KININOGEN/UROKINASE RECEPTORS IN TUMOR ANGIOGENESIS

许多恶性实体瘤的生长严重依赖于新血管的增殖，为快速分裂的细胞提供氧气和营养。认识到抑制肿瘤血管生成导致这些恶性肿瘤的消退和控制动物转移性扩散，导致了通过靶向其血管系统来控制癌症的主要努力。尿激酶受体（uPAR）不仅在恶性细胞上高表达，而且在调节细胞迁移和细胞相关蛋白水解活性的粘附中起关键作用，这些都是血管生成所必需的。最近，我们发现了一种新的抗血管生成分子，cleaved high molecular weight kininogen （HKa）。我们已经证明，HKa与内皮细胞上uPAR的2/3结构域结合，并与一种主要的粘附蛋白（玻璃体连接蛋白）竞争占用。当HKa结合时，也促进PK与钾激肽激酶的结合和激活，钾激肽激酶通过血浆钾激肽激酶将前尿激酶裂解为尿激酶，并随后增强纤溶酶原与细胞相关的纤溶酶的形成。我们现在假设HKa-uPAR的相互作用可能是控制血管生成的关键，而特定的肽序列可能是肿瘤血管生成的抑制剂。因此，我们将使用转染细胞和表面等离子体共振来研究HKa与uPAR的结合，重点是确定HKa的哪些结构域和亚结构域是结合位点。我们将利用单克隆抗体、重组片段（缺失突变体）和合成肽，根据HKa的晶体和核磁共振结构域3和5的分子同源性模型来确定HKa与尿激酶结合的确切序列。然后，我们将测试这些多肽对血管生成的四种体外成分的影响，即内皮粘附、迁移、增殖和细胞表面介导的蛋白质水解。初步研究表明，在纳摩尔浓度下，由HKa衍生的缺失突变体或合成肽抑制内皮细胞向玻璃体连接蛋白的迁移和内皮细胞的增殖。这些研究将扩展使用定点诱变和优化的环状肽来确定最小序列。我们将选择最有效的肽，并在碱性成纤维细胞生长因子刺激的鸡蛋绒毛膜尿囊膜（CAM）上进行体内测试，其中结构域5在30 nM处抑制血管生成，以及CAM上生长的肿瘤细胞。然后，我们将在免疫缺陷小鼠体内生长的人类肿瘤细胞上测试多肽。最后，我们将在Lewis肺肿瘤模型中测试这些新的抗血管生成肽阻止肿瘤生长转移的能力。我们期望这些研究将鉴定出具有生物活性的肽，这些肽可以作为设计拟肽血管生成抑制剂的先导化合物。

The growth of many malignant solid tumors is critically dependent on the proliferation of new vessels to supply the rapidly dividing cells with oxygen and nutrients. Recognition that inhibition of tumor angiogenesis leads to regression of these malignancies and controls metastatic spread in animals has led to a major effort to control cancer by targeting its vasculature. The urokinase receptor (uPAR) is not only highly expressed on malignant cells, but plays a critical role in regulating cell migration and adhesion of cell-associated proteolytic activity, which are all essential for angiogenesis. Recently, we have identified a new anti-angiogenic molecule, cleaved high molecular weight kininogen (HKa). We have shown that HKa binds to domain 2/3 of uPAR on endothelial cells and competes for occupancy with a major adhesive protein, vitronectin. HKa, when bound, also facilitates the binding and activation of PK to kallikrein, which cleaves pro-urokinase to urokinase by plasma kallikrein, and the subsequent enhancement of the formation of cell-associated plasmin from plasminogen. We now hypothesize that HKa-uPAR interactions may be critical in the control of angiogenesis, and that specific peptide sequences would be inhibitors of tumor angiogenesis. We will therefore study the binding of HKa to uPAR using both transfected cells and surface plasmon resonance with an emphasis on establishing which domains and subdomains of HKa are the sites of binding. We will use monoclonal antibodies, recombinant fragments (deletion mutants) and synthetic peptides derived from molecular homology models of domains 3 and 5 of HKa based on the crystal and NMR structures to identify the exact sequences responsible for HKa binding to urokinase. We will then test the effects of these polypeptides on four of the in vitro components of angiogenesis, namely, endothelial adhesion, migration, proliferation and cell surface-mediated proteolysis. Preliminary studies show that at nanomolar concentrations, deletion mutants or synthetic peptides derived from HKa inhibit migration of endothelial cells to vitronectin and endothelial cell proliferation. These studies will be extended using site-directed mutagenesis and optimized cyclic peptides to identify the minimum sequences. We will select the most potent peptides and test them in vivo on basic fibroblast growth factor stimulated chicken egg chorioallantoic membrane (CAM), where domain 5 at 30 nM inhibits angiogenesis, as well as tumor cells growing on the CAM. We will then test the polypeptides on human tumor cells which can grow in immunologically deficient mice. Finally, we will test these new anti-angiogenic peptides for their ability to prevent cancer growth metastasis in the Lewis lung tumor model. We expect that these studies will identify biologically active peptides which can serve as lead compounds in designing peptidomimetic angiogenic inhibitors.