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(Circulation Research. 2009;104:978.)
© 2009 American Heart Association, Inc.

Molecular Medicine

Akt-Mediated Transactivation of the S1P₁ Receptor in Caveolin-Enriched Microdomains Regulates Endothelial Barrier Enhancement by Oxidized Phospholipids

Patrick A. Singleton, Santipongse Chatchavalvanich, Panfeng Fu, Junjie Xing, Anna A. Birukova, Jennifer A. Fortune, Alexander M. Klibanov, Joe G. N. Garcia, Konstantin G. Birukov

From the Section of Pulmonary and Critical Medicine (P.A.S., S.C., P.F., J.X., A.A.B., J.G.N.G., K.G.B.), Department of Medicine, Division of Biomedical Sciences, University of Chicago, Ill; and Departments of Chemistry and Biological Engineering (J.A.F., A.M.K.), Massachusetts Institute of Technology, Cambridge.

Correspondence to Dr Patrick A. Singleton, Department of Medicine, GCIS Building W403E, University of Chicago, Chicago, IL 60637. E-mail psinglet{at}medicine.bsd.uchicago.edu

Endothelial cell (EC) barrier dysfunction results in increased vascular permeability, leading to increased mass transport across the vessel wall and leukocyte extravasation, the key mechanisms in pathogenesis of tissue inflammation and edema. We have previously demonstrated that OxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine) significantly enhances vascular endothelial barrier properties in vitro and in vivo and attenuates endothelial hyperpermeability induced by inflammatory and edemagenic agents via Rac and Cdc42 GTPase dependent mechanisms. These findings suggested potential important therapeutic value of barrier-protective oxidized phospholipids. In this study, we examined involvement of signaling complexes associated with caveolin-enriched microdomains (CEMs) in barrier-protective responses of human pulmonary ECs to OxPAPC. Immunoblotting from OxPAPC-treated ECs revealed OxPAPC-mediated rapid recruitment (5 minutes) to CEMs of the sphingosine 1-phosphate receptor (S1P₁), the serine/threonine kinase Akt, and the Rac1 guanine nucleotide exchange factor Tiam1 and phosphorylation of caveolin-1, indicative of signaling activation in CEMs. Abolishing CEM formation (methyl-β-cyclodextrin) blocked OxPAPC-mediated Rac1 activation, cytoskeletal reorganization, and EC barrier enhancement. Silencing (small interfering RNA) Akt expression blocked OxPAPC-mediated S1P₁ activation (threonine phosphorylation), whereas silencing S1P₁ receptor expression blocked OxPAPC-mediated Tiam1 recruitment to CEMs, Rac1 activation, and EC barrier enhancement. To confirm our in vitro results in an in vivo murine model of acute lung injury with pulmonary vascular hyperpermeability, we observed that selective lung silencing of caveolin-1 or S1P₁ receptor expression blocked OxPAPC-mediated protection from ventilator-induced lung injury. Taken together, these results suggest Akt-dependent transactivation of S1P₁ within CEMs is important for OxPAPC-mediated cortical actin rearrangement and EC barrier protection.

Key Words: OxPAPC • Akt • S1P receptor • caveolin-enriched microdomain • endothelial barrier enhancement

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