Akt-Mediated Transactivation of the S1P ₁ Receptor in Caveolin-Enriched Microdomains Regulates Endothelial Barrier Enhancement by Oxidized Phospholipids

Abstract

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.

Stimulation of Rho signaling by pathologic mechanical stretch is a “second hit” to Rho-independent lung injury induced by IL-6

“…To reduce the content of endogenous RhoA, cells were treated with gene-specific small interfering (si)RNA duplexes. Predesigned standard purity siRNA sets (Homo sapiens) were ordered from Dharmacon (Lafayette, CO), and transfection of EC with siRNA was performed as previously described (30). After 48 h of transfection, cells were used for experiments or harvested for Western blot verification of specific protein depletion.…”

Mechanotransduction by GEF-H1 as a novel mechanism of ventilator-induced vascular endothelial permeability

“…For in vivo experiments, predesigned standard purity Stealth GEF-H1-specific mouse siRNA sets were purchased from Invitrogen (Carlsbad, CA). Polymer-based administration of nonspecific or specific siRNA conjugated with polycation polyethilenimine (PEI-22) shown to promote lung-specific DNA and siRNA delivery was used (59,62). The optimal concentration of siRNA was determined in the series of preliminary experiments.…”

Atrial natriuretic peptide attenuates LPS-induced lung vascular leak: role of PAK1

“…For in vivo experiments, predesigned standard purity Stealth PAK1-specific mouse siRNA sets were purchased from Invitrogen (Carlsbad, CA). Polymer-based administration of nonspecific or PAK1-specific siRNA conjugated with polycation polyethylenimine (PEI-22) was shown to promote lung-specific DNA and siRNA delivery (48,49) and was used as described elsewhere (45). The optimal concentration of siRNA was determined in the following series of preliminary experiments.…”