The antiangiogenic activity of cleaved high molecular weight kininogen is mediated through binding to endothelial cell tropomyosin

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...

Chimeric Glutathione S-Transferases Containing Inserts of Kininogen Peptides

“…In this study, we presented the design of engineered GST proteins carrying inserts of the HGK-rich motif derived from a sequence within D5 of HK. HK D5 contains endothelial cell-binding sites and inhibits angiogenesis through its ability to cause apoptosis of proliferating endothelial cells and to inhibit endothelial cell proliferation and migration (6,10,(12)(13)(14)(15)(16). Moreover, HGK peptides were found to block tumor metastasis (6,10,(12)(13)(14)(15)(16).…”

“…HK D5 contains endothelial cell-binding sites and inhibits angiogenesis through its ability to cause apoptosis of proliferating endothelial cells and to inhibit endothelial cell proliferation and migration (6,10,(12)(13)(14)(15)(16). Moreover, HGK peptides were found to block tumor metastasis (6,10,(12)(13)(14)(15)(16). Although the D5 peptide region within the context of intact high molecular weight kininogen or recombinant D5 induces endothelial cell apoptosis with an IC 50 in the low nanomolar range, the isolated D5 peptides express at least 2 logs lower activity, presumably due to enhanced flexibility and a failure to maintain the appropriate conformation for meaningful interactions with endothelial cells (6,10,(12)(13)(14)(15)(16).…”

“…Moreover, HGK peptides were found to block tumor metastasis (6,10,(12)(13)(14)(15)(16). Although the D5 peptide region within the context of intact high molecular weight kininogen or recombinant D5 induces endothelial cell apoptosis with an IC 50 in the low nanomolar range, the isolated D5 peptides express at least 2 logs lower activity, presumably due to enhanced flexibility and a failure to maintain the appropriate conformation for meaningful interactions with endothelial cells (6,10,(12)(13)(14)(15)(16). In this study, we demonstrated that constraining flexibility of these peptides through insertion into the GST backbone enhances their activity.…”

“…HK D5 contains endothelial cell-binding sites and inhibits angiogenesis through its ability to cause apoptosis of proliferating endothelial cells (6,12,13) and to inhibit endothelial cell proliferation and migration (14). Moreover, a histidine-glycine-lysine (HGK) motif derived from this domain blocks tumor metastasis (6,10,(12)(13)(14)(15)(16).…”

“…HK D5 contains endothelial cell-binding sites and inhibits angiogenesis through its ability to cause apoptosis of proliferating endothelial cells (6,12,13) and to inhibit endothelial cell proliferation and migration (14). Moreover, a histidine-glycine-lysine (HGK) motif derived from this domain blocks tumor metastasis (6,10,(12)(13)(14)(15)(16). Although the exact mechanism of HGK peptide action has not been delineated (6,10,(12)(13)(14)(15)(16), this motif nevertheless represents an attractive target for the design of antitumor peptide therapeutics/drugs.…”