No abstract

Conformationally altered proteins and protein fragments derived from the extracellular matrix and hemostatic system may function as naturally occurring angiogenesis inhibitors. One example of such a protein is cleaved high molecular weight kininogen (HKa). HKa inhibits angiogenesis by inducing apoptosis of proliferating endothelial cells, effects mediated largely by HKa domain 5. However, the mechanisms underlying the antiangiogenic activity of HKa have not been characterized, and its binding site on proliferating endothelial cells has not been defined. Here, we report that the induction of endothelial cell apoptosis by HKa, as well as the antiangiogenic activity of HKa in the chick chorioallantoic membrane, was inhibited completely by antitropomyosin monoclonal antibody TM-311. TM-311 also blocked the high-affinity Zn 2؉ -dependent binding of HKa to both purified tropomyosin and proliferating endothelial cells. Confocal microscopic analysis of endothelial cells stained with monoclonal antibody TM-311, as well as biotin labeling of cell surface proteins on intact endothelial cells, revealed that tropomyosin exposure was enhanced on the surface of proliferating cells. These studies demonstrate that the antiangiogenic effects of HKa depend on high-affinity binding to endothelial cell tropomyosin.A ngiogenesis plays a central role in tumor progression (1, 2). This process is stimulated by growth factors such as vascular endothelial growth factor (3, 4) and basic fibroblast growth factor (bFGF; ref. 5) and inhibited by conformationally altered proteins or protein fragments often derived from the extracellular matrix (6) or hemostatic system (7). Although tumors remain dormant when the influences of pro-and antiangiogenic factors are balanced (8), triggering of an ''angiogenic switch'' may lead to a net increase in angiogenesis and tumor progression (9, 10).We recently reported that the two-chain form of human high molecular weight kininogen (HKa) inhibits angiogenesis by selectively inducing apoptosis of proliferating endothelial cells (11). These effects are mediated largely by kininogen domain 5 (11), particularly regions within the C terminus of this domain that mediate the binding of HKa to endothelial cells (12). However, the endothelial-binding site for HKa through which these effects are mediated has not been defined, and our previous studies failed to demonstrate an essential role for any of the known endothelial cell receptors for single-chain kininogen (HK) or HKa including the urokinase receptor (13), the receptor for the globular heads of C1q (14, 15), and cytokeratin 1 (16). Here, we report that the antiangiogenic activity of HKa depends on a high-affinity binding interaction with tropomyosin exposed on the surface of proliferating endothelial cells. Materials and MethodsMaterials. Two-chain HKa was purchased from Enzyme Research Laboratories (Bloomington, IN). Recombinant bFGF and vascular endothelial growth factor were from Becton-Dickinson Biosciences (Franklin Lakes, NJ). The antitropomyosin monoclo...

The low density lipoprotein receptor-related protein/ ␣ 2 -macroglobulin receptor (LRP/␣ 2 MR) mediates the internalization of numerous ligands, including prourokinase (pro-UK) and complexes between two-chain urokinase (tc-u-PA) and plasminogen activator inhibitor type-1 (PAI-1). It has been suggested that through its ability to internalize these ligands, LRP/␣ 2 MR may regulate the expression of plasminogen activator activity on cell surfaces; this hypothesis, however, has not been experimentally confirmed. To address this issue, we assessed the ability of LRP/␣ 2 MR to regulate plasminogen activator activity on human trophoblast cells, which express both LRP/␣ 2 MR and the urokinase receptor (uPAR). Trophoblasts internalized and degraded exogenous 125 I-pro-UK (primarily following its conversion to tc-u-PA and incorporation into tc-u-PA⅐PAI complexes) in an LRP/␣ 2 MR-dependent manner, which was inhibited by the LRP/␣ 2 MR receptor-associated protein. Receptor-associated protein also caused a ϳ50% reduction in cell surface plasminogen activator activity and delayed the regeneration of unoccupied uPAR by cells on which uPAR were initially saturated with pro-UK. Identical effects were caused by anti-LRP/␣ 2 MR antibodies. These results demonstrate that LRP/␣ 2 MR promotes the expression of cell surface plasminogen activator activity on trophoblasts by facilitating the clearance of tc-u-PA⅐PAI complexes and regeneration of unoccupied cell surface uPAR.