Metabolic Regulation of Endothelial Barrier Restoration Following Vascular Injury

项目摘要 内皮屏障功能障碍是急性呼吸窘迫综合征发病机制的中心因素 急性呼吸窘迫综合征（ARDS）和急性肺损伤（ALI）。近年来，在这方面取得了相当大的进展。 了解细胞内信号通路如何调节粘附分子的破坏和组装 连接（AJs）。AJs的再退火是一个代谢活跃的过程。然而，人们对它的作用知之甚少。 内皮代谢作为内皮屏障功能的调节剂和肺血管损伤的恢复。我们 支持性数据表明，内皮细胞对炎症激活的反应是上调内皮细胞的表达。 通过低氧诱导因子HIF 1 α及其关键下游代谢靶点PFK-FB 3， 糖酵解调节酶。这种糖酵解转变伴随着伴随的上调， 线粒体谷氨酰胺代谢，其补偿线粒体葡萄糖氧化的损失， 使细胞能够使用谷氨酰胺作为替代线粒体TCA循环燃料。我们观察到抑制PFK- FB 3阻止肺损伤后内皮屏障功能的恢复，从而强调了适应性作用 PFK-FB 3和增加糖酵解在内皮屏障恢复。基于这些发现，我们 肺微血管内皮细胞中糖酵解的诱导作为一种体内平衡机制， 恢复内皮屏障功能和肺液体平衡。在项目2中，我们将开展以下工作： 具体目的：（1a）我们将明确PFK-FB 3介导的糖酵解激活机制， 炎症和内皮细胞介导的肺内皮细胞代偿性β-氨基丁酸溶解 损伤，并确定这些代谢变化在修复内皮屏障中的必要作用;（1b）我们 将确定PFK-FB 3介导的糖酵解激活在再退火中的时空作用。 AJs和恢复内皮屏障完整性，（2）我们将确定内皮细胞的作用， 通过PFK-FB 3进行代谢重编程以恢复肺内皮屏障完整性和液体平衡 急性肺损伤模型中的炎症性肺损伤。利用最先进的代谢分析， 蛋白质结构和生物传感器以及新的遗传小鼠模型，我们将定义代谢 肺内皮炎性损伤激活的机制及其在肺修复中的作用 内皮屏障我们的长期目标是确定代谢靶点和开关， 加速内皮屏障的恢复和使肺液体平衡正常化以减轻急性肺损伤。

Project Summary Endothelial barrier dysfunction is a central factor in the pathogenesis of Acute Respiratory Distress Syndrome (ARDS) and Acute Lung Injury (ALI). In recent years, considerable advances have been made in the understanding of how intracellular signaling pathways modulate the disruption and assembly of adherens junctions (AJs). Re-annealing of AJs is a metabolically active process. Yet, little is known about the role of endothelial metabolism as a modulator of endothelial barrier function and restoration of lung vascular injury. Our Supporting Data demonstrate that endothelial cells respond to inflammatory activation with upregulation of signaling via the hypoxia-inducible factor HIF1α, and its crucial downstream metabolic target PFK-FB3, a critical regulatory enzyme for glycolysis. This glycolytic shift is accompanied by concomitant upregulation of mitochondrial glutamine metabolism, which compensates for the loss of mitochondrial glucose oxidation and enables cells to use glutamine as an alternate mitochondrial TCA cycle fuel. We observed that inhibition of PFK- FB3 prevents restoration of endothelial barrier function following lung injury, thus underscoring the adaptive role of PFK-FB3 and increased glycolysis during endothelial barrier restoration. Based on these findings, we posit that induction of glycolysis in lung microvessel endothelial cells serves as a homeostatic mechanism mediating the restoration of endothelial barrier function and lung fluid balance. In Project 2, we will pursue the following Specific Aims: (1a) We will define the mechanisms of PFK-FB3-mediated activation of glycolysis and compensatory glutaminolysis in lung endothelial cells as induced by inflammation and endothelial injury, and determine the requisite role of these metabolic shifts in repairing endothelial barrier; (1b) we will determine the spatial-temporal role of PFK-FB3-mediated activation of glycolysis in the re-annealing of AJs and restoring endothelial barrier integrity, and (2) We will determine the role of endothelial metabolic reprogramming via PFK-FB3 in restoring lung endothelial barrier integrity and fluid balance following inflammatory lung injury in models of ALI. Using state-of-the-art metabolic analyses, engineered protein constructs and biosensors as well as novel genetic mouse models, we will define the metabolic mechanisms activated by inflammatory injury of the lung endothelium and their role in restoring the lung endothelial barrier. Our long-term goal is to identify metabolic targets and switches that will promote and accelerate the recovery of the endothelial barrier and normalize lung fluid balance to mitigate acute lung injury.