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Biomedical subjects

Yoshikazu Yonemitsu

Publications and source records attributed to Yoshikazu Yonemitsu.

At least 19 recordsLinked to original sources
PubMed2006

Sprouty2 and Sprouty4 are essential for embryonic morphogenesis and regulation of FGF signaling.

Sprouty genes encode cytoplasmic membrane-associated proteins that inhibit receptor tyrosine kinase signaling. Four orthologs of Drosophila Sprouty (dSpry) (Sprouty1-4) have been identified in mammals. Physiological function of Sprouty1 and Sprouty2 has been investigated using gene targeting approaches, however to date detailed examination of Sprouty4 knockout (KO) mice has not been reported. In this study, Sprouty4 KO mice were generated and characterized. Although a significant fraction of Sprouty4 KO mice died shortly after birth due to mandible defects, the remainder were viable and fertile. Growth retardation was observed for most Sprouty4-deficient mice, with nearly all Sprouty4 KO mice having polysyndactyly. ERK activation was sustained in Sprouty4 KO mouse embryonic fibroblasts (MEFs) in response to FGF, but not to EGF. Sprouty2 and Sprouty4 double KO (DKO) mice were embryonic lethal and showed severe defects in craniofacial, limb, and lung morphogenesis. These findings suggest both redundant and non-redundant functions for Sprouty2 and Sprouty4 on embryonic development and FGF signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Critical roles of memory T cells and antidonor immunoglobulin in rejection of allogeneic bone marrow cells in sensitized recipient mice.

BACKGROUND: Allosensitization is a major risk factor for graft failure in clinical bone marrow transplantation, even with an human leukocyte antigen (HLA)-matched combination under radiation-based conditioning regimens. The critical components of immunological memory in donor bone marrow graft rejection in allosensitized hosts remain unclear at present. METHODS: C57BL/6-recipient mice, which had been intraperitoneally injected with splenocytes from donor C3H mice on day -35 (sensitized recipients), had been lethally irradiated with 10-Gy whole-body irradiation and were intravenously injected with T-cell-depleted bone marrow cells (TCD-BMC) from C3H mice or third-party SJL mice. RESULTS: Lethally irradiated recipient mice, which had been sensitized by donor splenocytes 5 weeks before the transplantation of TCD-BMC, completely rejected the donor-BMC in a donor-specific manner, whereas none of the nonsensitized recipient mice, all of which showed full allogeneic chimerism, rejected the donor TCD-BMC. Antibody-mediated T cell and/or Natural Killer (NK) cell depletion did not improve the ability of the sensitized recipients to overcome the rejection even when a megadose of TCD-BMC was administered to the sensitized recipients. Furthermore, BMC rejection occurred in sensitized B cell-deficient mice. In adoptive transfer experiments, naive mice, which received a transfer of purified T cells from sensitized mice, rejected the donor BMC, but not those from nonsensitized mice. Moreover, naive mice, which received a transfer of serum containing antidonor immunoglobulin, rejected the donor BMC. CONCLUSIONS: These findings suggest that alloreactive memory T cells and antidonor immunoglobulin independently function to reject donor BMC in sensitized recipients.

Adoptive Transfer↗
PubMed2006

Induction of efficient antitumor immunity using dendritic cells activated by recombinant Sendai virus and its modulation by exogenous IFN-beta gene.

Dendritic cell (DC)-based cancer immunotherapy has been paid much attention as a new and cancer cell-specific therapeutic in the last decade; however, little clinical outcome has been reported. Current limitations of DC-based cancer immunotherapy include sparse information about which DC phenotype should be administered. We here report a unique, representative, and powerful method to activate DCs, namely recombinant Sendai virus-modified DCs (SeV/DC), for cancer immunotherapy. In vitro treatment of SeV without any bioactive gene solely led DCs to a mature phenotype. Even though the expression of surface markers for DC activation ex vivo did not always reach the level attained by an optimized amount of LPS, superior antitumor effects to B16F1 melanoma, namely tumor elimination and survival, were obtained with use of SeV-GFP/DC as compared with those seen with LPS/DC in vivo, and the effect was enhanced by SeV/DC-expressing IFN-beta (SeV-murine IFN-beta (mIFN-beta)/DC). In case of the treatment of an established tumor of B16F10 (7-9 mm in diameter), a highly malignant subline of B16 melanoma, SeV-modified DCs (both SeV-GFP/DC and SeV-mIFN-beta/DC), but not immature DC and LPS/DC, dramatically improved the survival of animals. Furthermore, SeV-mIFN-beta/DC but not other DCs could lead B16F10 tumor to the dormancy, associated with strongly enhanced CD8+ CTL responses. These results indicate that rSeV is a new and powerful tool as an immune booster for DC-based cancer immunotherapy that can be significantly modified by IFN-beta, and SeV/DC, therefore, warrants further investigation as a promising alternative for cancer immunotherapy.

Adoptive Transfer↗
PubMed2006

Nonendothelial mesenchymal cell-derived MCP-1 is required for FGF-2-mediated therapeutic neovascularization: critical role of the inflammatory/arteriogenic pathway.

OBJECTIVE: Monocyte chemoattractant protein-1 (MCP-1) is a C-C chemokine that is known as an inflammatory/arteriogenic factor. Angiogenesis contributes to the inflammatory process; however, the molecular and cellular mechanisms of the links among the inflammatory pathway, arteriogenesis, and angiogenesis have not been well elucidated. METHODS AND RESULTS: Using murine models of fibroblast growth factor-2 (FGF-2)-mediated therapeutic neovascularization, we here show that FGF-2 targets nonendothelial mesenchymal cells (NEMCs) enhancing both angiogenic (vascular endothelial growth factor [VEGF]) and arteriogenic (MCP-1) signals via independent signal transduction pathways. Severe hindlimb ischemia stimulated MCP-1 expression that was strongly enhanced by FGF-2 gene transfer, and a blockade of MCP-1 activity via a dominant negative mutant as well as a deficiency of its functional receptor CCR2 resulted in the diminished recovery of blood flow attributable to adaptive and therapeutic neovascularization. Tumor necrosis factor (TNF)-alpha stimulated MCP-1 expression in all cell types tested, whereas FGF-2-mediated upregulation of MCP-1 was found only in NEMCs but not in others, a finding that was not affected by VEGF in vitro and in vivo. CONCLUSIONS: These results indicate that FGF-2 targets NEMCs independently, enhancing both angiogenic (VEGF) as well as inflammatory/arteriogenic (MCP-1) pathways. Therefore, MCP-1/CCR2 plays a critical role in adaptive and FGF-2-mediated therapeutic neovascularization.

Adaptation, Physiological↗
PubMed2006

The regulation of vascular endothelial growth factors (VEGF-A, -C, and -D) expression in the retinal pigment epithelium.

The vascular endothelial growth factor (VEGF) family plays an essential role in vascular development, angiogenesis and lymphangiogenesis. VEGF-A is a key regulator of endothelial cell functions and VEGF-C and VEGF-D are known to stimulate both angiogenesis and lymphangiogenesis. In a surgically removed subretinal vascular membrane of an age-related macular degeneration (AMD) patient, both VEGF-C and VEGF-D were confirmed, in addition to VEGF-A, to be markedly positive in the retinal pigment epithelium (RPE). There is no lymph vessel in ocular tissue, so it is possible that VEGF-C and VEGF-D expression in the RPE play some role in ocular angiogenesis, as well as VEGF-A. Next, we assessed the transition of VEGF-A, -C, and -D expression on several conditions, in human RPE. Hypoxia proverbially induced VEGF-A mRNA expression, meanwhile VEGF-C and VEGF-D mRNA expression was down-regulated. The Ca(2+) deprivation from culture medium strongly up-regulated VEGF-A and VEGF-D mRNA expression. Culture on plastic flasks precoated with poly-2-hydroxyethyl methacrylate up-regulated VEGF-D expression. Meanwhile, no significant change of VEGF-C mRNA expression was found in the blockade of cell-cell and/or cell-matrix adhesion. These findings suggest the possibility that VEGF-C and VEGF-D expression in RPE modify the ocular angiogenesis as angiogenic stimulators.

Aged↗
PubMed2006

Sphere formation of ocular epithelial cells in the ciliary body is a reprogramming system for neural differentiation.

It is well known that neural stem/progenitor cells of the central nervous system (CNS) can proliferate to form neurospheres (CNS-neurospheres) that are positive for nestin, an intermediate filament for neural progenitors. Retinal stem/progenitor properties were also isolated from the ciliary body (CB) of the eye where, as in the CNS, such stem/progenitors also form spheres and have been considered to expand only via expansion by their proliferation even from the single-cell level (called spheres of pigment cells from the ciliary margin: PCM-spheres). We here found a new and distinct process underlying the growth of CB cell-derived spheres (CB-spheres) that is unlike the mechanism of CNS- and PCM-sphere expansion; this new process is a cell proliferation-independent incorporation of neighbor spheres and cells cultured at high density (200 cells/mul). The majority of cells in CB-spheres consisted of nestin-negative epithelia-like cells and started to express nestin during the course of their expansion by high-density cultivation. The growth of CNS-neurospheres was sensitive to a cell-cycle inhibitor, whereas the growth of CB-spheres was not seriously affected by cell proliferation; rather, the spheres grew by incorporating other CB-spheres and nestin-negative adherent cells, the latter of which started to express nestin and lost the expression of epithelial markers after being incorporated. These results indicate that CB-spheres do not form by the accumulation of neural progenitors but rather by a reprogramming system from epithelia-like cells for neural differentiation, a clearly distinct mechanism from sphere formation by single-cell expansion of retinal stem/progenitor populations.

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

[How to design the translational research--special reference to gene therapy products].

The previous borders among basic research and clinical research are bridged by the system commonly termed as 'translational research(TR)'. TR encompasses i) the guidelines for drug development or for the identification of clinically relevant biomarkers; ii) experimental nonhuman studies conducted to show the 'proof of concept' for new therapeutic strategies; iii) initial clinical investigations for the development of new therapies; and iv) extensive clinical trial initiated in accordance with the above goals. Unfortunately, even though the current efforts by physicians and pharmaceutical companies, there are a number of hurdles that delay TR, especially in Japan.

Animals↗
PubMed2005

Diabetic microangiopathy in ischemic limb is a disease of disturbance of the platelet-derived growth factor-BB/protein kinase C axis but not of impaired expression of angiogenic factors.

Diabetic foot is caused by microangiopathy and is suggested to be a result of impaired angiogenesis. Using a severe hindlimb ischemia model of streptozotocin-induced diabetic mice (STZ-DM), we show that diabetic foot is a disease solely of the disturbance of platelet-derived growth factor B-chain homodimer (PDGF-BB) expression but not responses of angiogenic factors. STZ-DM mice frequently lost their hindlimbs after induced ischemia, whereas non-DM mice did not. Screening of angiogenesis-related factors revealed that only the expression of PDGF-BB was impaired in the STZ-DM mice on baseline, as well as over a time course after limb ischemia. Supplementation of the PDGF-B gene resulted in the prevention of autoamputation, and, furthermore, a protein kinase C (PKC) inhibitor restored the PDGF-BB expression and also resulted in complete rescue of the limbs of the STZ-DM mice. Inhibition of overproduction of advanced-glycation end product resulted in dephosphorylation of PKC-alpha and restored expression of PDGF-BB irrespective of blood sugar and HbA1c, indicating that advanced-glycation end product is an essential regulator for PKC/PDGF-BB in diabetic state. These findings are clear evidence indicating that diabetic vascular complications are caused by impairment of the PKC/PDGF-B axis, but not by the impaired expression of angiogenic factors, and possibly imply the molecular target of diabetic foot.

Angiogenesis Inducing Agents↗
PubMed2005

A free radical scavenger but not FGF-2-mediated angiogenic therapy rescues myonephropathic metabolic syndrome in severe hindlimb ischemia.

The therapeutic use of angiogenic factors shows promise in the treatment of critical limb ischemia; however, its potential for myonephropathic metabolic syndrome (MNMS), a fatal complication caused by arterial reconstruction, has not been elucidated. The objective of this study was to evaluate the effectiveness of recombinant Sendai virus-mediated gene transfer of fibroblast growth factor-2 (FGF-2) directly compared with that of a radical scavenger, MCI-186, in a rat model of MNMS. MNMS was surgically induced by aortic occlusion below renal arteries for 4 h, followed by 6 h of reperfusion. Administration of MCI-186 (twice; iv 5 min before induced ischemia and ip 5 min before reperfusion; 10 mg/kg, respectively), but not FGF-2 gene transfer (once, 48 h before induced ischemia), dramatically prevented the increase of serum biochemical markers as well as the edema of the gastrocnemius muscle. The effect of MCI-186 was accompanied by the marked suppression of the neutrophilic infiltration into the local (muscle) and remote (lung) organs. Although serum and muscular levels of a neutrophil-chemoattractant (growth-related oncogene/cytokine-induced neutrophil chemoattractant-1) were not affected by any treatment, the serum level of soluble intercellular adhesion molecule-1 was decreased by treatment with MCI-186 but not by treatment with FGF-2. These results suggest the distinct mechanism of MNMS from critical limb ischemia without reperfusion. Therefore, radical scavenging should be paid more attention than therapeutic angiogenesis when arterial circulation is reconstructed.

Angiogenesis Inducing Agents↗
PubMed2005

Platelet-derived growth factor-AA is an essential and autocrine regulator of vascular endothelial growth factor expression in non-small cell lung carcinomas.

It is widely accepted that angiogenesis is required for tumor progression. Vascular endothelial growth factor (VEGF) is a key molecule for tumor angiogenesis; however, its expressional regulation is not well understood during all stages of tumorigenesis. Using cell lines and surgical specimens of human non-small cell lung cancers (NSCLCs), we here show that platelet-derived growth factor-AA (PDGF-AA) is an essential autocrine regulator for VEGF expression. To directly assess the expression of PDGF-AA-dependent VEGF and its roles in tumorigenesis, we stably transfected established cell lines with their antisense genes. In addition, the levels of PDGF-AA and VEGF expression in surgical sections were measured and compared with clinicopathologic findings such as tumor size and patient prognosis. PDGF-AA tightly regulated VEGF expression and had a greater effect on tumor size and patient prognosis than did VEGF in both cell lines and surgical sections. PDGF-AA expression was not seen in the atypical adenomatous hyperplasia at all, whereas VEGF was occasionally seen. Furthermore, the frequency of VEGF expression was higher in advanced NSCLCs than in precancerous lesions, which was tightly correspondent to the results for PDGF-AA. These results indicate that PDGF-AA is an important regulator of the frequency and level of VEGF expression during the transition from a precancerous lesion to advanced cancer. The PDGF-AA/VEGF axis, therefore, may be a ubiquitous autocrine system for enhancing angiogenic signals, and PDGF-AA, and its related pathways could be a more efficient target of antiangiogenic therapy for cancers than VEGF and its pathways.

Animals↗
PubMed2005

Cytoplasmic expression and extracellular deposition of an antiangiogenic factor, pigment epithelium-derived factor, in human atherosclerotic plaques.

OBJECTIVE: To assess the expression and distribution of a neurotrophic/antiangiogenic factor, pigment epithelium-derived factor (PEDF), related to angiogenesis that is a possibly key event during atherogenesis in human atherosclerotic plaques. METHODS AND RESULTS: Twenty fresh aortic samples were used for reverse-transcription polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry (IHC). In addition, 80 stocked paraffin blocks of coronary arteries from 40 autopsy cases were also used. IHC revealed divergent staining patterns for PEDF in both the aortas and the coronary arteries tested, ie, "cytoplasmic staining" or "extracellular deposition," were observed, respectively. In the areas showing cytoplasmic staining, double PEDF was expressed in a majority of the foamy macrophages and in some smooth muscle cells, and the PEDF-positive cell frequency was positively correlated with that of microvessels in a cell-rich area in the coronary arteries (P<0.0001). Inversely, extracellular deposition of PEDF was seen in acellular areas and was negatively correlated with the number of microvessels (P=0.0003). CONCLUSIONS: These results suggest that PEDF may function as an antiangiogenic factor when it is deposited onto the extracellular matrix. Thus, PEDF may play a significant role in determining the balance of angiogenesis/ antiangiogenesis during atherogenesis.

Adult↗
PubMed2005

Angiogenesis and lymphangiogenesis and expression of lymphangiogenic factors in the atherosclerotic intima of human coronary arteries.

Little information regarding the development of lymphangiogenesis in coronary atherosclerosis is available. We immunohistochemically investigated the correlation among intimal neovascularization (CD34 for angiogenesis and lymphatic vessel endothelial hyaluronan receptor-1 [LYVE-1] and podoplanin for lymphangiogenesis), the expression of lymphangiogenic factors (vascular endothelial growth factor [VEGF]-C and VEGF-D), and the progression of atherosclerosis using 169 sections of human coronary arteries from 23 autopsy cases. The more the atherosclerosis advanced, the more often the neointimas contained newly formed blood vessels ( P < .0001). Vascular endothelial growth factor-C was expressed mostly in foamy macrophages and in some smooth muscle cells, whereas VEGF-D was abundantly expressed in both. The number of VEGF-C-expressing cells, but not that of VEGF-D-expressing cells, was increased as the lesion advanced and the number of intimal blood vessels increased ( P < .01). Lymphatic vessels were rare in the atherosclerotic intima (LYVE-1 vs CD34 = 13 vs 3955 vessels) compared with the number seen in the adventitia (LYVE-1 vs CD34 = 360 vs 6921 vessels). The current study suggests that VEGF-C, but not VEGF-D, may contribute to plaque progression and be a regulator for angiogenesis rather than lymphangiogenesis in coronary atherosclerotic intimas. Imbalance of angiogenesis and lymphangiogenesis may be a factor contributing to sustained inflammatory reaction during human coronary atherogenesis.

Aged↗
PubMed2005

The use of transformed Escherichia coli for experimental angiogenesis induced by regulated in situ production of vascular endothelial growth factor--an alternative gene therapy.

BACKGROUND: Defects in angiogenesis (blood vessel formation) are responsible for two most important causes of death in developed countries (ischemic heart disease and cancer). Vascular endothelial growth factor (VEGF) plays a pivotal role in physiological and pathological regulation of angiogenesis. In the last years several studies have indicated the possibilities of VEGF in the therapy of ischemic heart disease. However, especially VEGF gene therapy (naked DNA, plasmids and adenovirus mediated) is associated with adverse side effects regarding the expression regulation. AIM: To prepare bacterial strains producing VEGF using plasmids containing the VEGF cDNA for the use in experimental angiogenesis. METHODS AND RESULTS: Escherichia coli strain BL21(DE3) was transformed with Bluescript vector containing the inserts with cDNA sequences coding VEGF-A isoforms (VEGF121, VEGF164, VEGF189). Selection of recombinants was achieved by cultivating E. coli cells on ampicillin-added medium. The expression of target genes in the T7 expression system was induced by isopropyl-beta-D-thiogalactoside (IPTG). Polyacrylamide gel electrophoresis of the cell lysates showed the presence of polypeptides of molecular weight corresponding with known values of VEGF isoforms. Blood vessel formation induced by bacterial VEGF production was proved in vivo in mice seven days after intraperitoneal injection of transformed bacteria by light microscopy. CONCLUSION AND HYPOTHESIS: In summary, E. coli strain expressing VEGF was prepared and its biological effect confirmed. Bacteria, which produce angiogenic factors, provide a new modality for experimental angiogenesis and may be also suitable for clinical use. The in situ production of therapeutic proteins using optimalized prokaryotic expression systems can represent a useful tool for treatment based on molecular biomedicine. The main advantage of the described approach lies in the enhanced regulation control--bacterial expression can be regulated positively (induction by exogenous low molecular weight agents) and negatively (application of antibiotics). The hypothesis of alternative gene therapy should be proved in further studies.

Adenoviridae↗
PubMed2005

The effects of flavoxate hydrochloride on voltage-dependent L-type Ca2+ currents in human urinary bladder.

The effects of flavoxate hydrochloride (Bladderon, piperidinoethyl-3-methylflavone-8-carboxylate; hereafter referred as flavoxate) on voltage-dependent nifedipine-sensitive inward Ba(2+) currents in human detrusor myocytes were investigated using a conventional whole-cell patch-clamp. Tension measurement was also performed to study the effects of flavoxate on K(+)-induced contraction in human urinary bladder. Flavoxate caused a concentration-dependent reduction of the K(+)-induced contraction of human urinary bladder. In human detrusor myocytes, flavoxate inhibited the peak amplitude of voltage-dependent nifedipine-sensitive inward Ba(2+) currents in a voltage- and concentration-dependent manner (K(i) = 10 microM), and shifted the steady-state inactivation curve of Ba(2+) currents to the left at a holding potential of -90 mV. Immunohistochemical studies indicated the presence of the alpha(1C) subunit protein, which is a constituent of human L-type Ca(2+) channels (Ca(V)1.2), in the bundles of human detrusor smooth muscle. These results suggest that flavoxate caused muscle relaxation through the inhibition of L-type Ca(2+) channels in human detrusor.

Adult↗
PubMed2005

Ex vivo electroporation as a potent new strategy for nonviral gene transfer into autologous vein grafts.

Gene transfer to vein grafts has therapeutic potential to prevent late graft failure; however, certain issues, including efficacy and safety, have hindered the clinical application of this treatment modality. Here, we report the successful and efficient gene transfer of plasmid DNA via ex vivo electroporation into veins as well as into vein grafts. Two approaches were used: one involved transluminal in situ gene transfer using a T-shaped electrode (the "Lu" method), and the other was an adventitial ex vivo approach using an electroporation cuvette followed by vein grafting (the "Ad" method). The Lu method was carried out at 10 V, with optimal gene transfer efficiency in the in situ jugular veins of rabbits, and transgene expression was observed primarily in endothelial cells. However, when these veins were grafted into the arterial circulation, no luciferase activity was detected; this effect was probably due to the elimination of the gene-transferred cells as a result of endothelial denudation. In contrast, optimal and satisfactory gene transfer was obtained with the vein grafts subjected to the Ad method at 30 V, and transgene expression was seen primarily in adventitial fibroblasts. Gene transfer of endothelial nitric oxide synthase cDNA to the vein graft via the Ad method successfully limited the extent of intimal hyperplasia, even under hyperlipidemic conditions, at 4 wk after grafting. We thus propose that the Ad method via ex vivo electroporation may provide a novel, safe, and clinically available technique for nonviral gene transfer to sufficiently prevent late graft failure.

Animals↗