Vascular calcification (VC) shares some similarities to skeletal mineralisation, and involves hydroxyapatite deposition in arteries and cardiac muscle. Whilst VC has severe clinical consequences, the cellular mechanisms responsible are not fully elucidated. ATP and UTP (≥1 μM) inhibit bone mineralisation via P2 receptor-dependent (P2R) and independent mechanisms. The latter involves the hydrolysis of extracellular nucleotides by NPP1 to produce pyrophosphate (PPi), a key mineralisation inhibitor. This study investigated whether extracellular nucleotides also regulate VC. Vascular smooth muscle cells (VSMCs) were cultured in calcifying medium containing 2.5 mM phosphate for 14 days. We found that VSMCs express multiple P2Rs and expression was upregulated in calcifying conditions. The key source of extracellular ATP is controlled release from cells: removal of endogenous ATP by apyrase (an ecto-nucleotidase which hydrolyses ATP) resulted in a 45% increase in VSMC calcification. Culture with exogenous ATP and UTP (≥1 μM) decreased VSMC calcification by ≤80 and 90%, respectively (P<0.001). The selective agonists, 2-thioUTP and MRS2768, also inhibited VSMC calcification by <70% (P<0.001) suggesting a role for the P2Y2R in mediating these effects, Furthermore, the level of calcification is increased twofold in VSMCs from P2Y2R knockout mice. VC is associated with increased VSMC apoptosis and a transdifferentiation of VSMCs towards the osteogenic lineage. We observed that ATP/UTP increased VSMC number and decreased the expression of genes associated with osteoblast differentiation (e.g. Runx2, osterix, Ocn). CTP and GTP (≥10 μM), which are not P2R agonists but are hydrolysed by NPP1 to produce PPi, blocked VSMC calcification by ≤70%. Furthermore, in NPP1 knockout VSMCs, the inhibitory actions of ATP and UTP were tenfold less potent. These results indicate the P2R-independent mechanisms (involving PPi) contribute significantly to the inhibitory actions of extracellular nucleotides on VC. Taken together, our data suggest an important role for extracellular nucleotides, the P2Y2R and NPP1 in the regulation of VC.
Disclosure: The authors declared no competing interests. This work was supported by Arthritis Research UK (#19205).