Biomedical subjects

Tsuyoshi Takato

Publications and source records attributed to Tsuyoshi Takato.

At least 37 records · Page 2Linked to original sources

PubMed2004

Inflammatory signaling pathway containing TRAF6 contributes to neointimal formation via diverse mechanisms.

OBJECTIVE: The purpose of this study was to investigate the contribution of inflammatory signaling containing tumor necrosis factor receptor-associated factor 6 (TRAF6) to neointimal formation in a balloon injury model of rabbit carotid artery. METHODS: Male Japanese white rabbits fed a normal diet were used. We transferred the dominant negative (DN) form of TRAF6 to a rabbit carotid artery that was subjected to balloon injury by in vivo electroporation method, and then evaluated its effect on intimal lesion formation after balloon injury. RESULTS: An expression plasmid vector containing the TRAF6 DN sequence was successfully transferred to arterial wall cells, and its inhibitory effect on inflammatory signaling was confirmed by the marked suppression of nuclear factor-kappaB (NFkappaB) activity after injury. Morphometric analyses revealed significant inhibition of intimal lesion formation at 7 days after injury. Cell replication and accumulation of macrophages in the media were significantly decreased, and apoptosis was enhanced on day 2. Cell migration to the intima was suppressed on day 4. Extracellular signal-regulated kinase1/2 (ERK1/2) activity at 2 h after injury was also down-regulated. Interestingly, intimal cell replication was significantly blocked when TRAF6 DN was transfected at 7 days after injury. CONCLUSION: TRAF6 plays important roles in cell replication and migration, besides promotion of inflammatory cell infiltration and suppression of apoptosis.

Animals↗

PubMed2004

Osteoclast differentiation by RANKL requires NF-kappaB-mediated downregulation of cyclin-dependent kinase 6 (Cdk6).

UNLABELLED: This study investigated the involvement of cell cycle factors in RANKL-induced osteoclast differentiation. Among the G1 cell cycle factors, Cdk6 was found to be a key molecule in determining the differentiation rate of osteoclasts as a downstream effector of the NF-kappaB signaling. INTRODUCTION: A temporal arrest in the G1 phase of the cell cycle is a prerequisite for cell differentiation, making it possible that cell cycle factors regulate not only the proliferation but also the differentiation of cells. This study investigated cell cycle factors that critically influence differentiation of the murine monocytic RAW264.7 cells to osteoclasts induced by RANKL. MATERIALS AND METHODS: Growth-arrested RAW cells were stimulated with serum in the presence or absence of soluble RANKL (100 ng/ml). Expressions of the G1 cell cycle factors cyclin D1, D2, D3, E, cyclin-dependent kinase (Cdk) 2, 4, 6, and Cdk inhibitors (p18 and p27) were determined by Western blot analysis. Involvement of NF-kappaB and c-jun N-terminal kinase (JNK) pathways was examined by overexpressing dominant negative mutants of the IkappaB kinase 2 (IKK(DN)) gene and mitogen-activated protein kinase kinase 7 (MKK7(DN)) gene, respectively, using the adenovirus vectors. To determine the direct effect of Cdk6 on osteoclast differentiation, stable clones of RAW cells transfected with Cdk6 cDNA were established. Osteoclast differentiation was determined by TRACP staining, and cell cycle regulation was determined by BrdU uptake and flow cytometric analysis. RESULTS AND CONCLUSION: Among the cell cycle factors examined, the Cdk6 level was downregulated by RANKL synchronously with the appearance of multinucleated osteoclasts. Inhibition of the NF-kappaB pathway by IKK(DN) overexpression, but not that of the JNK pathway by MKK7(DN) overexpression, caused the decreases in both Cdk6 downregulation and osteoclastogenesis by RANKL. RAW cells overexpressing Cdk6 resist RANKL-induced osteoclastogenesis; however, cell cycle regulation was not affected by the levels of Cdk6 overexpression, suggesting that the inhibitory effect of Cdk6 on osteoclast differentiation was not exerted through cell cycle regulation. These results indicate that Cdk6 is a critical regulator of RANKL-induced osteoclast differentiation and that its NF-kappaB-mediated downregulation is essential for efficient osteoclast differentiation.

Animals↗

PubMed2004

Gene expression profile of human mesenchymal stem cells during osteogenesis in three-dimensional thermoreversible gelation polymer.

This study attempted to characterize the ability of thermoreversible gelation polymer (TGP) to induce differentiation of human mesenchymal stem cells (hMSC) into osteoblasts. Using a long oligo microarray system consisting of 3760 genes, we compared the expression profiles of the cells in 2-dimensional (2D) culture, 3D culture in collagen gel, and 3D culture in TGP with or without osteogenic induction. Compared to 2D culture, the gene expression profile of hMSC showed almost the same pattern in TGP without osteogenic induction, but 72% of genes (2701/3760) were up-regulated in collagen gel. With osteogenic induction, hMSC showed higher ALP activity and osteocalcin production in TGP as compared to 2D culture. Moreover, up-regulation and down-regulation of osteogenic genes were augmented in 3D culture in TGP as compared to 2D culture. As TGP is chemically synthesized and completely free from pathogen such as prion in bovine spongiform encephalopathy, these results suggest that TGP could be applied clinically to induce osteogenic differentiation of hMSC.

Alkaline Phosphatase↗

PubMed2004

Characterization of multipotent adult stem cells from the skin: transforming growth factor-beta (TGF-beta) facilitates cell growth.

Recently, adult stem cells have been isolated from the skin and designated as skin-derived precursors (SKPs). These SKPs, cultured in vitro, can give rise to neurons, glia, smooth muscle cells, and adipocytes. In the current study, we confirmed the clonal expansion of SKPs using a sphere-forming culture system in a medium containing methylcellulose. Among the growth factors, only transforming growth factor-beta (TGF-beta) was revealed to uniquely facilitate the sphere formation and proliferation of the SKPs in combination with EGF and bFGF. In addition, TGF-beta did not alter phenotypical characteristics of the SKPs under sphere-forming conditions. The effect of TGF-beta on sphere formation was not observed in neural stem cells, which expressed a different set of cell surface markers from SKPs, suggesting that SKPs have distinct features. Although the number of SKPs decreased with age, TGF-beta increased the sphere colony formation and proliferation in all ages. These results suggest that SKPs maintained in the presence of TGF-beta during culture are of potential use in cell-replacement therapies employing adult tissue sources.

Adipocytes↗

PubMed2004

Conduction performance of collateral vessels induced by vascular endothelial growth factor or basic fibroblast growth factor.

OBJECTIVE: In the present study, we delivered vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) gene to a rabbit model of hind limb ischemia utilizing an ex vivo method of gene transfer, and evaluated the functional performance of the developed collateral vessels. METHOD: The left femoral artery of a male Japanese White rabbit was excised to induce limb ischemia, and a section of skin was resected for culture of auto-fibroblasts. Twenty days later, the VEGF gene, bFGF gene or beta-galactosidase gene (LacZ) was adenovirally transferred to the cultured auto-fibroblasts (5x10(6) cells), and the next day, a pair of specifically infected fibroblasts (total 1x10(7) cells) was injected via the left internal iliac artery of the same rabbit. Pairs of transferred genes into the fibroblasts were as follows: LacZ/LacZ (control group), VEGF/LacZ (VEGF group), bFGF/LacZ (FGF group) and VEGF/bFGF (combination group). Twenty-eight days after cell administration, collateral development and its function were evaluated. RESULTS: Calf blood pressure ratio, resting blood flow of the left iliac artery and capillary density of ischemic muscle showed similar degrees of angiogenic effects in the VEGF and FGF groups, which were significantly greater than those in the control group. On the contrary, angiographic score, collateral conductance and smooth muscle cell (SMC)-positive vessel density in the FGF group were significantly greater than those in the VEGF group. In the combination group, collateral conductance showed synergistic effects, and in vivo blood flow and smooth muscle cell-positive vessel density revealed additive effects of VEGF and bFGF. CONCLUSION: These findings suggested that bFGF-induced collateral development exceeded VEGF-induced collateral development in the induction of arteriogenesis, and that combined gene delivery of VEGF and bFGF produced additive or synergistic effects of collateral development as compared with the effects induced by transfer of each gene alone.

Adenoviridae↗

PubMed2004

The combination of SOX5, SOX6, and SOX9 (the SOX trio) provides signals sufficient for induction of permanent cartilage.

OBJECTIVE: To regenerate permanent cartilage, it is crucial to know not only the necessary conditions for chondrogenesis, but also the sufficient conditions. The objective of this study was to determine the signal sufficient for chondrogenesis. METHODS: Embryonic stem cells that had been engineered to fluoresce upon chondrocyte differentiation were treated with combinations of factors necessary for chondrogenesis, and chondrocyte differentiation was detected as fluorescence. We screened for the combination that could induce fluorescence within 3 days. Then, primary mesenchymal stem cells, nonchondrogenic immortalized cell lines, and primary dermal fibroblasts were treated with the combination, and the induction of chondrocyte differentiation was assessed by detecting the expression of the cartilage marker genes and the accumulation of proteoglycan-rich matrix. The effects of monolayer, spheroid, and 3-dimensional culture systems on induction by combinations of transcription factors were compared. The effects of the combination on hypertrophic and osteoblastic differentiation were evaluated by detecting the expression of the characteristic marker genes. RESULTS: No single factor induced fluorescence. Among various combinations examined, only the SOX5, SOX6, and SOX9 combination (the SOX trio) induced fluorescence within 3 days. The SOX trio successfully induced chondrocyte differentiation in all cell types tested, including nonchondrogenic types, and the induction occurred regardless of the culture system used. Contrary to the conventional chondrogenic techniques, the SOX trio suppressed hypertrophic and osteogenic differentiation at the same time. CONCLUSION: These data strongly suggest that the SOX trio provides signals sufficient for the induction of permanent cartilage.

Adult↗

PubMed2004

Distinct osteogenic mechanisms of bones of distinct origins.

Mammalian bones have three distinct origins (paraxial mesoderm, lateral plate mesoderm, and neural crest) and undergo two different modes of formation (intra-membranous and endochondral). Bones derived from the paraxial mesoderm and lateral plate mesoderm mainly form through the endochondral process. During this process, hypertrophic chondrocytes play a vital role in inducing both osteogenesis and angiogenesis. One of the essential osteogenic factors secreted from hypertrophic chondrocytes is Indian hedgehog (Ihh). In contrast, bones derived from the neural crest mainly form through the intramembranous pro-cess and do not require Ihh. Thus, depending on their origin, bones have distinct signaling properties, which need to be considered in the research and application of bone biology.

Animals↗

PubMed2004

Aortic wall cell proliferation via basic fibroblast growth factor gene transfer limits progression of experimental abdominal aortic aneurysm.

OBJECTIVE: Our previous study demonstrated that high flow conditions stimulated cell proliferation in the aortic wall in a rat model of abdominal aortic aneurysm (AAA), and we speculated that there is a possible relation between medial cell density and aortic wall integrity. In the present study we delivered the basic fibroblast growth factor (bFGF) gene to the aortic wall of a rat AAA model and evaluated the effects of growth factor-enhanced smooth muscle cell (SMC) proliferation on aneurysm progression. METHODS: AAA was induced in rats by means of infusion of porcine pancreatic elastase. Immediately after elastase infusion the abdominal aorta was filled with an expression plasmid vector containing the bFGF gene (bFGF group) or LacZ gene (control group); then gene transfer to the aortic wall was carried out with an in vivo electroporation method. The animals were killed 7 days after treatment, and the aneurysm was measured. The numbers of SMCs, macrophages, and endothelial cells were counted with immunostaining, and cell replication was evaluated with bromodeoxyuridine (BrdU) staining. RESULTS: Aneurysm diameter in the bFGF group was significantly smaller than that in the control group (4.6 +/- 0.3 mm vs 6.5 +/- 1.4 mm; P <.01). The numbers of medial SMCs and BrdU-incorporated cells in the bFGF group were significantly greater than those in the control group (SMC, 101 +/- 34 per high-power field [hpf] vs 80 +/- 31/hpf; P <.05, BrdU, 107 +/- 63/hpf vs 50 +/- 33/hpf; P <.05), whereas no difference was detected in the numbers of macrophages and endothelial cells between the 2 groups. CONCLUSIONS: Delivery of bFGF to the aortic wall induced significant enhancement of medial SMC proliferation, without an increase in inflammatory infiltration, then successfully limited aneurysm enlargement. These findings suggest that increased medial cellularity inhibits aneurysm formation, which possibly offers a clue for developing a new strategy for treatment of AAAs.

Animals↗

PubMed2004

Bone morphogenetic protein 2-induced osteoblast differentiation requires Smad-mediated down-regulation of Cdk6.

Because a temporal arrest in the G(1) phase of the cell cycle is thought to be a prerequisite for cell differentiation, we investigated cell cycle factors that critically influence the differentiation of mouse osteoblastic MC3T3-E1 cells induced by bone morphogenetic protein 2 (BMP-2), a potent inducer of osteoblast differentiation. Of the G(1) cell cycle factors examined, the expression of cyclin-dependent kinase 6 (Cdk6) was found to be strongly down-regulated by BMP-2/Smads signaling, mainly via transcriptional repression. The enforced expression of Cdk6 blocked BMP-2-induced osteoblast differentiation to various degrees, depending on the level of its overexpression. However, neither BMP-2 treatment nor Cdk6 overexpression significantly affected cell proliferation, suggesting that the inhibitory effect of Cdk6 on cell differentiation was exerted by a mechanism that is largely independent of its cell cycle regulation. These results indicate that Cdk6 is a critical regulator of BMP-2-induced osteoblast differentiation and that its Smads-mediated down-regulation is essential for efficient osteoblast differentiation.

Animals↗

PubMed2004

Absence of platelet-activating factor receptor protects mice from osteoporosis following ovariectomy.

While platelet-activating factor (PAF) is produced in various diseases associated with bone resorption, its functions in bone metabolism remain unknown. Using PAF receptor-deficient mice, we evaluated the role of PAF in the development of bone resorption following ovariectomy, a model of postmenopausal osteoporosis. Through observations of bone mineral density and histomorphometric parameters, it was found that bone resorption was markedly attenuated in PAF receptor-deficient mice, indicating that PAF links estrogen depletion and osteoporosis in vivo. Osteoclasts expressed higher amounts of the enzymes required for PAF biosynthesis than osteoblasts. TNF-alpha and IL-1beta increased the acetyl-coenzyme A:lyso-PAF acetyltransferase activity in osteoclasts. Osteoclasts, but not osteoblasts, expressed the functional PAF receptor. PAF receptor stimulation prolonged the survival of osteoclasts in vitro. Furthermore, osteoclasts treated with a PAF receptor antagonist, and also those from PAF receptor-deficient mice, showed reductions in survival rate and Ca resorption activity. Consistently, in organ cultures, bone resorption was significantly suppressed by a PAF receptor antagonist treatment or genetic PAF receptor deficiency. Thus, these results suggest that, through the inflammatory cytokines, estrogen depletion enhances PAF production as a unique autocrine factor for osteoclast functions. Inhibition of PAF function might pave the way for a new strategy to prevent postmenopausal bone loss without disturbing osteoblast functions.

Animals↗

PubMed2004

[Regenerative medicine of bone and cartilage].

Regenerative medicine, which takes advantage of the unique capacity of stem cells, is a novel medical strategy to cure irreversible organ failure. There are three important elements in regenerative medicine: cells, scaffolds and signals. Although substantial progress regarding each element has been made in the past few years, it still falls short of clinical applications. In the geriatric field, osteoporosis, osteoarthritis and periodontitis are the major targets of regenerative medicine. They are usually not life-threatening, but often severely affect QOL of elderly patients. Since elderly people have already reduced number of stem cells in bone and cartilage, we need to know the sufficient conditions for osteogenesis and chondrogenesis by comprehensively screening various conditions. We developed a marker gene system expressing green fluorescence protein (GFP) under the control of osteoblast- and chondrocyte-specific promoters. This system helps us monitor osteoblast and chondrocyte differentiation easily, precisely and non-invasively. Using this system, we are now trying to find the sufficient conditions for osteogenesis and chondrogenesis, and to discover osteogenic and chondrogenic small compounds.

Aged↗

PubMed2004

[The relationship between osteoporosis and oral diseases].

Few studies have been defined on the relationship between osteoporosis and oral diseases. The correlation among osteoporosis, periodontal diseases and dental loss is now well investigated. These investigations will lead to the elucidation of the osteoporotic bone diseases in the oral region. In this manuscript, the cases of oral diseases in elderly patients are shown.

Aged↗

PubMed2003

Cooperative roles of Fyn and cortactin in cell migration of metastatic murine melanoma.

Src family kinases are major regulators of various integrin-mediated biological processes, although their functional roles and substrates in cancer metastasis are unknown. We explored the roles of Src family tyrosine kinases in cell migration and the spread of K-1735 murine melanoma cell lines with low or high metastatic potential. Corresponding to elevated cell motility and spreading ability, Fyn was selectively activated among Src family kinases, and the cell motility was blocked by an inhibitor of Src family kinases. Significant tyrosine phosphorylation of cortactin, stable complex formation between activated Fyn and cortactin, and co-localization of cortactin with Fyn at cell membranes were all observed only in cells with high metastatic potential. Both integrin-mediated Fyn activation and hyperphosphorylation of cortactin were observed 2-5 h after stimulation in highly metastatic cells, and they required de novo protein synthesis. We demonstrate that cortactin is a specific substrate and cooperative effector of Fyn in integrin-mediated signaling processes regulating metastatic potential.

Animals↗

PubMed2003

Targeting of iNOS with antisense DNA plasmid reduces cytokine-induced inhibition of osteoblastic activity.

Proinflammatry cytokines, tumor necrosis factor-alpha combined with interleukin-1beta, induce excessive production of nitric oxide (NO) and its cytotoxic metabolite peroxynitrite (ONOO-) via inducible nitric oxide synthase (iNOS) in murine osteoblasts. In this study, to properly estimate the effects of antisense DNA of iNOS on osteoblastic activity, we produced transformed cell lines with antisense plasmid that specifically targets the iNOS gene for potential long-lasting inhibition. Transformed antisense cell lines were identified by 1) the detection of antisense transcripts, 2) the attenuated expression of iNOS protein, 3) the reduction of NO synthase activity, and 4) the level of NO production. These cell lines targeting iNOS, which showed decreased production of both NO and ONOO-, prevented the inhibition of osteoblastic differentiation as was assayed by the mRNA expression of type I collagen, alkaline phosphatase, osteocalcin, and Core binding factor in the presence of proinflammatory cytokines. Present results indicate that the antisense DNA plasmid of iNOS is potent to reduce the cytokine-induced inhibition of osteoblastic activity.

Alkaline Phosphatase↗

PubMed2003

Secondary correction of the wide nasal root in bilateral nasal deformity associated with cleft lip in five Oriental patients.

Secondary correction of bilateral nasal deformity associated with a cleft lip is common. However, few reports have referred to the correction of the wide nasal root. In this study we describe a technique other than osteotomy for the correction of the wide nasal root used in five Oriental patients with bilateral nasal deformity associated with cleft lip. Satisfactory results were obtained, and two representative cases are described.

Adolescent↗

PubMed2003

A randomized controlled trial of therapeutic exercise for clicking due to disk anterior displacement with reduction in the temporomandibular joint.

Therapeutic exercise is a new concept of treatment for patients with clicking due to anterior disk displacement with reduction (ADDWR). In order to investigate the efficacy of the exercise, we designed a randomized controlled clinical trial to compare with no-treatment controls. The subjects were patients who complained of painless unilateral reciprocal clicking and were diagnosed as having ADDWR by Magnetic Resonance Imaging (MRI). Forty-two patients were randomly assigned. Three months after the randomization, the success rate was 61.9% (13/21 joints) in the exercise group and 0% (0/21 joints) in the control group, with a significant difference between the two groups (p=0.0001). However, captured disks in the successful cases were seen in only 23.1% on MRI examination. In conclusion, the therapeutic exercise for clicking due to ADDWR is significantly effective in reducing the clicking, and thought to be much more conservative and cost-effective than splint therapy or surgery.

Adolescent↗

PubMed2003

Global expression analysis of N-methyl-N'-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide microarrays.

Rat stomach carcinomas induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are widely used as a model for differentiated-type human stomach carcinomas. Here, we analyzed expression profiles in five MNNG-induced rat stomach carcinomas by the high-density oligonucleotide microarray containing approximately 8000 probe sets. 244 and 208 genes were up- and down-regulated, respectively, by 3-fold and over in four or five carcinomas. Up-regulated genes included those involved in the extracellular matrix remodeling (i.e. Collagen types I, III, V, MMP3), immune response (i.e. lysozyme, complements) and in ossification (i.e. Osteoblast-specific factor). Genes down-regulated included those related to hydrocarbon metabolism (i.e. aldose A, aldehyde dehydrogenase), gastric juice (ion transporter genes) and mucous production (Mucin 5) and gastric hormones (gastrin and somatostatin). The expression profile of the MNNG-induced rat stomach carcinomas shared many features with human stomach carcinomas while cyclin D1 was down-regulated in rat stomach carcinomas but up-regulated in human stomach carcinomas. When the expression profile of the MNNG-induced rat stomach carcinomas was compared with those of two kinds of rat mammary carcinomas, only 13 genes were commonly altered. These results showed that MNNG-induced stomach carcinomas possessed infiltrating capacity and had lost differentiated phenotypes of the stomach, in the same way as human stomach carcinomas, and could be used as a good model for them from the viewpoint of molecular expression profile.

Animals↗

PubMed2002

Cell density and growth-dependent down-regulation of both intracellular calcium responses to agonist stimuli and expression of smooth-surfaced endoplasmic reticulum in MC3T3-E1 osteoblast-like cells.

A two-dimensional intracellular Ca(2+) ([Ca(2+)](i)) imaging system was used to examine the relationship between [Ca(2+)](i) handling and the proliferation of MC3T3-E1 osteoblast-like cells. The resting [Ca(2+)](i) level in densely cultured cells was 1.5 times higher than the [Ca(2+)](i) level in sparsely cultured cells or in other cell types (mouse fibroblasts, rat vascular smooth muscle cells, and bovine endothelial cells). A high resting [Ca(2+)](i) level may be specific for MC3T3-E1 cells. MC3T3-E1 cells were stimulated with ATP (10 microM), caffeine (10 mM), thapsigargin (1 microM), or ionomycin (10 microM), and the effect on the [Ca(2+)](i) level of MC3T3-E1 cells was studied. The percentage of responding cells and the degree of [Ca(2+)](i) elevation were high in the sparsely cultured cells and low in densely cultured cells. The rank order for the percentage of responding cells and magnitude of the Ca(2+) response to the stimuli was ionomycin > thapsigargin = ATP > caffeine and suggests the existence of differences among the various [Ca(2+)](i) channels. All Ca(2+) responses in the sparsely cultured MC3T3-E1 cells, unlike in other cell types, disappeared after the cells reached confluence. Heptanol treatment of densely cultured cells restored the Ca(2+) response, suggesting that cell-cell contact is involved with the confluence-dependent disappearance of the Ca(2+) response. Immunohistological analysis of type 1 inositol trisphosphate receptors and electron microscopy showed distinct expression of inositol trisphosphate receptor proteins and smooth-surfaced endoplasmic reticulum in sparsely cultured cells but reduced levels in densely cultured cells. These results indicate that the underlying basis of confluence-dependent [Ca(2+)](i) regulation is down-regulation of smooth-surfaced endoplasmic reticulum by cell-cell contacts.

3T3 Cells↗