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An alphav beta5 Integrin-Dependent Programmed Cell Death Pathway in Activated Endothelial Cells
Integrins that bind to vitronectin are highly expressed in neovasculature within diseases with an angiogenic component, where they play an important regulatory role. We have designed a strategy to identify molecules associated to the cytoplasmic domains of transmembrane receptors and have successfully applied this approach to search for signaling proteins relevant to ±v=5 function. Our system was based on the screening of random phage libraries for the selection of peptides binding to the =5 cytoplasmic domain. We showed that one of the selected peptides (VVISYSMPD) induces apoptosis upon cell internalization. Next, we established that the cell death process induced by VVISYSMPD is sensitive to modulation by growth factors and by protein kinase C (PKC), and it cannot be triggered in =5 null cells. Finally, we determined that the VVISYSMPD peptide is a mimic of annexin V. Our results brought about a functional link between the ±v=5 integrin, annexin V, and a novel programmed cell death mechanism. This =5-dependent cell death pathway was designated as “endothanatos” (death from the inside). Here we seek to understand the structural basis for the regulation of PKC-mediated cell survival in order to determine the mechanism of action of annexin V in this process. We hypothesize that (i) additional molecules associate with ±vß5, annexin V, and PKC and (ii) that the transient association of these molecules can promote or inhibit cell death. We also propose to determine whether or not this cell death process is active during tissue remodeling and angiogenesis, given that ±v=5 is highly expressed in vivo upon endothelial cell stimulation with factors that regulate these processes. We wish to address the following questions: 1. Are there inside-out and outside-in signaling events that can be associated to VVISYSMPD-induced cell death? 2. Do additional molecules associate with ±vß5, annexin V, and PKC, and can the transient association of these molecules promote or inhibit cell death in the context of tissue remodeling? Completion of these Aims will allow us to characterize the mechanism by which annexin V regulates endothelial cell death. Uncovering the mechanistic basis for a receptor-mediated process controlling cell death during angiogenesis and tissue remodeling would be significant relevance for our understanding of endothelial cell biology and angiogenesis in breast cancer.
Breast cancer is the most common malignant tumor affecting women in the US. It has been estimated that up to 1 in 8 women will develop the disease during their lifetime. Breast cancers cannot grow beyond one mm size without their blood supply for nourish and oxygenation. Thus, we propose to attack breast cancers by blocking the formation of new blood vessels that sustain tumor growth. The formation of the tumor blood vessels network depends on the cells that form the lining of the blood vessels. These lining cells are not malignant; in order to be fed, the tumor recruits and activates them. Once activated, they quickly multiply, adhere, migrate, and form more blood vessels to feed the tumor. Such recruitment depends on certain adhesive receptors that become abundant in the surface of the activated blood vessels cells. Under normal conditions, these receptors allow the cells to interact with their environment. Such interaction is possible because these surface receptors (named 'integrins” because they “integrate” the cells to their environment) cross the membrane of the cells and have two ends: one end is located outside the cell while the other end is located in the inside of the cell. However, when breast cancer occurs, such system is subverted to allow the blood vessels to receive the recruiting signals from the tumor. Therefore, integrins play and important role in the formation of new blood vessels to feed the breast cancer: they receive instructions from the tumor through the end outside the cell and they transmit these instructions to their end located inside the cells of the blood vessels. In order to interfere with the communication between the tumor and the blood vessels we isolated very small pieces of protein (named “peptides”) that can zero in and bind to the region of the receptors that is located inside the cells. By loading the cells that form the lining of the blood vessels with these “peptides”, we can disturb the recruitment of blood vessels nourishing tumor cells. Our studies may bring new strategies to destroy blood vessels in breast tumors, leading to the elimination of primary and metastatic disease with no side-effects in normal organs.