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Biomedical subjects

Takashi Shimada

Publications and source records attributed to Takashi Shimada.

At least 55 records · Page 3Linked to original sources
PubMed2004

Chondrogenic and osteogenic differentiation of adipose-derived stem cells isolated from GFP transgenic mice.

Recent studies suggest that human adipose tissue contain pluripotent cells similar to bone marrow-derived stromal cells (BSCs). Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have previously demonstrated that BSCs differentiate into a variety of cell lineages both in vitro and in vivo). In the present study, we extend this approach to characterize adipose-derived stromal cells (ASCs)). These cells derived from human are sometimes called processed lipoaspirate (PLA) cells. ASCs were prepared from inguinal fat pads of GFP transgenic mice after extensive washing with PBS and treatment with collagenase. After the primary culture in control medium (DMEM + 10% FBS), the cells were incubated in either chondrogenic medium (DMEM + 1% FBS + insulin + ascorbate 2-phosphate + TGF-beta 1) or osteogenic medium (DMEM + 10%FBS + dexamethasone + ascorbate-2-phosphate + beta-glycerophosphate) for two to four weeks. Chondrogenic differentiation was assessed by Alcian blue staining, while osteogenic differentiation was by von Kossa and Alkaline phosphatase staining. ASCs incubated in chondrogenic medium induced Alcian blue positive cells. Incubation with osteogenic medium became positive for von Kossa and Alkaline phosphatase staining. No osteochondrogenic differentiation was observed in cells incubated with control medium. This cell population can be easily identified through fluorescence microscope, it should be an ideal source of ASCs for further experiments of stem cell biology and tissue engineering.

Adipocytes↗
PubMed2004

Fibroblast growth factor (FGF)-23 in patients with primary hyperparathyroidism.

OBJECTIVE: We aimed to determine the serum level of fibroblast growth factor-23 (FGF-23) in patients with primary hyperparathyroidism (pHPT) to understand its physiological role in the disorder. PATIENTS AND METHODS: Ninety-eight patients with pHPT who underwent parathyroidectomy formed the study group. We also measured serum FGF-23 in 11 of these patients on postoperative day 6. RESULTS: Serum FGF-23 levels was significantly higher in pHPT patients than in healthy controls (35.6+/-17.8 ng/l vs 28.9+/-11.2 ng/l (mean+/-s.d.); P<0.001 (Pearson's correlation coefficient)), but there was no significant difference in the serum FGF-23 level between pHPT patients with normal renal function (creatinine clearance (Ccr) of >or=70 ml/min) and healthy controls. Serum FGF-23 correlated positively with serum calcium (P<0.0001) and intact parathyroid hormone (PTH) (P<0.01), and negatively with Ccr (P<0.001), serum phosphate (P<0.05), and serum 1,25-dihydroxyvitamin D (1,25(OH)(2)D) (P<0.05). Multiple linear regression analysis of factors potentially determining serum FGF-23 levels in pHPT patients showed serum calcium (P<0.01) and Ccr (P<0.001) to be significant predictors. The serum levels of FGF-23 did not change after parathyroidectomy despite the normalization of serum calcium values. Multiple linear regression analysis revealed that serum FGF-23 was not a significant predictor of serum phosphate or 1,25(OH)(2)D in pHPT patients. CONCLUSIONS: FGF-23 may not play a significant role in regulating phosphate or 1,25(OH)(2)D in pHPT patients, especially in those with normal renal function. Further studies are warranted to determine the role of FGF-23 in renal insufficiency or failure.

Adult↗
PubMed2004

Effects of a fermented vegetable product on fat deposition and bone metabolism in ovariectomized rats.

We examined the effects of a fermented vegetable product (FVP), fermented by yeast as well as lactic acid bacteria, on body weight and fat deposition after ovariectomy in 10-wk-old Sprague-Dawley rats. The rats were divided into the following 5 groups: (1) ovari-ectomized rats fed as usual (OVX); (2) ones receiving FVP at a daily dose of 100 mg/kg (LOW); (3) ones receiving FVP at a daily dose of 1000 mg/kg (HIGH); (4) ones receiving a calcium-free diet (Ca-FREE); (5) sham-operated rats (SHAM). A slight increase in body weight was observed in the LOW group compared with the OVA group. The rate of increase in total fat content was lowest in the LOW group, but both bone weight and strength were similar to those in the other ovariectomized groups. In conclusion, the low dose of FVP turned out to reduce fat content without affecting bone weight and strength.

Adipose Tissue↗
PubMed2003

FGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis.

UNLABELLED: We analyzed the effects of an FGF-23 injection in vivo. FGF-23 caused a reduction in serum 1,25-dihydroxyvitamin D by altering the expressions of key enzymes for the vitamin D metabolism followed by hypophosphatemia. This study indicates that FGF-23 is a potent regulator of the vitamin D and phosphate metabolism. INTRODUCTION: The pathophysiological contribution of FGF-23 in hypophosphatemic diseases was supported by animal studies in which the long-term administration of recombinant fibroblast growth factor-23 reproduced hypophosphatemic rickets with a low serum 1,25-dihydroxyvitamin D [1,25(OH)2D] level. However, there is no clear understanding of how FGF-23 causes these changes. MATERIALS AND METHODS: To elucidate the molecular mechanisms of the FGF-23 function, we investigated the short-term effects of a single administration of recombinant FGF-23 in normal and parathyroidectmized animals. RESULTS: An injection of recombinant FGF-23 caused a reduction in serum phosphate and 1,25(OH)2D levels. A decrease in serum phosphate was first observed 9 h after the injection and was accompanied with a reduction in renal mRNA and protein levels for the type IIa sodium-phosphate cotransporter (NaPi-2a). There was no increase in the parathyroid hormone (PTH) level throughout the experiment, and hypophosphatemia was reproduced by FGF-23 in parathyroidectomized rats. Before this hypophosphatemic effect, the serum 1,25(OH)2D level had already descended at 3 h and reached the nadir 9 h after the administration. FGF-23 reduced renal mRNA for 25-hydroxyvitamin D-1alpha-hydroxylase and increased that for 25-hydroxyvitamin D-24-hydroxylase starting at 1 h. In addition, an injection of calcitriol into normal mice increased the serum FGF-23 level within 4 h. CONCLUSIONS: FGF-23 regulates NaPi-2a independently of PTH and the serum 1,25(OH)2D level by controlling renal expressions of key enzymes of the vitamin D metabolism. In conclusion, FGF-23 is a potent regulator of phosphate and vitamin D homeostasis.

Animals↗
PubMed2003

Cardiomyocyte regeneration from circulating bone marrow cells in mice.

We investigated the role of circulating bone marrow cells (BMC) in cardiomyocyte regeneration. BMC, isolated from transgenic mice expressing enhanced green fluorescent protein (GFP), were transplanted into lethally irradiated C57BL6 mice. Five weeks after bone marrow transplantation (BMT), flow cytometric analysis for GFP-positive cells confirmed reconstitution of transplanted bone marrow. Bone marrow transplant mice subsequently underwent left coronary artery ligation (myocardial infarction) or sham-operation, and were killed at 1 mo or 3 mo after operation. Infarct size was similar in bone marrow transplant mice at 1 mo (47.1 +/- 5.9%) and at 3 mo (45.3 +/- 7.8%), and echocardiography at 2 and 8 wk revealed decreasing left ventricular function. In infarcted heart, GFP-positive cells that expressed desmin and troponin T-C were identified by confocal microscopy. GFP and troponin T-C double-positive cells were predominantly in the peri-infarcted region (1 mo, 365 +/- 45 cells/50 sections; 3 mo: 458 +/- 100 cells/50 sections; p < 0.05 versus noninfarct, infarct, and sham-operated regions). Furthermore, BMC mobilization and differentiation into cardiomyocytes was found to be complete within 1 mo after myocardial infarction. These results demonstrate that circulating BMC undergo mobilization and differentiation in cardiac cells after myocardial infarction. Future studies are required to determine the molecular signaling mechanisms responsible for this phenomenon.

Animals↗
PubMed2003

Establishment of modified chimeric mice using GFP bone marrow as a model for neurological disorders.

Chimeric mice stably reconstituted with bone marrow cells represent a good model for analysis of the mechanism of bone marrow cell infiltration in the brain. However, in preparing chimeric mice, irradiation of the recipient mice is necessary to kill their own bone marrow before transplantation, which induces gliosis and inflammatory response by activation of astrocytes and microglia in the brain. Here, we determined the most suitable dose of irradiation associated with the least brain damage before transplantation for reconstitution of chimeric mice, using FACS analysis. Our mouse model of 10 Gy body/5 Gy head irradiation should be useful for investigating the mechanism(s) of microglial activation in various neurological disorders such as stroke, Alzheimer's disease and Parkinson's disease.

Animals↗
PubMed2003

Myeloproliferative disease in transgenic mice expressing P230 Bcr/Abl: longer disease latency, thrombocytosis, and mild leukocytosis.

P230 Bcr/Abl has been associated with indolent myeloproliferative disease (MPD). We generated transgenic mice expressing P230Bcr/Abl driven by the promoter of the long terminal repeat of the murine stem cell virus of the MSCV neo P230 BCR/ABL vector. Two founder mice exhibited mild granulocytosis and marked thrombocytosis and developed MPD. The disease of one founder mouse, no. 13, progressed to extramedullary myeloblastic crisis in the liver at 12 months old. The other founder mouse, no. 22, was found to have chronic-phase MPD with large populations of megakaryocytes and granulocytes in an enlarged spleen. The transgenic progeny of no. 22 clearly exhibited MPD at 15 months old. These results showed that P230Bcr/Abl had leukemogenic properties and induced MPD. The phenotype of the MPD caused by P230Bcr/Abl was characterized by mild granulocytosis, a high platelet count, infiltration of megakaryocytes in some organs, and a longer disease latency compared with the MPD caused by P210Bcr/Abl.

Animals↗
PubMed2003

Generation of a chimeric mouse reconstituted with green fluorescent protein-positive bone marrow cells: a useful model for studying the behavior of bone marrow cells in regeneration in vivo.

Studies have indicated that bone marrow contains both hematopoietic stem cells and mesenchymal stem cells that can differentiate into a variety of mesenchymal tissues, such as bone, cartilage, muscle, and adipose tissue. Therefore, bone marrow cells are thought to be very useful for cell and gene therapy for various diseases. However, the multipotentiality of these cells remains unclear. To address this issue, we established a chimeric model mouse stably reconstituted with green fluorescent protein (GFP)-marked bone marrow cells. We injected bone marrow cells from GFP-transgenic C57BL/6 mice into the tail veins of recipient wild-type C57BL/6 mice that had been irradiated with a lethal dose of 10 Gy from a cesium source. Microscopic examination and fluorescence-assisted cell sorter (FACS) analysis showed that bone marrow cells, including mesenchymal cells, were almost completely reconstituted with GFP+ cells 5 weeks after transplantation. FACS analysis with lineage-specific antibodies confirmed that the GFP+ cells could differentiate into all types of blood cells. To confirm the usefulness of this mouse model, we studied the role of circulating bone marrow-derived cells in healing of damaged intestine. We performed amputation and anastomosis of the jejunum 10 cm from the pyloric region of the stomach. On the third day after operation, a large number of GFP+ cells were infiltrated in the area of anastomosis, and these cells were positive for CD45 and F4/80 antigens. In 7 days, several cells became negative for CD45 and F4/80 and positive for alpha smooth muscle actin antigen, which is specific for smooth muscle. This finding suggested that bone marrow-derived cells had differentiated into smooth muscle. Because reconstituted bone marrow cells as opposed to injected bone marrow cells, behave naturally, this model is ideal for studying the multipotentiality of bone marrow cells in vivo.

Animals↗
PubMed2003

Bone formation following transplantation of genetically modified primary bone marrow stromal cells.

Bone marrow stromal cells contain mesenchymal stem cells that can differentiate into a variety of mesenchymal tissues; in the presence of BMP-2, for example, they differentiate into osteoblasts. We constructed replication-deficient adenoviral vectors encoding human BMP-2 (BMP-2/Ad) or BMP-4 (BMP-4/Ad) and used them to transduce primary bone marrow stromal cells from the femurs of four-week-old female C3H mice, which then expressed and processed functional BMP-2 or BMP-4 protein. Enzyme assays and histochemical staining showed both groups of cells to possess alkaline phosphatase activity, a marker of differentiation into osteoblasts, though the activity was higher in cells transduced with BMP-2/Ad. When BMP-2/Ad-transduced cells were injected into the thigh muscles of immunocompetent C3H mice, ossicle development was detected on radiographs within four weeks after injection. Moreover, histological analysis indicated that newly developed ossicles contain mature osseous components, including cortical bone and bone marrow, within eight weeks. Thus, syngeneic transplantation of genetically modified primary bone marrow stromal cells induced bone formation in immunocompetent mice, perhaps indicating its potential for use in the development of therapeutic protocols aimed at enhancing bone formation.

Adenoviridae↗
PubMed2003

Lentivirus-mediated expression of angiostatin efficiently inhibits neovascularization in a murine proliferative retinopathy model.

Ischemic retinal diseases, such as diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration, are a major cause of blindness worldwide. Angiostatin is an internal peptide fragment of plasminogen that inhibits endothelial proliferation in vitro and tumor growth in vivo. We now demonstrate that HIV vector encoding angiostatin (HIV-angiostatin) can inhibit retinal neovascularization in a mouse model of proliferative retinopathy. Intravitreal injections of HIV-angiostatin led to stable expression of the angiostatin gene in retinal tissue. Retinal neovascularization was histologically quantitated by a masked protocol. Retinal neovascularization in the eye injected with HIV-angiostatin was reduced in 90% (9/10; P=0.025) of animals, compared with the eye injected with phosphate-buffered saline. Reduction of histologically evident neovascular nuclei per 6-microm section averaged 68%, with maximal inhibitory effects of 87%. Neovascularization was not reduced in the eyes injected with HIV vector encoding enhanced green fluorescent protein. This is the first report that HIV-angiostatin can reduce neovascular cell nuclei in a murine proliferative retinopathy model. These data suggest that the anti-angiogenic activity of angiostatin has therapeutic potential for the treatment of retinal neovascularization.

Angiostatins↗
PubMed2003

Amplification, up-regulation and over-expression of DVL-1, the human counterpart of the Drosophila disheveled gene, in primary breast cancers.

Wnt proteins form a family of highly conserved, secreted signaling molecules that regulate cell-to-cell interactions during embryogenesis. Wnt genes and Wnt signaling are also implicated in cancer. It has been shown that Wnt proteins bind to receptors of the frizzled family on the cell surface. Through several cytoplasmic relay components including DVL-1, the human counterpart of the Drosophila disheveled gene, the signal is transduced to beta-catenin, which then enters the nucleus and forms a complex with T-cell factor (TCF) to activate transcription of Wnt target genes. We describe here the amplification of DVL-1 in 13 of 24 primary breast cancers examined, and increased expression of this gene in 11 of those tumors in comparison to corresponding non-cancerous breast tissues. Immunohistochemical staining demonstrated that DVL-1 protein was prominent in the cytoplasm of cancer cells, but not in normal epithelial cells of the mammary duct or in myoepithelial cells. These data indicate that amplification and increased expression of the DVL-1 gene may play some role in human breast carcinogenesis through derangement of the Wnt signaling pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Genetic diagnosis of Werdnig-Hoffmann disease: a problem for application to prenatal diagnosis.

We report a floppy infant with Werdnig-Hoffmann disease (spinal muscular atrophy: SMA type 1) and Klinefelter syndrome. After genetic counseling with parents, a genetic diagnosis using DNA from the infant's peripheral blood mononuclear cells was performed. The parents' deletion of exons 7 and 8 of the survival motor neuron (smn) gene and exons 4 and 5 of the neuronal apoptosis inhibitory protein (naip) gene were noted in the infant, so he was confirmed to have SMA type 1. The parents wanted to receive a prenatal diagnosis on the next pregnancy. However this genetic test is achieved by confirming that a specific band can not be detected by PCR. Therefore, this method should be applied with great care to prenatal diagnosis using chorionic villi, which may be contaminated with maternal tissue.

Cyclic AMP Response Element-Binding Protein↗