Protective Role of Intracellular S1P in Lung Injury

描述（申请人提供）：鞘脂代谢产物如神经酰胺和鞘碱是细胞存活、增殖、血管生成、迁移和血管完整性的重要调节剂。我们的体内和体外研究表明，鞘氨醇-1-磷酸（S1P）是一种天然存在的生物活性脂质，是一种必需的血管生成因子，可调节血管内皮细胞（EC）的通透性，这是ALI的关键和决定性特征。S1P通过其g蛋白偶联的S1P1-5受体在细胞外起作用，有证据支持S1P在钙释放和细胞增殖中的细胞内作用。S1P介导的细胞反应受其合成、鞘氨醇激酶（SphKs）催化以及S1P磷酸酶和S1P裂解酶（S1PL）介导的降解所调节。在我们的研究过程中，我们观察到动物或人肺微血管内皮细胞（HLMVECs）中SphKs和S1PL表达的调节影响lps诱导的炎症反应和屏障功能，这表明这些鞘脂代谢酶在维持屏障功能和完整性方面发挥了关键作用。在这一竞争性更新中，我们将验证“内皮细胞的SphKs和S1PL调节细胞内S1P调节LPS介导的肺部炎症和屏障功能障碍”的假设。SA#1将定义SphK1、sphk2和S1PL在调节lps介导的炎症反应和HLMVECs屏障功能中的作用。我们将利用分子、生化和脂质组学相结合的方法来评估和量化细胞内S1P的积累，并确定其在脂多糖诱导的IL-6释放和内皮屏障功能障碍中的作用。SA#2将明确SphKs和S1PL调控lps诱导的肺内皮细胞炎症和屏障功能障碍的分子机制。SA#3将描述SphKs和S1PL中ALI相关的单核苷酸多态性（snp），并进行snp特异性关联研究。sa# 4将确定SphK激活剂和S1PL抑制剂在lps诱导的小鼠肺损伤模型中作为潜在治疗剂的有效性。总之，在这一竞争更新中提出的研究将极大地推进和阐明SphKs和S1PL在ALI病理生物学中的关键作用，并促进鞘脂代谢的新型治疗药物的开发，以改善败血症诱导的肺部炎症和损伤。

DESCRIPTION (provided by applicant): Sphingolipid metabolites such as ceramides and sphingoid bases are important modulators of cell survival, proliferation, angiogenesis, migration, and vascular integrity. Our in vivo and in vitro studies indicate that sphingosine-1-phosphate (S1P), a naturally occurring bioactive lipid, is an essential angiogenic factor that regulates vascular endothelial cell (EC) permeability, a critical and defining feature of ALI. S1P acts extracellularly through its G-protein coupled S1P1-5 receptors and there is evidence that supports an intracellular role of S1P in calcium release and cell proliferation. S1P mediated cellular responses are regulated by its synthesis, catalyzed by sphingosine kinases (SphKs), and degradation mediated by S1P phosphatases, and S1P lyase (S1PL). In the course of our investigations, we observed that modulation of SphKs and S1PL expression in animals or human lung microvascular endothelial cells (HLMVECs) affected LPS-induced inflammatory responses and barrier function suggesting a key role for these sphingolipid metabolizing enzymes in maintenance of barrier function and integrity. In this competing renewal, we will test the hypothesis that "Modulation of intracellular S1P by SphKs and S1PL of the endothelium regulates LPS- mediated lung inflammation and barrier dysfunction". SA#1 will define the role of SphK1 and 2 and S1PL in modulating LPS-mediated inflammatory responses and barrier function in HLMVECs. We will utilize a combination of molecular, biochemical, and lipidomics approach to evaluate and quantify intracellular S1P accumulation and determine its role in LPs-induced IL-6 release and barrier dysfunction in the endothelium. SA#2 will define molecular mechanisms by which SphKs and S1PL regulate LPS-induced inflammation and barrier dysfunction in lung ECs. SA#3 will characterize ALI associated single nucleotide polymorphism (SNPs) in SphKs and S1PL and conduct SNP-specific association studies. SA#4 will determine the effectiveness of SphK activator(s) and S1PL inhibitor(s) as potential therapeutic agents in murine models of LPS-induced lung injury. Together, studies proposed in this competing renewal will greatly advance and clarify the key roles of SphKs and S1PL in ALI pathobiology and facilitate the development of novel therapeutic agents of sphingolipid metabolism in ameliorating sepsis-induced lung inflammation and injury. PUBLIC HEALTH RELEVANCE: Acute lung injury (ALI) is a devastating inflammatory lung injury which shares increased morbidity and mortality with enhanced vascular permeability as a defining feature. Sphingolipids such as sphingosine-1-phosphate (S1P), represent a novel biomarker and therapy for the vascular leak in ALI via stabilization or restoration of lung endothelial integrity. We propose to evaluate the enzymes of sphingolipid pathway(s) as modulators of S1P generation to improve management of patients with ALI.