Adventitial versus intimal liposome-mediated ex vivo transfection of canine saphenous vein grafts with endothelial nitric oxide synthase gene Manju Kalra,

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Adventitial versus intimal liposome-mediated ex vivo transfection of canine saphenous vein grafts with endothelial nitric oxide synthase gene Manju Kalra, MD, Corey J. Jost, MD, Sandra R. Severson, AA, Virginia M. Miller, PhD Journal of Vascular Surgery Volume 32, Issue 6, Pages 1190-1200 (December 2000) DOI: 10.1067/mva.2000.109211 Copyright © 2000 Society for Vascular Surgery and The American Association for Vascular Surgery, a Chapter of the International Society for Cardiovascular Surgery Terms and Conditions

Fig. 1 A, Cross-sectional area (CSA ) and B, blood flow in ecNOS-transfected canine saphenous vein grafts on postoperative day 3. Results are presented as mean ± SEM of measurements in ecNOS-transfected grafts normalized to contralateral control grafts (transfected with plasmid alone). Dotted line represents measurement in control vein grafts (100%; absolute values shown in Table) (n = 6 in each group). Journal of Vascular Surgery 2000 32, 1190-1200DOI: (10.1067/mva.2000.109211) Copyright © 2000 Society for Vascular Surgery and The American Association for Vascular Surgery, a Chapter of the International Society for Cardiovascular Surgery Terms and Conditions

Fig. 2 Representative ethidium bromide–stained 2% agarose gel demonstrating a 321-bp PCR product in DNA isolated from canine saphenous vein grafts transfected with ecNOS. Primers used in the PCR reaction annealed to the 5′ promoter region of the plasmid and the 3′ region of the cloned ecNOS gene. Ten microliters of PCR product loaded per lane. Lane 1, 100 bp DNA marker; Lane 2, plasmid alone; Lane 3, positive control signal at 321 bp in the plasmid plus ecNOS gene; Lane 4, water. Lanes 5-10, control vein grafts transfected with plasmid alone, endothelial/adventitial transfection: 10 μg/mL alone, 10 μg/mL with LipofectAMINE PLUS, and 100 μg/mL with LipofectAMINE PLUS. Lanes 11-16, vein grafts transfected with plasmid-containing ecNOS gene, endothelial/adventitial transfection: 10 μg/mL alone, 10 μg/mL with LipofectAMINE PLUS, and 100 μg/mL with LipofectAMINE PLUS. Journal of Vascular Surgery 2000 32, 1190-1200DOI: (10.1067/mva.2000.109211) Copyright © 2000 Society for Vascular Surgery and The American Association for Vascular Surgery, a Chapter of the International Society for Cardiovascular Surgery Terms and Conditions

Fig. 3 Quantification of ecNOS mRNA (total ecNOS mRNA – transfected + endogenous) by RT-PCR followed by PCR MIMIC in canine saphenous vein grafts transfected with ecNOS. Results are presented as mean ± SEM of values in ecNOS-transfected grafts normalized to contralateral control grafts (transfected with plasmid alone). Dotted line represents mRNA level in control vein grafts (100%; absolute values shown in Table). *P <.01 versus all other transfection groups (n = 6 in each group). Journal of Vascular Surgery 2000 32, 1190-1200DOI: (10.1067/mva.2000.109211) Copyright © 2000 Society for Vascular Surgery and The American Association for Vascular Surgery, a Chapter of the International Society for Cardiovascular Surgery Terms and Conditions

Fig. 4 Calcium-dependent activity of NOS in canine vein grafts transfected with ecNOS. Results are presented as mean ± SEM of levels in ecNOS-transfected grafts normalized to contralateral control grafts (transfected with plasmid alone). Dotted line represents calcium-dependent NOS activity in control vein grafts (100%; absolute values shown in Table) (n = 6 in each group). Journal of Vascular Surgery 2000 32, 1190-1200DOI: (10.1067/mva.2000.109211) Copyright © 2000 Society for Vascular Surgery and The American Association for Vascular Surgery, a Chapter of the International Society for Cardiovascular Surgery Terms and Conditions

Fig. 5 NOx levels in dogs with ecNOS transfection of saphenous vein grafts. Results are presented as mean ± SEM. A, Peripheral venous NOx levels on day 3 normalized to preoperative baseline levels (represented by the dotted line ). B, Arterial NOx levels in ecNOS-transfected grafts on day 3 at explant, normalized to contralateral control grafts (transfected with plasmid alone). Dotted line represents NOx level in control vein grafts (100%; absolute values shown in Table) (n = 6 in each group). Journal of Vascular Surgery 2000 32, 1190-1200DOI: (10.1067/mva.2000.109211) Copyright © 2000 Society for Vascular Surgery and The American Association for Vascular Surgery, a Chapter of the International Society for Cardiovascular Surgery Terms and Conditions

Fig. 6 Representative light micrographs (magnification ×100) showing cell proliferation (brown nuclei ) identified by nuclear antigen Ki-67 (MIB1) immunohistochemistry in canine saphenous vein graft transfected through the adventitia with liposome and 100 μg/mL plasmid containing ecNOS gene (A). B, Control graft transfected with liposome and plasmid alone. Journal of Vascular Surgery 2000 32, 1190-1200DOI: (10.1067/mva.2000.109211) Copyright © 2000 Society for Vascular Surgery and The American Association for Vascular Surgery, a Chapter of the International Society for Cardiovascular Surgery Terms and Conditions

Fig. 7 Quantification of cell proliferation in ecNOS-transfected canine saphenous vein grafts on day 3. The MIB1 proliferative index (positive cells/total cells × 100) was calculated for a section of each graft, as well as separately in the medial and adventitial layers. Results are presented as mean ± SEM of indices in ecNOS-transfected grafts normalized to contralateral control grafts (transfected with plasmid alone). Dotted line represents cell proliferation index in control vein grafts (100%; absolute values shown in Table) (n = 6 in each group). Journal of Vascular Surgery 2000 32, 1190-1200DOI: (10.1067/mva.2000.109211) Copyright © 2000 Society for Vascular Surgery and The American Association for Vascular Surgery, a Chapter of the International Society for Cardiovascular Surgery Terms and Conditions