New blood vessels can be formed by one of two related but distinct mechanisms -- vasculogenesis or angiogenesis. Vasculogenesis is defined as the development of blood vessels from angioblast precursor cells, whereas angiogenesis is the formation of new blood vessels from existing vasculature [1]. The formation of new blood vessels facilitates the physiological processes of embryonic development, female reproduction and wound healing [2]. Pathological angiogenesis is known to play a role in solid tumor formation, metastasis, diabetic retinopathy, macular degeneration, psoriasis and in inflammation-related diseases such as rheumatoid arthritis and ulcerative colitis [2]. Much attention has been paid to deregulated angiogenesis in cancer as the expansion of solid tumors beyond a minimal size is critically dependent on the formation of new blood vessels to supply oxygen, nutrients and growth factors [2]. Indeed, a high degree of vascularization in certain tumors indicates a poor clinical prognosis and increased risk of metastasis [3]. Angiogenesis involves endothelial cell (EC) migration from preexisting blood vessels, formation of adhesive sites between migrating cells and the extracellular matrix (ECM), and assembly of ECs into vessels [4]. Focal adhesion (FA) proteins play a role in each of these processes [5]. A focal adhesion is defined as a site of close interaction between the cell and its substrate [6]. In the case of ECs, this is the site where the cells are in close contact with the basement membrane of the blood vessel [7].;FAs are enriched in matrix receptors of the integrin family that connect the ECM with the cytoskeleton of an EC. Integrins are a family of heterodimeric transmembrane proteins comprising at least 18 alpha and 8 beta subunits, which form 24 known alphabeta-heterodimers depending on cell type and cellular function. Ligand binding to the extracellular integrin domain induces conformational changes and integrin clustering for activation of signaling cascades and recruitment of multiprotein complexes to FAs [8, 9]. At least seven members of the integrin family play important roles in EC biology: alphavbeta3, alphavbeta5, alphavbeta1, alpha5beta1, alpha3beta1, alpha2beta1, and alpha1beta1 [10]. In the developing vasculature and during angiogenesis, alphavbeta3 integrin is a major receptor expressed by ECs at FAs [4]. Historically, the largest amount of data pointed to alphavbeta3 integrin, a receptor for both fibronectin and vitronectin, and alphavbeta5, a vitronectin receptor, as major players in blood vessel formation. Indeed, blockade of alphavbeta3 or alphavbeta5 integrins with antagonists disrupts tumor and experimental angiogenesis [11-13]. However, genetic ablation of alphav, beta3, or beta3/beta5 integrin subunits in mice has relatively no effect on angiogenesis [14, 15]. In fact, mice lacking beta3 or beta3/beta5 integrin subunits show enhanced tumor growth and angiogenesis [14]. While the importance of alphavbeta3 in the vasculature is recognized, its exact role remains controversial and further research is required to determine its functions.;My project is designed to determine whether alphavbeta3 integrin mediates vimentin IF-FA association (Chapter 3) and how these interactions regulate not only the functions of alphavbeta3 integrin but also the adhesive and migratory behavior of ECs (Chapter 5), since the adhesion and migration of ECs plays important roles in both normal functions of the vasculature as well as during times of vascular remodeling. I have also investigated the role of plectin in linking the IF cytoskeleton to beta3 integrin and how microtubule motor proteins play a role in the recruitment of 6 vimentin IF to FAs in ECs (Chapter 4). Finally, I have done some studies on the effect of specific ECM ligands on integrin cross-talk, and how this cross-talk affects integrin recruitment to FAs and vimentin IF interaction with these FAs (Chapter 6).;In summary, my research demonstrates the importance of IF association with beta3 integrin in ECs, the mechanisms by which this association is made, and finally, the consequences of this association to EC function. Specifically, I have shown that IF recruitment to FAs in ECs requires beta3 integrin and plectin, and is mediated by microtubule motors. In CHO cells, which lack beta3 integrin but contain vimentin, IF are localized around the nucleus whereas in CHO cells expressing beta3 integrin (CHOwtbeta3), vimentin IFs extend to FAs at the cell periphery. This recruitment is regulated by tyrosine residues in the beta3 integrin tail. Moreover, CHOwtbeta3 cells exhibit significantly greater adhesive strength and motility than CHO or CHO cells expressing mutated beta3 integrin proteins. These differences require an intact vimentin network. Therefore, vimentin IF recruitment to the cell surface is tightly regulated and modulates the strength of adhesion of cells to their substrate as well as their ability to migrate over the substrate. (Abstract shortened by UMI.)
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