Abstract 196: OxPAPC Attenuates Thrombin-induced Endothelial Barrier Dysfunction via modulation of Small GTPase Activities
Increased levels of oxidized phospholipids generated by oxidation of shed cell membranes and apoptotic blebs have been detected in the local circulation and associated with inflammation, acute lung injury and cardiac ischemia. Previous studies have shown that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) activates small GTPases Rac and Cdc42, enhances endothelial peripheral actin cytoskeleton and enhances endothelial cell (EC) barrier properties. This study examined potential protective effects of oxidized phospholipids in a model of thrombin-induced EC barrier dysfunction, which is mediated by small GTPase Rho. Permeability changes in human pulmonary EC monolayers were assessed by measurements of transendothelial electrical resistance (TER). EC monolayer integrity was evaluated by immunofluorescent staining of actin and adherens junctions. Activation of small GTPases was measured by in vitro pull down assays. OxPAPC significantly attenuated thrombin-induced pulmonary EC hyperpermeability and markedly decreased thrombin-induced Rho activation and phosphorylation of its downstream target myosin light chain. These effects were accompanied by dramatic reduction in paracellular gap and stress fiber formation in acute phase of thrombin-induced EC barrier dysfunction (15 min). During recovery phase after thrombin challenge (50 min), OxPAPC accelerated recovery of transendothelial resistance and resealing of the paracellular gaps, and caused rapid reduction of stress fibers. These changes were accompanied by suppression of residual Rho activation, enhanced Rac activation and peripheral accumulation of Rac effector cortactin involved in actin polymerization. Pharmacological Rac inhibition or depletion using siRNA, as well as downregulation of Rac-specific guanine nucleotide exchange factor Tiam1 abolished positive OxPAPC effects on EC monolayer recovery. These results demonstrate for the first time barrier protective effects elicited by cell membrane-derived oxidized phospholipids in the model of thrombin-induced barrier dysfunction and suggest an important role for Tiam1-Rac mechanism in the EC barrier recovery associated with conditions of acute aseptic vascular injury.