Mechanical Injury of the Alveolar Epithelium

描述(申请人提供)：我们研究的广泛的长期目标是减少呼吸机诱导的肺损伤(VILI)的发生率，特征是气胸、进行性肺力学损害、肺泡细胞功能障碍以及肺液平衡和血-气屏障通透性的深刻变化。在之前的资助时期，我们确定了周期性和紧张性变形的大小，超过这个数值，我们测量了上皮细胞活性和屏障属性的急性变化。此外，我们在体外应用的损伤变形程度与临床使用的潮气量和呼气末正压有很好的相关性，这与呼吸机相关性肺损伤(VILI)的临床发病率有关。这些发现强调了使用仔细控制条件的实验模型来确定牵张诱导的肺泡上皮屏障破坏的机制，以及确定损伤干预的机会的潜在相关性。我们在资助的前6年建立的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。在这次竞争性更新中，我们的主要目标是确定特定的牵张诱导的机械和分子信号，这些信号在临床相关条件下调节肺泡上皮通透性，包括慢性持续循环和ARDS。在目标1中，我们假设败血症、高循环率和长拉伸持续时间的环境应激源都损害了原始细胞单层和完整肺的上皮单层屏障特性。在目标2中，我们假设拉伸通过激活丝裂原激活的蛋白激酶来诱导屏障改变，丝裂原激活的蛋白激酶启动依赖于核因子kappaB的细胞因子的表达和释放，最终增加单层通透性。在目标3中，我们将测试以下假设：急性(1小时)拉伸可重组细胞骨架，这直接通过将紧密连接(TJ)蛋白从肌动蛋白中分离出来而增加单层通透性，并通过启动激酶介导的TJ磷酸化和TJ复合体的分解来间接增加单层通透性。因此，我们在前几个资助期建立的基础上--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翻译--并朝着减少由于上皮过度扩张而导致的急性VILI的目标取得了进展。

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.