Inactivation Mechanisms of Microvascular Hyperpermeability

项目总结/摘要 血管通透性过高是炎症的标志。目前的治疗干扰了相关机制 出现高渗透性我们将重点关注终止高渗透性的机制， 高渗透性的负面影响是由于其持续超过保存器官所需的时间 功能我们将阐明积极终止高渗透性的机制。这项工作是基于a） 蛋白质运输机制的现有知识，B）VASP（血管扩张剂刺激的 磷蛋白）在细胞粘附和内皮屏障特性中的作用，c）我们证明eNOS 易位到胞质溶胶对于高渗透性的发生是必要的，d）我们的初步数据表明， 选择性刺激Epac 1（cAMP激活的交换蛋白）使eNOS返回细胞膜， e）我们的初步数据显示VASP是使eNOS返回质膜所必需的。我们将 测试中心假设，即导致高渗透性的激动剂信号传导启动延迟的 [cAMP]增加，导致VASP辅助eNOS和Epac 1转运至细胞 膜和触发失活的高渗透性。这一新的假设，其中障碍 恢复是一个通过信号机制运行的主动过程， 高渗透性，将通过两个特定目标（SA）进行测试。在每个特定目标中，我们将刺激eNOS 用Epac 1的选择性激活剂8-cPT-2-O-Me-cAMP转运至细胞膜，并将测量 渗透率作为终点。SA 1：检测内皮细胞通透性增高时胞浆eNOS的活性， 引起cAMP增加，导致高通透性失活。我们将评估 在锚eNOS条件下， 胞质溶胶或质膜以及在体内和体外刺激或抑制[cAMP]的方案。SA 2：至 确定磷酸化VASP作为eNOS和Epac 1转运至细胞的分子伴侣的作用 膜的在野生型内皮细胞（EC）和EC VASP-KO细胞中，我们将探讨 [NO]、VASP磷酸化、eNOS转位和高通透性失活。我们将 用8-cPT-2-O-Me-cAMP在体内和体外刺激eNOS向细胞膜转位。我们将评估 VASP磷酸化与eNOS和Epac 1共定位关系 连接测定（PLA）。将阐明抑制和终止高渗透性的过程。 微血管通透性调节机制的新进展，并将提供 为开发治疗血管炎症的新疗法奠定了基础。

PROJECT SUMMARY/ABSTRACT Vascular hyperpermeability is a hallmark of inflammation. Current therapy interferes with mechanisms involved in onset of hyperpermeability. We will focus on mechanisms that terminate hyperpermeability because negative effects of hyperpermeability are due to its persistence beyond the time required for preserving organ function. We will elucidate mechanisms that actively terminate hyperpermeability. This work is based on a) current knowledge of protein traffic mechanisms, b) the fundamental role of VASP (vasodilator stimulated phosphoprotein) in cell adhesion and endothelial barrier properties, c) our demonstration that eNOS translocation to cytosol is necessary for onset of hyperpermeability, d) our preliminary data demonstrating that selective stimulation of Epac1 (exchange protein activated by cAMP) returns eNOS to the cell membrane, and e) our preliminary data showing that VASP is required for returning eNOS to the plasma membrane. We will test the central hypothesis that the agonist signaling that leads to hyperpermeability initiates a delayed increase in [cAMP], which causes VASP-assisted translocation of eNOS and Epac1 to the cell membrane and triggers inactivation of hyperpermeability. This novel hypothesis, in which barrier restoration is an active process operating via signaling mechanisms distinct from those that cause hyperpermeability, will be tested through two Specific Aims (SA). In each Specific Aim, we will stimulate eNOS translocation to the cell membrane with 8-cPT-2-O-Me-cAMP, a selective activator of Epac1, and will measure permeability as an end-point. SA1: To test whether the activity of cytoplasmic eNOS during hyperpermeability causes an increase in cAMP that leads to inactivation of hyperpermeability. We will assess inactivation of hyperpermeability and measure [cAMP] as a function of NO and time under conditions that anchor eNOS to cytosol or plasma membrane and protocols that stimulate or inhibit [cAMP] in vivo and in vitro. SA2: To determine the role of phosphorylated VASP as a molecular partner in eNOS and Epac1 translocation to cell membrane. In wild-type endothelial cells (EC) and in EC VASP-KO cells, we will explore the relationships among [NO], VASP phosphorylation, eNOS translocation and inactivation of hyperpermeability. We will stimulate eNOS translocation to cell membrane with 8-cPT-2-O-Me-cAMP in vivo and in vitro. We will assess the relationship between VASP phosphorylation and co-localization of eNOS and Epac1 using proximity ligation assay (PLA). The elucidation of the processes that inactivate and terminate hyperpermeability will be novel advances in knowledge of regulatory mechanisms of microvascular permeability and will provide the basis for developing new therapies for treating vascular inflammation.