Fig. 3.

p-SMAD 1/5/9 staining was increased in arterioles of the subcutaneous fat in patients with calciphylaxis (a–d). p-SMAD staining was detected in smooth muscles cells in the medial layer as well as the smooth muscle actin (SMA)-negative cells in the intima (e–h). Von Kossa staining revealed the presence of a calcified vessel in subcutaneous tissue (a, b). High levels of nuclear p-SMAD 1/5/9 were detected in cells in an adjacent section of the same vessel (c, d). Representative p-SMAD 1/5/9-positive cells are indicated by arrows in d. Anti-SMA antibody was used to indicate the location of smooth muscle cells in the arteriole in a section from a patient with calciphylaxis (e, f). Staining for p-SMAD 1/5/9 in an adjacent section revealed nuclear p-SMAD in smooth muscle cells in the media layer (indicated by asterisks in f and h) as well as in SMA-negative cells in the intimal layer (indicated by arrows in h). Black boxes in (a, c, e, g) indicate the magnified regions shown in (b, d, f, h) respectively. Black bars in (a, c, e, g) indicate 100 μm. Black bars in (b, d, f, h) indicate 20 μm.