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Inactivation Mechanisms of Microvascular Hyperpermeability

微血管通透性过高的失活机制

基本信息

批准号：
10335153
负责人：
Walter N. Duran
金额：
$ 63.74万
依托单位：
RBHS-NEW JERSEY MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-04 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10335153
关键词：
Actins Agonist Back Biological Assay Blood Vessels Caveolae Cell Adhesion Cell membrane Cells Cessation of life Compartment syndromes Concentration Camps Cyclic AMP Cyclic GMP Cytosol Data Development Edema Embryo Endothelial Cells Endothelium Excision Feedback Gene Deletion In Vitro Inflammation Inflammatory Knowledge Ligation Location Measures Methods Microvascular Permeability Molecular Muscle NOS3 gene Nitric Oxide Nitrosation Organ Preservation Pathologic Pathology Permeability Phosphorylation Physicians Process Production Property Protein Family Proteins Protocols documentation Recovery Research Personnel Role Signal Pathway Signal Transduction Stimulus Surgeon Testing Therapeutic Time Tissues Vascular Diseases Vascular Permeabilities Work base improved in vivo in vivo evaluation novel novel therapeutics prevent response restoration targeted treatment therapy development tissue repair vascular inflammation vasodilator-stimulated phosphoprotein

项目摘要

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.

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。