Abstract. Inhibitors of angiogenesis are potential anti-cancer agents in that they deprive tumors of the blood necessary for growth and metastasis. The anti-angiogenic efficacy of tinzaparin, a known anticoagulant low molecular weight heparin (LMWH), was examined in vitro in endothelial cell tube formation assay and in vivo in the chick chorioallantoic membrane model. The observed anti-angiogenic effects of tinzaparin were shown to be dose-related and dependent on the relatively higher molecular weight tinzaparin fragments. These experiments demonstrated that tinzaparin is a potent inhibitor of angiogenesis (ED 90-100 range, 0.05-0.1 mg) regardless of the angiogenic factor and suggest that its effect is mediated via cellular release of tissue factor pathway inhibitor (TFPI). This was evident by the reversal of either tinzaparin or r-TFPI anti-angiogenesis efficacy by a specific monoclonal TFPI antibody. The ED 90-100 for the inhibition of angiogenesis for r-TFPI ranged from 0.01 to 0.03 mg in the chorioallantoic membrane model regardless of the pro- angiogenic factor. In addition, either tinzaparin or r-TFPI inhibited the growth of colon carcinoma tumors, human fibro- sarcoma tumors, and human lung carcinoma in the chorio- allantoic membrane tumor implant model. Thus, the LMWH tinzaparin, in addition to its anticoagulant effects, may offer important benefits in treatment of cancer and other disorders supported by pathologic angiogenesis. Introduction Angiogenesis is an essential feature of normal biologic processes, such as growth, development, reproduction, and repair of damaged tissue (1-3). Endogenous promoters and inhibitors regulate the complex process of angiogenesis (2). The later stages involve proliferation and organization of endothelial cells (ECs) into tube-like structures. Vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) are important promoters of this process; these promoters exert their effects by binding to cell surface receptors. Human ECs in culture can form tube-like structures with lumens, and they represent an in vitro model system for the study of the angiogenesis process. Pathologic angiogenesis, which may occur when a normal control mechanism is defective, contributes to the growth and metastasis of tumors (3), as well as to inflammatory and certain ocular diseases. Drugs that inhibit angiogenesis may be effective in the treatment of these human disorders (3). The mechanisms by which anti-angiogenic drugs exert their effects can vary widely, acting on different points in the complex process of tumor angiogenesis. Potential points of control include blocking the action of endogenous stimulators; inhibiting the growth, migration, and tube formation of ECs; and inhibiting the turnover of the capillary basement membrane (1). The role of heparin in angiogenesis modulation and its potential anti- cancer effect has been previously described, but without clear delineation of its pro- versus anti-angiogenic effect, as well as its mechanism of action (4,5). Tinzaparin, a low molecular weight heparin (LMWH), has an average molecular weight of 6.5 kDa; it is produced by enzymatic degradation of heparin and has proven efficacy in the treatment of deep vein thrombosis and pulmonary embolism (6,7). Tinzaparin's antithrombotic activity is mediated by binding to microvascular tissues and activation of antithrombin III, a potent anticoagulant (8). Tinzaparin also causes release of tissue factor pathway inhibitor (TFPI), an important endo- genous inhibitor of TF/VIIa (9). Several clinical trials have shown improved survival of cancer patients following heparin therapy (10-12). In one double-blind, multicenter clinical trial, tinzaparin was shown to be effective in the treatment of proximal deep vein thrombosis in a patient population that included a large percentage of cancer patients (6). These clinical data suggest that tinzaparin may have some benefit in the treatment of cancer patients. The present study was under- taken to elucidate the mechanisms through which tinzaparin may affect tumor angiogenesis and to assess its efficacy in inhibiting the angiogenesis processes. Materials and methods Endothelial tube formation assay. Human umbilical vein ECs (HUVECs) were obtained from Clonetics (Walkersville, MD, USA). Cells were grown to 80-90% confluence in endothelial growth medium (EGM) containing recombinant human endo- ONCOLOGY REPORTS 12: 683-688, 2004 683 Anti-angiogenic mechanisms and efficacy of the low molecular weight heparin, tinzaparin: Anti-cancer efficacy SHAKER A. MOUSA and SEEMA MOHAMED Pharmaceutical Research Institute, Albany College of Pharmacy, Albany, NY 12208, USA Received April 16, 2004; Accepted June 9, 2004 _________________________________________ Correspondence to: Dr Shaker A. Mousa, Pharmaceutical Research Institute, Albany College of Pharmacy, 106 New Scotland Avenue, Albany, NY 12208, USA E-mail: mousas@acp.edu Key words: angiogenesis, heparin, growth factors, cancer, low molecular weight heparin, tissue factor pathway inhibitor, factor Xa