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Tumor Biology |
Department of Genetics and Pathology, Rudbeck Laboratory, S-751 85 Uppsala [H. L., J. D., L. C-W.]; The Ludwig Institute for Cancer Research, Biomedical Center, S-751 24 Uppsala [T. S., A. Ö.]; and Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Biomedical Center, S-751 23 Uppsala [K. Y., I. B.], Sweden
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Endogenous angiogenesis inhibitors are fragments of abundant proteins, which by proteolytic cleavage gain new characteristics and specifically inhibit endothelial cell function. Examples of endogenous inhibitors are angiostatin, a fragment of plasminogen (4) , and endostatin, a fragment derived from the COOH-terminal noncollagenous part of collagen XVIII (5, 6, 7) . Angiostatin and endostatin have been shown to arrest tumor expansion in a synergistic manner (8) . Furthermore, a Mr 29,000 fragment of fibronectin (9) and a Mr 16,000 fragment of prolactin have been shown to possess antiangiogenic characteristics (10) . Other strategies for inhibition of endothelial cell function include the use of agents that inhibit VEGF3 or VEGF receptor signal transduction (11) . Furthermore, synthetic peptides that disrupt the binding of integrins to specific extracellular matrix proteins interfere with endothelial cell survival (12) .
The cleaved and latent forms of the serpin antithrombin have been shown recently to have antiangiogenic properties (13) . Antithrombin is a heparin-binding protein and the major plasma inhibitor of coagulation proteases, primarily thrombin and factor Xa (14 , 15) . Antithrombin inhibits its target proteases by exposing its reactive-site loop as a pseudosubstrate for the protease (14) . Cleavage at the reactive site triggers the insertion of the reactive-site loop into the main ß-sheet of antithrombin. As a consequence of this insertion, the protease is translocated to the opposite pole of the inhibitor and concurrently inactivated. The reactive-site loop of antithrombin can be cleaved, and thereby inactivated, by a number of proteases (16) . Moreover, an inactive form of antithrombin, denoted as latent, is produced by heat treatment of the inhibitor (15) . In cleaved and latent forms of antithrombin, the reactive-site loop is no longer present on the surface of the antithrombin but is inserted into the main ß-sheet of the molecule (17) .
We show that latent antithrombin efficiently inhibited tumor angiogenesis in a mouse fibrosarcoma model, when administered s.c. at only 1 mg/kg/day. Latent antithrombin did not affect the activation of FGF receptor 1 but inhibited cell migration toward FGF-2 and activation of FAK, and induced an increase in apoptosis of endothelial cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of Antithrombin.
Reactive loop-cleaved antithrombin was obtained by digestion of
purified human plasma antithrombin (19)
for 5 min at
37°C with thermolysin (20)
, followed by chromatography
on a 5 ml of HiTrap Heparin-Sepharose column (Amersham Pharmacia
Biotech, Uppsala, Sweden). Cleaved antithrombin appeared at about 0.3
M NaCl, appreciably before uncleaved antithrombin, which
appeared at about 1 M NaCl. Its behavior in SDS-PAGE was
characteristic of that of reactive-loop-cleaved antithrombin
(21)
. SDS-PAGE under nondenaturing conditions showed no
aggregates. The thrombin-inhibitory activity of the cleaved
antithrombin was <2% of that of uncleaved antithrombin
(16)
. Latent antithrombin was prepared by incubation of
plasma antithrombin (4 g/l) for 24 h at 60°C in 10
mM Tris/HCl, 0.5 M sodium citrate, pH 7.4
(15)
. The latent inhibitor was purified by heparin
affinity chromatography and eluted at about 0.3 M NaCl. It
migrated indistinguishably from native antithrombin in SDS-PAGE under
nonreducing and reducing conditions. SDS-PAGE under nondenaturing
conditions showed evidence of <5% of aggregates. The ability of the
latent form to inhibit thrombin was <2% of that of native
antithrombin (15)
.
CAM Assay.
The procedure of the CAM assay followed essentially that described
previously (22
, 23)
. The CAM was exposed by making a
1 x 1-cm window in the shell of 10-day fertilized hen
eggs. Filter discs (Whatman, Inc.) were saturated with 3 mg/ml
cortisone acetate (dissolved in 70% ethanol, which was allowed to
evaporate before addition of growth factors and inhibitors; Sigma) and
soaked in buffer (30 µl for each filter) with or without FGF-2
(Boehringer Mannheim; 0.2 µg/filter) and different forms of
antithrombin (3, 0.3 or 0.03 µg/filter), and the disc was added to an
avascular part of the CAM. After 3 days of incubation, the membrane was
inspected in a light microscope (Nikon Eclipse TE 300; x2.5 or x4).
Animal Studies.
The animal work was approved by the local board of animal
experimentation and performed according to the United Kingdom
Coordinating Committee on Cancer Research guidelines (24)
.
The animals were anesthetized with Isoflurane (Forene; Abbott) during
all manipulations. Female C57BL6/J mice (M&B, Ejby, Denmark), 6–8
weeks of age, were acclimated and caged in groups of five. T241
fibrosarcoma cells, 0.5 x 106 in
50 µl of DMEM, were injected s.c. into the left flank of the mouse.
Animals carrying palpable tumors within 4 days after injection were
randomized to 10-day treatment with 1 mg/kg/day of latent antithrombin,
thermolysin-cleaved antithrombin, native antithrombin, or vehicle
(PBS), given as a daily s.c. injection in the right flank. The tumors
were measured with a caliper once a day, in a double-blind procedure,
and their volumes were calculated by the formula 
/6 x width2 x length. ANOVA was
used for statistical analysis. After 10 days of treatment, the mice
were sacrificed with a lethal dose of pentobarbitone and perfused with
4% paraformaldehyde in Millonig’s phosphate buffer (pH 7.4). The
tumors were then embedded in paraffin according to standard
histological procedures and cut into sections 4 µm thick.
Immunohistochemical Staining of Tumor Sections.
PCNA was detected with use of the monoclonal antibody PC10 (0.5
µg/ml; Santa Cruz). Sections were pretreated for 2 x 7 min in a microwave oven at 750 W in 10 mM citrate buffer
(pH 6.0), followed by immunohistochemical staining using PC10 on a
Ventana NexES immunostainer with a diaminobenzidine peroxidase kit
(Ventana Medical Systems, Tucson, AZ). The percentage of PCNA-positive
cells in 2000 counted cells was estimated. Replacement of the primary
antibody with an irrelevant mouse IgG served as a negative control.
TUNEL for detection of apoptotic cells was performed as described (25) . Peroxidase-coupled Fab fragments raised against dUTP-digoxigenin (Roche) and diaminobenzidine peroxidase substrate were used for detecting positive reactions. Omission of terminal deoxynucleotidyl transferase enzyme served as a negative control. Sections were counterstained in Mayer’s hematoxylin, dehydrated, and mounted in Mountex resin (Histolab, Göteborg, Sweden). At least 1000 cells per tissue section were counted.
Tumor Angiogenesis.
For analysis of tumor angiogenesis, hematoxylin-stained tumor sections
were coded, and perfused vascular structures were counted at x400,
using an eyepiece grid of 10 x 10 squares. To adjust
for the presence of apoptotic, necrotic, or hemorrhagic areas, the
presence or absence of viable tumor tissue in the uppermost square to
the far right of the grid was noted and used in the calculations of
vascular parameters. Sampling of vision fields and stereological
quantification were performed, using the vascular parameters described
by Wassberg et al. (26)
.
Proliferation Assay.
PAE/FGFR-1 cells were seeded into 24-well dishes (2 x 104 cells/well). After 3 h, the medium was
changed to Ham’s F-12 containing 0.1% FCS. After an additional
12 h, FGF-2 (20 ng/ml), antithrombin (10 µg/ml), or FGF-2 (20
ng/ml) in combination with antithrombin at 1 or 10 µg/ml was added,
and added again at days 2 and 4. Numbers of cells in triplicates were
scored after 5 days of incubation, using a Coulter counter.
Annexin V Assay.
PAE/FGFR-1 cells cultured in Ham’s F-12, 10% FCS were incubated with
different forms of antithrombin (3 µg/ml). After 8 h, the medium
was changed to Ham’s F-12, 0.1% FCS and fresh antithrombin with or
without FGF-2 (10 ng/ml). After 18 h of incubation, the cells were
prepared according to the Annexin-V-FLUOS kit (Boehringer Mannheim).
Briefly, cells were trypsinized, stained with fluorescein-conjugated
Annexin V and 2.5 µg/ml propidium iodide, and analyzed with a flow
cytometer (FACSCalibur), with 488 nm excitation and a collecting light
scatter, green and red fluorescence. Apoptotic cells were defined as
cells with enhanced Annexin V fluorescence simultaneously exhibiting
normal propidium iodide staining. The frequency of necrotic cells (with
strongly increased propidium iodide staining) showed no differences
between the different experimental conditions used.
TUNEL Assay.
BCE cells were cultured in DMEM containing 10% NCS with 1 ng/ml FGF-2.
After 24 h, the medium was changed to starvation medium, DMEM with
0.25% NCS, containing latent antithrombin (3 µg/ml) or native
antithrombin (3 µg/ml). Forty h later, the cells were harvested,
fixed, and prepared according to the In situ Cell Death
Detection kit-Fluorescein kit with fluorescein-conjugated dUTP (Roche
Diagnostics). The percentage of TUNEL-positive cells in 1000 counted
cells was determined.
Migration Assay.
The migration assay was performed in a modified Boyden chamber
(27)
, using micropore nitrocellulose filters (8 µm
thick, 8-µm pores) coated with type-1 collagen solution at 100
µg/ml (Vitrogen 100; Collagen Corp.). Endothelial cells were
preincubated with latent antithrombin (3 µg/ml) for 30 min,
trypsinized, and resuspended at a concentration of 5.5 x 105 cells/ml in Ham’s F-12 medium
containing 0.1% FCS. The cell suspension was placed in the upper
chamber, and serum-free medium containing 0.25% BSA and 5 ng/ml FGF-2
or 5 µg/ml latent antithrombin, individually or in combination, was
placed below the filter. FCS at 10% was used as a positive control.
After 4 h at 37°C, the medium was removed, and cells adhering to
the filter were fixed in pure methanol and stained with Giemsa stain.
Cells on the lower side of the filter were counted in three separate
microscopic fields. Samples were analyzed in triplicate on three
separate occasions.
Immunoprecipitation and Immunoblotting.
PAE/FGFR-1 cells were serum starved overnight and stimulated or not
with FGF-2 (100 ng/ml), latent antithrombin (3 µg/ml) individually or
in combination, for 10 min at 37°C. The cells were lysed in
NP40-containing buffer, and samples were separated by SDS-PAGE and
transferred to Hybond-C Extra (Amersham Pharmacia Biotech). The
membranes were immunoblotted with anti-phosphoFAK antibodies (Biosource
International) and subsequently with anti-FAK antibodies (Biosource
International). Immunoreactive proteins were visualized by a
chemiluminescence detection system (28)
.
Detection of Actin Reorganization and Paxillin Localization.
Serum-starved PAE/FGFR-1 on glass coverslips were incubated with or
without FGF-2 (100 ng/ml) and latent antithrombin (3 µg/ml) for 20
min at 37°C. The cells were fixed in 3.7% paraformaldehyde,
permeabilized with 0.2% Triton X-100, rinsed, and incubated either
with FITC-labeled phalloidin in PBS (0.66 µg/ml; Ref.
29
) or with paxillin antibodies (Santa Cruz) and
tetramethylrhodamine isothiocyanate-coupled secondary antibody.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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or even at an equimolar concentration (Table 1)
. VEGF-induced angiogenesis in the CAM was similarly inhibited by
latent antithrombin (data not shown). In contrast, native and
thermolysin-cleaved antithrombin preparations had only a weak
inhibitory effect on growth factor-induced angiogenesis (Fig. 1
and
Table 1
).
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A, the tumor volume in the PBS-treated animals was about
three times that in mice treated with latent antithrombin. The tumors
in mice treated with thermolysin-cleaved antithrombin were only
slightly smaller than those in the control animals. Native antithrombin
also inhibited tumor expansion, although less efficiently than latent
antithrombin (Fig. 2B)
. Thus, at 1 mg/kg/day, latent
antithrombin was the most efficient of the different forms of
antithrombin in halting tumor expansion in this model, which is in
agreement with the results obtained in the CAM angiogenesis assay (Fig. 1)
.
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A,
were examined with regard to proliferation, apoptosis, and
angiogenesis. As shown in Fig. 2
C, there was no difference
in the proliferative index between tumors from animals treated with
latent antithrombin and with vehicle. The number of apoptotic tumor
cells was determined by TUNEL labeling, which specifically labels DNA
strand breaks, indicative of apoptosis (25)
. The number of
TUNEL-positive cells increased significantly from 1.5 to 3.1% with
latent antithrombin treatment (Fig. 2C)
. As shown in Table 2
, treatment with 1 mg/kg/day of latent antithrombin led to a decrease in
the vessel length density of the tumors, as compared with control
(P < 0.05) and to a tendency toward a
decrease in both the volumetric and surface density of the vessels. As
a control, the mean vessel section area, boundary length, and section
diameter were estimated and were found not to vary among the different
conditions (data not shown).
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shows that FGF-2 treatment induced an increase in the number of cells
to 340% of that in the controls in 5 days. In cultures treated with
FGF-2 together with latent antithrombin, the number increased to only
230%. In cultures treated with latent antithrombin alone, the number
of cells was slightly decreased compared with the basal condition. The
VEGF-induced increase in the number of PAE cells expressing VEGF
receptor-2 was similarly diminished by latent antithrombin (data not
shown). In contrast, FGF-2-induced proliferation of COS (monkey kidney
epithelial) cells was not affected by latent antithrombin (data not
shown).
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, presumably as a result of growth factor-induced survival. Treatment
with latent antithrombin led to a nearly 2-fold increase in Annexin
V-positive cells. Addition of FGF-2 to cells treated with latent
antithrombin gave slight protection against apoptosis, but comparison
of the cultures treated with FGF-2 with and without latent antithrombin
showed that the survival signal by FGF-2 was strongly counteracted by
latent antithrombin. Primary BCE cells also underwent apoptosis when
exposed to latent antithrombin for 40 h, as assessed by TUNEL
labeling (data not shown). The number of TUNEL-positive cells increased
from 1.5 to 4.2% among cells treated with latent antithrombin. As a
control, native antithrombin was added to the BCE cell medium. This led
to an increase in TUNEL-positive cells to 2.9% of the cell population.
Thus, native antithrombin was less efficient than latent antithrombin
in inducing endothelial cell apoptosis but was still active in this
respect. In contrast, T241 cell cultures did not show increased
apoptosis when treated with latent antithrombin under similar
conditions (data not shown).
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, the relative number of migrating cells increased from 100% (control,
BSA-treated) to 150% when stimulated with FGF-2. When latent
antithrombin was added together with FGF-2 during the Boyden chamber
assay, the number of migrating cells decreased to 105%. Latent
antithrombin in itself did not affect migration under these conditions.
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showed that treatment with FGF-2 for 20 min led to
formation of dense, actin-containing membrane structures, denoted as
edge ruffles. In cells treated with latent antithrombin together with
FGF-2 for 20 min, actin stress fibers were present, but the ruffle
formation was almost attenuated.
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shows that latent antithrombin treatment
abolished FGF-2-induced FAK tyrosine phosphorylation, without affecting
the FAK protein levels. Treatment with latent antithrombin alone, but
not with native antithrombin, led to increased levels of tyrosine
phosphorylated FAK. Induction of FGFR-1 tyrosine kinase activity by
FGF-2 in PAE/FGFR-1 cells was unaffected by treatment with latent
antithrombin (Fig. 6C)
. The basal level of FGFR-1 activation
in endothelial cells treated with latent antithrombin alone was
slightly increased.
PAE/FGFR-1 cells were further stained with an antiserum recognizing
paxillin, which is localized in focal adhesion contacts (Fig. 6D)
. FGF-2 stimulation of the cells for 20 min led to the
appearance of a punctate radial pattern, typical for focal adhesion
contacts. Coincubation of the cells with FGF-2 and latent antithrombin
prevented formation of focal adhesion contacts. There was no effect on
formation of focal adhesion contacts in cells treated with latent
antithrombin alone. Together, these results indicate that latent
antithrombin exerts its antiangiogenic effects by negative regulation
of the actin cytoskeleton, FAK, and focal adhesion formation.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, the unchanged proliferation index, and the increased
apoptosis of tumor cells (Fig. 2C)
, together with the
results from the CAM assay, imply that latent antithrombin exerts its
antitumoral effects through inhibition of angiogenesis. Native
antithrombin also decreased tumor growth, although less efficiently,
whereas tumors in animals treated with thermolysin-cleaved antithrombin
were similar in size to those in the control animals. The latent but
not the cleaved form of antithrombin was also found to be a potent
angiogenesis inhibitors in the CAM. However, native antithrombin did
not effectively inhibit neoangiogenesis in the CAM (Fig. 1)
. In a recent publication by O’Reilly et al. (13) , pancreatic elastase-cleaved and latent antithrombin were shown to be equally potent in arresting expansion of SK-NAS neuroblastoma and Lewis lung carcinoma when administered at 25 mg/kg/day, whereas native antithrombin had no effect in the tumor models investigated. Our results indicate that cleaved antithrombin has a lower antiangiogenic and tumor-inhibitory ability than latent antithrombin. This difference may have been obscured in the study by O’Reilly et al. (13) by the 25-fold higher dose and the longer duration of the treatment. The different effects of cleaved and latent antithrombin are compatible with the marked difference in their three-dimensional structures. Most strikingly, in cleaved antithrombin, residues 402–407 form strand 1 of ß-sheet C, and residues 394–401 are located in the region originally occupied by the uncleaved reactive-site loop (on "top" of the molecule in the standard orientation) but are highly mobile (17) . In contrast, in latent antithrombin, sheet C is disrupted, and the segment between residues 394 and 407 forms an exposed loop on the "side" of the protein (33) . Nevertheless, we cannot exclude the possibility that the use of different proteases, in preparing cleaved antithrombin namely elastase in the study by O’Reilly et al. (13) and thermolysin in the present work may have been partly responsible for the different effects in the two investigations. The native antithrombin used in the present study was prepared in-house from human plasma, and according to criteria such as heparin binding and thrombin-inhibitory ability (19) , the preparation was homogeneous, native antithrombin. Conversion of a certain amount of the native inhibitor to the latent form in the mice, as suggested for antithrombin in humans (32) , is a possibility that would be in agreement with the lack of effect of native antithrombin on CAM angiogenesis.
Latent antithrombin binds heparin with a low affinity
(Kd of
10-4 M; Ref.
34
), which implies that binding to heparin or heparan
sulfate is not critical in the antiangiogenic effects of latent
antithrombin. Furthermore, latent antithrombin does not bind thrombin,
which would indicate that the effect is not mediated through
interaction with thrombin bound to the endothelial cell receptor,
thrombomodulin (35)
. However, it is possible that the
receptor for plasma clearance of antithrombin-protease complexes, the
low density lipoprotein receptor-related protein, may be involved. This
receptor has been shown to bind such complexes but not cleaved
antithrombin (36)
. It cannot be excluded that lipoprotein
receptor-related protein binds latent antithrombin, which would explain
the difference in antiangiogenic effect between latent and cleaved
antithrombin observed in the present study.
Our data show that activation of FAK in the FGF-stimulated endothelial cells was attenuated in the presence of latent antithrombin. FAK activation has been shown to correlate with cell survival (37) . Thus, the increased rate of apoptosis that we observed in the latent antithrombin-treated endothelial cells could be explained on the basis of the block in FAK signaling. Moreover, we observed an increase in FAK activation in endothelial cells treated with latent antithrombin alone. The mechanism of FAK perturbment might involve displacement of integrin-binding to the extracellular matrix, e.g., by presentation of a high affinity binding site in latent antithrombin. However, there is no apparent integrin-binding site, such as an R-G-D tripeptide, in the antithrombin sequence (38) . Cells from mice lacking FAK expression as a result of gene inactivation fail to migrate, possibly on account of a defect in focal adhesion turnover (31) . We observed a decrease in migration and decreased formation of focal adhesions in response to FGF-2 when cells were treated with latent antithrombin. The regulation of actin filament organization was also disturbed in the latent antithrombin-treated cells. Regulation of the actin cytoskeleton involves Rho family GTPases, which have also been implicated in the regulation of FAK and paxillin phosphorylation during cell adhesion (39) . Thus, it is possible that Rho GTPases are primary targets of latent antithrombin and that FAK activation is subsequently perturbed.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by Grant 3820-B99-04XBC
(to L. C. W.) from the Swedish Cancer Foundation, a grant from the
Novo Nordisk Foundation, a grant from the Göran Gustafsson
Foundation, and Grant 4212 (to I. B.) from the Swedish Medical
Research Council. 
2 To whom requests for reprints should be
addressed, at Department of Genetics and Pathology, Rudbeck Laboratory,
S-751 85 Uppsala, Sweden. Phone: 46-18-471-43-63; Fax:
46-18-55-89-31; E-mail: Lena.Welsh{at}genpat.uu.se
3 The abbreviations used are: VEGF, vascular
endothelial growth factor; FGF, fibroblast growth factor; FGFR, FGF
receptor; FAK, focal adhesion kinase; PAE, porcine aortic endothelial;
BCE, bovine capillary endothelial; NCS, newborn calf serum; CAM,
chorioallantoic membrane; PCNA, proliferating cell nuclear antigen;
TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end
labeling.
Received 5/30/00. Accepted 10/ 2/00.
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  B. Richard, R. Swanson, S. Schedin-Weiss, B. Ramirez, G. Izaguirre, P. G. W. Gettins, and S. T. Olson Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin J. Biol. Chem., May 23, 2008; 283(21): 14417 - 14429. [Abstract] [Full Text] [PDF]
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W. Zhang, R. Swanson, Y. Xiong, B. Richard, and S. T. Olson Antiangiogenic Antithrombin Blocks the Heparan Sulfate-dependent Binding of Proangiogenic Growth Factors to Their Endothelial Cell Receptors: EVIDENCE FOR DIFFERENTIAL BINDING OF ANTIANGIOGENIC AND ANTICOAGULANT FORMS OF ANTITHROMBIN TO PROANGIOGENIC HEPARAN SULFATE DOMAINS J. Biol. Chem., December 8, 2006; 281(49): 37302 - 37310. [Abstract] [Full Text] [PDF]
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-32P and analyzed by
SDS-PAGE. D, cells were treated as in A,
fixed, and stained using antibodies against paxillin, followed by a
tetramethylrhodamine isothiocyanate-coupled secondary antibody. Note
the punctate pattern in FGF-2-treated cultures (arrows),
which is missing in cells treated with FGF-2 together with latent
antithrombin. Right, quantification by counting the
number of focal contacts in five separate microscopic fields.
Bars, SE.