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Inhibition of vascular smooth muscle cell calcification by ATP analogues

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Abstract

Arterial medial calcification (AMC) has been associated with phenotypic changes in vascular smooth muscle cells (VSMCs) that reportedly makes them more osteoblast-like. Previous work has shown that ATP/UTP can inhibit AMC directly via P2 receptors and indirectly by NPP1-mediated hydrolysis to produce the mineralisation inhibitor, pyrophosphate (PPi). This study investigated the role of P2X receptors in the inhibitory effects of extracellular nucleotides on VSMC calcification. We found that Bz-ATP, α,β-meATP and β,γ-meATP inhibited calcification by up to 100%. Culture in a high-phosphate medium (2 mM) was associated with increased VSMC death and apoptosis; treatment with Bz-ATP, α,β-meATP and β,γ-meATP reduced apoptosis to levels seen in non-calcifying cells. Calcification was also associated with alterations in the protein levels of VSMC (e.g. SM22α and SMA) and osteoblast-associated (e.g. Runx2 and osteopontin) markers; Bz-ATP, α,β-meATP and β,γ-meATP attenuated these changes in protein expression. Long-term culture with Bz-ATP, α,β-meATP and β,γ-meATP resulted in lower extracellular ATP levels and an increased rate of ATP breakdown. P2X receptor antagonists failed to prevent the inhibitory effects of these analogues suggesting that they act via P2X receptor-independent mechanisms. In agreement, the breakdown products of α,β-meATP and β,γ-meATP (α,β-meADP and methylene diphosphonate, respectively) also dose-dependently inhibited VSMC calcification. Furthermore, the actions of Bz-ATP, α,β-meATP and β,γ-meATP were unchanged in VSMCs isolated from NPP1-knockout mice, suggesting that the functional effects of these compounds do not involve NPP1-mediated generation of PPi. Together, these results indicate that the inhibitory effects of ATP analogues on VSMC calcification and apoptosis in vitro may be mediated, at least in part, by mechanisms that are independent of purinergic signalling and PPi.

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Funding

The authors are grateful for the funding from the British Heart Foundation (grant number PG/15/13/31296) and the Biotechnology and Biological Sciences Research Council (BBSRC) in the form of Institute Strategic Programme grants (BB/J004316/1 and BB/P013732/1).

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Author notes

  1. Jessal J. Patel

    Present address: School of Life & Medical Sciences, University of Hertfordshire, Hatfield, UK

Authors and Affiliations

  1. Department of Comparative Biomedical Sciences, Royal Veterinary College, London, NW1 0TU, UK

    Jessal J. Patel, Lucie E. Bourne, Caroline P. D. Wheeler-Jones & Isabel R. Orriss

  2. Sanford Burnham Prebys Medical Discovery Institute, La Jolla, USA

    José Luis Millán

  3. Department of Cell and Developmental Biology, University College London, London, UK

    Timothy R. Arnett

  4. The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, UK

    Vicky E. MacRae

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  1. Jessal J. Patel
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Correspondence to Isabel R. Orriss.

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Patel, J.J., Bourne, L.E., Millán, J.L. et al. Inhibition of vascular smooth muscle cell calcification by ATP analogues. Purinergic Signalling 15, 315–326 (2019). https://doi.org/10.1007/s11302-019-09672-3

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