Ann Thorac Surg 1998;66:449-454
© 1998 The Society of Thoracic Surgeons
Original articles: cardiovascular
Inhibitory effect of photooxidation on intimal and medial thickening of saphenous vein
Jyotirmay Chanda, MDa,
Ryosei Kuribayashi, MDa,
Ke-Xiang Liu, MDa,
Yoshiki Shibata, MDa
a Department of Cardiovascular Surgery, Akita University School of Medicine, Akita, Japan
Accepted for publication March 16, 1998.
Address reprint requests to Dr Chanda, Bejpara, Sreedhar Tank Rd, Jessore 7400, Bangladesh
e-mail: (eximpak{at}bdmail.net [Attention: Dr J Chanda])
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Abstract
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Background. The inhibitory effect of short-term photooxidation on medial and neointimal proliferation of human saphenous vein was investigated.
Methods. Culture medium–filled surgically prepared saphenous vein segments were photooxidized in 0.01% methylene blue solution for 5 minutes. Photooxidized and nonphotooxidized saphenous veins were checked for viability of endothelial cells by culturing vein segments for 21 days followed by histologic and immunohistochemical studies.
Results. Endothelial cells of saphenous vein segments remained unaffected after photooxidation. Both the intima and media of nonphotooxidized veins became highly cellular and thickened because of the proliferation and migration of smooth muscle cells. Like precultured fresh saphenous vein, intimal (0.031 ± 0.017 mm; p = 0.0067) and medial thicknesses (0.702 ± 0.123 mm; p < 0.0001) and proliferating cell nuclear antigen–positive cell count (14 ± 8/mm2; p = 0.0005) of cultured photooxidized veins were significantly less than those of cultured nonphotooxidized veins (intimal thickness, 0.059 ± 0.041 mm; medial thickness, 0.997 ± 0.228 mm; proliferating cell nuclear antigen positive cell count, 34 ± 16/mm2.
Conclusions. Methylene blue–induced short-term photooxidation is effective in inhibition of intimal and medial thickening of saphenous vein.
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Introduction
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Coronary vein graft disease contributes to significant morbidity and mortality after coronary artery bypass grafting and is responsible for the recurrence of angina pectoris, myocardial infarction, and compromised ventricular function [1]. The reasons for ongoing intimal proliferation in vein grafts are not evident [2]. Vein grafts respond to two major stimuli: (1) injury because of manipulation and ischemia at the time of the operation and (2) increased intraluminal pressure and shear stress exerted on the venous wall by the arterial environment [3]. Progression of medial and neointimal thickening with superimposed atherosclerosis in aortocoronary saphenous vein grafts is frequent and is the predominant cause of late graft closure after coronary artery bypass grafting [4, 5]. Proliferating cell nuclear antigen (PCNA), a nuclear protein required for DNA synthesis by DNA polymerase, is thought to be one of the principal components of the final common pathway regulating cell proliferation [6]. It has been postulated that perivascular fibroblasts may infiltrate injured media of arterialized saphenous vein grafts, differentiate to myofibroblasts (acquiring alpha smooth muscle actin), and contribute to vein graft remodeling [7].
Transformation of vein graft biology toward an adaptive response that does not predispose the vessel to accelerated atherosclerosis can yield vein grafts resistant to long-term graft failure [3]. To suppress the PCNA in the maximal number of smooth muscle cells and the activity of perivascular fibroblasts without affecting the viability of endothelial cells, we have photooxidized the adventitial surface of surgically prepared saphenous vein for a short period. In this study we have investigated the inhibitory effect of methylene blue–induced photooxidation on medial thickening and neointimal growth of freshly isolated saphenous vein cultured in vitro.
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Material and methods
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Surplus surgically prepared saphenous vein (5 to 12 cm in length) was obtained from 17 patients (mean age, 69.3 years; range, 56 to 75 years; 16 men and 1 woman) who were undergoing coronary artery bypass grafting. Ethical permission was obtained from the relevant authority. Surgically prepared vein segments had been subjected to side branch ligation, adventitial stripping, and uncontrolled manual distention with the patient’s heparinized blood followed by storage in the same medium for 40 to 180 minutes. Vein segments were obtained after completion of the last proximal anastomosis and collected in tissue culture medium RPMI 1640 with HEPES buffer, 25 mmol/L, and L-glutamine (Gibco/BRL, Life Technologies, Inc, Paisley, UK), containing penicillin-streptomycin, 100 µg/mL (Gibco); gentamicin, 2.5 µg/mL (Schering-Plough, Osaka, Japan); and amphotericin B, 5 µg/mL (Fungizone; Gibco) and transferred to the laboratory at room temperature.
On arrival at the laboratory before photooxidation, a vascular clip was put at one end of the vein segment, followed by filling the vein with culture medium without pressure and closing the other end of the vein with another vascular clip. Culture medium–filled vein segments were placed in a solution of photoactive dye (0.01% methylene blue in phosphate-buffered saline (PBS), pH 7.4) (methylene blue; Wako Chemical Co, Osaka, Japan). The solution was exposed to light of a 1000-W slide projector under continuous stirring for 5 minutes at 2°C. Immediately after photooxidation, vein segments were washed in PBS, opened longitudinally, and cut into 5-mm lengths. The viability of the endothelial cells of the treated and untreated vein segments was checked with immunofluorescent staining on frozen sections.
All photooxidized and nonphotooxidized vein segments (n = 30 segments of each group; size, 5 x 5 mm) were incubated in the same organ culture system developed by Soyombo and coworkers [8]. The culture medium consisted of tissue culture medium RPMI 1640 with HEPES buffer, 25 mmol/L, and L-glutamine (Gibco), containing 30% fetal bovine serum (Gibco); penicillin-streptomycin, 100 µg/mL (Gibco); gentamicin, 2.5 µg/mL (Schering-Plough); and amphotericin B, 5 µg/mL (Gibco), and maintained for 21 days at 37°C in a humidified atmosphere of 5% carbon dioxide in an air incubator. The tissue culture medium was replaced every 3 days.
At the end of the culture period, vein segments were washed in PBS. A portion (about one third) of each vein segment was prepared for frozen sample to be stained with fluorescein isothiocyanate (FITC)-labeled Ulex europaeus agglutinin I (UAE-I) for identification of endothelial cells (described subsequently). The remaining part of the vein was fixed overnight in 10% formaldehyde in PBS followed by processing and paraffin embedding, from which serial sections of 2 to 3 µm were cut at five levels from each segment and mounted on glass slides. To study the morphology of the vein, hematoxylin and eosin, Masson’s trichrome, and elastin van Gieson stains were used. The von Kossa stain was used to identify calcium phosphate. Intimal (luminal to the internal elastic lamina) and medial wall thicknesses (whole wall exterior to the internal elastic lamina) of each section were measured at five points of elastin van Gieson–stained slides with 40x magnification.
For immunohistochemistry, deparaffinized (with xylene) and rehydrated (with graded alcohol) tissue sections were preincubated with 0.1% hydrogen peroxide in PBS for 5 minutes and with 10% normal swine serum (Gibco) for 20 minutes in a humidified chamber to eliminate the endogenous peroxidase activity and to block nonspecific binding sites, respectively, before incubation with monoclonal antibodies.
To identify endothelial cells, sections were incubated overnight with rabbit anti–human von Willebrand factor serum (1:40 dilution; Dako A/S, Glostrup, Denmark) at 4°C. Bound monoclonal antibodies were detected using horseradish peroxidase (HRP)–conjugated goat anti-rabbit antibodies (IgG) (1:2,500 dilution; MBL, Nagoya, Japan) for 1 hour at 37°C. For identification of smooth muscle cells, tissue sections were incubated overnight with mouse anti–alpha smooth muscle actin monoclonal antibody (clone 1A4,1:10 dilution; Immunon, Pittsburgh, PA) at 4°C followed by incubation with HRP-conjugated goat anti-mouse antibodies (IgG) (1:100 dilution; Tagoimmunochemicals, Biosource International, Camarillo, CA) for 1 hour at 37°C. Both slides were exposed for 7 minutes to 0.04% solution of 3,3'-diaminobenzinidine (Sigma Chemical Co., St. Louis, MO) containing 0.03% hydrogen peroxide in 0.05 mol/Tris/HCL buffer (pH 3.0) for color development. Finally, slides were washed in running water for 20 minutes and counterstained with hematoxylin and eosin.
For detection of PCNA, incubation of the sections with mouse anti-PCNA/cyclin monoclonal antibody (clone PC10, 1:25 dilution; Novocastra Laboratories, Newcastle- upon-Tyne, UK) was carried out at 4°C overnight. After washes in PBS, biotinylated anti-mouse IgG (Dako) was applied at a dilution of 1:50 and incubated for 30 minutes at room temperature followed by incubation with strepavidin-peroxidase (1:500 dilution, Dako) at room temperature for 30 minutes. Slides were incubated with peroxidase-conjugated 3,3'-diaminobenzinidine (1:500 dilution, Dako) in Tris buffer (pH 7.6) for 5 to 7 minutes at room temperature and counterstained with hematoxylin and eosin. Total number of strongly positive cells in each section was counted with 400x magnification.
To assess the effect of photooxidation on viability (live or dead) of endothelial cells and adventitial tissue of saphenous vein, the Live/Dead Reduced Biohazard Viability Cytotoxicity Kit (L-7013, Molecular Probes, Inc, Eugene, OR) was used. Working solution of the dye was prepared by pipetting 5 µL of component A and 5 µL of component B into a common 2.5-mL volume of Hank’s balanced salt solution (1:500 dilution of each, HBSS, Gibco). Immediately after completion of the photooxidation, segments of photooxidized and nonphotooxidized veins were incubated in the diluted dye in complete darkness for 15 minutes at room temperature followed by washing in HBSS, fixing in freshly prepared 4% glutaraldehyde (glutaraldehyde EM 25%; TAAB Laboratories Equipment Ltd, Reading, UK) in HBSS for more than 15 minutes at room temperature, and finally washing in HBSS. After that, vein specimens were submerged in OCT compound (Tissue Tek; Miles Inc, Elkhart, IN) and quickly frozen in liquid nitrogen. The frozen samples were cut at 5 µm on a cryostat, mounted on glass slides, and observed using an epifluorescent microscope (Olympus BH2, Tokyo, Japan). At an excitation wavelength of 485 ± 11 nm, live (green fluorescent) and dead (red fluorescent) cells were viewed simultaneously. The fluorescence intensity of live cells was significantly lower than that of dead cells. To confirm the nature of the luminal cells (whether endothelial or not) detected with the aforementioned technique, frozen sections of treated and untreated vein segments were prepared and fixed for 10 minutes in 3.5% paraformaldehyde in freshly prepared PBS and stained with FITC-labeled UAE-I (1:20 dilution, Vector Laboratories, Burlingame, CA) in Dulbecco balanced salt solution (Gibco) and observed using an epifluorescent microscope. Endothelial cells were further confirmed for von Willebrand factor in paraffin sections of all treated and untreated vein segments before and after culture (as described previously).
Values are shown as the mean ± standard deviation. Values were compared by two-tailed unpaired t test with a p value of less than 0.01 considered statistically significant.
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Results
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The Live/Dead Cell Cytotoxicity assay demonstrated that the most superficial cells detected on the luminal surface of the precultured vein segments remained viable after photooxidation (Fig 1), whereas most cells of the adventitial and outer part of the medial layer of this group of veins were dead. The most superficial cells detected on the luminal surface of the precultured vein segments of both groups with the Live/Dead Cell Cytotoxicity assay were endothelial in nature as the adjacent sections stained positively with UAE-I and anti–von Willebrand factor stains. The endothelial layer of both groups of veins was intact (Fig 2) and viable 21 days after culture. There was no calcification (von Kossa–negative) in both photooxidized and nonphotooxidized veins after culture.
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Fig 1. Cryosection of photooxidized (A) and freshly isolated (B) vein segments stained with Live/Dead Reduced Biohazard Viability Cytotoxicity Kit. Live endothelial cells are stained green (solid arrow) and dead endothelial cells (open arrow) are red. The fluorescence intensity of live cells is significantly lower than that of dead cells. Notice photooxidation for 5 minutes does not affect the viability of the endothelial cells in comparison with those of freshly isolated vein. (x100 before 35% reduction.)
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Fig 2. Light photomicrographs show that the endothelial layer (arrow) of both photooxidized (A) and nonphotooxidized (B) vein segments remains intact after culture for 21 days. (Anti–von Willebrand factor stain; x100 before 32% reduction.)
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Both the intima and media of nonphotooxidized veins became highly cellular and thickened after culture, and most of these cells were smooth muscle cells in origin (Fig 3). Intimal and medial thicknesses of nonphotooxidized veins after culture were significantly higher than those of the same group of veins before culture (Table 1). There was remarkably less increase in thicknesses of both the intima and media in the photooxidized veins (see Fig 3) when cultured for 21 days. Intimal and medial thicknesses of cultured photooxidized veins did not significantly differ from those of freshly isolated veins (see Fig 3; Table 1).
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Fig 3. Histologic features show the neointima formation (A) and migration of smooth muscle cells (arrow) (B) into the neointima of nonphotooxided saphenous vein after culture for 21 days. Neointima (between arrows, luminal to the internal elastic lamina) is lightly stained (A). Neointima is absent above the internal elastic lamina (arrow) in a photooxidized saphenous vein cultured for 21 days (C), and smooth muscle cells are absent in the intima (D) of this vein segment. (A and C, elastin van Gieson; B and D, anti–alpha smooth muscle actin monoclonal antibody stains; x100 before 32% reduction.)
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Immunohistochemical detection of PCNA was used for determining the cell progression through the cell cycle in paraffin-embedded tissue. Cells that stained positive for PCNA were scanty both in the freshly isolated and cultured photooxidized vein segments but were abundant in cultured nonphotooxidized veins (Fig 4). In comparison with the precultured photooxidized veins, though insignificant, there was a decrease in the number of H&E-stained cells in the cultured photooxidized veins (data not shown). This is why the PCNA index [8] (total PCNA-positive cells divided by total cell count times 100%) was not used to present the PCNA data in this report. Count of PCNA-positive cells of cultured photooxidized veins did not significantly differ from that of freshly isolated veins (see Table 1). However, the PCNA count in cultured nonphotooxidized veins was significantly higher than that of cultured photooxidized veins (see Table 1).
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Fig 4. Light photomicrographs show that the number of proliferating cell nuclear antigen-positive cells (arrow) is much less in the cultured photooxidized saphenous vein (A) than those in nonphotooxidized saphenous vein segments (B) cultured for 21 days. (Anti-proliferating cell nuclear antigen stain; x100 before 32% reduction.)
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Comment
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Our study demonstrates that 0.01% methylene blue–induced photooxidation of the adventitial surface for 5 minutes suppresses the intimal and medial growth of surgically prepared saphenous vein in vitro. Because PCNA is one of the principal components of the final common pathway regulating cell proliferation [6], and PCNA count did not substantially increase (Table 1) in photooxidized veins after culture, conceivably through the suppression of PCNA in smooth muscle cells by photooxidation, the growth of intimal and medial layers of saphenous vein was inhibited.
Photooxidation of amino acids sensitized by methylene blue was described by Weil and associates [9]. Mechanic [10] has used a dye-mediated (methylene green) photooxidation technique for stabilization of heterologous tissue, and this tissue is used for fabrication of Photofix 
Pericardial Bioprosthesis (Carbomedics, Austin, TX) [11]. Bernstein and Mechanic [12] have noted that photooxidation converts the collagen fibrils to insoluble products, which are resistant to extreme methods of collagen denaturation and to solubilization by partial digestion with proteolytic enzymes. We have used the idea of the cross-linking effect of photooxidation on the outer tissue of saphenous vein to convert the maximal number of smooth muscle cells to nonproliferating, nonviable (inert) cells that are resistant to degradation as well.
To date, despite the extensive experimental studies, at the clinical level no clear-cut strategy of pharmacologic [13–17], surgical [18], and gene therapies [3, 6, 19] for prevention of smooth muscle cell proliferation and neointimal growth of saphenous vein grafts exists. It is too early to judge the efficacy of polytetrafluoroethylene tube [20] as a prosthetic coronary graft substitute for patients undergoing coronary artery bypass grafting. Photoradiation, a therapeutic measure in vascular stenosis was proposed by Dartsch and colleagues [21].
Methylene blue is occasionally applied to the adventitia of blood vessels during coronary artery bypass grafting and other vascular procedures to assist in the orientation of the vessel. Barber and associates [22] reported that surgical marking of blood vessels with methylene blue has an adverse effect on vascular reactivity. The concentration (0.01%) of methylene blue we used was 100 times less than that (1.0%) [22] used as a surgical marker. Moreover our exposure time (5 minutes) of saphenous vein to methylene blue was nine times less than that (45 minutes) used in the experiment of Barber and colleagues [22]. The Live/Dead Cell Cytotoxicity assay demonstrated that photooxidation of freshly isolated saphenous vein in 0.01% methylene blue for 5 minutes does not affect the viability of endothelial cells of the vein (Fig 1). There is no easy technique available to demonstrate both the live and dead cells simultaneously on the surface of a tissue in situ. The Live/Dead Cell Cytotoxicity assay is usually used to detect live and dead cells simultaneously in cell suspension. It is reasonable to consider that the dye must react with the cells of a superficial layer of tissue and develop fluorescence accordingly. In our experiment, with our technique, we found that the dye not only reacts with superficial cells, but also penetrates the tissue. This procedure is fast and easy to perform, and it served our purpose. It should be noted that our main purpose of photooxidation was to convert the vein to less bioactivity without interfering with the endothelium (very similar to an imaginary endothelial cell–seeded artificial biologic vascular prosthesis of autologous origin). The reduced number of live smooth muscle cells would react less actively. Therefore, even the absence of vasoreactivity in photooxidized saphenous vein does not contradict our theme.
Increases in thicknesses of intima and media and number of PCNA-positive cells of the cultured photooxidized veins, though insignificant compared with those of fresh precultured veins (Table 1), indicate that not all smooth muscle cells were dead after photooxidation. On the contrary, the presence of significantly decreased numbers of PCNA-positive cells in the cultured photooxidized veins in comparison with those in the cultured nonphotooxidized veins (see Table 1) suggests that photooxidation arrests the capability of proliferation in substantial numbers of smooth muscle cells of saphenous vein.
One may anticipate that under hemodynamic stress functioning smooth muscle cells of photooxidized vein would proliferate and migrate into the intima. It is conceivable that proliferation and migration of smooth muscle cells in photooxidized veins would be less intensive and slower than those of nonphotooxidized vein grafts. It has been hypothesized that during surgical injury adventitial fibroblasts modulate their phenotype to that of myofibroblasts and that this causes medial thickening of saphenous vein grafts [7]. Perhaps inactivation of adventitial fibroblasts with photooxidation might negatively influence the medial thickening of saphenous vein grafts.
We conclude that the methylene blue–induced photooxidation procedure is less time-consuming and easy to perform even in the operating room, and that fewer numbers of cells remain able to proliferate after the procedure. Conceivably, in addition to intraoperative photooxidation, other surgical measures [18] and postoperative treatment with cholesterol-lowering pharmacologic agents [15, 16] would enhance the patency rate of autologous saphenous vein grafts after implantation. In vitro study cannot exactly interpret the situation in vivo, and a large animal study is necessary to make any valid conclusions regarding the procedure.
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Inhibitory effect of methylene blue-induced photooxidation on intimal thickening of vein graft
Ann. Thorac. Surg.,
July 1, 1999;
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84 - 88.
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Abstract
Introduction
