Alveolar epithelial carbohydrate metabolism in acute lung injury

急性肺损伤（acute lung injury，ALI）是一种以急性发作、严重缺氧和急性呼吸道损伤为特征的炎症性肺部疾病。 肺水肿;其在患者中表现为急性呼吸窘迫综合征（ARDS）。在 目前，ARDS的具体治疗方法基本上是未知的。肺泡上皮细胞排列在 肺泡表面和II型肺泡上皮细胞（AT II）对损伤性损伤敏感 会导致急性肺损伤ALI中的炎症和免疫反应与组织的急剧变化相关 代谢，这可能是有害的或保护。一个关键的保护机制包括增强 通过稳定转录因子缺氧诱导因子（HIF）介导的肺糖酵解; 然而，特定的代谢过程，特别是涉及特定酶促糖酵解步骤的代谢过程（以及 中间底物的增加或减少）影响AT II细胞在损伤发作期间的反应， 大部分未知。我们提出了一个新的概念， 磷酸果糖激酶-2/果糖-2，6-二磷酸酶（PFKFB 3）及其转录调节因子HIF 1A是 介导AT II细胞的抗炎活性。我们假设过度的炎症和细胞损伤 细胞内乳酸和丙酮酸的增加，这是由HIF 1A驱动的 糖酵解和肺泡II型细胞中PFKFB 3的活化。利用这些天生的保护途径 可能为ALI的新治疗靶点提供基础。在这次重新提交，我们将：1.确定是否 HIF 1A是AT II细胞中PFKFB 3激活所必需的（具体目标1）; 2.确定是否激活 AT II细胞中的PFKFB 3减轻了由于ALI引起的炎症。我们将揭示PFKFB 3的下游效应 激活，特别是糖酵解通量的增加，以及糖酵解终产物 乳酸盐/丙酮酸盐淬灭AT II细胞中的过度炎症（特定目的2a）; 3.我们要瞄准肺泡- 正如我们假设的那样，上皮PFKFB 3在ALI期间的治疗作用，PFKFB 3的激活将增强 由于AT II细胞中的主要抗炎作用，糖酵解通量和减轻炎症（具体目的 2b）。为了辨别肺泡上皮细胞的作用，我们利用原代肺泡II型细胞和组织特异性细胞培养。 了解动物。呼吸机诱导的肺损伤和酸吸入将用作ALI的小鼠模型。到 离体肺损伤模型，我们将利用体外循环拉伸系统。药理和遗传学 方法将用于研究HIF 1A和PFKFB 3在体外或ALI在体内的功能作用。我们将 体外靶向AT II细胞，并提供基于糖酵解的增强递送的机制数据 活化剂2-6果糖二磷酸被纳米颗粒包裹。我们将描述上皮糖酵解 通量（代谢物、PFKFB 3和LDH活性、NADH/NAD测量）和炎症反应。

Acute lung injury (ALI) is an inflammatory lung disease characterized by its acute onset, severe hypoxia and pulmonary edema; which manifests itself in patients as acute respiratory distress syndrome (ARDS). At present, specific therapeutic approaches for ARDS are essentially unknown. Alveolar epithelial cells line the alveolar surface and type II alveolar epithelial cells (AT II) are known for their susceptibility to injurious insults that can lead to ALI. Inflammatory and immune responses in ALI are associated with dramatic shifts in tissue metabolism, which can either injurious or protective. A key protective mechanism involves enhanced pulmonary glycolysis, mediated through stabilization of the transcription factor hypoxia-inducible factor (HIF); however, how specific metabolic processes, notably those involving specific enzymatic glycolytic steps (and increase or decreases in intermediate substrates) affect the response of AT II cells during injury onset are largely unknown. We have developed a novel concept that in response to acute lung injury phosphofructokinase-2/fructose-2,6-bisphosphatase (PFKFB3 ) and its transcriptional regulator HIF1A are mediating anti-inflammatory activities of AT II cells. We hypothesize that excessive inflammation and cell injury in ALI are dampened by increases in intracellular lactate and pyruvate, which are induced by HIF1A-driven glycolysis and activation of PFKFB3 in alveolar type II cells. Harnessing those innate protective pathways could provide the base for novel therapeutic targets for ALI. In this resubmission we will: 1. Determine whether HIF1A is required for PFKFB3 activation in AT II cells (Specific Aim 1); 2. Determine whether the activation of PFKFB3 in AT II cells dampens inflammation due to ALI. We will uncover downstream effects of PFKFB3 activation, specifically the augmentation of glycolytic flux, and whether the glycolytic end products lactate/pyruvate quench excessive inflammation in AT II cells (Specific Aims 2a); 3. We will target alveolar- epithelial PFKFB3 therapeutically during ALI as we hypothesize, that activation of PFKFB3 will enhance the glycolytic flux and attenuate inflammation due to a dominant anti-inflammatory effect in AT II cells (Specific Aim 2b). To discern the role of the alveolar epithelium we utilize primary alveolar type II cells and tissue specific know out animals. Ventilator induced lung injury and acid aspiration will be used as murine models of ALI. To model lung injury ex vivo we will utilize an in vitro cyclic stretch system. Pharmacologic and genetic approaches will be utilized to study the functional role of HIF1A and PFKFB3 in vitro or ALI in vivo. We will target the AT II cells ex vivo and provide mechanistic data based on enhanced delivery of the glycolysis activator 2-6 fructose bisphosphate encapsulated by nanoparticles. We will characterize the epithelial glycolytic flux (metabolites, PFKFB3 and LDH activity, NADH/NAD measurement) and inflammatory response.