Effects of shear stress on regulation of epithelial permeability

描述(由申请人提供)： 这项K08申请的目标是促进基本技能的发展，使PI能够成为一名成功的院士并获得独立科学研究员的地位。呼吸道上皮起着屏障的作用，是呼吸道表面液体含量的调节器，也是调节呼吸道生理的细胞因子和其他产物的来源。除了周期性伸展和收缩，呼吸道上皮细胞还暴露在管腔切应力下，定义为单位表面积的摩擦力，由气流产生。在生理和病理条件下，呼吸道上皮细胞感受到的剪应力是不断变化的。我们有令人兴奋的新数据表明，气道上皮细胞中的剪切力导致气道上皮通透性的变化。因此，气道上皮屏障的动态调节可能是上皮细胞将管腔信息转化为上皮下反应的重要机制。我们的初步研究表明，切应力诱导TRPV4激活，随后L型电压门控通道激活，细胞内钙随后增加。呼吸道上皮细胞内钙的增加通过调节肌动蛋白重排和NO的产生导致上皮细胞旁通透性的协调变化，以及通过调节AQP5的丰度导致跨膜通透性的变化。我们建议研究切应力在改变细胞旁和跨膜通透性中的作用，并剖析调节这些影响的潜在机制。在SA#1中，我们将使用原代培养的人呼吸道上皮细胞(NHBE)和体外分离的小鼠气管来研究剪切力对呼吸道上皮通透性的影响。在SA#2中，我们将通过药物抑制和遗传操作来确定剪切诱导的钙通道在调节上皮细胞通透性中的作用和机制(TRPV4，L型通道)。在SA#3中，我们将研究剪切诱导的钙增加对NO产生的作用及其对通透性的影响。通过这些免费的技术和课程，PI将开发新的技能并生成关于机械剪切应力对呼吸道上皮细胞调节和功能的影响的新数据，这是一个很少被检查的区域，可能对正常的肺功能非常重要，以及剪切变化的条件，包括运动和哮喘。(摘要结束)

DESCRIPTION (provided by applicant): The goal of this K08 application is to facilitate development of essential skills that will allow the PI to become a successful academician and achieve independent scientific investigator status. The respiratory epithelium serves as a barrier, a regulator of the content of airway surface liquid, and a source of cytokines and other products that regulate airway physiology. In addition to cyclic extensions and contraction, airway epithelial cells are exposed to lumenal shear stress, defined as the frictional force per unit surface area, generated by airflow. The shear stress sensed by airway epithelia is continuously changing under both physiologic and pathologic conditions. We have exciting novel data suggesting that shear stress in airway epithelial cells leads to changes in airway epithelial permeability. Therefore, dynamic modulation of the airway epithelial barrier could be an important mechanism by which epithelial cells transduce lumenal information into subepithelial responses. Our preliminary studies indicate that shear stress induces TRPV4 activation with subsequent L-type voltage gated channel activation with subsequent increases in intracellular calcium. The increase in intracellular calcium in airway epithelial cells leads to concerted changes in epithelial paracellular permeability by modulating actin rearrangement and NO production, as well as in transmembrane permeability by modulating AQP5 abundance. We propose to study the role of shear stress in altering both paracellular and transmembrane permeability as well as dissect the underlying mechanisms regulating these effects. In SA#1we will investigate the effects of shear stress on airway epithelial permeability using primary cultured human airway epithelial cells (NHBE) and.ex vivo isolated mouse trachea. In SA#2 we will define the role for and the mechanisms (TRPV4, L-type channel) of shear-induced calcium flux in modulating epithelial permeability using pharmacologic inhibition and genetic manipulation. In SA#3 we will study the role od shear-induced increases in calcium on NO production and its effect on permeability. Through these complimentary techniques and coursework, the PI will develop new skills and generate novel data regarding the effects of mechanical shear stress on airway epithelial cell regulation and function, a little-examined area likely to be of immense importance to normal lung function, as well as conditions of altered shear including exercise and asthma. (End of Abstract)