The process of arterial calcification shares many similarities to skeletal mineralisation, and involves the deposition of hydroxyapatite in the arteries. However, the cellular mechanisms responsible have yet to be fully elucidated. Accumulating evidence suggests that aerobic glycolysis (the Warburg effect), plays a critical role in meeting the demand for energy and biosynthetic precursors during proliferation and differentiation in numerous cell types. Therefore we addressed the hypothesis that vascular smooth muscle cell (VSMC) calcification requires aerobic glycolysis to produce energy and the necessary biosynthetic precursors.Methods
Calcification of murine aortic VSMCs was induced by 3 mM Pi for 7 days. Calcium deposition was determined using alizarin red staining and a modified o-cresolphthalein method. VSMCs were cultured with the fluorescent glucose analogue 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)−2-Deoxyglucose (2-NBDG) to determine changes in glucose uptake. Gene expression was analysed by qRT-PCR.Results
Calcium deposition was significantly increased in VSMCs cultured in 3 mM Pi versus control conditions (124%, p<0.001). Calcified VSMCs also showed increased mRNA expression of Runx2, Phospho1, Ocn and PiT-1 (p<0.001), recognised osteogenic markers of arterial calcification. Furthermore 3 mM Pi treatment increased glucose uptake (98%, p<0.05) and Glut-1 mRNA expression (1.47 fold, p<0.001). Glycolysis converts glucose to pyruvate which is subsequently converted to either (i) acetyl-CoA by the pyruvate dehydrogenase complex (PDH) or (ii) lactate by lactate dehydrogenase (LDH). Notably, decreased VSMC calcification was observed in cells treated with sodium dicholoroacetate, an inducer of PDH activity (1 mM; 40%; p<0.01) and citric acid, synthesised in the mitochondria from acetyl CoA (1 mM; 72%, p<0.001). Treatment with the LDH inhibitor sodium oxamate (20 mM) or sodium lactate (50 mM) to induce pyruvate production also inhibited VSMC calcification (68% and 53% respectively, p<0.05). Activation of the Wnt pathway – an established regulator of Warburg metabolism – using the selective GSK3 inhibitor CHIR99021 (1 nM) significantly increased VSMC calcification (417%, p<0.001). However, co-?treatment with sodium oxamate (20 mM) significantly blunted the pro-calcification effect of CHIR99021 (69%, p<0.01).Conclusion
Together these data suggest that arterial calcification requires glucose metabolism through a mechanism involving Wnt signalling. Interruption of the glycolysis pathway may therefore represent a novel therapeutic target for clinical intervention.