Kininogen/Urokinase Receptors in Tumor Angiogenesis

描述（由申请人提供）：大多数恶性肿瘤的生长严重依赖于新微血管的增殖，以向这些快速生长的细胞提供氧气和营养。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扩散。HK是细胞表面介导的防御系统（接触系统）的关键成员，该系统与补体系统一样，旨在对病理生理损伤做出反应。我们鉴定的uPAR作为一个网站的HKA结合内皮细胞显示，HK和血浆激肽释放酶是重要的尿激酶原尿激酶的转换，从而形成纤溶酶的内皮细胞表面。用纤溶酶“武装”的内皮细胞可以通过蛋白水解细胞外基质（ECM）或通过激活基质金属蛋白酶而迁移到组织中。通过与uPAR结合，HKa还置换粘附蛋白玻连蛋白，从而允许内皮细胞与ECM分离。HK的抗粘附和促纤溶功能使我们证明了HK的促血管生成作用以及HK的HKa和D5的抗血管生成活性。该项目的长期目标是通过了解HK及其裂解产物-HKa，缓激肽和D5-对内皮受体的作用机制以及受体连接的后果，我们将能够调节体内血管生成。我们推测，HKA和D5的抗血管生成活性是由于锌依赖性结合uPAR，启动信号，然后干扰细胞周期。我们推测，促血管生成活性是由释放缓激肽，并独立调节。具体目标是：1）我们推测HKa和D5的抗血管生成活性除了诱导细胞凋亡和影响细胞周期蛋白外，还调节内皮细胞向血管管的分化。我们将使用一个三维（3D）胶原蛋白凝胶系统，在体外研究这一过程。2)我们推测，HKA和D5，这是负责激肽原的抗血管生成作用，都直接结合，并通过uPAR发挥其下游作用。由于uPAR是已知的结合和信号通过整合素，我们假设，HKa将破坏的途径，涉及连续磷酸化的Syk激酶，粘着斑激酶和桩蛋白。3)完整的HK是促血管生成的，并通过释放缓激肽发挥作用，这是单克隆抗体C11 C1通过阻止HK与细胞受体结合来抑制血管生成的能力的原因。我们将测试受体是B1还是B2，以及NO是否参与其中。我们将测试干扰HK与细胞受体结合的单克隆抗体（mAb C11 C1）是否会抑制肿瘤血管生成。4)由于mAb C11 C1和D5均抑制卵内内皮细胞粘附、迁移、增殖和血管生成，我们将测试D5或衍生自D5和mAb C11 C1的肽是否在三种鼠模型中抑制体内血管生成以及相关的肿瘤生长和转移; 5）我们将检测晚期和可能早期非-肺的小细胞癌，其水平不同于在正常对照受试者中发现的水平。成功完成这一建议可能会导致开发新的药物治疗癌症的特点是肿瘤诱导的血管生成。

DESCRIPTION (provided by applicant): The growth of most malignant tumors is critically dependent on the proliferation of new microvessels to supply these rapidly growing cells with oxygen and nutrients. Inhibition of tumor angiogenesis leads to regression of these malignancies and controls metastatic spread in animals. HK is a key member of the cell surface mediated defense system (contact system) which, like the complement system, is designed to respond to pathophysiological insults. Our identification of uPAR as a site for HKa binding on endothelial cells has shown that HK and plasma kallikrein are important in the conversion of prourokinase to urokinase and, thus, formation of plasmin on the endothelial cell surface. Endothelial cells "armed" with plasmin can migrate into tissues by proteolyzing the extracellular matrix (ECM) or by activating matrix metalloproteinases. By binding to uPAR, HKa also displaces the adhesive protein, vitronectin, thereby allowing the endothelial cells to detach from the ECM. The antiadhesive and profibrinolytic function of HK has led us to demonstrate the proangiogenic role of HK and the antiangiogenic activity of HKa and D5 of HK. The long-term goal of this project is that by understanding the mechanism of the action of HK and its cleavage products--HKa, bradykinin, and D5--on endothelial receptors and the consequence of ligation of the receptors, we will be able to regulate angiogenesis in vivo. We hypothesize that the antiangiogenic activity of HKa and D5 is due to zinc-dependent binding to uPAR, which initiates signaling which then interferes with the cell cycle. We postulate that proangiogenic activity is exerted by released bradykinin and is independently regulated. The specific aims are: 1) We hypothesize that the antiangiogenic activity of HKa and D5 in addition to inducing apoptosis and effecting cell cycle proteins modulates endothelial cell differentiation into vascular tubes. We will use a three-dimensional (3D) collagen gel system to study this process in vitro. 2) We postulate that HKa and D5, which are responsible for the antiangiogenic effects of kininogen, both bind directly and exert their downstream effects through uPAR. Since uPAR is known to bind to and signal through integrins, we postulate that HKa will disrupt the pathway that involves successive phosphorylation by Syk kinases, focal adhesion kinase and paxcillin. 3) Intact HK is proangiogenic and functions by liberating bradykinin, accounting for the ability of monoclonal antibody C11C1 to inhibit angiogenesis by preventing the binding of HK to cell receptors. We will test whether the receptor is B1 or B2 and whether NO is involved. We will test whether a monoclonal antibody (mAb C11C1) that interferes with the binding of HK to cell receptors will inhibit tumor angiogenesis. 4) Since both mAb C11C1 and D5, which inhibit endothelial cell adhesion, migration, proliferation and angiogenesis in ovo, we will test whether D5 or peptides derived from D5 and mAb C11C1 will inhibit angiogenesis in vivo in three murine models and associated tumor growth and metastasis; 5) We will test whether the HK light chain containing D5 exists in the urine of patients with advanced and possibly early non-small cell cancer of the lung at levels different from those found in normal control subjects. Successful completion of this proposal could result in the development of new agents for the treatment of cancer is characterized by tumor-induced angiogenesis.