Epithelial glycocalyx degradation mediates surfactant dysfunction in acute respiratory distress syndrome

项目总结/摘要 急性呼吸窘迫综合征（ARDS）是ICU中呼吸衰竭的常见原因，其特征在于 表面活性物质功能障碍和肺顺应性受损。我们的初步数据表明， 上皮糖萼，一层覆盖在上皮细胞顶端表面的糖胺聚糖，被破坏 在多种不同的小鼠ARDS模型中。此外，我们发现，特定的酶降解的 上皮糖萼足以引起肺顺应性降低和微肺不张， 由受损的表面活性剂功能介导。我们还将这些发现应用于人类， 从热湿交换过滤器获得的收集的空气空间流体，所述热湿交换过滤器是用于加湿的装置， 作为机械通气患者常规护理的一部分。令人兴奋的是，我们证明了 肺泡上皮细胞糖萼降解，通过测量气液中糖胺聚糖的水平， 预测机械通气持续时间和低氧血症程度。在本提案中，我们将确定 上皮糖萼降解引起表面活性剂功能障碍的机制，并证实 这些发现在人类ARDS患者中的翻译相关性， 空气流动。在具体目标1中，我们将确定与上皮糖萼结合的表面活性蛋白 使用肝素亲和层析对小鼠支气管肺泡灌洗液进行。然后我们将使用一个 硫酸乙酰肝素糖阵列，以确定负责此的特定硫酸乙酰肝素寡糖片段 互动这种相互作用的功能重要性将在小鼠中进行测试， 合成的表面活性剂蛋白结合寡糖（以竞争性地取代来自天然的表面活性剂） 肺泡上皮糖萼），并使用基于设计的 体视学、肺力学测量和约束固着液滴表面测量。在具体目标2中， 我们将进行一项试点观察性临床研究，在该研究中，我们将收集热湿交换过滤器， 从ARDS患者的呼吸机获得的高级肺力学数据（峰值压、平台压、驱动压） 患者然后，我们将使用这些数据来检验我们的假设，即糖胺聚糖的量流入 使用一种新的即时检测方法（二甲基亚甲基蓝）测量的空域与肺呈负相关 ARDS患者的依从性。鉴于目前没有可用的临床测试可以预测过程 这些发现有可能对ICU中的患者护理产生迅速的影响。本研究 将在高度合作的肺科学和重症监护医学部进行， 科罗拉多大学和丹佛健康医学中心。我们预计，该项目将产生的数据， 我将推动转化型ARDS研究领域的发展，并为我的K 08奖奠定基础 申请并最终过渡到独立调查员地位。

PROJECT SUMMARY/ABSTRACT Acute respiratory distress syndrome (ARDS), a common cause of respiratory failure in the ICU, is characterized by surfactant dysfunction and impaired lung compliance. Our preliminary data demonstrate that the alveolar epithelial glycocalyx, a layer of glycosaminoglycans that coat the apical surface of the epithelial cells, is damaged in multiple different murine models of ARDS. Additionally, we found that specific enzymatic degradation of the epithelial glycocalyx is sufficient to cause decreased lung compliance and microatelectasis, which appears to be mediated by impaired surfactant function. We have also translated these findings to humans using noninvasively collected airspace fluid obtained from heat moisture exchange filters, which are devices that are used to humidify the airways as part of the usual care of mechanically ventilated patients. Excitingly, we demonstrated that alveolar epithelial glycocalyx degradation, measured by levels of glycosaminoglycans in the airspace fluid, is predictive of duration of mechanical ventilation and degree of hypoxemia. In this proposal, we will determine the mechanisms by which epithelial glycocalyx degradation causes surfactant dysfunction and confirm the translational relevance of these findings in human ARDS patients using noninvasively collected airspace fluid. In Specific Aim 1, we will identify the surfactant protein that binds to the epithelial glycocalyx using heparin affinity chromatography conducted on mouse bronchoalveolar lavage fluid. We will then utilize a heparan sulfate glycoarray to identify the specific heparan sulfate oligosaccharide fragment responsible for this interaction. The functional importance of this interaction will then be tested in mice by administering chemically synthesized surfactant protein-binding oligosaccharides (to competitively displace surfactant from the native alveolar epithelial glycocalyx) and measuring the effects on lung structure and function using design-based stereology, lung mechanics measurements, and constrained sessile drop surfactometry. In Specific Aim 2, we will conduct a pilot observational clinical study in which we will collect both heat moisture exchange filters and advanced lung mechanics data (peak pressure, plateau pressure, driving pressure) from the ventilators of ARDS patients. We will then use this data to test our hypothesis that the quantity of glycosaminoglycans shed into the airspace, measured using a novel point-of-care assay (dimethylmethylene blue), is inversely related to lung compliance in ARDS patients. Given that there are currently no available clinical tests that can predict the course of ARDS, these findings have the potential to become rapidly impactful to patient care in the ICU. This research will be carried out in the highly collaborative Division of Pulmonary Sciences and Critical Care Medicine at the University of Colorado and Denver Health Medical Center. We anticipate that this project will generate data that will both advance the field of translational ARDS research and serve as the foundation for my K08 award application and eventual transition to independent investigator status.