© 1997 American Heart Association, Inc.
Articles |
Thrombin Receptor-Mediated Increase of Two Matrix Metalloproteinases, MMP-1 and MMP-3, in Human Endothelial Cells
From INSERM U. 311, Etablissement de Transfusion Sanguine de Strasbourg, Strasbourg Cédex, France (E.D.-C., F.L., J.-P.C., C.K.-S.); and Laboratoire d'Oncologie Moléculaire, Institut de Recherche contre les Cancers de l'Appareil Digestif, Hôpitaux Universitaires de Strasbourg, Strasbourg, France (C.O.).
Correspondence to Claudine Klein-Soyer, Etablissement de Transfusion Sanguine, INSERM U. 311, 10, rue Spielmann, B.P. 36, 67065 Strasbourg Cédex, France. E-mail claudine.soyer{at}etss.u-strasbg.fr
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Abstract |
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Abstract Matrix metalloproteinases (MMPs) are responsible for the degradation of extracellular matrix components and are secreted by a variety of cells including human endothelial cells. Because
-thrombin is known to interact with matrix components and
has been shown to activate latent MMP-2 in human umbilical vein
endothelial cells, we investigated whether human
-thrombin could also regulate other MMPs secreted by the human
saphenous vein or mammary artery endothelial cells
(EC). After treatment of EC with increasing concentrations of thrombin
for different periods of time, a significantly higher
gelatinolytic activity of both MMP-1 and MMP-3 was
observed in addition to MMP-2 activation. The effect of thrombin was
time and dose-dependent, reaching a maximum at 24 hours. After
treatment with 5 NIH U/ml thrombin for 24 hours, Western blotting
revealed 9.5- and 4.4-fold increases over control values for MMP-3 and
MMP-1, respectively. The synthetic thrombin receptor agonist peptide
SFLLRNPNDKYEPF fully reproduced the action of thrombin, whereas
chemical inactivation of the catalytic site of thrombin abolished its
effect on MMP-1 and MMP-3. Thrombin and SFLLRNPNDKYEPF both induced
MMP-3 mRNA synthesis but had no significant influence on constitutive
MMP-1 mRNA levels. These results demonstrate that thrombin not only
activates latent MMP-2 but also modulates MMP-1 and MMP-3
production in EC, this latter effect being mediated by the
G-protein-coupled thrombin receptor. Hence, our present data
provide evidence to support the suspected role of thrombin in tissue
remodeling and angiogenesis.
Key Words: interstitial collagenase • stromelysin-1 • angiogenesis • -thrombin • thrombin receptor agonist peptide
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Introduction |
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The key localization of the vascular endothelium between circulating blood and neighboring tissues confers to it numerous and important regulatory functions. Thus, the endothelium has an essential and complex role in the maintenance of blood fluidity and constitutes a selectively permeable barrier between the biologically active substances carried by the blood and the organs of the body. Endothelial cells synthesize an underlying extracellular matrix, which contributes to the preservation of vascular integrity. A number of events can lead to dysfunction of the endothelium and, particularly, to disruption of the equilibria between pro- and anticoagulant factors and between growth-promoting and growth-inhibiting factors.1 2 Angiogenesis, the sprouting of new vessels from preexisting vessels, is one such event that occurs in both physiologic and in pathologic situations.3 4 5 One of the mechanisms involved in the regulation of angiogenesis is extracellular matrix degradation.6 This process is controlled by several enzymes and, specifically, by a family of matrix-degrading metalloproteinases (MMPs), which possess a zinc molecule at their catalytic site and are divided into three subclasses according to their substrate specificity.7 8 The different subclasses of MMPs are released by endothelial cells constitutively or after stimulation: (1) interstitial collagenase or MMP-1 (EC 3.4.24.7), (2) MMP-2 also called gelatinase A or 72-kDa type IV collagenase (EC 3.4.24.24) and MMP-1 also called gelatinase B or 92-kDa type IV collagenase (EC 3.4.24.35), and (3) stromelysin-1 or MMP-3 (EC 3.4.24.17).9
In both physiologic and pathologic situations, the
endothelium can be exposed to thrombin, a
multifunctional serine protease that promotes pleiotropic responses.
The central enzyme of the coagulation system,10 thrombin,
also displays mitogenic activity toward vascular
cells.11 12 Thrombin can interact with the extracellular
matrix, where it remains functionally active and leads to the release
of sequestered active growth factors, thus, contributing to
inflammatory and the wound-healing processes.13 14 Among
stimuli known to regulate the activity of MMPs, thrombin has recently
been shown to activate progelatinase A in vascular
endothelial cells.15 Hence, to investigate
whether thrombin could have an influence on other MMPs secreted by
these cells, we examined the effects of human -thrombin on the
regulation of MMPs produced by human saphenous vein or mammary artery
endothelial cells.
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Materials
Cell culture media (Ml99 with Hank's salts and RPMI 1640), L-glutamine, trypsin-EDTA, PBS Dulbecco's formulation, fungizone, and antibiotics (penicillin and streptomycin) were from GIBCO (Paisley, United Kingdom). Tissue culture grade 35-mm-diameter Petri dishes were from Corning Glass Works (New York, NY) and 75 cm2 culture flasks from Falcon, Becton Dickinson Company (Lincoln Park, NJ). Pooled heat-inactivated human serum, hepatitis B- and human immunodeficiency virus-free, and human serum albumin were from the Etablissement de Transfusion Sanguine de Strasbourg, France.
Type A gelatin (porcine skin, 300 blooms), ß-casein (bovine milk),
phenylmethylsulfonyl fluoride, 1,10-phenanthroline, Coomassie
brilliant blue G 250, aprotinin (bovine lung), and
2-[N-morpholino]ethane-sulfonic acid were from
Sigma-Aldrich Corporation (St. Louis, Mo). Brij-35 was from ICN
Biochemicals (Costa Mesa, Calif). Human -thrombin (3000 National
Institute of Health units/mg/mL) was prepared according to Ngai
and Chang,16 and r-Hirudin (10 000
anti-thrombin units/mg/mL) was from Transgène (Strasbourg,
France). D-phenylalanyl-prolyl-arginine chloromethyl ketone
dihydrochloride (PPACK) was obtained from Calbiochem (La Jolla, Calif),
and the thrombin receptor antagonist peptide MSRPACPNDKYE
(MSRP..) was from Bachem (Bubendorf, Switzerland). The thrombin
receptor agonist peptide SFLLRNPNDKYEPF (SFLL..) corresponding to amino
acids 42 to 55 of the human thrombin receptor and the inactive control
peptide FSLLRNPNDKYEPF (FSLL..) were purchased from Neosystem
(Strasbourg, France). DNA probes for MMP-1 and MMP-3 were kindly
supplied by Professor Paul Basset (INSERM U. 184, Illkirch,
France).
Electrophoresis supplies were obtained from Bio-Rad Laboratories
(Hercules, Calif). Immobilon-P transfer membranes were from Millipore
Corporation (Bedford, Mass), and the Enhanced Chemiluminescent
detection kit ECL(TM) and Hybond-N nylon transfer membranes were
purchased from Amersham (Les Ulis, France). Monoclonal antibodies to
matrix metalloproteinases, anti-MMP-1 (clone: 4l-1E5, IgG2a) and
anti-MMP-3 (clone: 55-2A4, IgG1), were from Oncogene Science
(Uniondale, NY). Affinity purified peroxidase conjugated goat
anti-mouse IgG and normal goat serum were from Jackson Immunoresearch
Laboratories (West Grove, Pa). The RNA isolation kit RNA Now(TM) was from
Biogentex (Seabrook, Tex), G3PDH amplimers were from Clontech (Palo
Alto, Calif), and the cDNA synthesis kit Ready-to-Go, the Klenow
fragment of DNA polymerase I, G25 superfine Sephadex, and Random
Priming hexamers were from Pharmacia (Uppsala, Sweden).
3-(N-morpholino)propanesulfonic acid (MOPS) was obtained
from Euromedex (Souffelweyersheim, France), and
[32P]dCTP (3000 Ci/mmol) from NEN Dupont de Nemours
(Boston, Mass). Other chemicals were of analytical grade from Merck
(Darmstadt, Germany).
Cell Culture
Human saphenous vein or mammary artery
endothelial cells (EC) were isolated from vessel
fragments obtained during coronary bypass surgery and cultured
as previously described.17 18 Cryopreserved EC were grown
on Petri dishes coated with human fibronectin in M199/RPMI 1640 (v/v)
medium supplemented with 30% human serum, 100 U/mL penicillin, 100
µg/mL streptomycin, 0.25 µg/mL fungizone, 2 mM
glutamine at 37°C in a 5% CO2/95% air atmosphere. The
endotoxin concentration of the culture media never exceeded 0.04
ng/mL to avoid any constitutive activation of EC, and the cells
were used from passage two to six. Conditioned media were obtained by
incubating confluent EC for 24 hours with 1.2 mL of medium containing
0.01% (w/v) human serum albumin and 200 kallikrein inhibiting
units (KIW) aprotinin, a protease inhibitor not effective
against thrombin, in the presence of the appropriate concentrations of
the test compounds, after extensive rinsing of the cells with PBS to
eliminate any traces of serum. At the end of the incubation period, the
conditioned media were centrifuged for 4 minutes at 13 000
g in a Microfuge (Eppendorf, Hamburg, Germany) to remove
cellular debris, and the samples were frozen at -80°C until use.
When cell extracts were prepared after supernatant collection, the
monolayers were rinsed with PBS, solubilized in 0.5% (v/v) Triton
X-100 in PBS, and frozen at -80°C.
Preparation of PPACK-Thrombin
PPACK-thrombin, irreversibly inactivated at the
catalytic site, was prepared by incubation of -thrombin with a
5-fold molar excess of PPACK in 5 mM
2-[N-morpholino]ethane-sulfonic acid buffer, pH 6.5,
containing 0.75 M NaCl for 1 hour at 37°C, followed by removal of
unreacted PPACK by dialysis against the same buffer for 48 hours at
4°C with frequent buffer changes.
Substrate Gel Zymography
Gelatinolytic activities of MMPs in
EC-conditioned media were analyzed by zymography on gelatin or
ß-casein containing polyacrylamide gels as previously
described.19 Active and latent species both can be
visualized by this technique. The conditioned media were supplemented
with 5x concentrated sample buffer and made 1% (w/v) SDS and 10%
(v/v) glycerol; then 10 µl of the mixture was applied to 7.5%
polyacrylamide gels copolymerized with 0.12% (w/v) gelatin or
ß-casein. After electrophoresis, the gels were washed twice in 2.5%
(v/v) Triton X-100 for 2 hours to remove SDS and renature the MMPs.
After two further 5-minute washes in substrate buffer (50 mM Tris, pH
7.6, 5 mM CaCl2, 20 mM NaCl, 0.02% (v/v) Brij-35), gelatin
gels were incubated in the same buffer and ß-casein gels were
incubated in 50 mM Tris, pH 7.6, 10 mM CaCl2 for 16 hours
at 37°C. The gels were stained for 3 hours at room temperature in
30% (v/v) methanol and 10% (v/v) acetic acid containing 0.5% (w/v)
Coomassie brilliant blue G 250 and then destained in the same solution
in the absence of dye until visualization of the areas of gelatin or
ß-casein degradation.
Western Blotting
Aliquots (30 to 50 µl) of conditioned media were denatured
with SDS under reducing conditions and applied to 7.5%
polyacrylamide gels. After separation by electrophoresis, the
samples were electroblotted onto Immobilon-P transfer membranes, and
the proteins of interest were revealed with specific anti-MMP-1 (clone:
41-1E5, IgG2a) and anti-MMP-3 (clone: 55-2A4, IgG1), commercial
monoclonal antibodies (1 µg/mL). The characteristics of these
antibodies as given by the manufacturer are the following: anti-MMP-1
is directed against the epitope formed by amino acids 332 to 350 of
human MMP-1, and anti-MMP-3 was obtained by immunizing mice with
pro-MMP-3 purified from conditioned media of rheumatoid synovial
fibroblasts; its epitope is unknown. Both antibodies recognize latent
and active forms of the respective MMPs. Horseradish
peroxidase-conjugated goat anti-mouse IgGs were used after being
diluted to 1/5000 in 5% normal goat serum. The enhanced
chemiluminescent detection method was used according to the
manufacturer's instructions, using horseradish peroxidase-conjugated
goat anti-mouse IgGs, diluted to 1/5000 in 5% normal goat serum.
Preliminary experiments were performed to ascertain the molecular
weights of the bands revealed by the anti-MMP-1 and anti-MMP-3
antibodies. Samples were run on gels of different
acrylamide concentrations (7.5, 10, and 12%) in the
presence of adequate protein molecular weight standards. Anti-MMP-1
revealed one band around 52 kDa, the expression of which varied after
thrombin exposure; anti-MMP-3 revealed a band around 57 kDa, which
varied according to experiment. Both antibodies cross-reacted with a
constant band at 66 kDa, corresponding to human serum albumin
present in the conditioned medium (data not shown).
RNA Extraction and Northern Blot Analyses
EC were seeded in 75 cm2 tissue culture flasks
coated with fibronectin under standard culture conditions. At
confluence, the cell monolayers were washed with PBS, and 6 mL of
culture medium containing 0.01% (w/v) human serum albumin, 200
kallikrein inhibiting units of aprotinin, and appropriate
concentrations of test compounds was added. After a 12-hour incubation
at 37°C, total RNA was isolated by the RNA Now procedure. The probe
for G3PDH was obtained by polymerase chain reaction20
amplification of EC total RNA, using commercial primers that generated
an amplification product of 983 base pairs. Polymerase chain
reaction conditions were 30 cycles, each consisting of 1 minute of
denaturation at 94°C, 1.5 minutes of hybridization at 60°C, and 2
minutes of elongation at 72°C. After separation of the amplification
product by 1.2% agarose gel electrophoresis and staining with
ethidium bromide, the band of interest was carefully cut out with a
scalpel blade and purified by phenol extraction.21 The
human type-1 collagenase (MMP-1) cDNA probe was a
1.5-kilobase fragment inserted into the
EcoRI/XbaI sites of pBluescript, whereas the
human stromelysin-1 (MMP-3) cDNA probe was a 1.8-kilobase fragment
inserted into the EcoRI/SK+ sites of
pBluescript.22 All probes were 32P-labeled by
random priming23 and purified on Sephadex-G25
columns.21
Total cellular RNA (8 µg) from each sample was resolved on denaturing MOPS-formaldehyde-agarose gels, transferred to Hybond N nylon membranes, and cross-linked by heating for 2 hours at 80°C. The quality of the RNA was confirmed by the presence of intact 28S and 18S ribosomal RNA bands on methylene blue-stained membranes. Prehybridization was performed at 42°C in 50 mM sodium phosphate buffer, pH 6.5, containing 50% (v/v) formamide, 5x standard sodium citrate (SSC) (1xSSC=0.15 M NaCl, 0.015 M sodium citrate), 0.2% (w/v) polyvinylpyrrolidone/0.2% (w/v) Ficoll 400 (PF), 0.1% (w/v) SDS, 1% (w/v) glycine, and 500 µg/mL salmon sperm DNA. Radiolabeled probes were added at a concentration of 1x106 cpm/mL and hybridized for 16 hours at 42°C in 20 mM sodium phosphate buffer, pH 6.5, containing 50% formamide, 5xSSC, 1xPF, 0.1% (w/v) SDS, 10% (w/v) dextran sulfate, and 100 µg/mL salmon sperm DNA. After hybridization, the blots were washed once for 10 minutes and twice for 30 minutes at 42°C in 2xSSC/0.1% (w/v) SDS once for 30 minutes at 55°C in 0.1xSSC/0.1% (w/v) SDS and then once at 65°C in 0.1xSSC/0.1% (w/v) SDS. Radioactive bands were initially visualized on a phosphorimager (PhosphorImager, Molecular Dynamics, Sunnyvale, Calif) and further exposed to a Min RE 100 Kodak film (Rochester, NY) overnight or longer if necessary. After first hybridization with two probes, the blots were dehybridized after autoradiography by incubation in 0.01x standard sodium phosphate EDTA (SSPE)1xSSPE=0.15 M NaCl, 0.2M NaH2PO4, 0.025 M EDTA) containing 0.1% (w/v) SDS for 10 minutes at 100°C and further hybridized with the remaining probes.
Image Analysis
Quantification of gelatinolytic or
caseinolytic areas and Northern blot autoradiographic bands
was performed by measuring the integrated optical density (OD) using
Visiolab 1000 software (Biocom, Les Ulis, France).
Statistical Analyses
Results were compared by variance analysis followed by
the Newman-Keul's test using the statistical software STAT-ITCF
(ITCF-Boigneville, France).
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Results |
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Effects of Thrombin on MMP Expression by Human Saphenous Vein or Mammary Artery Endothelial Cells
MMP activity has been detected by gel substrate zymography. This technique allows detection of latent and activated forms of MMPs as treatment with SDS unfolds the structure of latent MMPs and exposes the zinc molecule. Analysis of serum-free conditioned media from nonstimulated human saphenous vein or mammary artery endothelial cells on gelatin substrate gels showed the presence of basal gelatinolytic activities at 72 kDa, 64/62 kDa, and 57/52 kDa (Fig 1A
).
The 72-kDa lysis area corresponded to latent MMP-2 (gelatinase A) and a
faint 64/62 kDa doublet to the activated form of the enzyme,
whereas bands at 57/52-kDa bands were attributed to latent MMP-1
(interstitial collagenase) and/or to latent
MMP-3 (stromelysin-1). A time course study showed that latent MMP-2
expression increases slightly over time and reaches a maximum at 24
hours, and the 64/62-kDa and 57/52-kDa bands increase up to 48 hours.
Zymography analysis was not performed for more than 48 hours,
because the absence of serum in the incubation medium leads to
extensive cell detachment after this time.
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Stimulation of EC with increasing concentrations of thrombin (1, 5, and
10 NIH U/mL) induced a dose-dependent activation of latent MMP-2 and a
rise in MMP-1/3 expression (Fig 1A
), the latter attaining a maximum of
4-fold over control values at 24 hours and 5 NIH U/mL of thrombin (Fig 1B). In further experiments, the usual conditions were an incubation
time of 24 hours and a thrombin concentration of 5 NIH U/mL.
ß-caseinolysis analysis of conditioned media from EC
submitted to increasing concentrations of thrombin (0.1 to 10 NIH U/mL)
for 24 hours confirmed the rise in expression of an MMP of size 
57
kDa (Fig 1C, bottom). A maximum activity of 3.2 ± 0.5-fold over
control values was reached at 5 NIH U/mL thrombin (mean±SEM, n=4;
Fig 1C, top). Only the latent form of MMP-2 was present in cell
extracts from nonstimulated EC (data not shown). When the zinc ion
chelator 1,10-phenanthroline was added to the substrate buffer,
gelatinolytic bands at 92, 72, 64/62, and 57/52 kDa
could no longer be observed, thus attesting to the metallo-dependence
of the proteases (Fig 1D). Although under these conditions
gelatinolytic activity was observed at 35 kDa
(Fig 1D, right), this band disappeared if the serine protease
inhibitor phenylmethylsulfonyl fluoride or the
specific thrombin inhibitor hirudin was added to the
substrate buffer, suggesting that it was caused by thrombin (data not
shown).
Effect of Thrombin on MMP-1 and MMP-3 Expression as Detected by
Western Blotting
The sensitivity of gelatin zymography for the detection of MMP-1
and MMP-3 is weak, and whereas ß-casein is known to be an
MMP-3-specific substrate, it does not allow discrimination between
MMP-1 and MMP-3. Therefore, Western blotting experiments were performed
using specific anti-MMP-1 and anti-MMP-3 antibodies. In 24-hour
conditioned media from untreated EC, MMP-1 was constitutively
expressed, whereas MMP-3 was only marginally present (130±28
versus 54±12 integrated milli-OD, mean±SEM, n=14). To allow
comparison between separate experiments, the constitutive activities of
MMP-1 and MMP-3 were set as 100% and the values after thrombin
treatment expressed as a percentage of the control. Exposure to
increasing concentrations of thrombin (0.1 to 10 NIH U/mL) for 24 hours
led to increases in both MMP-1 and MMP-3, as revealed by antibodies
against the latent and activated forms of the enzymes (Figs 2A and B). These rises in MMP-1 and MMP-3
reached maximum levels of 4.4±0.6-fold and 9.5±2.5-fold,
respectively, over control values at 5 NIH U/mL thrombin (mean±SEM,
n=4).
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Chemical agents such as organomercurials are able to unfold the structure of latent MMPs, thus exposing the zinc atom and allowing autocatalytic cleavage, which leads to the production of the lower molecular weight active forms of these enzymes. Hence, in some experiments, conditioned media were treated with aminophenyl mercuric acetate for 1 hour at 37°C before gelatin zymography. This treatment abolished the lysis areas in the range 57/52 kDa in both controls and thrombin-stimulated media, therefore, confirming that MMP-1 and MMP-3 were produced in their latent forms (data not shown).
Effects of Thrombin Receptor Peptides and PPACK-Thrombin on MMP-1
and MMP-3 Expression
The effects of thrombin on the expression of MMP-1 and MMP-3 by EC
could be the result of the proteolytic activity of the enzyme and be
mediated by the interaction of thrombin with its seven transmembrane
domain G-protein-coupled receptor.24 25 This receptor is
proteolytically activated by thrombin and can also be
activated by synthetic peptides mimicking the new amino
terminus.24 26 Therefore, we incubated EC with the
synthetic thrombin receptor agonist peptide
42SFLLRNPNDKYEPF55,27 28 the
sequence of which corresponds to the new amino terminus of the receptor
generated after cleavage by thrombin. Increasing concentrations of
SFLL.. (50 to 500 µg/ml) induced a dose-dependent augmentation
of MMP-1 and MMP-3 expression by EC as detected by Western blotting
(Figs 3A and B). At 100 µM SFLL..,
MMP-1 expression reached a maximum of 2.4±0.5-fold over control values
(mean±SEM, n=3), equivalent to 80% of the response induced by 2 NIH
U/mL thrombin. Higher concentrations of SFLL.. gave no further increase
in MMP-1 levels. As in the case of thrombin stimulation, the increase
in MMP-3 induced by SFLL.. was 2-fold higher than the rise in MMP-1,
attaining a maximum at 100 to 250 µmol SFLL.. and corresponding to
86% of the response obtained with 2 NIH U/mL thrombin. The inactive
control peptide with the first two amino acids reversed, FSLL.., failed
to increase MMP-1 or MMP-3 levels at concentrations up to 250 µM. A
thrombin receptor antagonist peptide MSRPACPNDKYE has been
shown to inhibit the platelet aggregation induced by
SFLL..29 Treatment of EC with 100 µM MSRP.. before the
addition of thrombin (5 NIH U/mL) resulted in a 30% decrease in the
MMP-1 and MMP-3 induction observed in the presence of thrombin alone.
Finally, chemical inactivation of the proteolytic site of thrombin by
PPACK totally abolished its effects on MMP-1 (Fig 3A) and MMP-3 (Fig 3B).
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MMP-1 and MMP-3 mRNA Levels in EC Exposed to Thrombin or the
Thrombin Receptor Agonist Peptide
MMP-1 and MMP-3 mRNA levels were estimated from Northern blots
obtained from confluent EC monolayers stimulated for 12 hours with
thrombin (5 or 10 NIH U/mL), the thrombin receptor agonist peptide
SFLL.. (100 µM), or the inactive control peptide FSLL.. (100 µM;
Fig 4A). Relative mRNA levels were
compared by laser densitometry using the phosphorimager. Values for
MMP-1 and MMP-3 were normalized against those for G3PDH, and
modulations induced by thrombin, SFLL.., or FSLL.. were expressed as
percentages of the control values for untreated cells (Fig 4B). MMP-3
mRNA was calculated as the MMP-3/G3PDH ratio x1000, as the control
expression was zero. At the concentrations used, neither thrombin nor
SFLL.. significantly modified the constitutive expression of MMP-1 mRNA
in EC, the maximal increase at 10 NIH U/mL thrombin, or 100 µM SFLL..
not exceeding 30% of control values. Whereas no MMP-3 mRNA could be
detected in untreated EC, thrombin and SFLL.. both induced significant
expression of MMP-3 transcripts. FSLL.. gave results similar to those
of control cells.
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Discussion |
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In the study presented herein, we demonstrate that thrombin is able to increase the expression of interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3) in cultured human saphenous vein and mammary artery endothelial cells. This effect of thrombin is mediated through interaction with its proteolytically activable G-protein-coupled receptor, as similar results were obtained using the receptor agonist peptide. The increase in MMP-3 was associated with an induction of MMP-3 transcripts, whereas the rise in MMP-1 increase appeared to be regulated at a posttranscriptional level because MMP-1 mRNA expression was not significantly modified by thrombin.
Basement membrane and extracellular matrix degradation occur in a wide range of physiologic and pathologic processes including embryogenesis, ovulation, bone remodeling, wound healing, tumor invasion, and metastasis. On account of the strategic location of the endothelium between the circulating blood and neighboring tissues, angiogenesis is associated with almost all of these processes. Matrix breakdown is mediated by proteolytic enzymes, among which MMPs play a critical role.7 8 30 31 In the present work, we show that thrombin, the pivotal enzyme of the blood coagulation system, over a range of concentrations including those regulating physiologic responses such as platelet activation (0.01-0.1 U/mL) or factor V activation (0.1-0.8 U/mL),32 33 modulates the activity of the MMPs constitutively synthesized by human saphenous vein and mammary artery endothelial cells. Unstimulated EC did not express the 92-kDa MMP-9 known as progelatinase B, unless traces of serum or endotoxin were present. Thus, thrombin was unable to promote the expression of progelatinase B in EC when thorough rinsing of EC was performed and when endotoxin-free reagents and media were used.
Thrombin was able to transform the 72-kDa progelatinase A (MMP-2) produced by vein or artery EC into its 64/62-kDa activated forms, thus confirming previous results obtained by Zucker et al15 with HUVEC. This process was concentration- and time-dependent. In the present work, we found EC from human adult veins or arteries to express constitutively MMP-1 and, to a lesser extent, MMP-3. Whereas MMP-3 has not to date been constitutively detected in HUVEC,34 it was present in conditioned media from human femoral vein EC,9 which confirms the notion that stromelysin-1/MMP-3 is not widely expressed in normal tissues but is mainly associated with inflammatory or malignant pathologies.7 35 36 Concomitant with MMP-2 activation, there was a significant increase in MMP-1/3. However, in a few cases, EC did not constitutively express detectable MMP-1/3 (2 in 15 batches); under these conditions thrombin, in contrast to phorbol 12-myristate 13-acetate, was unable to induce any MMP-1/3 gelatinolytic activity (data not shown). Western blotting experiments with specific anti-MMP-1 and anti-MMP-3 antibodies were used and established that thrombin stimulated the production of both MMPs by EC and to a higher extent that of MMP-3.
The cellular effects of thrombin are thought to be mediated, at least in part, by a specific receptor24 25 belonging to the G-protein-coupled receptor superfamily. Its activation mechanism involves specific proteolytic cleavage by thrombin of the amino-terminal extracellular domain, which unmasks a new amino terminus capable of serving in turn as a new ligand for the receptor. This thrombin receptor is the first member of a new family of proteolytically activated receptors and can also be activated by synthetic peptides mimicking the new amino terminus.24 26 EC are known to possess the thrombin receptor,37 38 where it is thought to be involved in processes such as phospholipase stimulation, of protein kinase C, mitogen-activated protein kinase activation, and cell growth stimulation (reviewed in 39 . Our experiments favor involvement of the thrombin receptor in the modulation of MMP-1/3 expression. First, treatment of EC with the receptor agonist peptide SFLL.. corresponding to the first 14 amino acids of the new amino terminus or with a shorter 6-amino acids peptide (data not shown) fully reproduces the effects of thrombin and induces a dose-dependent augmentation of MMP-1 and MMP-3 expression. Second, a thrombin receptor antagonist peptide able to inhibit the platelet aggregation induced by SFLL..29 decreases by 30% the increase in MMP-1 and MMP-3 production observed in the presence of thrombin alone. Finally, this effect is specific as the inactive control peptide FSLL.. is unable to modify MMP-1 or MMP-3 levels. Thus, the overall results demonstrate that in contrast to gelatinase A activation, which requires direct proteolytic cleavage of the proenzyme by thrombin,15 the increases in MMP-1 and MMP-3 activity induced by thrombin are mediated through its EC receptor.
Thrombin can activate a wide variety of cells, and the majority of responses seem to be mediated by a proteolytically activated-type receptor of apparently identical structure in different cell types.39 However, thrombin receptor regulation including its desensitization, internalization, recycling, and restoration presents cellular specificities.28 33 40 41 To determine the time of interaction necessary for thrombin to induce a maximal increase in MMP-1/3 expression, experiments were performed by blocking its action at different time points with the potent thrombin inhibitor, hirudin.42 Minimal activation required 15 minutes of exposure of EC to thrombin, and maximal expression was obtained after 6 hours (data not shown). This time course of MMP-1/3 expression on exposure of EC to thrombin is in agreement with the mobilization to the cell surface of an intracellular pool of receptors that could then interact with the excess thrombin present in our experiments (5 NIH U/mL, ~45 nM). The existence of such an intracellular pool of intact thrombin receptors has already been described in HUVEC, which recover 90% of thrombin responsiveness within 5 hours, independent of protein synthesis.41
The promoter regions of the genes for MMP-1 and MMP-3 share many common regulatory elements, and it has been shown that they can be coordinately regulated by several agents including cytokines.8 Therefore, in addition to detecting the proteins, we investigated whether thrombin, which concomitantly increases MMP-1 and MMP-3 expression, also regulated both MMPs at a transcriptional level. Northern blot analyses revealed that MMP-1 transcript levels did not significantly increase after treatment of EC with thrombin or with the receptor agonist peptide SFLL... In contrast, MMP-3 mRNA was not detectable in control EC or in EC treated with the inactive peptide FSLL.. but was significantly induced in the presence of thrombin or SFLL... Our results for the detection of MMP-1 and MMP-3 mRNA in unstimulated EC are consistent with those of other authors for HUVEC or femoral vein EC. However, unlike these authors who demonstrate that MMP-1 increase is associated to MMP-1 mRNA upregulation after treatment of EC with phorbol 12-myristate 13-acetate or tumor necrosis factor,9 we did not detect any significant MMP-1 mRNA upregulation after treating EC with thrombin, despite an increase of MMP-1. Thus, although thrombin induced a coordinated increase in MMP-1 and MMP-3 protein levels, its effects on MMP-1 and MMP-3 transcription were dissociated. It seems likely that the regulation of MMP-1 expression occurred at a posttranscriptional level, either by protein stabilization or by increased translation. The promoter regions of the MMP-1 and MMP-3 genes contain AP-1 cis-acting elements capable of regulating gene expression in response to agents such as cytokines, growth factors, or oncogenes.7 Thrombin is known to induce c-fos proto-oncogene expression in HUVEC,43 and thrombin receptor-activating peptide stimulates c-fos gene expression in vascular smooth muscle cells,27 whereas activation of the thrombin receptor in a human astrocytoma cell line leads to an increase in c-fos and c-jun mRNA, which in turn stimulates AP-1 transactivation.44 Therefore, the MMP-3 mRNA induction resulting from the activation of proteolytically activated receptors on EC by thrombin or SFLL.. could possibly be mediated by AP-1 transactivation. Other elements must nevertheless be involved to account for the differential regulation of MMP-1 and MMP-3 mRNA levels by thrombin as stimulation of EC with phorbol 12-myristate 13-acetate, a known inducer of c-fos and c-jun,45 induces and/or increases the expression of both MMP-1 and MMP-3.9
The results presented herein might be pertinent to some physiologic or pathologic situations. High concentrations of thrombin can be attained whenever blood coagulation or thrombosis is initiated during tissue repair, atherosclerosis, or vascular surgery. As a consequence of the ubiquity of the endothelium in the body, thrombin is also present in all tissues that are remodeled during embryogenesis, angiogenesis, wound healing, or tumor growth. Enzymes of the fibrinolytic system (tissue plasminogen activator, urokinase-type plasminogen activator, and plasminogen activator inhibitor(s)) are strongly involved in these processes46 and can be regulated by thrombin, particularly in EC.47 48 49 50 Thus, in addition to the role of thrombin in tissue remodeling through regulation of the fibrinolytic system, the present results demonstrate that thrombin can also participate by regulating the MMP system. The increase in MMP-1/3 activity induced by thrombin might be relevant to these processes, as it has been shown that at concentrations of up to 20 nM, thrombin can bind to the subendothelial extracellular matrix, where it remains functionally active and protected from circulating inhibitors.51 Thrombin accelerates dermal wound healing in rats14 52 and increases proteoglycan release from articular cartilage,53 this latter process being associated with a rise in MMP-3 mRNA. However, as thrombin does not seem to modify the expression of the tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2, at least in HUVEC in vitro,15 available results would suggest that in the presence of thrombin the MMP balance is in favor of a degradative process. In conclusion, an understanding of the mechanisms involved in the modulation of MMPs by thrombin is important in view of the development of pharmacologic inhibitors of angiogenesis and tumor growth.
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Acknowledgments |
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The authors are grateful to Mrs. Marlène Grunert, Simone Schuhler, Martine Moralès, and Francine Noël for excellent technical assistance, to Professor P. Basset from IGBMC-LGME-U.184 Illkirch C.U. Strasbourg for providing the cDNA probes for MMP-1 and MMP-3, and to the Service de Chirugie Cardiovasculaire des Hôpitaux Universitaires de Strasbourg for providing the human saphenous vein and mammary artery fragments. We also thank Miss Juliette Mulvihill for reviewing the English and Mrs Claudine Helbourg for expert typing of the manuscript. Part of this work was presented at the annual conference of the European Vascular Biology Association, May 30 to June 2, 1996, Göteborg, Sweden.
Received October 17, 1996; accepted February 3, 1997.
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