Adult respiratory distress (ARDS) is characterized by high microvascular permeability to plasma fluid and protein. It is widely accepted that the endothelium is the main barrier to exchange between the luminal and tissue compartments, and that in ARDS, the barrier of the endothelium is impaired. Recently, it has been proposed that in ARD, endothelial junction integrity is deranged and leads to edema. However, the mechanisms which maintain and regulate barrier are unknown. Several cell adhesive proteins, termed cadherins, have been described in epithelium and in endothelium, but their roles in barrier maintenance/regulation are known. In epithelia, the function of these molecules have been identified by inserting anti-cadherin antibodies in junctions to disrupt barrier. Using this approach in endothelia and epithelia, we have determined that N-cadherin helps maintain large vessel endothelial barrier, and that uvomorulin (E-cadherin) helps form the epithelial barrier. The object of this proposal is to identify the pulmonary microvascular cadherins, demonstrate their roles in barrier maintenance, and examine how they regulated and organized, and how this organization is deranged during ARDS. We will 1) identify cadherins by western blotting, fluorescence and protein sequencing and 2) demonstrate the role of cadherins in endothelial barrier using cell-column chromatography as a sensitive barrier assay, and 3) using cadherin antibodies as junctional probes, will examine the roles of cadherins in endothelial barrier in experimental models of ARDS. Preliminary western blotting data shows at least 2 types of cadherins in both large and microvessel endothelium (ME) epithelial barrier depends on the presence of uvomorulin. Importantly, using our cell-column approach, we have shown that the barrier maintained by large vessel endothelial cells depends on homotypic binding mediated by N-cadherin and suggests that N-cadherin may constitute part of the endothelial junction. Our data suggest that cadherin regulation may involves reversible association with the cytoskeleton which may provide a means of regulating junctional barrier. Improved understanding of the mechanisms responsible for barrier may aid in understanding and treating inflammatory conditions like ARDS.
