Crosstalk between S1P Receptor 1/S1P1 and P-Selectin/Selp in Regulation of Inflammatory Vascular Permeability

项目总结： 新冠肺炎大流行戏剧性地突显了ARDS的一种独特的血管内型，其特征是 深刻的内皮细胞(EC)渗透性、血栓形成和微血管病变(1；2)，以及未得到满足的重要需求 例如严重缺乏任何FDA批准的有效疗法来阻止或逆转致命的血管泄漏 ARDS/VILI。肺内皮细胞屏障完整性的丧失会导致血管渗漏、肺泡泛滥，这是一个关键特征。 ARDS多器官衰竭和死亡率。项目4研究人员已经证明了鞘氨醇-1-磷酸- 多功能脂质调节剂磷酸(S1P)及其类似物Tysiponate(Tysiponate)有效地减少血管 结扎高表达的G蛋白偶联受体S1PR1的渗漏和炎性肺损伤 肺的ECS。S1PR1连接通过RAC迅速启动信号通路，重组EC细胞骨架 GTP酶信号转导，增强连接完整性，降低血管通透性。相比之下，结扎术中的 S1PR3受体诱导Rho GTPase信号传导到细胞骨架，从而增加肺通透性。此外， S1PR3被释放到VILI或LPS暴露小鼠的循环微囊中，作为一种新的ARDS双功能蛋白. 预示生存的征兆。S1PR1和S1PR3启动子活性、表达与下游信号转导 受选择性启动子去甲基化的机械应力的病理生理水平的影响，以及受 与ARDS风险相关的启动子SNP。我们鉴定SELPLG是一种新的ARDS易感基因 BLACKS编码P-选择素糖蛋白配体1(PSGL1)，通过P-选择素(SELP)结合是至关重要的 参与了PMN的贩运。我们令人兴奋的数据表明S1PR/S1PR和S1PR的信号相互关联 PSGL1/SELPLG在EC血管渗漏调节中的作用鉴于ROS在肺血管渗漏中的主要作用 PMN贩运，具体目标#1(SA#1)，在强大的核心B援助下，将成为ROS- 敏感转录因子(HIF-1α/2α，NRF2)、启动子SNPs(在黑人中占优势)和DNA甲基化 S1PR1/S1PR3和SELPLG/SELP的遗传/表观遗传调控位点。利用令人信服的初步 数据和关键核心D支持，SA#2将研究AKT硝化、蛋白质的新屏障调节作用 去泛素化(UCHL1)，RAC GTP酶激活剂DOCK1，以及肌动蛋白结合FA蛋白，lamellipodin，in S1PR1和S1PR3信令。SA#3将表征S1PR1/S1PR3对PSGL1分泌的双重影响 和P-选择素从EC韦贝尔-帕拉德小体上动员，向EC细胞骨架发出信号，以及 改变EC屏障反应。在临床前大鼠和猪脂多糖/VILI中利用无与伦比的核心C专业知识 模型，SA#4将评估携带S1PR3的TySIPosome的治疗效果 SiRNA或UCHL1激活剂(CD19H28N205S)，并直接评估竞争性抑制物的疗效 P-选择素-PSGL-1结合，TSGL-Ig生物学(串联P-选择素糖蛋白配基-免疫球蛋白)。 因此，项目4研究将解决未得到满足的对EC中与ARDS相关的增加的新见解的需求 用于针对S1P1/3/PSGL1的新型ARDS疗法，以恢复肺循环的完整性。

PROJECT SUMMARY: The COVID-19 pandemic has dramatically highlighted a distinct vascular endotype of ARDS, characterized by profound endothelial cell (EC) permeability, thrombosis and microangiopathy (1; 2), and important unmet needs such as the critical absence of any effective FDA-approved therapies to halt or reverse lethal vascular leak in ARDS/VILI. Loss of lung EC barrier integrity results in vascular leakage, alveolar flooding, and is a critical feature of ARDS multi-organ failure and mortality. Project #4 investigators have demonstrated that sphingosine-1-phos- phate (S1P), a multifunctional lipid mediator, and its analog, Tysiponate (TySIP), effectively reduce vascular leakage and inflammatory lung injury via ligation of S1PR1, a G-protein-coupled receptor highly expressed in lung ECs. S1PR1 ligation rapidly initiates a signaling cascade that reorganizes the EC cytoskeleton via Rac GTPase signaling, enhances junctional integrity, and decreases vascular permeability. In contrast, ligation of the S1PR3 receptor induces Rho GTPase signaling to the cytoskeleton to increase lung permeability. Furthermore, S1PR3 is released into circulating microvesicles in VILI- or LPS-exposed mice serving as a novel ARDS bi- omarker that predicts survival. Both S1PR1 and S1PR3 promoter activity, expression and downstream signaling are influenced by pathophysiologic levels of mechanical stress with selective promoter demethylation, and by promoter SNPs associated with ARDS risk. We identified SELPLG as a novel ARDS susceptibility gene in Blacks, which encodes P-selectin glycoprotein ligand 1 (PSGL1) that via P-selectin (Selp) binding is critically involved in PMN trafficking. Our exciting data suggest interconnected signaling of S1PR/S1PR and PSGL1/SELPLG in regulation of EC vascular leak. Given the major role of ROS in pulmonary vascular leak and PMN trafficking, Specific Aim #1 (SA #1), with strong Core B assistance, will characterize the role of ROS- sensing transcription factors (HIF-1α/2α, NRF2), promoter SNPs (predominant in Blacks) and DNA methylation sites in genetic/epigenetic regulation of S1PR1/S1PR3 and SELPLG/SELP. Leveraging compelling preliminary data and critical Core D support, SA #2 will examine the novel barrier-regulatory roles for AKT nitration, protein deubiquitination (UCHL1), the Rac GTPase activator DOCK1, and the actin-binding FA protein, lamellipodin, in S1PR1 and S1PR3 signaling. SA #3 will characterize the dual influences of S1PR1/S1PR3 on PSGL1 secretion and P-selectin membrane mobilization from EC Weibel-Palade bodies, on signaling to the EC cytoskeleton, and altered EC barrier responses. Leveraging unparalleled Core C expertise in preclinical rat and porcine LPS/VILI models, SA #4 will evaluate the therapeutic effectiveness of the TySIPosome encargoed with either S1PR3 siRNAs or an UCHL1 activator, (CD19H28N205S), and directly assess the efficacy of a competitive inhibitor of P-selectin-PSGL-1 binding, the TSGL-Ig biologic (Tandem P-Selectin Glycoprotein Ligand-Immunoglobulin). Thus, Project #4 studies will address the unmet need for novel insights into ARDS-associated increases in EC permeability and for novel ARDS therapies that target S1P1/3/PSGL1 to restore integrity of the lung circulation.