Cytoskeletal Regulation of Lung Endothelial Pathobiology

描述（由申请人提供）： 修订后的A1 PPG申请仍然是高度转化的PPG，重点关注内皮细胞（EC）细胞骨架在急性呼吸窘迫综合征（ARDS）和呼吸机诱导的肺损伤（VILI）病理生物学中的关键作用。ARDS是一种死亡率约为30- 35%的毁灭性疾病，其定义为肺血管通透性增加，这是ARDS死亡率的主要影响因素。我们提出的临床相关PPG研究将定义EC信号传导驱动细胞骨架重塑的分子机制，通过产生细胞旁间隙（增加肺血管通透性）破坏血管完整性，或通过外周细胞骨架重塑和形成细胞骨架驱动的板状伪足和促进EC间隙闭合的局灶性粘连恢复EC屏障完整性。这些生物物理事件的分析代表了本PPG的主题基础，将由一个杰出的翻译团队进行，该团队由天才和互动的基础和医生-科学家研究者组成，他们将利用临床相关的生物活性/生物物理刺激（VEGF、TNFα、LPS、 机械应力）。项目#1将在细胞旁间隙调节的背景下对多功能非肌肉肌球蛋白轻链激酶（nmMLCK）亚型进行复杂的结构、功能和遗传分析，并将其作为肺血管屏障恢复的靶点。由于EC屏障调节增强是由外周肌动蛋白重塑决定的，因此项目2将提供关于nmMLCK与关键肌动蛋白结合蛋白（corneum、Ena/VASP样或EVL、c-Abl）在EC屏障恢复反应（外周细胞骨架重塑、板状脂质动力学、间隙闭合）中相互作用的新信息。粘着斑蛋白是复杂的参与者，通过双向信号传导到细胞骨架，参与ARDS的病理生物学和缓解。项目#3将探讨FA蛋白、整合素β4和桩蛋白在细胞骨架连接至粘着斑动力学（组装/拆卸）和炎症诱导EC间隙的层状脂质介导闭合中的作用，以及单核苷酸多态性（SNP）和翻译后修饰（PTM）对FA结构/功能的影响。在四个高度互动的核心的支持下，我们的编程方法被编织成独特的PPG功能：i）测试设计用于减弱高度可药物化的肺EC渗透性途径的新型ARDS/VILI疗法（MLCK抑制剂、S1 P、HGF、整联蛋白β4抗体）和，ii）关键PPG EC屏障调节基因中ARDS相关SNP的询问，研究非常重要，因为非洲裔个体是ARDS生存率降低的高风险人群。通过利用亚利桑那大学杰出的科学环境以及与伊利诺伊大学科学家的长期合作，这种“模型翻译PPG”非常适合提供对肺血管屏障调节的全面机制理解，并促进治疗靶点的开发，以恢复受损肺循环的完整性。

DESCRIPTION (Provided by applicant): This revised A1 PPG application remains a highly translational PPG focused on the critical role of the endothelial cell (EC) cytoskeleton in the pathobiology of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). ARDS, a devastating disorder with a mortality of ~30-35%, is defined by increases in lung vascular permeability, a major influence on ARDS mortality. The clinically-relevant PPG studies we have proposed will define the molecular mechanisms by which EC signaling drives cytoskeletal remodeling to either disrupt vascular integrity via production of paracellular gaps (that increases lung vascular permeability) or to restore EC barrier integrity via peripheral cytoskeletal remodeling and formation of cytoskeletal-driven lamellipodia and focal adhesions that promote EC gap closure. The analysis of these biophysical events represent the thematic underpinnings of this PPG and will be conducted by an outstanding translational team of gifted and interactive basic and physician-scientist investigators who will utilize clinically relevant bioactive/biophysical stimuli (VEGF, TNFα, LPS, mechanical stress) both in vitro and in preclinical models of ARDS and VILI. Project #1 will perform sophisticated structure, function and genetic analyses of the multi-functional non-muscle myosin light chain kinase (nmMLCK) isoform in the context of paracellular gap regulation and as a target for lung vascular barrier restoration. As EC barrier-regulatory enhancement is determined by peripheral actin remodeling, Project #2 will provide novel information regarding the interactions between nmMLCK and key actin-binding proteins (cortactin, Ena/VASP-like or EVL, c-Abl) in EC barrierrestorative responses (peripheral cytoskeletal remodeling, lamellipodial dynamics, gap closure). Focal adhesion (FA) proteins are complex participants in both the pathobiology and resolution of ARDS via bidirectional signaling to the cytoskeleton. Project #3 will interrogate the role of the FA proteins, integrin β4 and paxillin, in cytoskeletal linkages to focal adhesion dynamics (assembly/disassembly) and lamellipodialmediated closure of inflammation-induced EC gaps as well as the influence of single nucleotide polymorphisms (SNPs) and post-translational modifications (PTMs) on FA structure /function. Supported by four highly interactive cores, our programmatic approaches are woven into unique PPG features: i) testing of novel ARDS/VILI therapies designed to attenuate the highly druggable lung EC permeability pathway (MLCK inhibitors, S1P, HGF, integrin β4 antibodies) and, ii) the interrogation of ARDS-associated SNPs in key PPG EC barrier-regulatory genes, studies of enormous importance as African descent individuals are a population at high risk for reduced survival in ARDS. By leveraging the outstanding scientific environment at the University of Arizona and long standing collaborations with University of Illinois scientists, this "model translational PPG" is exceptionally suited to provide comprehensive mechanistic understanding of lung vascular barrier regulation, and facilitate the development of therapeutic targets to restore the integrity of the injured pulmonary circulation.