Mechanics and Deformation Responses of Lung Cells

描述(由申请人提供)：急性肺损伤(ALI)和成人呼吸窘迫综合征(ARDS)每年在北美夺走约75,000人的生命。虽然机械通气是一种挽救生命的治疗方法，但它可能会在肺部特别容易因变形压力而受伤的时候造成伤害。不像可以铸造的骨折，一个人不能固定受伤的肺。因此，即使是所谓的肺保护性呼吸机设置也不能使他们免受进一步的损害。在过去的三年里，我们研究了细胞应激失败的决定因素，作为肺部对变形应激的先天免疫反应的核心驱动因素。我们已经证明，暴露在大容量机械通气和跨肺压力下的肺细胞会经历可逆的质膜损伤，这种损伤可以触发促炎信号级联反应。在这个相互竞争的更新应用中，我们将识别和测试旨在保护肺泡上皮细胞免受变形损伤或促进伤口修复的治疗方法，并确定它们的作用机制。在令人信服的初步数据的指导下，我们将把重点放在渗透压作为肺保护干预的努力上，并在呼吸机诱导的肺损伤的实验模型中测试浓盐和糖溶液的效果。这些实验有三个具体目的：1)检测渗透压对肺细胞变形损伤敏感性和细胞修复的影响；2)检测渗透压对I型和II型肺泡上皮细胞(AEC)体积、表面积和质膜张力调节的影响；3)研究嘌呤能信号在渗透应激介导的肺泡上皮修复中的重要性。实验将在麻醉的机械通风啮齿动物身上进行，在隔离的灌流肺中进行，并在组织培养中会损伤和/或变形的细胞上进行。研究终点包括通过活标本显微镜评估的受伤和/或修复细胞的数量，肺部炎症的测量，以及肺和细胞结构和功能的测量。我们将使用光学陷阱来表征AEC中质膜绳索的长度张力关系，并将这些数据解释为膜面积和张力调节以及渗透压力对它们的影响的生物物理读数。我们将获得的知识将提供有关疾病机制以及进行临床试验的风险和好处的信息。与公共卫生相关。我们的团队研究了细胞应激失败的决定因素，作为肺部对变形应激的先天免疫反应的核心驱动因素。在初步实验中，我们已经确定渗透应激是机械通气的潜在细胞保护辅助因素。该提案旨在确定这种干预在呼吸机诱导的肺损伤的临床前模型中的有效性，并剖析其作用机制。

DESCRIPTION (provided by applicant): Acute lung injury (ALI) and the Adult Respiratory Distress Syndrome (ARDS) claim approximately 75,000 lives in North America each year. While mechanical ventilation is a life saving treatment, it may nevertheless cause harm by straining lungs at a time they are particularly prone to injury from deforming stress. Unlike a broken bone which can be casted, one cannot immobilize injured lungs. Therefore, even so-called lung protective ventilator settings may not spare them from further damage. For the past three years we have studied the determinants of cellular stress failure as a central driver of the lungs' innate immune response to deforming stress. We have shown that cells of lungs, which are exposed to mechanical ventilation with high volumes and trans-pulmonary pressure, experience reversible plasma membrane wounds and that such wounds can trigger proinflammatory signaling cascades. In this competing renewal application we will identify and test therapies designed to protect alveolar epithelial cells from deformation injury or to promote wound repair and we will define their mechanisms of action. Guided by compelling preliminary data we will focus our efforts on osmotic stress as lung-protective intervention and test the efficacy of concentrated salt and sugar solutions in experimental models of ventilator induced lung injury. The proposed experiments will serve three specific aims: 1) To test the effect of osmotic stress on the susceptibility of lung cells to deformation injury and on the probability of cell repair; 2) To examine the effect of osmotic stress on the regulation of volume, surface area and plasma membrane tension in type I and type II alveolar epithelial cells (AEC); 3) To examine the importance of purinergic signaling on osmotic stress mediated alveolar epithelial repair. Experiments will be carried out on anesthetized mechanically ventilated rodents, in isolated perfuse lungs and on cells that will be injured and/or deformed in tissue culture. Study endpoints include the number of wounded and/or repaired cells as assessed by live specimen microscopy, measures of lung inflammation, as well as measures of lung and cell structure and function. We will use optical traps to characterize the length tension relationships of plasma membrane tethers in AEC's and interpret the data as a biophysical readout of membrane area and tension regulation and the effects of osmotic stress on them. The knowledge we will gain, will inform about disease mechanisms and the risks and benefits of conducting a clinical trial. PUBLIC HEALTH RELEVANCE. Our group investigates the determinants of cellular stress failure as a central driver of the lungs' innate immune response to deforming stress. In preliminary experiments we have identified osmotic stress as potential cytoprotective adjunct to mechanical ventilation. The proposal seeks to establish the efficacy of this intervention in preclinical models of ventilator induced lung injury and to dissect its mechanisms of action.