Tropomyosin in the antiangiogenic activity of HKa

描述（由申请人提供）：高分子量激肽原（HK）是一种丰富的血浆糖蛋白，在钾likrein-激肽系统中起核心作用。血浆激肽激酶（klikrein）对HK的切割导致缓激肽的释放和双链高分子量激肽原（HKa）的产生。我们已经报道了HKa和重组HKa结构域5（在HK切割后暴露）以Zn2+依赖的方式诱导增殖的内皮细胞选择性凋亡，并抑制血管生成。基于分子模拟研究表明HKa结构域5与内皮抑素具有结构同源性，并有报道表明内皮抑素通过与原肌球蛋白的相互作用与内皮细胞结合，我们确定了原肌球蛋白是否参与了HKa的抗血管生成活性。我们观察到抗原肌球蛋白抗体阻断了HKa诱导的内皮细胞凋亡，以及HKa与增殖内皮细胞的结合。该抗体在体内也阻断了HKa的抗血管生成作用，其他抗原肌球蛋白抗体也具有这种作用。内皮细胞表达至少5种原肌球蛋白的异构体，使用共聚焦显微镜、生物素化内皮细胞表面蛋白的免疫沉淀和酸洗脱方法的研究表明，原肌球蛋白暴露在增殖的内皮细胞表面。对HKa与原肌球蛋白结合的直接测量表明，HKa与所研究的所有原肌球蛋白亚型具有高亲和力结合，这表明HKa与这些蛋白中的同源区域结合。最后，对凝乳胰蛋白酶消化的原肌球蛋白在HKa-sepharose上进行亲和纯化，并在HKa上筛选环状随机肽库，初步确定了原肌球蛋白内的HKa结合区。在本应用中，我们拟1)比较亚融合、增殖和融合内皮细胞中不同原肌球蛋白异构体的表达和亚细胞分布，2)确定体内原肌球蛋白是否暴露在血管生成内皮细胞表面，以及在这种情况下是否作为HKa的结合位点，3)确定HKa在原肌球蛋白中的结合位点，并评估其功能重要性。这些研究挑战了细胞骨架成分被认为无法进入细胞外环境的范式，并应提供有关内皮原肌球蛋白生物学及其在血管生成和HKa抗血管生成活性中的作用的新信息。

DESCRIPTION (provided by applicant): 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.