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Hemagglutinating virus of Japan-liposome–mediated gene transfer of endothelial cell nitric oxide synthase inhibits intimal hyperplasia of canine vein.

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Presentation on theme: "Hemagglutinating virus of Japan-liposome–mediated gene transfer of endothelial cell nitric oxide synthase inhibits intimal hyperplasia of canine vein."— Presentation transcript:

1 Hemagglutinating virus of Japan-liposome–mediated gene transfer of endothelial cell nitric oxide synthase inhibits intimal hyperplasia of canine vein grafts under conditions of poor runoff  Takuya Matsumoto, MD, Kimihiro Komori, MD, PhD, Yoshikazu Yonemitsu, MD, PhD, Ryuichi Morishita, MD, PhD, Katsuo Sueishi, MD, PhD, Yasufumi Kaneda, MD, PhD, Keizo Sugimachi, MD, PhD, FACS  Journal of Vascular Surgery  Volume 27, Issue 1, Pages (January 1998) DOI: /S (98) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

2 Fig. 1 Dissecting microscopic (a and b) and light microscopic (c) findings in pAct-NLS-lac Z gene-transferred canine vein grafts under 100 mm Hg of distending pressure. Vessel segments were harvested 4 days after gene transfer and reacted with X-Gal solution. a, Intense blue stain is diffusely visible on graft wall, whereas no apparent positive signal is recognized on host artery. Original magnification, ×6. b, High-powered view of X-Gal–stained graft wall. Intense blue signal is detected in all layers of vein graft. Original magnification, ×18. c, Light microscopic findings of X-Gal–stained canine vein graft, the same one as a and b. Intense blue stain is visible in both medial (m) and adventitial (ad) layer. Counterstained with nuclear fast red. Original magnification, ×200. Journal of Vascular Surgery  , DOI: ( /S (98) ) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

3 Fig. 2 Immunohistochemical detection of bovine endothelial cell nitric oxide (ecNOS) in pUC-CAGGS-ecNOS gene-transferred canine vein graft. Vessel segments were harvested 4 days after gene transfer and fixed with neutralized 10% formaldehyde. Five μm thin sections embedded in paraffin were examined immunohistochemically. Original magnification, ×100. Counterstained with methyl green. a, Immunohistochemical localization of bovine ecNOS protein in canine vein graft treated with pUC-CAGGS-ecNOS. Frequent positive signals are recognized in medial and adventitial cells (arrows). b, Serial section that was reacted with nonimmunized rabbit serum, as a primary antibody for the negative control. No signal was observed. c, Mock transfected (pUCCAGGS control vector transferred) canine vein graft reacted with antibovine ecNOS antibody. No signal was observed. Journal of Vascular Surgery  , DOI: ( /S (98) ) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

4 Fig. 3 NADPH diaphorase assay for vein graft. Vessel segments, harvested 4 days after gene transfer, were irrigated with phosphate-buffered saline solution and immediately reacted in NADPH solution for 30 minutes at 37° C. Reaction was stopped by washing with phosphate buffered saline solution followed by fixation with neutralized 10% formaldehyde. This experiment was repeated three times and produced similar results. a, Dissecting microscopic findings in untreated control vein. Weak and vague blue stain is visible around luminal surface, indicating endogenous endothelial ecNOS activity. Spotty blue stain (arrow) caused by endogenous NOS activity of vasa vasorum is also seen at adventitial layer. Original magnification, ×3. b, Mock transfected vein graft as a control. Almost no blue signal, except for adventitial spots (arrow), is evident. Original magnification, ×12. c, Bovine ecNOS cDNA-transferred canine vein graft. Intense blue stain is recognized in all layers of graft. Original magnification, ×12. Journal of Vascular Surgery  , DOI: ( /S (98) ) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

5 Fig. 4 Light microscopic features of midportion of vein grafts. Light microscopic findings indicated endothelial cell nitric oxide synthase (ecNOS; a) gene-transferred, buffer-treated (b), or mock-transfected (c) canine vein grafts implanted under conditions of poor runoff. Four weeks after gene transfer and graft implantation, the grafts were perfusion-fixed, harvested, and embedded in paraffin. Five μm sections were examined under a light microscope. ecNOS gene-transferred vein graft (a) shows greater reduced neointimal area (n, arrows) than seen in buffer-treated (b, arrows) or mock-transfected (c, arrows) vein grafts. lu, lumen; ad, adventitia. Elastica van Gieson's staining. Original magnification, ×100. Journal of Vascular Surgery  , DOI: ( /S (98) ) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

6 Fig. 5 Quantitative analyses of intimal thickening of canine vein grafts. Two different methods were used. Four weeks after gene transfection, the grafts were dissected gently, perfusion-fixed, and embedded in paraffin. We then measured 5 μm of five interval sections per vessel segment, stained with elastica van Gieson's stain. a, Measurements using an ocular cytometer placed on ocular lens of light microscope (eight-point method). b, Measurements using image analyzer (computer method). Both quantifications clarified that ecNOS gene transfer significantly reduced the neointimal lesion, compared with findings with buffer treatment and mock transfer. Journal of Vascular Surgery  , DOI: ( /S (98) ) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

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