The role of cadherins, a multigene family of calcium dependent cell adhesion molecules, in regulating vascular integrity and angiogenesis is not well-defined. Three well characterized cadherins, E- (epithelial), N- (neual), and P- (placenta) cadherin have been shown to promote maintenance of cell junctions in established tissues as well as cell sorting during embryogenesis. As most of the cadherin family members share a conserved cytoplasmic domain, we designed a degenerate PCR cloning approach to isolate additional members of the family. Using this method, a novel member of the cadherin gene superfamily, cadherin 5, was cloned and characterized. Using in situ hybridization and immunostaining with cadherin 5 specific reagents, we found that cadherin 5 is expressed specifically by endothelial cells. This led to the central hypothesis that cadherin 5 plays an integral role in the formation and maintenance of endothelial structures. We postulate that cadherin 5 function is regulated by its interaction with cytoplasmic binding proteins called caterins. Based on our preliminary data, we predicted that when inflammatory cells migrate through existing vascular structures, the cadherin/catenin interaction is modified by a signal transduction event, causing loss of cadherin/catenin binding and leading to the opening of endothelial junctions. This hypothesis will be tested in Aim 1 by characterizing the subcellular distribution of cadherin 5, the caterins and defined junctional proteins in an in vitro neutrophil transmigration assay using laser scanning confocal microscopy. In Aim 2, the interaction of cadherin 5 and its binding proteins during endothelial junctional events will be defined biochemically. In particular, the role of phosphorylation in modifying this interaction will be studied. As well as maintaining existing endothelial junctions, cadherin 5 dependent cell adhesion may be essential for the organization and formation of vascular structures. Structure-function studies are proposed in Aim 3 to define the cadherin 5 structure involved in cell adhesion and to determine its importance in angiogenesis.

The proposed studies will lay the groundwork for studying the cadherin signal transduction pathway in endothelial cells. In addition, they will provide insight into the regulation of endothelial junctional events during inflammation and metastasis as well as normal development and angiogenesis.

K01AR001991

1996-04-10

2000-03-31