KININOGEN/UROKINASE RECEPTORS IN TUMOR ANGIOGENESIS

许多恶性实体肿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会导致这些恶性肿瘤的消退，并控制动物体内的转移扩散，这导致了通过靶向肿瘤血管系统来控制癌症的重大努力。尿激酶受体(UPAR)不仅在恶性肿瘤细胞上高表达，而且在调节细胞迁移和细胞相关蛋白水解酶活性的黏附中起着关键作用，而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--裂解高分子激肽原(Hka)。我们已经证明，Hka与内皮细胞上uPAR的2/3结构域结合，并与主要的黏附蛋白Vitronectin竞争占据。当Hka结合时，也促进了PK与激肽释放酶的结合和激活，激肽释放酶将尿激肽原裂解为尿激酶，从而促进了纤溶酶原形成细胞相关的纤溶酶。我们现在假设，Hka-uPAR的相互作用可能在血管生成的控制中起关键作用，并且特定的肽序列将是肿瘤血管生成的抑制因子。因此，我们将使用转基因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的部位。我们将使用单抗、重组片段(缺失突变体)和基于晶体和核磁共振结构的Hka结构域3和5的分子同源模型衍生的合成肽来鉴定与Hka结合的确切序列。然后，我们将测试这些多肽对体外血管生成的四个组成部分的影响，即内皮细胞黏附、迁移、增殖和细胞表面介导的蛋白降解。初步研究表明，在纳摩尔浓度下，Hka来源的缺失突变体或合成肽可以抑制内皮细胞向Vitronectin的迁移和内皮细胞的增殖。这些研究将利用定点突变和优化环肽来确定最小序列。我们将选择最有效的多肽并在体内测试它们在碱性成纤维细胞生长因子刺激的鸡卵绒毛尿囊膜(CAM)上的作用，其中结构域5在30 nm处抑制血管生成，以及肿瘤细胞在CAM上生长。然后，我们将在人类肿瘤细胞上测试这些多肽，这些肿瘤细胞可以在免疫缺陷的小鼠身上生长。最后，我们将测试这些新的抗血管生成多肽在Lewis肺癌模型中防止肿瘤生长转移的能力。我们期望这些研究将确定生物活性多肽，这些活性多肽可以作为先导化合物来设计模拟多肽的血管生成抑制剂。

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.