The endothelium is acutely responsive to changes in fluid shear and messengers of vascular damage, such as thrombin. These agents can alter the selectivity of the endothelium's barrier function, the controlled movement of compounds and cells in and out of the vascular space, by eliciting changes in the intercellular junctional components. VE-cadherin complexes, which form the calcium-dependent intercellular junctions of endothelial cells, are targets of signaling pathways activated by thrombin receptors and fluid shear sensors. This proposal focuses upon defining the shared and unique signaling mechanisms activated by thrombin and fluid shear that regulate the assembly, stability and signaling capabilities of VE-cadherin complexes at endothelial cell-cell junctions. The hypotheses are as follows: I. Thrombin receptor and fluid shear regulate the reorganization o endothelial cell-cell junctions by stimulating the tyrosine phosphorylation of the VE-cadherin-associated proteins beta-catenin, gamma-catenin and p120-catenin. Regulation of alpha-catenin association with the complex alters the linkage of the VE-cadherin complexes to the actin cytoskeleton. II. Fluid shear and thrombin receptor regulation of the controlled tyrosine phosphorylation/dephosphorylation of beta-catenin, gamma-catenin and p120-catenin is mediated by changes in the activities or associations of the protein tyrosine phosphatase SHP-2 and the protein tyrosine kinases c-src, fyn, lyn and yes with the VE-cadherin complexes. Techniques of immunofluorescence, immunoblotting, immunoprecipitation, far western analysis as well as immune complex enzyme assays will be used to quantitate changes in the expression, associations, phosphorylation an activities of the proteins found in VE-cadherin complexes, including SHP-2, c-src, fyn, lyn and yes. Transient transfection of wild type and mutant constructs of SHP-2 and the src-family kinases as well as antisense protocols to ablate the activities of the protein tyrosine phosphatase and kinases will be used to examine the underlying mechanisms that regulate changes at endothelial cell junctions. The proposed research will provide foundation for examining problems of chronic vascular disease where the dysfunctional endothelium no longer appropriately regulates vascular permeability and vessel repair.
