Regulatory Mechanisms for Resolution of Inflammatory Microvascular Leakage

解决炎症性微血管渗漏的调节机制

基本信息

批准号：
8496100
负责人：
JEROME W BRESLIN
金额：
$ 35.58万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8496100
关键词：
Abbreviations Actins Adhesions Admission activity Adult Respiratory Distress Syndrome Binding Biochemical Cells Clinical Clinical Treatment Coupled Data Development Disease Edema Endothelial Cells Endothelium Extravasation Family Family member Feedback Foundations Functional disorder Glycocalyx Health Care Costs Hemorrhagic Shock Hospitals Image Incidence Infection Inflammation Inflammation Mediators Inflammatory Injury Intensive Care Intercellular Junctions Lead Life Maintenance Measurement Mediating Membrane Mesentery Methods Microcirculation Microvascular Permeability Modeling Molecular Molecular Biology Molecular Target Monomeric GTP-Binding Proteins Multiple Organ Failure N-terminal Outcome Pathway interactions Patients Permeability Phosphorylation Physiological Plasma Proteins Polyamines Rattus Recovery Resolution Resuscitation Rho-associated kinase Rnd3 protein Role Sepsis Syndrome Serine Serine Phosphorylation Site Severities Signal Transduction Site Stimulus Testing Text Therapeutic Intervention Tissues Trauma base cadherin 5 cell behavior cellular imaging cost farnesylation in vivo innovation intravital microscopy knowledge of results meetings monolayer mortality mutant neutrophil novel restoration rho theories therapy development trauma care venule

项目摘要

DESCRIPTION (provided by applicant): Microvascular hyperpermeability is a hallmark of the systemic inflammatory response syndrome (SIRS) present in 93% of intensive care patients. SIRS severely complicates resuscitation of trauma victims and worsens clinical outcomes, frequently progressing to multiple organ failure. The estimated annual US health care cost burden associated with SIRS is $16.7 billion. Despite advances in understanding of inflammation- induced microvascular hyperpermeability, the mechanisms that restore microvascular permeability to normal following an inflammatory challenge are unknown. Moreover, existing clinical treatments are not effective for restoration of microvascular barrier integrity once the inflammatory cascade has been initiated. We propose a novel paradigm of signaling mechanisms responsible for restoration of microvascular integrity following inflammation caused by traumatic injury. Our preliminary data show that administration of a polyamine-coated, cell permeable form of Rnd3, a Rho family small GTPase, reduces microvascular hyperpermeability in a rat model of hemorrhagic shock. We also have observed with live cell imaging of endothelial cells that lamellipodia formation and turnover represent a previously uncharacterized cell behavior important for normal endothelial barrier integrity. We hypothesize that Rnd3 promotes endothelial barrier restoration after inflammatory challenges by inhibiting RhoA/ROCK-mediated cell contraction and activating Rac1-mediated enhancement of intercellular junction integrity. Our specific aims are to: 1) Test the prediction that Ser phosphorylation and membrane targeting of Rnd3 are required for Rnd3 to reduce microvascular hyperpermeability; 2) To test the prediction that Rnd3 promotes negative feedback inhibition of ROCK-mediated cell contraction, which enhances endothelial barrier integrity; 3) To test the prediction that Rnd3 enhances Rac1-mediated lamellipodia formation and stabilization of VE-cadherin at junctions, promoting endothelial barrier integrity. The proposed novel, integrated approach capitalizes on a refined and relevant rat model of hemorrhagic shock combined with intravital microscopy of the in vivo mesenteric microcirculation and isolated venule methods to assess microvascular permeability. We will explore the central role of Rnd3 by employing an innovative method to deliver cell permeable Rnd3 protein to the mesenteric microcirculation. Cultured endothelial cell monolayer permeability models, imaging of GFP-actin and GFP-VE-cadherin dynamics in live endothelial cells, and biochemical studies will support the in vivo and isolated venule studies. The results of this study will enable us to develop a new theory of how endothelial barrier function can be restored during inflammation, which will serve as the foundation for novel therapies. Discovery of targets that can be used to resolve microvascular hyperpermeability will revolutionize the treatment of trauma patients, and will also create new opportunities to treat edema associated with a wide range of diseases.

描述（由申请人提供）：微血管通透性过高是 93% 重症监护患者中存在的全身炎症反应综合征 (SIRS) 的标志。 SIRS 使创伤受害者的复苏变得严重复杂化，并使临床结果恶化，经常进展为多器官衰竭。据估计，美国每年与 SIRS 相关的医疗保健费用负担为 167 亿美元。尽管对炎症引起的微血管通透性过高的理解取得了进展，但在炎症挑战后将微血管通透性恢复到正常的机制尚不清楚。此外，一旦炎症级联反应开始，现有的临床治疗就无法有效恢复微血管屏障的完整性。我们提出了一种新的信号机制范例，负责在创伤性损伤引起的炎症后恢复微血管完整性。我们的初步数据表明，给予多胺包被的细胞渗透性 Rnd3（一种 Rho 家族小 GTP 酶）可降低失血性休克大鼠模型的微血管通透性过高。我们还通过内皮细胞的活细胞成像观察到，片状伪足的形成和周转代表了以前未表征的细胞行为，这对正常内皮屏障的完整性很重要。我们假设 Rnd3 通过抑制 RhoA/ROCK 介导的细胞收缩和激活 Rac1 介导的细胞间连接完整性增强来促进炎症挑战后内皮屏障的恢复。我们的具体目标是：1）测试 Rnd3 需要 Ser 磷酸化和膜靶向 Rnd3 来降低微血管通透性过高的预测； 2）测试Rnd3促进ROCK介导的细胞收缩的负反馈抑制的预测，从而增强内皮屏障的完整性； 3) 测试 Rnd3 增强 Rac1 介导的片状伪足形成和连接处 VE-钙粘蛋白的稳定性，促进内皮屏障完整性的预测。所提出的新颖的综合方法利用了精致且相关的失血性休克大鼠模型，结合体内肠系膜微循环的活体显微镜和孤立的小静脉方法来评估微血管通透性。我们将通过采用创新方法将细胞渗透性 Rnd3 蛋白递送至肠系膜微循环来探索 Rnd3 的核心作用。培养的内皮细胞单层通透性模型、活内皮细胞中 GFP-肌动蛋白和 GFP-VE-钙粘蛋白动力学成像以及生化研究将支持体内和分离微静脉研究。这项研究的结果将使我们能够开发一种关于如何在炎症期间恢复内皮屏障功能的新理论，这将成为新疗法的基础。发现可用于解决微血管通透性过高的靶点将彻底改变创伤患者的治疗，也将为治疗与多种疾病相关的水肿创造新的机会。