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Purinergic Signalling

, Volume 15, Issue 3, pp 315–326 | Cite as

Inhibition of vascular smooth muscle cell calcification by ATP analogues

  • Jessal J. Patel
  • Lucie E. Bourne
  • José Luis Millán
  • Timothy R. Arnett
  • Vicky E. MacRae
  • Caroline P. D. Wheeler-Jones
  • Isabel R. OrrissEmail author
Original Article
  • 55 Downloads

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.

Keywords

Arterial medial calcification Vascular smooth muscle cells ATP analogues P2X receptors 

Notes

Funding information

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).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Comparative Biomedical SciencesRoyal Veterinary CollegeLondonUK
  2. 2.School of Life & Medical SciencesUniversity of HertfordshireHatfieldUK
  3. 3.Sanford Burnham Prebys Medical Discovery InstituteLa JollaUSA
  4. 4.Department of Cell and Developmental BiologyUniversity College LondonLondonUK
  5. 5.The Roslin Institute and Royal (Dick) School of Veterinary StudiesUniversity of EdinburghEdinburghUK

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