Mechanical Injury of the Alveolar Epithelium

描述（由申请人提供）：我们研究的广泛长期目标是降低呼吸机诱导的肺损伤（VILI）的发生率，其特征为气胸、肺力学进行性损害、肺泡细胞功能障碍以及肺液体平衡和血气屏障通透性的深刻变化。在之前的资助期间，我们确定了周期性和紧张性变形幅度，在此之上，我们测量了上皮细胞活力和屏障特性的急性改变。此外，我们在体外应用的损伤变形幅度与临床上使用的潮气量和呼气末正压相关，这些潮气量和呼气末正压与呼吸机诱导的肺损伤（VILI）的临床发病率相关。这些发现强调了使用实验模型与仔细控制条件的潜在相关性，以确定负责拉伸诱导的肺泡上皮屏障破坏的机制，并确定损伤干预的机会。我们在最初6年的资助中建立的VILI体外模型仅限于暴露于1小时变形期的健康大鼠肺泡上皮细胞单层。在这项竞争性更新中，我们的主要目标是确定在临床相关条件下（包括慢性连续循环和ARDS）调节肺泡上皮通透性的特定牵张诱导的机械和分子信号。在目标1中，我们假设败血症、高循环速率和长拉伸持续时间等环境压力源均会损害原代细胞单层和完整肺部的上皮单层屏障特性。在目的2中，我们假设牵张通过激活丝裂原活化蛋白激酶诱导屏障改变，丝裂原活化蛋白激酶启动核因子κ B依赖性细胞因子表达和释放，最终增加单层通透性。在目标3中，我们将测试急性（<1小时）拉伸重组细胞骨架的假设，这直接通过从肌动蛋白中解离紧密连接（TJ）蛋白来增加单层通透性，间接通过启动激酶介导的TJ磷酸化和TJ复合物分解。因此，我们建立在以前的资助期间建立的基础上-既加深了我们对基本机制的理解，又加强了我们研究结果的临床转化-并朝着我们减少由于上皮过度扩张引起的急性VILI的目标取得进展。

DESCRIPTION (provided by applicant): The broad long-term objective of our research is to reduce the incidence of ventilator-induced lung injury (VILI), characterized by pneumothorax, progressive impairment in pulmonary mechanics, alveolar cell dysfunction, and profound changes in lung fluid balance and blood-gas barrier permeability. In the previous funding periods we identified cyclic and tonic deformation magnitudes above which we measured acute alterations in epithelial cell viability and barrier properties. Moreover, the injurious deformation magnitudes we applied in vitro correlated well with tidal volumes and positive end-expiratory pressures used clinically that have been associated with clinical morbidity in ventilator induced lung injury (VILI). These findings underscore the potential relevance of using experimental models with carefully controlled conditions to identify the mechanisms responsible for stretch-induced disruption of the alveolar epithelial barrier, and to identify opportunities for injury intervention. The in vitro model for VILI that we established during the first 6 years of funding is limited to healthy rat alveolar epithelial cell monolayers exposed to 1 hr periods of deformation. In this competitive renewal our major objective is to determine specific stretch-induced mechanical and molecular signals that modulate alveolar epithelial permeability during clinically relevant conditions - including chronic continuous cycling and ARDS. In Aim 1 we hypothesize that the environmental stressors of sepsis, high cycling rates and long stretch durations each compromise epithelial monolayer barrier properties in both primary cell monolayers and intact lungs. In Aim 2 we hypothesize that stretch induces barrier alterations by activation of mitogen- activated protein kinases, which initiate nuclear factor kappa B dependent cytokine expression and release to ultimately increase monolayer permeability. In Aim 3 we will test the hypotheses that acute (<1 hr) stretch reorganizes the cytoskeleton, which increases monolayer permeability directly by dissociating tight junctional (TJ) proteins from actin, and indirectly by initiating kinase-mediated TJ phosphorylation and TJ complex disassembly. Thus, we build on the foundation established during previous funding periods - both to deepen our understanding of basic mechanisms and to enhance the clinical translation of our findings - and progress towards our goal of reducing acute VILI due to epithelial over-distension.