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

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. CONCLUSION: 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.

A single direct injection into the left ventricular wall of an adeno-associated virus 9 (AAV9) vector expressing extracellular superoxide dismutase from the cardiac troponin-T promoter protects mice against myocardial infarction.

BACKGROUND: Localized administration of a highly efficient gene delivery system in combination with a cardiac-selective promoter may provide a favorable biosafety profile in clinical applications such as coronary artery bypass graft surgery, where regions of myocardium can be readily injected to protect them against the potential threat of future ischemic events. METHODS: Adeno-associated virus (AAV) vectors expressing luciferase or enhanced green fluorescent protein (eGFP) packaged into AAV serotypes 1, 2, 6, 8 and 9 were injected into the left ventricular (LV) wall of adult mice to determine the time course, magnitude and distribution of gene expression. An AAV9 vector expressing extracellular superoxide dismutase (EcSOD) from the cardiac troponin T (cTnT) promoter was then directly injected into the LV wall of adult mice. Myocardial infarction was induced 4 weeks after injection and infarct size was determined by triphenyltetrazolium chloride and phthalo blue staining. RESULTS: Serotypes AAV 9, 8, 1 and 6 provided early onset of gene expression in the heart with minimal extra-cardiac gene expression. AAV9 provided the highest magnitude of gene expression. Immunostaining for eGFP showed expression spanning the anterior to posterior walls from the mid ventricle to the apex. A single direct injection of the AAV9 vector bearing EcSOD ( n = 5) decreased the mean infarct size by 50% compared to the eGFP control group (n = 8) (44 ± 7% versus 22 ± 5%; p = 0.04). CONCLUSIONS: AAV serotype 9 is highly efficient for cardiac gene delivery, as evidenced by early onset and high-level gene expression. AAV9-mediated, cardiac selective overexpression of EcSOD from the cTnT promoter significantly reduced infarct size in mice.

Blockade of interleukin-17A results in reduced atherosclerosis in apolipoprotein E-deficient mice.

BACKGROUND: T cells play an important role during the immune response that accompanies atherosclerosis. To date, the role for interleukin (IL)-17A in atherogenesis is not well defined. Here, we tested the hypothesis that atherosclerosis-prone conditions induce the differentiation of IL-17A-producing T cells, which in turn promote atherosclerosis. METHODS AND RESULTS: IL-17A was found to be elevated in the plasma and tissues of apolipoprotein E-deficient (Apoe(-/-)) mice. IL-17A-expressing T cells were significantly increased in the aortas, spleen, and lamina propria of aged Apoe(-/-) mice compared with age-matched C57BL/6 mice. IL-17A(+) T cells resided in both adventitia and aortas of aged Apoe(-/-) mice fed a chow diet. Elevated levels of IL-17A(+) T cells were also detected in the aortas of 21-week-old Apoe(-/-) mice fed a Western diet for 15 weeks. IL-17A(+) T cells were characterized as predominantly CD4(+) T helper 17 (Th17) cells and gammadelta(+) T cells. Blockade of IL-17A in Apoe(-/-) mice by use of adenovirus-produced IL-17 receptor A reduced plaque burden in Apoe(-/-) mice fed a Western diet for 15 weeks. In addition, the treatment diminished circulating IL-6 and granulocyte colony-stimulating factor levels and limited CXCL1 expression and macrophage content within the aortas. Conversely, IL-17A treatment of whole aorta isolated from Apoe(-/-) mice promoted aortic CXCL1 expression and monocyte adhesion in an ex vivo adhesion assay. CONCLUSIONS: These results demonstrate that atherosclerosis-prone conditions induce the differentiation of IL-17A-producing T cells. IL-17A plays a proatherogenic inflammatory role during atherogenesis by promoting monocyte/macrophage recruitment into the aortic wall.

Borderzone contractile dysfunction is transiently attenuated and left ventricular structural remodeling is markedly reduced following reperfused myocardial infarction in inducible nitric oxide synthase knockout mice.

OBJECTIVES: We sought to determine the effect of inducible nitric oxide synthase (iNOS) expression on regional contractile function and left ventricular (LV) remodeling after reperfused myocardial infarction (MI). BACKGROUND: Inducible nitric oxide synthase is known to contribute to global LV dysfunction after a large MI, but the mechanisms underlying this dysfunction remain unclear. METHODS: We used immunohistochemistry to investigate the distribution of iNOS expression in wild-type (WT) and iNOS knockout (KO) mice early (day 1) and late (day 28) after reperfused MI. We also used serial cardiac magnetic resonance imaging at baseline and at 1, 7, and 28 days after MI to assess LV volumes, ejection fraction (EF), regional circumferential strain (E(cc)), and day 1 infarct size. RESULTS: At baseline, LV volumes and EF were similar between groups. Day 1 infarct size was also similar between groups. Immunohistochemistry revealed that iNOS expression was abundant throughout the heart in WT mice on day 1 after MI, particularly near the infarct borderzone. On day 7 after MI, E(cc) in KO mice was significantly improved in some borderzone sectors compared with WT. The LV volumes were significantly lower in KO mice at days 7 and 28 compared with WT. The EF on days 7 and 28 was significantly higher in KO mice compared with WT. The circumferential extent of wall thinning was also significantly reduced in KO versus WT mice at days 7 and 28. CONCLUSIONS: Expression of iNOS contributes importantly to post-infarction contractile dysfunction and subsequent LV remodeling, suggesting new strategies to combat heart failure resulting from large MI.

Topoisomerase inhibition accelerates gene expression after adeno-associated virus-mediated gene transfer to the mammalian heart.

Utility of adeno-associated virus 2 (AAV2) vectors for cardiac gene therapy is limited by the prolonged lag phase before maximal gene expression. Topoisomerase inhibition can induce AAV2-mediated gene expression in vivo, but with variable success in different tissues. In this study, we demonstrate that topoisomerase inhibition can accelerate AAV2-mediated gene expression in the mouse heart. We used an AAV2 vector expressing firefly luciferase and monitored expression kinetics using non-invasive bioluminescence imaging. In the group receiving vector alone, cardiac luciferase activity was evident from week 2 onward and increased progressively to reach a steady plateau by 9 weeks postinjection. In the group receiving vector and camptothecine (CPT), luciferase expression was evident from days 2 to 4 onward and increased rapidly to reach a steady plateau by 3-4 weeks postinjection, nearly three times faster than in the absence of CPT (P<0.05). Southern blot analysis of AAV2 genomes in cardiac tissue showed rapid conversion of the AAV2 genome from its single-stranded to double-stranded form in CPT-treated mice. Non-invasive determinations of luciferase expression correlated well with in vitro luciferase assays. Direct injection of the AAV2 vector and long-term luciferase gene expression had no detectable effects on normal cardiac function as assessed by magnetic resonance imaging.

Topoisomerase Inhibition Accelerates Gene Expression after Adeno-associated Virus-mediated Gene Transfer to the Mammalian Heart.

Utility of adeno-associated virus 2 (AAV2) vectors for cardiac gene therapy is limited by the prolonged lag phase before maximal gene expression. Topoisomerase inhibition can induce AAV2-mediated gene expression in vivo, but with variable success in different tissues. In this study, we demonstrate that topoisomerase inhibition can accelerate AAV2-mediated gene expression in the mouse heart. We used an AAV2 vector expressing firefly luciferase and monitored expression kinetics using non-invasive bioluminescence imaging. In the group receiving vector alone, cardiac luciferase activity was evident from week 2 onward and increased progressively to reach a steady plateau by 9 weeks postinjection. In the group receiving vector and camptothecine (CPT), luciferase expression was evident from days 2 to 4 onward and increased rapidly to reach a steady plateau by 3-4 weeks postinjection, nearly three times faster than in the absence of CPT (P<0.05). Southern blot analysis of AAV2 genomes in cardiac tissue showed rapid conversion of the AAV2 genome from its single-stranded to double-stranded form in CPT-treated mice. Non-invasive determinations of luciferase expression correlated well with in vitro luciferase assays. Direct injection of the AAV2 vector and long-term luciferase gene expression had no detectable effects on normal cardiac function as assessed by magnetic resonance imaging.

Evidence that increased 12-lipoxygenase expression impairs pancreatic beta cell function and viability.

Leukocyte type 12-lipoxygenase (12-LO) is an enzyme specifically expressed in the beta cells of the pancreas. 12-LO oxidizes fatty acids such as arachidonic acid and linoleic acids to their respective hydroperoxides. Increased concentration of lipid hydroperoxides causes oxidative stress and this could lead to cellular dysfunction. Increased expression of 12-LO in beta cells has been observed with use of inflammatory cytokines and during the prediabetic phase of beta cell dysfunction in the Zucker diabetic fatty rat model. Also mice deficient in 12-LO expression show a decreased incidence of immune-mediated diabetes. To further understand the role of 12-LO in beta cell metabolism, we over-expressed mouse leukocyte type 12-LO in INS-1 cells (transformed rat beta cell line) using an adeno-associated virus (AAV) vector system. In 12-LO over-expressing cells, cell-associated 12-HETE levels increased approximately 5- and approximately 3-fold in the culture supernatant. In the cells over-expressing 12-LO, glucose-stimulated insulin secretion (GSIS) decreased by 25-30% one hour after exposure to high glucose (15mM). By 2h, GSIS decreased by 50-54% at high glucose levels. These data suggest that increased 12-LO products can reduce the synthesis, processing or secretion of insulin in beta cells. We next examined the effect of 12-LO over-expression on mitogen-activated protein kinases (MAPK) by Western blot analyses using antibodies specific for different phospho-MAP kinases. Over-expression of 12-LO led to an activation of c-Jun N-terminal kinase while it markedly reduced Erk1 and 2 phosphorylation (4-fold). Further, over-expression of 12-LO led to induction of apoptosis in beta cells as determined by DNA ladder assay. These results suggest that increased 12-LO plays a key role in altering beta cell metabolism. Thus, increased 12-LO expression can have a detrimental effect on pancreatic beta cell function and viability, suggesting that blockade of 12-LO activity or expression could provide a novel way to protect beta cells from inflammatory injury.