Kininogen/Urokinase Receptors in Tumor Angiogenesis

描述(申请人提供)：大多数恶性肿瘤的生长严重依赖于新的微血管的增殖，为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，并控制动物体内的转移扩散。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，旨在应对病理生理侮辱。我们鉴定uPAR是Hka与内皮细胞结合的一个部位，结果表明HK和血浆激肽释放酶在尿激酶原转化为尿激酶，从而在内皮细胞表面形成纤溶酶的过程中起着重要作用。“武装”纤溶酶的内皮细胞可以通过分解细胞外基质(ECM)或激活基质金属蛋白酶而迁移到组织中。通过与uPAR结合，Hka还取代了黏附蛋白Vitronectin，从而允许内皮细胞从ECM分离。HK的抗黏附和纤溶功能使我们证实了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及其切割产物--Hka、缓激肽和D5--对内皮细胞受体的作用机制以及受体连接的结果，我们将能够在体内调控血管生成。我们假设Hka和D5的抗血管生成活性是由于锌依赖与uPAR的结合，这启动了信号转导，然后干扰了细胞周期。我们推测促血管生成活性是由释放的缓激肽发挥作用的，并且是独立调节的。具体目的是：1)我们假设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内皮细胞分化。我们将使用三维(3D)胶原凝胶系统在体外研究这一过程。2)我们推测与激肽原抗血管生成作用有关的Hka和D5均直接结合并通过uPAR发挥其下游作用。由于已知uPAR与整合素结合并通过整合素传递信号，我们推测Hka将破坏涉及Syk激酶、粘着斑激酶和帕克西林连续磷酸化的通路。3)完整的HK是促血管生成的，通过释放缓激肽发挥作用，这可能是C11C1通过阻止HK与细胞受体结合而抑制血管生成的原因。我们将测试受体是B1还是B2，以及是否涉及NO。我们将测试干扰HK与细胞受体结合的单抗(MAbC11c1)是否会抑制肿瘤血管生成。4)由于D5和D5都能抑制血管内皮细胞的黏附、迁移、增殖和血管生成，因此我们将在三个小鼠模型中检测D5或D5衍生的多肽是否会抑制体内血管生成及相关的肿瘤生长和转移；5)我们将检测晚期和可能早期的非小细胞肺癌患者尿液中含有D5的轻链是否存在于与正常对照组不同的水平。这项提议的成功完成可能会导致开发新的药物来治疗以肿瘤诱导血管生成为特征的癌症。

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.