BETA-GLYCEROPHOSPHATE ACCELERATES CALCIFICATION IN CULTURED BOVINE VASCULAR SMOOTH-MUSCLE CELLS

Calcification is a common feature of advanced atherosclerotic lesions and is being reemphasized as a clinically significant element of vascu lar disease. However, the scarcity of in vitro models of vascular calc ification preclude studying its molecular and cellular mechanism. In t he present study, we describe an in vitro calcification system in whic h diffuse calcification can be induced by culturing bovine vascular sm ooth muscle cells (BVSMC) in the presence of beta-glycerophosphate, as corbic acid, and insulin in a manner analogous to in vitro mineralizat ion by osteoblasts. Calcification was confirmed by von Kossa staining and Ca-45 accumulation. Factor analysis revealed that beta-glycerophos phate is the most important factor for this calcification process, sug gesting 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 demons trating that levamisole, a specific inhibitor of ALP, inhibited BVSMC calcification in a dose-dependent manner. Bisphosphonates such as etid ronate and pamidronate potently inhibited BVSMC calcification, suggest ing that hydroxyapatite formation may be involved. Importantly, expres sion 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 a nalyzing the molecular and cellular mechanisms of vascular calcificati on.