Ann Thorac Surg 1999;68:84-88
© 1999 The Society of Thoracic Surgeons
Original Articles
Inhibitory effect of methylene blue-induced photooxidation on intimal thickening of vein graft
Ke-Xiang Liu, MDa,
Fumio Yamamoto, MDa,
Satoshi Sekine, MDa,
Yoshikazu Goto, MDa,
Keiji Seki, MDa,
Katsuyuki Kondoh, MDa,
Yan Fu, MDb
a Department of Cardiovascular Surgery, Akita University School of Medicine, Akita, Japan
b Department of Biochemistry, Akita University School of Medicine, Akita, Japan
Address reprint requests to Dr Liu, Department of Cardiovascular Surgery, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan
e-mail: kxliu{at}cvs.med.akita-u.ac.jp
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Abstract
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Background. We have previously speculated that methylene blue-induced photooxidation of adventitial surface for 5 minutes can completely inhibit the intimal and medial growth of surgically prepared saphenous vein in vitro. In this study, inhibitory effect of methylene blue-induced photooxidation on intimal thickening of vein graft in vivo was investigated.
Methods. Jugular vein grafts were photooxidized in 0.01% methylene blue solution for 5 minutes, and interposed into arterial circulation for 4 weeks in rabbits. Vein grafts were studied by morphometry and immunohistochemistry.
Results. The intimal thickening of photooxidized vein grafts were suppressed significantly compared with those in the nonphotooxidized group. Proliferated cell nuclear antigen (PCNA) index (total PCNA-positive cells/total cell count x 100%) of vein graft was significantly higher in the nonphotooxidized group than those in the photooxidized group.
Conclusions. Methylene blue-induced photooxidation is effective in the inhibition of intimal thickening of vein graft interposed in the arterial circulation for 4 weeks in vivo.
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Introduction
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Coronary saphenous vein grafts in humans have a more limited patency rate than artery grafts. It is generally accepted that the patency rate is about 80% to 90% at 1 year and 50% to 70% at 5 to 10 years. Its stenosis or occlusion results in the recurrence of angina pectoris and myocardial infarction, and contributes to morbidity and mortality after coronary artery surgery [1, 2]. Many clinical and experimental studies have demonstrated that intimal and medial thickening may critically narrow the graft lumen in itself or may promote atheroma formation leading to further stenosis [3, 4]. Proliferation of vascular smooth muscle cells may be a key contributor to neointimal formation and medial thickening [5, 6].
The photochemical action of dyes on amino acids has been studied by various researchers [7–9]. Weil and associates first demonstrated photooxidation of methylene blue on amino acid in 1951 [7]. Bernstein and Mechanic have noted that photochemical action of dyes converts the collagen fibrils to insoluble products toward pepsin digestion [10]. Moore and associates also described that the resistance of photooxidized bovine pericardial tissue to pepsin digestion and the use of dye-mediated photooxidation increased the stability of pericardial tissue that retain certain physical properties of natural tissue [11].
We have reported previously that 0.01% methylene blue-induced photooxidation of adventitial surface for 5 minutes inhibited completely the intimal and medial growth of surgically prepared saphenous vein in vitro by suppressing the proliferating activity of smooth muscle cells and adventitial fibroblasts without affecting the viability of endothelial cells [12]. In the current study, we demonstrate that 0.01% methylene blue-induced photooxidation of adventitial surface can significantly reduce intimal thickening of vein graft interposed in the arterial circulation for 4 weeks in the rabbit.
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Material and methods
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An autologous jugular vein graft model was used in adult male Japanese white rabbits weighting 3 to 3.5 kg. The animals were randomly assigned to two groups of 8 rabbits each. Anesthesia was induced with intramuscular ketamine (20 mg/kg) and xylazine (4 mg/kg), then was maintained with inhaled sebofuroren (1.5% with O2/N2O 2:1). The left carotid artery and jugular vein were dissected via a vertical midline neck incision, and all side branches were carefully ligated with 4-0 silk suture and divided. After intravenous heparinization (1.5 mg/kg), a 2–3-cm segment of jugular vein was removed and flushed with saline solution containing heparin (5 U/mL) and papaverine (0.5 mg/mL), then immersed in this solution for 5 minutes in the control group. In the experimental group, vascular clips were placed at the proximal and distal end of the jugular vein after intravenous heparinization (1.5 mg/kg). The segment of jugular vein filling heparinized blood was removed and 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 while stirring continued for 5 minutes at 2°C. Immediately after photooxidation, vein segment was washed in saline solution and clips removed, then flushed with saline solution containing heparin (5 U/mL) and papaverine (0.5 mg/mL). An adjacent section of the left common carotid artery was also free of surrounding connective tissues. Flow through the vessels was interrupted proximally and distally with microvascular clips. The jugular vein graft was anastomosed into the carotid artery in a reverse end-to-side interposed fashion by using continuous 8-0 polypropylene sutures. The carotid artery was tied close to both anastomosis and divided in between. Skin was closed with 4-0 silk suture. Animal care complied with the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985).
The rabbits were killed at 4 weeks after operative procedure. The interposed vein graft containing an adjacent section of the left carotid artery and opposite jugular vein were harvested, respectively. After the vessel was gently flushed with heparinized saline solution, the distal end of the artery was closed with vascular clip, and the graft was distended with 10% formaldehyde until the diameter was approximately the same as that in vivo. The proximal end of the artery was closed with vascular clip and then the specimens were immersed in 10% formaldehyde for 12 hours. The anastomotic site of the vein graft was discarded, and tissue specimens were prepared for histologic and immunohistochemical analysis. We implanted autologous jugular vein graft into the common carotid artery of 16 rabbits. Vein graft occlusion occurred in 3 rabbits because of a surgical technical mistake. The first mistake occurred in the nonphotooxidation group, followed by two in the photooxidation group.
To study the morphometry of the species, 2–3-µm serial sections were cut from each paraffin block and stained with hematoxylin and eosin (H&E), and elastin van Gieson (EVG) stain. von Kossa stain was used to identify calcium phosphate.
For immunohistochemistry, deparaffinized and rehydrated tissue sections were blocked with 0.1% hydrogen peroxide (H2O2), incubated with 10% normal swine serum (Gibco, Grand Island, NY) for 20 minutes. After washing in PBS, the sections were incubated with primary antibodies overnight at 4°C. Smooth muscle cells were detected with mouse anti-alpha smooth muscle actin monoclonal antibody (clone 1A4, 1:10 dilution; Immunon, Pittsburgh, PA). Tissue sections were incubated with the monoclonal antibody overnight at 4°C followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti–mouse antibodies (IgG) (1:100 dilution; Tagoimmuno-chemicals, Biosource International, Camarillo, CA) for 1 hour at 37°C. To identify endothelial cells, anti–human von Willebrand factor serum (anti–vWf) (1:40 dilution; Dako A/S, Glostrup, Denmark) was used. Bound monoclonal antibodies were detected using HRP-conjugated goat anti–rabbit antibodies (IgG) (1:2500 dilution; MBL, Nagoya, Japan). Then, slides were exposed to 0.04% solution of 3,3'-diaminobenzinidine (Sigma Chemical Co, St. Louis, MO) containing 0.03% H2O2 in 0.05 mol/Tris/HCl buffer (pH 3.0) for color development, washed in running water for 20 minutes, and counterstained with H&E.
For detection of proliferated cell nuclear antigen (PCNA) in vein grafts of the control and experimental groups, incubation of the sections with mouse anti-PCNA/cyclin monoclonal antibody (clone PC 10, 1:25 dilution; Novocastra Laboratories, Newcastle upon Tyne, UK) was carried out at 4°C overnight. After being washed 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 (Dako) in Tris buffer (pH 7.6) for 5–7 minutes at room temperature and counterstained with H&E.
Morphometry
Morphometry was performed using elastin van Gieson-stained cross-sectional histological sections in each segment. Because the external elastic lamina of interposed jugular vein graft is difficult to identify, we measured the wall area of neointima for the comparative studies of the two groups. Using a profile projector (V-16; Nikon Co, Nippon, Japan), 50x magnified profiles of luminal surface contour and the margin between intima and media were drawn on plastic sheets. These contours were digitized (Cosmozone-1; Nikon Co) to obtain the area (mm2) encompassed by the luminal contour (LA) and by the outer margin of intima (IA), respectively. The neointimal area from the lumen to the limit between intima and media was calculated as IA minus LA.
Proliferative index
Cell proliferation was quantified with the use of the PCNA stained slides, and quantifications were performed for each vessel. Six fields of each section containing intima and media were counted with 400x magnification. The proliferative index was expressed as the percent of PCNA-positive nuclei.
Statistical analysis
All data were expressed as mean standard deviation of the mean. Statistics were carried out with unpaired Student’s t test. Statistically significant differences were considered to exist at p < 0.01.
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Results
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The structure of controlled jugular vein in Japanese white rabbits consists of a single layer of endothelial cells, a very narrow medial layer, and a wide adventitial layer (Fig 1A). The intima of nonphotooxidized vein grafts became remarkably thickened when implanted in the carotid arterial circulation for 4 weeks (Fig 1B). As compared with nonphotooxidized vein grafts, there was significantly less increase in thickness of the intima in the photooxidized vein grafts implanted in the carotid arterial circulation for 4 weeks (Fig 1C, Table 1).
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Fig 1. Light micrographs of elastin van Gieson stain. (A) Normal jugular vein. (B) Nonphotooxidized vein graft demonstrates remarkably thickening intima after implant for 4 weeks. (C) Photooxidized vein graft shows a thin neointima after implant for 4 weeks; the intimal thickening is less significant than that in nonphotooxidized vein graft (original magnification x100).
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To identify cells of thickening intima, anti–von Willebrand factor and anti–smooth muscle actin stain were used. The stain results showed the cells of intima and partial media of vein grafts of the both nonphotooxidized and photooxidized groups are positive for 
-SM actin (Fig 2), and the endothelial layer of both nonphotooxidized vein graft (Fig 2A) and photooxidized vein graft are positive for anti–von Willebrand factor (Fig 3). von Kossa stain was negative in vein grafts of both the nonphotooxidized and photooxidized vein implanted in the carotid arterial circulation for 4 weeks.
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Fig 3. Light micrographs of anti–von Willebrand factor stain show that the endothelial layer of both nonphotooxidized vein graft (A) and photooxidized vein graft (B) are positive for anti–von Willebrand factor (original magnification x200).
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Immunohistochemical detection of PCNA was used for determining the cell progression through the cell cycle in paraffin-embedded tissue. Many PCNA-positive cells were found in the nonphotooxidized vein graft (Fig 4A), and PCNA-positive cells in the photooxidized vein graft (Fig 4B) are significantly less common than in nonphotooxidized vein graft. PCNA index (total PCNA-positive cells/total cell count x 100%) of vein graft was significantly higher in the nonphotooxidized group than those in the photooxidized group (Table 1).
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Fig 4. Light photomicrographs of anti–PCNA stain. Many PCNA-positive cells are found in nonphotooxidized vein graft (A). PCNA-positive cells in photooxidized vein grafts (B) decrease significantly more than those in nonphotooxidized vein graft (original magnification x200).
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Comment
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In this study, we demonstrated that 0.01% methylene blue-induced photooxidation of adventitial surface of jugular vein for 5 minutes suppresses significantly intimal thickening of jugular vein graft interposed in the arterial circulation for 4 weeks in the rabbits.
Vein graft intimal thickening in the arterial circulation is an important etiologic factor leading to vein graft stenosis and occlusion in coronary bypass surgery. Many clinical and experimental studies have demonstrated that migration and proliferation of vascular smooth muscle cells may be a key contributor to neointimal formation and medial thickening. Under normal physiological conditions, the majority of vascular smooth muscle cells are in the normal quiescent state, and cell growth is controlled by a balance between endogenous growth-promoting factors and proliferation-inhibition factors. Vein graft injury incurred during routine surgery intervention may activate intimal proliferation by promoting the transition of vascular smooth muscle cells from a contractile to a synthetic phenotype [13, 14]. Mann and associates demonstrated that the transformation of vein graft biology toward an adaptive response using antisense oligodeoxynucleotide blockage of medial smooth muscle proliferation can yield vein graft resistant to long-term graft failure [15]. In this study, we photooxidized the adventitial surface of the jugular vein of the rabbits in 0.01% methylene blue to convert the majority of vascular smooth muscle cells to less bio-active and nonproliferating cells. Our results confirm that methylene blue-induced photooxidation is effective on inhibiting intimal hyperplasia of vein graft in vivo. Immunohistochemical detection of PCNA was used for determining the cell progression through the cell cycle in paraffin-embedded tissue because PCNA is one of the principle components of the final common pathway regulating cell proliferation [16]. PCNA index was significantly lower in the photooxidized vein grafts compared with those in the nonphotooxidized vein grafts. It is conceivable that migration and proliferation of vascular smooth muscle cells would be less in the photooxidized vein grafts.
We previously have demonstrated that 0.01% methylene blue-induced photooxidation of adventitial surface of saphenous vein for 5 minutes does not affect the viability of endothelial cells using the Live/Dead Cytotoxicity Kit [12]. In this study, we utilized anti–von Willebrand factor stain to identify endothelial cell. The stain results showed that the cells of the surface layer of intima of grafts in both the photooxidized and nonphotooxidized vein were stained by antibody to factor XIII-related antigen (Fig 2A). It is reasonable to assume that the endothelial cells also persist and take part in the neointimal form in the photooxidized vein graft. Simultaneously, we proved that 0.01% methylene blue-induced photooxidation does not induce calcium deposition in the vein graft interposed in the arterial circulation for 4 weeks in vivo using von Kossa stain.
The photooxidation of proteins in the presence of methylene blue causes a rapid destruction of histidine and tryptophan residues and a slower destruction of tyrosine, cysteine, and methionine residues [7, 8]. Bernstein and Mechanic have demonstrated that photooxidation of a collagen solution resulted in the formation of a protein mass "no longer soluble under the most extreme denaturing" and also insoluble toward pepsin digestion [10]. Moore and associates also described that the resistance of photooxidized bovine pericardial tissue to pepsin digestion and the use of dye-mediated photooxidation increased the stability of pericardial tissues that retain certain physical properties of natural tissue [11]. Our main purpose was to reduce neointimal formation by converting the maximal number of smooth muscle cells of vein graft to nonproliferating, nonviable cell using the effect of methylene blue-induced photooxidation. Our results have demonstrated that 0.01% methylene blue-induced photooxidation for 5 minutes suppressed significantly intimal thickening of vein graft interposed in the arterial circulation, although it can not convert all smooth muscle cells to cells of losing bio-activity. One question worth asking is why methylene blue-induced photooxidation can not completely inhibit neointimal formation of vein graft in the current study compared with those in a previous study in vitro [12]. One may anticipate that some endogenous growth-promoting factors, such as platelet-derived growth factor and other mitogens released by platelets, injured endothelial cells, and smooth muscle cells, would simulate survival of smooth muscle cells proliferating and migrating into intima, and hemodynamic stress functioning would increase the activity of remaining bio-active smooth muscle cells proliferating and migrating into the intima in vivo. Because intimal hyperplasia of a vein graft in vivo is multifactorial [17, 18], one would expect that a combination of methylene blue-induced photooxidation procedure and several other therapeutic modalities [17, 19, 20] would enhance the patency rate of autologous vein grafts interposing arterial circulation.
In conclusion, taking into account our results, it appears methylene blue-induced photooxidation can inhibit neointimal formation of vein graft interposed in arterial circulation in vivo. The procedure of methylene blue-induced photooxidation is simple, and easy to perform. It may be performed even in the intraoperative procedure because it takes only 5 minutes.
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References
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-
Bourassa M.G. Fate of venous grafts. J Am Coll Cardiol 1991;17:1081-1083.[Medline]
-
Grondin C.M., Campeau L., Thornton J.C., et al. Coronary artery bypass grafting with saphenous vein. Circulation 1989;79(Suppl):124-129.
-
Fonkalsrud E.W., Sanchez M., Zerubavel R. Morphological evaluation of canine autogenous vein grafts in the arterial circulation. Surgery 1978;84:253-264.[Medline]
-
Zwolak R.M., Kirkman T.R., Clowes A.W. Atherosclerosis in rabbit vein grafts. Artherosclerosis 1989;9:374-379.[Abstract/Free Full Text]
-
Dilley R.J., McGeachie J.K., Teannant M. The role of cell proliferation and migration in the development of a neo-intimal layer in veins grafted into arteries in rats. Cell Tissue Res 1992;269:281-287.[Medline]
-
Ip J.H., Fuster V., Badimon L., et al. Syndromes of accelerated atherosclerosis. J Am Coll Cardiol 1990;15:1667-1687.[Abstract]
-
Weil L., Gordon W.G., Buchert A.R. Photooxidation of aminoacids in the presence of methylene blue. Arch Biochem Biophys 1951;33:90-109.
-
Weil L. On the mechanism of the photo-oxidation of amino acids sensitized by methylene blue. Arch Biochem Biophys 1965;110:57-68.
-
Jori G., Galiazzo G., Marzotto A., et al. Dye-sensitized selective photooxidation of methionine. Biochem Biophys Acta 1968;154:1-9.[Medline]
-
Bernstein P.H., Mechanic G.L. A natural histine-based imminium cross-link in collagen and its location. J Biol Chem 1980;255:10414-10422.[Free Full Text]
-
Moore M.A., Bohachevsky I.K., Cheung D.T., et al. Stabilization of pericardial tissue by dye-mediated photooxidation. J Biomed Mater Res 1994;28:611-618.[Medline]
-
Chanada J., Kuribayashi R., Liu K.X., et al. Inhibitory effect of photooxidation on intimal and medial thickening of saphenous vein. Ann Thorac Surg 1998;66:449-454.[Abstract/Free Full Text]
-
Fuster V., Badimon L., Badimon J.J., et al. The pathogenesis of coronary artery disease and the acute coronary syndromes. N Engl J Med 1992;326:242-250.[Medline]
-
Ross R. The pathogenesis of atherosclerosis. Nature 1993;362:801-809.[Medline]
-
Mann M.J., Gibbons G.H., Kernoff R.S., et al. Genetic engineering of vein grafts resistant to atherosclerosis. Proc Natl Acad Sci USA 1995;92:4502-4506.[Abstract/Free Full Text]
-
Morishita R., Gibbons G.H., Ellison K.E., et al. Single intraluminal delivery of antisense cdc2 kinase and proliferation-cell nuclear antigen oligonucleotides results in chronic inhibition of neointimal hyperplasia. Proc Natl Acad Sci USA 1993;90:8474-8478.[Abstract/Free Full Text]
-
Ferns G.A., Raines E.W., Sprugel K.H., et al. Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF. Science 1991;253:1129-1132.[Abstract/Free Full Text]
-
Edelman E.R., Nugent M.A., Smith L.T., et al. Basic fibroblast growth factor enhances the coupling of intimal hyperplasia and proliferation of vasa vasorum in injured rat arteries. J Clin Invest 1992;89:465-473.
-
Benezra M., Ben-Sasson S.A., Regan J., et al. Antiproliferative activity to vascular smooth muscle cells and receptor binding of heparin-mimicking polyaromatic anionic compounds. Arterioscler Thromb 1994;14:1992-1999.[Abstract/Free Full Text]
-
Tanaka K., Honda M., Kuramochi T., et al. Prominent inhibitory effects of tranilast on migration and proliferation of and collagen synthesis by vascular smooth muscle cells. Atherosclerosis 1994;107:179-185.[Medline]
Accepted for publication January 25, 1999.
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Abstract
Introduction
