1. Volume 15, Issue 2, Pages 295-302 (February 2007)
VEGF-specific Short Hairpin RNA–expressing Oncolytic Adenovirus Elicits Potent Inhibition of Angiogenesis and Tumor Growth 
Ji Young Yoo, Joo-Hang Kim, Young-Guen Kwon, Eok-Cheon Kim, Nam Kyu Kim, Hye Jin Choi, Chae-Ok Yun 
Molecular Therapy 
Volume 15, Issue 2, Pages (February 2007)
DOI: /sj.mt
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Design and characterization of VEGF-specific siRNAs. (a) Location of two VEGF-specific siRNAs examined in this study. (b) Sequences for generating two shRNA targeting VEGF, shVEGF-1, and shVEGF-2. (c) shRNA-mediated in vitro knockdown of VEGF gene. Cells were transfected for 48 h with pshVEGF-1 or pshVEGF-2, and the knockdown of endogenous expression was measured by reverse transcriptase–polymerase chain reaction for VEGF.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Quantification of VEGF. Human U343 glioma cells were infected with (a) Ad-ΔB7 or Ad-ΔB7-shVEGF and (b) Ad-ΔE1, YKL-1, Ad-ΔB7, or Ad-ΔB7-shVEGF at a MOI of 10 and 20. VEGF concentration was measured in the culture supernatant at 24 and 48 h after infection by ELISA. (c) Various human cancer cell lines were transduced with Ad-ΔE1 or Ad-ΔE1-shVEGF at 10–500 MOI. VEGF concentration was measured in the culture supernatant 96 h after transduction by ELISA. Each value represents the mean±SE of at least three independent experiments.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Characterization of replication-incompetent Ad expressing VEGF-specific shRNA. (a) Inhibition of tube formation by Ad-ΔE1-shVEGF. Changes in cell morphology were captured using an inverted microscope (original magnification: × 40, × 200). (b) Inhibition of vessel sprouting by Ad-ΔE1-shVEGF. Representative aortic rings were photographed. Original magnification: × 40. (c) The area covered by the tube network was quantified using Image-Pro Plus software. Experiments were repeated three times and values are means of triplicates of representative of three independent experiment; bars, ±SE. *P<0.05 versus Ad-ΔE1 treatment. (d) Vessel sprouting index. *P<0.001 versus Ad-ΔE1 treatment. The assay was scored from 0 (least positive) to 5 (most positive) and the data are presented as mean (n=6). Each data point was assayed in sextuplets.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
VEGF-specific shRNA–expressing Ads inhibit vascularization in the Matrigel plug assay. (a) Representative images of Matrigel plugs removed from mice showing reduction in plug vascularization by Ad-ΔE1-shVEGF- and Ad-ΔB7-shVEGF-treated cells. (b) Immunohistochemical staining of sections of Matrigel plugs with anti-CD31 antibody. Original magnification: × 400. (c) Mean number±SE of blood vessels per group. *, **P<0.001 versus Ad-ΔE1 or Ad-ΔB7 treatment.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Antitumor effects of shVEGF–expressing oncolytic Ad. (a) Subcutaneous tumors derived from U343 human glioma cells were treated with Ad-ΔB7 (▪), Ad-ΔB7-shVEGF (○), or PBS (⧫). Values represent the means±SE for eight animals per group. (b) Overall survival. The survival of Ad-treated mice was evaluated over a period of 70 days.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
Antiangiogenic effects of VEGF-specific shRNA–expressing oncolytic Ad, Ad-ΔB7-shVEGF, in U343 human glioma xenografts. (a) Hematoxylin and eosin staining. Blood vessel density was assessed by immunohistochemical staining for CD31; hematoxylin counterstained (CD31). TdT-mediated dNTP nick end labeling (TUNEL) staining of apoptotic cells (indicated by an arrow) in tumor tissue; methyl green counter stained (TUNEL). Immunohistochemical staining of Ad hexon protein to localize Ad in tumor tissue; hematoxylin counterstained (Ad hexon). Ad-infected cells stain brown as indicated by an arrow. Original magnification: × 400. (b) The mean microvessel density for each treatment group. Three tumors per group were analyzed, and all data are shown as mean±SE. *P<0.001 versus PBS or Ad-ΔB7 treatment. (c) VEGF contents in tumors. Each data point represents mean VEGF levels for each individual tumor (seven mice per group) and the mean VEGF level for each group is represented by a line. VEGF level is expressed as picograms per milligram of total protein.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

8. Figure 7
Time course and magnitude of the VEGF gene silencing effect of Ad-ΔE1-shVEGF or Ad-ΔB7-shVEGF. (a) VEGF expression in U343 cells treated with Ad-ΔE1-shVEGF or Ad-ΔB7-shVEGF. Each value represents the mean±SE of at least three independent experiments. (b) VEGF expression in U343 xenograft models injected with PBS, Ad-ΔE1-shVEGF, or Ad-ΔB7-shVEGF. VEGF level is expressed as picograms per milligram of total protein. Results represent the mean±SE of three animals per group.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions