Shioi A, Nishizawa Y, Jono S, Koyama H, Hosoi M, Morii H
Second Department of Internal Medicine, Osaka City University Medical School, Japan.
Arterioscler Thromb Vasc Biol. 1995 Nov;15(11):2003-9. doi: 10.1161/01.atv.15.11.2003.
Calcification is a common feature of advanced atherosclerotic lesions and is being reemphasized as a clinically significant element of vascular disease. However, the scarcity of in vitro models of vascular calcification preclude studying its molecular and cellular mechanism. In the present study, we describe an in vitro calcification in which diffuse calcification can be induced by culturing bovine vascular smooth muscle cells (BVSMC) in the presence of beta-glycerophosphate, ascorbic acid, and insulin in a manner analogous to in vitro mineralization by osteoblasts. Calcification was confirmed by von Kossa staining and 45Ca accumulation. Factor analysis revealed that beta-glycerophosphate is the most important factor for this calcification process, suggesting that alkaline phosphatase (ALP) may be involved. As predicted, high levels of ALP expression were detected by ALP assay and Northern blot analysis. Functional significance of ALP was confirmed by demonstrating that levamisole, a specific inhibitor of ALP, inhibited BVSMC calcification in a dose-dependent manner. Bisphosphonates such as etidronate and pamidronate potently inhibited BVSMC calcification, suggesting that hydroxyapatite formation may be involved. Importantly, expression of osteopontin mRNA was dramatically increased in calcified BVSMC compared with uncalcified control cells. These data suggest that beta-glycerophosphate can induce diffuse calcification by an ALP-dependent mechanism and that this in vitro calcification system is useful for analyzing the molecular and cellular mechanisms of vascular calcification.
钙化是晚期动脉粥样硬化病变的一个常见特征，并且正作为血管疾病临床上的一个重要因素而被重新强调。然而，血管钙化的体外模型稀缺，这妨碍了对其分子和细胞机制的研究。在本研究中，我们描述了一种体外钙化模型，其中通过在β-甘油磷酸、抗坏血酸和胰岛素存在的情况下培养牛血管平滑肌细胞（BVSMC），可以以类似于成骨细胞体外矿化的方式诱导弥漫性钙化。通过冯·科萨染色和45Ca积累证实了钙化。因子分析表明，β-甘油磷酸是该钙化过程中最重要的因素，提示碱性磷酸酶（ALP）可能参与其中。正如所预测的，通过ALP测定和Northern印迹分析检测到高水平的ALP表达。通过证明ALP的特异性抑制剂左旋咪唑以剂量依赖性方式抑制BVSMC钙化，证实了ALP的功能意义。双膦酸盐如依替膦酸和帕米膦酸强烈抑制BVSMC钙化，提示可能涉及羟基磷灰石的形成。重要的是，与未钙化的对照细胞相比，钙化的BVSMC中骨桥蛋白mRNA的表达显著增加。这些数据表明，β-甘油磷酸可以通过依赖ALP的机制诱导弥漫性钙化，并且这种体外钙化系统对于分析血管钙化的分子和细胞机制是有用的。
