Antiangiogenic Effects of Latent Antithrombin Through Perturbed Cell-Matrix Interactions and Apoptosis of Endothelial Cells1

[CANCER RESEARCH 60, 6723–6729, December 1, 2000] Antiangiogenic Effects of Latent Antithrombin through Perturbed Cell-Matrix Interactions and Apoptosis of Endothelial Cells1

Helena Larsson, Tobias Sjo¨blom, Johan Dixelius, Arne O¨ stman, Karin Ylinenja¨rvi, Ingemar Bjo¨rk, and Lena Claesson-Welsh2 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. O¨ .]; and Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Biomedical Center, S-751 23 Uppsala [K. Y., I. B.], Sweden

ABSTRACT The cleaved and latent forms of the serpin antithrombin have been shown recently to have antiangiogenic properties (13). Antithrombin Antithrombin is a plasma protein of the serpin superfamily that may is a heparin-binding protein and the major plasma inhibitor of coag- occur as several conformational variants. The native form of antithrombin ulation proteases, primarily thrombin and factor Xa (14, 15). Anti- is a major regulator of blood clotting. In the present study, we have identified the mechanism underlying the antiangiogenic action of a heat- thrombin inhibits its target proteases by exposing its reactive-site loop denatured form, denoted latent antithrombin. Fibroblast growth factor as a pseudosubstrate for the protease (14). Cleavage at the reactive site (FGF)-induced angiogenesis in the chick embryo and angiogenesis in triggers the insertion of the reactive-site loop into the main ␤-sheet of mouse fibrosarcoma tumors were inhibited by treatment with latent antithrombin. As a consequence of this insertion, the protease is antithrombin at 1 mg/kg/day. Thermolysin-cleaved and native antithrom- translocated to the opposite pole of the inhibitor and concurrently bin were less efficient in these respects. Treatment with latent antithrom- inactivated. The reactive-site loop of antithrombin can be cleaved, and bin induced apoptosis of cultured endothelial cells and inhibited cell thereby inactivated, by a number of proteases (16). Moreover, an migration toward FGF-2. Under these conditions, FGF-2-stimulated FGF inactive form of antithrombin, denoted as latent, is produced by heat receptor kinase activity was unaffected. However, actin reorganization, treatment of the inhibitor (15). In cleaved and latent forms of anti- activation of focal adhesion kinase, and focal adhesion formation were thrombin, the reactive-site loop is no longer present on the surface of disturbed by latent antithrombin treatment of FGF-2-stimulated endothe- ␤ lial cells. These data indicate that latent antithrombin induces apoptosis of the antithrombin but is inserted into the main -sheet of the molecule endothelial cells by disrupting cell-matrix interactions through uncou- (17). pling of focal adhesion kinase. 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 INTRODUCTION FGF receptor 1 but inhibited cell migration toward FGF-2 and activation of FAK, and induced an increase in apoptosis of endothelial Angiogenesis, the formation of new capillaries, is a crucial process cells. during embryogenesis, in wound healing, and in the female reproduc- tive organs (1). A growing number of diseases, including cancer and inflammatory disorders, are characterized by excessive, deregulated MATERIALS AND METHODS angiogenesis (2), attributable to increased production of growth factors or to decreased production of angiogenesis inhibitors (3). Cell Culture. The PAE cell line overexpressing FGFR-1 (18) was cultured Endogenous angiogenesis inhibitors are fragments of abundant in Ham’s F-12 medium supplemented with 10% FCS. Primary BCE cells, kindly provided by Dr. Rolf Christofferson (Uppsala University, Uppsala, proteins, which by proteolytic cleavage gain new characteristics and Sweden) were cultured in DMEM containing 10% NCS and 3 ng/ml FGF-2 specifically inhibit endothelial cell function. Examples of endogenous (Boehringer Mannheim). The T241 fibrosarcoma cell line, kindly provided by inhibitors are angiostatin, a fragment of plasminogen (4), and endosta- Dr. Lars Holmgren (Karolinska Institute, Stockholm, Sweden), was cultured in tin, a fragment derived from the COOH-terminal noncollagenous part DMEM supplemented with 10% FCS. Media and sera were from Life Tech- of collagen XVIII (5–7). Angiostatin and endostatin have been shown nologies. to arrest tumor expansion in a synergistic manner (8). Furthermore, a Preparation of Antithrombin. Reactive loop-cleaved antithrombin was

Mr 29,000 fragment of fibronectin (9) and a Mr 16,000 fragment of obtained by digestion of purified human plasma antithrombin (19) for 5 min at prolactin have been shown to possess antiangiogenic characteristics 37°C with thermolysin (20), followed by chromatography ona5mlofHiTrap (10). Other strategies for inhibition of endothelial cell function include Heparin-Sepharose column (Amersham Pharmacia Biotech, Uppsala, Swe- the use of agents that inhibit VEGF3 or VEGF receptor signal trans- den). Cleaved antithrombin appeared at about 0.3 M NaCl, appreciably before uncleaved antithrombin, which appeared at about 1 M NaCl. Its behavior in duction (11). Furthermore, synthetic peptides that disrupt the binding SDS-PAGE was characteristic of that of reactive-loop-cleaved antithrombin of integrins to specific extracellular matrix proteins interfere with (21). SDS-PAGE under nondenaturing conditions showed no aggregates. The endothelial cell survival (12). thrombin-inhibitory activity of the cleaved antithrombin was Ͻ2% of that of uncleaved antithrombin (16). Latent antithrombin was prepared by incubation Received 5/30/00; accepted 10/2/00. of plasma antithrombin (4 g/l) for 24 h at 60°C in 10 mM Tris/HCl, 0.5 M The costs of publication of this article were defrayed in part by the payment of page sodium citrate, pH 7.4 (15). The latent inhibitor was purified by heparin charges. This article must therefore be hereby marked advertisement in accordance with affinity chromatography and eluted at about 0.3 M NaCl. It migrated indistin- 18 U.S.C. Section 1734 solely to indicate this fact. 1 This study was supported by Grant 3820-B99-04XBC (to L. C. W.) from the Swedish guishably from native antithrombin in SDS-PAGE under nonreducing and Cancer Foundation, a grant from the Novo Nordisk Foundation, a grant from the Go¨ran reducing conditions. SDS-PAGE under nondenaturing conditions showed ev- Gustafsson Foundation, and Grant 4212 (to I. B.) from the Swedish Medical Research idence of Ͻ5% of aggregates. The ability of the latent form to inhibit thrombin Council. was Ͻ2% of that of native antithrombin (15). 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; CAM Assay. The procedure of the CAM assay followed essentially that Fax: 46-18-55-89-31; E-mail: [email protected]. described previously (22, 23). The CAM was exposed by making a 1 ϫ 1-cm 3 The abbreviations used are: VEGF, vascular endothelial growth factor; FGF, fibro- window in the shell of 10-day fertilized hen eggs. Filter discs (Whatman, Inc.) blast growth factor; FGFR, FGF receptor; FAK, focal adhesion kinase; PAE, porcine were saturated with 3 mg/ml cortisone acetate (dissolved in 70% ethanol, aortic endothelial; BCE, bovine capillary endothelial; NCS, newborn calf serum; CAM, chorioallantoic membrane; PCNA, proliferating cell nuclear antigen; TUNEL, terminal which was allowed to evaporate before addition of growth factors and inhib- deoxynucleotidyl transferase-mediated dUTP nick end labeling. itors; Sigma) and soaked in buffer (30 ␮l for each filter) with or without FGF-2 6723

(Boehringer Mannheim; 0.2 ␮g/filter) and different forms of antithrombin (3, Migration Assay. The migration assay was performed in a modified Boy- 0.3 or 0.03 ␮g/filter), and the disc was added to an avascular part of the CAM. den chamber (27), using micropore nitrocellulose filters (8 ␮m thick, 8-␮m After 3 days of incubation, the membrane was inspected in a light microscope pores) coated with type-1 collagen solution at 100 ␮g/ml (Vitrogen 100; (Nikon Eclipse TE 300; ϫ2.5 or ϫ4). Collagen Corp.). Endothelial cells were preincubated with latent antithrombin Animal Studies. The animal work was approved by the local board of (3 ␮g/ml) for 30 min, trypsinized, and resuspended at a concentration of animal experimentation and performed according to the United Kingdom 5.5 ϫ 105 cells/ml in Ham’s F-12 medium containing 0.1% FCS. The cell Coordinating Committee on Cancer Research guidelines (24). The animals suspension was placed in the upper chamber, and serum-free medium contain- were anesthetized with Isoflurane (Forene; Abbott) during all manipulations. ing 0.25% BSA and 5 ng/ml FGF-2 or 5 ␮g/ml latent antithrombin, individ- Female C57BL6/J mice (M&B, Ejby, Denmark), 6–8 weeks of age, were ually or in combination, was placed below the filter. FCS at 10% was used as acclimated and caged in groups of five. T241 fibrosarcoma cells, 0.5 ϫ 106 in a positive control. After4hat37°C, the medium was removed, and cells 50 ␮l of DMEM, were injected s.c. into the left flank of the mouse. Animals adhering to the filter were fixed in pure methanol and stained with Giemsa carrying palpable tumors within 4 days after injection were randomized to stain. Cells on the lower side of the filter were counted in three separate 10-day treatment with 1 mg/kg/day of latent antithrombin, thermolysin- microscopic fields. Samples were analyzed in triplicate on three separate cleaved antithrombin, native antithrombin, or vehicle (PBS), given as a daily occasions. s.c. injection in the right flank. The tumors were measured with a caliper once Immunoprecipitation and Immunoblotting. PAE/FGFR-1 cells were se- a day, in a double-blind procedure, and their volumes were calculated by the rum starved overnight and stimulated or not with FGF-2 (100 ng/ml), latent formula ␲/6 ϫ width2 ϫ length. ANOVA was used for statistical analysis. antithrombin (3 ␮g/ml) individually or in combination, for 10 min at 37°C. The After 10 days of treatment, the mice were sacrificed with a lethal dose of cells were lysed in NP40-containing buffer, and samples were separated by pentobarbitone and perfused with 4% paraformaldehyde in Millonig’s phos- SDS-PAGE and transferred to Hybond-C Extra (Amersham Pharmacia Bio- phate buffer (pH 7.4). The tumors were then embedded in paraffin according tech). The membranes were immunoblotted with anti-phosphoFAK antibodies to standard histological procedures and cut into sections 4 ␮m thick. (Biosource International) and subsequently with anti-FAK antibodies (Bio- Immunohistochemical Staining of Tumor Sections. PCNA was detected source International). Immunoreactive proteins were visualized by a chemilu- with use of the monoclonal antibody PC10 (0.5 ␮g/ml; Santa Cruz). Sections minescence detection system (28). were pretreated for 2 ϫ 7 min in a microwave oven at 750 W in 10 mM citrate Detection of Actin Reorganization and Paxillin Localization. Serum- buffer (pH 6.0), followed by immunohistochemical staining using PC10 on a starved PAE/FGFR-1 on glass coverslips were incubated with or without Ventana NexES immunostainer with a diaminobenzidine peroxidase kit (Ven- FGF-2 (100 ng/ml) and latent antithrombin (3 ␮g/ml) for 20 min at 37°C. The tana Medical Systems, Tucson, AZ). The percentage of PCNA-positive cells in cells were fixed in 3.7% paraformaldehyde, permeabilized with 0.2% Triton 2000 counted cells was estimated. Replacement of the primary antibody with X-100, rinsed, and incubated either with FITC-labeled phalloidin in PBS (0.66 an irrelevant mouse IgG served as a negative control. ␮g/ml; Ref. 29) or with paxillin antibodies (Santa Cruz) and tetramethylrho- TUNEL for detection of apoptotic cells was performed as described (25). damine isothiocyanate-coupled secondary antibody. Peroxidase-coupled Fab fragments raised against dUTP-digoxigenin (Roche) and diaminobenzidine peroxidase substrate were used for detecting positive RESULTS reactions. Omission of terminal deoxynucleotidyl transferase enzyme served as a negative control. Sections were counterstained in Mayer’s hematoxylin, FGF-2- and VEGF-induced Angiogenesis in the CAM Is Inhib- dehydrated, and mounted in Mountex resin (Histolab, Go¨teborg, Sweden). At ited by Latent Antithrombin. We examined the effects of different least 1000 cells per tissue section were counted. forms of antithrombin on growth factor-induced angiogenesis in the Tumor Angiogenesis. For analysis of tumor angiogenesis, hematoxylin- CAM. FGF-2 induced a branching network of capillaries, which was stained tumor sections were coded, and perfused vascular structures were efficiently inhibited by coincubation with latent antithrombin at a counted at ϫ400, using an eyepiece grid of 10 ϫ 10 squares. To adjust for the 10-fold molar excess (Fig. 1) or even at an equimolar concentration 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 (Table 1). VEGF-induced angiogenesis in the CAM was similarly noted and used in the calculations of vascular parameters. Sampling of vision inhibited by latent antithrombin (data not shown). In contrast, native 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 ϫ 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 ␮g/ml), or FGF-2 (20 ng/ml) in combination with antithrombin at 1 or 10 ␮g/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 ␮g/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 ␮g/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 ␮g/ml) or native antithrombin (3 ␮g/ml). Forty h later, the cells were harvested, fixed, and prepared according to the In situ Cell Death Detection kit-Fluorescein kit Fig. 1. Effects of latent, native, and thermolysin-cleaved antithrombin on FGF-2- induced angiogenesis in the CAM. FGF-2-induced angiogenesis was efficiently inhibited with fluorescein-conjugated dUTP (Roche Diagnostics). The percentage of by coincubation with latent antithrombin but only weakly by thermolysin-cleaved or TUNEL-positive cells in 1000 counted cells was determined. native antithrombin, as quantified in Table 1. 6724

Table 1 Inhibitory effect of different forms of antithrombin (AT) on chick CAM Latent Antithrombin Induces Apoptosis. The decrease in the ␮ angiogenesis induced by FGF-2 (0.2 g/filter) number of endothelial cells in cultures treated with latent antithrombin The score, from 0 (low) to 3 (high), was based on the number of vessel branch points, according to the method of Friedlander et al. (23). Average values for five to six embryos could be attributable to induction of apoptosis. To investigate this were recorded. The variability was Ͻ15%. possibility, serum-starved PAE/FGFR-1 cultures were incubated over- Stimulator Inhibitor Angiogenesis score No. of embryos night in the presence or absence of latent antithrombin and subse- Buffer 0.3 6 quently stained for Annexin V to detect early plasma membrane FGF-2 2 6 changes indicative of the apoptotic process (30). In cultures treated FGF-2 Latent AT, 3 ␮g 0.1 5 with FGF-2, the fraction of Annexin V-positive cells decreased in FGF-2 Latent AT, 0.3 ␮g 0.5 5 FGF-2 Latent AT, 0.03 ␮g 1.9 5 comparison with that in the controls (Fig. 4), presumably as a result of FGF-2 Native AT, 3 ␮g 1.5 6 growth factor-induced survival. Treatment with latent antithrombin FGF-2 Cleaved AT, 3 ␮g 1.6 6

and thermolysin-cleaved antithrombin preparations had only a weak inhibitory effect on growth factor-induced angiogenesis (Fig. 1 and Table 1). Fibrosarcoma Growth in Mice Is Inhibited by Treatment with Antithrombin. The effects of different forms of antithrombin on the growth of syngeneic T241 fibrosarcoma tumors in C57BL6/J mice were studied. Mice carrying palpable tumors on the left flank were treated with latent, native, or thermolysin-cleaved antithrombin (1 mg/kg) daily by s.c. injections in the right flank. After 10 days of treatment, when the control animal tumor size had reached 2 cm3, the animals were sacrificed. As shown in Fig. 2A, 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). Samples of tumors from the same untreated and latent antithrombin-treated animals, as referred to in Fig. 2A, were examined with regard to proliferation, apoptosis, and angiogenesis. As shown in Fig. 2C, 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 Ͻ 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). Effect of Latent Antithrombin on Endothelial Cell Prolifera- tion. The molecular mechanisms underlying the effect of latent antithrombin on angiogenesis were further studied with use of endothelial Fig. 2. Latent antithrombin inhibits tumor growth via decreased angiogenesis and increased apoptosis. A, female C57BL6/J mice were inoculated s.c. with 0.5 ϫ 106 T241 cells in culture. PAE cells expressing FGFR-1 were used to analyze cells in PBS. When tumors were palpable (treatment day 0), animals were randomly the effect of latent antithrombin on FGF-2-induced cell growth. Fig. 3 assigned to receive 10-day treatment with 1 mg/kg/day latent antithrombin (AT; n ϭ 4), thermolysin-cleaved AT (n ϭ 6), or PBS (n ϭ 6) by s. c. injection. Thermolysin-cleaved shows that FGF-2 treatment induced an increase in the number of cells AT did not affect tumor volume at treatment day 10 (P ϭ 0.57), whereas latent AT caused to 340% of that in the controls in 5 days. In cultures treated with a significant reduction as compared with control (P Ͻ 0.05; ANOVA). Mean tumor FGF-2 together with latent antithrombin, the number increased to only volumes are shown; bars, SE. B, T241-bearing animals were treated with 1 mg/kg/day latent AT (n ϭ 7), native AT (n ϭ 6), or PBS control (n ϭ 6). At treatment day 10, 230%. In cultures treated with latent antithrombin alone, the number administration of latent AT had significantly reduced tumor volume (P Ͻ 0.05), whereas of cells was slightly decreased compared with the basal condition. The native AT had not (P ϭ 0.37; ANOVA) as compared with PBS control. Mean tumor VEGF-induced increase in the number of PAE cells expressing VEGF volumes are shown; bars, SE. C, latent AT-treated and PBS control tumors from A were sectioned and stained for proliferating cells using antibodies against PCNA and for receptor-2 was similarly diminished by latent antithrombin (data not apoptotic cells using TUNEL. Quantification was done by calculating the percentage of shown). In contrast, FGF-2-induced proliferation of COS (monkey positive nuclei in randomly chosen, nonnecrotic fields of vision. There was no difference in the proliferative index in latent AT-treated versus control tumors. The apoptotic index kidney epithelial) cells was not affected by latent antithrombin (data increased upon treatment with latent AT as compared with control (P Ͻ 0.05, two-tailed not shown). Student’s t test). 6725

Table 2 Effects of latent antithrombin (AT) on tumor angiogenesis number of cells migrating through the filter, toward the potential Latent antithrombin decreased the length and volumetric and surface densities of tumor stimulator or inhibitor on the opposite side of the filter, during 4 h blood vessels. Perfused blood vessel profiles in sections from tumors from animals treated with PBS or latent antithrombin (Fig. 2A) were scored using an unbiased counting frame of incubation at 37°C was counted. As shown in Fig. 5, the relative and analyzed as described by Wassberg et al. (26). number of migrating cells increased from 100% (control, BSA- Control Latent AT treated) to 150% when stimulated with FGF-2. When latent anti- Parametera (n ϭ 5) (n ϭ 4) Change P thrombin was added together with FGF-2 during the Boyden cham- Ϫ2 Ϯ Ϯ Ϫ Ͻ Lv (mm ) 82.3 9.3 52.5 4.9 37% 0.05 ber assay, the number of migrating cells decreased to 105%. Latent Ϫ3 Ϯ Ϯ Ϫ Vv (10 ) 30.1 5.8 18.5 1.7 39% 0.159 Ϫ1 Ϯ Ϯ Ϫ antithrombin in itself did not affect migration under these condi- Sv (mm ) 5.4 0.7 3.4 0.3 37% 0.068 a tions. Lv, length of vessels/tumor volume (length density); Vv, volume of vessels/tumor volume (volumetric density); Sv, surface area of vessels/tumor volume (surface density). Parameters were determined as described (26).

Fig. 4. Effects of latent antithrombin on apoptosis. PAE cells overexpressing FGFR-1 were incubated with latent antithrombin (3 ␮g/ml). After 8 h, the medium was changed to Ham’s F-12 supplemented with 0.1% FCS, together with fresh latent antithrombin (3 ␮g/ml) with or without FGF-2 (10 ng/ml). After 18 h of incubation, apoptotic cells were Fig. 3. Effects of latent antithrombin on FGF-2 induced proliferation. Equal numbers detected by Annexin V staining and quantified by fluorescence activated cell sorter of PAE cells overexpressing FGFR-1 were cultured for 5 days with and without FGF-2 analysis. The fraction of Annexin V-positive cells in the control, serum-starved cultures (20 ng/ml) and latent antithrombin (10 ␮g/ml). The number of cells in triplicate wells was (on average, 10.5%) was set to 1. Bars, SE. determined with a Coulter counter. Mean values of three different experiments are shown; bars, SE. led to a nearly 2-fold increase in Annexin V-positive cells. Addition of FGF-2 to cells treated with latent antithrombin gave slight protec- tion 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). Latent Antithrombin Inhibits Endothelial Cell Migration and Focal Adhesion Formation. The ability of PAE/FGFR-1 cells to migrate in a mini-Boyden chamber in the presence or absence of latent antithrombin was examined (27). Cells preincubated with latent antithrombin for 30 min were seeded on one side Fig. 5. Effects of latent antithrombin on FGF-2 induced migration. PAE cells overexpressing FGFR-1 were analyzed for their ability to migrate in a mini-Boyden chamber of a collagen-coated nitrocellulose filter, and FGF-2 with or with- toward FGF-2, in the presence or absence of latent antithrombin (3 ␮g/ml). Mean values out latent antithrombin was added to the wells under the filter. The of three different experiments are shown; bars, SE. 6726

Fig. 6. Effects of latent antithrombin on actin ruffles, FAK, and focal adhesion formation by latent antithrombin. A, PAE cells overexpressing FGFR-1 cultured on coverslips were incubated for 20 min with and without FGF-2 (100 ng/ml) and latent antithrombin (3 ␮g/ml). Cells were fixed and stained with rhodamine-coupled phalloidin. B, cells in 5-cm dishes were treated with FGF-2 (100 ng/ml), native or latent antithrombin (3 ␮g/ml) as indicated for 10 min at 37°C. Aliquots of total cell lysates were separated by SDS-PAGE and transferred to membrane that was immunoblotted with phospho-specific FAK antibodies (upper panel) and FAK antibodies (lower panel). C, cells were treated with FGF-2 and latent antithrombin as in B, lysed, and immunoprecipitated with antiserum against FGFR-1. Samples were pro- cessed for in vitro kinase assay in the presence of ␥-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.

With the aim of identifying the molecular mechanisms underlying PAE/FGFR-1 cells were further stained with an antiserum recog- the effect of latent antithrombin on endothelial cell migration, actin nizing paxillin, which is localized in focal adhesion contacts (Fig. reorganization was studied. Staining with rhodamine-coupled phalloi- 6D). FGF-2 stimulation of the cells for 20 min led to the appearance din (Fig. 6A) showed that treatment with FGF-2 for 20 min led to of a punctate radial pattern, typical for focal adhesion contacts. formation of dense, actin-containing membrane structures, denoted as Coincubation of the cells with FGF-2 and latent antithrombin pre- edge ruffles. In cells treated with latent antithrombin together with vented formation of focal adhesion contacts. There was no effect on FGF-2 for 20 min, actin stress fibers were present, but the ruffle formation of focal adhesion contacts in cells treated with latent formation was almost attenuated. antithrombin alone. Together, these results indicate that latent anti- The cytoplasmic tyrosine kinase FAK is localized in focal adhe- thrombin exerts its antiangiogenic effects by negative regulation of sions and has a critical role in the regulation of cell migration (31). We the actin cytoskeleton, FAK, and focal adhesion formation. examined the effect of latent antithrombin on FAK tyrosine phosphorylation by immunoblotting with an anti-phosphoFAK antibody. Fig. DISCUSSION 6B shows that latent antithrombin treatment abolished FGF-2-induced FAK tyrosine phosphorylation, without affecting the FAK protein Latent antithrombin, which is inactive as a protease inhibitor, is levels. Treatment with latent antithrombin alone, but not with native created by heat denaturation, but a slow conversion of native anti- antithrombin, led to increased levels of tyrosine phosphorylated FAK. thrombin to the latent form may also take place under in vivo condi- Induction of FGFR-1 tyrosine kinase activity by FGF-2 in PAE/ tions (32). We have shown in the present study that latent antithrom- FGFR-1 cells was unaffected by treatment with latent antithrombin bin is effective in halting tumor growth at the low dose of 1 mg/kg/ (Fig. 6C). The basal level of FGFR-1 activation in endothelial cells day. The observed effects on blood vessel density (Table 2), the treated with latent antithrombin alone was slightly increased. unchanged proliferation index, and the increased apoptosis of tumor 6727

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2000 American Association for Cancer Research. ANTIANGIOGENIC EFFECT OF ANTITHROMBIN cells (Fig. 2C), together with the results from the CAM assay, imply in the antithrombin sequence (38). Cells from mice lacking FAK that latent antithrombin exerts its antitumoral effects through inhibi- expression as a result of gene inactivation fail to migrate, possibly on tion of angiogenesis. Native antithrombin also decreased tumor account of a defect in focal adhesion turnover (31). We observed a growth, although less efficiently, whereas tumors in animals treated decrease in migration and decreased formation of focal adhesions in with thermolysin-cleaved antithrombin were similar in size to those in response to FGF-2 when cells were treated with latent antithrombin. the control animals. The latent but not the cleaved form of antithrom- The regulation of actin filament organization was also disturbed in the bin was also found to be a potent angiogenesis inhibitors in the CAM. latent antithrombin-treated cells. Regulation of the actin cytoskeleton However, native antithrombin did not effectively inhibit neoangio- involves Rho family GTPases, which have also been implicated in the genesis in the CAM (Fig. 1). regulation of FAK and paxillin phosphorylation during cell adhesion In a recent publication by O’Reilly et al. (13), pancreatic elastase- (39). Thus, it is possible that Rho GTPases are primary targets of cleaved and latent antithrombin were shown to be equally potent in latent antithrombin and that FAK activation is subsequently perturbed. arresting expansion of SK-NAS neuroblastoma and Lewis lung carcinoma when administered at 25 mg/kg/day, whereas native anti- ACKNOWLEDGMENTS thrombin had no effect in the tumor models investigated. Our results indicate that cleaved antithrombin has a lower antiangiogenic and We thank Charlotte Wikner, Mari-Anne Carlsson, and Helena Hermelin for excellent technical assistance. 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 REFERENCES higher dose and the longer duration of the treatment. The different effects of cleaved and latent antithrombin are compatible with the 1. Risau, W. Mechanisms of angiogenesis. Nature (Lond.), 386: 671–674, 1997. 2. Folkman, J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat. marked difference in their three-dimensional structures. Most strik- Med., 1: 27–31, 1995. ingly, in cleaved antithrombin, residues 402–407 form strand 1 of 3. Veikkola, T., Karkkainen, M., Claesson-Welsh, L., and Alitalo, K. Regulation of ␤ angiogenesis via vascular endothelial growth factor receptors. 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Helena Larsson, Tobias Sjöblom, Johan Dixelius, et al.

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