Spatiotemporal Control of Glycolysis Modulates ATP Generation and Enhances Restoration of Endothelial Barrier Function Following Inflammatory Injury

Abstract

BackgroundAcute inflammatory injury is characterized by dysfunction of the vascular endothelium, resulting in disruption of endothelial adherens junctions, increased vascular permeability and leukocyte infiltration. While the mechanisms of assembly and disassembly of endothelial adherens junctions have been extensively studied, little is known about the bioenergetics of the barrier repair process. The glycolysis regulatory enzyme PFKFB3 has recently been identified as a key mediator of endothelial metabolism and function and could thus serve as a potential mediator of barrier repair.Methods and ResultsTo investigate how the endothelial metabolic profile changes during inflammation, we performed a Seahorse bioenergetic flux assay on Human Lung Microvascular Endothelial cells (HLMVECs). Glycolysis, as measured by the extracellular acidification rate, increased by 36% following treatment with the inflammatory mediator TNFα, while oxidative phosphorylation was not affected. Pharmacological inhibition of PFKFB3 abolished TNFα mediated upregulation of glycolysis. PFKFB3 activity was also necessary for barrier restoration of HLMVECs following injury, as measured by a transendothelial electrical resistance (TER) assay (p<0.0001). Whole cell ATP levels, measured by live cell microscopy using the fluorescent ATP biosensor Perceval HR, increased 31% following TNFα treatment (p<0.0001), largely concentrated at the cell periphery. We hypothesized that this regional ATP generation is PFKFB3 dependent. To address this question, we engineered a novel construct in which PFKFB3 is targeted to the cell membrane in an inducible manner and thus regulating metabolism in a spatiotemporal manner. Importantly, PFKFB3 membrane targeting enhanced endothelial barrier restoration by increasing the extent and rate of recovery (p<0.0001) following barrier disruption.ConclusionsThis data suggests that spatial and temporal regulation of PFKFB3 mediated glycolysis is important for restoration of the endothelial barrier after inflammatory injury. A better understanding of the endothelial barrier bioenergetics could allow for the development of novel therapeutic approaches to treat diseases in which endothelial barrier function is acutely compromised.Support or Funding InformationAHA 18PRE34070092 National Institute of Health P01HL060678This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Circadian Influences on Myocardial Ischemia-Reperfusion Injury and Heart Failure

“…13 Endothelial PER2 influences mitochondrial oxygen consumption and ATP production during hypoxia. 175–177 Additionally, hypoxia-induced changes in cox4.2 mRNA, known for enhancing oxygen efficiency, were disrupted in Per2 knockdown endothelial cells, affecting ATP levels. 178 In Per2 −/− mice, ischemia-induced increases in complex 4 activity were reduced.…”