Differential Roles of Sphingosine 1-Phosphate Receptor PTMs and SNPs in Regulation of Ventilator-Induced Lung Injury (VILI)

项目总结： 肺内皮细胞(EC)屏障完整性的丧失，导致血管渗漏和肺泡泛滥，是一种 呼吸机相关性肺损伤(VILI)的病理生物学特征。项目#3团队的研究是 首次证明多功能脂质介体1-磷酸鞘氨醇(S1P)是一种有效的 结扎G蛋白偶联的S1P减少炎性肺损伤血管渗漏的实验研究 受体在肺内皮细胞中高表达。我们已经证明，通过S1P或S1P连接S1P1受体 S1P类似物，迅速刺激通过rac1重组肺EC细胞骨架的信号级联 GTP酶激活，增强连接完整性，降低血管通透性。S1P管理 降低VILI、内毒素、缺血/再灌流引起的小鼠和犬肺血管高通透性， 辐射或创伤性脑死亡，氧合和肺泡水肿形成明显改善。在……里面 相反，S1P3受体的结扎诱导Rho GTP酶信号传递到细胞骨架以增加肺 渗透性。此外，S1P3由VILI或LPS释放到循环微囊中，是一种新的 预测生存的ARDS生物标志物。我们已公布的和初步的数据表明，生理和 机械应力的病理生理水平对S1PR1和S1PR3启动子活性有选择性影响， 表达和下游信号转导。这包括选择性的启动子去甲基化反应 机械应力和启动子单核苷酸多态对启动子活性的影响。 在项目3中，我们将协助翻译关于S1P1/S1P3在欧共体应对措施中的作用的信息 机械应力和VILI，并评估S1P1和S1P3，下游效应器(rac1和RhoA)， 关键字S1P1和S1P3翻译后修改(PTM)的影响以及S1PR1和S1PR1的贡献 S1PR3 SNP与VILI风险和严重性的关系。特定目标(SA)#1将详细说明S1PR1和S1PR3启动子调控 机械应力诱导的启动子甲基化的元件、位置及S1PR1和S1PR3的影响 SNPs启动子对机械应激诱导的S1PR1和S1PR3 5‘启动子的调控和表达。我们的 已发表的研究还表明，VILI是ROS产生和蛋白质硝化的主要刺激因素 S1P3是一个特定的目标。SA#2将描述机械应力引起的功能后果 Ptms(硝化和磷酸化)参与S1P1/S1P3异常调节的rac1和RhoA信号级联反应 会改变肺血管屏障的完整性。这些研究将包括编码SNP的S1PR1和S1PR3的影响， 对S1P3硝化作用、持续激活的硝化RhoA和持续失活的Rho1、AS的评价 新型有效的VILI生物标志物和信号分子。最后，SA#3将评估潜在的VILI 治疗靶点包括新的S1P1特异性激动剂，以及rac1和RhoA硝化屏蔽肽。 总之，项目3的研究将验证针对这一复杂信号通路的新疗法 VILI病理生物学的解决和降低VILI/ARDS发病率和死亡率的先进策略。

PROJECT SUMMARY: The loss of lung endothelial cell (EC) barrier integrity, resulting in vascular leakage and alveolar flooding, is a critical feature of the pathobiology of ventilator-induced lung injury (VILI). Studies by the Project #3 team were the first to demonstrate that sphingosine 1-phosphate (S1P), a multifunctional lipid mediator, is an effective therapy for reducing vascular leakage in inflammatory lung injury via ligation of S1P1, a G-protein-coupled S1P receptor highly expressed in lung ECs. We have demonstrated that ligation of the S1P1 receptor by S1P or S1P analogues, rapidly stimulates a signaling cascade that reorganizes the lung EC cytoskeleton via Rac1 GTPase activation, enhances junctional integrity and decreases vascular permeability. S1P administration reduces murine and canine lung vascular hyper-permeability evoked by VILI, LPS, ischemia/reperfusion, radiation, or traumatic brain death with marked improvement in oxygenation and alveolar edema formation. In contrast, ligation of the S1P3 receptor induces Rho GTPase signaling to the cytoskeleton to increase lung permeability. Furthermore, S1P3 is released into circulating microvesicles by VILI or LPS and serves as a novel ARDS biomarker that predicts survival. Our published and preliminary data indicate that physiologic and pathophysiologic levels of mechanical stress have selective effects on S1PR1 and S1PR3 promoter activity, expression and downstream signaling. This includes selective promoter demethylation responses to excessive mechanical stress and the influence of promoter single nucleotide polymorphisms (SNPs) on promoter activity. In Project #3, we will facilitate the translation of information on the role of S1P1/S1P3 in EC responses to mechanical stress and VILI, and evaluate the role of S1P1 and S1P3, downstream effectors (Rac1 and RhoA), the influence of key S1P1 and S1P3 post-translational modifications (PTMs), and the contribution of S1PR1 and S1PR3 SNPs to VILI risk and severity. Specific Aim (SA) #1 will detail S1PR1 and S1PR3 promoter regulatory elements, sites of promoter methylation induced by mechanical stress and the influence of S1PR1 and S1PR3 promoter SNPs on mechanical stress-induced S1PR1 & S1PR3 5' promoter regulation and expression. Our published studies have also indicated that VILI is a major stimulus for ROS generation and protein nitration with S1P3 a specific target. SA #2 will characterize the functional consequences of mechanical stress-induced PTMs (nitration and phosphorylation) involved in S1P1/S1P3 dysregulated Rac1 and RhoA signaling cascades that alter lung vascular barrier integrity. These studies will include effects of S1PR1 and S1PR3 coding SNPs, evaluation of S1P3 nitration, persistently activated nitrated RhoA, and persistently inactivated nitrated Rac1, as novel and effective VILI biomarkers and signaling molecules. Finally, SA #3 will evaluate potential VILI therapeutic targets including novel S1P1-specific agonists, and Rac1 and RhoA nitration-shielding peptides. Together, Project #3 studies will validate novel therapies targeting this complex signaling pathway in the resolution of VILI pathobiology and advance strategies to reduce the morbidity and mortality of VILI/ARDS.