cGMP Compartmentalization in Pulmonary Endothelial Barrier Dysfunction

肺内皮屏障功能障碍中的 cGMP 区室化

• 批准号: 7483124
• 负责人: Eric Peter Schmidt
• 金额: $ 5.89万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7483124
• 关键词: 2' -phosphodiesterase; Acute Lung Injury; Air; Animal Model; Atrial Natriuretic Factor; Attenuated; Binding; Blood; Blood Vessels; Cations; Caveolae; Cell Fractionation; Cell Line; Cell membrane; Complex; Confocal Microscopy; Cyclic AMP; Cyclic GMP; Cytosol; Data; Dependence; Detection; Disruption; Dose; Endothelial Cells; Environmental air flow; Failure; Filtration; Functional disorder; Gene Transfer; Goals; Guanylate Cyclase; Immunoprecipitation; In Vitro; Infection; Injury; Knockout Mice; Late Effects; Lead; Liquid substance; Lung; Mechanical ventilation; Mechanics; Mediating; Membrane; Mus; Natriuretic Peptides; Nitric Oxide; Nitric Oxide Synthase; Oxadiazoles; Particulate; Pathway interactions; Permeability; Peroxonitrite; Phosphodiesterase Inhibitors; Preparation; Production; Pulmonary Edema; Quinoxalines; Role; Signal Transduction; Soluble Guanylate Cyclase; Specificity; Stretching; Structure of parenchyma of lung; Tidal Volume; Time; Trauma; Vascular Endothelium; Ventilator-induced lung injury; base; caveolin 1; indexing; inhibitor/antagonist; insight; methylxanthine; mortality; mouse model; phosphoric diester hydrolase; prevent; research study; response

DESCRIPTION (provided by applicant): Failure of the lung endothelial barrier leads to pulmonary edema and acute lung injury (All). Mechanical ventilation contributes to all mortality by subjecting the lung to excessive ventilatory stretch (VS), causing ventilator induced lung injury (VILI). In animal models of VILI, pulmonary endothelial barrier dysfunction is associated with increased endogenous nitric oxide (NO) production. NO activates endothelial soluble guanyl cyclase (sGC), producing cyclic guanosine monophosphate (cGMP). The effect of endothelial cGMP on VS-induced endothelial barrier function is unclear. Our preliminary experiments in a mouse model of VILI suggest that VS activates endothelial sGC to increase cGMP. After VS onset, sGC inhibition or stimulation attenuates or exacerbates barrier dysfunction, respectively, suggesting an injurious role for cGMP. In contrast, sGC activation before VS is barrier protective. Increasing endothelial cGMP production by non- sGC mechanisms (particulate guanylyl cyclase, pGC) is protective of the endothelial barrier, regardless of timing with the onset of VS. These and other data suggest an intracellular compartmentalization of sGC- mediated cGMP synthesis that is altered by VS to change the effect of cGMP signaling on barrier function, possibly by translocation of sGC from the cytosol to a caveolar membrane domain. The goal of this application is to determine the effect of cGMP on VILI with a specific focus on cGMP compartmentalization. In Aim 1, the effect of sGC-produced cGMP on endothelial barrier function will be further characterized in an isolated perfused mouse lung preparation. The dose-response and specificity of sGC activation on cGMP production and endothelial barrier dysfunction will be determined. In addition, the timing of the VS-induced switch from cGMP protection to harm will be determined. In Aim 2, the effect of lung VS on the subcellular compartmentalization of endothelial sGC will be determined in both intact mouse lungs and cyclically- stretched endothelial cells by confocal microscopy and subcellular fractionation. The barrier effect of sGC- caveolae interaction will be determined in isolated, perfused caveolin-1 -/-mouse lungs. VS-induced interactions of sGC with phosphodiesterase 2, a key effector of cGMP-mediated endothelial barrier dysfunction, will be explored using specific inhibitors, immunoprecipitation, and cAMP measuremnts. Relevance: Acute lung injury from infection or trauma causes lung failure because the endothelial cells lining blood vessels allow fluid to leak into the air spaces. Mechanical ventilatory support is vital, but over- distension of lung tissue can worsen injury through changes in endothelial molecules such as cGMP. Understanding how cGMP worsens endothelial leakiness could lead to better therapies of acute lung injury.

描述(申请人提供)：肺内皮细胞屏障失效导致肺水肿和急性肺损伤(ALL)。机械通气通过使肺承受过度的呼吸机牵张(VS)，导致呼吸机诱导的肺损伤(VILI)，从而导致所有的死亡。在VILI的动物模型中，肺内皮屏障功能障碍与内源性一氧化氮(NO)的产生增加有关。NO激活内皮细胞可溶性鸟苷环化酶(SGC)，产生环鸟苷一磷酸(CGMP)。内皮细胞cGMP对血管紧张素转换酶诱导的内皮屏障功能的影响尚不清楚。我们在VILI小鼠模型上的初步实验表明，VS激活内皮sGC以增加cGMP。VS发作后，sGC抑制或刺激分别减弱或加重屏障功能障碍，提示cGMP的损伤作用。相反，在VS前激活sGC具有屏障保护作用。通过非sGC机制(颗粒鸟苷酸环化酶，PGC)增加内皮细胞cGMP的产生是对内皮屏障的保护，与VS的发病时间无关。这些和其他数据表明，sGC介导的cGMP合成在细胞内发生了区域化，VS改变了cGMP信号对屏障功能的影响，可能是通过将sGC从胞浆移位到空泡膜域。本应用程序的目标是确定cGMP对VILI的影响，并特别关注cGMP的区划。在目标1中，sGC产生的cGMP对内皮屏障功能的影响将在分离的灌流小鼠肺标本中进一步表征。SGC激活对cGMP产生和内皮屏障功能障碍的剂量效应和特异性将被确定。此外，VS诱导的cGMP保护转换为伤害的时间将被确定。在目标2中，将通过共聚焦显微镜和亚细胞分离技术在完整的小鼠肺和周期性拉伸的内皮细胞中检测肺VS对内皮细胞sGC亚细胞区域化的影响。SGC-Caveolae相互作用的屏障效应将在分离、灌流的Caveolin-1/-小鼠肺中确定。VS诱导的sGC与磷酸二酯酶2(cGMP介导的内皮屏障功能障碍的关键效应因子)之间的相互作用将使用特定的抑制剂、免疫沉淀和cAMP测量方法来探索。相关性：感染或创伤引起的急性肺损伤会导致肺衰竭，因为血管内皮细胞允许液体泄漏到空气中。机械通气支持是至关重要的，但肺组织过度扩张会通过改变内皮细胞分子如cGMP而加重损伤。了解cGMP如何加重内皮细胞渗漏可能会导致更好的急性肺损伤治疗方法。