1. Adenovirus-mediated intra-arterial delivery of cellular repressor of E1A-stimulated genes inhibits neointima formation in rabbits after balloon injury 
Yaling Han, MD, PhD, Liang Guo, MD, PhD, Chenghui Yan, MD, PhD, Peng Guo, MS, Jie Deng, MS, Xiaoyan Mai, MS, Jian Kang, BSMed, Shaohua Li, MD, PhD 
Journal of Vascular Surgery 
Volume 48, Issue 1, Pages (July 2008)
DOI: /j.jvs
Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

2. Fig 1
Expression of cellular repressor of E1A genes (CREG) in normal and balloon-injured rabbit carotid arteries and its correlation with vascular smooth muscle cell (SMC) differentiation and proliferation. a, The segments of the rabbit common carotid artery (CCA) were harvested at 3, 7, 14, or 28 days (n = 5 for each time point) after balloon injury. Paraffin sections were prepared and immunostained for CREG and the SMC differentiation markers SM α-actin and SM myosin heavy chain (SM MHC). Cell proliferation was assessed by 5-bromo-2-deoxyuridine (BrdU) incorporation and detected with an anti-BrdU antibody. Specific staining was detected with a DAB chromogen kit and appears brown. b and c, Western blot analysis was performed for CREG, SM MHC, and SM α-actin in homogenates of the vascular tissue. β-Actin serves as loading control. The blots were scanned by densitometry and plotted as the ratio of CREG, SM α-actin, or SM MHC to β-actin. CREG expression was significantly down-regulated at 3 and 7 days after balloon injury vs day 0 (P < .01) and restored after 14 days. Concurrently, SM α-actin and SM MHC levels were markedly decreased at the same time compared with the baseline (P < .01 and .05, respectively; n = 5 for each time point).
Journal of Vascular Surgery  , DOI: ( /j.jvs )
Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

3. Fig 2
Adenovirus-mediated transfer of green fluorescent protein (GFP)/cellular repressor of E1A genes (CREG) to the injured artery. a, Fluorescent micrographs show GFP expression in the intima and media of the carotid artery at 3 days after balloon injury and adenovirus-GFP transduction. Infusion with saline alone serves as no treatment (NT) control. Arrowheads point to GFP-positive cells. Arrows point to the autofluorescence of the elastic lamina. b and c, Western blot analysis for GFP expression over time in adenovirus-GFP–transduced arteries. β-Actin serves as loading control (n = 5). d and e, Western blot analysis for CREG expression in the arteries transduced with adenovirus (Ad)-CREG, Ad-GFP, or the saline control (NT). β-Actin serves as the loading control. CREG expression in the saline control and the Ad-GFP group was significantly decreased at 3 days after balloon injury compared with that of the uninjured arteries (P < .01). CREG transduction dramatically increased the CREG level in the vessel wall (P < .01 vs the adenovirus-GFP and saline control; n = 5 for each group).
Journal of Vascular Surgery  , DOI: ( /j.jvs )
Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

4. Fig 3
Adenovirus-mediated cellular repressor of E1A genes (CREG) transfer to the balloon-injured artery inhibits intimal hyperplasia. a and b, In these representative micrographs of arterial sections stained with hematoxylin and eosin, arrowheads define the neointima. The areas of the neointima and the media in the cross sections of the artery were measured morphometrically and plotted. The neointima area and the intima/media ratio of adenovirus-CREG–transduced arteries were much smaller than those of the no treatment saline (NT) and adenovirus green fluorescent protein (Ad-GFP) controls at 28 days after balloon injury (P < .01); n = 5 for each group. c and d, Angiography of common carotid arteries was performed in Ad-CREG, Ad-GFP, and saline treated groups. Arrows define the minimal lumen diameter. The diameter stenosis rate of Ad-CREG–transduced arteries was significantly reduced compared with that of NT and Ad-GFP groups at 28 days after vascular injury (P < .01); n = 5 for each group. d, Data are presented with the standard deviation.
Journal of Vascular Surgery  , DOI: ( /j.jvs )
Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

5. Fig 4
Cellular repressor of E1A genes (CREG) transfer inhibits smooth muscle cell (SMC) proliferation and dedifferentiation but does not affect endothelial repair. a, Representative micrographs of carotid arteries display 5-bromo-2-deoxyuridine (BrdU)-labeled cells at 3 and 7 days after balloon injury and adenoviral infection. Adenovirus-mediated (Ad)-CREG treatment resulted in a significantly reduced number of BrdU-labeled cells at days 3 and 7 after vascular injury (P < .01 vs no treatment (NT) with saline and Ad-green fluorescent protein [GFP]). b, Immunostaining of injured arteries for SM α-actin and SM myosin heavy chain (MHC) at 7 days after treatment with Ad-CREG, Ad-GFP, or NT. c and d, Western blot analysis for SM α-actin, SM MHC and β-actin in the homogenates of injured arteries at 7 days after balloon injury. The blots were scanned by densitometry and plotted as the ratio of SM α-actin or SM MHC to β-actin; n = 5. Ad-CREG treatment increased the expression of SM α-actin and SM MHC at days 7 after vascular injury (P < .01 vs NT and Ad-GFP). d, Data are presented with the standard deviation. e, Representative micrographs show the injured artery immunostained for CD31 at 14 and 28 days after adenoviral transduction. Saline infusion alone serves as NT control.
Journal of Vascular Surgery  , DOI: ( /j.jvs )
Copyright © 2008 The Society for Vascular Surgery Terms and Conditions