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.

中文翻译：

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ATP类似物对血管平滑肌细胞钙化的抑制作用。

动脉内侧钙化 (AMC) 与血管平滑肌细胞 (VSMC) 的表型变化有关，据报道这使它们更像成骨细胞。先前的研究表明，ATP/UTP 可以通过 P2 受体直接抑制 AMC，并通过 NPP1 介导的水解间接抑制 AMC，从而产生矿化抑制剂焦磷酸盐（PP _i）。本研究调查了 P2X 受体在细胞外核苷酸对 VSMC 钙化的抑制作用中的作用。我们发现 Bz-ATP、α,β-meATP 和 β,γ-meATP 抑制钙化高达 100%。在高磷酸盐培养基（2 mM）中培养与增加的 VSMC 死亡和细胞凋亡有关；用 Bz-ATP、α,β-meATP 和 β,γ-meATP 处理可将细胞凋亡降低至非钙化细胞中的水平。钙化还与 VSMC（例如 SM22α 和 SMA）和成骨细胞相关（例如 Runx2 和骨桥蛋白）标志物的蛋白质水平的改变有关。Bz-ATP、α,β-meATP 和 β,γ-meATP 减弱了蛋白质表达的这些变化。Bz-ATP、α,β-meATP 和 β,γ-meATP 的长期培养导致细胞外 ATP 水平降低和 ATP 分解率增加。P2X 受体拮抗剂未能阻止这些类似物的抑制作用，表明它们通过 P2X 受体非依赖性机制起作用。一致地，α,β-meATP 和 β,γ-meATP（分别为α,β-meADP 和亚甲基二膦酸盐）的分解产物也剂量依赖性地抑制 VSMC 钙化。此外，Bz-ATP、α,β-meATP 和 β,γ-meATP 在从 NPP1 敲除小鼠中分离出来的 VSMC 中的作用没有改变，这表明这些化合物的功能作用不涉及 NPP1 介导的 PP 生成。_我。总之，这些结果表明 ATP 类似物在体外对 VSMC 钙化和细胞凋亡的抑制作用可能至少部分是由独立于嘌呤能信号传导和 PP _i的机制介导的。