Stretch-Induced Changes in Endothelial Contractility

描述(由申请人提供)：呼吸机诱导的肺损伤(VILI)是机械通风引起的过度肺细胞拉伸引起的发病率和死亡率的常见原因。高通透性肺水肿是VILI的主要特征，但其发病机制尚不清楚。这些变化可能涉及对内皮细胞(EC)的直接损伤或“应激失败”，以及涉及EC信号通路的微妙事件，EC信号通路由基于肌动蛋白的系统调节，影响细胞收缩和应激纤维形成。我们假设，暴露于过度循环拉伸的内皮细胞会发生表型变化，导致收缩蛋白表达增加，从而增强激动剂驱动的收缩和通透性。利用体外模型，人肺血管内皮细胞(HPAEC)和人微血管内皮细胞(HMEC)将暴露在循环拉伸(5%和18%径向伸长)中。在特定的目标#1中，我们将描述拉伸对宏观和微观肺内皮细胞的时间和应变依赖效应。在特定的目标#2，使用Affymetrix基因芯片，我们将确定改变收缩蛋白基因表达模式的周期拉伸修改。EC收缩通常受EC肌球蛋白轻链激酶(MLCK)的调节，MLCK是由赞助商的实验室首次克隆的一种酶，它驱动肌球蛋白轻链磷酸化，肌球蛋白轻链磷酸化是肌球蛋白收缩的前体。在具体目标#3中，将通过测量MLCK活性和阐述药物抑制剂的作用以及MLCK突变结构的过度表达来研究MLCK在拉伸后EC屏障功能障碍中的作用。这些研究确定了EC对周期拉伸的生化和基因组反应，可能会导致针对VILI的新治疗策略。

DESCRIPTION (provided by applicant): Ventilator-induced lung injury (VILI) is a common cause of the morbidity and mortality associated with excessive lung cell stretch produced by mechanical ventilation. High permeability pulmonary edema is a cardinal feature of VILI, however, the mechanisms involved are unclear. These changes likely involve direct damage to endothelial cells (EC) or "stress failure", as well as subtle events involving EC signaling pathways which modulate by an actin-based system that effects cell contraction and stress fiber formation. We hypothesize that endothelial cells exposed to excessive cyclic stretch undergo phenotypic changes that result in increased contractile protein expression resulting in enhanced agonist-driven contractility and permeability. Utilizing an in vitro model, human pulmonary artery endothelial cells (HPAEC) and human microvascular endothelial cells (HMEC) will be exposed to cyclic stretch (5% and 18% radial elongation). In specific aim #1, we will characterize the time- and strain-dependent effect of stretch on macro and micro lung EC. In specific aim #2, using Affymetrix cDNA microarrays, we will determine altered contractile protein gene expression patterns modified by cyclic stretch. EC contraction is often regulated by EC myosin light chain kinase (MLCK), an enzyme first cloned by the sponsor's laboratory that drives myosin light chain phosphorylation, a precursor of actomyosin contraction. In specific aim #3, the role of MLCK in EC barrier dysfunction after stretch will be investigated with measures of MLCK activity and elaboration of the effects of pharmacologic inhibitors as well as and overexpression of MLCK mutant constructs. These studies, which define EC biochemical and genomic response to cyclic stretch, may result in new therapeutic strategies that target VILI.