Adeno-associated virus serotype 9-mediated overexpression of extracellular superoxide dismutase improves recovery from surgical hind-limb ischemia in BALB/c mice

Autor: R. John Lye, John M. Sanders, Brian H. Annex, Amina Saqib, Konkal Matt R. Prasad, Brent A. French, Arabindra B. Katwal
Rok vydání: 2011
Předmět:
Zdroj: Journal of Vascular Surgery. 54:810-818
ISSN: 0741-5214
DOI: 10.1016/j.jvs.2011.03.278
Popis: ObjectiveNeovascularization 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.MethodsAdeno-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 × 1011 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.ResultsEcSOD 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.ConclusionAAV9-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.Clinical RelevanceAtherosclerosis remains a major cause of morbidity and mortality in the Western world. Extensive research has been conducted to attempt to harness the ability to facilitate the growth of blood vessels to limit the complications that follow an arterial occlusion. In no area of medicine is this needed greater than in peripheral arterial disease (PAD), where patients continue to experience high rates of poor wound healing and amputation. Gene therapy has been attempted in humans, with mixed results to date. Our study used a preclinical model of surgically induced hind-limb ischemia in a strain of mice that have a low rate of endogenous perfusion recovery from ischemia and a high risk of necrosis. We used an adeno-associated virus serotype 9 (AAV9) as the vector to deliver the gene expressing extracellular superoxide dismutase (EcSOD). Intramuscular EcSOD demonstrated a beneficial effect in this preclinical model. The AAV9 was able to induce high levels of gene expression without inducing detectable inflammation. This study may serve as a foundation for the future investigation of EcSOD, AAV9, or both, in PAD.
Databáze: OpenAIRE
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