Singleton Patrick A, Chatchavalvanich Santipongse, Fu Panfeng, Xing Junjie, Birukova Anna A, Fortune Jennifer A, Klibanov Alexander M, Garcia Joe G N, Birukov Konstantin G
Department of Medicine, Division of Biomedical Sciences, Section of Pulmonary and Critical Medicine, University of Chicago, Ill 60637, USA.
Circ Res. 2009 Apr 24;104(8):978-86. doi: 10.1161/CIRCRESAHA.108.193367. Epub 2009 Mar 12.
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 (S1P(1)), 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-beta-cyclodextrin) blocked OxPAPC-mediated Rac1 activation, cytoskeletal reorganization, and EC barrier enhancement. Silencing (small interfering RNA) Akt expression blocked OxPAPC-mediated S1P(1) activation (threonine phosphorylation), whereas silencing S1P(1) 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 S1P(1) receptor expression blocked OxPAPC-mediated protection from ventilator-induced lung injury. Taken together, these results suggest Akt-dependent transactivation of S1P(1) within CEMs is important for OxPAPC-mediated cortical actin rearrangement and EC barrier protection.
内皮细胞（EC）屏障功能障碍会导致血管通透性增加，进而使跨血管壁的物质运输增多以及白细胞渗出，这是组织炎症和水肿发病机制中的关键环节。我们之前已经证明，氧化型1-棕榈酰-2-花生四烯酰-sn-甘油-3-磷酸胆碱（OxPAPC）在体外和体内均能显著增强血管内皮屏障特性，并通过Rac和Cdc42 GTP酶依赖性机制减轻炎症和致水肿因子诱导的内皮细胞高通透性。这些发现提示了具有屏障保护作用的氧化磷脂具有潜在的重要治疗价值。在本研究中，我们检测了与富含小窝蛋白的微区（CEMs）相关的信号复合物在人肺内皮细胞对OxPAPC的屏障保护反应中的作用。对经OxPAPC处理的内皮细胞进行免疫印迹分析显示，OxPAPC介导了鞘氨醇-1-磷酸受体（S1P(1)）、丝氨酸/苏氨酸激酶Akt和Rac1鸟嘌呤核苷酸交换因子Tiam1快速募集（5分钟）至CEMs，并使小窝蛋白-1磷酸化，这表明CEMs中发生了信号激活。消除CEM形成（甲基-β-环糊精）可阻断OxPAPC介导的Rac1激活、细胞骨架重排以及内皮细胞屏障增强。沉默（小干扰RNA）Akt表达可阻断OxPAPC介导的S1P(1)激活（苏氨酸磷酸化），而沉默S1P(1)受体表达则可阻断OxPAPC介导的Tiam1募集至CEMs以及Rac1激活和内皮细胞屏障增强。为了在具有肺血管高通透性的急性肺损伤小鼠体内模型中证实我们的体外研究结果，我们观察到选择性沉默小窝蛋白-1或S1P(1)受体表达可阻断OxPAPC介导的对呼吸机诱导的肺损伤保护作用。综上所述，这些结果表明CEMs内Akt依赖性的S1P(1)反式激活对于OxPAPC介导的皮质肌动蛋白重排和内皮细胞屏障保护至关重要。
