotype and a contractile phenotype.5 Contractile-state cells have a muscle-like appearance and predominate in normal vessels. However, these cells can revert to the proliferative phenotype, being 1 important early event in atherosclerosis. For example, after mechanical injury of the endothelium, medial VSMCs migrate into the intima and replicate.6 The proliferative, or synthetic, phenotype is fibroblast-like, being characterized by a rapidly dividing state of VSMCs, loss of contractile properties, and production of extracellular matrix components like elastin or collagen.7 8
Fibrous plaques modify arterial biomechanical
parameters compared with normal vessels and cause at least
instability and sometimes even rupture accompanied by thrombosis and
occlusion.3 9 Collagen matrix reorganization is
mediated by
2ß1-integrin (very
late acting [VLA]2) in many cell types,10 11
including VSMCs.12 Two additional -subunits
(1 and 3) can form
complexes with the ß1-subunit
(CD29)13 to form VLA1 and VLA3 that also function
as collagen receptors.14 15 16
CD9 antigen, 1 of the leukocyte differentiation antigens, is a 24-kDa surface-membrane glycoprotein17 present on platelets, early B cells, activated T cells, eosinophils, and basophils, as well as nonhematopoietic cells like endothelial cells, fibroblasts, or glial cells.18 CD9 belongs to the tetraspanin, or transmembrane 4, superfamily (TM4-SF) that comprises at least 18 cell surface proteins with high sequence homology. Tetraspanins are characterized by the presence of 4 conserved, membrane-spanning, hydrophobic domains and 2 extracellular loops, with both NH2 and COOH termini localized intracellularly (for reviews, see References 18 and 1918 19 ). Recent reports have shown that CD9 and other members of the tetraspanin family, like CD53, CD63, or CD81 (TAPA-1) associate with VLA integrins in some cell types.20 21 22 23 The function of CD9 and other tetraspanins is still largely unknown, but CD9 is thought to play a role in cell differentiation and migration.24 An inverse correlation between metastatic potential and CD9 expression has been shown,25 and there is strict regulation of CD9 expression in the course of lymphocyte and megakaryocyte differentiation and maturation.26 Moreover, anti-CD9 monoclonal antibodies (MoAbs) inhibit VSMC migration27 and augment neutrophil adherence to the endothelium28 or pre-B cell adhesion to bone marrow fibroblasts.29 These results led us to investigate the role of CD9 in some functions of VSMCs related to atherosclerosis.
In the current study, we investigated the modulation of CD9 and CD29 expression in human arterial SMCs having different phenotypes. Because many of the biological events leading to atherosclerotic development are VLA-integrin dependent, we looked for an association of CD9 with VLA integrins in VSMCs. By coimmunoprecipitation experiments and collagen gel contraction assays, we showed both a physical and a functional association between this tetraspanin and some VLA proteins. These findings suggest that CD9 could be a marker of proliferative VSMCs and could modulate some of the integrin-dependent functions of these cells.
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Materials
Aprotinin, antipain, benzamidine, chymostatin, acidic PMSF, and protein G–agarose were from Sigma Chemical Co. Cell culture media RPMI 1640 and M199, HEPES, L-glutamine, penicillin, streptomycin, amphotericin B, G418 sulfate, trypsin-EDTA solution (trypsin 0.5 g/L and EDTA 0.2 g/L), and collagenase were from Gibco. Endotoxin-free human serum was from the Etablissement Régional de Transfusion Sanguine (Strasbourg, France). The serum was prepared from a pool of 13 to 15 healthy donors negative for hepatitis B virus and HIV and was complement-inactivated for 30 minutes at 56°C. Triton X-100 and Tween 20 were from Merck. ECL reagents and Hyperfilms were from Amersham. MoAb ALMA1 and ALMA3 (anti-CD9 MoAbs) were obtained after immunization of mice with human washed platelets, fusion with the myeloma cell line P3x653.Ag8, and screening against GST-CD9 fusion proteins. Final hybridoma clones were isotyped by using an isotyping kit (Boehringer Mannheim). Other MoAbs used included the following: Z81.1 (anti-CD81), H5C6 (anti-CD63), 4B4 (Coulter), and P4C10 (Sigma; anti–ß1-integrin). MOPC21 (Sigma) and MARK1 (Jackson) were used as isotypic control MoAbs. Biotinylated C9 MoAb (anti-CD29) was kindly provided by Dr Zardi (Genoa, Italy). MoAbs to
-integrins were all from Immunotech: HP2B6
(anti-1), Gi9
(anti-2), C3
(anti-3), HP2/1
(anti-4), SAM1
(anti-5), and GoH3
(anti-6).
Cell Culture
Human VSMCs were collected from fragments of kidney arteries
obtained during nephrectomy and cultured with the explant
technique.30 The culture medium was M199/RPMI
1640 (1:1, vol/vol) containing 10 mmol/L HEPES, 2 mmol/L
L-glutamine, antibiotics (100 U/mL penicillin and 100
µg/mL streptomycin), amphotericin B (0.25 µg/mL), and 10% human
serum. The cells were frozen at the second passage and used in
experiments from the third to the 10th passage. Plates were precoated
with type I collagen (0.03 mg/mL) from rat tail, prepared as previously
described by Mauch et al.31 For Chinese hamster
ovary (CHO) cells, the plates were left uncoated, and the medium
contained 0.5 g/L G418 sulfate but not M199. CHO cells expressing high
levels of human CD9 at their surface were obtained after transfection
of the full-length cDNA as reported previously.24
All the media and solutions used had an endotoxin content <0.04 ng/mL,
as tested by a Limulus chromogenic assay.
Hydrated Collagen Gel Contraction Assays
VSMCs were included in tridimensional (3D) collagen gels as
described by Lee et al.12 In brief, cells were
detached and added to neutralized type I collagen (2.5 mg/mL), prepared
as described for plating collagen, at a final concentration of
105 cells/mL. The collagen gel suspensions (2 mL
each) were incubated in 6-well plates (Costar) for 1 hour at 37°C to
polymerize the collagen, culture medium was then added, and the gels
were gently cut away from the sides with a scalpel and lifted off the
bottom of the well. At different times thereafter, the diameter of the
gels was measured with Starwise image analysis software
(Imstar). The same protocol was used for CHO cells with a cellular
density of 7.5x105 cells/mL and a 1 mg/mL
collagen concentration.
Flow Cytometric Analysis
VSMCs in collagen gels (contractile phenotype) were
treated for 35 minutes at 37°C with 0.5 mL collagenase
(350 U/mL). The cells were rinsed once with PBS containing 1% human
albumin and resuspended in FMF medium (RPMI 1640, 5%
inactivated goat serum, and 0.2%
NaN3). Subconfluent VSMCs in culture flasks
(proliferative phenotype) were quickly detached by
trypsin/EDTA, washed once, and resuspended in FMF as above. We
controlled the experimental conditions so that neither trypsin nor
collagenase treatments altered the expression of CD9 (data
not shown). The cells were then first incubated with purified MoAbs at
20 µg/mL for 30 minutes at 4°C, washed once with PBS, resuspended
in FMF containing 10 µg/mL FITC-coupled goat anti-mouse second
antibody, and incubated for 30 minutes at 4°C in the dark. After
dilution in PBS, cells were analyzed in a FACsort
cytofluorometer (Becton Dickinson). Quantification of surface antigen
density was achieved with a calibration kit (QUANTUM 26 premixed, Flow
Cytometry Standards Corp), and results were expressed as molecules of
equivalent soluble fluorochrome.
Immunoprecipitation and Western Blotting
Detached VSMCs (2x106 per condition) were
washed 3x with PBS and resuspended in lysis buffer (1% Triton or 1%
CHAPS in PBS, 1 µg/mL aprotinin, 1 µg/mL antipain, 1 µg/mL
benzamidine, 1 µg/mL chymostatin, and 0.1 µg/mL acidic PMSF). After
a 25-minute incubation on ice, cells were centrifuged for 15
minutes at 10 000 rpm at 4°C. The supernatant was precleared for 2
hours at 4°C under agitation by incubation in lysis buffer containing
0.01% nonimmune mouse serum and protein G–agarose. After
centrifugation for 10 minutes at 3000 rpm, the
supernatant was incubated for 1 hour on ice with 30 µg/mL of MoAb and
precipitated with protein G–agarose in detergent buffer. After a
1-hour incubation at 4°C under agitation, cells were washed 3x in
lysis buffer and twice in 10 mmol/L Tris buffer. The proteins were
analyzed by SDS–polyacrylamide gel electrophoresis
under nonreduced conditions. They were then transferred to a
polyvinylidene difluoride (PVDF) membrane (Millipore) and
incubated in 0.1% casein in PBS plus 0.05% Tween (PBS-T). After 3
washes in PBS-T, the membrane was incubated for 1 hour with specific
biotinylated MoAbs (10 µg/mL) in PBS-T and 0.2% BSA under constant
agitation at room temperature. The membrane was washed 3x with PBS-T
and incubated with streptavidin–horseradish peroxidase. After 3 washes
with PBS-T, the bands were revealed by using the ECL chemiluminescence
kit (Amersham).
Statistical Analysis of Results
All results are expressed as mean±SEM. Student's t
test was used, with P<0.05 considered significant.
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Results |
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In Vitro Models of Contractile and Proliferative Phenotypes
To obtain an in vitro model of the proliferative phenotype, we used cells subcultured on a collagen type I–coated surface. Previous studies have shown a phenotypic reversion to the contractile phenotype with an increase in the volume occupied by myofilaments and in the expression of smooth muscle
-actin mRNA as the cultures become
confluent.32 To avoid this phenotypic reversion,
we used VSMCs at densities between 103 and
4x103 cells/cm2. At this
density, VSMCs had a marked fibroblast-like appearance and exhibited a
doubling time of 24 to 36 hours. To obtain an in vitro model of the
contractile phenotype, VSMCs were cultured in 3D collagen gels
for 6 days. In this case, VSMCs had a less dendritic and a more
elongated morphology compared with proliferating cells and expressed
smooth muscle -actin, whereas the expression of this marker was
almost undetectable in proliferating VSMCs (data not shown).
Expression of CD9 and CD29 in Proliferative Versus Contractile
Phenotypes
To investigate whether CD9 and/or CD29 could be a potential marker
of the phenotypic state of VSMCs, we measured and compared surface
expression of both markers by using quantitative flow cytometry. At the
cellular density used for the proliferating VSMCs
(103 to 4x103
cells/cm2), the amount of CD9 was twice that of
proliferating cultures having higher or lower cell densities (<1000 or
4x103 to 2x104
cells/cm2, data not shown). We showed that CD9 is
downregulated when VSMCs approach the contractile phenotype in
3D collagen gels (Figures 1
and 2A).
CD81, a tetraspanin closely related to CD9, was similarly regulated.
However, the expression of the tetraspanin CD63 was unchanged under the
same experimental conditions (Figure 1). The change from the
proliferative phenotype to the contractile phenotype
was accompanied by a 51% decrease in CD9 surface expression (Figure 2A) and by a 44% downregulation of CD29
surface expression (Figure 2B). The absolute levels of both markers in
a given phenotype were very similar, with an 1:1 CD9 to CD29
ratio.
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Coimmunoprecipitation of CD9/CD29 Complexes
To investigate a potential association between CD9 and
ß1-integrins, we performed
coimmunoprecipitation experiments. Under nonstringent conditions with
1% CHAPS, the anti-CD29 MoAb P4C10 coprecipitated a 24-kDa band
corresponding to CD9, as revealed by Western blotting with biotinylated
anti-CD9 MoAb (Figure 3A). Even under
more stringent conditions with 1% Triton X-100, coprecipitation of CD9
with P4C10 was still present but to a much weaker extent. In the
reciprocal experiment (Figure 3B), the anti-CD9 MoAb ALMA1
coprecipitated a 110-kDa band corresponding to
ß1-integrin under nonstringent conditions but
not in Triton X-100 lysates (data not shown). The isotype control MoAb
MOPC21 coprecipitated neither CD9 (Figure 3A) nor
ß1-integrin (Figure 3B). The coprecipitation
with MoAbs to 1 to
6-integrins with biotinylated ALMA1 (anti-CD9)
showed an association of CD9 with 2 and
3-integrins (Figure 4A) and a weak association with
5. The same experiment with biotinylated C9
(anti-ß1 integrin) shows that
2, 3, and
5 are the main -integrins associated with
ß1-integrin in VSMCs (Figure 4B).
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Effect of MoAbs Against CD9 and CD29 on Contraction of Hydrated
Collagen Gels
The contraction of hydrated collagen gels was used as a model for
the reorganization of the extracellular collagen matrix occurring in
the vessel wall. The possible role of CD9 and CD29 in this process was
investigated by measuring the effect of anti-CD9 and anti-CD29 MoAbs on
the decrease in diameter of hydrated collagen gels with time in
culture. The anti-CD9 MoAb ALMA1 stimulated gel contraction by
decreasing the delay of onset of contraction compared with untreated
cells (Figure 5). Therefore, the extent
of contraction was doubled after 72 hours, and there was still 80%
stimulation when compared with untreated cells after 96 hours. ALMA3,
another anti-CD9 MoAb, produced the same effect (data not shown).
Inversely, the anti-CD29 MoAb 4B4 slowed the gel contraction process,
with 50% inhibition after 72 hours and 36% after 96 hours. The
anti-CD29 MoAb P4C10 produced a similar effect (data not shown). When
the 2 MoAbs ALMA1 and 4B4 were added together, there was no significant
difference versus untreated cells after 24 hours. A significant
stimulation of gel contraction was found after 72 hours, with a 70%
increase compared with control. At 96 hours, the effect was
superimposable with that obtained with the anti-CD9 MoAb alone.
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), irrelevant antibody (MARK1); (
), anti-CD9 MoAb (ALMA1);
(
), anti-CD29 MoAb (4B4); and (
), anti-CD9 plus anti-CD29 MoAbs.
All antibodies were used at 5 µg/mL.
Effect of Human CD9 Transfection on Contraction of Hydrated
Collagen Gels
CD9-transfected CHO cells were used in contraction assays to
confirm that CD9 is implicated in collagen matrix reorganization.
Overexpression of CD9 more than doubled the extent of gel contraction
(130% increase after 216 hours) compared with mock-transfected cells
(Figure 6). Moreover, the delay of onset
of contraction was reduced by 30%, and the speed of contraction was
increased by 43% (46.8 versus 32.8 µm/h, P<0.02,
n=4).
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) and mock-transfected CHO cells ( |
Discussion |
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The basis for this study originates from results suggesting that CD9 may be implicated in cell migration27 and adhesion.28 29 We thus wanted to investigate the possibility that CD9 plays a role in vascular proliferative diseases. In view of the major role played by VLA integrins in cell-matrix interaction occurring during vascular diseases, we simultaneously measured the expression of CD9 and VLA integrins in VSMCs. The questions asked were the following: (1) Is CD9 expressed in human VSMCs? (2) Is this expression modulated in different phenotypes? (3) Is there an association of CD9 and ß1-integrins? (4) What are the functional implications of the expression of CD9 in VSMCs? We first showed that CD9 is expressed in human VSMCs, confirming previous reports.20 26 Quantitative flow cytometry allowed us to estimate a basal expression of 5x105 CD9 molecules per VSMC. We also showed that the ß1-integrin CD29 is expressed in our cells in amounts that are comparable to those of CD9.
Various in vitro models have been proposed to reproduce the contractile
phenotype of VSMCs in culture: these include serum starvation
and/or the use of heparin,33 attachment to
laminin34 or Matrigel,35 or
culture of VSMCs in 3D collagen gels. Collagen gels have been shown to
cause differentiation of several cell types.36 37
Moreover, fibrillar collagen inhibits arterial SMC
proliferation,38 which is consistent with
the low proliferative index of VSMCs in the media of the normal
arterial wall. Thus, a 3D culture system may well induce
the differentiation of VSMCs toward the contractile phenotype
by growth arrest and cell differentiation caused by the surrounding
collagen. In our experience, cells placed in 3D collagen gels showed at
least a 100-fold increase in smooth muscle -actin expression
compared with proliferative VSMCs (data not shown). We thus compared
the expression of CD9 and CD29 in proliferating VSMCs versus cells
placed in 3D collagen gels.
We wished to investigate whether the expression of CD9 and CD29 was
modified during these phenotypic changes. The expression of CD9 and
CD29 in the proliferative phenotype is twice that in the
contractile phenotype (Figure 2
). However, this phenotypic
modulation was not a nonspecific consequence of the experimental
procedures used to obtain the different phenotypes, since the
expression of another tetraspanin, CD63, was not modified from 1
phenotype to the other (Figure 1). We can also notice that
CD81, a tetraspanin known to associate with CD9 and CD29 in many cell
types,18 19 is similarly upregulated when
approaching the proliferative phenotype. The
anti–ß1-MoAb used does not discriminate
between the various VLA subtypes, and preliminary results show that
variations in the expression of individual VLA integrins seem to occur.
We also found that the CD9 to CD29 ratio is close to 1 for both
phenotypes. Our experiments show an expression of
5x105 molecules of CD9 per VSMC in
proliferating cells. This high level of CD9 expression and its
phenotypic modulation, which parallels that of CD29, suggest that CD9
and VLA integrins could play a critical role in some of the cellular
processes involved in the phenotypic change of VSMCs. Moreover, in
other cell types, CD9 and other tetraspanins as well as CD29 have been
implicated in cellular processes known to occur in the development of
atherosclerotic lesions, including cell
adhesion,28 29
motility,27 39 and
proliferation.40 This finding supports a
potential role for CD9 and CD29 in atherosclerotic and
restenotic processes.
An association between CD9 and VLA integrins in this cell type is
supported by coprecipitation of CD9 and
ß1-integrin in VSMCs (Figure 3). CD9 and other
members of the tetraspanin family, like CD53, CD63, CD81, and CD82,
have already been shown to associate with
ß1-integrins in several cell types but never
before in vascular cells. CD9 has been reported to associate with VLA3,
VLA4, and VLA6 but not with VLA1
integrins.20 21 41 42 Interaction with VLA5 seems
so far restricted to pre-B cells23 and
interaction with VLA2 to human epidermal
keratinocytes.43 In
coprecipitation experiments, we showed an association between CD9 and
VLA2 and VLA3 and a weak association of CD9 with VLA5 (Figure 4).
Previous studies by Lee et al12 showed that VSMCs
express abundant levels of VLA1, VLA2, VLA3, and VLA5, whereas the cell
surface expression of VLA4 is low and VLA6 is almost undetectable. In
our cells, the pattern of VLA surface expression was
VLA2>VLA3>VLA5
VLA1=VLA4=VLA6, as tested by quantitative flow
cytometry (data not shown). It is well possible in this case that the
VSMCs cultured on type I collagen could induce the overexpression of
2 and 3-integrins,
which are known to be collagen receptors.13
Additional studies will be necessary to determine the importance of the
extracellular matrix on the association of CD9 with distinct VLAs in
VSMCs.
Collagen matrix reorganization is implicated in the stiffening of
atherosclerotic tissue.44 Contraction assays of
hydrated collagen gels suggest that the CD9/CD29 association could
result in a functional coupling. We showed that anti-CD29
function–blocking antibodies inhibit the contraction of the collagen
gel by VSMCs, probably by inhibiting the VLA2 integrin in accord with
the results of Lee et al.12 In contrast, the 2
anti-CD9 MoAbs tested led to an earlier onset of gel contraction. In
view of the opposing effect of CD9 and VLA integrin MoAbs (Figure 5),
we tested the effects of both antibodies together. The anti-CD9 MoAb
counteracted the effect of the anti–ß1-integrin MoAb on gel
contraction. Moreover, transfection of CD9 into CHO cells led to
increased speed and shorter delay of onset of collagen gel contraction
mediated by these cells (Figure 6). These results implicate CD9 in the
reorganization of the extracellular matrix and suggest that the
association to VLA integrins may serve to either amplify or regulate
the ability of VLA to reorganize collagen lattices. However, the
cellular effect of anti-CD9 MoAbs is largely unknown. In the absence of
any evidence for a direct interaction of CD9 with collagen, we can
speculate that CD9 is a regulating subunit of VLA integrins implicated
in collagen reorganization. This regulation could occur at 2 levels.
CD9 could interfere with integrins in their binding to extracellular
ligands and/or in the transduction of integrin-dependent intracellular
signals. In the latter hypothesis, the anti-CD9 MoAb could stimulate
intracellular events through its binding to CD9, leading to a
modulation of ß1-integrin–dependent
contraction. In this respect, CD9 could act as a "molecular
facilitator" of integrin signaling complexes, as proposed by Maecker
et al.19
The pathways of cell activation after CD9 stimulation are largely
unknown, except in the case of platelet activation. In
platelets, anti-CD9 antibodies lead to the stimulation of the
Fc
R by the Fc region of the antibody, whereas the variable part
is bound on CD9.45 This induces an increase in
intracellular calcium, which is therefore only partially dependent on
CD9. Using fura 2–loaded VSMCs, we found no increase in intracellular
Ca2+ after stimulation with anti-CD9 MoAbs. We
also showed by flow cytometry the absence of FcRs in VSMCs (data not
shown). Therefore, the effect of anti-CD9 MoAbs on gel contraction is
not due to an Fc-dependent increase in Ca2+.
However, this does not exclude the possibility that binding of the MoAb
to CD9 could have other intracellular effects. Recently, a direct
effect of an anti-CD9 F(ab')2 fragment has been
reported involving the activation of the nonreceptor tyrosine kinase
p72syk.46 Activation of a
tyrosine kinase could thus be implicated in the stimulatory effect of
the anti-CD9 antibody in collagen gel contraction. VLA integrins, when
activated by fibrillary proteins such as collagen or
fibronectin, phosphorylate tyrosine kinases such as
p125FAK.47 CD9, through its
ability to physically associate with VLA integrins, could interfere
with p125FAK tyrosine
phosphorylation. The hyaluronan receptor RHAMM is
another example of a nonintegrin membrane
receptor48 that is able to mimic
2ß1 stimulation by
restoring the phosphorylated state of
p125FAK or other tyrosine kinases.
In conclusion, we propose that CD9 could be a potential marker of the atherosclerotic state of blood vessels. Indeed, our study shows an increase in CD9 surface expression in cultured VSMCs when they switch from the contractile to the proliferative phenotype. There are precedents linking overexpression of tetraspanin molecules in the course of the atherosclerotic process. Indeed, Xu et al49 have shown that CD9 is upregulated during monocyte/macrophage differentiation. The mRNA for CD63, another member of the tetraspanin family, is increased in atherosclerotic lesions or in aged vessels.50 The hypothesis that CD9 is a potential marker of the atherosclerotic state of vessels will have to be confirmed by comparing CD9 expression in normal and atherosclerotic tissues. CD9, by its association with VLA integrins, could play an important role in atherosclerosis by modulating the collagen matrix reorganization, a process involved in the thickening of the blood vessel.44 Indeed, we showed in this study that MoAbs directed against CD9 can counteract the inhibition of gel contraction induced by anti-CD29 MoAbs, which is considered a good model of collagen matrix reorganization in atherosclerosis. Moreover, independent studies have shown that anti-CD9 antibodies can counteract VSMC migration,27 another key cellular event occurring in atherosclerotic lesions,1 2 probably by the inhibition of the migration signal induced by VLA integrins.51 The stimulation of collagen matrix reorganization and the inhibition of VSMC migration by MoAbs against CD9 speak for CD9 as a potential target in the treatment of proliferative diseases like atherosclerosis or restenosis after coronary angioplasty.
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Acknowledgments |
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This work was partially supported by grants from the Groupe de Recherche et d'Etude sur les Maladies Artérielles (GREMA, to A.S. and A.B.). Dr David O. Azorsa benefited from a postdoctoral grant of the Eli Lilly–Université Louis Pasteur–Centre National de la Recherche Scientifique program. We thank Dr Kenneth Takeda for his constructive analysis of the manuscript.
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Footnotes |
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Part of this work was presented at the Satellite Symposium of the 11th International Symposium on Atherosclerosis, "Smooth Muscle Cell Proliferation and Differentiation," Bordeaux, France, October 2–4, 1997.
Received January 15, 1998; accepted April 21, 1998.
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