Effects of stretch and shear stress on alveolar epithelial cell cytoskeleton

急性肺损伤患者经常接受正压机械通气，以改善气体交换。然而，在充满液体的肺泡的周期性关闭和再开放过程中，机械通气可能会产生很大的剪切力，而相对宽松的充气肺泡则周期性地过度扩张。这可能会导致或加重呼吸机所致的肺损伤，从而对急性肺损伤患者产生负面影响。在肺泡上皮细胞中，角蛋白中间丝是主要的结构蛋白。角蛋白IF在维持上皮细胞的机械完整性方面发挥着重要作用，在体外，它们能够在不改变其结构完整性的情况下承受广泛的应变条件。在体内对剪应力的反应中，IF已被证明经历了对剪应力的适应性变化。本应用旨在研究角蛋白IF对环状结构的响应 通过三个相互关联的特定目标来确定角蛋白IF的这些变化对肺泡上皮细胞功能的影响。具体目的#1.确定在野生型和角蛋白8基因敲除小鼠中，周期性拉伸和/或剪切应力是否会引起角蛋白IF的改变，从而改变肺泡液的重吸收。特定目的#2.确定周期性剪切应力和/或拉伸诱导的角蛋白IF的改变(拆解)是否由肺泡上皮细胞中蛋白激酶C依赖的磷酸化所介导。具体目的#3.确定周期性拉伸和/或剪切应力是否通过泛素-蛋白酶体途径引起肺泡上皮细胞泛素化的改变和角蛋白IF网络的调节降解，从而导致肺细胞损伤。这些研究的完成将为循环拉伸的机制提供新的见解--以及 剪切力致肺泡上皮损伤。

Patients with acute lung injury are often placed on positive-pressure mechanical ventilation to improve gas exchange. However, mechanical ventilation may generate large shear forces during the cyclic closure and reopening of fluid filled alveoli, while the relatively spared, air-filled alveoli are cyclically overdistended. This may cause or worsen ventilator induced lung injury which may have a negative impact in patients with acute lung injury. In alveolar epithelial cells keratin intermediate filaments (IF) are the major structural proteins. Keratin IF are known to play an important role in maintaining the mechanical integrity of epithelial cells, and in vitro they are able to withstand a wide range of strain conditions without alterations in their structural integrity. In response to shear stress in vivo, IF have been shown to undergo adaptive changes in response to shear stress. This application proposes to study the response of keratin IFs to cyclic stretch, and cyclic and continuous shear stress in alveolar epithelial cells, and determine the effect of these changes in keratin IF on alveolar epithelial function via three interrelated specific aims. Specific aim#1. To determine whether cyclic stretch and/or shear stress-induced changes in keratin IFs alter alveolar fluid reabsorption in wild-type and keratin 8 knockout mice. Specific aim #2. To determine whether cyclic shear stress and/or stretch-induced changes (disassembly) in keratin IFs are mediated by protein kinase C-dependent phosphorylation in alveolar epithelial cells. Specific aim #3. To determine whether cyclic stretch and/or shear stress causes changes in ubiquitination and the regulated degradation of keratin IF networks by the ubiquitin-proteasome pathway in alveolar epithelial cells leading to lung cell injury. Completion of these studies will provide new insights into mechanisms responsible for cyclic stretch-and shear stress-induced alveolar epithelial lung injury.