Effects of stretch and shear stress on alveolar epithelial cell cytoskeleton

急性肺损伤患者常采用正压机械通气以改善气体交换。然而，机械通气可能在充满流体的肺泡的周期性闭合和重新打开期间产生大的剪切力，而相对空闲的、充满空气的肺泡周期性过度扩张。这可能导致或加重呼吸机诱导的肺损伤，这可能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（IF）是主要的结构蛋白。已知角蛋白IF在维持上皮细胞的机械完整性中起重要作用，并且在体外它们能够承受广泛的应变条件而不改变其结构完整性。在体内对剪切应力的响应中，IF已经被证明响应于剪切应力而经历适应性变化。本申请提出研究角蛋白IFs对环状寡核苷酸的反应。 拉伸，和循环和连续的切应力在肺泡上皮细胞，并确定这些角蛋白IF的变化对肺泡上皮功能的影响，通过三个相互关联的具体目标。具体目标#1。确定周期性拉伸和/或剪切应力诱导的角蛋白IF变化是否改变野生型和角蛋白8基因敲除小鼠的肺泡液体重吸收。具体目标#2为了确定是否周期性剪切应力和/或拉伸诱导的角蛋白IF的变化（拆卸）介导的蛋白激酶C依赖的磷酸化在肺泡上皮细胞。具体目标#3确定周期性拉伸和/或剪切应力是否引起肺泡上皮细胞中泛素化的变化以及通过泛素-蛋白酶体途径调节的角蛋白IF网络降解，从而导致肺细胞损伤。这些研究的完成将为周期性拉伸的机制提供新的见解， 切应力诱导的肺泡上皮肺损伤。

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.