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.
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