[Show abstract][Hide abstract] ABSTRACT: Targeting therapeutic gene expression to the skeletal muscle following intravenous (IV) administration is an attractive strategy for treating peripheral arterial disease (PAD), except that vector access to the ischemic limb could be a limiting factor. As adeno-associated virus serotype 9 (AAV-9) transduces skeletal muscle at high efficiency following systemic delivery, we employed AAV-9 vectors bearing luciferase or enhanced green fluorescent protein (eGFP) reporter genes to test the hypothesis that increased desialylation of cell-surface glycans secondary to hindlimb ischemia (HLI) might help offset the reduction in tissue perfusion that occurs in mouse models of PAD. The utility of the creatine kinase-based (CK6) promoter for restricting gene expression to the skeletal muscle was also examined by comparing it with the cytomegalovirus (CMV) promoter after systemic administration following surgically induced HLI. Despite reduced blood flow to the ischemic limbs, CK6 promoter-driven luciferase activities in the ischemic gastrocnemius (GA) muscles were ∼34-, ∼28- and ∼150-fold higher than in the fully perfused contralateral GA, heart and liver, respectively, 10 days after IV administration. Furthermore, luciferase activity from the CK6 promoter in the ischemic GA muscles was ∼twofold higher than with CMV, while in the liver CK6-driven activity was ∼42-fold lower than with CMV, demonstrating that the specificity of ischemic skeletal muscle transduction can be further improved with the muscle-specific promoters. Studies with Evans blue dye and fluorescently labeled lectins revealed that vascular permeability and desialylation of the cell-surface glycans were increased in the ischemic hindlimbs. Furthermore, AAV9/CK6/Luc vector genome copy numbers were ∼sixfold higher in the ischemic muscle compared with the non-ischemic muscle in the HLI model, whereas this trend was reversed when the same genome was packaged in the AAV-1 capsid (which binds sialylated, as opposed to desialylated glycans), further underscoring the importance of desialylation in the ischemic enhancement of transduction displayed by AAV-9. Taken together, these findings suggest two complementary mechanisms contributing to the preferential transduction of ischemic muscle by AAV-9: increased vascular permeability and desialylation. In conclusion, ischemic muscle is preferentially targeted following systemic administration of AAV-9 in a mouse model of HLI. Unmasking of the primary AAV-9 receptor as a result of ischemia may contribute importantly to this effect.Gene Therapy advance online publication, 28 March 2013; doi:10.1038/gt.2013.16.
[Show abstract][Hide abstract] 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.
Full-text · Article · Jul 2011 · Journal of vascular surgery: official publication, the Society for Vascular Surgery [and] International Society for Cardiovascular Surgery, North American Chapter
[Show abstract][Hide abstract] ABSTRACT: Atherosclerotic occlusion of vessels outside of the heart is commonly referred to as peripheral arterial disease (PAD). The lower extremity is the most common site of PAD and its development is associated with the same risk factors involved in general atherosclerosis. However, there is emerging evidence that other risk factors may play a key role in the development of PAD. Over the past decade polymorphism in a number of genes has been shown to contribute to the risk of developing PAD. These genes can be classified into proartherosclerosis or proatherothrombosis based on the known gene function. Moreover, they can be categorized as "novel" polymorphism when the function of the genes is not known or when the specific gene within an associated genetic locus is not known. It is intriguing that not only are gene polymorphisms associated with PAD being identified, but more recently studies are now finding gene polymorphisms that may be important in development of this syndrome only in the contest of certain environmental factors such as diabetes. Currently how these gene-environment interactions contribute to the pathogenesis of PAD is poorly understood but will likely play a critical role in future understanding of this complex disease.
Full-text · Article · Mar 2011 · Current Diabetes Reports