In vitro angiogenesis inhibition with selective compounds targeting the key glycolytic enzyme PFKFB3

Abnormal glycolytic metabolism contributes to angiogenic sprouting involved in atherogenesis. We investigated the potential anti-angiogenic properties of specific 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) inhibitors in endothelial cells (ECs). ECs were treated with PFKFB3 inhibitors (named PA-1 and PA-2) and their effects on metabolic and functional characteristics of ECs were investigated. The anti-glycolytic compound 3-(pyridinyl)- 1-(4-pyridinyl)- 2-propen-1-one (3PO) was used as reference compound. PFKFB3 expression and activity (IC₅₀ about 3-21 nM) was inhibited upon treatment with both compounds. Glucose uptake and lactate export were measured using commercial assays and showed a partial reduction up to 40%. PFKFB3 inhibition increased intracellular lactate accumulation, and reduced expression of monocarboxylate transporters-1 (MCT1) and MCT4. Furthermore, endothelial cell migration and proliferation assays demonstrated significant reduction upon treatment with both compounds. Matrix- metalloproteinase (MMP) activity, measured by gelatin zymography, and expression was significantly reduced (up to 25%). In addition, PA compounds downregulated the expression of VCAM-1, VE-cadherin, VEGFa, VEGFR2, TGF-β, and IL-1β, in inflamed ECs. Finally, PA-1 and PA-2 treatment impaired the formation of angiogenic sprouts measured by both morphogenesis and spheroid-based angiogenesis assays. Our data demonstrate that the anti-glycolytic PA compounds may affect several steps involved in angiogenesis. Targeting the key glycolytic enzyme PFKFB3 might represent an attractive therapeutic strategy to improve the efficacy of cancer treatments, or to be applied in other pathologies where angiogenesis is a detrimental factor.