After an acute phase of inflammation or injury, restoration of the endothelial barrier is important to regain vascular integrity and to prevent edema formation. However, little is known about mediators that control restoration of endothelial barrier function. We show here that oxidized phospholipids that accumulate at sites of inflammation and tissue damage are potent regulators of endothelial barrier function. Oxygenated epoxyisoprostane-containing phospholipids, but not fragmented oxidized phospholipids, exhibited barrier-protective effects mediated by small GTPases Cdc42 and Rac and their cytoskeletal, focal adhesion, and adherens junction effector proteins. Oxidized phospholipid-induced cytoskeletal rearrangements resulted in a unique peripheral actin rim formation, which was mimicked by coexpression of constitutively active Cdc42 and Rac, and abolished by coexpression of dominant-negative Rac and Cdc42. Thus, oxidative modification of phospholipids during inflammation leads to the formation of novel regulators that may be critically involved in restoration of vascular barrier function.

Ventilator-induced lung injury is a life-threatening complication of mechanical ventilation at high-tidal volumes. Besides activation of proinflammatory cytokine production, excessive lung distension directly affects blood-gas barrier and lung vascular permeability. To investigate whether restoration of pulmonary endothelial cell (EC) monolayer integrity after agonist challenge is dependent on the magnitude of applied cyclic stretch (CS) and how these effects are linked to differential activation of small GTPases Rac and Rho, pulmonary ECs were subjected to physiologically (5% elongation) or pathologically (18% elongation) relevant levels of CS. Pathological CS enhanced thrombin-induced gap formation and delayed monolayer recovery, whereas physiological CS induced nearly complete EC recovery accompanied by peripheral redistribution of focal adhesions and cortactin after 50 minutes of thrombin. Consistent with differential effects on monolayer integrity, 18% CS enhanced thrombin-induced Rho activation, whereas 5% CS promoted Rac activation during the EC recovery phase. Rac inhibition dramatically attenuated restoration of monolayer integrity after thrombin challenge. Physiological CS preconditioning (5% CS, 24 hours) enhanced EC paracellular gap resolution after step-wise increase to 18% CS (30 minutes) and thrombin challenge. These results suggest a critical role for the CS amplitude and the balance between Rac and Rho in mechanochemical regulation of lung EC barrier.

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We have previously described differential effects of physiologic (5%) and pathologic (18%) cyclic stretch (CS) on agonist-induced pulmonary endothelial permeability. This study examined acute and chronic effects of CS on agonist-induced intracellular signaling and cell morphology in the human lung macro-and microvascular endothelial cell (EC) monolayers. Endothelial permeability was assessed by analysis of morphological changes, parameters of cell contraction and measurements of transendothelial electrical resistance. Exposure of both microvascular and macrovascular EC to 18% CS for 2 -96 hrs increased thrombin-induced permeability and monolayer disruption. Interestingly, the ability to promote thrombin responses was present in EC cultures exposed to 48-96 hrs of CS even after replating onto non-elastic substrates. In turn, physiologic CS preconditioning (72 hrs) attenuated thrombin-induced paracellular gap formation and MLC phosphorylation in replated EC cultures. Long term preconditioning at 18% CS (72 hrs) increased the content of signaling and contractile proteins including Rho GTPase, MLC, MLC kinase, ZIP kinase, PAR1, caldesmon and HSP27 in the pulmonary microvascular and macrovascular cells. We conclude that short term CS regulates EC permeability via modulation of agonist-induced signaling, whereas long term CS controls endothelial barrier at both post-translational level and via magnitude-dependent regulation of pulmonary EC phenotype, signaling and contractile protein expression.

IntroductionOxidized phospholipids appear in the pulmonary circulation as a result of acute lung injury or inflammation. We have previously shown that oxidized phospholipids exhibit barrier-protective effects on pulmonary endothelial cell (EC) monolayers. However, effects of oxidized phospholipids on EC focal adhesion (FA) and adherence junction (AJ) remodeling have not been yet fully explored.GoalTo study molecular mechanisms of adherens junction and focal adhesion remodeling mediated by oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC).MethodsAll experiments were performed on human pulmonary artery endothelial cells (HPAEC). Intracellular protein localization was analyzed by immunocytochemistry. Subcellular localization of the proteins of interest was determined using subcellular proteome extraction kit. Protein phosphorylation profile was assessed by Western blot analysis. Protein-protein interactions were analyzed by coimmunoprecipitation assays.ResultsEnhancement of EC barrier in response to OxPAPC was accompanied by dynamic remodeling of focal adhesions and adherence junctions. Immunofluorescent analysis of OxPAPC-stimulated EC revealed dramatic translocation and peripheral and enhanced peripheral staining for AJ proteins beta-catenin and VE-cadherin. In addition, OxPAPC treatment increased tyrosine phosphorylation of FA proteins FAK at Tyr-576 and paxillin at Tyr-118, which was associated with peripheral redistribution of FA and AJ complexes. Furthermore, subcellular fractionation analysis showed increase of VE-cadherin, beta-catenin, and GIT2 in membrane fraction after OxPAPC challenge. Remarkably, coimmunoprecipitation studies indicated increased interaction of paxillin with FA components FAK, vinculin and GIT2, and AJ protein beta-catenin upon HPAEC stimulation with OxPAPC. Complementary experiments with immunoprecipitation beta-catenin followed by probing for paxillin confirmed these results.ConclusionsThe results of these studies characterize OxPAPC-induced focal adhesion remodeling and determine for the first time the specific interactions between focal adhesion and adherens junction protein complexes in endothelial barrier-protective responses to OxPAPCGrant support: HL076259, HL075349 for K.G.B., AHA-SDG for A.A.B.

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