Department of Pharmacology, Medical School, University of Patras, 25110 Patras, Greece
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ABSTRACT |
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Thrombin has been reported to be
a potent angiogenic factor both in vitro and in vivo, and many of the
cellular effects of thrombin may contribute to activation of
angiogenesis. In this report we show that thrombin-treatment of human
endothelial cells increases mRNA and protein levels of
v
3-integrin. This thrombin-mediated effect is specific, dose dependent, and requires the catalytic site of
thrombin. In addition, thrombin interacts with
v
3 as demonstrated by direct binding of
v
3 protein to immobilized thrombin. This
interaction of thrombin with
v
3-integrin,
which is an angiogenic marker in vascular tissue, is of functional
significance. Immobilized thrombin promotes endothelial cells
attachment, migration, and survival. Antibody to
v
3 or a specific peptide antagonist to
v
3 can abolish all these
v
3-mediated effects. Furthermore, in the
chick chorioallantoic membrane system, the antagonist peptide to
v
3 diminishes both basal and the
thrombin-induced angiogenesis. These results support the pivotal role
of thrombin in activation of endothelial cells and angiogenesis and may
be related to the clinical observation of neovascularization within thrombi.
attachment; migration; apoptosis; reverse transcription-polymerase chain reaction
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INTRODUCTION |
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THE FREQUENCY OF BLOOD COAGULATION in cancer patients, known for more than 130 years, is supported by clinical, laboratory, and histopathological evidence. This is explained at the molecular and cellular level by the thromboplastic activity of circulating tumor cells, the existence of "a cancer coagulative factor," the activation of factor X, the generation of prothrombinase by tumor cells, and the encircling of cancerous tissue by fibrin deposits (38, 50). In addition, the possibility of a relation between blood clotting mechanisms and tumor progression and development of metastases was postulated as early as 1878 by Billroth (7) on the basis of the observation that cancer cells exist within thrombi. This finding was interpreted as evidence that tumor cells spread by thromboembolism. More recently, large epidemiological studies have provided evidence that the standardized incidence ratio for certain types of cancer is as high as 6.7 within a year following a thromboembolic episode (3, 41). These clinical data are in line with animal experiments where thrombin-treated B16 melanoma cells show a dramatic increase in their metastatic potential in the lung of rats (37). These observations have led to experimental use of heparin, aspirin, and warfarin for the prevention and treatment of tumors in animal models and humans (23, 50).
We proposed earlier (33, 47) that the tumor-promoting effect of thrombin/thrombosis may be related to our finding that thrombin is a potent promoter of angiogenesis, a process essential for tumor growth and metastasis. The angiogenic action of thrombin was shown to be receptor-mediated and independent of fibrin formation. The importance of thrombin and its receptors in embryonic development and angiogenesis is also supported by the findings of Griffin et al. (21), who showed that the expression of protease-activated receptor-1 (PAR-1) by endothelial cells rescues the fetal vessel fragility and bleeding of mouse embryo engineered to lack PAR-1. Recently, we have shown that thrombin has a synergistic effect with the vascular endothelial growth factor (VEGF) by upregulating its receptors in cultured endothelial cells (46). This finding, in connection to data showing that thrombin increases the secretion of VEGF from human prostate cancer cells (32), may result in mutual stimulation of endothelial and cancer cells for activation of angiogenesis and tumor progression. The role of thrombin receptors in angiogenesis and tumor progression is also supported by the findings of Even-Ram et al. (19), who showed that the metastatic ability of human breast cancer cells is related to the number of thrombin receptors on these cells.
In this paper we present evidence that thrombin interacts with
v
3-integrin at the molecular and cellular
level in endothelial cells. Integrin
v
3
is known to be expressed in vascular cells during angiogenesis and
remodeling and in tumor cells, where it contributes to malignant
phenotype (18). We demonstrate that thrombin-treatment of
endothelial cells upregulates the expression of mRNA and protein of
v
3-integrin, resulting in the
potentiation of cell migration toward vitronectin, the
v
3 ligand. In addition, we show that
immobilized thrombin, like vitronectin, can serve as a ligand for
v
3-integrin. This interaction of thrombin
with
v
3 is shown to be of functional
significance in vitro and in vivo. Immobilized thrombin facilitates
endothelial cell attachment, migration, and survival via
v
3-integrin interaction. In addition, we
show that
v
3-integrin is involved in the
thrombin-promoting angiogenesis in the chick chorioallantoic membrane system.
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METHODS |
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Endothelial cell culture. Human umbilical vein endothelial cells (HUVECs) were obtained by established methods (25) from freshly delivered umbilical cords from caesarean births. Cells were cultured as described previously (46) and used for experiments from passages 3-5.
RNA isolation and semiquantitative RT-PCR.
After reaching confluence and 3 days after the last medium change,
HUVECs were incubated with serum-free M199 medium containing 0.5%
bovine serum albumin (SFM199-0.5% BSA) alone or with thrombin (kindly provided by Dr. J. Fenton II, Albany, NY) or with hirudin (Sigma) or DIP-thrombin (thrombin that is chemically inactivated at the
active site by diisopropylphosphofluoridate; kindly provided by Dr. J. Fenton II). After the indicated time periods, total cellular
RNA was purified by the guanidinium thiocyanate-phenol-chloroform method (12). RT-PCR was performed by using the Promega
access RT-PCR system (Madison, WI) according to the manufacturer's
protocol. Primer sequences (all synthesized by Research and Technology
Institute, Heraclion, Greece) were as follows: 3
(24) (sense, 5'-GTGCTGACGCTAACTGACC-3'; antisense,
5'-CATGGTAGTGGAGGCAGAGT-3'; expected size of PCR product, 284 bp) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control
(sense, 5'-TGAAGGTCGGAGTCAACGGATTTG-3'; antisense, 5'-CATGTGGGCCATGAGGTCCACCAC-3'; expected size of PCR product, 967 bp).
The RT-PCR profile consisted of 45 min at 48°C for reverse transcription and 5 min of initial denaturation at 94°C, followed by
26 cycles of 1 min of denaturation at 94°C, 1 min of annealing at
54°C, 1 min of polymerization at 72°C, and, finally, 10 min of
extension at 72°C. Ten microliters of the RT-PCR products were separated in 1% (wt/vol) agarose gels and stained with ethidium bromide. The gels were then photographed and scanned to quantitate the
obtained RT-PCR products. Densitometry analysis was performed by using
image analysis software (ImagePC; Scion), and the ratio of
3 to GAPDH in each lane was calculated. Results are
representative of three independent experiments and are expressed as
means ± SE. Statistical analysis was performed with Student's
t-test.
Immunoprecipitation and Western blot analysis.
Three days after the last medium change, confluent endothelial
monolayers on gelatin were incubated with SFM199-0.5% BSA alone or with thrombin for 24 h. Viability of endothelial cells was tested by trypan blue exclusion, and live cell number was estimated for
each experimental group. Cells were then lysed at 4°C by scraping in
lysis buffer containing 10 mM Tris · HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM
N-ethylmaleimide, 0.1 U/ml aprotinin, and 10 µg/ml
leupeptin (all from Sigma). For immunoprecipitation, cell extracts were
precleared with normal mouse IgG (Sigma) for 1 h and
immunoprecipitated with protein A-Sepharose conjugated with mouse
monoclonal antibody against human
v
3-integrin (clone LM609; Chemicon
International). The supernatants were then reimmunoprecipitated with
protein A-Sepharose conjugated with goat polyclonal antibody against
human actin (Santa Cruz Biotechnology), a highly conserved protein that
is expressed in all eukaryotic cells. Immunoprecipitated protein
(
v
3, actin) were then separated on 8%
SDS-PAGE under nonreducing conditions. After electrophoresis, proteins
on gels were transferred on nitrocellulose membranes. The membranes
obtained after blocking with 10% skim milk were first incubated with
anti-
3 mouse monoclonal antibody CD61 (Chemicon
International) or the anti-actin goat polyclonal antibody for 2 h
at room temperature and with a rabbit anti-mouse IgG or rabbit
anti-goat IgG conjugated with horseradish peroxidase for 1 h. The
blots were then developed by using enhanced chemiluminescence (ECL;
Amersham Pharmacia Biotech) according to the manufacturer's protocol.
Films were then scanned to quantitate the obtained
3-subunit and actin bands, and the ratio of
3 to actin was calculated. Results are representative of
three independent experiments and expressed as means ± SE.
Statistical analysis was performed with Student's t-test.
Cell attachment assay.
Forty-eight-well plates were coated overnight with the indicated
concentrations of thrombin or DIP-thrombin. The wells were blocked for
60 min with 0.5% BSA in PBS at 37°C. HUVECs were briefly trypsinized, followed by washes with serum-free M199 medium. Cells were
suspended 5 × 105 ml1 in
SFM199-0.5% BSA and incubated in the presence or absence of the
cyclic peptide EMD 121974 (c-RGD), cyclic peptide EMD 135981 (c-RAD),
or mouse anti-
v
3 (LM609) antibody for 15 min at 37°C. The c-RGD peptide (cycled
Arg-Gly-Asp-D-Phe-Nme-Val) is a highly active and specific
v
3-integrin antagonist (17),
whereas the c-RAD (cycled Arg-
-Ala-Asp-D-Phe-Val)
peptide is inactive. Cell suspensions (1 × 105 cells/well) were then added to the wells, and the
plates were incubated at 37°C for 60 min. The nonadherent cells were
aspirated, and the adhered cells were rinsed twice with cold PBS before
being fixed and stained with Diff-Quick (Baxter Healthcare). The
average area covered by adhered cells was measured in triplicate wells with a computerized digital image analyzer (ver. 4.12, MCID software, Brock University, St Catherines, ON, Canada). Each experiment was
repeated at least two times. Results are means ± SE expressed as
pixels area × 10
3.
Cell migration.
HUVECs migration was assessed by the modified Boyden's chamber assay,
i.e., in Transwell cell culture chambers (Corning and Costar).
Polycarbonate filters with 8-µm pores were used to separate the upper
and the lower chamber. When chemotaxis (directional motility) analysis
was performed, the indicated proteins were added to the lower chamber.
Endothelial cells were added to the upper compartment of the chamber at
density of 1 × 105 cells/100 µl in
SFM199-0.5% BSA and incubated for 6 h at 37°C, allowing
cells to migrate in the lower chamber. In some experiments, HUVECs were
pretreated with thrombin (1 IU/ml) for 12 h and then washed,
trypsinized, and added to the upper chambers. In haptotactic cell
motility assay, the undersurface of the membrane filter was precoated with the indicated concentrations of thrombin, DIP-thrombin, the ECM proteins vitronectin or gelatin, or heat-denatured BSA. To
modulate the migration toward the immobilized proteins, lower chambers
were filled with SFM199-0.5% BSA containing the c-RGD or c-RAD
peptide or anti-v
3 antibody (LM609).
After incubation for 6 h, the cells on the filters were fixed and
stained with Diff-Quick reagents. The nonmigrated cells (cells in upper
surface) were removed by wiping with cotton swabs. The filters then
with the migrated cells were cut off and mounted on glass slides. To examine the effect of soluble thrombin on the migration toward various
immobilized matrix proteins (vitronectin or gelatin), we added thrombin
(1 IU/ml) to the lower chambers. The cells on the lower surface were
counted manually under microscope in six predetermined fields at high
magnification. Each experiment was repeated at least two times, and
results are means ± SE expressed in terms of number of cells per
high-magnification microscopic field (cells/HMMF). Statistical analysis
was performed with Student's t-test.
Cell survival.
To determine the capability of immobilized thrombin to support cell
survival, we plated HUVECs suspended in SFM199-0.5% BSA on dishes
that had been precoated with thrombin, DIP-thrombin, or 0.5%
heat-denatured BSA. Where indicated, soluble c-RGD or c-RAD peptides or
anti-v
3 antibody was added to cells.
Apoptotic cell death was monitored 6 h later by measuring DNA
fragmentation using the Cell Death Detection ELISA kit (Roche Molecular
Biochemicals). Each experiment was repeated at least two times, and
results are means ± SE expressed as optical density at 402 nm
(OD402). Statistical analysis was performed with Student's
t-test.
Solid-phase ligand-binding assay.
Microtiter wells were coated with thrombin or DIP-thrombin in PBS
overnight at room temperature. The wells were blocked with 1% BSA in
Ca2+/Mg2+ TBS (150 mM NaCl, 25 mM
Tris · HCl, pH 7.4, 1 mM CaCl2, 1 mM MgCl2) at room temperature for 2 h. The
v
3 protein (Chemicon International) was
overlaid in Ca2+/Mg2+ TBS with 10 mM octyl
glucoside (Sigma) and incubated with rotation at 4°C overnight. To
modify the binding of the purified integrin to thrombin, we
preincubated the
v
3 protein with c-RGD
peptide, anti-
v
3 antibody (LM609), or
EDTA for 30 min at 4°C. Unbound integrin molecules were removed by
three washes with Ca2+/Mg2+ TBS-0.05% Tween
20. The bound
v
3 was incubated with
monoclonal mouse anti-
v
3 antibody (LM609)
for 2 h at room temperature. After extensive washes with
Ca2+/Mg2+ TBS-0.05% Tween 20, the bound
antibodies were detected by using rabbit anti-mouse IgG (Chemicon
International) conjugated with horseradish peroxidase. Substrate
solution consisting of hydrogen peroxide and tetramethylbenzidine was
added to the wells. The reactions were stopped with 2 N sulfuric acid,
and absorbance was measured at 492 nm (OD492). Background
absorbance observed in the wells coated with BSA was deducted from the
values obtained. Each experiment was repeated at least two times, and
results are means ± SE expressed as OD492.
Statistical analysis was performed with Student's t-test.
Chick chorioallantoic membrane assay. The in vivo chick chorioallantoic membrane (CAM) angiogenesis model was used as described previously (31). Briefly, biochemical evaluation of angiogenesis was performed by determining the extent of collagenous protein biosynthesis in the CAM lying directly under the disks applied at day 9 of chick embryo development. Both control and test disks contained radiolabeled proline (0.5 µCi/disk), and test disks also contained thrombin or c-RGD or c-RAD peptides, or the combination. After 48 h, the tissue under the disks was subjected to collagenase digestion. The resulting radiolabeled tripeptides, corresponding to basement membrane collagen and other collagenous material synthesized by the CAM, were counted and expressed as cpm/mg protein. For each egg, collagenous protein biosynthesis under the disk containing the test material was expressed as a percentage of that under the control disk in the same egg. Results are means ± SE expressed as % of control. Statistical analysis was performed with Student's t-test.
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RESULTS |
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Thrombin upregulates v
3 mRNA and
protein levels in HUVECs.
We employed a sensitive quantitative RT-PCR technique to examine
3 gene expression in HUVECs. Primers for
3 and GAPDH were chosen so that they would correspond to
the regions where sequence homologies between the two primers are
relatively low and would generate products of differing lengths. This
allowed us to perform RT-PCR with the housekeeping gene GAPDH into the
same reaction tubes with
3. Results presented in this
report show that each set of primers worked well in specifying their
corresponding mRNA. Single bands at about 284 and 967 bp were obtained
for
3 and GAPDH mRNA, respectively. Titration curves of
RT-PCR products were employed for determining the quantitative range in
which the reactions proceeded exponentially (Fig.
1A). Signal intensities of the
products obtained were plotted as functions of RNA template amount and
cycle number. Thus we established the optimal conditions for RT-PCR,
which were performed with 500 ng of total RNA for 26 cycles. As shown
in Fig. 1B, treatment of HUVECs with thrombin (1.0 IU/ml)
resulted in a significant increase in the message for
3
compared with untreated cells. The upregulation of
3
mRNA expression was evident 8 h after thrombin stimulation. In
addition, thrombin increased
3 mRNA in a dose-dependent
fashion. As shown in Fig. 1C, thrombin at 1 IU/ml increased
3 mRNA expression to maximal levels. At 3 IU/ml, the
stimulatory effect of thrombin declined to control levels. This
bell-shaped effect of thrombin is observed in many of the cellular
actions of thrombin, including angiogenesis (47). The
specificity of thrombin was examined with the use of hirudin, which
inhibits thrombin by binding both the catalytic and the anion-binding
exocite. When hirudin (1 IU/ml) combined with thrombin (1 IU/ml), the
thrombin-upregulating effect was abolished (Fig. 1D). In
addition, DIP-thrombin, which is catalytically inactive, had no effect
on
3 mRNA levels (Fig. 1D).
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Endothelial cell adhesion on immobilized thrombin is mediated by
v
3-integrin.
The upregulation of
v
3 by thrombin raises
the question as to whether this thrombin-induced angiogenic phenotype
of endothelial cells is manifested in other integrin-mediating effects
related to angiogenesis such as attachment, migration, and survival.
Quantitative cell attachment assays were used to characterize the
endothelial cell-thrombin interactions. As shown in Fig.
2A, immobilized thrombin supported endothelial cell adhesion in a concentration-dependent manner
up to 1 µg/well. Above this concentration we observed the characteristic bell-shaped curve, known to occur in many of the actions
of thrombin. DIP-thrombin (the chemically inactivated thrombin at the
active site) also increased cell attachment when immobilized on a solid
surface. This excluded the involvement of the catalytic site of
thrombin and the requirement for a proteolytic activation of thrombin
receptors on endothelial cells. Furthermore, it was demonstrated that
cell attachment on immobilized thrombin or DIP-thrombin was
v
3 dependent. When HUVECs were pretreated with 10 µg/ml LM609 antibody or 10 µg/ml c-RGD, the attachment of
endothelial cells on thrombin or DIP-thrombin was abolished (Fig.
2B). The inactive c-RAD peptide had no effect (Fig. 2B).
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Immobilized thrombin promotes
v
3-dependent endothelial cell migration.
The
v
3-integrin is known to have a
critical role in cell migration and survival (28, 34).
Furthermore,
v
3 antagonists have been
shown to exert their antiangiogenic effects in vivo by blocking
survival signals mediated by this integrin (8, 44). In
view of the above findings, we examined whether thrombin affects
v
3-integrin-dependent endothelial cell
migration and survival.
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Immobilized thrombin promotes
v
3-dependent endothelial cell survival.
When HUVECs were plated on immobilized BSA under serum-free conditions,
significant levels of apoptosis were detected after 6 h of
incubation (Fig. 5). In contrast, and
similar to what was reported previously (44), plating of
HUVECs on immobilized anti-
v
3 antibody
protect cells from apoptosis (data not shown). We found that
immobilized thrombin or DIP-thrombin mimics the effects of anti-
v
3 antibody and similarly promoted
endothelial cell survival. Decreased levels of apoptosis were
detected in cells adherent on plates coated with thrombin or
DIP-thrombin (Fig. 5). When soluble
anti-
v
3 antibody or c-RGD peptide was
present, a significant increase in the apoptosis of cells
plated on immobilized thrombin was observed (Fig. 5). In contrast, the
inactive peptide c-RAD was without effect on thrombin-induced cell
survival (Fig. 5).
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Thrombin binds to purified
v
3-integrin.
We demonstrated a direct
thrombin-
v
3-integrin interaction in a
solid-phase ligand binding assay, using a commercially available purified
v
3 preparation. As shown in Fig.
6A, soluble
v
3-integrin can bind to immobilized
thrombin or DIP-thrombin in a concentration-dependent manner, and the
binding is saturable. The specificity of the interaction was
established by the fact that when
v
3 was
neutralized by anti-
v
3 monoclonal
antibody (LM609) or antagonized by c-RGD peptide, the binding was
abolished. When EDTA was present, the binding was also abolished,
suggesting that this process was cation dependent (Fig. 6B).
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Integrin v
3 is involved in
thrombin-induced angiogenesis.
We have used collagenous protein biosynthesis (CPB) as a biochemical
index of angiogenesis (31). As shown in Fig.
7, thrombin (1 IU/disk) caused 100%
increase of CPB, which is in line with our previous results
(47). The c-RGD (100 µg/disk) peptide when used alone
caused an inhibition of 30% compared with controls. The combination of
thrombin with c-RGD peptide decreased the thrombin-promoting angiogenesis to 28%. The inactive c-RAD peptide had no effect. These
results suggest that
v
3 antagonists can
modulate the overall angiogenic effect of thrombin.
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DISCUSSION |
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In this report we have shown in endothelial cells that thrombin
increases the mRNA and protein levels of
v
3-integrin in a time- and
concentration-dependent fashion. The transduction mechanisms involved
for this upregulation are unknown. We are in the process of exploring
these mechanisms and the requirement of thrombin receptor activation.
Upregulation of
v
3 by thrombin has also
been demonstrated previously in human colon adenocarcinoma cells
(11) and in cultured smooth muscle cells
(43). Furthermore, it has been shown that thrombin induces
adhesion of tumor cells, neutrophils, and monocytes to endothelial cell
by increasing the expression of the integrin
IIb
3 in these cells (27, 36, 48).
We have also demonstrated that thrombin serves as a ligand to
v
3. In a cell-free system, immobilized
thrombin binds to purified human
v
3-integrin. Soluble
anti-
v
3 antibody, c-RGD, or EDTA blocked
this interaction. These findings are in line with the results of Byzova
and Plow (9), who have shown that prothrombin can serve as
a ligand for activated
v
3 on vascular
endothelial cells and smooth muscle cells. This was proposed as an
explanation for a previously unrecognized interface between the
adhesive and procoagulant properties of these cells.
The interaction of thrombin with the
v
3-integrin of endothelial cells is of
functional significance. As shown in Fig. 2, immobilized thrombin
supported endothelial cell attachment. This adhesion apparently was
mediated via RGD sequence of the thrombin molecule, and the chemically
inactivated DIP-thrombin behaves the same as, if not better than,
native thrombin probably because DIP-thrombin has more of the RGD
sequence exposed. The endothelial cell attachment to thrombin or
DIP-thrombin was specific and dependent on
v
3. A selective antagonist peptide to
v
3 (c-RGD), but not the inactive analog
(c-RAD), as well as a monoclonal antibody against
v
3 (LM609) prevented the adhesion to
thrombin. Thrombin as well as prothrombin contains an RGD sequence
within its catalytic domain. It was shown by Stubbs and Bode
(45) by analysis of the crystal structure of thrombin that
the RGD sequence is part of its active site and lies at the bottom of
the S1 specificity pocket. On the basis of that finding, it was
postulated that the RGD domain is not accessible on the surface of the
thrombin molecule. This proposal was further supported by the work of
Bar-Shavit and colleagues (5, 6), who reported that
cleavage products of thrombin or chemically modified thrombin, but not
native thrombin, increased the adhesion of endothelial cells in an
RGD-dependent manner. Contrary to these results, we found that
endothelial cells adhered to native thrombin. The source of our
thrombin preparation was the same as that used in the work of
Bar-Shavit and colleagues (5, 6). Similar results were
also obtained when a commercially available purified native
-thrombin (Sigma) was used (data not shown). The preparation of
thrombin used was fully active in two assay systems: upregulating
v
3-integrin and promoting angiogenesis in
the CAM assay. Both thrombin preparations supported endothelial cell
adhesion in a dose-dependent manner up to 1 µg/well. Above that
concentration of thrombin, the effect is diminished. This dose-dependent effect of thrombin may explain the discrepancy with the
results of Bar-Shavit and colleagues (5, 6), although in
their work the thrombin concentrations used were not obvious. In their
work, they found that DIP-thrombin,
-thrombin, and
N-
-tosyl-L-lysylchloromethylketone-thrombin were poorly active in endothelial cell attachment. On the contrary, NO2-thrombin is active and its action is antagonized by
soluble native thrombin, which they claimed to be inactive.
The bell-shaped effect of thrombin is a general phenomenon for many of
the actions of thrombin and is not well understood. Receptor-mediated
effects of thrombin may be related to desensitization and recycling of
thrombin receptors by thrombin (30). In our experiments of
endothelial cell attachment and migration to thrombin, one possible
explanation might be that increasing thrombin concentration favors
thrombin-thrombin interactions, thus hiding binding sites. Another
explanation might be that thrombin at higher concentrations binds to
low-affinity sites in the cell surface and activates intracellular
mechanisms that antagonize the effects of high-affinity sites. This
proposal is supported by the results of Sower et al. (42).
They have shown that increased concentrations of the thrombin peptide
TP508 (corresponding to the binding region of thrombin) stimulated a
nonproteolytically activated receptor component. This caused an
increased expression of annexin V, which has been shown to inhibit
protein kinase C (40). Protein kinase C inhibition may be
the explanation for the effects of high thrombin concentrations. It has
also been shown that various types of cells respond to thrombin in a
biphasic way. Zain et al. (49) and Ahmad et al. (2) have shown that a low concentration of thrombin
induces mitogenesis in tumor cells, whereas a high concentration
impairs cell growth. The apoptosis-inducing effects of thrombin
in neurons and astrocytes were only visible at high concentrations. On
the contrary, at low concentrations, thrombin was mitogenic for these nervous tissue cells and protected them from oxidative stress, cytotoxicity of -amploid, hypoxia, and withdrawal of growth
factors (15, 16). Opposing concentration-dependent effects
of thrombin were also shown in rat glioma cells by Schafberg et al.
(39).
Thrombin also acts as an haptotactic factor for endothelial cells. As
shown in Fig. 3, endothelial cells migrated toward immobilized thrombin
or DIP-thrombin, and this effect was also specific and v
3 dependent. The presence of c-RGD or
LM609 (but not the inactive c-RAD peptide) cancelled out the
haptotactic effect of thrombin. Furthermore, thrombin mimics the effect
of other matrix proteins such as vitronectin and gelatin in enhancing
endothelial cells migration. However, when soluble thrombin is present,
this binding is abolished, most likely because of saturation of
v
3 binding sites of endothelial cells by
thrombin. The fact that the promotion of endothelial cells migration by
thrombin is an
v
3-mediated event is also
supported by the results of the experiments of Fig. 4B. When
endothelial cells are pretreated with thrombin for 12 h, for
upregulation of
v
3 expression, their
ability to migrate toward vitronectin or gelatin is substantially
increased. We cannot rule out the possibility that thrombin may also
interact with other members of the integrin family, because it occurs
in human melanoma cells in which thrombin interacts with
v
5 integrin (20).
Experiments are in progress to assess this possibility. The association
of endothelial cells with thrombin via
v
3
integrin supports their survival under serum-free conditions. The data presented in Fig. 5 indicate that the protection from apoptosis caused by thrombin or DIP-thrombin is cancelled out when c-RGD or LM609
is present. This is in line with in vivo results by others (8), who have shown that integrin
v
3 antagonists promote tumor regression
by inducing apoptosis in angiogenic blood vessels.
The intracellular signaling events triggered by
v
3-thrombin interaction is under
investigation. Recent reports showing that integrin engagement leads to
activation of MAP kinases and that this pathway is mediated by the
stimulation of p125FAK tyrosine phosphorylation (10,
35). Thrombin, like VEGF, increases pp125FAK
tyrosine phosphorylation in platelets, mesangial cells, and endothelial cells (1, 13, 22, 29). It is likely, therefore, that the
stimulation of endothelial cells attachment, migration, and survival by
thrombin via
v
3 involves an extensive
network of signaling events distal to p125FAK. Components
of focal adhesions and of endothelial cell-to-cell junctions are both
linked to the actin of cytoskeleton and may be functionally integrated
through common signaling events in the migration and survival of
endothelial cells.
The aforementioned v
3-mediated effects of
thrombin are most likely contributing to angiogenic action of thrombin,
providing an explanation for the angiogenesis par excellence occurring
within thrombi. A very common clinical observation is that after
thrombosis in a large vein, the thrombus is recanalized with new
vessels seen with angiography. It is known that while thrombin in the plasma is rapidly inactivated by anti-thrombin, the thrombin trapped within the thrombi is protected and is slowly released during thrombolysis. Most likely, this trapped thrombin acts as angiogenic factor by attracting endothelial cells, mediating their angiogenic phenotype. In addition, thrombin is also known to interact with various
constituents of the ECM. ECM-immobilized thrombin is protected from
inactivation by its circulating inhibitors and induces many cellular
responses (26). Binding of thrombin to the subendothelial ECM through a short anchorage binding site leaves the majority of the
molecule functional and available for cellular interaction (4).
The experimental findings discussed here provide evidence at the
molecular and cellular level that many processes involved in
angiogenesis are promoted by thrombin through
v
3-integrin. In the CAM system, which is
used as a model for studying angiogenesis in vivo by many
investigators, we have shown that
v
3
antagonist can downregulate the angiogenesis-promoting effect of
thrombin. The results present in this paper along with our previous
findings (46, 47) establish the role of thrombin as an
angiogenic factor. Many of the actions of thrombin on endothelial cells
can contribute to their angiogenic phenotype and provide an explanation
for the long-known association between thrombosis and tumor progression.
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ACKNOWLEDGEMENTS |
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We thank Dr. Simon Goodman and Dr. Alfred Jonczyk (Merck, Germany) for providing the cyclic peptides EMD 121974 and EMD 135981.
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FOOTNOTES |
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This work was supported by grant from the Greek Ministry of Research and Technology.
Address for reprint requests and other correspondence: M. E. Maragoudakis, Dept. of Pharmacology, Medical School, Univ. of Patras, 25110 Patras, Greece (E-mail: maragoud{at}med.upatras.gr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
July 17, 2002;10.1152/ajpcell.00162.2002
Received 10 April 2002; accepted in final form 11 July 2002.
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