BACKGROUND/AIMS: Increase in vascular permeability is a cardinal feature of all inflammatory diseases and represents an imbalance in vascular contractile forces and barrier-restorative forces, both of which are highly dependent on actin cytoskeletal dynamics. In addition to the involvement of key vascular barrier-regulatory, actin-binding proteins, such as nmMLCK and cortactin, we recently demonstrated a role for a member of the Ena-VASP family known as Ena-VASP-like (EVL) in promoting vascular focal adhesion (FA) remodeling and endothelial cell (EC) barrier restoration/preservation. METHODS: To further understand the role of EVL in EC barrier-regulatory processes, we examined EVL-cytoskeletal protein interactions in FA dynamics in vitro utilizing lung EC and in vivo murine models of acute inflammatory lung injury. Deletion mapping studies and immunoprecipitation assays were performed to detail the interaction between EVL and cortactin, and further evaluated by assessment of changes in vascular EC permeability following disruption of EVL-cortactin interaction. RESULTS: Initial studies focusing on the actin-binding proteins, nmMLCK and cortactin, utilized deletion mapping of the cortactin gene (CTTN) to identify cortactin domains critical for EVL-cortactin interaction and verified the role of actin in promoting EVL-cortactin interaction. A role for profilins, actin-binding proteins that regulate actin polymerization, was established in facilitating EVL-FA binding. CONCLUSION: In summary, these studies further substantiate EVL participation in regulation of vascular barrier integrity and in the highly choreographed cytoskeletal interactions between key FA and cytoskeletal partners.