Biomedical subjects

Yasufumi Kaneda

Publications and source records attributed to Yasufumi Kaneda.

At least 19 recordsLinked to original sources

PubMed2007

Inactivated Sendai virus particles eradicate tumors by inducing immune responses through blocking regulatory T cells.

UV-inactivated, replication-defective Sendai virus particles [hemagglutinating virus of Japan envelope (HVJ-E)] injected into murine colon carcinoma (CT26) tumors growing in syngeneic BALB/c mice eradicated 60% to 80% of the tumors and obviously inhibited the growth of the remainder. Induced adaptive antitumor immune responses were dominant in the tumor eradication process because the effect was abrogated in severe combined immunodeficient mice. Murine and human dendritic cells underwent dose-dependent maturation by HVJ-E in vitro. Profiles of cytokines secreted by dendritic cells after HVJ-E stimulation showed that the amount of interleukin-6 (IL-6) released was comparable to that elicited by live HVJ. Real-time reverse transcription-PCR and immunohistochemistry revealed that HVJ-E induced a remarkable infiltration of dendritic cells and CD4+ and CD8+ T cells into tumors. In addition, CT26-specific CTLs were induced with the evidence of enhanced CD8+ T-cell activation in a CD4+CD25- T cell-dependent manner. On the other hand, conditioned medium from dendritic cells stimulated by HVJ-E rescued CD4+CD25- effector T-cell proliferation from Foxp3+CD4+CD25+ regulatory T cell (Treg)-mediated suppression and IL-6 was presumably dominant for this phenomenon. We also confirmed such rescue in mice treated with HVJ-E in vivo. Moreover, antitumor effect of HVJ-E was significantly reduced by an in vivo blockade of IL-6 signaling. This is the first report to show that HVJ-E alone can eradicate tumors and the mechanism through which it induces antitumor immune responses. Because it can enhance antitumor immunity and simultaneously remove Treg-mediated suppression, HVJ-E shows promise as a novel therapeutic for cancer immunotherapy.

Animals↗

PubMed2007

Rapid transport of plasmid DNA into the nucleolus via actin depolymerization using the HVJ envelope vector.

BACKGROUND: Although nuclear transport of therapeutic genes is an essential requirement of human gene therapy, factors required for nuclear entry of DNA remain to be elucidated. Non-viral vector systems have led to numerous improvements in the efficiency of delivery of exogenous DNA into cells. However, nuclear transport of plasmid is difficult to achieve. METHODS: We examined nuclear translocation efficiency of Cy3-labeled plasmid DNA (Cy3-pDNA) delivered by the hemagglutinating virus of Japan envelope (HVJ-E) vector, Lipofectamine or microinjection. We also examined the effect of actin depolymerization on nuclear transport of Cy3-pDNA. RESULTS: Cy3-pDNA reached the nucleus, particularly in the nucleolus, in 30 min after fusion-mediated delivery using the HVJ-E vector, while the DNA was retained in the cytoplasm during the observed period after the delivery by cationic liposomes. HVJ-E treatment transiently depolymerized actin filaments, and acceleration of nucleolar entry of microinjected DNA was achieved when treated with either empty HVJ-E or cytochalasin D, an inhibitor of actin depolymerization, prior to microinjection. CONCLUSIONS: These results suggest that plasmid DNA can be transported rapidly from the cytoplasm to the nucleolus when actin filaments are depolymerized. Thus, the HVJ-E vector can accelerate the transport of DNA to the nucleolus by actin depolymerization.

Actins↗

PubMed2006

Needleless intranasal administration of HVJ-E containing allergen attenuates experimental allergic rhinitis.

Allergic rhinitis (AR) is one of the most common chronic diseases. Although current medications are highly effective in controlling its symptoms, they do not reverse the allergen-specific hypersensitivities that underlie the disease. Immunoglobulin E is a key mediator of AR, and preventing its production is clinically important. In this study, we developed an efficient needleless intranasal protein delivery system using the hemagglutinating virus of Japan envelope vector (HVJ-E). Intranasal delivery of ovalbumin (OVA) once a week for 3 weeks using this system enhanced OVA-induced interferon-gamma production by murine splenocytes. This treatment also attenuated the OVA-induced release interleukin-4 (IL-4) and IL-5 from splenocytes and the production of plasma OVA-specific immunoglobulin E in OVA-sensitive AR model mice. Thus, allergen-containing HVJ-E may be useful for noninvasive treatment of AR.

Administration, Intranasal↗

PubMed2006

Transfection of human hepatocyte growth factor gene ameliorates secondary lymphedema via promotion of lymphangiogenesis.

BACKGROUND: Lymphedema is a disorder of the lymphatic vascular system characterized by impaired lymphatic return and swelling of the extremities. Treatment for this disabling condition remains limited and largely ineffective. The goal of the present study was to investigate the therapeutic efficacy of hepatocyte growth factor (HGF) in animal models of lymphedema. METHODS AND RESULTS: Immunofluorescent analysis demonstrated that canine primary lymphatic endothelial cells (cLECs) were positive for lymphatic-specific markers (vascular endothelial growth factor receptor-3, LYVE-1, podoplanin, and Prox1) and the HGF receptor c-Met. Treating cLECs with human recombinant HGF resulted in a dose-dependent increase in cell growth and migration and increased activity of extracellular signal-regulated kinase and Akt. In human LECs, c-Met also was expressed, and treatment with HGF increased cell growth and migration in a dose-dependent manner. Transfection of human HGF plasmid DNA in cLECs also increased the c-fos promoter activity. Furthermore, weekly HGF gene transfer in a rat tail lymphedema model by disruption of lymphatic vessels resulted in a decrease in lymphedema thickness. Although expression of the endothelial cell marker PECAM-1 was increased in both HGF- and vascular endothelial growth factor 165-injected groups, expression of LEC markers (LYVE-1 and Prox1) was increased only in the HGF-injected group. CONCLUSIONS: These data demonstrate that expression of HGF via plasmid transfer improves lymphedema via promotion of lymphangiogenesis. Further studies to determine the clinical utility of this approach would be of benefit to patients with lymphedema.

Animals↗

PubMed2006

Hypertension accelerated experimental abdominal aortic aneurysm through upregulation of nuclear factor kappaB and Ets.

In this study, we focused on the effect of hypertension on the transcription factors nuclear factor kappaB (NFkappaB) and ets in the mechanisms of abdominal aortic aneurysm (AAA), and we investigated how hypertension affects the progression of AAA. AAA was produced by elastase perfusion in hypertensive rats and normotensive rats. The size of AAA rapidly increased in hypertensive rats as compared with normotensive rats. Western blot analysis demonstrated that the expression of matrix metalloproteinase (MMP)-2, -3 , -9, and -12, as well as intercellular adhesion molecule, was increased in hypertensive AAA rats, accompanied by upregulation of NFkappaB and ets. Moreover, in situ zymography showed that the activity of MMPs was increased in the aorta of a hypertensive AAA model as compared with that in a normotensive AAA model. Interestingly, transfection of chimeric decoy oligodeoxynucleotide (ODN) resulted in significant inhibition of aortic dilatation both in normotensive and hypertensive rats at 4 weeks after transfection. Destruction of elastic fibers was also significantly inhibited by transfection of chimeric decoy ODN in both hypertensive rats and normotensive rats. The expression of MMP-2, -3, -9, and -12, as well as intercellular adhesion molecule, was significantly attenuated by the chimeric decoy ODN, accompanied by inhibition of the migration of macrophages. Also, the effect of chimeric decoy ODN was confirmed in an organ culture. The present study demonstrated that hypertension accelerated the progression of experimental AAA through upregulation of NFkappaB and ets. Inhibition of NFkappaB and ets could be a novel therapeutic strategy to treat AAA in hypertensive patients.

Animals↗

PubMed2006

Inhibitory effects of NFkappaB decoy oligodeoxynucleotides on neointimal hyperplasia in a rabbit vein graft model.

Autologous vein remains the commonly used conduit for bypass grafts; however, neointimal hyperplasia is known to be one of the major disease processes in vein graft failure. In this study, we focused on the important role of NFkappaB which controls the expression of numerous genes for various cytokines and adhesion molecules in the mechanism of graft failure. Thus, we investigated the inhibitory effect of NFkappaB decoy oligodeoxynucleotides (ODN) on vein graft failure in a rabbit hypercholesterolemic model. Jugular vein to carotid artery interposition grafts in rabbits were transfected intraoperatively with NFkappaB decoy ODN (40 micromol/l) by ex-vivo pressure-mediated transfection (300 mm Hg, 10 min). Treatment with NFkappaB decoy ODN significantly suppressed intimal hyperplasia 4 weeks after vein implantation as compared to scrambled decoy ODN, and increased medial thickness, leading to a significant reduction in intima-to-media ratio. Treatment with NFkappaB decoy ODN significantly inhibited the recruitment of macrophages and the proliferation of vascular smooth muscle cells (VSMC), while apoptosis in VSMC was significantly increased by NFkappaB decoy ODN. In addition, a study of vascular reactivity demonstrated that transfection of grafts with NFkappaB decoy ODN significantly improved endothelium-mediated vasorelaxation as compared to scrambled decoy ODN. Here, we demonstrated that inhibition of NFkappaB activation using decoy ODN inhibited the development of neointimal hyperplasia, followed by suppression of inflammatory changes and accumulation of VSMC in the neointima of rabbit vein grafts. The present study raises the possibility of a novel strategy for prevention of graft failure.

Animals↗

PubMed2006

The effect of VEGF on blood vessels and blood cells during Xenopus development.

Vascular endothelial growth factor (VEGF) is known to play an essential role in vascular development. We have overexpressed VEGF122 or VEGF170, which are equivalent to mouse VEGF120 and VEGF164, in developing Xenopus embryos. Overexpression of VEGF170 but not VEGF122 demonstrated an absence of expression of hematopoietic markers alpha-globin and GATA-1 but only in the posterior portion of the blood island. Interestingly, strong signals of endothelial markers, msr, fli-1, and tie-2, were detectable in those regions, instead of hematopoietic markers. These results suggested both that injection of VEGF170 resulted in disturbance of vasculogenesis in the posterior portion of the blood island, with excessive production of endothelial cells at the expense of blood cells, and that the anterior and posterior portions of the VBI may have distinct characteristics.

Animals↗

PubMed2006

Mouse model of human infertility: transient and local inhibition of endometrial STAT-3 activation results in implantation failure.

Embryo implantation involves a series of biochemical reactions and its failure is an important therapeutic target of infertility treatment. We established an infertile mouse model using transient and local suppression of signal transducer and activator of transcription-3 (STAT-3) activity by STAT-3 decoy transfer into the uterine cavity during implantation, resulting in <30% implantation. This infertility is caused by suppression of decidualization, which is indispensable for implantation, and independent of progesterone. These conditions may mimic clinically unexplained infertility. Our results suggest that STAT-3 could be a useful target for diagnosis and therapy of human implantation failure.

Animals↗

PubMed2006

Gene transfer of hepatocyte growth factor gene improves learning and memory in the chronic stage of cerebral infarction.

There is no specific treatment to improve the functional recovery in the chronic stage of ischemic stroke. To provide the new therapeutic options, we examined the effect of overexpression of hepatocyte growth factor (HGF) in the chronic stage of cerebral infarction by transferring the HGF gene into the brain using hemagglutinating virus of Japan envelope vector. Sixty rats were exposed to permanent middle cerebral artery occlusion (day 1). Based on the sensorimotor deficits at day 7, the rats were divided equally into control vector or HGF-treated rats. At day 56, rats transfected with the HGF gene showed a significant recovery of learning and memory in Morris water maze tests (control vector 50+/-4 s; HGF 33+/-5 s; P<0.05) and passive avoidance task (control vector 132.4+/-37.5 s; HGF 214.8+/-26.5 s; P<0.05). Although the total volume of cerebral infarction was not related to the outcome, immunohistochemical analysis for Cdc42 and synaptophysin in the peri-infarct region revealed that HGF enhanced the neurite extension and increased synapses. Immunohistochemistry for glial fibriary acidic protein revealed that the formation of glial scar was also prevented by HGF gene treatment. Additionally, the number of the arteries was increased in the HGF group at day 56. These data demonstrated that HGF has a pivotal role for the functional recovery after cerebral infarction through neuritogenesis, improved microcirculation, and the prevention of gliosis. Our results also provide evidence for the feasibility of gene therapy in the chronic stage of cerebral infarction.

Animals↗

PubMed2006

Simple and highly efficient method for transient in vivo gene transfer to mid-late pregnant mouse uterus.

Up- and down-regulation of various genes in the placenta, decidua and amnion has been reported during the mid-late period of pregnancy and in pregnancy-related complications, such as preeclampsia and preterm labor. However, whether this gene regulation at the feto-maternal interface directly influences the physiology/pathophysiology of disease remains unknown. In order to study this problem, transient gene transfer into the pregnant uterus at mid-late term would be a useful tool. We injected exogenous plasmid entrapped using a commercially available Hemagglutinating Virus of Japan Envelope (HVJ-E) vector system (GenomONE Neo, Ishihara Sangyo) into the extra-amniotic space of the upper part of the pregnant mouse uterus on day 14.5 post-coitus (p.c.). Luciferase activity driven by the cytomegalovirus promoter was detectable for 3 days after transfection in the upper, middle and lower part of the uterus. beta-Galactsidase activity was localized in the basal lamina of the placenta, the decidual membrane and the fetal membrane. Exogenous plasmid was not transmitted to the fetus. The course of pregnancy was not disturbed by this procedure; rupture of membranes, intrauterine fetal growth restriction and preterm birth were not observed. Thus, we demonstrated that this transient gene transfer method is highly efficient and minimally invasive, and expect that this procedure will be a useful tool to analyze the pathophysiology of pregnancy-related disorders.

Animals↗

PubMed2006

HVJ-based non-viral gene transfer method: successful gene therapy using HGF and VEGF genes in experimental ischemia.

VEGF and HGF are pleiotropic factors that regulate cell growth, cell motility, and morphogenesis of various types of cells. The receptors of these growth factors are expressed in neurons and endothelial cells, and are identified as neurotrophic, neuroprotective, and angiogenic factors. Indeed, gene therapy using viral vectors encoding the VEGF or HGF gene has been reported to be effective for preventing the expansion of ischemic injury. However, the safety issue of viral vectors is a major problem in clinical application. To overcome this problem, we have developed an HVJ-based non-viral vector, which achieves high-efficiency transfection rates of viral vectors with the safety of liposomes. This review discusses the feasibility of gene therapy using an HVJ-based non-viral vector containing the VEGF or HGF gene for cerebral ischemia.

Animals↗

PubMed2006

Development of high-throughput functional screening of therapeutic genes, using a hemagglutinating virus of Japan envelope vector.

Isolation of effective therapeutic genes is critical for the advancement of gene therapy for various diseases, including vascular diseases and cancers. The goal of the present study was to screen a human cDNA library, using a hemagglutinating virus of Japan envelope (HVJ-E) vector, to isolate candidate genes with potent therapeutic potential. The advantages of a high-throughput functional screening system based on the HVJ-E vector include (1) rapid preparation of the vector containing the DNA library, (2) effective fusion-mediated transfer of the plasmids to various cells with minimal toxicity, and (3) easy cloning of candidate genes by transformation of Escherichia coli. These advantages resulted in a lower probability of damage to isolated clones and in minimization of the time needed to screen for candidate genes. Screening of a human heart library for candidate genes to regulate endothelial cell growth identified three growth-stimulating genes, as evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and c-fos promoter activity, the products of which were more potent than vascular endothelial growth factor. Similarly, two growth-inhibiting genes were identified, the effects of which were similar to angiostatin. Overall, this novel system will help advance our understanding of cell biology and promote the utility of human gene therapy.

Cell Line↗

PubMed2006

Non-viral vectors for cancer therapy.

Cancers are diverse and often resistant to therapeutic strategies. Gene therapy has yet to meet the promise of a breakthrough in cancer therapy. There are several barriers to overcome in cancer gene therapy. One of the biggest challenges is the design of appropriate vectors. Numerous viral and non-viral methods for gene transfer have been developed for human gene therapy, but both viral and non-viral vectors have limitations and advantages. In this review article, recent improvements in the development of non-viral vectors for delivering gene therapy for the treatment of cancer will be discussed.

Animals↗

PubMed2006

Dnmt3a2 targets endogenous Dnmt3L to ES cell chromatin and induces regional DNA methylation.

DNA methylation is involved in fundamental cellular processes such as silencing of genes and transposable elements, but the underlying mechanism of regulation of DNA methylation is largely unknown. DNA methyltransferase 3-like protein (Dnmt3L), a member of the Dnmt3 family of proteins, is required during the establishment of DNA methylation patterns in germ cells. Dnmt3L does not possess enzymatic activity. Rather, in vitro analysis indicates that Dnmt3L stimulates DNA methylation by both Dnmt3a and Dnmt3b through direct binding to these proteins. In the current study, we demonstrated that in vivo, Dnmt3L physically and functionally interacted with the Dnmt3 isoform Dnmt3a2. In wild-type embryonic stem (ES) cells, but not in cells lacking Dnmt3a, endogenous Dnmt3L was concentrated in chromatin foci. In ES cells deficient in both Dnmt3a and Dnmt3b, Dnmt3L was distributed diffusely throughout the nucleus and cytoplasm, and ectopic expression of Dnmt3a2, but not Dnmt3a or Dnmt3b, restored wild-type Dnmt3L localization. We showed that endogenous Dnmt3L physically interacted with Dnmt3a2, but not Dnmt3a or Dnmt3b, in ES cells and embryonic testes. We also found that specific CpG sites were demethylated upon depletion of either Dnmt3a or Dnmt3L, but not Dnmt3b, in ES cells. These results provide evidence for a physical and functional interaction between Dnmt3L and Dnmt3a2 in the nucleus. We propose that Dnmt3a2 recruits Dnmt3L to chromatin, and induces regional DNA methylation in germ cells.

Animals↗

PubMed2006

Tumor necrosis factor-a antisense transfer remarkably improves hepatic graft viability.

BACKGROUND: Cold ischemia/reperfusion injury of the hepatic graft, an unsolved problem in liver transplantations, is attributed to the release of inflammatory cytokines, especially the tumor necrosis factor- (TNF) alpha, from activated Kupffer cells (KC). Therefore, the specific inhibition of TNF-alpha could improve the viability of the hepatic graft upon reperfusion. METHODS: We assessed the efficacy of TNF-alpha antisense (TNF-AS) oligodeoxynucleotides (ODNs) delivery to KC in a rodent liver transplantation model. RESULTS: Seventy-one percent of the animals that received 6 hours preserved grafts in baths of lactated Ringer's solution (4 degrees C) and were treated with TNF-AS survived for over 14 days. Eighty percent of the animals treated with vehicle, sense ODNs, or balanced salt saline (BSS) died. Four hours after reperfusion of the liver, a significant reduction was noted in livers treated with TNF-AS in the release of cytosolic enzymes from the hepatocytes and the serum TNF-alpha (P<0.05). The expressions of TNF-alpha on KC and of intercellular adhesion molecule-1 on sinusoidal endothelial cells were completely suppressed in TNF-AS-treated livers. CONCLUSIONS: TNF-AS delivery improves the viability of the hepatic graft, and this technique may solve hepatic graft nonfunction in a clinical setting.

Animals↗

PubMed2006

Targeted chemotherapy against intraperitoneally disseminated colon carcinoma using a cationized gelatin-conjugated HVJ envelope vector.

The hemagglutinating virus of Japan envelope (HVJ-E; Sendai virus) vector derived from inactivated HVJ particles can be used to deliver DNA, proteins, and drugs into cells both in vitro and in vivo. HVJ-E is capable of delivering bleomycin, an anticancer drug, to various cancer cell lines, thereby producing 300-fold greater cytotoxicity than administration of bleomycin alone. In a mouse model of peritoneally disseminated colon cancer, we injected HVJ-E containing the luciferase gene into the peritoneum. Unexpectedly, luciferase gene expression was not observed within the tumor deposits or any organs. However, when combined with cationized gelatin (CG), CG-HVJ-E produced a high level of luciferase gene expression primarily within the tumor deposits. Forty-eight hours after introducing colon cancer cells into the peritoneum of experimental mice, CG-HVJ-E with or without bleomycin was injected into the abdominal cavity. Following six injections of bleomycin-incorporated CG-HVJ-E, complete responses were observed in 40% of the mice examined. All of the mice that received either empty CG-HVJ-E or bleomycin alone died within 40 days of having cancer cells introduced into the peritoneum. When the mice with complete responses were rechallenged with colon cancer cells from the same cell line, no tumors developed. Thus, CG-HVJ-E may suppress peritoneal dissemination of cancer.

Animals↗

PubMed2006

Construction of a novel DNA decoy that inhibits the oncogenic beta-catenin/T-cell factor pathway.

The oncogenic beta-catenin/T-cell factor (TCF) signal is a common trigger inducing expressions of various cancer-related genes and is activated in various types of human malignancy. The aim of this study was to create an effective double-stranded DNA decoy that would interfere with endogenous TCF hyperactivity in tumor cells. We first established the TCF-activated model using nontumor human embryonic kidney 293 (HEK293) cells by introducing a beta-catenin cDNA. Based on a consensus TCF-binding sequence in the cyclin D1 and c-myc promoters, several double-stranded oligodeoxynucleotides were designed and tested for their ability to inhibit TCF activity in the HEK293 model. Among them, the 18-mer oligodeoxynucleotide stably formed double-stranded DNA and efficiently inhibited TCF activity. FITC-labeled oligodeoxynucleotide was efficiently incorporated into the nucleus at 6 hours and remained within cells for up to 72 to 96 hours. When compared with scrambled oligodeoxynucleotide, we found that the 18-mer TCF decoy significantly inhibited TCF activity and promoter activities of the downstream target genes, such as cyclin D1, c-myc, and matrix metalloproteinase 7 in HCT116 colon cancer cells. Reverse transcription-PCR assays indicated that mRNA expression of these genes decreased with treatment of the TCF decoy. Proliferation assay showed that the TCF decoy significantly inhibited growth of HCT116 tumor cells, but not of nontumor HEK293 cells. Our data provide evidence that the TCF decoy reduced both TCF activity and transcriptional activation of downstream target genes. Thus, this TCF decoy is potentially an efficient and nontoxic molecular targeting therapy for controlling malignant properties of cancer cells.

Base Pair Mismatch↗

PubMed2006

Anti-oxidant gene therapy by NF kappa B decoy oligodeoxynucleotide.

Oxidative stress to cardiovascular cells induced by an interaction of multiple cytokines and adhesion molecules has been postulated to be responsible for cardiovascular disease. Since nuclear factor-kappaB (NFkappaB) also plays a pivotal role in the coordinated transactivation of cytokine and adhesion molecule genes, we utilized oligodeoxynucleotides (ODNs) as "decoy" cis-elements that block the binding of nuclear factors to promoter regions of targeted genes, resulting in the inhibition of gene transactivation. Indeed, transfection of NFkappaB decoy ODNs into coronary artery effectively prevented transactivation of essential cytokine and adhesion molecule protein expression, and thereby protected the myocardium from infarction. Transfection of NFkappaB decoy ODNs into balloon-injured carotid artery or porcine coronary artery markedly reduced neointimal formation. Thus, a clinical trial using NFkappaB decoy ODNs to treat restenosis was started in 2002. Recently, the therapeutic target utilizing NFkappaB decoy has been expanded to glomerulonephritis, rheumatoid arthritis, atopic dermatitis and osteoporosis. Moreover, the clinical trials to treat RA patients were initiated in 2003 and a Phase I/IIa human clinical trial using NFkappaB decoy ODNs to treat atopic dermatitis was initiated in December 2001. Topical application of NFkappaB decoy ODNs exhibited marked therapeutic effects on the facial skin condition of patients with atopic dermatitis. The covalently modified ODNs were developed by enzymatically ligating two identical molecules, thereby preventing their degradation by exonucleases. Indeed, the modified decoy ODNs possess increased nuclease resistance and are transported more efficiently into cells. Although there are still unresolved issues, decoy ODN drugs should become a reality.

Animals↗