Figure 2.

Effects of statins and ascorbic acid on AoSMC culture calcification. Human AoSMC were seeded in plastic plates in 5% FBS/DMEM. Additions of statins and ascorbic acid (AsA) started after cells grew to confluent layer in 5 mM b-glycerophoshate/5% FBS/DMEM supplemented (B, C) or nonsupplemented (A) with 25 mcM forskolin. Medium was changed every two or three days. After 10 day incubation cell culture Calcium content (A, B) was measured in 0.6 N HCl whole cell layer extracts (48 hours incubation at 37°C). Cell-associated alkaline phosphatase activity (C) was analyzed by fluorescent assay after incubation with 25 mcg/ml 4-MUP (fluorescent ALP substrate, Sigma). Experimental procedures are described in more details in Materials and Methods section and in the Figure 1 legend. * and ~, indicate significant differences from unsupplemented controls and from ascorbiate-free samples, respectively (P<0.05) in two-tailed t-test.