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Helper-dependent adenovirus is superior to first-generation adenovirus for expressing transgenes in atherosclerosis-prone arteries.

ABSTRACT: Vascular gene transfer is a powerful tool for investigating and treating vascular diseases; however, its utility is limited by brevity of transgene expression and vector-associated inflammation. Helper-dependent adenovirus (HDAd), an advanced-generation adenovirus that lacks all viral genes, is superior to first-generation adenovirus (FGAd) in normal rabbit arteries. We compared HDAd to FGAd in arteries of cholesterol-fed rabbits, a model of early atherogenesis in which transgene expression might be decreased, and inflammation increased.Carotid arteries of chow- and cholesterol-fed rabbits were infused with FGAd, HDAd, or medium. HDAd expressed a transgene at least as well in arteries of cholesterol-fed rabbits as in arteries of chow-fed rabbits and expressed more durably than FGAd. In arteries of cholesterol-fed rabbits, HDAd stimulated less intimal growth, lipid deposition, and inflammation than FGAd. Neither vector affected phenylephrine-induced contraction or nitroprusside-mediated relaxation; however, both vectors decreased maximal acetylcholine-stimulated vasorelaxation. The relative absence of intimal growth in HDAd arteries could interfere with the utility of this model for testing atheroprotective genes; however, both coinfusion of FGAd and extension of cholesterol feeding yielded larger intimal lesions, on which atheroprotective genes could be tested.HDAd is superior to FGAd for expression of transgenes in atherosclerosis-prone arteries.

SUBMITTER: Jiang B

PROVIDER: S-EPMC3098319 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Helper-dependent adenovirus is superior to first-generation adenovirus for expressing transgenes in atherosclerosis-prone arteries.

Jiang Bo B Qian Kun K Du Liang L Luttrell Ian I Chitaley Kanchan K Dichek David A DA

Arteriosclerosis, thrombosis, and vascular biology 20110331 6

<h4>Objective</h4>Vascular gene transfer is a powerful tool for investigating and treating vascular diseases; however, its utility is limited by brevity of transgene expression and vector-associated inflammation. Helper-dependent adenovirus (HDAd), an advanced-generation adenovirus that lacks all viral genes, is superior to first-generation adenovirus (FGAd) in normal rabbit arteries. We compared HDAd to FGAd in arteries of cholesterol-fed rabbits, a model of early atherogenesis in which transge ...[more]

PMID: 21454808

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Project description:Background Platelet-endothelial interactions are thought to contribute to early atherogenesis. These interactions are potentiated by oxidative stress. We used in vivo molecular imaging to test the hypothesis that platelet-endothelial interactions occur at early stages of plaque development in obese, insulin-resistant nonhuman primates, and are suppressed by NADPH-oxidase-2 inhibition. Methods and Results Six adult rhesus macaques fed a Western-style diet for a median of 4.0 years were studied at baseline and after 8 weeks of therapy with the NADPH-oxidase-2-inhibitor apocynin (50 mg/kg per day). Six lean control animals were also studied. Measurements included intravenous glucose tolerance test, body composition by dual-energy X-ray absorptiometry, carotid intimal medial thickness, carotid artery contrast ultrasound molecular imaging for platelet GPIbα (glycoprotein- Ibα) and vascular cell adhesion molecule-1, and blood oxidative markers on mass spectrometry. Compared with lean controls, animals on a Western-style diet were obese (median body mass: 16.0 versus 8.7 kg, P=0.003; median truncal fat: 49% versus 20%, P=0.002), were insulin resistant (4-fold higher insulin-glucose area under the curve on intravenous glucose tolerance test, P=0.002), had 40% larger carotid intimal medial thickness (P=0.004), and exhibited oxidative signatures on proteomics. In obese but not lean animals, signal enhancement on molecular imaging was significantly elevated for GPIbα and vascular cell adhesion molecule-1. The signal correlated modestly with intimal medial thickness but not with the degree of insulin resistance. Apocynin significantly (P<0.01) reduced median signal for GPIbα by >80% and vascular cell adhesion molecule-1 signal by 75%, but did not affect intimal medial thickness, body mass, or intravenous glucose tolerance test results. Conclusion In nonhuman primates, diet-induced obesity and insulin resistance leads to platelet-endothelial adhesion at early atherosclerotic lesion sites, which is associated with the expression of pro-inflammatory adhesion molecules. These responses appear to be mediated, in part, through oxidative pathways.

Project description:AimTo construct a tricistronic hepatitis C virus (HCV) replicon with double internal ribosome entry sites (IRESes) of only 22 nucleotides for each, substituting the encephalomyocarditis virus (EMCV) IRESes, which are most often used as the translation initiation element to form HCV replicons.MethodsThe alternative 22-nucleotide IRES, RNA-binding motif protein 3 IRES (Rbm3 IRES), was used to form a tricistronic HCV replicon, to facilitate constructing HCV-harboring stable cell lines and successive antiviral screening using a luciferase marker. Briefly, two sequential Rbm3 IRESes were inserted into bicistronic pUC19-HCV plasmid, consequently forming a tricistronic HCV replicon (pHCV-rep-NeoR-hRluc), initiating the translation of humanized Renilla luciferase and HCV non-structural gene, along with HCV authentic IRES initiating the translation of neomycin resistance gene. The sH7 cell lines, in which the novel replicon RNA stably replicated, were constructed by neomycin and luciferase activity screening. The intracellular HCV replicon RNA, expression of inserted foreign genes and HCV non-structural gene, as well as response to anti-HCV agents, were measured in sH7 cells and cells transiently transfected with tricistronic replicon RNA.ResultsThe intracellular HCV replicon RNA and expression of inserted foreign genes and HCV non-structural gene in sH7 cells and cells transiently transfected with tricistronic replicon RNA were comparable to those in cells stably or transiently transfected with traditional bicistronic HCV replicons. The average relative light unit in pHCV-rep-NeoR-hRluc group was approximately 2-fold of those in the pUC19-HCV-hRLuc and Tri-JFH1 groups (1.049 × 10(8) ± 2.747 × 10(7) vs 5.368 × 10(7) ± 1.016 × 10(7), P < 0.05; 1.049 × 10(8) ± 2.747 × 10(7) vs 5.243 × 10(7) ± 1.194 × 10(7), P < 0.05), suggesting that the translation initiation efficiency of the first Rbm3 IRES in the two sequential IRESes was stronger than the HCV authentic IRES and EMCV IRES. The fold changes of 72 h/4 h relative light units in the pHCV-rep-NeoR-hRluc and pUC19-HCV-hRLuc groups were similar (159.619 ± 9.083 vs 163.536 ± 24.031, P = 0.7707), and were both higher than the fold change in the Tri-JFH1 group 159.619± 9.083 vs 140.811 ± 9.882, P < 0.05; 163.536 ± 24.031 vs 140.811 ± 9.882, P < 0.05), suggesting that the replication potency of the Rbm3 IRES tricistronic replicon matched the replication of bicistronic replicon and exceeded the potency of EMCV IRES replicon. Replication of tricistronic replicons was suppressed by ribavirin, simvastatin, atorvastatin, telaprevir and boceprevir. Interferon-alpha 2b could not block replication of the novel replicon RNA in sH7 cells. After interferon stimulation, MxA mRNA and protein levels were lower in sH7 than in parental cells.ConclusionTricistronic HCV replicon with double Rbm3 IRESes could be applied to evaluate the replication inhibition efficacy of anti-HCV agents.

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Helper-dependent adenovirus is superior to first-generation adenovirus for expressing transgenes in atherosclerosis-prone arteries.

Publications

Helper-dependent adenovirus is superior to first-generation adenovirus for expressing transgenes in atherosclerosis-prone arteries.

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