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

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Presentation on theme: "Adeno-associated virus serotype 9-mediated overexpression of extracellular superoxide dismutase improves recovery from surgical hind-limb ischemia in."— Presentation transcript:

1 Adeno-associated virus serotype 9-mediated overexpression of extracellular superoxide dismutase improves recovery from surgical hind-limb ischemia in BALB/c mice  Amina Saqib, MBBS, Konkal-Matt R. Prasad, PhD, Arabindra B. Katwal, MBBS, John M. Sanders, BS, R. John Lye, PhD, Brent A. French, PhD, Brian H. Annex  Journal of Vascular Surgery  Volume 54, Issue 3, Pages (September 2011) DOI: /j.jvs Copyright © 2011 Society for Vascular Surgery Terms and Conditions

2 Fig. 1 Bioluminescence imaging is shown of vector-injected mice. A, Mice were injected with adeno-associated virus (AAV)/cytomegalovirus (CMV)/luciferase (Luc) in the tibialis anterior and gastrocnemius muscles (5 × 1010 viral genomes/muscle). Luciferase expression in these mice was monitored for up to 6 weeks by in vivo bioluminescence imaging using an in vivo imaging system (IVIS; see Materials and Methods). B, Graph shows the time course of in vivo AAV serotype 9 (AAV9)–mediated luciferase expression in mice injected with AAV9/CMV/Luc in tibialis anterior and gastrocnemius muscles. The mean values of bioluminescence as average radiance (photons/s-cm2-sr) were obtained from the regions of interest and plotted against time. C, In vitro luciferase assays were performed on protein extracts from skeletal muscles, liver, heart, lungs, and spleen at 6 weeks after the indicated vector injection. Luciferase activity was expressed as relative light units (RLU) per milligram of protein and was plotted on a log scale. The error bars show the standard error of the mean. D, Wild-type mice received a single intramuscular injection of AAV/CMV/Luc or AAV/CMV/β-galactosidase (β-gal; 1 × 1011 viral genomes) in tibialis anterior muscles. β-gal staining after 7 days shows efficient transduction of skeletal muscle fibers. Error bars show the standard deviation. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

3 Fig 2 Quantitation of adeno-associated virus (AAV) serotype 9 (AAV9)–mediated extracellular superoxide dismutase (EcSOD) expression is shown in normal hind limb muscles. A, Mice were injected intramuscularly with AAV/cytomegalovirus (CMV)/luciferase (Luc) or AAV/CMV/EcSOD (1 × 1011 viral genomes/mouse) into the tibialis anterior and gastrocnemius muscles. At 14 days after vector administration, EcSOD expression was detected by Western blot analysis. Levels of EcSOD expression were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B, Expression of EcSOD expression by Western blot analysis was 7.9-fold higher in the EcSOD-treated group than in controls. *P < Error bars show the standard error of the mean. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

4 Fig 3 Quantitation of adeno-associated virus (AAV) serotype 9–mediated extracellular superoxide dismutase (EcSOD) expression in ischemic hind-limb muscles: A, Mice were injected intramuscularly with AAV/cytomegalovirus (CMV)/luciferase (Luc) or AAV/CMV/EcSOD (1 × 1011 viral genomes/mouse) into the tibialis anterior and gastrocnemius muscles at the time of hind-limb ischemia surgery. At 14 days after vector administration EcSOD expression was detected by Western blot analysis. Levels of EcSOD expression were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein. B, EcSOD expression by Western blot analysis was twofold higher in the EcSOD-treated ischemic muscles than in controls (n = 10 per group). *P < .05). Error bars show the standard error of the mean. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

5 Fig 4 Perfusion is improved in hind limbs treated with extracellular superoxide dismutase (EcSOD). Mean perfusion ratio was 33.7% ± 1.7% for EcSOD and 32.8% ± 1.9% for control groups at baseline on day 0 after hind-limb ischemia surgery and vector injection. The EcSOD group demonstrated significantly better perfusion recovery at day 14 than the control group (*P < .01). AAV, Adeno-associated virus; CMV, cytomegalovirus; Luc, luciferase. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

6 Fig 5 Necrosis scores were improved in mice treated with extracellular superoxide dismutase EcSOD. A, Nine of 10 mice showed some degree of necrosis in the adeno-associated virus (AAV)/cytomegalovirus (CMV)/luciferase (Luc) control group 7 days after vector injection, including 3 mice with grade 3 necrosis. B, In the AAV/CMV/EcSOD-treated group, only six mice showed some degree of necrosis, and none had grade 3 necrosis. *P < .05. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

7 Fig 6 A, Quantitative assessment of apoptosis expressed as the percentage of TUNEL-positive nuclei/total number of nuclei (n = 10 per group). *P < B, Quantitative analysis of capillary density expressed as the number of capillaries per muscle fiber (n = 10 per group). *P < .02. Error bars show the standard error of the mean. AAV, Adeno-associated virus; CMV, cytomegalovirus; EcSOD, extracellular superoxide dismutase; Luc, luciferase; TUNEL, terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

8 Fig 9 The preservation of filamentous actin (green) is markedly improved in ischemic limbs treated with (C) extracellular superoxide dismutase (EcSOD) than in (A) control ischemic limbs, whereas the levels of total actin (orange) are comparable in (D) EcSOD-treated and (B) control groups. AAV, Adeno-associated virus; CMV, cytomegalovirus; Luc, luciferase. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

9 Fig 10 Levels of cyclic guanosine monophosphate (cGMP; pmol/mg tissue) in ischemic tissues from control and extracellular superoxide dismutase (EcSOD)-treated mice at 14 days after ischemia induction are significantly increased compared with the control group (n = 3 per group). *P < .01. The error bars show the standard error of the mean. AAV, Adeno-associated virus; CMV, cytomegalovirus; Luc, luciferase. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

10 Fig 7, (online only) Assessment of nonischemic skeletal muscle after adeno-associated virus (AAV)/cytomegalovirus (CMV)/extracellular superoxide dismutase (EcSOD) injection: Hematoxylin and eosin (H&E) staining of the (A) control and (B) EcSOD-injected skeletal muscles revealed no detectable inflammation. Similarly, (C) CD45 and (E) CD68 immunostaining of (D) control and (F) EcSOD-injected muscles did not indicate leukocytic infiltrates. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

11 Fig 8, (online only) Immunostaining for CD45+ leukocytes in (A) ischemic controls and (B) extracellular superoxide dismutase (EcSOD)-injected ischemic skeletal muscles. CD68 staining in (C) controls and (D) EcSOD-injected ischemic skeletal muscles showed similar results. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

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