Background-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. Methods and Results-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. Conclusions-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.

Low-intensity ultrasound with echo-enhanced Optison induced efficient gene transfer unlike that with Albunex or Levovist.

Recently, there have been numerous reports on the application of non-thermal ultrasound energy for treating various diseases in combination with drugs. Furthermore, the introduction of microbubbles and nanobubbles as carriers/enhancers of drugs has added a whole new dimension to therapeutic ultrasound. Non-thermal mechanisms for effects seen include various forms of energy due to cavitation, acoustic streaming, micro jets and radiation force which increases possibilities for targeting tissue with drugs, enhancing drug effectiveness or even chemically activating certain materials. Examples such as enhancement of thrombolytic agents by ultrasound have proven to be beneficial for acute stroke patients and peripheral arterial occlusions. Non-invasive low intensity focused ultrasound in conjunction with anti-cancer drugs may help to reduce tumor size and lessen recurrence while reducing severe drug side effects. Chemical activation of drugs by ultrasound energy for treatment of atherosclerosis and tumors is another new field recently termed as "Sonodynamic therapy". Lastly, advances in molecular imaging have aroused great expectations in applying ultrasound for both diagnosis and therapy simultaneously. Microbubbles or nanobubbles targeted at the molecular level will allow medical doctors to make a final diagnosis of a disease using ultrasound imaging and then immediately proceed to a therapeutic ultrasound treatment.

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Bone morphogenetic proteins (BMPs) are morphogens implicated in embryonic and regenerative odontogenic differentiation. Gene therapy has the potential to induce reparative dentin formation for potential pulp capping. We have optimized the gene transfer of Growth/differentiation factor 11 (Gdf11)/Bmp11 plasmid DNA into dental pulp stem cells by sonoporation in vivo. Dental pulp tissue treated with plasmid pEGFP or CMV-LacZ in 5-10% Optison (Molecular Biosystems Inc., San Diego, CA) and stimulated by ultrasound (1 MHz, 0.5 W/cm(2), 30 sec) showed significant efficiency of gene transfer and high level of protein production selectively in the local region, within 500 microm of the amputated site of the pulp tissue. The Gdf11 cDNA plasmid transferred into dental pulp tissue by sonoporation in vitro, induced the expression of dentin sialoprotein (Dsp), a differentiation marker for odontoblasts. The transfection of Gdf11 by sonoporation stimulated a large amount of reparative dentin formation on the amputated dental pulp in canine teeth in vivo. These results suggest the possible use of BMPs using ultrasound-mediated gene therapy for endodontic dental treatment.

US with MBs greatly increases gene transfer to in vivo and in vitro corneal cells. This noninvasive gene transfer method may be a useful tool for clinical gene therapy.

In the gastrula stage embryo, the epiblast migrates toward the primitive streak and ingresses through the primitive groove. Subsequently, the ingressing epiblast cells undergo epithelial-mesenchymal transition (EMT) and differentiate into the definitive endoderm and mesoderm during gastrulation. However, the developmental mechanisms at the end of gastrulation have not yet been elucidated. Histological and genetic analyses of the ventral ectodermal ridge (VER), a derivative of the primitive streak, were performed using chick and mouse embryos. The analyses showed a continued cell movement resembling gastrulation associated with EMT during the early tailbud stage of both embryos. Such gastrulation-like cell movement was gradually attenuated by the absence of EMT during tail development. The kinetics of the expression pattern of noggin (Nog) and basal membrane degradation adjacent to the chick and the mouse VER indicated a correlation between the temporal and/or spatial expression of Nog and the presence of EMT in the VER. Furthermore, Nog overexpression suppressed EMT and arrested ingressive cell movement in the chick VER. Mice mutant in noggin displayed dysregulation of EMT with continued ingressive cell movement. These indicate that the inhibition of Bmp signaling by temporal and/or spatial Nog expression suppresses EMT and leads to the cessation of the ingressive cell movement from the VER at the end of gastrulation.