Dept. of Physiology and Biophysics, Case Western Reserve Univ., School of Medicine, 2109 Adelbert Rd., Cleveland, OH 44106, USA.
Am J Physiol Cell Physiol. 2010 Mar;298(3):C702-13. doi: 10.1152/ajpcell.00419.2009. Epub 2009 Dec 16.
Vascular calcification is a multifaceted process involving gain of calcification inducers and loss of calcification inhibitors. One such inhibitor is inorganic pyrophosphate (PP(i)), and regulated generation and homeostasis of extracellular PP(i) is a critical determinant of soft-tissue mineralization. We recently described an autocrine mechanism of extracellular PP(i) generation in cultured rat aortic vascular smooth muscle cells (VSMC) that involves both ATP release coupled to the ectophosphodiesterase/pyrophosphatase ENPP1 and efflux of intracellular PP(i) mediated or regulated by the plasma membrane protein ANK. We now report that increased cAMP signaling and elevated extracellular inorganic phosphate (P(i)) act synergistically to induce calcification of these VSMC that is correlated with progressive reduction in ability to accumulate extracellular PP(i). Attenuated PP(i) accumulation was mediated in part by cAMP-dependent decrease in ANK expression coordinated with cAMP-dependent increase in expression of TNAP, the tissue nonselective alkaline phosphatase that degrades PP(i). Stimulation of cAMP signaling did not alter ATP release or ENPP1 expression, and the cAMP-induced changes in ANK and TNAP expression were not sufficient to induce calcification. Elevated extracellular P(i) alone elicited only minor calcification and no significant changes in ANK, TNAP, or ENPP1. In contrast, combined with a cAMP stimulus, elevated P(i) induced decreases in the ATP release pathway(s) that supports ENPP1 activity; this resulted in markedly reduced rates of PP(i) accumulation that facilitated robust calcification. Calcified VSMC were characterized by maintained expression of multiple SMC differentiation marker proteins including smooth muscle (SM) alpha-actin, SM22alpha, and calponin. Notably, addition of exogenous ATP (or PP(i) per se) rescued cAMP + phosphate-treated VSMC cultures from progression to the calcified state. These observations support a model in which extracellular PP(i) generation mediated by both ANK- and ATP release-dependent mechanisms serves as a critical regulator of VSMC calcification.
血管钙化是一个多方面的过程，涉及到钙化诱导物的增加和钙化抑制剂的减少。无机焦磷酸盐 (PP(i)) 是一种这样的抑制剂，细胞外 PP(i) 的调节生成和动态平衡是软组织矿化的关键决定因素。我们最近描述了一种在培养的大鼠主动脉血管平滑肌细胞 (VSMC) 中产生细胞外 PP(i) 的自分泌机制，该机制涉及到 ATP 释放与外切核苷酸酶/焦磷酸酶 ENPP1 的偶联，以及由质膜蛋白 ANK 介导或调节的细胞内 PP(i) 外流。我们现在报告说，cAMP 信号的增加和细胞外无机磷酸盐 (P(i)) 的升高协同作用，诱导这些 VSMC 的钙化，这与细胞外 PP(i) 积累能力的逐渐降低相关。PP(i) 积累的减少部分是由 cAMP 依赖性的 ANK 表达减少介导的，同时伴有 cAMP 依赖性的 TNAP 表达增加，TNAP 是一种组织非选择性碱性磷酸酶，可降解 PP(i)。cAMP 信号的刺激不会改变 ATP 释放或 ENPP1 的表达，并且 cAMP 诱导的 ANK 和 TNAP 表达的变化不足以诱导钙化。单独升高细胞外 P(i)只会引起轻微的钙化，而不会引起 ANK、TNAP 或 ENPP1 的显著变化。相比之下，与 cAMP 刺激相结合，升高的 P(i) 会导致支持 ENPP1 活性的 ATP 释放途径减少；这导致 PP(i) 积累率显著降低，从而促进了强烈的钙化。钙化的 VSMC 的特征是多种平滑肌分化标志物蛋白的持续表达，包括平滑肌 (SM) α-肌动蛋白、SM22α 和钙调蛋白。值得注意的是，添加外源性 ATP(或 PP(i) 本身)可以防止 cAMP + 磷酸盐处理的 VSMC 培养物进展为钙化状态。这些观察结果支持了这样一种模型，即通过 ANK 和 ATP 释放依赖性机制介导的细胞外 PP(i) 的产生是 VSMC 钙化的关键调节剂。
