Rat aortic rings were prepared using the same method as in the above section. Aortic rings were embedded in NO gels with hMSCs (106 cells/ml; n = 2) and maintained at 37°C and 5% CO2 in EGM-2 for 14 days. Expressions of pericyte-related markers including CYP1B1, S1PR1, NG2, and PDGFRβ were evaluated by immunocytochemistry as described above. Expression of angiogenesis-related markers was additionally evaluated by immunostaining using primary rabbit anti-Flk1 antibody (1:100; Santa Cruz Biotechnology) with secondary biotinylated goat anti-rabbit IgG (Vector Labs) antibodies. To visualize aortic rings, Cy5-conjugated streptavidin (Vector Labs) was treated with fluorescein isothiocyanate–conjugated lectin (Sigma-Aldrich). To determine cells of human origin, human mitochondria were stained with primary anti-human mitochondria antibodies (1:20; Millipore). Z-stack or single-plane images were obtained using an LSM780 confocal microscope (Zeiss), and the images were analyzed using ZEN imaging software (ZEN 2012; Zeiss).

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