Alam Masih-ul, Kirton John Paul, Wilkinson Fiona L, Towers Emily, Sinha Smeeta, Rouhi Mansour, Vizard Thomas N, Sage Andrew P, Martin David, Ward Donald T, Alexander Marie Yvonne, Riccardi Daniela, Canfield Ann E
Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Michael Smith Building, Oxford Road, Manchester M139PT, UK.
Cardiovasc Res. 2009 Feb 1;81(2):260-8. doi: 10.1093/cvr/cvn279. Epub 2008 Oct 13.
Vascular calcification (VC) is highly correlated with increased morbidity and mortality in advanced chronic kidney disease (CKD) patients. Allosteric modulation of the calcium-sensing receptor (CaR) by calcimimetics inhibits VC in animal models of advanced CKD. Here, we investigated the expression of the CaR in the vasculature and tested the ability of calcimimetics to prevent vascular smooth muscle cell (VSMC) calcification in vitro.
Immunohistochemical staining demonstrated that CaR protein is present in VSMC in normal, non-calcified human arteries. In contrast, low levels of CaR immunoreactivity were detected in atherosclerotic, calcified arteries. Immunfluorescence and immunoblotting revealed that CaR protein was also expressed by human and bovine VSMC in vitro. Acute stimulation of VSMC with increased Ca2+ stimulated extracellular signal-regulated kinase (ERK1/2) phosphorylation, suggesting that the VSMC CaR is functional. VSMC CaR expression decreased when these cells deposited a mineralized matrix or following 24 h incubation in mineralization medium with increased (i.e. 1.8 or 2.5 mM) Ca2+. Culturing VSMC in mineralization medium containing 1.8 and 2.5 mM Ca2+ or with the membrane-impermeant CaR agonist Gd3+ enhanced mineral deposition compared with that observed in 1.2 mM Ca2+. Over-expression of dominant-negative (R185Q) CaR enhanced, whereas the calcimimetic R-568 attenuated, VSMC mineral deposition.
These results demonstrate that: (i) VSMCs express a functional CaR; (ii) a reduction in CaR expression is associated with increased mineralization in vivo and in vitro; (iii) calcimimetics decrease mineral deposition by VSMC. These data suggest that calcimimetics may inhibit the development of VC in CKD patients.
血管钙化（VC）与晚期慢性肾脏病（CKD）患者发病率和死亡率的增加高度相关。拟钙剂对钙敏感受体（CaR）的变构调节在晚期CKD动物模型中可抑制VC。在此，我们研究了CaR在脉管系统中的表达，并测试了拟钙剂在体外预防血管平滑肌细胞（VSMC）钙化的能力。
免疫组织化学染色显示，CaR蛋白存在于正常、未钙化的人动脉的VSMC中。相比之下，在动脉粥样硬化、钙化动脉中检测到低水平的CaR免疫反应性。免疫荧光和免疫印迹显示，人及牛的VSMC在体外也表达CaR蛋白。用增加的Ca2+急性刺激VSMC可刺激细胞外信号调节激酶（ERK1/2）磷酸化，提示VSMC CaR具有功能。当这些细胞沉积矿化基质时或在含增加（即1.8或2.5 mM）Ca2+的矿化培养基中孵育24小时后，VSMC CaR表达下降。与在1.2 mM Ca2+中观察到的情况相比，在含1.8和2.5 mM Ca2+的矿化培养基中培养VSMC或用膜不透性CaR激动剂Gd3+培养可增强矿物质沉积。显性负性（R185Q）CaR的过表达增强了VSMC矿物质沉积，而拟钙剂R-568则减弱了这种沉积。
这些结果表明：（i）VSMC表达功能性CaR；（ii）CaR表达的降低与体内和体外矿化增加相关；（iii）拟钙剂减少VSMC的矿物质沉积。这些数据表明，拟钙剂可能抑制CKD患者VC的发展。
