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

项目概要： 肺内皮细胞（EC）屏障完整性的丧失，导致血管渗漏和肺泡灌洗，是一种严重的肺损伤。 呼吸机诱导的肺损伤（VILI）病理生物学的关键特征。项目#3团队的研究是 第一个证明鞘氨醇1-磷酸（S1 P），一种多功能脂质介质，是有效的 通过连接G蛋白偶联的S1 P1-S1 P1减少炎性肺损伤中血管渗漏的治疗 受体在肺内皮细胞中高度表达。我们已经证明，通过S1 P或 S1 P类似物，快速刺激信号级联，通过Rac 1重组肺EC细胞骨架 GT3激活增强连接完整性并降低血管通透性。S1 P给药 降低由VILI、LPS、缺血/再灌注引起的鼠和犬肺血管通透性过高， 放射或创伤性脑死亡，氧合和肺泡水肿形成显著改善。在 相反，S1 P3受体的连接诱导Rho GT3信号传导至细胞骨架，以增加肺 磁导率此外，S1 P3通过VILI或LPS释放到循环微泡中，并作为一种新的免疫调节剂。 预测存活率的ARDS生物标志物。我们发表的和初步的数据表明， 机械应力的病理生理水平对S1 PR 1和S1 PR 3启动子活性具有选择性影响， 表达和下游信号传导。这包括选择性启动子去甲基化反应， 机械应力和启动子单核苷酸多态性（SNPs）对启动子活性的影响。 在项目#3中，我们将促进关于S1 P1/S1 P3在EC响应中的作用的信息的翻译， 机械应力和VILI，并评估S1 P1和S1 P3，下游效应子（Rac 1和RhoA）， 关键S1 P1和S1 P3翻译后修饰（PTM）的影响，以及S1 PR 1和 S1 PR 3 SNP与VILI风险和严重程度的关系具体目标（SA）#1将详细说明S1 PR 1和S1 PR 3启动子调控 机械应力诱导的启动子甲基化位点及S1 PR 1和S1 PR 3的影响 启动子SNP对机械应力诱导的S1 PR 1和S1 PR 3 5'启动子调控和表达的影响。我们 已发表的研究也表明VILI是ROS产生和蛋白质硝化的主要刺激物 S1 P3是一个特定的目标。SA #2将表征机械应力诱导的功能后果 PTM（硝化和磷酸化）参与S1 P1/S1 P3失调的Rac 1和RhoA信号级联反应 从而改变肺血管屏障的完整性。这些研究将包括S1 PR 1和S1 PR 3编码SNP的影响， 评价S1 P3硝化、持续活化的硝化RhoA和持续失活的硝化Rac 1， 新的和有效的VILI生物标志物和信号分子。最后，SA #3将评估潜在VILI 治疗靶点，包括新的S1 P1特异性激动剂和Rac 1和RhoA硝化屏蔽肽。 总之，项目#3研究将验证针对这种复杂信号通路的新疗法， 解决VILI病理生物学问题并提出降低VILI/ARDS发病率和死亡率的策略。

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.