Fig. 1.

DCA induces VSMC calcification. A) Effects of DCA on VSMC viability and proliferation, as determined by a) MTS assay and b) BrdU incorporation. Results shown are from experiments using VSMC from 3 mice, and performed in triplicate. B&C) Effects of DCA on VSMC calcification. VSMC were cultured in osteogenic media with DCA (0–5 mM) for up to 21 days. Calcification was determined by a) ALP staining at 14 days; and b) Alizarin Red staining at 21 days; and C) Quantitative measurement of calcium content at 21 days in parallel sets of experiments. Representative images of 3 independent experiments performed in duplicate are shown (n = 3, *p < 0.05). D) Effects of DCA on the expression of smooth muscle and osteogenic marker genes. VSMC were exposed to DCA for 14 days, expression of smooth muscle markers SM22α, α-actin (SMA) and osteogenic markers ALP, ColIa, OC and Runx2 was determined by RT-PCR. Representative results from 3 independent experiments performed in duplicate are shown. E) Effects of DCA on arterial calcification ex vivo. Aortic rings from wild type mice were cultured in osteogenic media with DCA (0, 1 and 5 mM) for 21 days, and stained by H&E for histology (i, iii, v) and Alizarin Red (ii, iv, vi) for calcification. Higher magnification images of the boxed areas in a are shown in b. Results shown are representative images from 4 independent experiments.