Attenuation of IH by the HDAC6 Inhibition in a Rat Restenosis Model
Balloon angioplasty was performed in rat carotid arteries to induce intimal hyperplasia (IH) and restenosis (lumen narrowing), and vehicle (equal-amount dimethylsulfoxide) or tubastatin A (2 mg/rat) or RGFP (2 mg/rat) was applied in a Triblock hydrogel. Arteries were collected 14 days later for morphometric analysis. (A and C) Representative van Gieson– and hematoxylin and eosin–stained cross sections, respectively. Neointimal thickness is indicated between arrowheads; arrow (green) points to external elastic lamina (EEL). (B and D) Quantification of IH (intima/media area [I/M] ratio), lumen area, and EEL length. Data are quantified as fold changes versus vehicle control (normalized value as 1); mean ± SEM; n = 3 to 5 animals; *p < 0.05 versus control (one-sample Student’s t-test). (E, F) α-SMA immunostaining and quantification. A threshold of fluorescence intensity was set with ImageJ software to exclude the adventitia layer. Fluorescence in each image was then normalized to the total number of 4′,6′-diamidino-2-phenylindole (DAPI)-stained nuclei in the medial and neointimal layers, which was manually counted. Data presentation: mean ± SEM; n = 3 to 5 animals; *p < 0.05 (1-way analysis of variance). Abbreviations as in Figures 1 and 2.