Surgical marking pen dye inhibits saphenous vein cell proliferation and migration in saphenous vein graft tissue Shinsuke Kikuchi, MD,a,b Richard D. Kenagy, PhD,a Lu Gao, MD,a Thomas N. Wight, PhD,c Nobuyoshi Azuma, MD,b Michael Sobel, MD,a,d and Alexander W. Clowes, MD,a Seattle, Wash; and Asahikawa, Japan Objective: Markers containing dyes such as crystal violet (CAS 548-62-9) are routinely used on the adventitia of vein bypass grafts to avoid twisting during placement. Because little is known about how these dyes affect vein graft healing and function, we determined the effect of crystal violet on cell migration and proliferation, which are responses to injury after grafting. Methods: Fresh human saphenous veins were obtained as residual specimens from leg bypass surgeries. Portions of the vein that had been surgically marked with crystal violet were analyzed separately from those that had no dye marking. In the laboratory, they were split into easily dissected inner and outer layers after removal of endothelium. This cleavage plane was within the circular muscle layer of the media. Cell migration from explants was measured daily as either (1) percentage of migration-positive explants, which exclusively measures migration, or (2) number of cells on the plastic surrounding each explant, which measures migration plus proliferation. Cell proliferation and apoptosis (Ki67 and TUNEL staining, respectively) were determined in dye-marked and unmarked areas of cultured vein rings. The dose-dependent effects of crystal violet were measured for cell migration from explants as well as for proliferation, migration, and death of cultured outer layer cells. Dye was extracted from explants with ethanol and quantified by spectrophotometry. Results: There was significantly less cell migration from visibly blue compared with unstained outer layer explants by both methods. There was no significant difference in migration from inner layer explants adjacent to blue-stained or unstained sections of vein because dye did not penetrate to the inner layer. Ki67 staining of vein in organ culture, which is a measure of proliferation, progressively increased up to 6 days in nonblue outer layer and was abolished in the blue outer layer. Evidence of apoptosis (TUNEL staining) was present throughout the wall and not different in blue-stained and unstained vein wall segments. Blue outer layer explants had 65.9 6 8.0 ng dye/explant compared with 2.1 6 1.3 for nonblue outer layer explants. Dye applied in vitro to either outer or inner layer explants dose dependently inhibited migration (IC50w10 ng/explant). The IC50s of crystal violet for outer layer cell proliferation and migration were 0.1 and 1.2 mg/ mL, whereas the EC50 for death was between 1 and 10 mg/mL. Conclusions: Crystal violet inhibits venous cell migration and proliferation, indicating that alternative methods should be considered for marking vein grafts. (J Vasc Surg 2015;-:1-7.) Clinical Relevance: More than 300,000 coronary artery and peripheral vein grafts are performed annually with use of human saphenous veins. About 30% of these grafts fail within a year in part because of intimal hyperplasia, which arises in response to vein graft injury. We have found that the dye in marking pens surgeons use to mark the vein graft adversely affects the health of the cells in the graft: it inhibits venous cell migration and proliferation and increases cell death. Limiting the use of such dyes may reduce early vein graft injury during harvest and preparation and may have the potential to reduce subsequent vein graft failure in patients.
From the Department of Surgery, University of Washington, Seattlea; the Department of Vascular Surgery, Asahikawa Medical University, Asahikawa, Japanb; the Matrix Biology Program, Benaroya Research Institute at Virginia Mason, Seattlec; and the Division of Vascular Surgery, VA Puget Sound Health Care System and University of Washington, Seattle.d NIH grants R01 HL30946 (A.W.C., M.S.) and R41 HL106967 (T.N.W.), Department of Veterans Affairs, Veterans Health Administration Merit Review Agency (M.S.). Author conflict of interest: none. Presented in part as a poster at the Arteriosclerosis, Thrombosis, and Vascular Biology meeting, Toronto, Canada, May 1-3, 2014. Reprint requests: Richard D. Kenagy, PhD, Center for Cardiovascular Biology, University of Washington, PO Box 358050, 850 Republican St, Seattle, WA 98109 (e-mail: [email protected]). The editors and reviewers of this article have no relevant financial relationships to disclose per the JVS policy that requires reviewers to decline review of any manuscript for which they may have a conflict of interest. 0741-5214 Copyright Ó 2015 by the Society for Vascular Surgery. http://dx.doi.org/10.1016/j.jvs.2014.10.017
Vein grafts are frequently used for aortocoronary and peripheral vascular reconstructions. However, severe luminal narrowing is a primary cause of graft failure.1 This typically develops within the first year in approximately 25% to 30% of all grafts because of pathologic remodeling and intimal hyperplasia. Whereas much research is aimed at determining the pathologic mechanisms of luminal narrowing, technical factors such as surgical graft preparation are also important.2 For example, other factors, such as conduit distention,3 vein conduit storage,4 and stretching during surgical harvest,5 can adversely affect the function of vein wall cells. Surgical marking pens containing dyes such as crystal violet are routinely used on the adventitia of vein bypass grafts to avoid twisting of the vein grafts during placement. It continues to be assumed that these pens are innocuous despite 1
2 Kikuchi et al
evidence that dyes used in these pens can inhibit endothelium-dependent vasodilation6,7 and the smooth muscle contractile response.8 To further explore possible deleterious effects of marking pens on vein grafts, we have studied the effect of crystal violet on venous cell migration, proliferation, and apoptosis. These are the major biologic activities that contribute to intimal hyperplasia in both large and small animal models of vein graft healing.9-15 METHODS Procurement of human saphenous veins and migration assay. Human saphenous vein remnants were obtained from patients undergoing coronary artery bypass or peripheral vascular bypass surgeries under protocols approved by the University of Washington or the Benaroya Research Institute Institutional Review Board, and all subjects gave informed consent as indicated in these protocols. Veins were either endoscopically or openly harvested. All were distended to identify untied branches and were stored in heparinized buffered saline before being placed into Dulbecco’s modified Eagle’s medium (DMEM) with 10 mM HEPES, pH 7.4, for transport to the laboratory for dissection. The use of a surgical skin marker was decided by the surgical team. The human saphenous vein was dissected free of extraneous tissue, and sections that were marked intraoperatively with blue dye were separated from unmarked sections. A visually distinct, white, luminal layer was easily dissected from the remaining vein after removal of endothelium by gentle wiping with a cottontipped swab. The identity of the natural cleavage plane of vein dissection was confirmed by immunohistochemical staining for smooth muscle a-actin of the dissected layers. This demonstrated that both outer and inner layers contain smooth muscle a-actin-positive cells and that the cleavage plane goes through the circular muscle layer. The layers, hereafter called inner layer and outer layer, respectively, were cut into 2.5-mm2 explants by a McIlwain tissue chopper (customized to cut sizes >1 mm2). Explants were placed into 25-cm2 flasks (15/flask), and 1.2 mL of DMEM with 20% fetal bovine serum (DMEM/20% FBS) was added. Medium was changed three times per week. Explants were examined daily for 8 days, and cell migration was quantified as both the percentage of total explants with at least one migrating cell (a measure of only migration) and the average number of cells on plastic surrounding each explant (a measure of the combination of migration and proliferation).16 Extraction and quantification of dye. To determine the amount of dye adsorbed to the different vein samples during the surgical marking, explants prepared as described before were divided into four groups: (1) visibly bluestained outer layer explants, (2) inner layer explants luminally adjacent to blue-stained outer layer explants, (3) outer layer explants with no visible blue dye, and (4) inner layer explants luminally adjacent to outer layer explants with no visible blue dye. Crystal violet was quantified spectrophotometrically (OD590, the lmax of crystal violet) with use of crystal violet standards dissolved in ethanol.
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Pens obtained from the surgical services providing specimens for this study were confirmed to contain crystal violet, and dye extracted from vein graft specimens displayed a lmax of 590 nm. This assay is linear up to 50 mg/mL crystal violet. Blue dye was extracted by immersing five explants of each group in 100 mL of absolute ethanol and incubating at room temperature for 30 minutes. Extraction efficiency of crystal violet added to unstained explants was 80% with a first extraction and >90% with a third extraction. Whereas crystal violet may be tightly bound to tissue components such as DNA,17 most dye is available for extraction. Dose-response effect of crystal violet on cell migration. To determine the effects of crystal violet on migration of cells from the explants, 1 mL of crystal violet or buffer alone (0-500 ng/mL) was added to explants (in the absence of culture medium) from unstained sections of vein and incubated for 5 minutes. Separate experiments using nonblue outer layer or adjacent inner layer explants were performed as indicated in the figure legends. Some explants were then extracted to measure bound dye; other explants were placed in a 25-cm2 flask after one wash with DMEM/HEPES (15 explants per condition per vein for the number of veins indicated in the figure legends). Explants were given growth medium as before, and the percentage of migration-positive explants was determined at day 8. Measurement of proliferation and apoptosis in organ culture. After removal of loose connective tissue, veins were cut into rings w3 mm in length and placed in 20% FBS/DMEM (one/2 mL). The medium was changed every other day, and the tissue was fixed in 10% neutral buffered formalin at 0, 2, 4, and 6 days. Blue areas were marked with India ink before processing and embedding in paraffin because the blue dye is removed during processing. We measured cell proliferation by immunohistochemical staining of 5-mm sections with Ki67 (0.5 mg/mL SP6; Abcam, Inc, Cambridge, Mass) using the avidin-biotin-peroxidase method (Vector Laboratories, Burlingame, Calif) with rabbit immunoglobulin G as a negative control. For measurement of apoptosis, the terminal deoxynucleotidyl transferase (TdT)emediated dUTP-biotin nick end labeling (TUNEL) assay was performed with a kit (ApopTag; Millipore, Jaffrey, NH) following the manufacturer’s instructions but specifically using TdT at 1:32, citrate buffer (pH 4.5) rather than proteinase K, and omission of TdT as a negative control. To compare blue dyeestained sections to unstained sections, the percentage of Ki67-or TUNEL-positive nuclei was determined in adventitia, outer media, and inner media/intima for the portion of the perimeter marked by India ink (ie, blue dye) as well as for the same perimeter length on the opposite side of the vein (unstained). Adventitia plus outer media is equivalent to the outer layer designation, and inner media/intima is the same as the inner layer designation. Dose-response effects of crystal violet on outer layer cell proliferation, migration, and death. Cultured outer layer cells were obtained by the explant method from
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Fig 1. Cell migration from blue vs nonblue saphenous vein explants. Data are the mean 6 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 ¼ 11 veins) and inner layer (C and D, n ¼ 8 veins). *P < .001 vs blue outer layer or inner layer.
saphenous vein unstained outer layer explants in 20% FBS/ DMEM. When cells near the explants become confluent, we found that the optimal medium is smooth muscle cell growth medium (Cell Applications, Inc, San Diego, Calif), which was also used for subsequent growth of passaged cells. Cells were used at passage 6. For proliferation and death assays, 48,000 cells were seeded in 5% FBS in bovine basal medium (Cell Applications, Inc) in duplicate six-well plates. The next day, cells were counted with a hemocytometer, and the medium in remaining wells was changed to Cell Applications smooth muscle cell growth medium for proliferation studies or transferred to 2% serum for studies of cell death. Cells were then exposed for 3 days to a range of concentrations of crystal violet (0, 0.01, 0.1, 1.0, 10 mg/mL) and then counted again. Preliminary experiments demonstrated that cell number is maintained but not increased with 2% FBS alone. For migration assays, cells were preincubated for 30 minutes with crystal violet (0, 0.01, 0.1, 1.0, 10 mg/mL) and then seeded into the upper well of a 48-well microchemotaxis chamber at 30,000 per well (Neuro Probe, Inc, Gaithersburg, Md) in bovine basal medium in the presence of the same concentrations of dye. Polycarbonate filters with 8-mm pores coated with 100 mg/mL monomeric
Table. Amount of surgeon-applied marking pen dye extracted from explants Extracted blue dye (ng/explant) Outer layer explant Visibly unmarked explant Visibly blue explant Inner layer explant Visibly unmarked explant Visibly blue explant
2.1 6 1.3 65.9 6 8.0a 0.5 6 0.3 2.1 6 1.5
Data are the mean 6 standard error of the mean of the amount of dye in explants from seven to 11 veins (outer layer) or four or five veins (inner layer). a P < .01 vs unmarked explant.
bovine skin collagen were used, and bottom chambers contained 10% serum. After 5 hours at 37 C, the cells that had migrated through the filters were fixed and counted at 400. Statistical analysis. Comparisons of measures between unstained and blue-stained explants and between various concentrations of dye were made with analysis of variance with repeated measures or the Wilcoxon test with a Bonferroni correction as needed (SPSS, v20; SPSS Inc, Chicago, Ill). A P # .05 was considered statistically
4 Kikuchi et al
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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 6 standard error of the mean of inner medial/intimal (B), outer medial (C), and adventitial (D) percentage Ki67-positive nuclei. n ¼ 5 veins at days 0 and 6, 3 at days 2 and 4. *P < .05 blue vs nonblue.
significant. Data are presented as the mean 6 standard error of the mean. RESULTS Cells began migrating from saphenous vein explants after a lag phase of about 3 days, with outer layer cells initiating migration more quickly than inner layer cells. Migration was significantly less from blue-stained compared with unstained outer layer explants by day 5. There was a reduction of >80% migration at day 8 by both measures of migration (Fig 1, A and B). Inner layer explants separated into those adjacent to blue-stained outer layer and those adjacent to unstained outer layer exhibited equal cell migration (Fig 1, C and D), which is consistent with the visual observation that the staining from the surgeon’s marking pen almost never penetrated into the inner layer. Of the hundreds of veins dissected in this laboratory, only one vein had lightly visible staining of the inner layer from the surgeon’s marking pen. Indeed, although bluestained outer layer explants had more than 30-fold the amount of dye seen in visibly unstained outer layer explants, there was no difference in amount of blue dye detected in inner layer explants, whether they were adjacent to a blue-stained outer layer or an unstained outer layer (Table). The effect of surgeon-applied blue dye on cell proliferation and apoptosis in vein rings was determined by
comparing Ki67 and TUNEL staining in the blue-stained perimeter of vein compared with the same perimeter length of unstained vein on the opposite side of the vein ring. These were whole, undissected vein segments. The percentages of Ki67-and TUNEL-positive cells were counted separately in the inner media/intima, outer media, and adventitia separated as illustrated in Fig 2, A. In the unstained portion of the vein, proliferation increased progressively between days 2 and 6, with adventitia showing the highest proliferation, followed by the outer media, then the inner media/intima. In contrast, there was no proliferation observed in the blue-stained side of the vein except in the inner media/intima, which is again consistent with a lack of penetration of blue dye into the inner layer (Fig 2, B-D). In contrast, TUNEL-positive cells were observed at all times primarily in the adventitia (Fig 3, A), but no difference was noted between dye-positive and dye-negative areas of the vessel (Fig 3, B-D). To rule out the possibility of a difference in response to the dye between outer layer and inner layer cells, we also performed crystal violet dose-response studies on unstained outer layer and adjacent inner layer explants (Fig 4). Inhibition of migration from both types of explants occurred with an IC50 of w10 ng/explant. Dose-response curves for crystal violet for proliferation, migration, and death of cultured outer layer cells were also determined. At the lowest concentrations, it inhibited proliferation, and at
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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 6 standard error of the mean of inner medial/intimal (B), outer medial (C), and adventitial (D) percentage TUNEL-positive nuclei. N ¼ 5 veins at days 0 and 6, 3 at days 2 and 4.
the highest concentrations, it induced cell death. The IC50 was w0.1 mg/mL for proliferation and w1.2 mg/mL for migration; for death, the EC50 was between 1 and 10 mg/mL (Fig 5, A-C). DISCUSSION We have found that crystal violet, the dye used in many surgical marking pens, has a strong inhibitory effect on migration and proliferation of cells in the vein ex vivo. These data add to a growing body of evidence that commonly used marking pens have deleterious effects on the veins used for grafting, such as inhibition of both vasoconstrictor and vasodilator function.6-8 Surgical marking pens have dyes, such as crystal violet and methylene blue, dissolved in either ethanol or polyethylene glycol. In this study, we focused on the effect of crystal violet, a dye in pens used in Seattle, and did not address effects of the diluent. Whereas ethanol also has been shown to inhibit smooth muscle cell proliferation and migration,18,19 expected inhibition of cell migration from outer layer explants based on levels of dye measured in surgeon-treated veins was 99.9% (Fig 4) compared with observed inhibition of 86.1% (Fig 1, A), suggesting little if any effect of diluents on cell migration in this case. The inhibition of cell migration from explants is probably not directly linked to inhibition of cell proliferation or increased cell death. Whereas the IC50 for adventitial
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.
fibroblast proliferation is 10-fold lower than that for migration (0.1 vs 1.2 mg/mL, respectively), inhibitors of proliferation such as hydroxyurea do not appreciably alter migration from explants,20 and nonproliferating vascular cells migrate after injury.21 Nor does the inhibition of
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6 Kikuchi et al
Fig 5. Dose-response effect of crystal violet on proliferation (A), death (B), and migration (C) of outer layer cells. n ¼ 4 experiments performed in duplicate (A and C) or quadruplicate (B). *,#P < .05 vs 0 mg/mL. HPF, High-power field (400).
migration appear to relate to cell death. For if we assume that the 2.5-mm2 adventitial explants have a volume of w1 mm3, the measured amount of dye/explant achieves a concentration of nearly 70 mg/mL. This is well beyond the EC50 for death (
From the Department of Surgery, University of Washington, Seattlea; the Department of Vascular Surgery, Asahikawa Medical University, Asahikawa, Japanb; the Matrix Biology Program, Benaroya Research Institute at Virginia Mason, Seattlec; and the Division of Vascular Surgery, VA Puget Sound Health Care System and University of Washington, Seattle.d NIH grants R01 HL30946 (A.W.C., M.S.) and R41 HL106967 (T.N.W.), Department of Veterans Affairs, Veterans Health Administration Merit Review Agency (M.S.). Author conflict of interest: none. Presented in part as a poster at the Arteriosclerosis, Thrombosis, and Vascular Biology meeting, Toronto, Canada, May 1-3, 2014. Reprint requests: Richard D. Kenagy, PhD, Center for Cardiovascular Biology, University of Washington, PO Box 358050, 850 Republican St, Seattle, WA 98109 (e-mail: [email protected]). The editors and reviewers of this article have no relevant financial relationships to disclose per the JVS policy that requires reviewers to decline review of any manuscript for which they may have a conflict of interest. 0741-5214 Copyright Ó 2015 by the Society for Vascular Surgery. http://dx.doi.org/10.1016/j.jvs.2014.10.017
Vein grafts are frequently used for aortocoronary and peripheral vascular reconstructions. However, severe luminal narrowing is a primary cause of graft failure.1 This typically develops within the first year in approximately 25% to 30% of all grafts because of pathologic remodeling and intimal hyperplasia. Whereas much research is aimed at determining the pathologic mechanisms of luminal narrowing, technical factors such as surgical graft preparation are also important.2 For example, other factors, such as conduit distention,3 vein conduit storage,4 and stretching during surgical harvest,5 can adversely affect the function of vein wall cells. Surgical marking pens containing dyes such as crystal violet are routinely used on the adventitia of vein bypass grafts to avoid twisting of the vein grafts during placement. It continues to be assumed that these pens are innocuous despite 1
2 Kikuchi et al
evidence that dyes used in these pens can inhibit endothelium-dependent vasodilation6,7 and the smooth muscle contractile response.8 To further explore possible deleterious effects of marking pens on vein grafts, we have studied the effect of crystal violet on venous cell migration, proliferation, and apoptosis. These are the major biologic activities that contribute to intimal hyperplasia in both large and small animal models of vein graft healing.9-15 METHODS Procurement of human saphenous veins and migration assay. Human saphenous vein remnants were obtained from patients undergoing coronary artery bypass or peripheral vascular bypass surgeries under protocols approved by the University of Washington or the Benaroya Research Institute Institutional Review Board, and all subjects gave informed consent as indicated in these protocols. Veins were either endoscopically or openly harvested. All were distended to identify untied branches and were stored in heparinized buffered saline before being placed into Dulbecco’s modified Eagle’s medium (DMEM) with 10 mM HEPES, pH 7.4, for transport to the laboratory for dissection. The use of a surgical skin marker was decided by the surgical team. The human saphenous vein was dissected free of extraneous tissue, and sections that were marked intraoperatively with blue dye were separated from unmarked sections. A visually distinct, white, luminal layer was easily dissected from the remaining vein after removal of endothelium by gentle wiping with a cottontipped swab. The identity of the natural cleavage plane of vein dissection was confirmed by immunohistochemical staining for smooth muscle a-actin of the dissected layers. This demonstrated that both outer and inner layers contain smooth muscle a-actin-positive cells and that the cleavage plane goes through the circular muscle layer. The layers, hereafter called inner layer and outer layer, respectively, were cut into 2.5-mm2 explants by a McIlwain tissue chopper (customized to cut sizes >1 mm2). Explants were placed into 25-cm2 flasks (15/flask), and 1.2 mL of DMEM with 20% fetal bovine serum (DMEM/20% FBS) was added. Medium was changed three times per week. Explants were examined daily for 8 days, and cell migration was quantified as both the percentage of total explants with at least one migrating cell (a measure of only migration) and the average number of cells on plastic surrounding each explant (a measure of the combination of migration and proliferation).16 Extraction and quantification of dye. To determine the amount of dye adsorbed to the different vein samples during the surgical marking, explants prepared as described before were divided into four groups: (1) visibly bluestained outer layer explants, (2) inner layer explants luminally adjacent to blue-stained outer layer explants, (3) outer layer explants with no visible blue dye, and (4) inner layer explants luminally adjacent to outer layer explants with no visible blue dye. Crystal violet was quantified spectrophotometrically (OD590, the lmax of crystal violet) with use of crystal violet standards dissolved in ethanol.
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Pens obtained from the surgical services providing specimens for this study were confirmed to contain crystal violet, and dye extracted from vein graft specimens displayed a lmax of 590 nm. This assay is linear up to 50 mg/mL crystal violet. Blue dye was extracted by immersing five explants of each group in 100 mL of absolute ethanol and incubating at room temperature for 30 minutes. Extraction efficiency of crystal violet added to unstained explants was 80% with a first extraction and >90% with a third extraction. Whereas crystal violet may be tightly bound to tissue components such as DNA,17 most dye is available for extraction. Dose-response effect of crystal violet on cell migration. To determine the effects of crystal violet on migration of cells from the explants, 1 mL of crystal violet or buffer alone (0-500 ng/mL) was added to explants (in the absence of culture medium) from unstained sections of vein and incubated for 5 minutes. Separate experiments using nonblue outer layer or adjacent inner layer explants were performed as indicated in the figure legends. Some explants were then extracted to measure bound dye; other explants were placed in a 25-cm2 flask after one wash with DMEM/HEPES (15 explants per condition per vein for the number of veins indicated in the figure legends). Explants were given growth medium as before, and the percentage of migration-positive explants was determined at day 8. Measurement of proliferation and apoptosis in organ culture. After removal of loose connective tissue, veins were cut into rings w3 mm in length and placed in 20% FBS/DMEM (one/2 mL). The medium was changed every other day, and the tissue was fixed in 10% neutral buffered formalin at 0, 2, 4, and 6 days. Blue areas were marked with India ink before processing and embedding in paraffin because the blue dye is removed during processing. We measured cell proliferation by immunohistochemical staining of 5-mm sections with Ki67 (0.5 mg/mL SP6; Abcam, Inc, Cambridge, Mass) using the avidin-biotin-peroxidase method (Vector Laboratories, Burlingame, Calif) with rabbit immunoglobulin G as a negative control. For measurement of apoptosis, the terminal deoxynucleotidyl transferase (TdT)emediated dUTP-biotin nick end labeling (TUNEL) assay was performed with a kit (ApopTag; Millipore, Jaffrey, NH) following the manufacturer’s instructions but specifically using TdT at 1:32, citrate buffer (pH 4.5) rather than proteinase K, and omission of TdT as a negative control. To compare blue dyeestained sections to unstained sections, the percentage of Ki67-or TUNEL-positive nuclei was determined in adventitia, outer media, and inner media/intima for the portion of the perimeter marked by India ink (ie, blue dye) as well as for the same perimeter length on the opposite side of the vein (unstained). Adventitia plus outer media is equivalent to the outer layer designation, and inner media/intima is the same as the inner layer designation. Dose-response effects of crystal violet on outer layer cell proliferation, migration, and death. Cultured outer layer cells were obtained by the explant method from
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Fig 1. Cell migration from blue vs nonblue saphenous vein explants. Data are the mean 6 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 ¼ 11 veins) and inner layer (C and D, n ¼ 8 veins). *P < .001 vs blue outer layer or inner layer.
saphenous vein unstained outer layer explants in 20% FBS/ DMEM. When cells near the explants become confluent, we found that the optimal medium is smooth muscle cell growth medium (Cell Applications, Inc, San Diego, Calif), which was also used for subsequent growth of passaged cells. Cells were used at passage 6. For proliferation and death assays, 48,000 cells were seeded in 5% FBS in bovine basal medium (Cell Applications, Inc) in duplicate six-well plates. The next day, cells were counted with a hemocytometer, and the medium in remaining wells was changed to Cell Applications smooth muscle cell growth medium for proliferation studies or transferred to 2% serum for studies of cell death. Cells were then exposed for 3 days to a range of concentrations of crystal violet (0, 0.01, 0.1, 1.0, 10 mg/mL) and then counted again. Preliminary experiments demonstrated that cell number is maintained but not increased with 2% FBS alone. For migration assays, cells were preincubated for 30 minutes with crystal violet (0, 0.01, 0.1, 1.0, 10 mg/mL) and then seeded into the upper well of a 48-well microchemotaxis chamber at 30,000 per well (Neuro Probe, Inc, Gaithersburg, Md) in bovine basal medium in the presence of the same concentrations of dye. Polycarbonate filters with 8-mm pores coated with 100 mg/mL monomeric
Table. Amount of surgeon-applied marking pen dye extracted from explants Extracted blue dye (ng/explant) Outer layer explant Visibly unmarked explant Visibly blue explant Inner layer explant Visibly unmarked explant Visibly blue explant
2.1 6 1.3 65.9 6 8.0a 0.5 6 0.3 2.1 6 1.5
Data are the mean 6 standard error of the mean of the amount of dye in explants from seven to 11 veins (outer layer) or four or five veins (inner layer). a P < .01 vs unmarked explant.
bovine skin collagen were used, and bottom chambers contained 10% serum. After 5 hours at 37 C, the cells that had migrated through the filters were fixed and counted at 400. Statistical analysis. Comparisons of measures between unstained and blue-stained explants and between various concentrations of dye were made with analysis of variance with repeated measures or the Wilcoxon test with a Bonferroni correction as needed (SPSS, v20; SPSS Inc, Chicago, Ill). A P # .05 was considered statistically
4 Kikuchi et al
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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 6 standard error of the mean of inner medial/intimal (B), outer medial (C), and adventitial (D) percentage Ki67-positive nuclei. n ¼ 5 veins at days 0 and 6, 3 at days 2 and 4. *P < .05 blue vs nonblue.
significant. Data are presented as the mean 6 standard error of the mean. RESULTS Cells began migrating from saphenous vein explants after a lag phase of about 3 days, with outer layer cells initiating migration more quickly than inner layer cells. Migration was significantly less from blue-stained compared with unstained outer layer explants by day 5. There was a reduction of >80% migration at day 8 by both measures of migration (Fig 1, A and B). Inner layer explants separated into those adjacent to blue-stained outer layer and those adjacent to unstained outer layer exhibited equal cell migration (Fig 1, C and D), which is consistent with the visual observation that the staining from the surgeon’s marking pen almost never penetrated into the inner layer. Of the hundreds of veins dissected in this laboratory, only one vein had lightly visible staining of the inner layer from the surgeon’s marking pen. Indeed, although bluestained outer layer explants had more than 30-fold the amount of dye seen in visibly unstained outer layer explants, there was no difference in amount of blue dye detected in inner layer explants, whether they were adjacent to a blue-stained outer layer or an unstained outer layer (Table). The effect of surgeon-applied blue dye on cell proliferation and apoptosis in vein rings was determined by
comparing Ki67 and TUNEL staining in the blue-stained perimeter of vein compared with the same perimeter length of unstained vein on the opposite side of the vein ring. These were whole, undissected vein segments. The percentages of Ki67-and TUNEL-positive cells were counted separately in the inner media/intima, outer media, and adventitia separated as illustrated in Fig 2, A. In the unstained portion of the vein, proliferation increased progressively between days 2 and 6, with adventitia showing the highest proliferation, followed by the outer media, then the inner media/intima. In contrast, there was no proliferation observed in the blue-stained side of the vein except in the inner media/intima, which is again consistent with a lack of penetration of blue dye into the inner layer (Fig 2, B-D). In contrast, TUNEL-positive cells were observed at all times primarily in the adventitia (Fig 3, A), but no difference was noted between dye-positive and dye-negative areas of the vessel (Fig 3, B-D). To rule out the possibility of a difference in response to the dye between outer layer and inner layer cells, we also performed crystal violet dose-response studies on unstained outer layer and adjacent inner layer explants (Fig 4). Inhibition of migration from both types of explants occurred with an IC50 of w10 ng/explant. Dose-response curves for crystal violet for proliferation, migration, and death of cultured outer layer cells were also determined. At the lowest concentrations, it inhibited proliferation, and at
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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 6 standard error of the mean of inner medial/intimal (B), outer medial (C), and adventitial (D) percentage TUNEL-positive nuclei. N ¼ 5 veins at days 0 and 6, 3 at days 2 and 4.
the highest concentrations, it induced cell death. The IC50 was w0.1 mg/mL for proliferation and w1.2 mg/mL for migration; for death, the EC50 was between 1 and 10 mg/mL (Fig 5, A-C). DISCUSSION We have found that crystal violet, the dye used in many surgical marking pens, has a strong inhibitory effect on migration and proliferation of cells in the vein ex vivo. These data add to a growing body of evidence that commonly used marking pens have deleterious effects on the veins used for grafting, such as inhibition of both vasoconstrictor and vasodilator function.6-8 Surgical marking pens have dyes, such as crystal violet and methylene blue, dissolved in either ethanol or polyethylene glycol. In this study, we focused on the effect of crystal violet, a dye in pens used in Seattle, and did not address effects of the diluent. Whereas ethanol also has been shown to inhibit smooth muscle cell proliferation and migration,18,19 expected inhibition of cell migration from outer layer explants based on levels of dye measured in surgeon-treated veins was 99.9% (Fig 4) compared with observed inhibition of 86.1% (Fig 1, A), suggesting little if any effect of diluents on cell migration in this case. The inhibition of cell migration from explants is probably not directly linked to inhibition of cell proliferation or increased cell death. Whereas the IC50 for adventitial
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.
fibroblast proliferation is 10-fold lower than that for migration (0.1 vs 1.2 mg/mL, respectively), inhibitors of proliferation such as hydroxyurea do not appreciably alter migration from explants,20 and nonproliferating vascular cells migrate after injury.21 Nor does the inhibition of
JOURNAL OF VASCULAR SURGERY --- 2015
6 Kikuchi et al
Fig 5. Dose-response effect of crystal violet on proliferation (A), death (B), and migration (C) of outer layer cells. n ¼ 4 experiments performed in duplicate (A and C) or quadruplicate (B). *,#P < .05 vs 0 mg/mL. HPF, High-power field (400).
migration appear to relate to cell death. For if we assume that the 2.5-mm2 adventitial explants have a volume of w1 mm3, the measured amount of dye/explant achieves a concentration of nearly 70 mg/mL. This is well beyond the EC50 for death (
