Fig. 5. TIMP4 represses calcification of VSMCs. VSMCs were transiently transfected with pCMV-TIMP4 and incubated in the presence of 3 mM Pi for 2 days (A, B). VSMCs were transfected with siTIMP4-1, 2 and 3 and incubated for 2 days (C). Protein levels of TIMP4, Dlx5, and CBFA1 measured by Western blot analysis (A, C, upper panel). Protein levels of TIMP4, Dlx5, and CBFA1 were measured by densitometry analysis (A, C, lower panel). Alizarin red S staining (B). Data represent the mean±standard error of the mean of 3 individual experiments.

TIMP, tissue inhibitor of metalloproteinase; VSMC, vascular smooth muscle cell; Pi, inorganic phosphate; Dlx5, distal-less homeobox 5; CBFA1, core-binding factor α-1.

Statistical significance determined relative to a control by the Student's t-test (^*p<0.05; ^†p<0.01; ^‡p<0.001), and compared with phosphate-treated cells by the Student's t-test (^§p<0.05; ^∥p<0.01).