Mechanistic role of membrane pore formation in lung-endothelial barrier failure due to blood-borne pathogens.

项目摘要 意义总体目标是确定急性呼吸窘迫的机制和治疗方法 急性呼吸窘迫综合征（ARDS）是由条件致病菌铜绿假单胞菌（PA）的脓毒性菌血症引起的。 这些机制仍然不明确。我们的前提是，血源性PA通过快速地 内化在肺内皮细胞中。因此，存在Gasdermin D机制的激活， 膜孔形成Ca 2+通过孔进入内皮细胞胞质溶胶，使f-肌动蛋白不稳定， 从而引起屏障失效--ARDS的主要原因。 Approach.我们将通过实时共聚焦显微镜来评估两个特定目标的前提 (RCM)活鼠肺以及其他常规方法。在SA 1中，我们将确定LPS的作用 肺内皮的转染，PA相关LPS内化的模型。在SA 2中，我们将确定 通过静脉PA注射建模的菌血症的肺内皮效应，以及通过 腹膜内PA感染。将在遗传操作的小鼠中评价前提，以评价（i） gasdermin D和膜孔形成的其他假设;（ii）ESCRT-1介导的孔修复机制。 III系统;（iii）孔诱导的Ca 2+和f-肌动蛋白机制在屏障失效中的作用;（iv）PA的影响 在肺内皮细胞的内化;和（v）内皮肌动蛋白增强细胞的功效- 渗透性蛋白是治疗PA腹膜炎所致ARDS的有效药物。 冲击我们的研究将首次揭示肺内皮孔形成作为一种 内皮屏障功能衰竭是脓毒症诱导的肺外呼吸窘迫综合征的基础， 感染PA。PA的内皮内化将被理解为这一过程中的关键机制。 病理将首次揭示内皮孔形成的动力学和机制。 针对内皮孔形成（因此屏障失效）的分子策略将被评价为 治疗由于PA败血症引起的ARDS。将在机制和 治疗由于PA诱导的孔隙形成引起的脓毒症诱导的ARDS。

PROJECT SUMMARY Significance. The overall goals are to define mechanisms and therapies for the acute respiratory distress syndrome (ARDS) due to septic bacteremia of the opportunistic pathogen, Pseudomonas aeruginosa (PA). These mechanisms remain undefined. Our premise is that blood-borne PA cause ARDS by rapidly internalizing in the lung endothelium. Consequently, there is activation of the gasdermin D mechanism of membrane pore formation. Ca2+ enters the endothelial cytosol through the pores, destabilizing f-actin, and thereby inducing barrier failure – the major cause of ARDS. Approach. We will evaluate the premise in two Specific Aims by means of real-time confocal microscopy (RCM) of live mouse lungs, as well as other general approaches. In SA1, we will determine the effects of LPS transfection of the lung endothelium, a model of PA-associated LPS internalization. In SA2, we will determine lung-endothelial effects of bacteremia, modeled by intravenous PA injection, and extra-pulmonary sepsis by intraperitoneal PA infection. The premise will be evaluated in genetically manipulated mice to evaluate (i) the gasdermin D and other hypotheses of membrane pore formation; (ii) mechanisms of pore repair by the ESCRT- III system; (iii) the role of pore-induced Ca2+ and f-actin mechanisms in barrier failure; (iv) effects of PA internalization in the lung endothelium; and (v) the efficacy of endothelial actin enhancement by cell- permeable proteins as effective therapy against ARDS due to PA peritonitis induced. Impact. Our studies will for the first time, reveal the importance of lung-endothelial pore formation as a mechanism of the endothelial barrier failure that underlies sepsis-induced ARDS due to extra-pulmonary infection by PA. The endothelial internalization of PA will be understood as the critical mechanism in this pathology. The dynamics and mechanisms of endothelial pore formation will be revealed for the first time. Molecular strategies directed against endothelial pore formation, therefore barrier failure, will be evaluated as therapies for ARDS due to PA sepsis. Outstandingly novel understanding will be achieved in mechanisms and therapies of sepsis-induced ARDS due to PA-induced pore formation.