The Role of ex-vivo Gene Therapy of Vein Grafts with Egr-1 Decoy in the Suppression of Intimal Hyperplasia M. Peroulis, J. Kakisis, A. Kapelouzou, A.

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The Role of ex-vivo Gene Therapy of Vein Grafts with Egr-1 Decoy in the Suppression of Intimal Hyperplasia M. Peroulis, J. Kakisis, A. Kapelouzou, A. Giagini, S. Giaglis, G. Mantziaras, N. Kostomitsopoulos, P. Karayannacos, A. Macheras European Journal of Vascular and Endovascular Surgery Volume 40, Issue 2, Pages 216-223 (August 2010) DOI: 10.1016/j.ejvs.2010.04.013 Copyright © 2010 European Society for Vascular Surgery Terms and Conditions

Figure 1 Nuclear localization of FITC–ODN in jugular vein graft of rabbit model. Fluorescent photomicrographs indicate concentrated green FITC–ODN signals (A), that correspond to nuclei labeled with the DAPI coloration (B), as seen in detail in the merged image (C). (Original magnification ×20, scale bar: 200μm). European Journal of Vascular and Endovascular Surgery 2010 40, 216-223DOI: (10.1016/j.ejvs.2010.04.013) Copyright © 2010 European Society for Vascular Surgery Terms and Conditions

Figure 2 Egr-1 mRNA expression in qRT–PCR. Egr-1 expression was 60% and 57% lower in the decoy treated grafts compared to the untreated and mutant ODN treated grafts, respectively (n=6 for each group, ** P<0.001). European Journal of Vascular and Endovascular Surgery 2010 40, 216-223DOI: (10.1016/j.ejvs.2010.04.013) Copyright © 2010 European Society for Vascular Surgery Terms and Conditions

Figure 3 a. Photomicrographs showing representative cross sections of vein grafts treated with Egr-1 decoy ODN (A), mutant ODN (B), vehicle alone (C) and vein grafts without any treatment (D) 1.5 month after placement in ipsilateral carotid artery (immunohistochemical staining for Ki-67, original magnification ×20, scale bar: 200μm). b. Inhibitory effect of Egr-1 decoy transfection on proliferation (Ki-67 positive cells) at 1.5 and 3 months after placement of vein grafts treated with Egr-1 decoy ODN (A), mutant ODN (B), vehicle alone (C) and vein grafts without any treatment (D) (n=6 for each group, * P<0.05). European Journal of Vascular and Endovascular Surgery 2010 40, 216-223DOI: (10.1016/j.ejvs.2010.04.013) Copyright © 2010 European Society for Vascular Surgery Terms and Conditions

Figure 4 Light micrographs of normal vein (a), Egr-1 decoy ODN-treated vein grafts (b) and mutant decoy ODN-treated vein grafts (c) in hypercholesterolemic rabbits 3 months after grafting. An intimal and medial hyperplasia is seen in the vein grafts compared to the normal vein (hematoxylin-eosin stain: original magnification ×5. scale bar: 500μm). European Journal of Vascular and Endovascular Surgery 2010 40, 216-223DOI: (10.1016/j.ejvs.2010.04.013) Copyright © 2010 European Society for Vascular Surgery Terms and Conditions

Figure 5 Intimal and medial wall thickness at 1.5 and 3 months after placement of vein grafts treated with Egr-1 decoy ODN (A), mutant ODN (B), vehicle alone (C) and vein grafts without any treatment (D) in cholesterol-fed rabbits. Intimal hyperplasia of Egr1 decoy ODN versus mutant ODN, vehicle alone and the untreated vein grafts was 50%, 42% and 41% reduced respectively at 1.5 months and 32%, 30% and 36% reduced respectively at 3 months (n=6 for each group, * P<0.05). Gray bars: intimal thickness (μm), black bars: medial thickness (μm). European Journal of Vascular and Endovascular Surgery 2010 40, 216-223DOI: (10.1016/j.ejvs.2010.04.013) Copyright © 2010 European Society for Vascular Surgery Terms and Conditions

Figure 6 Intimal and medial cross-sectional wall area at 1.5 and 3 months after placement of vein grafts treated with Egr-1 decoy ODN (A), mutant ODN (B), vehicle alone (C) and vein grafts without any treatment (D) in cholesterol-fed rabbits. The intimal area of Egr1 decoy ODN versus mutant ODN, vehicle alone and the untreated vein grafts was 54%, 45% and 41% reduced respectively at 1.5 months and 39%, 35% and 40% reduced respectively at 3 months (n=6 for each group,** P<0.001,*** P<0.0001). Gray bars: intimal area (mm2), black bars: medial area (mm2). European Journal of Vascular and Endovascular Surgery 2010 40, 216-223DOI: (10.1016/j.ejvs.2010.04.013) Copyright © 2010 European Society for Vascular Surgery Terms and Conditions

Figure 7 Effect of Egr-1 decoy transfection in lumenal area 1.5 and 3 months after placement of vein grafts treated with Egr-1 decoy ODN (A), mutant ODN (B), vehicle alone (C) and vein grafts without any treatment (D) in cholesterol-fed rabbits. The lumen area of Egr-1 decoy ODN versus scrambled ODN, vehicle alone and the untreated vein grafts was 28%, 32% and 35% increased respectively at 1.5 months and 39%, 29% and 44% increased respectively at 3 months (n=6 for each group, * P<0.05, ** P<0.001). European Journal of Vascular and Endovascular Surgery 2010 40, 216-223DOI: (10.1016/j.ejvs.2010.04.013) Copyright © 2010 European Society for Vascular Surgery Terms and Conditions