Brilliant blue FCF is a nontoxic dye for saphenous vein graft marking that abrogates response to injury Kyle M. Hocking, PhD, Weifeng Luo, PhD, Fan Dong.

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Brilliant blue FCF is a nontoxic dye for saphenous vein graft marking that abrogates response to injury Kyle M. Hocking, PhD, Weifeng Luo, PhD, Fan Dong Li, PhD, Padmini Komalavilas, PhD, Colleen Brophy, MD, Joyce Cheung-Flynn, PhD Journal of Vascular Surgery Volume 64, Issue 1, Pages 210-218 (July 2016) DOI: 10.1016/j.jvs.2014.12.059 Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 1 Brilliant blue FCF (FCF) does not impair contractility in porcine saphenous veins (PSVs). Segments of PSVs (n = 3-15) were either left untreated (Ctrl), marked with a surgical skin marker (SSM) or a solution of FCF (2.6 mM, in 5% propylene glycol and water) with a cotton swab in a longitudinal line and incubated at room temperature in PlasmaLyte for 15 minutes, or treated with 50% isopropanol (IPA) or 1% methylene blue (MB) for 15 minutes. Treated segments were then incubated, cut into rings, suspended in a muscle bath, and treated with KCl (110 mM). Force generated was converted to stress. The error bars show the standard deviation; n.s., not significant. *P < .05 in paired t-tests. Journal of Vascular Surgery 2016 64, 210-218DOI: (10.1016/j.jvs.2014.12.059) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 2 Cytotoxicity of vein graft marking dyes on human umbilical venous smooth muscle cells (HUVSMCs). A, Phase contrast images of HUVSMCs either left untreated or treated with gentian violet (1%) dissolved in 50% isopropanol (GV/IPA), 50% isopropanol (IPA), 1% gentian violet (GV), or 2.6 mM brilliant blue FCF (FCF) for 10 minutes (left panels) and then stained with trypan blue (right panels). Minimal dead cells were detected in untreated and FCF-treated cells (black arrows), whereas all cells took up trypan blue after brief exposure to 50% IPA. GV alone also chemically fixed the cells and could not be washed off. Because the cells were already stained, no trypan blue staining was performed on these cells. The white arrows indicate precipitation of GV due to low organic solvent content. B, HUVSMCs seeded in 96-well plates were left untreated (−) or treated for 12 hours with staurosporine (+; 800 nM), methylene blue (MB; 5 and 10 mM), GV (50 and 100 μM), IPA (1%- 20%), FCF (50 μM), brilliant blue G (BBG; 50 μM), and allura red (Red; 50 μM). Cytotoxicity of the treatments was evaluated by measuring release of protease in the medium using a dead-cell protease assay and expressed as percentage of dead cells in each well. Each data point represents an average of triplicate wells for each treatment in each independent experiment. Data represent mean ± standard deviation; n = 5-7; *P < .05 in Tukey multiple comparison tests. Journal of Vascular Surgery 2016 64, 210-218DOI: (10.1016/j.jvs.2014.12.059) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 3 Brilliant blue FCF (FCF) restores functional viability after stretch injury in porcine saphenous vein (PSV). Pig saphenous veins were either untreated (Ctrl; n = 5-10) or stretched to twice their resting length (Stretched; n = 10). Stretched segments were then either left untreated, marked with a solution of FCF (2.6 mM, in 5% propylene glycol and water) with a cotton swab in a longitudinal line, or treated in 50 μM brilliant blue G (BBG) or 50 μM allura red (Red). The segments were then incubated at room temperature for 15 minutes in PlasmaLyte and cut into rings, suspended in a muscle bath, and treated with KCl (110 mM). Force generated was converted to stress. Results are presented as mean ± standard deviation. *P < .05, **P < .005 in paired t-tests. Journal of Vascular Surgery 2016 64, 210-218DOI: (10.1016/j.jvs.2014.12.059) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 4 Brilliant blue FCF (FCF) inhibits intimal thickening in porcine saphenous veins (PSVs) in organ culture. Rings from pig saphenous veins were either left untreated (Ctrl) or treated in the presence of FCF (50 μM), brilliant blue G (BBG; 50 μM), and allura red (Red; 50 μM) in organ culture for 14 days. Veins were stained with Verhoeff-van Gieson stain, and intimal layer thickening was measured. Results are presented as mean ± standard deviation. #P < .05 vs preculture; *P < .05 vs control; ƚP < .05 vs FCF in paired t-tests. Journal of Vascular Surgery 2016 64, 210-218DOI: (10.1016/j.jvs.2014.12.059) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 5 Brilliant blue FCF (FCF) inhibits P2X7R-mediated cytosolic Ca2+ fluxes in rat aorta. Rat aortic rings (n = 4-8) were suspended in the FluoroPlex muscle bath, either left untreated (control) or treated with FCF or the other P2X7R antagonists adenosine 5′-triphosphate periodate oxidized sodium salt (oATP), KN-62, or brilliant blue G (BBG) before contraction with 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP). Concurrent force generation (A) and cytosolic Ca2+ flux (B) were measured. Results are presented as mean ± standard deviation. *P < .0, **P < .001. ***P < .0001 vs control in paired t-tests. Journal of Vascular Surgery 2016 64, 210-218DOI: (10.1016/j.jvs.2014.12.059) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 6 Brilliant blue FCF (FCF) inhibits P2X7R-mediated contraction in human saphenous vein (HSV). A, Rings of HSV (n = 6) were suspended in the muscle bath, either left untreated (control) or treated with 50 μM FCF for 30 minutes before contraction with 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP). B, Representative muscle bath force tracings of BzATP-induced contraction in HSV. Results are presented as mean ± standard deviation. *P = .006 vs control in a paired t-test. C and D, Expression of P2X7R in HSV as detected by immunohistochemistry using preabsorbed (C) or normal P2X7R-specific antibody (D). P2X7Rs were stained red. Scale bar = 200 μm. L, Lumen; M, medial. Journal of Vascular Surgery 2016 64, 210-218DOI: (10.1016/j.jvs.2014.12.059) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 7 Model of P2X7R activation during vein graft preparation injury. Surgical harvest and preparation cause vein graft injury (1), leading to release of adenosine triphosphate (ATP, 2). ATP activates the P2X7 receptor on neighboring cells, propagating the response to injury (3). Brilliant blue FCF (FCF) may mitigate the effect of P2X7R activation (4) by inhibiting membrane pore formation, [Ca2+]i flux, and additional release of extracellular ATP. Agonists, red; inhibitors, blue. BBG, Brilliant blue G; BzATP, 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate; oATP, adenosine 5′-triphosphate periodate oxidized sodium salt. Journal of Vascular Surgery 2016 64, 210-218DOI: (10.1016/j.jvs.2014.12.059) Copyright © 2016 Society for Vascular Surgery Terms and Conditions