Calcification of human vascular smooth muscle cells: associations with osteoprotegerin expression and acceleration by high-dose insulin

Arterial medial calcifications occur often in diabetic individuals as part of the diabetic macroangiopathy. The pathogenesis is unknown, but the presence of calcifications predicts risk of cardiovascular events. We examined the effects of insulin on calcifying smooth muscle cells in vitro and measured the expression of the bone-related molecule osteoprotegerin (OPG). Human vascular smooth muscle cells (VSMCs) were grown from aorta from kidney donors. Induction of calcification was performed with β-glycerophosphate. The influence of insulin (200 μU/ml or 1,000 μU/ml) on calcification was judged by measuring calcium content in the cell layer and by von Kossa staining. OPG was measured in the medium by ELISA. Histochemistry was used for determination of alkaline phosphatase (ALP). Bone sialoprotein (BSP) and OPG mRNA expressions were done by RT-PCR. β-Glycerophosphate was able to induce calcification in human smooth muscle cells from a series of donors after variable time in culture. Decreased OPG amounts we...