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

ABSTRACT: 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.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.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.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.

SUBMITTER: Saqib A

PROVIDER: S-EPMC3166979 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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

Saqib Amina A Prasad Konkal-Matt R KM Katwal Arabindra B AB Sanders John M JM Lye R John RJ French Brent A BA Annex Brian H BH

Journal of vascular surgery 20110702 3

<h4>Objective</h4>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.<h4>Methods</h4>Adeno-associated virus serotype 9 (AAV9) vectors expressing EcSOD or lucifera ...[more]

PMID: 21723687

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Project description:OBJECTIVE:Peripheral arterial disease can cause not only ischemia but also skeletal muscle damage. It has been known that macrophages (MPs) play an important role in coordinating muscle repair; however, phenotype transition of monocyte-MP in ischemic muscle has not been well defined. Hence, the purpose of this study was to examine the temporal recruitment of MPs and to explore their therapeutic effect on ischemic muscle regeneration. METHODS:Unilateral femoral artery excision was performed on C57BL/6 mice. Myeloid cells were isolated from the ischemic muscles, characterized using flow cytometry. Bone marrow-derived MPs were injected (2 × 106 cells) into the ischemic gastrocnemius muscle 24 hours after injury. Blood flow recovery was measured using laser speckle imaging. Functional outcome was evaluated by assessing the contractile force of ischemic muscles. Histologic analysis included quantification of myofiber size, collagen deposition, number of inflammatory and MyoD-expressing cells, and capillary density. RESULTS:Neutrophils and inflammatory monocytes-MPs were present at day 1 after injury. The mature MPs then remained elevated as the dominant population from day 5 to day 21 with the observation of regenerating fibers. Functional measurements revealed that the force production was significantly enhanced after treatment with proinflammatory M1 MPs (94.9% vs 77.9%; P < .05), and this was consistent with increased myofiber size, capillary- fiber ratio, and perfusion (78.6% vs 39.9%; P < .05). Moreover, the percentage of MyoD-expressing nuclei was significantly higher at day 4, indicating that M1 MPs may hasten muscle repair. Whereas early delivery of anti-inflammatory M2 MPs improved myofiber size, this was accompanied by persistent fibrosis suggesting ongoing tissue remodeling, and lower force production was observed. CONCLUSIONS:We demonstrated the dynamics of myeloid cells in skeletal muscle after ischemic insult, and the administration of exogenous M1 MPs in a temporally coordinated manner successfully improved angiogenesis and skeletal muscle regeneration. Our results suggested that cell therapy using MPs may be a promising adjunctive therapeutic approach for peripheral arterial disease.

Project description:Background Circadian rhythm disorders, often seen in modern lifestyles, are a major social health concern. The aim of this study was to examine whether circadian rhythm disorders would influence angiogenesis and blood perfusion recovery in a mouse model of hind limb ischemia. Methods and Results A jet-lag model was established in C57BL/6J mice using a light-controlled isolation box. Control mice were kept at a light/dark 12:12 (12-hour light and 12-hour dark) condition. Concentrations of plasma vascular endothelial growth factor and circulating endothelial progenitor cells in control mice formed a circadian rhythm, which was diminished in the jet-lag model (P<0.05). The jet-lag condition deteriorated tissue capillary formation (P<0.001) and tissue blood perfusion recovery (P<0.01) in hind limb ischemia, which was associated with downregulation of vascular endothelial growth factor expression in local ischemic tissue and in the plasma. Although the expression of clock genes (ie, Clock, Bmal1, and Cry) in local tissues was upregulated after ischemic injury, the expression levels of cryptochrome (Cry) 1 and Cry2 were inhibited by the jet-lag condition. Next, Cry1 and Cry2 double-knockout mice were examined for blood perfusion recoveries and a reparative angiogenesis. Cry1 and Cry2 double-knockout mice revealed suppressed capillary density (P<0.001) and suppressed tissue blood perfusion recovery (P<0.05) in the hind limb ischemia model. Moreover, knockdown of CRY1/2 in human umbilical vein endothelial cells was accompanied by increased expression of WEE1 and decreased expression of HOXC5. This was associated with decreased proliferative capacity, migration ability, and tube formation ability of human umbilical vein endothelial cells, respectively, leading to impairment of angiogenesis. Conclusions Our data suggest that circadian rhythm disorder deteriorates reparative ischemia-induced angiogenesis and that maintenance of circadian rhythm plays an important role in angiogenesis.

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

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

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