The formation of new vessels plays a fundamental role in processes such as embryonic development, wound healing, tumor growth and ischemia-reperfusion. In those processes the interaction between the endothelial cells and proteins of the extracellular matrix is essential. We have developed a model of capillary tube formation in a double layer of fibrin in vitro. In fibrin the critical structure for this function is the N terminal 15-42 aminoacids of the beta chain. This action is mediated by associating with VE-cadherin. We found that for p120ctn to bind the cytoplasmic juxtamembrane domain of VE-cadherin a highly conserved octapeptide of that region should be present. The interaction between VE-cadherin and p120ctn seems to modulate cell growth and adhesiveness. We proposed to further characterize the role of the VE-cadherin -p120ctn interaction in regulating cellular growth. We will analyze the growth characteristics of CHO cells, devoid of cadherin, transfected with either wild type or mutant VE- cadherin unable to bind p120ctn. We will determine whether genes that are known to regulate cellular growth are targets of p120ctn taking advantage of our system where we know whether p120ctn is free or bound. We will also try to identify using microarrays techniques whether there are genes that are expressed differently when p120ctn is bound or unbound to VE-cadherin. We also propose to characterize the role of the interaction between VE-cadherin and p120ctn in capillary tube formation in the double layer of fibrin in vitro. We propose that the interaction between VE-cadherin and p120ctn is important for the angiogenesis process and that p120ctn may be involved in the transduction of a signal upon fibrin engaging the extracellular domain of VE- cadherin. Our hypothesis is that the relationship between VE-cadherin and p120ctn is dynamic during capillary tube formation. We will study their co- localization and phosphorylation state during capillary tube formation by immunohistochemical methods and immunoprecipitation methods. We will also assay whether downregulation of p120ctn abrogates tube formation. Finally we will determine whether wild type cadherin can restore the ability to form capillary tubes to endothelial cells that do not expressed VE-cadherin and do not form capillaries. If so we will determine whether our mutant VE-cadherin unable to bind p120ctn confers the same properties. Our hypothesis is that the interaction between VE-cadherin and p120ctn is needed for capillary tubes to form and therefore we predict that our mutant VE-cadherin unable to bind p120ctn will not restore the ability on those cells to form tubes. These studies will help elucidate the basic biochemical and biological responses by which the interaction between VE-cadherin and p120ctn may modulate angiogenesis.
| 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 |
