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Introduction: Acute Lung Injury (ALI) is characterized by endothelial barrier dysfunction of the lung vasculature, leading to disassembly of endothelial adherens junctions, increased vascular permeability and fluid accumulation in the lungs. Little is known about the bioenergetics of the barrier disruption and repair processes. The glycolysis regulatory enzyme PFKFB3 has recently been identified as a key mediator of endothelial glucose metabolism and could thus serve as a potential mediator of barrier repair. Methods and Results: To quantitatively measure metabolic changes in response to an inflammatory stimulus, we performed a Seahorse bioenergetic flux assay using human lung microvascular endothelial cells (HLMVECs). Glycolysis, as measured by the extracellular acidification rate, increased acutely by 36% (from 9.1 to 12.4 mpH/min) following treatment with the inflammatory mediator TNFα. This upregulation was abolished in the presence of PFK15, a specific inhibitor of PFKFB3. PFKFB3 activity was also necessary for barrier restoration of HLMVECs following injury, as measured by a transendothelial electrical resistance (TER) assay (p<0.0001). Live cell microscopy of endothelial cells expressing the ATP biosensor Perceval HR revealed that ATP is generated at cell junctions following TNFα treatment. Additionally, controlled translocation of PFKFB3 to the plasma membrane resulted in enhanced barrier restoration after injury, both in terms of the extent of barrier integrity as well as the rate of recovery (p<0.0001). Finally, PFKFB3 was pharmacologically inhibited in vivo following lung injury with the bacterial toxin LPS in mice. This inhibition resulted in significantly impaired recovery of lung vascular barrier function as measured by an Evans blue albumin (EBA) permeability assay (p<0.001). Endothelial cell-specific overexpression of PFKFB3 in mice by liposomal delivery resulted in an enhanced rate of recovery from LPS mediated injury. Lung vascular leakiness decreased from 29.0 to 18.3 ng EBA/g body weight at two days following the LPS injury (p<0.001), thus achieving pre-injury levels of barrier function. Conclusions: Glycolytic ATP generation regulated by PFKFB3 is essential 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 which enhance barrier function in severe diseases such as acute lung injury. [ABSTRACT FROM AUTHOR]

Differentiating stem cells must adapt their mitochondrial metabolism to fit the needs of the mature differentiated cell. In a recent study, we observed that during differentiation to an endothelial phenotype, pluripotent stem cell mitochondria are removed by mitophagy, triggering compensatory mitochondrial biogenesis to replenish the mitochondrial pool. We identified the mitochondrial phosphatase PGAM5 as the link between mitophagy and transcription of the mitochondrial biogenesis regulator PPARGC1A/PGC1α in the nucleus. Swapping of mitochondria through the coupled processes of mitophagy and mitochondrial biogenesis lead to enhanced metabolic reprogramming in the differentiated cell.