Suppression of PFKFB3-driven glycolysis restrains endothelial-to-mesenchymal transition and fibrotic response.

Endothelial-to-mesenchymal transition (EndoMT), the process wherein endothelial cells lose endothelial identity and adopt mesenchymal-like phenotypes, constitutes a critical contributor to cardiac fibrosis. The phenotypic plasticity of endothelial cells can be intricately shaped by alteration of metabolic pathways, but how endothelial cells adjust cellular metabolism to drive EndoMT is incompletely understood. Here, we identified 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) as a critical driver of EndoMT via triggering abnormal glycolysis and compromising mitochondrial respiration. Pharmacological suppression of PFKFB3 with salvianolic acid C (SAC), a phenolic compound derived from Salvia miltiorrhiza, attenuates EndoMT and fibrotic response. PFKFB3-haplodeficiency recapitulates the anti-EndoMT effect of SAC while PFKFB3-overexpression augments the magnitude of EndoMT and exacerbates cardiac fibrosis. Mechanistically, PFKFB3-driven glycolysis compromises cytoplasmic nicotinamide adenine dinucleotide phosphate (reduced form, NADPH) production via hijacking glucose flux from pentose phosphate pathway. Efflux of mitochondrial NADPH through isocitrate/α-ketoglutarate shuttle replenishes cytoplasmic NADPH pool but meanwhile impairs mitochondrial respiration by hampering mitochondrial iron-sulfur cluster biosynthesis. SAC disrupts PFKFB3 stability by accelerating its degradation and thus maintains metabolic homeostasis in endothelial cells, underlying its anti-EndoMT effects. These findings for the first time identify the critical role of PFKFB3 in triggering EndoMT by driving abnormal glycolysis in endothelial cells, and also highlight the therapeutic potential for pharmacological intervention of PFKFB3 (with SAC or other PFKFB3 inhibitors) to combat EndoMT-associated fibrotic responses via metabolic regulation.