© 1997 American Heart Association, Inc.
Articles |
Thrombospondin-1 Is a Potent Mitogen and Chemoattractant for Human Vascular Smooth Muscle Cells
From the Department of Clinical Pharmacology, Imperial College School of Medicine at St Mary's, London, UK.
Correspondence to Dr Mahendra Patel, Department of Clinical Pharmacology, Imperial College School of Medicine at St Mary's, St Mary's Hospital, London W2 1NY, England UK. E-mail m.k.patel{at}ic.ac.uk
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Abstract |
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Abstract Thrombospondin-1 (TSP-1) is a matricellular protein that is present in negligible amounts in normal human vasculature but occurs in significant amounts in diseased vessels. In this study, we examined the effect of TSP-1 on DNA synthesis, proliferation, and migration in human vascular smooth muscle cells grown from saphenous vein. TSP-1 (0.1 to 30 µg/mL) elicited a concentration-dependent increase in DNA synthesis under serum-free conditions. In combination with platelet-derived growth factor, TSP-1 induced a synergistic effect on DNA synthesis that was significantly higher than the additive effect of both agents. In proliferation assays, TSP-1 increased cell numbers by 50% relative to the serum-free controls over 14 days. In migration assays, conducted using modified Boyden chambers, TSP-1 (
10
µg/mL) elicited marked chemotaxis to a degree equivalent to
platelet-derived growth factor. The chemotactic response to TSP-1
(10 µg/mL) was abolished by the GRGDSP peptide but unaffected by the
control GRGESP peptide, whereas neither peptide inhibited DNA synthesis
stimulated by TSP-1. Inhibition of tyrosine kinase activity with
genistein or tyrphostin A23 abolished DNA synthesis induced by TSP-1,
and a neutralizing antibody to platelet-derived growth factor had
no effect on DNA synthesis. Similarly, migration in response to TSP-1
was largely inhibited by these tyrosine kinase inhibitors.
TSP-1 is a strong mitogen and chemoattractant for human vascular smooth
muscle cells under serum-free conditions. The novel finding that TSP-1
is mitogenic for human cells contrasts with previous
studies that have not shown any significant effect of TSP-1 itself on
the growth of animal-derived smooth muscle cells. TSP-1 may play an
important modulatory role in the local regulation of vascular smooth
muscle function in vascular pathologies in humans.
Key Words: thrombospondin-1 • DNA synthesis • proliferation • migration • human vascular smooth muscle
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Introduction |
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Two of the key events that occur during the formation of an atherosclerotic lesion and after vascular injury in blood vessels during surgical intervention are the migration of VSMCs from the media into the intima and the proliferation of some of these VSMCs.1 Numerous growth-regulatory molecules are involved in cell proliferation, including both stimulatory and inhibitory agents. Among the most important in terms of cell proliferation are PDGF,2 basic fibroblast growth factor,3 and TGFß1.4 5 These factors are capable of inducing VSMC proliferation and are not generally expressed in normal vessels, whereas they are upregulated during atherogenesis and vascular injury. PDGF, basic fibroblast growth factor, and TGFß1 are pleiotropic regulators in that they are also chemoattractants, and it is particularly interesting that some molecules have the ability to coordinate both the migration and proliferation of smooth muscle cells.
TSP-1 is an archetypal matricellular protein6 in that it
is an extracellular glycoprotein that binds to matrix
proteins in addition to cell-surface receptors or to molecules such as
cytokines or proteases, which themselves interact with the cell
surface. TSP-1 was originally isolated as a 420-kD protein released
from the 
granules of platelets that had been stimulated by
thrombin.7 8 9 Subsequent studies have identified that the
TSP gene family comprises five multimeric
isoforms10 11 12 13 14 15 that are expressed in a variety of tissues
but display different temporal and spatial
distributions.16 17 18
In the vasculature, TSP-1 is associated with atherosclerotic lesions,19 occluded grafts,20 and VSMC hyperplasia after vascular injury21 or hypertension.22 By contrast, only negligible amounts of TSP-1 are found in normal vasculature, suggesting some undefined role for TSP-1 in pathological changes in the vasculature. TSP-1 expression is rapidly and transiently increased after tissue damage and platelet activation. A number of cell types, including VSMCs,23 monocytes,24 platelets,9 endothelial cells,25 26 and fibroblasts,27 secrete TSP-1. Stimulation of quiescent rat VSMCs with PDGF has been shown to upregulate TSP-1 at the transcriptional level within 30 minutes28 and at the protein level within an hour.29 The kinetics of gene expression, along with its independence from de novo protein synthesis, have led to the classification of TSP-1 as an immediate early-response gene.28 The role of TSP-1 in the proliferative response of VSMCs to PDGF and the potentiation of mitogenesis induced by EGF30 31 has suggested a modulatory function for TSP-1 in VSMC proliferation. In this investigation, we have examined the effect of TSP-1 on DNA synthesis, proliferation, and migration of human VSMCs grown from saphenous vein.
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Methods |
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Materials
Cell-culture plastics, media, supplements, TSP-1 (purified from human platelets), recombinant human PDGF-BB, and TGFß1 were obtained from Life Technologies. TSP-1 was purified in the presence of Ca2+ by affinity chromatography, using heparin-agarose and gelatin-Sepharose to remove fibronectin, ß-thromboglobulin, platelet factor 4, and fibrinogen.32 33 34 Immunoblotting studies have been conducted to assess possible contamination of the human platelet-derived TSP-1 with either TGFß1 or PDGF. These experiments have failed to detect both PDGF and TGFß1 in the commercial TSP-1 employed in this investigation (data not shown).
Recombinant human TSP-1 expressed in a baculovirus expression system (Adams and Lawler, unpublished data, 1997) was the generous gift of Dr J. Adams (MRC Laboratory for Cell Biology, University College London, UK) and Dr J. Lawler (Harvard Medical School, Boston, Mass).
FCS was obtained from MB Meldrum Ltd. [methyl-3H]Thymidine (740 GBq/mmol) was purchased from ICN Flow. Polycarbonate filters were obtained from Poretics. The CytoTox 96 nonradioactive cytotoxicity assay kit was obtained from Promega. Neutralizing chicken antibody to TGFß1 (AB-101-NA) and nonimmune chicken immunoglobulins, as well as neutralizing rabbit antibody to PDGF (AB-20-NA) and nonimmune rabbit immunoglobulins, were purchased from R&D Systems Europe. Tyrphostin A23, GRGDSP, and GRGESP peptides were obtained from Calbiochem Novabiochem. Trichloroacetic and hydrochloric acids were obtained from Fisher Scientific. Bovine serum albumin fraction V was purchased from Boehringer Mannheim. All other reagents were obtained from Sigma Chemical Company.
Cell Culture
Human VSMCs were grown from saphenous vein, using an explant
technique as described previously.35 VSMCs were routinely
grown in DMEM buffered with 25 mmol/L HEPES and
supplemented with 15% (vol/vol) FCS, 4 mmol/L
l-alanyl-l-glutamine (Glutamax-I), penicillin (100 U/mL), streptomycin
(100 µg/mL), and gentamicin (25 µg/mL). Cell cultures
were maintained in a humidified atmosphere of 5% CO2
(vol/vol) in air at 37°C. VSMCs at the third passage were
employed for all the experiments described in this investigation. Cell
strains from different patients were selected for experimentation on
the basis of availability.
Immunocytochemical studies of human VSMCs have shown positive staining
for -actin, a smooth muscle marker,36 but negative
staining for factor VIII–related antigen, a marker for
endothelial cells.
Measurement of DNA Synthesis
DNA synthesis was determined by measuring the incorporation of
[methyl-3H]thymidine into acid-insoluble material as
described previously.37 Briefly, quiescent cells (in
96-well plates), which had been deprived of FCS for 120 hours, were
treated with supplemented serum-free medium comprising DMEM
supplemented with insulin (1 µmol/L), sodium selenite
(0.1 µmol/L), transferrin (63.5 nmol/L, 5
µg/mL), and 0.1% (wt/vol) bovine serum albumin
fraction V38 with or without defined growth factor(s).
[methyl-3H]Thymidine was added (1 µCi per well, 5
µCi/mL) between 24 and 30 hours after stimulation, and the experiment
was terminated. In the time course experiment, however,
[methyl-3H]thymidine (1 µCi per well, 5 µCi/mL) was
added for the last 6 hours of each time interval.
Proliferation Assay
A 14-day growth assay was performed with minor modifications to
the technique previously described.35 Briefly, cells were
seeded at 2.5x104 per well in 24-well plates (using DMEM
supplemented with 15% FCS) and allowed to attach overnight. The growth
medium was removed on the following day and the cells were washed twice
with Dulbecco's PBS without calcium and magnesium (PBS-A). The
cultures were rendered quiescent by incubating the cells for 72 hours
in NCTC 109 medium supplemented with 25 mmol/L HEPES,
4 mmol/L Glutamax I, 0.25%(wt/vol) bovine serum
albumin fraction V, and antibiotics. Cell counts were performed
on triplicate wells, using a model D industrial Coulter counter
(Coulter Electronics), after harvesting with 0.25% (wt/vol)
trypsin/1 mmol/L EDTA in PBS-A on days 0, 3, 7, 10, and 14
after growth arrest. Fresh growth medium (NCTC 109) supplemented with
TSP-1 (5 µg/mL, 1.19x10-8
mol/L) or PDGF-BB (20 ng/mL,
6.67x10-10 mol/L) was replenished at
these time points.
Cytotoxicity Assay
The nonradioactive assay employed in this investigation was
based on the measurement of lactate dehydrogenase released from the
cytosol of damaged cells. The assay was conducted according to the
supplier's protocol and measured the conversion of a tetrazolium salt
into a red formazan product in the presence of lactate
dehyrogenase.
Migration Assays
These assays were performed using blind-well chemotaxis chambers
(Neuroprobe). Briefly, VSMCs were trypsinized and resuspended in DMEM
supplemented with 15% FCS. The resultant cell suspension was
centrifuged (1000 rpm, 10 minutes) and resuspended in
serum-free DMEM supplemented with 0.1% (wt/vol) bovine serum
albumin fraction V. Viability was assessed using trypan blue
dye exclusion and a cell suspension prepared at a density of
2.25x105 viable cells per milliliter.
The upper and lower compartments of the blind-well chambers were separated by 13-mm gelatin-coated polycarbonate filters with 8-µm pores. The relevant control or chemoattractant (0.3 mL) was added to the lower compartment and cell suspension (0.4 mL, 2.25x105 cells per milliliter) added to the upper chamber unless otherwise stated. The migration assay was allowed to proceed for 5 hours at 37°C in a humidified atmosphere of 5%CO2 in air before the assay was terminated by fixing the cells in absolute ethanol. The filters were stained for 15 minutes in a 65 mmol/L toluidine blue solution, and the cells on the upper side of the filter were gently scraped off, leaving the migrated cells on the underside of the filters for counting. The cells present in four fields of view (x200 magnification) were counted on duplicate filters.
Statistics and Data Analysis
Data are presented as mean±SEM of the number of
observations. DNA synthesis and migration experiments were compared
using a paired t test. P<.05 was considered
significant. Concentration-response data were fitted to a logistic
function by nonlinear regression using a macro written by one of the
authors (A.D. Hughes) in Excel (Microsoft).
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Results |
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DNA Synthesis and Proliferation Studies
The induction of DNA synthesis by TSP-1 (5 µg/mL, 1.19x10-8 mol/L) was examined over a 96-hour period under serum-free conditions in human saphenous vein–derived VSMCs (Fig 1
). TSP-1
elicited maximum DNA synthesis over the 24 to 30 hour time interval in
these cells. The peak DNA synthesis in response to PDGF-BB (10
ng/mL, 3.33x10-10 mol/L) and
15% FCS also occurred over the same time period. In subsequent
experiments, DNA synthesis was assessed by using a pulse of
[methyl-3H]thymidine added during the 24 to 30 hour
period after stimulation of quiescent cells.
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TSP-1 induced a concentration-dependent increase in DNA synthesis under
serum-free conditions, with a 91.5±50.1% and 471.4±164% (n=6)
increase relative to the serum-free controls at 0.1 µg/mL
(2.38x10-10 mol/L) and 30
µg/mL (7.14x10-8 mol/L),
respectively (Fig 2). By comparison, an
approximate EC50 concentration of PDGF-BB (10 ng/mL)
increased DNA synthesis by 455.9±103.2% (n=6) in the same
experiments. Preliminary experiments using baculovirus-derived
recombinant human TSP-1 (10 µg/mL) have also shown an increase
in DNA synthesis in four different cell strains (data not shown). These
observations show that TSP-1 is a potent stimulus for DNA synthesis in
human saphenous vein–derived VSMCs and acts in a
concentration-dependent manner. In the subsequent DNA synthesis
experiments, human platelet-derived TSP-1 was employed at a
concentration of 5 µg/mL.
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The contribution of TGFß1 (which could be a very small
contaminant of the TSP-1 isolated from platelets) was examined in
DNA synthesis experiments. The effect of TGFß1 over a
range of concentrations from 1 pg/mL
(3.85x10-14 mol/L) to 10 ng/mL
(3.85x10-10 mol/L) was examined on DNA
synthesis under serum-free conditions. There was a maximal increase in
DNA synthesis of 47.3±13.2% at 100 pg/mL (Fig 3, n=17) which was reduced at higher
concentrations of TGFß1. Neutralizing antibody to
TGFß1 inhibited the action of TGFß1 such
that there was no significant difference relative to the nonimmune
immunoglobulins. There was, however, no significant difference between
the effect of TSP-1 on DNA synthesis in the presence of either
neutralizing TGFß1 antibody (10 µg/mL) or
nonimmune immunoglobulins (10 µg/mL). Furthermore,
immunoblotting data failed to detect
TGFß1 in the commercial TSP-1 employed in this
investigation (data not shown).
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The effect of TSP-1 on DNA synthesis was examined in combination with
PDGF-BB (Fig 4). Stimulation of quiescent
human VSMCs by TSP-1 (5 µg/mL) and PDGF-BB (10 ng/mL)
increased DNA synthesis by 354.4±53.2% (n=4) and 627.5±75.0% (n=4),
respectively, over the serum-free levels. In combination, TSP-1 and
PDGF-BB induced a dramatic 1955.9±238.9% (n=4) increase in DNA
synthesis, which was significantly greater than the additive effect of
each of these agents (P=.009). In these same
experiments, when DNA synthesis was maximally stimulated by PDGF-BB (30
ng/mL), the simultaneous addition of TSP-1 further
enhanced DNA synthesis relative to the effect of PDGF-BB (30
ng/mL) alone (P=.005, n=4; Fig 4).
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To determine whether TSP-1 could promote not only DNA synthesis but also an increase in cell number, a 14-day proliferation assay was conducted under serum-free conditions using NCTC 109 as the basal medium. TSP-1 (5 µg/mL) increased cell number by 50±6% (n=3) over 14 days relative to the serum-free control, in which the cell number remained virtually unchanged (1±3% increase, n=3). By comparison, growth medium supplemented with PDGF-BB (20 ng/mL) promoted a 95±33% increase in cell number (n=3) over 14 days in the same experiments.
The role of the RGD (Arg-Gly-Asp) sequence, which is present in the cell-binding domain of several extracellular matrix proteins (including TSP-1) and interacts with integrins, was assessed in the stimulation of DNA synthesis by TSP-1 in these human VSMCs. Neither the GRGDSP (100 µmol/L) nor the control GRGESP peptide (100 µmol/L) inhibited DNA synthesis induced by TSP-1 (n=4, data not shown).
The possible involvement of tyrosine kinases in mediating the action of
TSP-1 on DNA synthesis was investigated by using the tyrosine kinase
inhibitors genistein and tyrphostin A23. The combination of
either genistein (50 µmol/L) or tyrphostin A23 (100
µmol/L) with TSP-1 (5 µg/mL) abolished DNA synthesis
stimulated by TSP-1 (n=4; Fig 5). Both of
the tyrosine kinase inhibitors reduced the basal level of
DNA synthesis, but neither inhibitor exerted cytotoxic
effects on the cells at the concentrations used in these
experiments.
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The possible role of PDGF in mediating the action of TSP-1 on DNA
synthesis was further examined by using a neutralizing antibody to
PDGF. DNA synthesis stimulated by TSP-1 was unaffected by the presence
of a neutralizing antibody to PDGF (Fig 6). As expected, this neutralizing
antibody prevented the action of PDGF-BB (10 ng/mL) on the same
cells as shown in Fig 6.
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Migration Studies
TSP-1 induced a concentration-dependent increase in the migration
of human VSMCs, as shown in Fig 7. The
migratory response at 30 µg/mL was similar to that elicited by
the maximally effective concentration of PDGF-BB (2 ng/mL,
6.67x10-11 mol/L), which was used as a
positive control for these assays because it consistently
induces a strong migratory response in saphenous vein–derived
VSMCs.39
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To assess whether the response to TSP-1 was occurring as a result of
directed migration rather than random movement, a checkerboard
analysis was performed. Fig 8
shows the results of assays performed on four different cell strains
and normalized with respect to the migration induced by TSP-1 (10
µg/mL) in the lower chamber. The serum-free controls contained
12±4% (n=4) of the number of cells that had migrated in response to
TSP-1. Similarly, the presence of TSP-1 (10 µg/mL) in both the
upper and lower chambers, such that there was no directed gradient,
resulted in an equivalent degree of migration to the serum-free
controls. Furthermore, incubation of the cells with TSP-1 in the upper
chamber while having serum-free medium alone in the lower chamber also
induced virtually no migration. These data show that TSP-1 acts
primarily as a chemotactic rather than a chemokinetic agent in these
assays.
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In view of the possibility of trace TGFß1 contamination of the human platelet-derived TSP-1 employed in the migration experiments, the effect of TGFß1 on migration was examined in three VSMC strains. TGFß1 (over a range of concentrations from 1 pg/mL to 10 ng/mL) failed to promote migration in two of three cell strains studied and induced only a small response in the other cell strain. In view of the response seen with TSP-1 in these cell strains, possible TGFß1 contamination was considered to be of little importance in terms of mediating the chemotactic action of TSP-1.
The effect of the GRGDSP and GRGESP peptides was investigated on
migration of human VSMCs induced by TSP-1 (10 µg/mL,
2.38x10-8 mol/L). Chemotaxis
stimulated by TSP-1 was inhibited by the GRGDSP (100
µmol/L) but not the control GRGESP (100 µmol/L)
peptide (Fig 9). In contrast, neither
GRGDSP nor GRGESP had any significant effect on PDGF-BB–induced
chemotaxis (n=4).
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Considering the results of the DNA synthesis experiments with the
tyrosine kinase inhibitors, their effect on migration
induced by TSP-1 (10 µg/mL) was examined. Both genistein
(50 µmol/L) and tyrphostin A23 (100 µmol/L)
substantially inhibited TSP-1–induced migration by 58±11% and
71±9% (n=4), respectively (Fig 10).
In this case both agents also inhibited migration in response to
PDGF-BB (2 ng/mL), as would be expected in view of the tyrosine
kinase activity of PDGF receptors.
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Discussion |
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This study has demonstrated that TSP-1 is a powerful stimulus for DNA synthesis, proliferation, and chemotaxis in human saphenous vein–derived VSMCs. Human platelet-derived TSP-1 stimulated a similar magnitude of chemotaxis to that seen with optimal concentrations of PDGF-BB and only slightly less stimulation of DNA synthesis and proliferation than PDGF-BB in the same cell strains. The novel finding in this investigation, in contrast to previous studies using animal-derived VSMCs,30 31 in which TSP-1 has been shown to be mitogenic only in combination with other mitogens such as EGF, was that under serum-free conditions, TSP-1 alone acts as an effective mitogen for human VSMCs. Our data suggest that it is unlikely that these effects are due to contamination of TSP-1 with some other growth factor and that they may be attributable to species differences in receptor(s) for TSP-1 between human and animal cells, although this possibility remains to be substantiated.
The time course for [3H]thymidine incorporation induced by TSP-1 showed that in common with PDGF-BB and FCS, DNA synthesis was maximal between 24 and 30 hours after stimulation of quiescent human VSMCs. This finding contrasts with a previous study using rat mesangial cells that showed modest increases in DNA synthesis of 49% and 72% at 28 and 48 hours, respectively, in response to TSP-1 (10 µg/mL).40 By comparison, the same concentration of TSP-1 increased DNA synthesis by over 330% in the human VSMC cultures employed in this study. Moreover, there was no delayed increase in DNA synthesis in these human VSMCs, in contrast to previous findings using mesangial cells.
The DNA synthesis experiments with TSP-1 and PDGF-BB in combination proved particularly interesting, in that TSP-1, when added simultaneously with PDGF-BB, significantly increased PDGF-BB–stimulated levels of DNA synthesis in human VSMCs. The enhancement of DNA synthesis occurred at both submaximal and maximal levels of stimulation by PDGF-BB, suggesting that PDGF-BB and TSP-1 act through two independent but complementary pathways. Our findings with human VSMCs contrast with other reports regarding the effect of TSP-1 in combination with EGF or PDGF. In rat VSMCs, TSP-1 and EGF have been reported to elicit a synergistic effect on [3H]thymidine labeling.30 Similarly, in rat mesangial cells, TSP-1 in combination with either EGF or PDGF has been shown to exert an additive effect on DNA synthesis.40 Furthermore, the latter study showed that TSP-1 induced EGF production and significantly increased constitutive PDGF-AB production in a concentration-dependent manner and that anti-PDGF neutralizing antibody eliminated the effect of TSP-1 on mesangial cell growth.40 The experiments that we have performed with PDGF neutralizing antibody suggest that PDGF is not involved in mediating the effect of TSP-1 on DNA synthesis in these human VSMCs. Taken together with the DNA synthesis data from the TSP-1/PDGF-BB combination, this finding indicates that the action of TSP-1 in these human VSMCs is unlikely to occur through PDGF release. Furthermore, the neutralization and TSP-1/PDGF combination experiments on DNA synthesis, as well as the PDGF immunoblotting data, also exclude the possibility that the effect of TSP-1 on DNA synthesis is due to PDGF contamination of the TSP-1 preparation. This view is further supported by a limited number of observations with recombinant human TSP-1 that also increased DNA synthesis in human VSMCs.
In addition to its effect on DNA synthesis, platelet-derived TSP-1 also induced an increase in cell number in a 14-day proliferation assay. The degree of proliferative response to TSP-1 was not as great as that induced by PDGF-BB (20 ng/mL). The greater magnitude of response to PDGF-BB in these VSMCs parallels the findings in the DNA synthesis assays and may be attributable to the upregulation of PDGF-ß receptor during subculture, as previously observed in human skin fibroblasts.41
Since TSP-1 has previously been shown to bind and activate latent TGFß1,42 43 44 45 we have examined the effect of TGFß1 on DNA synthesis under serum-free conditions to assess its contribution, if any, to the mitogenic effect of TSP-1. TGFß1 induces a small increase in [3H]thymidine incorporation in these human VSMCs with a bell-shaped concentration-response relationship, ie, the response to TGFß1 is reduced at the higher concentrations. Bimodal effects of TGFß1 on VSMCs have been previously reported, with stimulation of DNA synthesis at low concentrations but inhibition at higher TGFß1 concentrations.5 Our findings are essentially in keeping with these data, although we have observed a smaller stimulatory response to TGFß1 at higher concentrations rather than the inhibition observed in other studies. The response of cells to TGFß1 has previously been reported to vary, depending on the species and density of cells, as well as the concentration of TGFß1.46 47 Our immunoblotting studies on the human platelet-derived TSP-1 used in this investigation have failed to detect TGFß1. In our studies, the TGFß1 neutralizing antibody failed to inhibit the effects of TSP-1 on DNA synthesis; this observation, together with the weak effects of TGFß1 on DNA synthesis in human VSMCs, makes it unlikely that TGFß1 contamination of TSP-1 accounts for the marked DNA synthesis seen in these studies in response to TSP-1.
The stimulation of chemotaxis by TSP-1 in these human saphenous vein–derived VSMCs is in accordance with previous findings in calf pulmonary arterial smooth muscle cells which showed that TSP-1 was an even more effective chemoattractant than PDGF or basic fibroblast growth factor.48 The TSP-1 concentration employed in this study (10 µg/mL, 2.38x10-8 mol/L) was lower than that employed with the calf pulmonary VSMCs (1x10-6 mol/L), which was shown to induce near-maximal migration of calf pulmonary VSMCs.48 The findings in this study with human VSMCs have shown that TSP-1 at much lower concentrations induces at least an equivalent degree of chemotaxis as the maximally effective concentration of PDGF-BB for these cells.39
TSP-1 has been reported to bind to a number of sites on the cell surface.49 50 51 52 53 54 The participation of the RGD sequence, in the C-terminal cell-binding domain of TSP-1 that interacts with integrins,55 was assessed in the induction of DNA synthesis by TSP-1. In this study, neither the GRGDSP (100 µmol/L) nor the control GRGESP (100 µmol/L) peptide inhibited DNA synthesis stimulated by TSP-1, indicating that an interaction of TSP-1 with integrins does not seem to be required for the effect on DNA synthesis. By contrast, the RGD peptide abolished VSMC migration induced by TSP-1, suggesting that chemotaxis was largely dependent on an integrin–TSP-1 interaction. In preliminary studies, we have observed that the RGD peptide inhibits VSMC attachment to a TSP-1–coated polystyrene substratum (data not shown). These data are consistent with the findings of Lawler et al55 that cell attachment to TSP-1 is mediated by RGD and calcium-dependent mechanisms. Cell attachment is an obligatory step in the chemotaxis assay, and the inhibition of migration by the RGD peptide may be attributable to interference with this process. The binding site responsible for the action of TSP-1 on DNA synthesis does not appear to be an integrin and remains to be defined.
The role of tyrosine phosphorylation in mediating the
action of TSP-1 was examined in these human VSMCs. The
inhibitory effects of genistein and tyrphostin A23 on DNA
synthesis and migration suggest that these actions of TSP-1 are largely
dependent on tyrosine phosphorylation. TSP-1 has
recently been reported to increase tyrosine
phosphorylation of focal adhesion kinase, paxillin, and
an 
90-kD protein in C32 human melanoma cells,56 and we
have recently shown that several
tyrosine-phosphorylated proteins are among the early
cellular signals seen in response to TSP-1.57 Integrin
activation is associated with tyrosine phosphorylation
of a number of cellular proteins,58 although we cannot
exclude the possibility that other TSP-1 receptors also participate in
this effect.
In summary, TSP-1 is a potent mitogen and chemoattractant for human VSMCs. These effects of TSP-1 probably involve actions at integrins, and in the case of DNA synthesis, other TSP-1 receptor(s). Both stimulation of chemotaxis and proliferation may be mediated intracellularly by increased tyrosine phosphorylation. DNA synthesis in response to TSP-1 does not appear to be mediated by the release of PDGF, and the implications of the marked species difference with regard to DNA synthesis between animal and human VSMCs may be particularly significant to human disease. The powerful chemotactic and mitogenic actions of TSP-1 suggest that it may play an important role in pathological events in the human vasculature.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by the Medical Research Council (M.K. Patel), Pfizer US (J.S. Lyn), and the British Heart Foundation (G.F. Clunn). The recombinant human TSP-1 expressed in a baculovirus expression system was the generous gift of Dr J. Adams (MRC Laboratory for Cell Biology, University College London, UK) and Dr J. Lawler (Harvard Medical School, Boston, Mass). We wish to thank Karen Gallagher for expert technical assistance in the maintenance of our VSMC cultures and the surgeons and theater staff at St Mary's Hospital for providing the vascular tissues for this investigation.
Received August 18, 1996; accepted December 27, 1996.
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