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.

K08HL130586

2018-01-01

2022-03-15