FIG 4.

SMC-specific IRF8 overexpression promotes neointima formation and SMC phenotypic switching in response to wire injury. (A) Schematic of the SMC-specific TG vector structure. The vector was first constructed with a mouse SM22α promoter, a 5′ HA tag, and an SV40 poly(A) signal. The mouse full-length IRF8 cDNA (mIRF8) was cloned into the vector between the EcoRI sites. (B) Arterial IRF8 protein levels in TG and WT mice determined by Western blotting. (C) HE-stained sections show the structures of nontransgenic (NTG) and SMC-specific IRF8 TG mouse carotid arteries at 14 days and 28 days after wire injury surgery. Black-framed areas in the upper panels are magnified and shown in the lower panels. Arrowheads, internal elastic lamina (IEL) and external elastic lamina (EEL), highlighted in dotted lines. The internal elastic lamina indicates the intimal-medial boundary, while the external elastic lamina represents the medial-adventitial boundary; M, media; NI, neointima. Intimal areas and intima/media ratios were quantified (n = 6 to 12 per group at each time point). Bars, 50 μm. (D) Immunofluorescent staining of α-SMA and SM22α (green in different rows) in nontransgenic and TG mouse arteries at 14 and 28 days after injury. DAPI (blue) staining marks the nuclei. The optical density values of α-SMA and SM22α fluorescence are also provided (n = 3 or 4 per group at each time point). White bar, 20 μm. (E) Levels of α-SMA, SM22α, smoothelin, OPN, and desmin protein in nontransgenic and TG mouse arteries were determined by Western blotting. Arteries harvested from the sham operation group and the injured group at 28 days postinjury were used. The expression levels were normalized to the expression level of GAPDH and quantified. Blots are representative of three independent experiments. In panels B to E, all of the values are presented as means ± SDs, and statistical significance is indicated. *, P < 0.05 compared with the nontransgenic group.