Protein Regulation and Cytoskeletal Dynamics of Pulmonary Endothelial Barrier Function

摘要 肺内皮屏障功能的丧失是导致富含蛋白质的液体从肺组织中渗漏的原因。 血管间隙进入通常充满空气的肺泡，导致低氧血症、呼吸衰竭和 急性呼吸窘迫综合征（ARDS）患者的发病率和死亡率显著增加。一个主要 这种屏障功能的决定因素是细胞骨架重排和力的产生， 形状和膜动力学导致细胞内间隙形成。该提案将从机制上 表征3种关键细胞骨架蛋白的功能作用，这些蛋白整合在一起决定肺 内皮细胞（EC）通透性：非肌肉肌球蛋白轻链激酶（MLCK）， 肌动蛋白-肌球蛋白棘轮效应中的细胞骨架重排和力的产生 结合蛋白（CTTN），一种多功能的适配器和支架蛋白， 许多动态的细胞骨架过程，并已知与MLCK在以下区域形成稳定的关联： 外周肌动蛋白聚合和重排反应屏障保护性刺激;和肌动蛋白 相关蛋白复合物阿普2/3，肌动蛋白聚合和分支的关键效应，在这些外周 已知与coronin相互作用的位点。这些蛋白质的具体作用及其相互作用， 一个复杂的细胞骨架动力学尚未完全确定。此外， 细胞骨架动力学对膜动力学和最终屏障完整性的影响在 特别是在肺血管系统中。我们假设CTTN、MLCK和Arp 2/3形成 整合的复合物调节肺EC细胞骨架结构和膜动力学， 确定屏障功能。这项建议将使用互补的和复杂的分子，生物化学， 和成像技术来表征这些蛋白质的作用和它们在调节肌动蛋白中的相互作用， 结构具体目标1将表征MLCK、CTTN和阿普2/3在生成 在屏障增强条件下调节肺EC渗透性的关键肌动蛋白结构，或 破坏具体目标2将探讨这些结构对膜的功能后果 动力学和屏障完整性，采用新的MLCK构建体，在coronin的推定位点突变， 结合以及蛋白质抑制剂和沉默RNA。Specific Aim 3将使用这些新试剂来 通过表征MLCK的综合功能效应，将我们的观察转化为临床前模型， CTTN和Arp 2/3在LPS诱导的急性肺损伤（ALI）小鼠模型中的作用。这些研究将提供新的 机械见解和对ALI/ARDS细胞机制的进一步理解，这将有助于 开发新的治疗靶点，以逆转或减轻这些破坏性病理性疾病的影响， 对患者造成的影响。

ABSTRACT Loss of pulmonary endothelial barrier function is responsible for the leakage of protein rich fluid from the vascular space into the normally air filled alveoli which leads to hypoxemia, respiratory failure and significant morbidity and mortality in patients with acute respiratory distress syndrome (ARDS). A major determinant of this barrier function is cytoskeletal rearrangement and force generation which in turn alters cell shape and membrane dynamics leading to intracellular gap formation. This proposal will mechanistically characterize the functional roles of 3 critical cytoskeletal proteins that integrate to determine lung endothelial cell (EC) permeability: non-muscle myosin light chain kinase (MLCK), an effector of cytoskeletal rearrangement and force generation through its catalytic action in the ratcheting of actin-myosin bonds; cortactin (CTTN), a multifunctional adapter and scaffolding protein which serves a regulatory role in many dynamic cytoskeletal processes and is known to form a stable association with MLCK in areas of peripheral actin polymerization and rearrangement in response to barrier protective stimuli; and the actin related protein complex Arp 2/3, a key effector of actin polymerization and branching at these peripheral sites that is known to interact with cortactin. The specific roles of these proteins and their interactions as part of a complex in cytoskeletal dynamics are not yet fully characterized. Furthermore the consequence of cytoskeletal dynamics on membrane kinetics and ultimately barrier integrity remains ill-defined in the endothelium particularly in the lung vasculature. We hypothesize that CTTN, MLCK and Arp2/3 form integrated complexes which regulate pulmonary EC cytoskeletal structure and membrane dynamics to determine barrier function. This proposal will use complementary and sophisticated molecular, biochemical, and imaging techniques to characterize the roles of these proteins and their interactions in regulating actin structure. Specific Aim 1 will characterize the integrated roles of MLCK, CTTN and Arp 2/3 in generating critical actin structures that regulate permeability in lung EC under conditions of barrier enhancement or disruption. Specific Aim 2 will explore the functional consequences of these structures on membrane dynamics and barrier integrity, employing novel MLCK constructs, mutated at the putative site of cortactin binding as well as protein inhibitors and silencing RNA. Specific Aim 3 will use these novel reagents to translate our observations to a pre-clinical model by characterizing the integrated functional effects of MLCK, CTTN, and Arp2/3 in a murine model of LPS-induced acute lung injury (ALI). These studies will provide novel mechanistic insights and an improved understanding of the cellular mechanisms of ALI/ARDS which will aid in the development of new therapeutic targets to reverse or lessen the impact of these devastating pathologic conditions on patients who suffer from them.