The formation of new blood vessels plays a fundamental role in biological processes such as embryonic development, wound healing, and tumor growth. During these processes, the interactions of endothelial cells (EC) with proteins of the extracellular matrix are essential for the induction and maintenance of neovascularization. In vivo, following injury of a vessel, a fibrin clot is formed that interacts with the EC and serves as a provisional matrix for formation of new blood vessels. We have studied endothelial cell differentiation in a fibrin gel """"""""sandwich"""""""" and observed that EC forms a network of capillary-like tubes, and the molecular determinants responsible for the morphogenesis in this system appears to be rather distinct. In fibrin II, the N-terminal region of the beta chain, a peptide 15-42, seems to express the biological activity which is mediated by associating VE-cadherin. We propose that endothelial cells respond in a fibrin sandwich by defined signals which originate from the direct association of apically fibrin, with the apical membrane via VE cadherin. In addition, the surface distribution of membrane cadherins, along with their association with specific catenins, may be crucial in the formation of capillary tubes. Based on these investigations, we propose to further characterize the domain of VE-cadherin that is responsible for its association with the N-terminal domain of fibrin, and to identify the VE-cadherin domain that intervenes in homophilic binding. Both interactions, heterophilic fibrin VE- cadherin and homophilic cell-cell play a fundamental role in the formation of capillaries. In these studies, we will construct mutants of VE-cadherin with specific deletions in the last two extracellular domains. CHO cells and endothelial cells devoid of VE-cadherin will be transfected with VE-cadherin mutants. Binding of the N-terminal fragment of fibrin, homophilic interactions and ability to form capillary-like structures in the fibrin assay will be analyzed and the results will be compared to those obtained with cells transfected with the wild type VE- cadherin. Pervious studies demonstrated the role of VE-cadherin- catenin complexes in EC homophilic binding and in this proposal, we will investigate the role of VE-cadherin association with beta-catenin, plakoglobin and p120 in hpmophilic binding and in the formation of capillary-like structures. VE-cadherin mutants with specific deletions of two cytoplasmic catenin binding domains will be prepared and functional studies will be done following transfection. Finally, we will also study the distribution of VE-cadherin and assembly of VE-cadherin-catenin complexes in cell monolayers and during the formation of capillaries. Immunocytochemical studies will also be done and analysis of the distribution of VE-cadherin and VE-cadherin- catenin complexes will be performed by using fluorescence microscopy, confocal microscopy and EM. These studies should elucidate the biochemical and biological responses that induce the assembly of EC into capillary-like structures as a response of the EC to the interaction with fibrin.
| Keller, Margaret A; Martinez, Jose; Baradet, Timothy C et al. (2005) Fibrinogen Philadelphia, a hypodysfibrinogenemia characterized by abnormal polymerization and fibrinogen hypercatabolism due to gamma S378P mutation. Blood 105:3162-8 |
| Ferber, Andres; Yaen, Christopher; Sarmiento, Edna et al. (2002) An octapeptide in the juxtamembrane domain of VE-cadherin is important for p120ctn binding and cell proliferation. Exp Cell Res 274:35-44 |
