Inhibition of vascular smooth muscle cell calcification by ATP analogues

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 (PP i). 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 PP i. 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 PP i .

Bone marrow niche ATP levels determine leukemia-initiating cell activity via P2X7 in leukemic models

“…Alternatively, we have treated both murine and human primary AML cells with different doses of ATP in vitro and revealed that the low dose of ATP (10-100 uM) indeed could enhance the proliferation of leukemia cells of WT, but not P2x7-KO, murine leukemia cells ( Figures S9A-S9B) or human ones ( Figures S9C-S9E). In contrast, the high dose of ATP (1 mM) inhibited leukemia cell growth ( Figures S9A-S9E), which might be resulted from nonspecific cytotoxicity (53)(54)(55). However, the apoptosis ( Figures S9F-S9J) and cell cycle ( Figures S9K-S9O) were not altered upon the treatment of low dose of ATP (the changes in high dose group may be due to nonspecific cytotoxicity), indicating that ATP mediated signaling may mainly sustain the self-renewal ability of leukemia cells and the blockade of the ATP-P2X7 pathway will be a potential strategy for the leukemia treatment.…”

In search of markers useful for evaluation of graft patency - molecular analysis of ‘muscle system process’ for internal thoracic artery and saphenous vein conduits

“…The P2X receptor is of interest to researchers in relation to blood vessel smooth muscle cells (VSMC) e.g. synthetic ATP analogues and P2X receptor agonists (Bz-ATP, α, β-meATP and β, γ-meATP) have been shown to inhibit VSMC calcification in vitro [43]. However, there is little information regarding the effect of P2RX1 on the patency of the blood vessel, so this requires further study.…”

Vitamin D receptor deficiency increases systolic blood pressure by upregulating the renin‑angiotensin system and autophagy

“…VSMCs were identified by negative platelet endothelial cell adhesion molecule1 (marker of endothelial cells) and vimentin (marker of fibroblasts), and positive α-smooth muscle actin (marker of VSMCs) expression. VSMCs from the 3rd to 5th passages were used in the present study ( 15 ).…”

“…TRIzol ® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to isolate total RNA from VSMCs according to the manufacturer's instructions. mRNA levels of SOD , Ang II and Ang II type 1 receptor ( AT1R ) in the VSMCs were detected using RT-qPCR ( 15 ). Briefly, first-strand cDNAs were reverse-transcribed from 2 µg of total RNAs using M-MLV (Moloney Murine Leukemia Virus Reverse Transcriptase) with oligo ( dT ); as the primer) and Rain in 1X M-MLV buffer.…”