Amyloid Endostatin Induces Endothelial Cell Detachment by Stimulation of the Plasminogen Activation System
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Vol. 1, 561–568, June 2003 Molecular Cancer Research 561 Amyloid Endostatin Induces Endothelial Cell Detachment by Stimulation of the Plasminogen Activation System Arie Reijerkerk,1 Laurent O. Mosnier,2 Onno Kranenburg,1 Bonno N. Bouma,2 Peter Carmeliet,4 Tom Drixler,1,3 Joost C.M. Meijers,2,5 Emile E. Voest,1 and Martijn F.B.G. Gebbink1 1Department of Medical Oncology, 2Thrombosis and Haemostasis Laboratory, Department of Haematology, and 3Department of Surgery, University Medical Center Utrecht, Utrecht, The Netherlands; 4Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium; and 5Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Abstract endostatin was purified from conditioned medium of murine Endostatin is a fragment of collagen XVIII that acts as an hemangioendothelioma (EOMA) cells as a proteolytically inhibitor of tumor angiogenesis and tumor growth. Anti- cleaved fragment of collagen XVIII. Generation of endostatin tumor effects have been described using both soluble can be achieved by cleavage of collagen by cathepsin L (4), and insoluble recombinant endostatin. However, differ- matrilysin, (5) or elastase (6). ences in endostatin structure are likely to cause differ- Endostatin has distinct antiangiogenic and anti-tumoral ences in bioactivity. In the present study, we have activities in several animal models (7). Tumor growth was investigated the cellular effects of insoluble endostatin. significantly inhibited by i.p. or s.c. injection of endostatin We previously found that insoluble endostatin shows all without induction of acquired drug resistance (1, 8). In addition, the hallmarks of amyloid aggregates and potently rat endostatin induced significant inhibition of carcinogen- stimulates tissue plasminogen activator-mediated induced mammary tumor growth (9). Endostatin is also formation of the serine protease plasmin. We here show bioactive using delivery approaches such as DNA vaccination that amyloid endostatin induces plasminogen activation and viral expression (10, 11). However, contrary to this, no by endothelial cells, resulting in vitronectin degradation antiangiogenic effect of endostatin was seen in other studies and plasmin-dependent endothelial cell detachment. despite high serum levels (12–15). This paradox has led to Endostatin-mediated stimulation of plasminogen activa- discussions about the efficacy of endostatin (16). Until now, this tion, vitronectin degradation, and endothelial cell paradox remains unsolved. Possibly, the outcome of endostatin detachment is inhibited by carboxypeptidase B, indicat- treatment will depend on its structure (see below) and/or the ing an essential role for carboxyl-terminal lysines. Our presence of factors provided at the site of action. results suggest that amyloid endostatin may inhibit A variety of molecular mechanisms have been proposed to angiogenesis and tumor growth by stimulating the underlie endostatin activity. Endostatin blocks vascular endo- fibrinolytic system. thelial growth factor (VEGF)-mediated signaling through KDR/ Flk-1 (17) and induces endothelial cell apoptosis, associated Introduction with decreased levels of anti-apoptotic proteins Bcl-2 and Angiogenesis, the formation of new capillaries from pre- BclXL (18). Endostatin can bind tropomyosin, an actin existing blood vessels, is important during various pathological stabilizing protein, and has been suggested to disrupt micro- processes, including inflammation and tumor growth. Anti- filament integrity ultimately causing endothelial cell apoptosis angiogenic therapy is being considered as a potentially (19). Direct effects of endostatin on cell adhesion may be powerful new therapy for cancer and other angiogenesis- caused by suppression of integrin function (20, 21). Other dependent diseases. Endostatin is one of the most potent studies have implicated that endostatin acts on the proteolytic inhibitors of angiogenesis and can induce tumor regression in system by binding and inhibiting active metalloproteinase mice (1). Clinical trials are currently ongoing (2, 3). Originally, (MMP)-2 (22, 23) or down-regulating the urokinase plasmi- nogen activator system (24). However, it is not clear to what extent these activities contribute to the antiangiogenic and anti- tumor effects of endostatin in vivo. Received 2/14/03; revised 4/16/03; accepted 4/22/03. A complicating factor is that endostatin is used in a soluble, The costs of publication of this article were defrayed in part by the payment of as well as in an insoluble form. Whereas both forms have page charges. This article must therefore be hereby marked advertisement in been reported to inhibit tumor growth, the regression of accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Grant support: Dutch Cancer Society (M.F.B.G.G.); the Fischer Stichting tumors has only been reported with insoluble endostatin (1). (A.R.); The Netherlands Heart Foundation; and Crucell N.V., Leiden, The Different structural forms of endostatin have distinct bioactiv- Netherlands. Note: J.C.M.M. is an established investigator of The Netherlands ities and may therefore produce their antiangiogenic effects Heart Foundation. A.R. and L.O.M. contributed equally to this work. Requests for reprints: M.F.B.G. Gebbink, Department of Medical Oncology— through distinct mechanisms. The structure of soluble, F02.126, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX globular endostatin has been elucidated (25, 26). Recently, Utrecht, The Netherlands. Phone: 31-30-250-6265; Fax: 31-30-252-3741. E-mail: [email protected] we found that endostatin is a protein with high propensity to Copyright D 2003 American Association for Cancer Research. form amyloid fibers through extensive cross-h sheet formation Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2003 American Association for Cancer Research. 562 Amyloid Endostatin Induces Endothelial Cell Detachment (27, 28). Moreover, we established that only insoluble endostatin but not soluble endostatin stimulates tissue plasminogen activator (t-PA)-mediated plasminogen activation and induces cell toxicity (27, 28). Here we have studied the effect of insoluble endostatin, to which we refer as amyloid endostatin, on endothelial cell-mediated plasmin formation and cell adhesion. Results Amyloid Endostatin Stimulates Plasmin Formation We previously showed that denatured endostatin forms amyloid fibrils and stimulates t-PA-mediated plasminogen activation in vitro (27). We show here that amyloid endostatin stimulates t-PA-mediated plasminogen activation in a dose- dependent manner (Fig. 1A). Stimulation of t-PA-mediated activation of plasminogen by amyloid endostatin was as potent as stimulation by fibrin fragments, the classical stimulator of t-PA-mediated plasminogen activation (Fig. 1B). No activation of plasminogen was observed in the absence of t-PA and amyloid endostatin alone did not convert the chromogenic substrate (not shown). Amyloid Endostatin Binds Plasminogen and t-PA Because endostatin is a stimulator of plasminogen activation by t-PA, we tested whether amyloid endostatin was able to bind plasminogen and t-PA. Both plasminogen and t-PA bound to immobilized amyloid endostatin in a dose-dependent and saturable manner (Fig. 2A). Binding of t-PA to amyloid endostatin [apparent dissociation constant (K )=1.6F 0.3 nM] FIGURE 1. Amyloid endostatin-stimulated t-PA-mediated plasmino- d gen activation is concentration dependent. Different concentrations of was similar to the binding of t-PA to fibrin degradation products amyloid endostatin (A) and fibrin fragments (B) were added to (Kd =2.3F 0.4 nM) (Fig. 2B). The affinity of plasminogen for plasminogen. Plasminogen activation was started by addition of t-PA. At the indicated time points, samples were taken and plasmin activity was amyloid endostatin (K =2.7F 0.6 nM) was about 10 times d analyzed using the chromogenic substrate S-2251. These figures show a higher than the affinity of plasminogen for fibrin fragments representative experiment out of five independent experiments. , 4 AM;,2AM;,1AM;,0.5AM;,0AM. (Kd = 28.4 F 5.8 nM). Carboxypeptidase Inhibits the t-PA-Stimulatory Activity of Amyloid Endostatin Amyloid Endostatin Stimulates Plasminogen Activation The binding of plasminogen and t-PA to fibrin is mediated by Endothelial Cells by the lysine-binding sites in the kringle domains of We next determined whether amyloid endostatin can plasminogen and t-PA and by carboxyl-terminal lysine residues stimulate plasminogen activation mediated by endothelial cells, that are generated in fibrin during plasmin digestion (29). a biological source of tissue-type plasminogen activator. Therefore, we investigated the importance of carboxyl-terminal Different concentrations of amyloid endostatin were added to lysines in amyloid endostatin-mediated plasmin formation. confluent monolayers of endothelial cells in the presence of To this end, we used the porcine pancreas carboxypeptidase 0.4 AM plasminogen. After 24 h, the medium was analyzed by B (CpB) that removes basic (arginine and lysine) amino acids Western blotting. Plasminogen cleavage by plasminogen from proteins. Treatment with CpB reduced amyloid endo- activator results in the formation of plasmin, which migrates statin-stimulated plasminogen activation (Fig. 3). The inhib- at approximately Mr 50,000 in gel electrophoresis. In contrast itory effect of CpB on amyloid endostatin was