High molecular weight kininogen (HK) is an abundant plasma glycoprotein that plays a central role in the kallikrein-kinin system. Cleavage of HK by plasma kallikrein results in release of bradykinin and generation of two-chain high molecular weight kininogen (HKa). We have reported that HKa and recombinant HKa domain 5 (which is exposed following HK cleavage) induce selective apoptosis of proliferating endothelial cells in a Zn2+-dependent manner, and inhibit angiogenesis. Based on molecular modeling studies suggesting that HKa domain 5 has structural homology to endostatin, and a report suggesting that endostatin bound to endothelial cells through interactions with tropomyosin, we determined whether tropomyosin was involved in the antiangiogenic activity of HKa. We observed that an anti-tropomyosin antibody blocked HKa-induced endothelial cell apoptosis, as well as the binding of HKa to proliferating endothelial cells. This antibody also blocked the antiangiogenic effects of HKa in vivo, and additional antitropomyosin antibodies shared these effects. Endothelial cells express at least 5 isoforms of tropomyosin, and studies employing confocal microscopy, immunoprecipitation of biotinylated endothelial cell surface proteins and acid elution approaches suggest that tropomyosin is exposed on the surface of proliferating endothelial cells. Direct measurement of the binding of HKa to tropomyosin demonstrated high affinity binding to all tropomyosin isoforms studied, suggesting that HKa bound to a homologous region within these proteins. Finally, affinity purification of chymotrypsin-digested tropomyosin on HKa-sepharose, as w ell as panning of a cyclic random peptide Library on HKa, led to tentative identification of HKa binding regions within tropomyosin. In this application, we propose to 1) compare the expression and subcellular distribution of different tropomyosin isoforms by subconfluent, proliferating and confluent endothelial cells, 2) determine whether tropomyosin is exposed on the surface of angiogenic endothelial cells in vivo, and whether it serves as a binding site for HKa in this setting, and 3) define the HKa binding site in tropomyosin, and assess its functional importance. These studies challenge the paradigm in which cytoskeletal components are considered inaccessible to the extracellular milieu, and should provide new information concerning the biology of endothelial tropomyosin, and its roles in angiogenesis and the antiangiogenic activity of HKa.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL076810-01
Application #
6774364
Study Section
Special Emphasis Panel (ZRG1-HP (02))
Program Officer
Hasan, Ahmed AK
Project Start
2004-04-01
Project End
2008-03-31
Budget Start
2004-04-01
Budget End
2005-03-31
Support Year
1
Fiscal Year
2004
Total Cost
$358,353
Indirect Cost
Name
Case Western Reserve University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Bu, Chunya; Gao, Lei; Xie, Weidong et al. (2009) beta2-glycoprotein i is a cofactor for tissue plasminogen activator-mediated plasminogen activation. Arthritis Rheum 60:559-68
Lin, Jim Jung-Ching; Li, Yan; Eppinga, Robbin D et al. (2009) Chapter 1: roles of caldesmon in cell motility and actin cytoskeleton remodeling. Int Rev Cell Mol Biol 274:1-68
Wang, Siying; Yu, Wen-Mei; Zhang, Wanming et al. (2009) Noonan syndrome/leukemia-associated gain-of-function mutations in SHP-2 phosphatase (PTPN11) enhance cell migration and angiogenesis. J Biol Chem 284:913-20
Lin, Jim Jung-Ching; Eppinga, Robbin D; Warren, Kerri S et al. (2008) Human tropomyosin isoforms in the regulation of cytoskeleton functions. Adv Exp Med Biol 644:201-22
Merkulov, Sergei; Zhang, Wan-Ming; Komar, Anton A et al. (2008) Deletion of murine kininogen gene 1 (mKng1) causes loss of plasma kininogen and delays thrombosis. Blood 111:1274-81
Schmaier, A H; McCrae, K R (2007) The plasma kallikrein-kinin system: its evolution from contact activation. J Thromb Haemost 5:2323-9
Eppinga, Robbin D; Li, Yan; Lin, Jenny L-C et al. (2006) Tropomyosin and caldesmon regulate cytokinesis speed and membrane stability during cell division. Arch Biochem Biophys 456:161-74
Sun, Danyu; McCrae, Keith R (2006) Endothelial-cell apoptosis induced by cleaved high-molecular-weight kininogen (HKa) is matrix dependent and requires the generation of reactive oxygen species. Blood 107:4714-20
An, Feng-Qi; Folarin, Hope Merlene; Compitello, Nicole et al. (2006) Long-term-infected telomerase-immortalized endothelial cells: a model for Kaposi's sarcoma-associated herpesvirus latency in vitro and in vivo. J Virol 80:4833-46
McCrae, Keith R; Donate, Fernando; Merkulov, Sergei et al. (2005) Inhibition of angiogenesis by cleaved high molecular weight kininogen (HKa) and HKa domain 5. Curr Cancer Drug Targets 5:519-28