Figure 5. AdvSca1-SM cells adopt a myofibroblast, and only rarely macrophage, phenotype in response to vascular injury.

Gli1-Cre^ERT-YFP mice were subjected to carotid arterial injury, and uninjured right and injured left carotids were harvested at 3 days, 7 days, and 3 weeks postinjury; fixed; and embedded in OCT. (A) Arterial sections were immunofluorescently stained for YFP (green), and in situ hybridization was used to detect periostin transcripts (red). Representative images from N = 3 at each time point. Note strong induction of periostin in AdvSca1-SM cells in response to injury. (B and C) Arterial sections were immunofluorescently stained for YFP (green) and CD68 (B; red) or MAC2 (C; red). Representative images from N = 3 per time point. Note that AdvSca1-SM cells do not coexpress either macrophage marker. Scale bars for panels A–C: 50 μm. M, arterial media; A, arterial adventitia; NI, neointima. Dashed lines indicate the external and internal elastic laminae. (D) Single-cell suspensions were isolated from uninjured and 7-day postinjured carotid arteries; cells were stained for SCA1, YFP, CD11c, MHCII, LY6G, CD11b, CD64, αSMA, and Aqua VI and analyzed by flow cytometry. Total macrophages (left graph) and YFP⁺ AdvSca1-SM cell-derived macrophages (middle right) were quantified as percentage of all live cells. Median expression of αSMA (middle left) and CD64 (right) by the YFP⁺ cell population was expressed as absolute value. Each point represents an individual carotid artery; N = 3. Data represent mean ± SEM; paired Student’s t tests (2 tailed); *P < 0.05; **P < 0.01.