Abstract 9817: Krüppel-Like Factor 4 Integrates Immunometabolism of Human Arterial Endothelial Cells

Abstract

Introduction: Inflammation and metabolic alterations are two common hallmarks of cardiovascular diseases. Yet, the molecular mechanisms integrating inflammation and cell metabolism remain elusive. Objective: We investigated whether Krüppel-like factor 4 (KLF4) serves as a link between cellular glucose metabolism and inflammation in human arterial endothelial cells. Methods and Results: Primary human pulmonary artery and aortic endothelial cells (PAEC and AoEC, respectively) were stimulated with tumor necrosis factor α (TNFα) or lipopolysaccharide (LPS). Results from fluorescent microscopy and Seahorse extracellular flux analysis revealed that TNFα and LPS enhanced glucose uptake by 2.2-fold, glycolysis by 2.3-fold, and mitochondrial respiration by 1.6-fold in PAEC and AoEC. The increase in glycolysis correlated with a 2-4-fold upregulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) and PFKFB4, two key regulatory enzymes of glycolysis. Silencing PFKFB3 but not PFKFB4 entirely prevented TNFα-induced enhancement of glycolysis with a concomitant metabolic shift to mitochondrial respiration. Mechanistically, TNFα and LPS stimulated KLF4 mRNA and protein expression (2-4-fold), which functions as a transcriptional repressor of PFKFB3 . KLF4 knockdown potentiated TNFα-induced stimulation of PFKFB3 expression by 1.6-fold and glycolysis by 2.2-fold, effects which were completely prevented by co-silencing of PFKFB3 . Functionally, KLF4 silencing exacerbated TNFα-induced vascular cell adhesion molecule 1 (VCAM1) expression (by 2-3-fold), E-selectin secretion (by 2.8-fold), and monocyte adhesion (by 1.6-fold) in PAEC, all of which were attenuated by over 50% when PFKFB3 was inhibited by siRNA or with 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3-PO). By contrast, KLF4 overexpression-mediated inhibition of inflammation was significantly counteracted in PAEC with co-overexpression of PFKFB3 . Conclusion: These results indicate that KLF4 inhibits inflammation by normalizing PFKFB3-mediated enhancement of glycolysis and that the KLF4-PFKFB3 signaling axis integrates immunometabolism in human arterial endothelial cells.