Abstract 14475: AAV9-Mediated Over-Expression of Extracellular Superoxide Dismutase Improves Recovery from Hindlimb Ischemia in BALB/c Mice
Introduction: We hypothesized that over-expression of extracellular superoxide dismutase (EcSOD) in skeletal muscle would increase capillary density and improve recovery following surgical hindlimb ischemia.
Methods: AAV vectors expressing EcSOD or luciferase (control) from the CMV promoter were cross-packaged into AAV9 capsids and injected IM into hindlimb muscles (1x1011 vp/limb) of 12 wk-old mice. In non-ischemic B6 mice (n=6 / group) EcSOD expression was assessed at day 14 post-injection by Western blotting for EcSOD. In BALB/c mice (n=10 / group) ischemia was induced after IM injection by ligating and excising the left femoral artery from above the inguinal ligament to its bifurcation at the saphenous and popliteal arteries. Capillary density and actin organization were assessed at day 14 post-ischemia by immunostaining for PE-CAM and phalloidin staining, respectively. Limb perfusion was measured by laser Doppler on days 0, 7 & 14 post ischemia and expressed relative to the non-ischemic limb. Limb necrosis was scored daily as Grade 0 (none) to Grade 4 (complete).
Results: In non-ischemic EcSOD treated B6 mice, EcSOD levels by Western were 1.53 fold higher than in controls (Panel A, p<0.05). In ischemic EcSOD treated BALB/c mice, actin organization was better preserved and capillary density was 1.94 fold higher (1638±20/mm2, Mean±SEM) than in controls (Panel B, 846±176/mm2, p<0.05). Recovery of perfusion ratio at day 14 post-ischemia in BALB/c mice was 1.44 fold higher in EcSOD vs. controls (p<0.05, Panel C). Increased capillary density was correlated (R = 0.654, p<0.05) with increased perfusion. Progression of limb necrosis was significantly slower in EcSOD vs. control mice (Panel D).
Conclusion: AAV9-mediated over-expression of EcSOD in skeletal muscle significantly improves recovery from hindlimb ischemia in mice, consistent with preserved actin organization, improved capillary density and higher perfusion ratio in treated limbs.
- © 2010 by American Heart Association, Inc.