β-Glycerophosphate Accelerates Calcification in Cultured Bovine Vascular Smooth Muscle Cells

Abstract 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 system in which diffuse calcification can be induced by culturing bovine vascular smooth muscle cells (BVSMC) in the presence of β-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 β-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 in...