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 atherosclerosisprone 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 A therosclerosis is the leading cause of cardiovascular disease worldwide. Defined as chronic inflammation of the artery wall, its progression from fatty streaks to more complex lesions and plaque rupture involves a complicated interplay between many different cell types and cytokine networks. Both innate and adaptive immune responses have been shown to regulate local and systemic inflammation during atherogenesis. 1,2 T cells are found within the adventitia of normal/noninflamed vessels as a result of a constitutive T-cell homing into the aorta. 3 Atherosclerosis-prone conditions accelerate T-cell recruitment into the aorta of apolipoprotein E-deficient (Apoe Ϫ/Ϫ ) mice in both the early and advanced stages of atherosclerosis. 3 The majority of aortic T cells are T-cell receptor ␣ ϩ CD4 ϩ cells, with few CD8 ϩ and ␥␦ ϩ T cells present. 1,4 Of the CD4 ϩ T cells, T helper 1 (Th1) cells predominate over T helper 2 (Th2) cells during early lesion formation and respond with an elevated production of interferon (IFN)-␥ and interleukin (IL)-6. In the later stages of the disease, a switch to a Th2 response and IL-4 production is evident in the atherosclerotic lesions of Apoe Ϫ/Ϫ mice. 5 Clinical Perspective on p 1755IL-17A is a member of the IL-17 family, which includes IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. 6 Many lymphocyte subsets secrete IL-17A in response to cytokine or monoclonal antibody stimulation, including CD4 ϩ ␣ ϩ (Th17 cells) CD8 ϩ , CD4 Ϫ CD8 Ϫ ␣ low , natural killer T cells, and ␥␦ ϩ T cells. 7 The expression of IL-17A is low under normal/noninflamed conditions, in which ␥␦ ϩ T cells are the largest IL-17A-producing T-cell subset. 6 In several murine models of autoimmune diseases, including multiple sclerosis, inflammatory bowel disease, and arthritis, serum IL-17A levels are elevated, and the T helper 17 (Th17) cell population is expanded and plays a highly pathogenic role. 8 Conversely, IL-17A is a protective cytokine in host responses against extracellular pathogens through the induction of proinflammatory cytokines such as IL-6, tumor
Newly-isolated serotypes of AAV readily cross the endothelial barrier to provide efficient transgene delivery throughout the body. However, tissue-specific expression is preferred in most experimental studies and gene therapy protocols. Previous efforts to restrict gene expression to the myocardium often relied on direct injection into heart muscle or intracoronary perfusion. Here, we report an AAV vector system employing the cardiac troponin T promoter (cTnT). Using luciferase and eGFP, the efficiency and specificity of cardiac reporter gene expression using AAV serotype capsids: AAV-1, 2, 6, 8 or 9 were tested after systemic administration to 1 week old mice. Luciferase assays showed that the cTnT promoter worked in combination with each of the AAV serotype capsids to provide cardiomyocyte-specific gene expression, but AAV-9 followed closely by AAV-8 was the most efficient. AAV9-mediated gene expression from the cTnT promoter was 640-fold greater in the heart compared to the next highest tissue (liver). eGFP fluorescence indicated a transduction efficiency of 96% using AAV-9 at a dose of only 3.15×1010 viral particles per mouse. Moreover, the intensity of cardiomyocyte eGFP fluorescence measured on a cell-by-cell basis revealed that AAV-mediated gene expression in the heart can be modeled as a Poisson distribution; requiring an average of nearly two vector genomes per cell to attain an 85% transduction efficiency.
Background Localized administration of a highly efficient gene delivery system in combination with a cardiac-selective promoter may provide an adequate biosafety profile in clinical applications such as coronary artery bypass graft surgery (CABG), where regions of myocardium can be readily injected to protect them against the potential threat of future ischemic events. Methods AAV vectors expressing firefly luciferase or 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 the extracellular isoform of superoxide dismutase (EcSOD) from the cTnT promoter was then directly injected into the LV wall of adult mice. Acute myocardial infarction (MI) was induced 4 weeks after injection and infarct size was determined by TTC 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% as compared to the eGFP control group (n=8) (44±7% vs 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.
Resolution of the covalently closed terminus of adeno-associated Virus (AAV) DNA is mediated by viral replication protein Rep78 or Rep68. In vitro studies with purified Rep proteins indicate that concurrent with this resolution is a covalent attachment of one of the proteins to the 5' end of the viral genome. The in vivo existence and fate of the covalently associated Rep protein during the virus life cycle has not yet been elucidated. In this report, we use immunoprecipitation analyses to demonstrate that the Rep78 protein is covalently attached to viral DNA in a preformed virion. The attached Rep78 is susceptible to antibody binding and protease digestion, and the DNA linkage is susceptible to nuclease digestion, therefore Rep78 is probably located on the outside of the particle. Rep proteins are also attached to double-stranded replicative-form monomer (RFM) DNA in extracts from AAV and adenovirus coinfected cells. Rep protein attachment to RFM and encapsidated AAV DNA suggest that the covalent complex is an intermediate in virus assembly. These observations are similar to those noted by others for the autonomous parvoviruses and provide additional insights into parvovirus assembly.
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.
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.
Following myocardial infarction (MI), contractile dysfunction develops not only in the infarct zone but also in noninfarcted regions of the left ventricle remote from the infarct zone. Inflammatory activation secondary to MI stimulates inducible nitric oxide synthase (iNOS) induction with excess production of nitric oxide. We hypothesized that the anti-inflammatory effects of selective A(2A)-adenosine receptor (A(2A)AR) stimulation would suppress inflammation and preserve cardiac function in the remote zone early after MI. A total of 53 mice underwent 60 min of coronary occlusion followed by 24 h of reperfusion. The A(2A)AR agonist (ATL146e, 2.4 microg/kg) was administered intraperitoneally 1, 3, and 6 h postreperfusion. Because of the 1-h delay in treatment after MI, ATL146e had no effect on infarct size, as demonstrated by contrast-enhanced cardiac MRI (n = 18) performed 24 h post-MI. ATL146e did however preserve global cardiac function at that time by limiting contractile dysfunction in remote regions [left ventricle wall thickening: 51 +/- 4% in treated (n = 9) vs. 29 +/- 3% in nontreated groups (n = 9), P < 0.01]. RT-PCR, immunohistochemistry, and Western blot analysis indicated that iNOS mRNA and protein expression were significantly reduced by ATL146e treatment in both infarcted and noninfarcted zones. Similarly, elevations in plasma nitrate-nitrite after MI were substantially blunted by ATL146e (P < 0.01). Finally, treatment with ATL146e reduced NF-kappaB activation in the myocardium by over 50%, not only in the infarct zone but also in noninfarcted regions (P < 0.05). In conclusion, A(2A)AR stimulation after MI suppresses inflammatory activation and preserves cardiac function, suggesting the potential utility of A(2A)AR agonists against acute heart failure in the immediate post-MI period.
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