Glycolysis blockade reduces synthesis and maturation of glycoproteins as well as leukocyte adhesion in inflamed aortic valve cells

Abstract Introduction Inflammation and metabolism are hallmarks of calcific aortic valve disease (CAVD). In this context, a recent study associates inflammation, hyper-glycolysis, and calcification of aortic valve interstitial cells (VICs). Inflammatory signals, through the JAK-STAT/HIF1-α axis, induce the production of cytokines and adhesion molecules, which are glycoproteins associated with pathogenically relevant processes, i.e., calcification and leukocyte adhesion to valve endothelium. Objective Our aim was to elucidate the role of inflammation and metabolic rewiring on the production and maturation of glycoproteins associated to CAVD pathogenesis and its consequences on aortic valve cell pathophysiology. Methods The study was performed in VICs and endothelial valve cells (VECs) explanted from human valves. Primary VICs and VECs cultures were exposed to inflammatory agonists. The role of glycolysis was performed by pharmacological blockade with 2-DG and glucose deprivation. Methods included metabolic analysis by Seahorse, gene expression analysis by qPCR, protein expression by Western blot and ELISA, and leukocyte adhesion assays. Results Since inflammation reprograms VIC metabolism to enhance glycolysis, thus mimicking the phenotype found in diseased valves, we first addressed the role of glycolysis on VICs exposed to an inflammatory conditions. Pharmacological blockade of glycolysis with 2-DG and glucose deprivation significantly impaired JAK-STAT-HIF-1-α signalling, the secretion of interleukin 6, as well as the expression and glycosylation of adhesion molecules such as ICAM-1 and VCAM-1 in inflamed VIC. Next, given the critical role of these molecules in VEC pathophysiology, we further examined whether glycolysis-dependent metabolic alterations also occur in VECs. Seahorse metabolic analysis and 2-DG treatment disclosed that, under inflammatory conditions, VECs undergo a distinct metabolic shift compared to VICs, relying on oxidative phosphorylation for ATP production in a glucose uptake-dependent manner. However, as found in VIC, the JAK-STAT-HIF1-α axis is also involved in mediating the effects induced by metabolic rewiring on cytokine production and adhesion molecule expression and glycosylation. Later, we investigated the functional relevance of glycolysis on immune cell adhesion and found that, both glucose uptake and ATP synthase activity were essential for leukocyte-VEC adhesion. Conclusions Glycolysis blockade impairs the synthesis and maturation of glycoproteins and subsequent leukocyte adhesion to inflamed valve cells, which is relevant to early stages of CAVD pathogenesis.