Biomedical subjects

R Morishita

Publications and source records attributed to R Morishita.

At least 19 recordsLinked to original sources

PubMed2006

Prevention of onset of Parkinson's disease by in vivo gene transfer of human hepatocyte growth factor in rodent model: a model of gene therapy for Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra (SNi). As neurotrophic factors support the survival and enhance the function of dopaminergic neurons, gene therapy using neurotrophic factors has become the center of interest. Thus, we focused on hepatocyte growth factor (HGF) as a neurotrophic and angiogenic growth factor. At 7 days before injection of 6-hydroxydopamine into the SNi, stereotaxic transfection of human HGF or lacZ plasmid was performed into the unilateral striatum of rats. Expression of human HGF in the injected sites could be detected in rats transfected with HGF plasmid DNA, using immunohistochemical staining. Consistently, human immunoreactive HGF protein could be detected at least up to 12 days after transfection. Interestingly, PD rats transfected with lacZ demonstrated amphetamine-induced rotational asymmetry. However, transfection of HGF plasmid DNA resulted in significant inhibition of abnormal rotation up to 24 weeks in a dose-dependent manner. Over 90% of dopaminergic neurons were lost in PD rats transfected with lacZ, whereas over 70% survived in rats transfected with HGF, as assessed by immunohistochemical staining. Overall, the present study demonstrated that overexpression of HGF prevented neuronal death in a PD rat model, providing a potential novel therapy for PD.

Animals↗

PubMed2006

Angiogenic and antifibrotic actions of hepatocyte growth factor improve cardiac dysfunction in porcine ischemic cardiomyopathy.

Impairment of cardiac function in ischemic cardiomyopathy has been postulated to be due to the decrease in blood flow and increase in collagen synthesis. Therefore, an approach to alter them directly by means of a growth factor may open up a new therapeutic concept in ischemic cardiomyopathy. From this viewpoint, hepatocyte growth factor (HGF) is a unique growth factor with angiogenic and antifibrotic effects. Thus, we examined the feasibility of gene therapy using HGF plasmid DNA for ischemic cardiomyopathy. Human HGF plasmid DNA at a dose of 0.4 or 4 mg was injected into ischemic myocardium of pigs induced by ameroid constrictor with the NOGA system. At 1 month after injection, the ischemic area was significantly reduced in the HGF group, accompanied by a significant increase in capillary density and regional myocardial perfusion in the ischemic area (P<0.01). In contrast, a significant decrease in fibrotic area was observed in the HGF group, associated with a significant decrease in collagen I, III and TGF-beta synthesis as compared to the control group (P<0.01). Consistently, cardiac function was significantly improved in the 4 mg HGF group as compared to the control group (P<0.05). Overall, the present in vivo experiments demonstrated that intramyocardial injection of human HGF plasmid DNA in ischemic cardiomyopathy resulted in a significant improvement in cardiac function through an increase in blood flow and decrease in fibrosis. These favorable outcomes suggest potential utility to treat patients with ischemic heart disease using HGF gene transfer. Currently, a phase I study using human HGF plasmid DNA is ongoing to test the validity of this concept.

Animals↗

PubMed2006

Acceleration of wound healing by combined gene transfer of hepatocyte growth factor and prostacyclin synthase with Shima Jet.

Although skin diseases are one of the target diseases for gene therapy, there has been no practical gene transfer method. First, we examined gene transfer efficiency of the spring-powered jet injector, Shima Jet, which was originally developed as a non-needle jet injector of insulin. Local gene expression was about 100 times higher when the luciferase plasmid was transferred by the Shima Jet than by a needle. Gene transfer of beta-galactosidase revealed gene expression in the epidermis. Based on these results, we then examined the potential of gene therapy using the Shima Jet for wound healing. An increase of cellular proliferation of the epidermis and the number of microvessels in the granulation tissue was observed after hepatocyte growth factor (HGF) gene transfer. An increase in blood flow around the wound was observed after prostacyclin synthase (PGIS) gene transfer. Moreover, promotion on wound healing was observed in HGF gene transferred group, and further promotion was observed in combined gene transferred group as assessed by measuring wound area. These results indicate that co-transfer of HGF and PGIS genes by the Shima Jet could be an effective strategy to wound healing.

Animals↗

PubMed2006

NFkappaB decoy oligodeoxynucleotides ameliorates osteoporosis through inhibition of activation and differentiation of osteoclasts.

The transcription factor, nuclear factor-kappa B (NFkappaB), is believed to play a pivotal role in osteoclast formation. In this study, we focused on NFkappaB decoy oligodeoxynucleotides (ODN) as a new therapeutic strategy to attenuate osteoporosis. Tartrate-resistant acid phosphatase (TRAP)-positive multinuclear osteoclasts formed in mononuclear cells including osteoclast precursors from neonatal rabbit bone marrow were increased in the presence of 1,25-dihydroxyvitamin D3, whereas transfection of NFkappaB decoy ODN decreased the number of TRAP-positive cells and attenuated RANKL and M-CSF-induced osteoclast formation. NFkappaB decoy ODN also inhibited the activity of osteoclasts, as assessed by pit formation. In rat ovariectomized model of estrogen deficiency, continuous administration of NFkappaB decoy ODN attenuated the increase of TRAP activity, accompanied by a significant increase in calcium concentration in tibia and femur and decrease in urinary deoxypyridinoline. In additional osteoporosis model using vitamin C-deficient rat, inhibition of NFkappaB by decoy ODN dramatically improved the bone length, weight, density as assessed by dual-energy X-ray absorptiometry. Overall, inhibition of NFkappaB by decoy strategy prevented osteoporosis through the inhibition of bone resorption. Targeting of NFkappaB might be potential therapy in various bone metabolic diseases.

Absorptiometry, Photon↗

PubMed2006

Liver-directed gene therapy of diabetic rats using an HVJ-E vector containing EBV plasmids expressing insulin and GLUT 2 transporter.

Insulin gene therapy in clinical medicine is currently hampered by the inability to regulate insulin secretion in a physiological manner, the inefficiency with which the gene is delivered, and the short duration of gene expression. To address these issues, we injected the liver of streptozotocin-induced diabetic rats with hemagglutinating virus of Japan-envelope (HVJ-E) vectors containing Epstein-Barr virus (EBV) plasmids encoding the genes for insulin and the GLUT 2 transporter. Efficient delivery of the genes was achieved with the HVJ-E vector, and the use of the EBV replicon vector led to prolonged hepatic gene expression. Blood glucose levels were normalized for at least 3 weeks as a result of the gene therapy. Cotransfection of GLUT 2 with insulin permitted the diabetic rats to regulate their blood glucose levels upon exogenous glucose loading in a physiologically appropriate manner and improved postprandial glucose levels. Moreover, cotransfection with insulin and GLUT 2 genes led to in vitro glucose-stimulated insulin secretion that involved the closure of K(ATP) channels. The present study represents a new way to efficiently deliver insulin gene in vivo that is regulated by ambient glucose level with prolonged gene expression. This may provide a basis to overcome limitations of insulin gene therapy in humans.

Animals↗

PubMed2006

Prevention of abdominal aortic aneurysms by simultaneous inhibition of NFkappaB and ets using chimeric decoy oligonucleotides in a rabbit model.

Abdominal aortic aneurysm (AAA) is one of the major vascular diseases caused by atherosclerosis. Because treatment for AAA mainly consists of surgery to prevent deaths from AAA rupture and there is a conspicuous absence of alternative therapeutic strategies, the development of minimally invasive treatment is needed. To develop a novel therapeutic approach, we examined the simultaneous inhibition of the transcription factors NFkappaB and ets, which regulate inflammation and matrix degradation, in a rabbit AAA model. In this study, we employed chimeric decoy oligodeoxynucleotides (ODN), containing the consensus sequences of both the NFkappaB- and ets-binding sites, to inhibit both the transcription factors simultaneously. Using a delivery sheet, we examined the inhibitory effect of chimeric decoy ODN on aortic dilatation. Ultrasound and angiographic analysis demonstrated that treatment with chimeric decoy ODN significantly prevented the progression of elastase-induced aortic dilatation. The inhibitory effect of chimeric decoy ODN on aortic dilatation was also confirmed by histological studies. Treatment with chimeric decoy ODN reduced the activities of matrix metalloproteinase (MMP)-2 and MMP-9 and markedly inhibited the proteolysis of elastin as compared to scrambled decoy ODN. Interestingly, treatment with chimeric decoy ODN also suppressed VCAM-1 and MCP-1 gene expression, leading to inhibition of macrophage infiltration in the adventitia and media. The present study in a rabbit model provides a novel strategy to treat AAA by the simultaneous inhibition of both NFkappaB and ets using chimeric decoy ODN. Further modification of chimeric decoy ODN would be useful to treat AAA as a decoy-based therapy.

Animals↗

PubMed2005

Inhibition of ets, an essential transcription factor for angiogenesis, to prevent the development of abdominal aortic aneurysm in a rat model.

The pathophysiology of abdominal aortic aneurysms (AAA) is considered to be complicated. As matrix degradation contributes to the progression of AAA, the destruction and degradation of elastin fibers caused by an increase in matrix metalloproteinases (MMPs) plays a pivotal role in the development of AAA. Although ets, an essential transcription factor for angiogenesis, regulates MMPs, the role of ets in the development of AAA has not yet been clarified. Thus, we evaluated the role of ets in a rat AAA model using a decoy strategy. Transfection of ODN into AAA was performed by transient aortic perfusion of elastase and by wrapping the AAA in a delivery sheet containing decoy ODN. The inhibitory effect of ets decoy ODN on ets binding activity was confirmed by gel mobility shift assay. MMPs expression was decreased in the aorta transfected with ets decoy ODN as compared to scrambled decoy ODN. Also, ultrasound study demonstrated that elastase-induced aneurismal dilation was significantly suppressed by transfection of ets decoy ODN at 4 weeks after treatment as compared to scrambled decoy ODN. Moreover, the destruction of elastin fibers was inhibited in the aorta transfected with ets decoy ODN, accompanied by a reduction of MMPs expression. An inhibitory effect of decoy ODN on MMP expression was confirmed by ex vivo experiments showing that transfection of decoy ODN into an organ culture of human aorta resulted in significant inhibition of the secretion of both MMP-1 and MMP-9. Here, we demonstrated that ets may play a pivotal role in the progression of AAA through the activation of MMPs in a rat model. Ets might be a potential target to develop pharmacotherapy/gene therapy to treat AAA through the inhibition of MMPs.

Animals↗

PubMed2004

Inhibition of neointimal hyperplasia after balloon injury by cis-element 'decoy' of early growth response gene-1 in hypercholesterolemic rabbits.

Early growth response factor-1 (Egr-1) is a transcription factor that is rapidly activated after vascular injury and thus might contribute to vascular proliferation and inflammation. We hypothesized that Egr-1 might therefore be a therapeutic target against restenosis. Hypercholesterolemic rabbits were intraluminally administered synthetic DNA as a 'decoy' against Egr-1 immediately after carotid artery balloon injury. Efficient transfection was confirmed by the delivery of a fluorescence-labeled decoy. Gel mobility-shift assay showed increased Egr-1 activity after balloon injury and its prevention by Egr-1 decoy transfection in vivo. Egr-1 decoy transfection attenuated early inflammation and proliferation and later neointimal hyperplasia. In addition, Egr-1 decoy transfection reduced gene expression and protein production of Egr-1-dependent genes such as platelet-derived growth factor-B, transforming growth factor-beta1, and monocyte chemoattractant protein-1. The Egr-1 pathway has an essential role in the pathogenesis of neointimal hyperplasia after balloon injury in hypercholesterolemic rabbits. This decoy strategy is a potential practical form of therapy for human restenosis.

Angioplasty, Balloon↗

PubMed2004

Transcription factor decoy for AP-1 reduces mesangial cell proliferation and extracellular matrix production in vitro and in vivo.

Diabetic nephropathy is characterized by an expansion of glomerular mesangium, caused by mesangial cell proliferation and excessive accumulation of extracellular matrix (ECM) proteins, which eventually leads to glomerulosclerosis and renal failure. Activator protein-1 (AP-1), a transcription factor, is implicated in the transcriptional regulation of a wide range of genes participating in cell proliferation and ECM production. This investigation was undertaken to test the hypothesis that AP-1 plays an important role in ECM gene expression, and to develop a molecular therapeutic strategy based on decoy oligodeoxynucleotides (ODN). In this report, we show that transfection with AP-1 decoy ODN strongly inhibits high glucose- and angiotensin II-induced cell proliferation and expression of ECM genes in cultured mesangial cells in vitro. Administration of AP-1 decoy ODN into streptozotocin-induced diabetic rat kidney in vivo using the hemagglutinating virus of Japan (HVJ)-liposome method virtually abolished TGF-beta1 and plasminogen activator inhibitor-1 expression. Our results collectively indicate that AP-1 activation is crucial for mesangial cell proliferation and ECM production in response to high glucose and angiotensin II. Moreover, use of stable AP-1 decoy ODN combined with the highly effective HVJ-liposome method provides a novel potential molecular therapeutic strategy for the prevention of diabetic nephropathy.

Angiotensin II↗

PubMed2004

Development of efficient plasmid DNA transfer into adult rat central nervous system using microbubble-enhanced ultrasound.

Although gene therapy might become a promising approach for central nervous system diseases, the safety issue is a serious consideration in human gene therapy. To overcome this problem, we developed an efficient gene transfer method into the adult rat brain based on plasmid DNA using a microbubble-enhanced ultrasound method, since microbubble-enhanced ultrasound has shown promise for transfecting genes into other tissues such as blood vessels. Using the microbubble-enhanced ultrasound method, luciferase expression was increased approximately 10-fold as compared to injection of naked plasmid DNA alone. Interestingly, the site of gene expression was limited to the site of insonation with intracisternal injection, in contrast to previous studies using viruses. Expression of the reporter gene, Venus, was readily detected in the central nervous system. The transfected cells were mainly detected in meningeal cells with intracisternal injection, and in glial cells with intrastriatal injection. There was no obvious evidence of tissue damage by microbubble-enhanced ultrasound. Overall, the present study demonstrated the feasibility of efficient plasmid DNA transfer into the central nervous system, providing a new option for treating various diseases such as tumors.

Animals↗

PubMed2004

Effect of nifedipine on endothelial function in normotensive smokers: potential contribution of increase in circulating hepatocyte growth factor.

Calcium antagonists are reported to have protective effects on the endothelium in vitro and in vivo. Especially, nifedipine, among many calcium antagonists, was shown to improve endothelial dysfunction in patients with hypertension. However, no report has determined whether the improvement of endothelial dysfunction by nifedipine is due to direct effects or indirect effects such as its hypotensive effect. Thus, in this study, we evaluated the direct effects of nifedipine on smoking-induced endothelial dysfunction, since cigarette smoking itself is a major factor in damage of endothelial cells, as well as hypertension. We examined whether nifedipine improves endothelial function in 10 normotensive smokers without any risk factors for atherosclerosis. The subjects were treated with 20 mg nifedipine monotherapy (n = 10) or placebo (n = 10) for 4 weeks. Nifedipine did not affect blood pressure and heart rate of normotensive smokers. We measured forearm blood flow (FBF) by strain-gauge plethysmography after 2 and 4 weeks of treatment. Changes in vasodilator response to reactive hyperaemia were significantly improved in nifedipine-treated subjects (P < 0.05), while there was no significant change in FBP response in control subjects. Response to nitroglycerin was not changed in either group. Moreover, to evaluate the mechanisms of the direct effects of nifedipine on the endothelium, we focused on hepatocyte growth factor (HGF), which is a novel angiogenic growth factor with an antiapoptotic action on endothelial cells. Interestingly, serum HGF concentration in smokers treated with nifedipine was significantly elevated both at 2 and 4 weeks (P < 0.05). Overall, these results demonstrated direct effects of nifedipine in the improvement of endothelial dysfunction in normotensive smokers. The increase in serum HGF concentration by nifedipine might contribute to the improvement of endothelial dysfunction.

Adult↗

PubMed2004

Antisense oligonucleotides as a powerful molecular strategy for gene therapy in cardiovascular diseases.

Antisense oligonucleotides (ODN) technology is one of the most promising therapeutic strategies to prevent the progress of diseases through inhibiting the specific gene expression. They are well established to serve as molecular tools for several biologic applications, from the study of single gene function up to complex target validations. From the theoretical simple action, sequence-specific inhibition of mRNA functions after complex formation and presumably enzymatic degradation of the target mRNA, they obviously carry a high therapeutic potential to treat human diseases. In addition to the potential for the treatment, antisense ODN may be applicable for investigations of the mechanism and stereochemistry of biochemical reactions, mapping of nucleic acid protein interactions, and diagnostic applications. However, the design of antisense ODN, is very difficult because many factors affecting their activity and stability must be considered. Especially, the modifications of ODN are very critical and many researchers are trying to establish ODN which have resistance to nucleolytic degradation, high affinity to complementary nucleic acid, high selectivity in binding with complementary nucleic acid, the ability to activate ribonuclease H that selectively degrades the RNA strand of ODN-RNA complex, cell permeability, and favorable pharmacokinetic and pharmacodynamic attributes. In this review we would like to introduce some modifications of ODN design and examples of our applications of antisense ODN in cardiovascular disease in animal models.

Animals↗

PubMed2003

Inhibition of NFkappaB activation using cis-element 'decoy' of NFkappaB binding site reduces neointimal formation in porcine balloon-injured coronary artery model.

Application of DNA technology to regulate the transcription of disease-related genes has important therapeutic potential. The transcription factor NFkappaB plays a pivotal role in the transactivation of inflammatory and adhesion molecule genes, leading to vascular lesion formation. Double-stranded DNA with high affinity for NFkappaB may be introduced as 'decoy' cis elements to bind NFkappaB and block the activation of genes mediating inflammation, resulting in effective drugs for treating intimal hyperplasia. In this study, we tested the feasibility of NFkappaB decoy therapy to treat neointimal formation in a porcine coronary artery balloon injury model as a pre-clinical study. An angioplasty catheter was introduced into the left anterior descending coronary artery of the pig to cause vascular injury. First, we tested the feasibility of transfection of FITC-labeled NFkappaB decoy ODN using a hydrogel balloon catheter. Fluorescence due to NFkappaB decoy ODN could be detected throughout the medial layer. Therefore, we transfected NFkappaB decoy ODN into the balloon-injured LAD using a hydrogel catheter. Histological evaluation demonstrated that the neointimal area in the balloon-injured artery was significantly reduced by NFkappaB decoy ODN as compared to scrambled decoy ODN at 1 week after single transfection, accompanied by a significant reduction in PCNA-positive stained cells (P < 0.01). Interestingly, the reduction of ICAM-positive staining was observed, accompanied by the inhibition of migration of macrophages. Of importance, intravascular ultrasound (IVUS) confirmed that neointimal area in the balloon-injured artery was significantly reduced by NFkappaB decoy ODN at 4 weeks after transfection (P < 0.01). Interestingly, the inhibition of neointimal area was only limited to the lesion transfected with NFkappaB decoy ODN, while other lesions without NFkappaB decoy ODN demonstrated a marked increase in neointimal formation. Here, we report the successful in vivo transfer of NFkappaB decoy ODN using a hydrogel catheter to inhibit vascular lesion formation in balloon-injured porcine coronary artery.

Angioplasty, Balloon, Coronary↗

PubMed2003

Transfection of NFkappaB-decoy oligodeoxynucleotides using efficient ultrasound-mediated gene transfer into donor kidneys prolonged survival of rat renal allografts.

Nuclear factor kappaB (NFkappaB) plays a pivotal role in the coordinated transactivation of a series of genes of cytokines and adhesion molecules that are highly involved in the onset of acute rejection in organ transplantation. We previously developed decoy cis-elements oligo deoxyribonucleic acid against NFkappaB (NFkappaB-decoy) that effectively inhibited the activation of major inflammatory mediators in vitro and in vivo. Accordingly, we hypothesized that transfection of NFkappaB-decoy into the donor kidney would prevent acute rejection and prolong graft survival, and thus provide effective therapy for renal acute rejection. To transfect NFkappaB-decoy, we employed a novel approach using ultrasound exposure with an echocardiographic contrast agent, Optison, and clearly demonstrated successful transfection of NFkappaB-decoy into renal tissue. The therapeutic effect of NFkappaB-decoy on renal allografts was then evaluated in a rat renal allograft model (Wistar-Lewis). In the control group, graft function significantly deteriorated with marked destruction of renal tissue, accompanied by increased production of major inflammatory mediators, and all animals died of renal failure by 9 days. In contrast, graft function (serum creatinine on day 2, NFkappaB-treated: 0.97+/-0.16 versus control: 1.84+/-0.23 mg/dl, P<0.01) and histological structure were well preserved with significantly decreased expression of NFkappaB-regulated cytokines and adhesion molecules, including IL-1, iNOS, MCP-1, TNF-alpha, and ICAM-1, in allografts transfected with NFkappaB-decoy. As a result, animal survival was significantly prolonged in this group as compared to controls (14.2+/-5.2 versus 7.1+/-1.2 days, P<0.01). Thus, we established a novel ultrasound-Optison-mediated gene transfection approach and demonstrated the significant prolongation of graft survival by the successful transfection of NFkappaB-decoy into the donor kidney in a rat renal allograft model.

Albumins↗

PubMed2002

HGF/NK4 inhibited VEGF-induced angiogenesis in in vitro cultured endothelial cells and in vivo rabbit model.

AIMS/HYPOTHESIS: As vascular endothelial growth factor (VEGF) plays a pivotal role in the development of diabetic retinopathy, inhibition of angiogenesis induced by VEGF is crucial to treat diabetic retinopathy. HGF (hepatocyte growth factor)/NK4, containing the N-terminal hairpin domain and the four subsequent kringle domains of HGF, is considered as a specific antagonist for HGF. Our aim was to explore the inhibitory effects of HGF/NK4 on angiogenesis induced by VEGF. METHODS: To analyze the in vivo angiogenesis, we used rabbit corneal micropocket assay. Proliferation and migration of human endothelial cells, expression of ets-1, an essential transcription factor for angiogenesis, and the phosphorylation of extracellular signal-regulated kinase (ERK) was examined with or without HGF/NK4. RESULTS: Using corneal micropocket assay, in vivo administration of HGF/NK4 inhibited angiogenesis induced by VEGF. HGF/NK4 inhibited proliferation and migration of human endothelial cells induced by VEGF in a dose-dependent manner. Interestingly, VEGF-mediated phosphorylation of ERK was significantly attenuated by HGF/NK4. Of importance, HGF/NK4 attenuated the increase in ets-1 protein stimulated by VEGF. Nevertheless, HGF/NK4 did not affect phosphorylation of VEGF receptor-2 [kinase domain region (KDR)/foetal liver kinase (Flk)-1]. Although tyrosine phosphatase inhibitor (Na(3)VO(4)), or okadaic acid, serine-threonin kinase inhibitor, did not prevent the inhibition of ERK phosphorylation by HGF/NK4, co-incubation of HGF/NK4 with VEGF significantly diminished mitogen-activated protein (MAP) ERK kinase (MEK) phosphorylation (p<0.01). CONCLUSIONS/INTERPRETATION: Overall, HGF/NK4 inhibited angiogenesis induced by VEGF through inhibition of phosphorylation of ERK and ets-1 expression in in vitro cultured endothelial cells and in vivo rabbit model.

Animals↗

PubMed2002

Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle.

Although clinical trials of stimulation of angiogenesis by transfection of angiogenic growth factors using naked plasmid DNA or adenoviral vector have been successful, there are still unresolved problems for human gene therapy such as low transfection efficiency and safety. From this viewpoint, it is necessary to develop safe and efficient novel nonviral gene transfer methods. As therapeutic ultrasound induces cell membrane permeabilization, ultrasound irradiation might increase the transfection efficiency of naked plasmid DNA into skeletal muscle. Thus, we examined the transfection efficiency of naked plasmid DNA using ultrasound irradiation with echo contrast microbubble (Optison) in vitro and in vivo experiments. First, we examined the feasibility of ultrasound-mediated transfection of naked plasmid DNA into skeletal muscle cells. Luciferase plasmid mixed with or without Optison was transfected into cultured human skeletal muscle cells using ultrasound (1 MHz; 0.4 W(2)) for 30 s. Interestingly, luciferase activity was markedly increased in cells treated with Optison, while little luciferase activity could be detected without Optison (P < 0.01). Electron microscopy demonstrated the transient formation of holes (less than 5 microM) in the cell surface, which could possibly explain the rapid migration of the transgene into the cells. Next, we studied the in vivo transfection efficiency of naked plasmid DNA using ultrasound with Optison into skeletal muscle. Two days after transfection, luciferase activity in skeletal muscle transfected with Optison using ultrasound was significantly increased about 10-fold as compared with plasmid alone. Successful transfection was also confirmed by beta-galactosidase staining. Finally, we examined the feasibility of therapeutic angiogenesis using naked hepatocyte growth factor (HGF) plasmid in a rabbit ischemia model using the ultrasound-Optison method. Five weeks after transfection, the angiographic score and the number of capillary density in rabbits transfected with Optison using ultrasound was significantly increased as compared with HGF plasmid alone (P < 0.01), accompanied by a significant increase in blood flow and blood pressure ratio (P < 0.01). Overall, the ultrasound transfection method with Optison enhanced the transfection efficiency of naked plasmid DNA in vivo as well as in vitro. Transfection of HGF plasmid by the ultrasound-Optison method could be useful for safe clinical gene therapy to treat peripheral arterial disease without a viral vector system.

Animals↗

PubMed2002

Molecular strategy using cis-element 'decoy' of E2F binding site inhibits neointimal formation in porcine balloon-injured coronary artery model.

Transcription factor E2F plays a pivotal role in the transactivation of cell cycle regulatory genes, leading to vascular lesion formation. Double-stranded DNA with high affinity for E2F as 'decoy' cis elements may block the activation of genes mediating the cell cycle, resulting in an effective therapeutic agent for treating intimal hyperplasia. In this study, we tested the feasibility of E2F decoy therapy to treat neointimal formation in a porcine coronary artery balloon injury model. An angioplasty catheter was inserted in the left anterior descending coronary artery of pigs to cause vascular injury. Initially, we tested the feasibility of transfection of FITC-labeled E2F decoy ODN using a hydrogel balloon catheter. Fluorescence due to E2F decoy ODN could be detected throughout the medial layer. Therefore, we transfected E2F decoy ODN into the balloon-injured artery using hydrogel catheter. Of importance, intravascular ultrasound (IVUS) and histological evaluation demonstrated that plaque area in the balloon-injured artery was significantly reduced by E2F decoy ODN compared with mismatched decoy ODN at 1 month after a single transfection (P < 0.01). In contrast, luminal and total vessel areas were significantly increased in vessels treated with E2F decoy ODN as compared with mismatched decoy. Endothelial function after angioplasty was not affected by E2F decoy transfection. Finally, we tested the acute toxicity of E2F decoy ODN in monkeys, and no apparent side-effects were detected. Here, we report the successful in vivo transfer of E2F decoy ODN using a hydrogel catheter to inhibit vascular lesion formation in balloon-injured porcine coronary artery without any apparent side-effects.

Angioplasty, Balloon, Coronary↗

PubMed2002

Prevention and regression of atopic dermatitis by ointment containing NF-kB decoy oligodeoxynucleotides in NC/Nga atopic mouse model.

Atopic dermatitis, a chronic inflammatory skin disease characterized by relapsing eczema and intense prurigo, requires effective and safe pharmacological therapy. In this study, we examined the efficacy of ointment containing NF-kB decoy oligodeoxynucleotides (ODN) on atopic dermatitis lesions in NC/Nga mice, which are characterized by the spontaneous onset of atopic dermatitis in conventional conditions. Topical administration of NF-kB decoy ODN twice a month resulted in a significant reduction in clinical skin condition score and marked improvement of histological findings. Reduction of the atopic skin condition by NF-kB decoy ODN was accompanied by a significant decrease in migration of mast cells into the dermis and an increase in apoptotic cells. Here, we demonstrated the successful treatment of atopic dermatitis with ointment containing NF-kB decoy ODN in a mouse model, promising new therapy for atopic dermatitis.

Administration, Cutaneous↗