Surgical marking pen dye inhibits saphenous vein cell proliferation and migration in saphenous vein graft tissue Shinsuke Kikuchi, MD, Richard D. Kenagy,

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Surgical marking pen dye inhibits saphenous vein cell proliferation and migration in saphenous vein graft tissue Shinsuke Kikuchi, MD, Richard D. Kenagy, PhD, Lu Gao, MD, Thomas N. Wight, PhD, Nobuyoshi Azuma, MD, Michael Sobel, MD, Alexander W. Clowes, MD Journal of Vascular Surgery Volume 63, Issue 4, Pages 1044-1050 (April 2016) DOI: 10.1016/j.jvs.2014.10.017 Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 1 Cell migration from blue vs nonblue saphenous vein explants. Data are the mean ± standard error of the mean of migration (percentage migration-positive explants, A and C; cells/explant, B and D) from explants of the outer layer (A and B, n = 11 veins) and inner layer (C and D, n = 8 veins). ∗P < .001 vs blue outer layer or inner layer. Journal of Vascular Surgery 2016 63, 1044-1050DOI: (10.1016/j.jvs.2014.10.017) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 2 Ki67 (proliferation) index in blue vs nonblue sections of veins in organ culture. Cross-section of human saphenous vein stained with Ki67 antibody and hematoxylin (A). For counting purposes, veins were divided into sections of inner media plus intima (IL), outer media (O), and adventitia (Adv). The dotted lines indicate the boundaries of inner media, outer media, and adventitia. Data are the mean ± standard error of the mean of inner medial/intimal (B), outer medial (C), and adventitial (D) percentage Ki67-positive nuclei. n = 5 veins at days 0 and 6, 3 at days 2 and 4. ∗P < .05 blue vs nonblue. Journal of Vascular Surgery 2016 63, 1044-1050DOI: (10.1016/j.jvs.2014.10.017) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 3 TUNEL staining (A) in blue (lower panel) vs nonblue (upper panel) sections of one representative cross section (100×) of a vein in organ culture (Adv, adventitia; L, lumen; M, media). Data are the mean ± standard error of the mean of inner medial/intimal (B), outer medial (C), and adventitial (D) percentage TUNEL-positive nuclei. N = 5 veins at days 0 and 6, 3 at days 2 and 4. Journal of Vascular Surgery 2016 63, 1044-1050DOI: (10.1016/j.jvs.2014.10.017) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 4 Dose-response effect of crystal violet on cell migration from venous outer (closed circles) and inner (open circles) layer explants. Each point represents data from 15 explants in one flask treated with or without the indicated amount of crystal violet normalized to the value of explants not receiving the dye for that particular vein (100%). The amount of crystal violet was determined by extraction from parallel explants. Journal of Vascular Surgery 2016 63, 1044-1050DOI: (10.1016/j.jvs.2014.10.017) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 5 Dose-response effect of crystal violet on proliferation (A), death (B), and migration (C) of outer layer cells. n = 4 experiments performed in duplicate (A and C) or quadruplicate (B). ∗,#P < .05 vs 0 μg/mL. HPF, High-power field (400×). Journal of Vascular Surgery 2016 63, 1044-1050DOI: (10.1016/j.jvs.2014.10.017) Copyright © 2016 Society for Vascular Surgery Terms and Conditions