Caspase-1, the Microvascular Endothelium, and Infection

Caspase-1、微血管内皮和感染

基本信息

批准号：
9043174
负责人：
JONATHON PETER AUDIA
金额：
$ 37.88万
依托单位：
UNIVERSITY OF SOUTH ALABAMA
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-04-01 至 2020-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9043174
关键词：
Adult Respiratory Distress Syndrome Affect Anti-Inflammatory Agents Anti-inflammatory Biological Assay Biology CASP1 gene Case Study Cell Death Cell physiology Cells Cellular Stress Clinical Trials Cysteine Data Endothelial Cells Endothelium Enzymes Eukaryotic Cell Failure Fluorescence Generations Glycolysis Glycolysis Inhibition Goals Health Homologous Gene Human Immune Immunoblotting Infection Inflammation Inflammatory Inflammatory Response Interleukin-1 beta Interleukin-18 Life Link Lung Measures Mediating Mitochondria Mitochondrial Proteins Modeling Molecular Mutagenesis Nitrogen Outcome Oxygen Pathogenesis Patient-Focused Outcomes Patients Pattern recognition receptor Peptide Hydrolases Phospholipase A2 Positioning Attribute Prevalence Process Pseudomonas aeruginosa Pulmonary Edema Recovery Reporter Resolution Respiration Rickettsia prowazekii Role Severities Signal Pathway Signal Transduction Site-Directed Mutagenesis Stem cells Stress Testing Toxin Ventilator Venus base biological adaptation to stress combat cytokine extracellular glycation inhibitor/antagonist interleukin-1beta-converting enzyme inhibitor macrophage monocyte mortality novel outcome prediction pathogen programs receptor research study respiratory response scaffold targeted treatment therapeutic development

项目摘要

DESCRIPTION (provided by applicant): Despite compelling experimental evidence linking inflammation to the pathogenesis of Acute Respiratory Distress Syndrome (ARDS), anti-inflammatory clinical trials have systematically failed to demonstrate beneficial effects in patients. This failure is often ascribed to the interrelated prospects that pan-suppression of inflammation is deleterious or that anti-inflammatories also inhibit protective stress responses. Thus, identifying novel targets to treat ARDS requires an understanding of the basic biology underlying key molecules that drive both pro-inflammatory responses in immune cells and protective stress adaptation programs in non-immune cells. Preliminary Data show that, in addition to inducing pro-inflammatory responses in macrophages, Caspase-1 protease activation protects Pulmonary Microvascular Endothelial Cell (PMVEC) and Pulmonary Arterial Endothelial Cell (PAEC) barrier function in response to infection. Additional data presented herein support a model in which Caspase-1 degrades glycolytic and mitochondrial proteins in PMVECs and PAECs as a protective strategy that limits accumulation of advanced N-glycation end products (AGEs) and reactive oxygen/nitrogen species (RS) induced by infection. Intriguingly, the model opportunistic pathogen (Pseudomonas aeruginosa) and vasculotropic pathogen (Rickettsia prowazekii) used in these studies both deploy homologous secreted phospholipase A2 toxins (ExoU) that inhibit Caspase-1 activation. The proposed experiments will test the Hypothesis that Caspase-1 activation in PMVECs and PAECs elicits degradation of glycolytic and mitochondrial proteins as an adaptive stress response to protect barrier function. Specific Aim 1 will elucidate mechanisms of Caspase-1 activation in PMVECs and PAECs by: 1.1) Defining the mechanisms by which PMVECs and PAECs sense infection at the level of Inflammasome signaling using a Split Venus fluorescence complementation reporter. 1.2) Determining mechanisms by which the ExoU secreted toxin inhibits Caspase-1 activation using phospholipase A2 signaling inhibitors. Specific Aim 2 will elucidate mechanisms of Caspase-1-induced stress responses in PMVECs and PAECs by: 2.1) Elucidating glycolytic and mitochondrial proteins degraded by Caspase-1 and validating targets by site-directed mutagenesis and enzyme function assays. 2.2) Determining whether Caspase-1 protects PMVEC and PAEC barrier function during infection. Molecular and pharmacologic approaches will be used to either down-regulate or up-regulate Caspase-1 followed by assessment of barrier function, AGEs, and RS. Inhibitors of glycolytic intermediates, AGEs, and/or RS will unveil their roles in barrier demise. Specific Aim 3 will correlate Caspase- 1 activation and mitochondrial function with ARDS patient outcomes by: 3.1) Measuring active Caspase-1 and mitochondrial respiration in immune cells and non-immune cells. 3.2) Correlating outcomes with patient mortality and ventilator-free days. Long-term impact on human health and translational potential lie in identifying targets for therapies to treat ARDS, which are currently lacking.

描述（由申请人提供）：尽管有令人信服的实验证据将炎症与急性呼吸窘迫综合征（ARDS）的发病机制联系起来，但抗炎临床试验未能系统地证明对患者有益，这种失败通常归因于以下相互关联的前景。炎症的泛抑制是有害的，或者抗炎药也会抑制保护性应激反应，因此，确定治疗 ARDS 的新靶点需要了解驱动促炎症反应的关键分子的基础生物学。免疫细胞和非免疫细胞中的保护性应激适应程序初步数据表明，除了诱导巨噬细胞中的促炎反应外，Caspase-1 蛋白酶激活还可以保护肺微血管内皮细胞 (PMVEC) 和肺动脉内皮细胞 (PAEC)。本文提供的其他数据支持 Caspase-1 降解 PMVEC 和 PAEC 中的糖酵解蛋白和线粒体蛋白的模型。作为一种限制感染诱导的晚期 N-糖基化终产物 (AGE) 和活性氧/氮 (RS) 积累的保护策略，有趣的是，模型中使用了机会性病原体（铜绿假单胞菌）和亲血管病原体（普氏立克次体）。两项研究都采用了抑制 Caspase-1 激活的同源分泌型磷脂酶 A2 毒素 (ExoU)。拟议的实验将进行测试。 PMVEC 和 PAEC 中的 Caspase-1 激活会引起糖酵解和线粒体蛋白降解作为保护屏障功能的适应性应激反应的假设。具体目标 1 将通过以下方式阐明 PMVEC 和 PAEC 中的 Caspase-1 激活机制： 1.1) 定义机制。 PMVEC 和 PAEC 使用 Split Venus 荧光互补报告基因在炎症体信号传导水平上感知感染 1.2)。使用磷脂酶 A2 信号抑制剂确定 ExoU 分泌的毒素抑制 Caspase-1 激活的机制将通过以下方式阐明 PMVEC 和 PAEC 中 Caspase-1 诱导的应激反应机制： 2.1) 阐明由 Caspase 降解的糖酵解和线粒体蛋白。 1 并通过定点诱变和酶功能测定验证靶标。2.2) 确定。 Caspase-1 在感染期间是否保护 PMVEC 和 PAEC 屏障功能将用于下调或上调 Caspase-1，然后评估屏障功能、AGE 和糖酵解中间体 AGE 抑制剂。和/或 RS 将揭示其在屏障消亡中的作用，具体目标 3 将通过以下方式将 Caspase-1 激活和线粒体功能与 ARDS 患者结果相关联。 3.1) 测量免疫细胞和非免疫细胞中的活性 Caspase-1 和线粒体呼吸 3.2) 将结果与患者死亡率和无呼吸机天数相关联，对人类健康和转化潜力的长期影响在于确定治疗目标。 ARDS，目前缺乏。