Epithelial glycocalyx degradation mediates surfactant dysfunction in acute respiratory distress syndrome

项目摘要/摘要 急性呼吸窘迫综合征(ARDS)是ICU呼吸衰竭的常见原因，其特征是 肺表面活性物质功能障碍和肺顺应性受损。我们的初步数据显示，肺泡 上皮糖萼是覆盖在上皮细胞顶端表面的一层糖胺多聚糖，受损。 在多种不同的ARDS小鼠模型中。此外，我们还发现，特定的酶降解 上皮性糖萼足以导致肺顺应性降低和微肺不张，这似乎是 受表面活性物质功能受损的影响。我们还将这些发现转化为人类使用非侵入性的 从热湿交换过滤器获得的收集的空域流体，这些过滤器是用于增湿的设备 作为机械通气患者日常护理的一部分。令人兴奋的是，我们证明了 肺泡上皮糖萼降解，通过空泡液中的糖胺多聚糖水平来衡量，是 预测机械通气时间和低氧血症程度。在这份提案中，我们将确定 上皮细胞糖基化降解导致表面活性物质功能障碍的机制和证实 这些发现在人类ARDS患者中使用非侵入性收集的翻译相关性 空域流体。在特定的目标1中，我们将鉴定与上皮糖催化剂结合的表面活性蛋白。 采用肝素亲和层析法对小鼠支气管肺泡灌洗液进行亲和层析。然后，我们将使用 用硫酸乙酰肝素糖链阵列鉴定与此有关的硫酸乙酰肝素低聚糖片段 互动。这种相互作用的功能重要性将在小鼠身上通过化学给药进行测试。 人工合成的表面活性蛋白结合寡糖(竞争性取代天然的表面活性物质 肺泡上皮细胞甘油三酯)，并使用基于设计的方法测量对肺结构和功能的影响 体视学、肺力学测量和限制性固着液滴表面测量术。在具体目标2中，我们 将进行一项试验性观察性临床研究，在该研究中，我们将收集热湿交换过滤器和 ARDS呼吸机的先进肺力学数据(峰值压力、平台压力、驾驶压力) 病人。然后，我们将使用这些数据来验证我们的假设，即糖胺聚糖的数量流入 空域，使用一种新的护理点检测方法(二甲基亚甲基蓝)进行测量，与肺呈负相关 急性呼吸窘迫综合征患者的依从性。鉴于目前还没有可用的临床测试来预测病程 对于ARDS，这些发现有可能迅速对ICU的患者护理产生影响。这项研究 将在高度合作的肺科学和重症监护医学司在 科罗拉多大学和丹佛健康医疗中心。我们预计该项目将生成以下数据 两者都将推进翻译ARDS研究领域，并成为我K08奖的基础 申请并最终过渡到独立调查员身份。

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.