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，在这方面取得了长足的进步。 了解细胞内信号通路如何调节黏附分子的破坏和组装 连接点(AJ)。AJ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和肺血管损伤修复的调节剂。我们的 支持数据显示内皮细胞对炎症激活的反应是通过上调 通过缺氧诱导因子α及其关键的下游代谢靶点pfk-fb3的信号转导。 糖酵解的调节酶。这种糖酵解转变伴随着随之而来的 线粒体谷氨酰胺代谢，它补偿了线粒体葡萄糖氧化和 使细胞能够使用谷氨酰胺作为线粒体TCA循环的替代燃料。我们观察到，抑制PFK- FB3阻止肺损伤后内皮细胞屏障功能的恢复，从而强调了适应性作用 在内皮屏障修复过程中，PFK-FB3的表达和糖酵解增加。基于这些发现，我们假设 诱导肺微血管内皮细胞糖酵解是一种动态平衡机制 内皮屏障功能和肺液平衡的恢复。在项目2中，我们将实现以下目标 具体目标：(1A)我们将确定PFK-FB3介导的糖酵解激活和 炎症和内皮诱导的肺内皮细胞代偿性谷氨酰胺分解 损伤，并确定这些代谢转变在修复内皮屏障中的必要作用；(1b)我们 将确定PFK-FB3介导的糖酵解激活在再退火中的时空作用 AJ和恢复内皮屏障完整性，以及(2)我们将确定内皮细胞的作用 应用PFK-FB3代谢重编程恢复肺内皮细胞屏障完整性和液体平衡 ALI模型中的炎性肺损伤。使用最先进的代谢分析，工程化 蛋白质结构和生物传感器以及新的小鼠遗传模型，我们将定义代谢 肺内皮细胞炎性损伤激活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目标和开关，以促进和 加速内皮细胞屏障的恢复，恢复肺液平衡，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.