Glycolysis Inhibition Regulates Endothelial Junctions by Perturbing Actin and Focal Adhesions

One of the central functions of the endothelium is to maintain a vascular barrier that prevents fluid leakiness and immune cell influx from the circulating blood into the tissue. The barrier integrity of the endothelium is largely controlled by adherens junctions (AJs) and the key AJ molecule VE-cadherin, which maintains cell cohesion via homotypic trans-interaction with VE-cadherin molecules on neighboring endothelial cells. Tension is required to maintain junction homeostasis, but little is known about the role of endothelial metabolism and bioenergetics in regulating junctional forces. Because glycolysis is the main source of ATP generation in endothelial cells, we examined the bioenergetic control of the mechanics of VE-cadherin junctions, by focusing on the glycolysis regulatory enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Results from traction force imbalance measurements and a VE-cadherin tension sensor revealed that inhibiting PFKFB3 significantly reduced the average junctional tension and the force on VE-cadherin complexes. The decrease in tension was largely due to mechanical changes distal from the cell-cell contacts. Specifically, inhibiting glycolysis perturbs focal adhesions and disrupts actin organization, directly impacting the net force on intercellular contacts. These findings identify a critical role of cellular metabolism for the mechanics and integrity of vascular endothelial junctions, by maintaining global cell mechanics.