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，以向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并控制动物中的转移性扩散，导致了通过靶向其脉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度表达，而且在调节细胞迁移和粘附的细胞相关蛋白水解活性中起关键作用，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，裂解高分子量激肽原（HKa）。 我们已经表明，HKa结合到结构域2/3的uPAR内皮细胞和竞争占用的主要粘附蛋白，玻连蛋白。 当结合时，HKa还促进PK与激肽释放酶的结合和活化，其通过血浆激肽释放酶将尿激酶原切割为尿激酶，以及随后增强从纤溶酶原形成细胞相关纤溶酶。 我们现在假设HKa-uPAR相互作用在控制血管生成中可能是至关重要的，并且特定的肽序列将是肿瘤血管生成的抑制剂。 因此，我们将研究结合HKA uPAR使用转染细胞和表面等离子体共振，重点是建立哪些域和亚域的HKA的结合位点。 我们将使用单克隆抗体，重组片段（缺失突变体）和合成肽衍生的结构域3和5的HKa的基础上的晶体和NMR结构的分子同源性模型，以确定确切的序列负责HKa结合尿激酶。 然后，我们将测试这些多肽对血管生成的四个体外组分的影响，即内皮细胞粘附、迁移、增殖和细胞表面介导的蛋白水解。初步研究表明，在纳摩尔浓度，缺失突变体或合成肽衍生自HKa抑制内皮细胞迁移到玻连蛋白和内皮细胞增殖。 这些研究将使用定点诱变和优化的环肽来扩展，以确定最小序列。 我们将选择最有效的肽，并在体内测试它们对碱性成纤维细胞生长因子刺激的鸡胚绒毛尿囊膜（CAM），其中域5在30 nM抑制血管生成，以及肿瘤细胞生长的CAM。 然后，我们将在可以在免疫缺陷小鼠中生长的人类肿瘤细胞上测试多肽。最后，我们将在刘易斯肺肿瘤模型中测试这些新的抗血管生成肽预防癌症生长转移的能力。 我们希望这些研究将确定生物活性肽，可以作为先导化合物在设计拟肽血管生成抑制剂。

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.