Alveolar epithelial carbohydrate metabolism in acute lung injury

急性肺损伤(ALI)是一种以急性起病、严重缺氧和肺部感染为特征的炎症性肺部疾病。 肺水肿；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。肺泡上皮细胞系 肺泡表面和II型肺泡上皮细胞(AT II)以其对伤害性侮辱的敏感性而闻名 这可能会导致阿里。ALI的炎症和免疫反应与组织的剧烈变化有关 代谢，这可能是有害的，也可能是保护性的。一个关键的保护机制涉及增强 肺糖酵解，通过稳定转录因子低氧诱导因子(HIF)； 然而，具体的代谢过程，特别是涉及特定的酶糖酵解步骤的代谢过程(和 中间底物的增加或减少)在损伤开始时影响AT II细胞的反应 很大程度上是未知的。我们开发了一种新的概念来应对急性肺损伤 Phosphofructokinase-2/fructose-2，6-bisphosphatase(PFKFB3)及其转录调控因子HIF1a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 在ALI中，细胞内乳酸和丙酮酸的增加受到抑制，这是由HIF1a驱动的 肺泡II型细胞中PFKFB3的糖酵解和活化。利用这些与生俱来的保护途径 可为ALI新的治疗靶点提供依据。在这次重新提交中，我们将：1.确定是否 HIF1a是激活AT II细胞中PFKFB3所必需的(特异性靶点1)；2.确定是否激活 AT II细胞中的PFKFB3抑制ALI所致的炎症反应。我们将揭示PFKFB3的下游影响 激活，特别是糖酵解通量的增加，以及糖酵解最终产物是否 乳酸/丙酮酸抑制AT II细胞过度炎症(特异性靶点2a)；3.我们将针对肺泡- 上皮性PFKFB3在ALI中的治疗作用正如我们假设的那样，PFKFB3的激活将增强 由于AT II细胞的主要抗炎作用，糖酵解流量和减轻炎症(特定目标 2B)。为了辨别肺泡上皮的作用，我们利用原代肺泡II型细胞和组织特异性 了解动物。呼吸机诱导的肺损伤和酸吸入可作为ALI的小鼠模型。至 体外肺损伤模型我们将使用体外循环拉伸系统。药理和遗传学 HIF1a和PFKFB3在体外或ALI体内的功能作用将被用来研究。我们会 体外靶向AT II细胞，并提供基于糖酵解增强递送的机制数据 纳米颗粒包裹的激活剂2-6果糖二磷酸。我们将描述上皮糖酵解的特征 通量(代谢物、PFKFB3和LDH活性、NADH/NAD测定)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.