1. Volume 16, Issue 12, Pages 1927-1936 (December 2008)
Endothelial-targeted Gene Transfer of Hypoxia-inducible Factor-1α Augments Ischemic Neovascularization Following Systemic Administration 
Reshef Tal, Aviv Shaish, Karen Rofe, Erez Feige, Nira Varda-Bloom, Arnon Afek, Iris Barshack, Livnat Bangio, Israel Hodish, Shoshana Greenberger, Michael Peled, Eyal Breitbart, Dror Harats 
Molecular Therapy 
Volume 16, Issue 12, Pages (December 2008)
DOI: /mt
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Stimulation of in vitro angiogenesis by hypoxia-inducible factor-1α (HIF-1α) adenoviruses. Human umbilical vein endothelial cells were left untreated (control) or infected with Ad-CMV-GFP, or Ad-CMV-wtHIF-1α, or Ad-CMV-Triple, or Ad-PPE-Triple. In vitro angiogenesis assay was performed 48 hours later. (a) Representative photographs of tube formation taken 8 hours after seeding on matrigel. (b) Tube formation was quantified by counting the number of capillary branches per high-power microscopic field (×40). The results are mean values ± SD of five random microscopic fields. *P < versus Ad-CMV-Triple mutant, and +P < versus Ad-CMV-wt, Ad-CMV-GFP, and control.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Endothelial and ischemia specificity of PPE1-3x promoter. (a–r) green fluorescent protein (GFP) (green, left), CD31 (red, middle) or merged (right) confocal fluorescent microscopic images of gastrocnemius muscle sections of mice subjected to either hindlimb ischemia or sham procedure, which were injected systemically 7 days later with either (a–f) saline, (g–l) Ad-CMV-GFP, or (m–r) Ad-PPE1-3x-GFP. Ad-CMV-GFP expression in both (g) nonischemic and (j) ischemic limbs was observed within muscle fibers without correlation to (h,k) CD31-stained endothelial cells as seen in the (i,l) merged images. Ad-PPE1-3x-GFP demonstrated specificity to ischemic condition, showing expression only in the (p) ischemic limb without expression in the (m) nonischemic limb. (p) Ad-PPE1-3x-GFP expression revealed colocalization with (q) CD31-stained endothelial cells as seen in yellow in the (r) merged image, indicating specific expression of Ad-PPE1-3x-GFP only in ischemic endothelial cells. White arrows point to endothelial cells with GFP and CD31 colocalization. Original magnification ×40.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Human hypoxia-inducible factor-1α (HIF-1α) transgene biodistribution following systemic administration. Real-time quantitative PCR of human HIF-1α mRNA expression relative to glyceraldehyde 3-phosphate dehydrogenase 5 days after systemic administration of adenoviruses in mice subjected to hindlimb ischemia, in liver, lung, spleen, heart, kidney, brain, small intestine, gonads, and ischemic and nonischemic hindlimb muscles, showing expression of Ad-PPE-Triple only in ischemic hindlimb muscle. Data for real-time quantitative PCR are expressed as mean values ± SE. n = 3–4 per group. *P < 0.05 versus Ad-CMV-wt and Ad-CMV-Triple.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Ad-PPE1-3x-Triple is less toxic than Ad-CMV-Triple after systemic administration. (a) Changes in body weights of mice were monitored following systemic injection of saline or adenovirus (n = 7–9 per group). (b) Serum bilirubin, (c) AST, and (d) ALT levels were measured 5 and 21 days following systemic injection to determine liver functions.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Hematoxylin and eosin staining of histological sections of mice livers 5 and 21 days following systemic injection. Arrows point to lymphocytic infiltration. Arrowhead points to hepatocyte ballooning. Original magnification ×40.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
Postischemic angiogenesis is augmented by systemic Ad-PPE1-3x-Triple treatment but not by Ad-CMV-Triple treatment. (a) Hindlimb ischemia was induced, followed 7 days later by tail-vein adenovirus injection. Blood flow in the ischemic limb was measured immediately after, and at 7, 14, 21, and 28 days after left femoral artery resection. (b) Blood flow is expressed as the ratio of the left (ischemic) to right (nonischemic) limb perfusion. Saline, n = 9; Ad-CMV-Luc, n = 8; Ad-CMV-wt, n = 9; Ad-CMV-Triple, n = 7; Ad-PPE-Triple, n = 8. *P < 0.01 versus Ad-PPE-Triple. (c) Representative laser Doppler blood perfusion images of the ischemic (left) and nonischemic (right) limbs at day 28 after surgery. In color-coded images, normal perfusion is depicted in red while low and/or no perfusion is depicted in blue. (d) Limb necrosis score at 21 days after femoral artery resection as an index of severity of limb ischemia: 0, normal; 1, pale foot or gait abnormalities; 2, necrosis in one toe; 3, necrosis in more than one toe; 4, toe amputation. *P < 0.05 versus Ad-PPE-Triple. Representative (e) CD31 staining and (f) quantitation of capillaries from sections of gastrocnemius muscles 28 days after femoral artery ligation. Original magnification ×40. *P < 0.01 versus Ad-PPE-Triple.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

8. Figure 7
Expression of mRNA of hypoxia-inducible factor-1α target genes within ischemic muscle [but not of vascular endothelial growth factor (VEGF serum protein)] is augmented following systemic Ad-PPE-Triple treatment. Hindlimb ischemia was induced, followed 7 days later by tail-vein adenovirus injection. n = 3–4 per group. (a–c) Real-time quantitative PCR for (a) VEGF, (b) platelet-derived growth factor B (PDGF-B), and (c) stromal cell–derived factor-1 (SDF-1) mRNAs within ischemic skeletal muscle 5 days following systemic administration of saline or adenoviruses. Data are expressed as multiples of increase in induction relative to saline treatment, and shown as mean values ± SE. *P < 0.05 versus all other groups. (d) Serum VEGF protein concentration (pg/ml) in mice 5 days after treatment with saline or adenoviruses. Data are expressed as mean values ± SE. *P < 0.05 versus all other groups.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions