Gene transduction into aortic wall using plasmid-loaded cationized gelatin hydrogel- coated polyester stent graft Hongshan Zhong, MD, Osamu Matsui, MD,

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Gene transduction into aortic wall using plasmid-loaded cationized gelatin hydrogel- coated polyester stent graft Hongshan Zhong, MD, Osamu Matsui, MD, Ke Xu, MD, Takahiro Ogi, MD, Jun-ichiro Sanada, MD, Yasuo Okamoto, PhD, Yasuhiko Tabata, PhD, Yoh Takuwa, MD Journal of Vascular Surgery Volume 50, Issue 6, Pages 1433-1443 (December 2009) DOI: 10.1016/j.jvs.2009.07.071 Copyright © 2009 Society for Vascular Surgery Terms and Conditions

Fig 1 a, Partially-covered polyester stent graft; b, X-ray film of the rabbit that underwent the implantation of three CG hydrogel-coated partially-covered stent grafts (black arrows) in the aorta via right femoral artery. Journal of Vascular Surgery 2009 50, 1433-1443DOI: (10.1016/j.jvs.2009.07.071) Copyright © 2009 Society for Vascular Surgery Terms and Conditions

Fig 2 Whole mount X-gal staining of vessel segments and grafts. X-gal staining-positive blue dots and spots were revealed in both the aorta (a) and the neointima on the pCAGGS-LacZ-loaded graft (b). Parts of positive signals are indicated by black arrowheads. No blue dot was detected in the control aorta (c) or the graft (d). Journal of Vascular Surgery 2009 50, 1433-1443DOI: (10.1016/j.jvs.2009.07.071) Copyright © 2009 Society for Vascular Surgery Terms and Conditions

Fig 3 X-gal staining and immunohistochemical staining of cryosections of the pCAGGS-LacZ-loaded stent graft implanted vessel segment. a, X-gal staining followed by fast-red nuclear counter staining showed blue positive dots (black arrows) in the neointima and the tissue ingrowth into the graft; b, Immunohistochemical staining using anti-α smooth muscle actin antibody. The α-smooth muscle actin-positive cells (brown color) were observed in the neointima, media, and vasa vasorum in the adventitia; c, Immunohistochemical staining using anti-macrophages (RbM2) antibody. RbM2-positive macrophages (brown color) were observed mainly in the neointima. Journal of Vascular Surgery 2009 50, 1433-1443DOI: (10.1016/j.jvs.2009.07.071) Copyright © 2009 Society for Vascular Surgery Terms and Conditions

Fig 4 Real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis of aortic segments and various organs for LacZ mRNA expression. LacZ mRNA (a) and β-actin mRNA as internal control (b). LacZ mRNA was detected only in the pCAGGS-LacZ-loaded, CG hydrogel-coated stent graft implanted vessel segment. No dissemination was observed to distal or proximal sections of the aorta or to remote organs. Positive control (PC) of pCAGGS-LacZ transfected Cos 7 cells; 1: Vessel segment with pCAGGS-LacZ-loaded stent graft; 2: Vessel segment with pCAGGS-loaded stent graft; 3: Vessel segment proximal to stent grafted (pCAGGS-LacZ) vessel segment; 4: Vessel segment distal to stent grafted (pCAGGS-LacZ) vessel segment; 5: Brain; 6: Heart; 7: Liver; and 8: Kidney. Journal of Vascular Surgery 2009 50, 1433-1443DOI: (10.1016/j.jvs.2009.07.071) Copyright © 2009 Society for Vascular Surgery Terms and Conditions

Fig 5 Time-dependent expression of LacZ in aortic segments and stent grafts after graft stent implantation. a, Whole mount X-gal staining of vessel segments and stent grafts at days 1, 3, and 7 after implantation of stent grafts loaded with pCAGGS-LacZ at the plasmid concentration of 10.0 mg/mL. Lower panel represents the magnified views of the blocked areas in the upper panel. Much greater numbers of X-gal staining-positive blue dots are observed in the vessel segment at day 1 after implantation compared with the segments at days 3 and 7. b, Quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis of LacZ mRNA expression in the aortic segment. The transduction efficiency at day 1 was significantly higher than at day 7 (*P < .05 by one-way analysis of variance (ANOVA) and Newman-Keuls multiple comparison test). Journal of Vascular Surgery 2009 50, 1433-1443DOI: (10.1016/j.jvs.2009.07.071) Copyright © 2009 Society for Vascular Surgery Terms and Conditions

Fig 6 Dose-dependent expression of LacZ in aortic segments and stent grafts after graft stent implantation. a, Whole mount X-gal staining of vessel segments and stent grafts at day 1 after implantation of graft stents loaded with pCAGGS-LacZ at the plasmid concentrations of either 0.1, 1.0, or 10.0 mg/mL. Lower panel represents the magnified views of the blocked areas in the upper panel. Larger numbers of X-gal staining-positive blue dots are observed in the vessel segment with stent grafts loaded with 10.0 mg/mL plasmid solution compared with 1.0 and 0.1 mg/mL plasmid solutions. b, Quantitative real-time reverse transcriptase polymerase change reaction (RT-PCR) analysis of LacZ mRNA expression in the aortic segment. The transduction efficiency in 10.0 mg/mL plasmid was significantly higher than 1.0 and 0.1 mg/mL plasmid (*P < .05 vs 0.1 mg/mL loading concentration and f P < .01 vs 1.0 mg/mL loading concentration by one-way analysis of variance (ANOVA) and Newman-Keuls multiple comparison test). Journal of Vascular Surgery 2009 50, 1433-1443DOI: (10.1016/j.jvs.2009.07.071) Copyright © 2009 Society for Vascular Surgery Terms and Conditions