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| Angiopoietin-2 Gene Transfer Induces Potent Neovascularization in the Hindlimb Ischemia Model: Interactions with VEGF |
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Back to 86th Annual Meeting Back to Program Outline Objective: Angiopoietins (Ang1 and 2), are ligands for the endothelial receptor, Tie2. Ang1 is an agonist, whereas Ang2 is a context-dependent antagonist releasing endothelial cells (EC) from the tonic inhibitory influence of Ang1, and facilitating EC activation in response to VEGF and the initiation of angiogenesis. In the rodent ischemic hindlimb model, we noted an early (day 1 to day 7) 2-fold rise in the ratio of Ang-2:Ang-1 mRNA and protein expression in the ischemic leg (I) versus the contralateral non-ischemic (NI) limb in response to ischemia induction (day 0). Therefore, we hypothesized that Ang2, would be the more effective proangiogenic factor in the hindlimb ischemia model. Methods: Sprague-Dawley rats (500-650g) received intramuscular injections (500µl) into the ischemic hindlimb at day 7, of plasmid DNA encoding for human Ang1 alone (500µg) (n=9), Ang2 alone (n=9), VEGF alone (n=9) or combination (1000µg, n=5/group) of Ang1+VEGF or Ang2+VEGF. Control animals (n=11) received null plasmid injections. As well, conditional transgenic mice were created overexpressing Ang1 or Ang2 under the control of a doxycycline regulated promoter. Limb perfusion was assessed with Laser doppler imaging expressed as a ratio of ischemic (I) to non-ischemic (NI) leg in both mice and rats. Angiographic score (rats) was determined on day 35. Results: Overexpression of Ang2 in mice, but not Ang1, increased ischemic limb perfusion (I/NI: 0.77±0.11 vs. 0.45±0.02 respectively, mean±SEM, p<0.01) compared to littermate controls (0.36±0.02, p<0.005). Single gene therapy with Ang2 or VEGF similarly enhanced perfusion (I/NI: 0.66±0.14 and 0.63±0.15 respectively, p<0.05), while Ang1 alone (0.55±0.13) or null plasmid (0.48±0.03) did not improve perfusion. The combination of Ang1 and VEGF gene transfer was not superior to VEGF alone (0.62±0.06), however, Ang2 and VEGF treatment resulted in a greater improvement in perfusion scores than either factor alone (0.74±0.05, p<0.05). Angiographic scores were also greater in Ang2, VEGF and Ang2+VEGF compared to control (p<0.05), whereas treatment with Ang1 alone showed no significant improvement. Conclusions: In the chronic hindlimb ischemia model, overexpression of Ang2 was superior to Ang1 in enhancing ischemic limb perfusion in transgenic mice. Ang2, but not Ang1 gene transfer in the rat produced anatomic and physiological evidence of increased collateral vessel formation. The apparent synergy between VEGF and Ang2 gene transfer suggests that this may be a promising therapeutic approach. Back to 86th Annual Meeting Back to Program Outline |
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