Background: The integrity of the endothelial cell barrier of the microvasculature is compromised during inflammation. The increased vascular permeability leads to tissue injury and organ dysfunction. In recent years, considerable advances have been made in the understanding of signalling mechanisms regulating the endothelial barrier integrity. The role of endothelial glycolysis as a modulator of endothelial barrier integrity is not yet well-studied. The aim of the present study was to investigate the effect of inflammation on endothelial glycolysis and its role in the maintenance of endothelial barrier integrity.

Methods: The study was carried out on cultured human umbilical vein endothelial cells (HUVECs). Inflammatory condition was simulated by treating cells with low concentrations (1 ng/mL) of TNFa for 24h. Endothelial barrier function was analysed by measuring the flux of albumen through endothelial monolayers cultured on filter membranes. Gene expression was analysed by qPCR-based assays. The capacity of endothelial cells for maximal ATP synthesis rate was investigated by the real-time live-cell imaging using FRET-based ATP-biosensor (live cell FRET). The cellular glucose uptake was measured by fluorescent microscopy using a fluorescent analogue of glucose (2-NBDG). Extracellular lactate accumulation was analysed by lactate-glow assay (Promega Inc.).

Results: Treatment of human endothelial cells with TNFa resulted in significant upregulation of glycolytic ATP synthesis rate accompanied by an enhanced expression of glycolytic enzymes 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and phosphofructokinase 1 (PFK1), and enhanced glucose uptake by endothelial cells (n=5; p<0.05 for all parameters tested). Moreover, TNFa treatment caused an enhanced accumulation of lactate in conditioned media accompanied by acidic pH of the medium. Treatmwnt of ECs with TNFa caused a 3-fold increase in endothelial permeability. Pharmacological inhibition of glycolysis either by partial replacement of glucose with 2-deoxy glucose (2DG) or an inhibition of PFKFB3 resulted in further worsening (a 5-fold increase in permeability) of TNFa-induced endothelial barrier failure. Similar effects were observed when major endothelial glucose transporter  GLUT1 was pharmacologically inhibited. Addition of pyruvate was not sufficient to ameliorate these effects. These effects of glycolysis inhibition on endothelial barrier were further verified immunostaining of endothelial cell-cell junctions.

Conclusion: The study demonstrates that TNFa upregulates endothelial glycolysis. Moreover, the data suggest that upregulation of glycolysis may serve as an endogenous metabolic adaptation to the TNFa-induced suppression of mitochondrial ATP synthesis, which protects endothelial barrier integrity.