Volume 9, Issue 5, Pages 689-698 (May 2004) Overexpression of p53 Increases Lumen Size and Blocks Neointima Formation in Porcine Interposition Vein Grafts  Song Wan, Sarah J George, Stuart A Nicklin, Anthony P.C Yim, Andrew H Baker  Molecular Therapy  Volume 9, Issue 5, Pages 689-698 (May 2004) DOI: 10.1016/j.ymthe.2004.02.005 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Ad-mediated overexpression of p53 in porcine interposition vein grafts in vivo. (A) En face β-galactosidase expression in an Adβ-gal-infected vein graft harvested 7 days postinfection. (B) Real-time PCR quantification of Ad virion DNA in vein grafts harvested at 7 and 28 days. UD, undetectable (n = 3 grafts/group). A representative real-time trace is shown; green and blue lines illustrate 7- and 28-day samples, respectively. Molecular Therapy 2004 9, 689-698DOI: (10.1016/j.ymthe.2004.02.005) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 2 p53 overexpression in vein grafts. (A) Immunohistochemical detection of p53 in Adβ-gal- and Adp53-transduced vein grafts at 7 and 28 days postgrafting. The scale bar represents 10 μm and is applicable to all images. Arrowheads indicate some of the cells with nuclei containing p53 protein. (B) Representative Western blot analysis and quantification by densitometry (from n = 3 samples/time point) of p53 expression in grafts. *p < 0.05 vs control. Molecular Therapy 2004 9, 689-698DOI: (10.1016/j.ymthe.2004.02.005) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Modulation of cell proliferation by overexpression of p53. PCNA was detected and quantified both in the media and in the neointimal regions in vein grafts harvested 7 and 28 days postinfection. *P < 0.05 vs Adβ-gal-infected grafts. Molecular Therapy 2004 9, 689-698DOI: (10.1016/j.ymthe.2004.02.005) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Induction of apoptosis by p53 overexpression in vivo. (A) Quantification of apoptosis induced by p53 measured by ISEL 7 days postgrafting (n = 4 grafts/group, *P < 0.05). (B) Representative sections from Adβ-gal- and Adp53-transduced grafts stained by ISEL for days 7 and 28 postgrafting and immunohistochemical staining for cleaved PARP. The scale bar represents 25 μm and is applicable to all images. Molecular Therapy 2004 9, 689-698DOI: (10.1016/j.ymthe.2004.02.005) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Effects of p53 on cell density. Cells were counted on hematoxylin and eosin-stained sections to analyze overall cell density in the media and neointima at both 7 and 28 days postgrafting. Molecular Therapy 2004 9, 689-698DOI: (10.1016/j.ymthe.2004.02.005) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 6 Effects of overexpression of p53 on lumen and vessel size. (A) In situ zymography to assess proteolytic activity in grafts. White zones represent areas of proteolytic activity. The yellow lines in 28-day samples indicate the medial:intimal boundaries. (B) Representative low-magnification images of Adβ-gal- and Adp53-infected vessels at 7 and 28 days postgrafting. The scale bar represents 100 μm and is applicable to all images. Molecular Therapy 2004 9, 689-698DOI: (10.1016/j.ymthe.2004.02.005) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 7 Effects of p53 overexpression on neointima formation. Neointimal thickness was quantified in 7- and 28-day grafts. *P < 0.05 vs Adβ-gal-infected grafts, n = 6. The scale bar represents 50 μm and is applicable to all images. Arrows indicate the intima. Molecular Therapy 2004 9, 689-698DOI: (10.1016/j.ymthe.2004.02.005) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 8 Three-month analysis of grafts. (A) En face images of whole grafts harvested 3 months postsurgery. (B) Analysis of neointima formation by EVG and (C) low-magnification images to show lumen sizes. Arrows in (B) indicate the neointima. Molecular Therapy 2004 9, 689-698DOI: (10.1016/j.ymthe.2004.02.005) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions