title: Adeno-associated virus serotype 9-mediated overexpression of extracellular superoxide dismutase improves recovery from surgical hind-limb ischemia in BALB/c mice.
abstract: OBJECTIVE
  Neovascularization is a physiologic repair process that partly depends on nitric oxide. Extracellular superoxide dismutase (EcSOD) is the major scavenger of superoxide. It is an important regulator of nitric oxide bioavailability and thus protects against vascular dysfunction. We hypothesized that overexpression of EcSOD in skeletal muscle would improve recovery from hind-limb ischemia.
  
  
  METHODS
  Adeno-associated virus serotype 9 (AAV9) vectors expressing EcSOD or luciferase (control) from the cytomegalovirus promoter were cross-packaged into AAV9 capsids and injected intramuscularly into the hind-limb muscles (1 × 10(11) viral genomes/limb) of 12-week-old mice. Ischemia was induced after intramuscular injections. Laser Doppler was used to measure limb perfusion on days 0, 7, and 14 after injection. Values were expressed as a ratio relative to the nonischemic limb. EcSOD expression was measured by Western blotting. Capillary density was documented by immunohistochemical staining for platelet endothelial cell adhesion molecule. Apoptosis was assessed by terminal deoxynucleotide transferase-mediated biotin-deoxy uridine triphosphate nick-end labeling and necrosis was visually evaluated daily.
  
  
  RESULTS
  EcSOD expression was twofold upregulated in EcSOD treated vs control ischemic muscles at day 14. Capillary density (capillaries/fiber) was 1.9-fold higher in treated (1.65 ± 0.02) vs control muscle (0.78 ± 0.17, P < .05). Recovery of perfusion ratio at day 14 after ischemia was 1.5-fold greater in EcSOD vs control mice (P < .05). The percentage of apoptotic nuclei was 1.3% ± 0.4% in EcSOD-treated mice compared with 4.2% ± 0.2% in controls (P < .001). Limb necrosis was also significantly lower in EcSOD vs control mice.
  
  
  CONCLUSIONS
  AAV9-mediated overexpression of EcSOD in skeletal muscle significantly improves recovery from hind-limb ischemia in mice, consistent with improved capillary density and perfusion ratios in treated mice.
authors: Division of Cardiovascular Medicine/Department of Medicine, University of Virginia, Charlottesville, VA 22908, USA.
authors: Amina
authors: A
authors: Saqib
authors: Konkal-Matt R
authors: KM
authors: Prasad
authors: Arabindra B
authors: AB
authors: Katwal
authors: John M
authors: JM
authors: Sanders
authors: R John
authors: RJ
authors: Lye
authors: Brent A
authors: BA
authors: French
authors: Brian H
authors: BH
authors: Annex
mesh_terms: D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002196:Capillaries / Q000201:enzymology / Q000503:physiopathology; D006152:Cyclic GMP / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics*; D004195:Disease Models, Animal; D015316:Genetic Therapy; D005822:Genetic Vectors; D006614:Hindlimb; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D007273:Injections, Intramuscular; D007511:Ischemia / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000503:physiopathology / Q000628:therapy*; D017078:Laser-Doppler Flowmetry; D049409:Luciferases, Firefly / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D018482:Muscle, Skeletal / Q000098:blood supply*; D009336:Necrosis; D018919:Neovascularization, Physiologic; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D020127:Recovery of Function; D012039:Regional Blood Flow; D013482:Superoxide Dismutase / Q000096:biosynthesis* / Q000235:genetics; D013997:Time Factors
chemical_list: D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011993:Recombinant Fusion Proteins; D049409:Luciferases, Firefly; C499090:Sod3 protein, mouse; D013482:Superoxide Dismutase; D006152:Cyclic GMP