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      Ultrasound-Microbubble-Mediated NF-κB Decoy Transfection Attenuates Neointimal Formation after Arterial Injury in Mice

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          Abstract

          Objective: Decoy transfection is a significant methodology for suppressing gene activation. The decoy can be synthesized easily; however, the major obstacle is the difficulty involved in effective in vivo delivery. Methods and Results: We used a fluorescein-labeled decoy to investigate the ultrasound-microbubble-mediated in vivo delivery in normal and injured mouse arteries. We showed that this approach could deliver the decoy into target tissues. In addition, we performed in vivo NF-ĸB decoy transfection into murine injured arteries using the ultrasound-microbubble method. Murine femoral arteries were injured using flexible wires to establish arterial injury. Pathologically, neointima/media areas in the NF-ĸB decoy transfection using ultrasound-microbubble group showed less than those in the control groups. Immunohistochemistry revealed that enhanced expression of inflammatory factors was observed in nontreated injured arteries, while the NF-ĸB decoy suppressed the expression. Conclusion: We revealed that ultrasound-microbubble delivery of the decoy is effective for transfection into target organs. We also indicated that NF-ĸB decoy transfection using this method has potential for the suppression of neointimal formation. Ultrasound-mediated transfection of the decoy can be beneficial for the clinical treatment of restenosis after coronary intervention and other cardiovascular diseases.

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          Most cited references 16

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          Local delivery of plasmid DNA into rat carotid artery using ultrasound.

          Although viral vector systems are efficient to transfect foreign genes into blood vessels, safety issues remain in relation to human gene therapy. In this study, we examined the feasibility of a novel nonviral vector system by using high-frequency, low-intensity ultrasound irradiation for transfection into blood vessels. Luciferase plasmid mixed with or without echo contrast microbubble (Optison) was transfected into cultured human vascular smooth muscle cells (VSMC) and endothelial cells (EC) with the use of ultrasound. Interestingly, luciferase activity was markedly increased in both cell types treated with Optison. We then transfected luciferase plasmid mixed with Optison by means of therapeutic ultrasound into rat artery. Two days after transfection, luciferase activity was significantly higher in carotid artery transfected with luciferase gene with Optison and ultrasound than with plasmid alone. In addition, we transfected an anti-oncogene (p53) plasmid into carotid artery after balloon injury as a model of gene therapy for restenosis. Two weeks after transfection, the intimal-to-medial area ratio in rats transfected with wild-type p53 plasmid complexed with Optison by means of ultrasound was significantly decreased as compared with control, accompanied by a significant increase in p53 protein. No apparent toxicity such as inflammation could be detected in blood vessels transfected with plasmid DNA with ultrasound and Optison. Overall, we demonstrated that an ultrasound transfection method with Optison enhanced transfection efficiency of naked plasmid DNA into blood vessels without any apparent toxicity. Transfection of p53 plasmid with the use of this method should be useful for safe clinical gene therapy without a viral vector system.
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            Nuclear factor kappaB: a potential therapeutic target in atherosclerosis and thrombosis.

            Cardiovascular diseases are the leading cause of morbidity and mortality in Western countries. Atherosclerosis, the background for many cardiovascular diseases, is characterized by the accumulation of lipid and fibrotic entities in large arteries and bears many similarities with chronic inflammatory diseases such as rheumatoid arthritis. Common features include extravasation of blood-derived leukocytes, as well as production of cytokines, chemokines and matrix-degrading enzymes. There are also many shared signaling pathways, including activation of the nuclear factor kappaB (NFkappaB) cascade. In the context of atherosclerosis, there are a range of candidate stimuli which can activate NFkappaB, including traditional risk factors, infectious agents, cytokines and cell-cell contact. Many inflammatory genes relevant to the pathogenesis of atherosclerosis are regulated by NFkappaB, the activated form of which is present in atherosclerotic plaques. Thus, it is essential to understand the role of this important signaling cascade in atherosclerosis, in a quest for more specific therapeutic targets.
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              Ultrasound-mediated transfection of mammalian cells.

              Mammalian cells were successfully transfected with plasmid DNA in vitro using ultrasound transmitted through the walls of cell culture flasks or plates. Primary rat fibroblasts or chondrocytes were exposed to ultrasound in the presence of plasmids containing lacZ or neo genes. The transfection efficiency was evaluated by counting the number of beta-galactosidase (beta-Gal) positive cells or neomycin-resistant colonies. Transfection efficiency was optimized by varying ultrasound conditions, ambient temperatures (room temperature or 37 degrees C), plasmid concentrations, and initial cell populations. Additional experiments were performed performed to elucidate the mechanism of the ultrasound-mediated transfection. Maximal gene transfection was seen with two ultrasound conditions: 1-MHz carrier frequency 411 +/- 189 kPascal continuous wave with 20 or 30 sec of exposure time, and 1 MHz carrier frequency 319 +/- 157 kPascal continuous wave with 40 or 60 sec of exposure time. Gene expression was negligible when transfection procedures were performed at room temperature. The average stable transfection rate was 0.34% of surviving cells with a plasmid concentration of 40 micrograms/ml in primary fibroblasts. The transient transfection rate was 2.4% of surviving cells for primary chondrocytes. Data suggest that increasing plasmid concentration will increase efficiency. Identical treatment with 3.5 MHz produced no transfection, implying that cavitation produced by the ultrasound pressure wave appeared to play a critical role in mediating transfection. Ultrasound-mediated transfection was effective for suspended cells as well as for plated cells. This transfection method is simple, easy to keep sterile, and convenient. Ultrasound-mediated transfection appears to be a promising method for gene transfer into mammalian cells.
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                Author and article information

                Journal
                JVR
                J Vasc Res
                10.1159/issn.1018-1172
                Journal of Vascular Research
                S. Karger AG
                1018-1172
                1423-0135
                2006
                December 2005
                21 December 2005
                : 43
                : 1
                : 12-18
                Affiliations
                aDepartment of Cardiovascular Medicine, Tokyo Medical and Dental University, Tokyo, and bDivision of Clinical Gene Therapy, Osaka University, Osaka, Japan
                Article
                89103 J Vasc Res 2006;43:12–18
                10.1159/000089103
                16244495
                © 2006 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 4, References: 20, Pages: 7
                Categories
                Research Paper

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