Brilliant blue FCF is a nontoxic dye for saphenous vein graft marking that abrogates response to injury

Abstract

Background: Injury to saphenous vein grafts during surgical preparation may contribute to the subsequent development of intimal hyperplasia, the primary cause of graft failure. Surgical skin markers currently used for vascular marking contain gentian violet and isopropanol, which damage tissue and impair physiologic functions. Brilliant blue FCF (FCF) is a nontoxic dye alternative that may also ameliorate preparation-induced injury.

Methods: Porcine saphenous vein (PSV) was used to evaluate the effect of FCF on physiologic responses in a muscle bath. Cytotoxicity of FCF was measured using human umbilical venous smooth muscle cells. Effect of FCF on the development of intimal hyperplasia was evaluated in organ culture using PSV. Intracellular calcium fluxes and contractile responses were measured in response to agonists and inhibitors in rat aorta and human saphenous vein.

Results: Marking with FCF did not impair smooth muscle contractile responses and restored stretch injury-induced loss in smooth muscle contractility of PSV. Gentian violet has cytotoxic effects on human umbilical venous smooth muscle cells, whereas FCF is nontoxic. FCF inhibited intimal thickening in PSV in organ culture. Contraction induced by 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate and intracellular calcium flux were inhibited by FCF, oxidized adenosine triphosphate, KN-62, and brilliant blue G, suggesting that FCF may inhibit the purinergic receptor P2X7.

Conclusions: Our studies indicated that FCF is a nontoxic marking dye for vein grafts that ameliorates vein graft injury and prevents intimal thickening, possibly due to P2X7 receptor inhibition. FCF represents a nontoxic alternative for vein graft marking and a potentially therapeutic approach to enhance outcome in autologous transplantation of human saphenous vein into the coronary and peripheral arterial circulation.

Figure 1. FCF does not impair contractility in porcine saphenous veins

Segments of porcine saphenous veins (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 in at room temperature in PlasmaLyte for 15min, or treated with 50% isopropanol (IPA) or 1% methlyene blue (MB) for 15 min 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. *P< 0.05 in paired t-tests.

Figure 2. Cytotoxicity of vein graft marking dyes on human umbilical venous smooth muscle cells (HUVSMC)

(A) Phase contract images of HUVSMC either left untreated or treated with gentian violet (GV; 1%) dissolved in 50% isopropanol (GV/IPA), 50% IPA, 1% GV, or 2.6mM FCF for 10 min (left panels) and then with 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. White arrows indicated precipitation of GV due to low organic solvent content. (B) HUVSMC seeded in 96-well plates were left untreated (−) or treated for 12 hrs with staurosporine (+; 800nM), MB (5 and 10mM), GV (50 and 100μM); IPA (1–20%); FCF (50μM); BBG (50μM); and allura red (Red; 50μM). Cytotoxicity of the treatments were evaluated by measuring release of protease in the medium using a dead-cell protease assay and expressed as % dead cell of each well. Each data point represents an average of triplicate wells for each treatment in each independent experiment. Data represents mean±SD; n=5–7; *P<0.05 in Tukey’s Multiple Comparison tests.

Figure 3. FCF restores functional viability after stretch injury in porcine saphenous vein

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) using a cotton swab in a longitudinal line, or treated in 50μM brilliant blue G (BBG) or 50μM Allura red (Red). The segments were then incubated at room temperature for 15 min 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±SD. *P< .05, **P<.005 in paired t-tests.

Figure 4. FCF inhibits intimal thickening in porcine saphenous veins in organ culture

Rings from pig saphenous veins were either left untreated (ctrl) or treated in the presence of FCF (50μM); BBG (50μM); and allura red (Red; 50μM) in organ culture for 14 days. Veins were stained using Verhoff Van Gieson stain and intimal layer thickening was measured. Results are presented as mean±SD. #P<0.05 vs pre-culture; *P<.05 vs control; ^ƚP<.05 vs FCF in paired t-tests.

Figure 5. 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 other P2X₇R antagonists oATP, KN62 or BBG prior to contraction with BzATP. Concurrent force generation (A) and cytosolic Ca²⁺ flux (B) were measured. Results are presented as mean±SD. *P<0.0, **P<0.001. ***P<0.0001 vs to control in paired t-tests.

Figure 6. FCF inhibits P2X₇R-mediated contraction in human saphenous vein

A. Rings of HSV (n=6) were suspended in the muscle bath, either left untreated (control) or treated with 50μM FCF for 30min prior to contraction with BzATP. B. Representative muscle bath force tracings of BzATP-induced contraction in HSV. Results are presented as mean±SD. *P=0.006 vs control in a paired t-test. Expression of P2X₇R in HSV as detected by immunohistochemistry using pre-absorbed (C) or normal P2X₇R-specific antibody (D). P2X₇R were stained red. Scale bar = 200 μm; L= lumen, M=medial.

Figure 7. Model of P2X₇R activation during vein graft preparation injury

Surgical harvest and preparation cause vein graft injury (1), leading to release of ATP (2). ATP activates the P2X₇ receptor on neighboring cells, propagating the response to injury (3). 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)