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

项目总结 意义重大。总体目标是确定急性呼吸窘迫的机制和治疗方法。 由条件致病菌铜绿假单胞菌(PA)的败血症引起的综合征(ARDS)。 这些机制仍未明确。我们的前提是血液传播的PA迅速引起ARDS 内化于肺内皮细胞。因此，存在Gasdermin D机制的激活 膜孔形成。CA2通过毛孔进入内皮细胞溶质，破坏f-肌动蛋白的稳定，并 从而导致屏障失效--这是ARDS的主要原因。 接近。我们将通过实时共聚焦显微镜在两个特定目标上对前提进行评估 (RCM)活体小鼠肺，以及其他一般方法。在SA1中，我们将确定内毒素的影响 肺内皮细胞转染法，PA相关的内毒素内化模型。在SA2中，我们将确定 静脉注射PA模拟菌血症和肺外脓毒症的肺内皮细胞效应 腹膜内PA感染。这一前提将在基因操纵的小鼠中进行评估，以评估(I) Gasdermin D和其他膜孔形成假说；(Ii)ESCRT修复孔的机制- III系统；(Iii)孔洞诱导的钙和f-肌动蛋白机制在屏障失效中的作用；(Iv)PA的作用 肺内皮细胞的内化；以及(V)细胞-肌动蛋白增强内皮细胞肌动蛋白的功效。 通透性蛋白是治疗PA腹膜炎所致ARDS的有效方法。 冲击力。我们的研究将首次揭示肺内皮细胞毛孔形成作为一种 脓毒症致肺外ARDS的内皮屏障功能障碍机制 金黄色葡萄球菌感染。PA的内皮内化将被理解为这一过程中的关键机制 病理学。将首次揭示内皮孔形成的动力学和机制。 针对内皮细胞孔形成的分子策略，因此屏障破坏，将被评估为 PA败血症所致ARDS的治疗。将在机制和机制方面实现卓越的新奇理解 PA诱导的气孔形成引起的脓毒症ARDS的治疗。

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.