Eur J Vasc Endovasc Surg 34, 333e339 (2007) doi:10.1016/j.ejvs.2007.04.002, available online at http://www.sciencedirect.com on
Photodynamic Therapy Reduces Intimal Hyperplasia in Prosthetic Vascular Bypass Grafts in a Pig Model J. Heckenkamp,1* S. Mellander,2 P. Fogelstrand,2 S. Breuer,1 J. Brunkwall1 and E. Mattsson2 1
Division of Vascular Surgery, Department of Vascular and Visceral Surgery, University of Cologne, Cologne, Germany, and 2Wallenberg Laboratory for Cardiovascular Research, Department of Vascular Surgery, Sahlgrenska University Hospital, Gothenburg, Sweden Background. Bypass surgery has a failing frequency of 30% during the first year, mainly due to intimal hyperplasia (IH). This negative effect is most pronounced in artificial grafts. Photodynamic therapy (PDT) is a technique in which light activates photosensitizer dyes to produce free-radicals resulting in an eradication of cells in the vascular wall. The aim of this study was to determine the effectiveness of PDT to reduce IH in a preclinical porcine PTFE bypass model. Material and methods. Ten pigs were used. After a pilot PDT dosimetry study (n ¼ 3) PTFE grafts were bilaterally placed into the circulation as bypasses from the common to the external iliac arteries (n ¼ 7). The right sides served as controls (C). Before implantation of the left grafts, the arterial connecting sites of the left distal anastomoses were PDTtreated. The arteries were pressurized at 180 mmHg for 5 minutes with the photosensitizer Methylene Blue (330 mg/ml), and thereafter endoluminally irradiated with laser light (l ¼ 660 nm, 100 mW/cm2, 150 J/cm2). After 4 weeks the specimens were retrieved and formalin fixed. Cross sections through the midportions of the distal anastomoses and the grafts were used for histology, immunohistochemistry to identify inflammatory cells and morphometric evaluation (n ¼ 7). Results. No systemic side effects and no graft occlusions were noted. PDT-treated anastomoses showed reduced IH in the mid-portions of the anastomoses (Area of IH: mm2/mm graft: C: 6970 1536, PDT: 2734 2560; P < 0.005) as well as in the grafts (C: 5391 4031, PDT: 777 1331; P < 0.02). The number of inflammatory cells per microscopic field was increased after PDT (C: 24 16, PDT: 37 15; P < 0.009). Conclusions. Adjuvant PDT, performed in an endovascular fashion, was a safe method to reduce prosthetic graftstenosis in a preclinical setting. This study underscores the clinical potential of PDT to inhibit the development of clinical bypass graftstenosis. Ó 2007 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved. Keywords: Photodynamic therapy; Intimal hyperplasia; Bypass grafts.
Introduction One of the major drawbacks to vascular bypass surgery is the incidence of graft stenosis especially at the site of the distal anastomosis, resulting, in part, from intimal hyperplasia (IH). IH results from vessel wall injury and involves the migration and proliferation of smooth muscle cells and myofibroblasts into the subintimal space of the vessel with excessive deposition of extracellular matrix proteins.1 Although several attempts have been made, there is so far no generally accepted treatment modality to clinically reduce the occurrence of graft stenosis. The major approaches used in the search for drugs to reduce *Corresponding author. J. Heckenkamp, MD, Division of Vascular Surgery, Department of Surgery, University of Cologne, JosephStelzmann-Str. 9, D-50924 Koeln, Germany. E-mail address:
[email protected]
restenosis, include anti-platelet agents, anti-thrombotic agents and those that interfere with cell proliferation and migration. However, only few of them have shown promising results in randomized clinical trials.2 Adjuvant radiotherapy and the implantation of drug-eluting stents to reduce restenosis after angioplasty and in-stent restenosis especially in the coronary system have been shown to reduce restenosis in clinical trials, but have not been used at prosthetic bypass anastomoses.3,4 The dominant focus of photodynamic therapy (PDT) research has been on the treatment of neoplasms. PDT has also gained interest as a local vascular therapeutic approach to inhibit IH.5e11 PDT is a photochemical process that uses light at a specific wavelength to activate otherwise relatively inert photosensitiser dyes for the production of free-radical moieties.12 These reactive species exert their cytotoxic effects by damaging cellular organelles and membranes, but have also been found to affect
1078–5884/000333 + 07 $32.00/0 Ó 2007 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.
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extracellular matrix molecules.13 Since IH is an iatrogenic, local obstruction, the precise and strictly local cytotoxic effects of PDT seem to be very well suited for endovascular therapy to inhibit the development of graft stenosis. PDT treatment of balloon injured carotid rat arteries reduces the amount of IH. With correct dosimetry of PDT, acute depletion of endothelial cells, medial smooth muscle cells, and adventitial fibroblasts has been observed in the targeted segment. This occurs without thrombus formation, development of aneurysms, or inflammatory cell infiltration.14,15 Research in vascular PDT applications has focused on the post-interventional arterial wound healing. However, PDT also has shown promising results to inhibit vein graft failure.16,17 The role of PDT to prevent prosthetic graft failure is unknown. Since it is acknowledged, that intimal hyperplasia in prosthetic grafts is, in part, due to arterial cell ingrowth. It can be hypothesized that a PDT-induced elimination of that cell population may favourably modulate the vascular wound healing response and therefore improve the patency rate of these grafts. This study was performed to investigate whether PDT has a role in reducing intimal hyperplasia in prosthetic grafts in a preclinical setting using a porcine iliac PTFE bypass model.
Materials and Methods Animal model Ten male castrated domestic pigs, weighing approximately 25 kg were used in the study. Anaesthesia was induced with intramuscular ketamine hydrocloride (10 mg/kg) and thiopentalnatrium (10 mg/kg) and maintained after endotracheal intubation with isoflurane. Animal protocols were approved by the ethical committee at Gothenburg University and conformed to guidelines set forth by national laws. The used photosensitizer and laser-light dosimetry was evaluated in a pilot study (3 pigs). Both iliac arteries were used for PDT and the specimens were harvested after 24 hours. PDT-treatment used fluence of 100, 150 and 200 J/cm2, the photosensitizer Methylene Blue was locally administered for 2 minutes at concentrations of 250 and 330 mg/ml, based on a small animal study.15 For the final study, a photosensitizer dose of 330 mg/ml and a light dose of 150 J/cm2 were used, based on the histological findings of the pilot study (Fig. 1). PTFE-grafts (polytetrafluoroethylene), 5 mm internal diameter (FlowLine Bipore, Jotec GmbH, Germany), Eur J Vasc Endovasc Surg Vol 34, September 2007
Fig. 1. Representative light micrographs of PDT-treated cross sections using different doses. (330 mg/ml Methylene Blue; a: 100 J/cm2, b: 150 J/cm2, c: 200 J/cm2). Specimens were harvested 24 hours after PDT. Note the remaining cells after PDT using 100 J/cm2, the few faint stained cells after 150 J/cm2 in the deeper layers of the media (b, see arrows) and the polynuclear cells in the upper layers of the media after 200 J/cm2 (c, see arrows).
were bilaterally placed into the circulation as bypasses from the common to the external iliac arteries as described before in 7 pigs.18 The geometry of the anastomoses was designed uniformly in all animals. Before graft-implantation the left arterial segment for the future distal anastomosis was pressurized at 180 mmHg for 5 minutes with the photosensitizer Methylene Blue (330 mg/ml) and endovascular PDT application performed as described previously. The control side was pressurized using saline solution.6 The segment was irradiated endoluminally with homogenous laser light, emitted by a diode laser (B and W Tek Inc., Newark, USA), to deliver 150 J/cm2
PDT Reduces IH in Prosthetic Vascular Bypass
at an irradiance of 100 mW/cm2 at a wavelength of 660 nm. Homogenous intraluminal light emission was achieved by coupling the laser to a 600 mm optical fiber with a 3 cm diffuser tip, introduced into a transparent balloon angioplasty catheter (OptaÔ Pro, Cordis, Germany, Fig. 2). Anastomoses were sutured end to side using 6-0 polypropylene sutures (Ethicon, Germany). The native iliac arteries were ligated to shunt the blood flow through the implanted grafts. Heparin was given intravenously (400 U/kg) before clamping and an additional dose of 200 U/kg was injected after two hours of surgery. Antibiotics were administered intramusculary (dihydrostreptomycin 6.5 mg/kg and benzylpenicillin 5 mg/kg) for a total of 3 days. After 4 weeks the animals were sacrificed under general anaesthesia by a potassium chloride overdose and the specimens were retrieved. Tissue samples and histomorphometry Both grafts were removed together with the native vessels en bloc and flushed with isotonic saline solution. Specimens from the mid portion of the distal anastomoses and the graft adjacent to these anastomoses were immersed in 4% formalin, buffered with sodium phosphate, for approximately 12 hours. Dehydration was performed in a graded series of ethanol (70, 95, 99.5%), xylene, and finally the specimens were embedded in paraffin. The specimens were stained with hematoxylin and eosin for descriptive histologic and morphometric analyses. Areas of intimal hyperplasia were quantified at the midportion of the anastomoses and the adjacent graft using light microscopy with Kontron Electronic image analysing system (KS 400 version 2.0, Carl Zeiss, Germany).
Fig. 2. Intraoperative figure showing the inserted and inflated balloon angioplasty catheter with the optical fiber emitting 660 nm monochromatic light. The left side of the picture is showing the distal part of the vessel, the crossing ureter is marked with an arrow.
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Immunohistochemistry For antigen retrieval, sections were treated with proteinase K (DAKO, Denmark) for 5 min. To block endogenous peroxidase and non-specific binding sites, the specimens were incubated for 5 min with 3% hydrogen peroxide and 15 min with 5% milk powder solution (Semper, Sweden). An anti-human monocyte/ macrophage antibody recognizing the intracytoplasmatic antigen (L1 or Calprotectin) in monocytes, tissue macrophages and granulocytes (1:100, clone MAC 387, Serotec, England) was used. The specimens were incubated with the primary antibody for 3 h at room temperature and for 30 min with the secondary antibody conjugated with horseradish peroxidase (EnVision kit, DAKO). DAB substrate (Vector Laboratories) was used for visualization. Nuclei were counterstained with hematoxylin. Control slides with non-immune IgG as primary antibody were included. A leukocyte count was performed in the vessel wall of the recipient artery by counting the positive cells at 4 different locations per specimen (clock-wise at 12, 3, 6 and 9) using light microscopy (400 magnification). The mean of the four values was used as one data point.18 Statistical evaluation All values are expressed as the mean SD. Differences in IH development and differences assessed using paired t-test with a P < 0.05 being significant. Results Pilot study No thrombosis was noted in any artery of the 3 pigs. Using a photosensitizer concentration of 250 mg/ml, complete cell eradication could not be achieved with any of the light doses. Using a Methylene Blue at 330 mg/ml, different histological results were noted. With a fluence of 100 J/cm2 a significant cell numbers were still present in the media (Fig. 1a). At 150 J/cm2 almost all medial cells were absent, with the exception of some faintly staining cells in the deeper layers of the media (Fig. 1b). At 200 J/cm2 some inflammatory appearing cells were noted in the luminal medial layers (Fig. 1c). Therefore, the dose of 150 J/cm2 was selected for the following experiments. PDT bypass study At the time of harvest, all animals appeared healthy and had no evidence of weight loss or wound Eur J Vasc Endovasc Surg Vol 34, September 2007
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infection. No bypass occlusion was observed in the time frame of 4 weeks. At the distal anastomosis the area of IH (mm2/mm graft) in controls was 6970 1536 whereas the area of IH after PDT was 2734 2560 (P < 0.005, Fig. 3). No gross thrombus was noted. The media consisted of normal appearing matrix tissue in both groups, but cellularity appeared to be reduced after PDT. The adventitia of both groups comprised loose connective tissue interspersed with fibroblast-like cells (Fig. 4). In the bypass graft the area of IH (mm2/mm length) in controls was 5391 4031, whereas the area of IH after PDT of the artery on the PDT-treated side was 777 1331 (P < 0.02, Fig. 5). The lesions in controls appeared typical of IH, with mesenchymal cells embedded in extracellular matrix (Fig. 6). The number of inflammatory cells (cells per microscopic field 400) in controls was 24 16 whereas the number of inflammatory cells after PDT was 37 15 (P < 0.009, Fig. 7). Discussion
Fig. 4. Representative light micrograph of a PDT-treated (a) and control (b) cross section of the distal anastomosis stained with hematoxylin and eosin 4 weeks after surgery. Note the elevated amount of intimal hyperplasia in the control anastomosis compared to the PDT-treated anastomosis (see arrows).
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The occurrence of anastomotic and graft intimal hyperplasia with subsequent graft failure in peripheral PTFE bypass graft surgery remains an unsolved problem. For the first time we have demonstrated, in a porcine model, that the adjuvant application of arterial PDT at the site of the distal anastomosis can reduce the amount of intimal hyperplasia at the anastomosis and within the graft. The mechanisms of vascular PDT to reduce restenosis in the arterial system after vascular
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Fig. 3. Area of intimal hyperplasia at the midportion of the anastomoses in control and PDT-treated specimens. The area is calculated as mm2 intimal hyperplasia/mm graft. Data are expressed as mean SD. P < 0.005 in controls vs. PDT. N ¼ 7 in both groups. Eur J Vasc Endovasc Surg Vol 34, September 2007
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Fig. 7. Inflammatory cells per microscopic field (400) in untreated and PDT-treated specimens. Data are expressed as mean SD. P < 0.009 in controls vs. PDT. N ¼ 7 in both groups.
Fig. 6. Representative light micrograph of a PDT-treated (a) and control (b) cross section of the distal bypass graft stained with hematoxylin and eosin 4 weeks after surgery. Note the elevated amount of intimal hyperplasia in the control grafts compared to the PDT-treated grafts (see arrows).
interventions have been investigated intensively in small animal models.8,15,19,20 PDT has also been applied to large animal models and in human clinical trials to investigate the role of PDT to inhibit restenosis after balloon angioplasty.5,11,21 These studies have demonstrated the safety and feasibility of vascular PDT in humans.
The established preclinical porcine model was well suited for this study because it closely mimics human healing in bypass grafts.18 As in humans, artificial vascular grafts implanted into pigs do not seem to form an endothelial lining. This is different to other experimental models, which in contrast to humans show artificial graft healing with an endothelial coverage.22,23 Since the distal anastomosis is the clinically relevant anastomosis for graft failure in most cases,24 only this anastomosis was PDT-treated and investigated in this study, with local application of the clinically available photosensitizer Methylene Blue. Despite the selectivity of confined light irradiation after systemic photosensitizer application, the site-specific delivery of a photosensitizer has conceptual appeal. First, it would achieve specific local drug concentrations. Second, concentrating the drug at the target site could minimize systemic adverse effects. All anastomoses were performed uniformly, since the geometry of the anastomoses influences biomechanical variables, which are important in the development of IH.25 Cells from the connected artery are considered to be the major source of graft IH. Four weeks after surgery, control grafts developed a considerable amount of IH at the site of the distal anastomosis as well as in the graft. In this time frame, IH did not cause graft failure. However a further progression of IH in this model might be expected, based on previous longterm studies.18 In the present study PDT treatment significantly reduced but did not eliminate IH, as seen in small animal models. Perhaps, achieving correct photosensitizer and light and dosimetry in arteries of bigger size is more difficult, compared to smaller vessels. In small animal models, 24 hours after PDT the arteries were found to be acellular.26 In the pilot study using the porcine iliac artery model, Eur J Vasc Endovasc Surg Vol 34, September 2007
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some remaining smooth muscle cells were noted in the deeper layers of the media 24 hours after PDT. This may be a result of suboptimal dosimetry or incomplete dead cell removal from the artery within this time frame of 24 hours due to the thickness of the vessel. Dosimetry for the final study was not increased further because of the inflammatory cells in upper medial layers in the pilot study (Fig. 3c) and it has been shown in small animals that a PDT overdose can also lead to photothrombosis of the vessel.27 These observations suggest that besides induction of cytotoxicity, a dose-dependant alteration of the extracellular environment also occurs. PDT has a small therapeutic window, which needs to be addressed intensively in human size arteries to avoid a too low PDT dose with subsequent development of IH and also to avoid too high doses with subsequent inflammatory cell attraction or thrombosis. In addition to dosimetry issues, it has been shown that not only arterial cells but also perivascular tissue cells and blood cells might contribute to IH in this model.18 These sources were not affected by PDT to the artery. Further research is necessary to optimize PDT to reduce prosthetic bypass graft failure and elucidate the mechanisms of reduced IH in this model. Our promising findings of the efficacy of PDT at reducing IH in prosthetic grafts are limited to a time frame of 4 weeks. Long-term observations and optimized photosensitizer and light dosimetry are necessary before possible application to man. Acknowledgements The authors thank Priv.-Doz. Dr. F. Adili for the helpful discussions. This study was funded by a grant from the state Northrhein-Westfalia, Germany. Jotec GmbH, Germany is acknowledged for its support for this study and for providing the PTFE grafts.
References 1 NEVILLE RF, SIDAWY AN. Myointimal hyperplasia: basic science and clinical considerations. Semin Vasc Surg 1998;11:142e148. 2 KUCHULAKAKANTI P, WAKSMAN R. Therapeutic potential of oral antiproliferative agents in the prevention of coronary restenosis. Drugs 2004;64:2379e2388. 3 WAKSMAN R, CHENEAU E, AJANI AE, WHITE RL, PINNOW E, TORGUSON R et al. Intracoronary radiation therapy improves the clinical and angiographic outcomes of diffuse in-stent restenotic lesions: results of the Washington Radiation for In-Stent Restenosis Trial for Long Lesions (Long WRIST) Studies. Circulation 2003;107:1744e1749. 4 KASTRATI A, MEHILLI J, VON BECKERATH N, DIBRA A, HAUSLEITER J, PACHE J et al. Sirolimus-eluting stent or paclitaxel-eluting stent vs balloon angioplasty for prevention of recurrences in patients with coronary in-stent restenosis: a randomized controlled trial. JAMA 2005;293:165e171.
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5 WAKSMAN R, LEITCH I, ROESSLER J, YAZDI H, SEABRON R, TIO F et al. Intracoronary photodynamic therapy reduces neointimal growth without suppressing re-endothelialization in a porcine model. Heart 2006;92:1138e1144. 6 JENKINS MP, BUONACCORSI GA, RAPHAEL M, NYAMEKYE I, MCEWAN JR, BOWN SG et al. Clinical study of adjuvant photodynamic therapy to reduce restenosis following femoral angioplasty. Br J Surg 1999;86:1258e1263. 7 PAI M, JAMAL W, MOSSE A, BISHOP C, BOWN S, MCEWAN J. Inhibition of in-stent restenosis in rabbit iliac arteries with photodynamic therapy. Eur J Vasc Endovasc Surg 2005;30:573e581. 8 ADILI F, SCHOLZ T, HILLE M, HECKENKAMP J, BARTH S, ENGERT A et al. Photodynamic therapy mediated induction of accelerated re-endothelialisation following injury to the arterial wall: implications for the prevention of postinterventional restenosis. Eur J Vasc Endovasc Surg 2002;24:166e175. 9 GABELER E, VAN HILLEGERSBERG R, STATIUS VAN EPS R, SLUITER W, MULDER P, VAN URK H. Endovascular photodynamic therapy with aminolaevulinic Acid prevents balloon induced intimal hyperplasia and constrictive remodelling. Eur J Vasc Endovasc Surg 2002;24:322e331. 10 VISONA A, ANGELINI A, GOBBO S, BONANOME A, THIENE G, PAGNAN A et al. Local photodynamic therapy with Zn(II)-phthalocyanine in an experimental model of intimal hyperplasia. J Photochem Photobiol B 2000;57:94e101. 11 MANSFIELD RJ, JENKINS MP, PAI ML, BISHOP CC, BOWN SG, MCEWAN JR. Long-term safety and efficacy of superficial femoral artery angioplasty with adjuvant photodynamic therapy to prevent restenosis. Br J Surg 2002;89:1538e1539. 12 DOUGHERTY TJ, GOMER CJ, HENDERSON BW, JORI G, KESSEL D, KORBELIK M et al. Photodynamic therapy. J Natl Cancer Inst 1998; 90:889e905. 13 OVERHAUS M, HECKENKAMP J, KOSSODO S, LESZCZYNSKI D, LAMURAGLIA GM. Photodynamic therapy generates a matrix barrier to invasive vascular cell migration. Circ Res 2000;86: 334e340. 14 LAMURAGLIA GM, CHANDRASEKAR NR, FLOTTE TJ, ABBOTT WM, MICHAUD N, HASAN T. Photodynamic therapy inhibition of experimental intimal hyperplasia: acute and chronic effects. J Vasc Surg 1994;19:321e331. 15 HECKENKAMP J, ADILI F, KISHIMOTO J, KOCH M, LAMURAGLIA GM. Local photodynamic action of methylene blue favorably modulates the postinterventional vascular wound healing response. J Vasc Surg 2000;31:1168e1177. 16 NIGRI GR, KOSSODO S, WATERMAN P, FUNGALOI P, LAMURAGLIA GM. Free radical attenuation prevents thrombosis and enables photochemical inhibition of vein graft intimal hyperplasia. J Vasc Surg 2004;39:843e849. 17 YAMAGUCHI A, WOODBURN KW, HAYASE M, ROBBINS RC. Reduction of vein graft disease using photodynamic therapy with motexafin lutetium in a rodent isograft model. Circulation 2000;102: III275eIII280. 18 MELLANDER S, FOGELSTRAND P, ENOCSON K, JOHANSSON BR, MATTSSON E. Healing of PTFE grafts in a pig model recruit neointimal cells from different sources and do not endothelialize. Eur J Vasc Endovasc Surg 2005;30:63e70. 19 NYAMEKYE I, BUONACCORSI G, MCEWAN J, MACROBERT A, BOWN S, BISHOP C. Inhibition of intimal hyperplasia in balloon injured arteries with adjunctive phthalocyanine sensitised photodynamic therapy. Eur J Vasc Endovasc Surg 1996;11:19e28. 20 YAMAGUCHI A, WOODBURN KW, HAYASE M, HOYT G, ROBBINS RC. Photodynamic therapy with motexafin lutetium (Lu-Tex) reduces experimental graft coronary artery disease. Transplantation 2001;71:1526e1532. 21 GONSCHIOR P, VOGEL-WIENS C, GOETZ AE, HUEHNS TY, BREGER F, GERHEUSER F et al. Endovascular catheter-delivered photodynamic therapy in an experimental response to injury model. Basic Res Cardiol 1997;92:310e319. 22 CLOWES AW, ZACHARIAS RK, KIRKMAN TR. Early endothelial coverage of synthetic arterial grafts: porosity revisited. Am J Surg 1987; 126:501e504.
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23 HERTZER NR. Regeneration of endothelium in knitted and velour dacron vascular grafts in dogs. J Cardiovasc Surg (Torino) 1981;22: 223e230. 24 LONGSTER PW, KLEINSTREUER C. Particle-hemodynamics modeling of the distal end-to-side femoral bypass: effects of graft caliber and graft-end cut. Med Eng Phys 2003;25:843e858. 25 LEI M, ARCHIE JP, KLEINSTREUER C. Computational design of a bypass graft that minimizes wall shear stress gradients in the region of the distal anastomosis. J Vasc Surg 1997;25: 637e646.
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26 STATIUS VAN EPS RG, CHANDRASEKAR NR, HASAN T, LAMURAGLIA GM. Importance of the treatment field for the application of vascular photodynamic therapy to inhibit intimal hyperplasia. Photochem Photobiol 1997;67:337e342. 27 ADILI F, STATIUS VAN EPS RG, LAMURAGLIA GM. Significance of dosimetry in photodynamic therapy of injured arteries: classification of biological responses. Photochem Photobiol 1999;70:663e668. Accepted 1 April 2007 Available online 21 May 2007
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