Bone Abstracts

Vascular calcification (VC) involves hydroxyapatite deposition in the arteries and cardiac muscle. VC is thought to share some outward similarities to skeletal mineralisation and has been associated with the transdifferentiation of vascular smooth muscle cells (VSMCs) to an osteoblast-like phenotype. We have previously shown that ATP, UTP and synthetic ATP-analogues (α, β-meATP, β, γ-meATP, Bz-ATP) (≥1 μM) act to potently inhibit both bone mineralisation and VC by ≤95%. This study compared the mechanisms by which extracellular nucleotides block these processes. Primary mouse VSMCs and osteoblasts were cultured for up to 21 days in calcifying (2.5 mM phosphate) and osteogenic (2 mM β-glycerophosphate, 50 μg/ml ascorbate) medium, respectively. Cells were treated with 1–100 μM ATP, UTP α, β-meATP, β, γ-meATP or Bz-ATP for the duration of the culture. Basal alkaline phosphatase (TNAP) activity was 12-fold higher in mineralising osteoblasts compared to calcifying VSMCs (P<0.001). The activity of NPPs, which generate pyrophosphate from ATP, was tenfold higher in calcifying VSMCs (P<0.001). In differentiating and mature osteoblasts, extracellular nucleotides (≥10 μM) inhibited TNAP activity by ≤50% (P<0.001). In contrast, ATP, UTP and ATP-analogues stimulated TNAP activity in calcifying VSMCs by ≤20-fold (P<0.001); at these concentrations VC was typically reduced by ≤90%. Prolonged treatment with ATP and related compounds (≤100 μM) had no effect on osteoblast number and viability. However, in calcifying VSMCs treatment with extracellular nucleotides increased cell number by ≤2.5-fold and reduced the percentage of dead cells by ≤70% (P<0.001). Since VC is also associated with increased apoptosis, extracellular nucleotides may prevent VC by promoting VSMC survival. Our results indicate that although the functional effects of extracellular nucleotides on bone mineralisation and VC are similar, the underlying cellular mechanisms are distinct. Notably it suggests that TNAP may not exert significant effects on VC. Our findings indicate that, despite outward similarities, there are key differences between the processes of bone mineralisation and vascular calcification.