© 2001 American Heart Association, Inc.
Vascular Biology |
Cartilage Oligomeric Matrix Protein (Thrombospondin-5) Is Expressed by Human Vascular Smooth Muscle Cells
From the Department of Medicine III (Cardiology) (R.R., M.F., D.I.A., K.R.K.), University of Tübingen, Tübingen, Germany; and Department of Pathology (H.C., J.L.), Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Mass.
Correspondence to Reimer Riessen, MD, Department of Medicine III (Cardiology), University of Tübingen, Otfried-Müller-Str 10, 72076 Tübingen, Germany. E-mail reimer.riessen{at}uni-tuebingen.de
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Abstract—Cartilage oligomeric matrix protein (COMP/thrombospondin [TSP]-5) belongs to the thrombospondin gene family and is an extracellular glycoprotein found predominantly in cartilage and tendon. To date, there is limited evidence of COMP/TSP-5 expression outside of the skeletal system. The aim of the present study was to investigate the expression of COMP/TSP-5 in cultured human vascular smooth muscle cells and human arteries. COMP/TSP-5 mRNA and protein expression was detected in cultured human vascular smooth muscle cells with both Northern blotting and immunoprecipitation. Serum, as well as transforming growth factor (TGF)-ß1 and TGF-ß3, stimulated COMP/TSP-5 mRNA expression. COMP/TSP-5 was detected in normal as well as atherosclerotic and restenotic human arteries with immunohistochemistry. The majority of COMP/TSP-5 was expressed in close proximity to vascular smooth muscle cells. In vitro attachment assays demonstrated strong adhesion of smooth muscle cells to COMP/TSP-5–coated surfaces, with the majority of cells spreading and forming stress fibers. In addition, COMP/TSP-5 supported the migration of smooth muscle cells in vitro. The present study shows that COMP/TSP-5 is present in human arteries and may play a role in the adhesion and migration of vascular smooth muscle cells during vasculogenesis and in vascular disease settings such as atherosclerosis.
Key Words: Cartilage oligomeric matrix protein • thrombospondin • extracellular matrix • smooth muscle cells
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Introduction |
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The thrombospondins (TSPs) are a family of
5 structurally related
glycoproteins. Of these 5 glycoproteins, only
TSP-1 and -2 have been investigated and detected in vascular tissues to
date. TSP-1 is a trimeric 450-kDa glycoprotein that was
originally isolated from the 
-granules of blood
platelets.1 Subsequent
studies revealed that TSP-1 is also expressed by a variety of other
cell types, including smooth muscle cells (SMCs),
endothelial cells (ECs), fibroblasts, and
macrophages.2 TSP-1
has been detected with immunostaining in
atherosclerotic and restenotic human
arteries.3 4 After
experimental arterial injury, TSP-1 mRNA expression is
upregulated within hours.5
Functionally, TSP-1 has been shown to (1) promote cell adhesion,
proliferation, and
migration,2 6 (2)
modulate
angiogenesis,7 8
(3) regulate plasmin
production,9 and (4)
activate transforming growth factor
(TGF)-ß1.9 10 TSP-2 is similar to TSP-1 in terms of its molecular architecture, but it shows a different pattern of expression in developing organisms.11 In cultured ECs, which express high levels of TSP-1, TSP-2 expression could not been detected.12 In vivo, however, TSP-2 expression has been demonstrated in ECs of normal blood vessels, as well as in SMCs of atherosclerotic arteries.12 Very little is known about the functions of TSP-2, but gene knock-out studies in mice indicate a role in collagen fibrillogenesis and angiogenesis.13
The aim of the present study was to investigate the expression of cartilage oligomeric matrix protein (COMP/TSP-5), a 524-kDa pentameric glycoprotein that is the fifth member of the thrombospondin gene family, in vascular cells.14 COMP/TSP-5 is an abundant component of cartilage and tendon.15 16 Mutations of COMP/TSP-5 in a region of the gene that encodes calcium-binding sites cause pseudoachondroplasia and multiple epiphyseal dysplasia, which are autosomal dominant chondrodysplasias characterized by short stature, early-onset osteoarthritis, and ligamentous laxity.17 In the first description of COMP/TSP-5 by Hedbom et al,15 its expression was found to be restricted to the skeletal system in bovine tissues and newborn rats. However, in a subsequent study, Oldberg et al18 noted a low level of COMP/TSP-5 mRNA expression in rat aortic tissue but did not pursue this finding further. The aim of the present study was to investigate whether COMP/TSP-5 is expressed in human vascular cells and whether it is a component of the normal and atherosclerotic human arterial wall.
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Methods |
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Reagents
Recombinant platelet-derived growth factor (PDGF)-AA, PDGF-BB, TGF-ß1, and TGF-ß3 were obtained from R&D Systems.
Cell Culture
Human arterial SMCs and ECs from vessel
specimens of the iliac artery of human donors for liver
transplantations were isolated and cultured as previously described in
detail.19 20 SMCs
were routinely cultured with a mixture of Weymouth’s MB 752/1 and
Nutrient Mixture Ham’s F-12, supplemented by 10% FCS, 100 U/mL
penicillin, and 100 µg/mL streptomycin (GIBCO BRL). Vascular SMCs
were identified by the characteristic "hill and valley" growth
pattern and positive immunocytochemical staining with a monoclonal
antibody against smooth muscle -actin (Progen). Before the addition
of growth factors, SMCs were kept in serum-free medium (Weymouth’s MB
752/1 and Nutrient Mixture Ham’s F12 supplemented with
insulin/transferrin/thyreoglobulin) for 3 days. Attachment and
migration assays were performed with human saphenous vein SMCs cultured
in low glucose Dulbecco’s modified Eagle’s medium (GIBCO BRL) with
10% FCS, 100 U/mL penicillin, and 100 µg/mL
streptomycin.
ECs were grown on collagen-coated plastic culture dishes (Iwaki Glass) and subcultured in Endothelial Cell Growth Medium Kit (PromoCell) containing bovine brain extract with EC growth factor (ECGF), 2% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin (GIBCO BRL). Cells were characterized as ECs by immunocytochemical staining with polyclonal antibodies against von Willebrand antigen (Boehringer Mannheim). Monolayers of confluent cells grew in the characteristic "cobblestone" pattern. SMCs and ECs were cultured at 37°C in 5% CO2 atmosphere and were used between passages 3 and 6.
cDNA Probes
For Northern blot analysis, the following
cDNA probes were used: human TSP-1 (1.1
kb),21 human TSP-2 (3.1 kb),
human-COMP/TSP-5 (2.4 kb),14
and human GAPDH (Clontech). All cDNAs were labeled nonradioactively
with digoxigenin-conjugated nucleotides using a random
primed labeling reaction (DIG-Highprime; Boehringer Mannheim).
Concentration of the labeled probe was estimated by dot blotting
according to the manufacturer’s instructions.
Northern Blot Analysis
Agarose/formaldehyde gels were loaded with 10 µg
RNA per lane, and RNA was resolved by electrophoresis. RNA was
transferred to a nylon membrane (Hybond N+;
Amersham) and crosslinked with ultraviolet radiation. Hybridization and
detection of digoxigenin-labeled probes (DIG-System; Boehringer
Mannheim) were based on previously published
protocols.22 23
Membranes were simultaneously hybridized with combinations
of cDNA probes (6 to 32 ng labeled cDNA each) to avoid repeated
stripping and reprobing of the membrane (resulting bands in
parentheses): human TSP-1 (7.0 kb), human TSP-2 (7.0 kb), human
COMP/TSP-5 (2.8 kb), and GAPDH (1.4 kb) as a control gene to check for
equal loading of the lanes. Hybridized cDNA was visualized on x-ray
film with a chemiluminescence reaction of an alkaline
phosphatase–conjugated anti-digoxigenin Fab fragment (dilution
1:15 000) with the luminescence reagent CSPD (dilution 1:100;
Boehringer Mannheim). After detection, the 28S and 18S rRNA
bands on the membranes were stained with 0.03% methylene blue (in 0.3
mol/L Na-acetate, pH 5.2) as an additional control for equal loading
and transfer of RNA.
Antibodies
The anti–COMP/TSP-5 antibody (F8, polyclonal
rabbit)24 used for
immunoprecipitations and staining of atherectomy specimens was purified
on a protein A-Sepharose column. The IgG was used at a concentration of
8 µg/mL. Negative controls were performed with preimmune serum IgG.
Polyclonal rabbit antibodies raised against human COMP/TSP-5
(generously supplied by Dr M. Paulsson, University of Cologne) were
purified, concentrated on a Sepharose A column, and used for blocking
of cell attachment.
Additional immunostainings were performed
with a monoclonal anti–-smooth muscle actin–alkaline phosphatase
antibody (dilution 1:300, clone 1A4; Sigma Chemical Co) and a
monoclonal anti–proliferating cell nuclear antigen (PCNA) antibody
(dilution 1:40, clone PC10; Sigma Chemical Co).
Metabolic Labeling of Cells and
Immunoprecipitation
Human smooth muscle cells and human chondrocytes (as
positive control cells) were seeded onto 6-well tissue culture dishes
and grown to near-confluence. Cells were washed 3 times with serum-free
medium and incubated in the medium for 6 hours. Cells were then washed
with methionine- and cysteine-free medium and switched to the same
medium containing 50 µCi 35S-methionine
and 35S-cysteine (New England Nuclear) and
labeled for 20 hours. Some wells contained either 10 ng/mL TGF-ß1 or
10% FCS. At the end of the labeling, conditioned medium was harvested,
centrifuged to clear off cell debris, and PMSF was added
to a final concentration of 1 mmol/L. Medium was then stored at
-20°C.
For immunoprecipitation, 40 µL of protein A-Sepharose was added to each sample and incubated for 20 minutes at 4°C. The samples were centrifuged, and the supernatant was incubated with 5 µL F8 serum and 40 µL protein A-Sepharose for 2 hours at 4°C with gentle agitation. After centrifugation, the pellets were washed 3 times with Tris-buffered saline containing 0.1% Tween 20. The pellets were boiled for 4 minutes in SDS sample buffer containing 10% ß-mercaptoethanol and analyzed by SDS-PAGE. The intensity of the bands were quantified with PhosphorImager scans.
Immunohistochemistry of Human Atherectomy
Specimens
Tissue specimens were retrieved through directional
atherectomy from 10 patients with peripheral artery disease
as previously
described.25 26
Three specimens were obtained from restenotic lesions between 9
and 12 months after previous balloon angioplasty. Seven specimens were
retrieved from sites not previously treated with
percutaneous revascularization and
were designated as primary lesions. In addition, mammary arteries from
5 patients were obtained during bypass surgery. Methanol-fixed and
paraffin-embedded slides were deparaffinized, endogenous
peroxidase was quenched with 3%
H2O2 in PBS, and
unspecific protein binding was blocked by incubation with normal goat
or horse serum (dilution 1:100). Primary antibodies were applied for 1
hour at room temperature. Slides were washed in PBS plus 0.1% Tween
20. Fast red served as a substrate for the alkaline
phosphatase–conjugated SMC -actin antibody (Signet). For all other
immunostainings, the Vectastain Universal Elite ABC Kit
(Vector) was used with diaminobenzidine as the chromogen. Slides were
counterstained with toluidine blue for 5 minutes, washed with distilled
water, and mounted with Permount (Fisher).
Attachment Assays
Human TSP-1 was isolated from platelets as
previously
described.27 28
Recombinant COMP/TSP-5 was obtained from transfected human embryonic
kidney cells (293 cells) (Hui Chen and Jack Lawler, manuscript in
preparation). Human fibronectin was purchased from Sigma Chemical Co.
To coat dishes (Immunolon 2 or 4; Dynatech) with COMP/TSP-5, TSP-1, or
fibronectin, the proteins (1 to 20 µg/mL) were first dissolved in
HEPES-buffered saline (HBS) (10 mmol/L HEPES, pH 7.2, 3
mmol/L KCl, 0.5 mmol/L MgCl2, 136
mmol/L NaCl with or without 1 mmol/L
CaCl2).29
Dishes were then incubated overnight at 4°C with the protein
solutions and washed 3 times with HBS with
CaCl2, and unspecific protein binding was
blocked for 1 hour at room temperature with 0.2% BSA solution
(fraction V, heat-denatured at 70°C for 1 hour; Sigma Chemical Co).
Specific binding to COMP/TSP-5 was blocked by adding a polyclonal
COMP/TSP-5-antibody (25 or 50 µg/mL) to the BSA solution. SMCs were
washed 3 times with HBS without CaCl2 and then
trypsinized with TPCK-trypsin solution (Worthington Biochemical
Corporation), dissolved in HBS without CaCl2 to
a final concentration of 0.1 mg/mL. After 3 to 5 minutes, proteolysis
was stopped with soybean trypsin inhibitor (SBTI, 0.5 mg/mL
in HBS with CaCl2, Sigma Chemical Co). Cells
were sedimented at 1000 rpm for 5 minutes and then washed 2 times in
SBTI solution. Subsequently, cells were resuspended in HBS with
CaCl2, the BSA solution was removed from the
dishes, and the cell solution was added. Plates were allowed to attach
for 2 hours at 37°C. In 1 set of experiments, half of the SMCs were
incubated with cycloheximide (20 µg/mL; Sigma Chemical Co) during
this attachment period and the preceding 2 hours to block
endogenous protein and ECM synthesis. Subsequently, dishes
were washed 3 times with HBS containing CaCl2.
Plates were frozen at -80°C for 30 minutes, and subsequently,
attached cells were quantified with CyQUANT (Molecular Probes)
according to the manufacturer’s instructions. Nonspecific cell
adhesion was determined by coating the dishes with heat-denatured BSA,
and this value was subtracted from the measured values. The reported
value is the average of 8 wells per experimental group. For
statistical analysis, ANOVA followed by Scheffé’s test was
used.
Staining of Cell Attachment
Laboratory-Tek chamber slides (Nunc) were coated with
TSP-1, COMP/TSP-5, or fibronectin (20 µg/mL in HBS with
CaCl2). SMCs were added and incubated for 2
hours at 37°C. Staining of the filamentous actin of the cells was
performed as previously
described.27 Briefly, cells
were fixed in 3.7% formaldehyde for 10 minutes, and
permeabilized for 10 minutes in 50 mmol/L MES, pH
6.1, 5 mmol/L MgCl2, 3 mmol/L EGTA,
100 mmol/L KCl, and 0.2% Triton X-100. TRITC-phalloidin (Sigma
Chemical Co) was applied at a concentration of 200 ng/mL for 1 hour at
room temperature. Cells were then washed, and slides were mounted.
Stained cells were examined with a fluorescence
microscope.
Migration Assay of SMCs Toward
COMP/TSP-5
Purified COMP/TSP-5 and TSP1 were coated at different
concentrations on the underside of the membrane of ChemoTx cell
migration chambers (Neuro Probe) at 4°C overnight. The membrane was
blocked with 1% BSA in HBS containing 2 mmol/L
CaCl2. SMCs were trypsinized and prepared for
migration assays using the protocol described here for attachment
assays and resuspended in HBS with CaCl2
containing 1% BSA. Cells were loaded on top of the membrane, and the
migration assay was allowed to proceed for 6 hours at 37°C. At the
end of the incubations, the cells that remained on the upper side of
the membrane were wiped off with Q-tips, and individual membranes were
cut out; the cells that had migrated through the membrane were
quantified with CyQUANT.
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Results |
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Expression of COMP/TSP-5 mRNA in Cultured Human SMCs
Expression of COMP/TSP-5 mRNA by human vascular SMCs was demonstrated by Northern blotting (Figure 1
). Serum, as well as TGF-ß1 and TGF-ß3, strongly
increased COMP/TSP-5 mRNA levels
(Figures 1A and 1B). COMP/TSP-5 mRNA levels reached a maximum
at 2 to 4 hours after the beginning of stimulation
(Figure 1C). In contrast, stimulation with PDGF-AA and -BB
did not consistently increase COMP/TSP-5 mRNA expression (not
shown). Hybridization with the full-length COMP/TSP-5 cDNA resulted in
the detection of a second band with a size of 
0.5 kb
(Figure 1). The intensity of the 0.5-kb band was proportional
to the intensity of the GAPDH housekeeping gene and was not affected by
stimulation with growth factors. The 0.5-kb band disappeared when
modified COMP/TSP-5 cDNA probes were used in which the untranslated
regions of the full-length cDNA were removed, indicating a nonspecific
hybridization of the untranslated regions of the full-length COMP/TSP-5
cDNA (not shown). Detection of the 0.5-kb band was used in the
following studies as a positive control for hybridization with the
COMP/TSP-5 probe. Expression of COMP/TSP-5 was compared with the
expression of the other TSP-genes
(Figure 1). SMCs expressed TSP-1 and -2 under serum-free
conditions. Serum or growth factors (PDGF-AA, PDGF-BB, TGF-ß1, or
TGF-ß3) did not further increase TSP-1 or -2 mRNA levels in human
SMCs.
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Expression of COMP/TSP-5 mRNA in Cultured Human
ECs
In human ECs, COMP/TSP-5 mRNA expression could not be
detected, even under stimulation with growth factors
(Figure 1D
). The presence of the 0.5-kb band in these
experiments confirmed the hybridization with the COMP/TSP-5 cDNA probe.
Hybridization with the TSP-2 probe revealed a very weak signal. In
contrast, cultured ECs strongly expressed TSP-1, but again, growth
factors had no stimulatory effect. Because ECs would not grow in
serum-free conditions, cells were grown in the presence of 2% serum.
This level of serum may have been sufficient to maximally stimulate
TSP-1 gene expression.
COMP/TSP-5 Protein Is Synthesized by Cultured
Human SMCs
Because COMP/TSP-5 is a constituent of bovine
serum, we have metabolically labeled the proteins that are
synthesized by cultured SMCs to establish that COMP/TSP-5 is secreted
by SMCs. Immunoprecipitation with the anti–COMP/TSP-5 antiserum F8
revealed the presence of a 110-kDa band in the media conditioned by
either SMCs or chondrocytes
(Figure 2). Western blotting confirmed that this band
corresponds to reduced COMP/TSP-5 (data not shown). COMP/TSP-5 protein
synthesis was detected under serum-free culture in both cell types and
was stimulated by the addition of 10% FCS or 10 ng/mL TGF-ß1.
Treatment with serum or 10 ng/mL TGF-ß1 increased the level of
COMP/TSP-5 protein expression in SMCs by 121% and 79%,
respectively.
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Immunohistochemical Staining of COMP/TSP-5 in
Human Arteries
Immunostaining for COMP/TSP-5 revealed
strong staining in the medial layer of normal internal mammary arteries
(Figure 3). In sections with intimal thickening,
subendothelial SMCs also showed COMP/TSP-5 staining. In
the endothelial layer, however, COMP/TSP-5 staining was
negative. Strong staining for COMP/TSP-5 was also found on the vast
majority (>90%) of -actin–positive SMCs in human primary
atherosclerotic and restenotic lesions
(Figure 4). A small portion of COMP/TSP-5–negative and
-actin–positive SMCs were located predominantly in relatively
hypocellular areas of primary atherosclerotic plaque. The COMP/TSP-5
staining appeared to be cell associated, with very little COMP/TSP-5
present in the extracellular matrix. COMP/TSP-5 staining on SMCs
was not related to the presence or absence of the proliferation marker
PCNA (not shown).
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Attachment and Migration of SMCs in Response to
COMP/TSP-5
SMCs attached to COMP/TSP-5 adsorbed to plastic dishes
in the presence of 1 mmol/L Ca2+ but
not to heat-treated BSA. The attachment of the SMCs increased with
increasing concentrations of COMP/TSP-5 and reached a maximum at 10
µg/mL
(Figure 5A). The specificity of COMP/TSP-5 attachment could
be confirmed by blocking with a polyclonal anti–COMP/TSP-5 antibody
(Figure 5B). The number of cells attached to
COMP/TSP-5–coated dishes was 4 times higher than that attached to
TSP-1–coated dishes
(Figure 5C). In contrast, SMC attachment to COMP/TSP-5 and
fibronectin was found to be comparable
(Figure 5D). Blocking of SMC protein and ECM synthesis by the
addition of cycloheximide to the culture medium did not significantly
alter attachment to COMP/TSP-5 or fibronectin
(Figure 5D). ECs also showed strong attachment to
COMP/TSP-5–coated dishes, which was 10 times higher than that to
TSP–1-coated dishes
(Figure 5C). Phalloidin staining demonstrated that most of
the SMCs (80%) spread on COMP/TSP-5–coated dishes (not shown).
Part of the SMCs displayed extended protrusions and an irregular cell
boundary, whereas others exhibited well-defined regular edges. SMCs
attached to fibronectin showed similar morphological features. In
contrast, SMCs grown on TSP-1 stayed
rounded.29
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COMP/TSP-5 supported the migration of SMCs
(Figure 6). Compared with TSP-1, COMP/TSP-5 seemed to be a
better substrate for SMC migration, especially at lower concentrations
(
50 µg/mL). For example, whereas very few cells migrated at 10
µg/mL TSP-1, this concentration of COMP/TSP-5 supported high levels
of SMC migration.
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Discussion |
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This study shows that COMP/TSP-5, the fifth member of the thrombospondin gene family, is a component of the human arterial wall and is predominantly produced by vascular SMCs. Previous studies have demonstrated the presence of COMP/TSP-5 in cartilage, tendon, bone, and synovium and have revealed a role in the pathogenesis of pseudoachondroplasia, multiple epiphyseal dysplasia, and rheumatoid arthritis.17 Expression of COMP/TSP-5 in vascular tissues has so far been reported only by Oldberg et al.18 These authors investigated the presence of COMP/TSP-5 mRNA in different rat tissues and found that aorta was the only tissue outside the skeletal system in which COMP/TSP-5 expression could be detected.
In the present study, we detected the expression of COMP/TSP-5 mRNA in human SMCs with Northern blotting. Protein expression in SMCs could be confirmed by metabolic labeling and immunoprecipitation with antibodies against COMP/TSP-5. Immunohistochemistry revealed that COMP/TSP-5 is a component of the normal, the atherosclerotic, and the restenotic human arterial wall. COMP/TSP-5 is localized predominantly in close proximity to SMCs and not in the free extracellular matrix. This association with SMCs was similar in normal, atherosclerotic, and restenotic arteries. Only a small portion of SMCs, mainly in the hypocellular areas of primary plaques, were COMP/TSP-5 negative. In addition, the presence of a cell proliferation marker (PCNA) was not associated with an altered COMP/TSP-5 staining pattern. COMP/TSP-5 expression in ECs was below our limit of detection, and COMP/TSP-5 staining of the endothelium was not observed on immunocytochemistry.
The distribution of COMP/TSP-5 in atherosclerotic and restenotic lesions differed markedly from the distribution of TSP-1, which was investigated in a previous study.4 TSP-1 staining was detected only in a small fraction of SMCs in these lesions and was mostly confined to the extracellular matrix of hypocellular atherosclerotic plaque. Animal studies suggest, however, that SMCs express high levels of TSP-1 in the very early phase (ie, hours to days) after vascular injury,4 5 30 which is usually missed when atherectomy specimens are analyzed. The expression of TSP-2 in arterial tissues has not been studied with immunohistochemical techniques due to a lack of specific antibodies. One study, however, demonstrated TSP-2 mRNA expression in human atherosclerotic plaque with the use of in situ hybridization.12
Our in vitro studies indicated that the expression of COMP/TSP-5 in human SMCs is differently regulated than the expression of TSP-1 and -2. COMP/TSP-5 mRNA expression in subconfluent human SMCs could be markedly stimulated with serum or TGF-ß1 and -3, whereas the high expression of TSP-1 and -2 even in serum-free medium was not further increased by serum or growth factors. In our study, serum, recombinant PDGF-AA/BB, and TGF-ß1/3 did not clearly and consistently increase the high baseline expression of TSP-1 and -2 in human SMCs grown in serum-free medium. Earlier studies have demonstrated an increased TSP-1 expression in response to serum, PDGF, and TGF-ß in rat SMCs.31 32 33 34 These differences might be due to several factors. (1) It is known that at least mouse and human TSP-1 promotor regions differ in their transcription factor binding sites and their serum responsive elements.16 35 (2) In the work by Majack et al,31 32 33 mostly PDGF and TGF-ß isolated from platelets were used, which might contain a mixture of different growth factor isoforms (eg, PDGF-AA, -AB, and -BB) and might also be contaminated by other growth factors (purity was >95%). In the study by Hugo et al,34 the source of PDGF-BB was not mentioned. In contrast, we used recombinant growth factors for all experiments. (3) In the studies by Majack et al33 and Liau and Chan,36 in which rabbit SMCs were used, TGF-ß increased TSP-1 expression only in confluent, density-arrested SMCs, not in sparse SMCs. For our experiments, we used subconfluent SMCs. TSP-2 lacks the serum-responsive elements found in the promotor region of TSP-1 and does not respond to serum stimulation after mild serum deprivation (0.4% serum for 24 hours).37 However, Laherty et al38 could demonstrate an induction of TSP-2 by serum when Swiss 3T3 cells were kept serum free under very stringent conditions (0.2% serum for 5 to 6 days). In our experiments, SMCs were kept in serum-free medium (Weymouth’s MB 752/1 and Nutrient Mixture Ham’s F-12 supplemented with insulin/transferrin/thyreoglobulin) for 3 days.
COMP/TSP-5 and TSP-1 also differed functionally in cell attachment and migration assays. SMCs attached to surfaces coated with COMP/TSP-5 and then spread and organized stress fibers. SMC attachment on COMP/TSP-5 was similar to attachment on fibronectin in terms of cell morphology and quantitative assays, whereas SMCs attached much less on TSP-1. COMP/TSP-5–coated membranes also supported the migration of SMCs.
In summary, the present study demonstrates that COMP/TSP-5 is a component of the normal and atherosclerotic human arterial wall and is expressed predominantly by vascular SMCs. COMP/TSP-5 in the normal media as well as the primary atherosclerotic arterial wall may facilitate SMC migration. COMP/TSP-5 differs in its distribution and its functional properties from the structurally related glycoprotein TSP-1. Further ultrastructural studies should clarify whether COMP/TSP-5, together with other extracellular matrix molecules such as collagen type IV, laminin, and heparan sulfate, is a component of the basement membrane that surrounds vascular SMCs and whether it, for example, sequesters calcium in close proximity to the cell membrane and its receptors.16 39
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Acknowledgments |
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This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (Ri 663/1-3); the Federal Ministry of Education, Science, Research and Technology (Fö 01 KS 9602); the Interdisciplinary Center for Clinical Research (IZKF), Tübingen; the University of Tübingen (Fortüne-Programm); and National Institutes of Health, National Heart, Lung, and Blood Institute grants HL-28749 and HL-49081. We thank Ulrike U. Herzog and Heike Runge for technical assistance. We also wish to thank Dr Jeffrey M. Isner (St Elizabeth’s Medical Center, Boston, Mass) for providing atherectomy specimens, Dr M. Paulsson (University of Cologne, Cologne, Germany) for supplying COMP/TSP-5-antibodies, and Dr Peter Libby (Brigham and Women’s Hospital, Boston, Mass) for supplying the human saphenous vein SMCs.
Received January 11, 2000; accepted August 7, 2000.
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