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 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2000 American Association for Cancer Research. ANTIANGIOGENIC EFFECT OF ANTITHROMBIN (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

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