Isolation of Signaling Molecules Involved in Angiogenic Pathways Mediated Alpha v Integrins

摘要

Angiogenic vasculature selectively expressed alphavBETA3 end alphavBETA5 integrins but they are not expressed in normal vasculature. They are cell adhesion molecules that play an important role in the regulation of angiogenesis. There are at least two cytokine-dependent pathways that lead to angiogenesis in vivo which can be distinguished by their dependency on specific alphav integrins. Here we aim to define what molecules are involved in alphavBETA3- and alphavBETA5-selective angiogenic signaling. We hypothesize that: i) different molecules associate with each of these integrins after angiogenesis is triggered by defined cytokines; ii) the assembly of specific molecules with the BETA3 or BETA5 cytoplasmic domains results in selective signaling. The strategy used to approach these questions is based on the panning of phage peptide libraries of BETA3 and BETA5 cytoplasmic domains. Selected peptides are used to characterize candidate molecules mimicked by the peptides using biochemistry techniques. Finally, by using micro-injection-based techniques and internalizable forms of the synthetic peptides, we studied the effect of the integrin cytoplasmic domain binding peptides in cell adhesion, migration, apoptosis and proliferation upon stimulation with factors that can activate endothelial cells in vitro. We established that a cell death process induced by BETA5- binding peptide is sensitive to modulation by growth factors and by protein kinase C (PKC), and it cannot be triggered in BETA5 null cells. Finally, we show that the BETA5-binding peptide is a mimic of annexin V. Our results suggest a functional link between the alphavBETA5 integrin, annexin V, and a novel programmed cell death mechanism. These studies will shed light into molecular basis of selective signal transduction pathways initiated by alphavBETA3 and alphavBETA5. New assays aimed to inhibit of angiogenesis, and ultimately, new strategies to treat breast cancer may result from this work.