1. Volume 121, Issue 5, Pages 1040-1047 (November 2001)
Gene therapy for gastric ulcers with single local injection of naked DNA encoding VEGF and angiopoietin-1 
Michael K. Jones, Hirofumi Kawanaka, Dolgor Baatar, Imre L. Szabó, Kouji Tsugawa, Rama Pai, Gou Young Koh, Ingune Kim, I.James Sarfeh, Andrzej S. Tarnawski 
Gastroenterology 
Volume 121, Issue 5, Pages (November 2001)
DOI: /gast
Copyright © 2001 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Accelerated ulcer healing by single injection of cDNA encoding Ang1 and VEGF. Percent reduction in ulcer size was measured at 14 days after ulcer induction and injection of either control plasmid (set to 100%), Ang1-M-H, VEGF165-M-H, or the combination of Ang1-M-H + VEGF165-M-H (n = 18 for each group).
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

3. Fig. 2
Histologic illustration of ulcer healing in representative groups at 14 days. Quantitative data are presented in the Results section. Single injection of plasmids expressing VEGF165 and Ang1 accelerates ulcer healing and scar formation. (A) In the control group (empty vector), a large ulcer is present with a moderate amount of granulation tissue. In the basal portion of the ulcer margin mucosa, several dilated glands are present (arrows). (B) Representative ulcer from the Ang1-treated group. Granulation tissue is more developed, compared with the control group, reaching about the height of the ulcer margin. Several tubes are budding into the granulation tissue (arrow). (C) In the VEGF-treated group, 33% of the ulcers were completely healed. Mucosal scar contained several dilated gastric glands (arrows), but restoration of the mucosal architecture in the scar, even if imperfect, is accomplished. (D) In the VEGF + Ang1–treated group, 44% of the ulcers were completely healed. Restoration of the mucosal architecture is almost complete except for a few slightly dilated glands (arrow). (E) In the VEGF-treated group, 67% of the ulcers were incompletely healed. In the unhealed ulcers, mucosa of the ulcer margin has increased height (vs. controls), and restoration of the mucosal architecture within the ulcer margin is relatively good. (F) In the VEGF + Ang1–treated group, 56% of the ulcers were incompletely healed. The granulation tissue was greatly increased vs. controls, almost reaching the height of the ulcer margin, and the mucosal architecture within the ulcer margin was well restored. Magnification 100×.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Gene expression from the VEGF165-M-H plasmid. (A) RT-PCR using total RNA obtained from the margin of ulcers 2 days after injection of either control plasmid or VEGF165-M-H. The primer set used specifically recognizes only the VEGF165-M-H transcript cDNA to amplify the 475 bp VEGF-Myc product (top arrow). The β-actin products (bottom arrow) indicate equal amounts of total RNA used in the RT-PCR. (B) Western blot of total protein from granulation tissue of ulcers 7 days after injection with either control plasmid or VEGF165-M-H. The blot was probed with anti-6xHis antibody. The arrow shows the VEGF165-M-H protein product, and asterisks show nonspecific bands that serve to indicate equal sample loading. Relative sizes are indicated to the left (arrows).
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Expression of the VEGF165-Myc-His and Ang1-Myc-His fusion proteins in granulation tissue at the ulcer base. (A) Frozen tissue sections were taken 7 days after ulcer induction and injection of control plasmid; (B) plasmid-encoding VEGF165; (C) plasmid-encoding Ang1. The sections were stained with the anti-6xHis antibody. The VEGF165-Myc-His fusion protein is expressed mainly in the pericytes of the microvessels, and in some endothelial cells, whereas the Ang1-Myc-His fusion protein is expressed in endothelial cells lining the microvessels and some adjacent pericytes. Photomicrographs were taken with an Omega (FITC/Tex red) filter in which a positive signal is green. Magnification 200×.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

6. Fig. 5
Photomicrographs of granulation tissue stained by H&E. (A) Granulation tissue of ulcers injected with control plasmid; (B) plasmid-encoding VEGF165; (C) or the combination of plasmids encoding both VEGF165 and Ang1. The microvessels of the granulation tissue from the ulcers injected with either the control plasmid or the plasmid-encoding VEGF165 contained primarily only endothelial cells with very few pericytes. In contrast, the microvessels of the granulation tissue from the ulcers injected with the combined plasmids expressing both VEGF165 and Ang1 appeared to be more well-formed and contained more pericytes (arrows). Pericytes were identified by their close adherence to the endothelium and characteristic shape of being flat toward the abluminal surface of the endothelium and convex toward the surrounding collagen.13 Magnification 400×.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions