Adenovirus-mediated gene transfer of human inducible nitric oxide synthase in porcine vein grafts inhibits intimal hyperplasia Melina R. Kibbe, MDa,

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Adenovirus-mediated gene transfer of human inducible nitric oxide synthase in porcine vein grafts inhibits intimal hyperplasia Melina R. Kibbe, MDa, Edith Tzeng, MDa, Susan L. Gleixner, BSa, Simon C. Watkins, PhDb, Imre Kovesdi, PhDc, Alena Lizonova, PhDc, Michel S. Makaroun, MDa, Timothy R. Billiar, MDa, Robert Y. Rhee, MDa Journal of Vascular Surgery Volume 34, Issue 1, Pages 156-165 (July 2001) DOI: 10.1067/mva.2001.113983 Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 1 Diagram depicts how IJ vein grafts were processed for morphometric and immunohistochemical analysis. DA, Distal anastomosis; DM, distal middle; M, middle; PM, proximal middle; PA, proximal anastomosis. Journal of Vascular Surgery 2001 34, 156-165DOI: (10.1067/mva.2001.113983) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 2 Cross sections of porcine IJ veins staining for β-galactosidase activity. Vein grafts were infected with AdlacZ and collected (A) 3 (n = 4), (B) 7 (n = 4), and (C) 21 (n = 8) days after infection and bypass. (D) Vein grafts infected with AdiNOS were collected at 3 days to serve as negative control. (Original magnification ×10.) Journal of Vascular Surgery 2001 34, 156-165DOI: (10.1067/mva.2001.113983) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 3 Nitrite, a stable end product of NO metabolism, was measured by Greiss reaction from the supernatant of porcine veins infected with AdiNOS or AdlacZ. Nitrite accumulation during the first 24 hours postinfection was measured and expressed as nanomoles per gram of tissue. Results represent average of all 16 pigs. Journal of Vascular Surgery 2001 34, 156-165DOI: (10.1067/mva.2001.113983) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 4 AdiNOS or AdlacZ infection of vein grafts resulted in minimal inflammation. Staining was performed for (A) neutrophils, (B) macrophages, and (C) lymphocytes by use of antibodies against myeloperoxidase, ED1, or CD45, respectively. Internal positive controls consist of porcine small bowel. Negative controls consist of sections stained with same protocol without primary antibody. Arrows indicate positive staining cells. n = 4 for 3 and 7 day groups. n = 8 for 21 day group. (Original magnification ×10.) L, Lumen. Journal of Vascular Surgery 2001 34, 156-165DOI: (10.1067/mva.2001.113983) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 5 Proliferative activity in vein grafts treated with AdiNOS (B, D) or AdlacZ (A, C) as determined by Ki67 staining. Vein grafts were collected at 3 (A, B) and 7 (C, D) days after bypass and stained with antibody to Ki67. E, Sections stained without the Ki67 antibody served as negative controls. n = 4 for each group. (Original magnification ×10.) L, Lumen. Journal of Vascular Surgery 2001 34, 156-165DOI: (10.1067/mva.2001.113983) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 6 iNOS gene transfer quantitatively decreased cellular proliferation rates at both 3- and 7-day time points as determined by Ki67 staining. Vein grafts were collected at their respective time points and stained for presence of Ki67. With a computer program, the total number of positive staining cells was determined, and this was adjusted to the total number of nuclei present. Five random areas were quantified for each section. A, Average proliferation rates of the entire graft for veins at 3 days. B, Proliferation rates reported by region at 3 days. C, Average proliferation rates of the entire graft at 7 days. D, Proliferation rates reported by region for veins at 7 days. n = 4 for each group. Journal of Vascular Surgery 2001 34, 156-165DOI: (10.1067/mva.2001.113983) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 7 iNOS gene transfer reduces IH in vein grafts. Vessels infected with AdiNOS and AdlacZ were collected at 3 (n = 4), 7 (n = 4), and 21 (n = 8) days. Images are cross sections of vein grafts. (Original magnification ×1.) Journal of Vascular Surgery 2001 34, 156-165DOI: (10.1067/mva.2001.113983) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 8 iNOS gene transfer reduces I/M in vein grafts. Vein grafts were infected with either AdiNOS or AdlacZ and collected at (A, B) 7 (n = 4) and (C, D) 21 (n = 8) days after bypass for morphometric analysis. A, Mean I/M averaged over entire length of vein graft at 7 days after bypass; B , I/M by graft region at 7 days; C, mean I/M averaged over entire length of vein graft at 21 days after bypass; D, I/M by graft region at 21 days. Journal of Vascular Surgery 2001 34, 156-165DOI: (10.1067/mva.2001.113983) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions