Figure 4. AdvSca1-SM cells contribute to injury-induced adventitial remodeling.

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), αSMA (red), and SCA1 (white). Representative images from N = 7 (3 days), N = 10 (7 days), and N = 11 (3 weeks). Scale bars: 50 μm. M, arterial media; A, arterial adventitia, NI, neointima. Dashed lines indicate the external and internal elastic laminae. Note a time-dependent expansion of AdvSca1-SM cells predominantly in the adventitia, with a concomitant loss of AdvSca1-SM cell SCA1 expression, but also migration into the arterial media. (B) Uninjured and 3- and 7-day postinjury carotid artery sections were immunofluorescently stained for YFP (green) to identify AdvSca1-SM cells. Sections were imaged for coexpression of YFP and label-free SHG for collagen deposition (red). Elastin autofluorescence is also observed on the green channel. Representative 40× images from N = 6 per condition. M, arterial media; A, arterial adventitia. (C–F) Quantification of stained images from A and B. (C) Total YFP⁺ cells. (D) Percentage of YFP⁺SCA1⁺ cells per total YFP⁺ cells. (E) Percentage of YFP⁺αSMA⁺ cells per total YFP⁺ cells. (F) Normalized SHG+ area (pixel); SHG signal was normalized the outer medial circumference. Data represent mean ± SEM; (C, E, and F) Kruskal-Wallis test followed by Dunn’s multiple-comparison test; (D) 1-way ANOVA with Bonferroni’s post hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.