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Biomedical subjects

Yasufumi Sato

Publications and source records attributed to Yasufumi Sato.

At least 19 recordsLinked to original sources
PubMed2006

All-trans retinoic acid induces in vitro angiogenesis via retinoic acid receptor: possible involvement of paracrine effects of endogenous vascular endothelial growth factor signaling.

A natural retinoid all-trans retinoic acid (ATRA) regulates a variety of important cellular functions via retinoic acid receptor (RAR). ATRA has therapeutically been used against various malignancies including acute promyelocytic leukemia. Recently ATRA has also been recognized to be beneficial against atherosclerotic vascular disorders. However, its effects on angiogenesis remain controversial. We therefore examined ATRA effects on in vitro angiogenesis in terms of capillary-like tube formation using human umbilical vein endothelial cells (HUVECs)/normal human dermal fibroblast (NHDF) coculture. ATRA as well as RAR agonist Am80 significantly induced capillary-like tube formation. The ATRA-induced tube formation was inhibited by coincubation with RAR antagonist LE540/LE135. HUVEC proliferation, but not its migration, was also induced by ATRA. The ATRA-induced tube formation was completely abolished by coincubation with vascular endothelial growth factor (VEGF) neutralizing antibody or with VEGF receptor (VEGFR)-2 (KDR) neutralizing antibody, but not VEGFR-1 (Flt-1) neutralizing antibody. ATRA and Am80 induced VEGF secretion in the coculture as well as VEGF secretion/mRNA expression in NHDFs. Transcription activity of human VEGF gene promoter in NHDFs was stimulated by ATRA, which was augmented by RAR overexpression. ATRA also induced VDGFR-2/KDR mRNA expression in HUVECs. Moreover, ATRA-induced secretion of hepatocyte growth factor as well as angiopoietin-2 in the coculture. Taken together, ATRA may have induced angiogenesis via RAR mainly by stimulation of HUVEC proliferation and enhancement of endogenous VEGF signaling and in part by induction of hepatocyte growth factor and angiopoietin-2 production. Retinoids may therefore be potential candidates for therapeutic angiogenesis against ischemic vascular disorders.

Angiopoietin-2↗
PubMed2006

Calcium-induced synergistic inhibition of a translational factor eEF2 in nerve growth cones.

Local protein synthesis in nerve growth cones has been suggested, but how it is controlled remains largely unknown. We found eukaryotic elongation factor-2 (eEF2), a key component of mRNA translation, in growth cones by immunocytochemistry. While phosphorylated eEF2 was weakly distributed in advancing growth cones, eEF2 phosphorylation was increased by high potassium-evoked calcium influx. In the growth cone, calcium elevation increased eEF2 kinase (EF2K), a calcim-calmodulin-dependent enzyme. Calcium also decreased the level of phosphorylated p70-S6 kinase (S6K), a kinase known to inhibit EF2K. Moreover, calcium elevation decreased total eEF2 in growth cones. Since phosphorylated eEF2 inhibits mRNA translation, calcium elevation appears to inhibit mRNA translation in growth cones by a synergistic mechanism involving regulation of EF2K, S6K, and eEF2 itself. Time-lapse imaging showed that calcium elevation induced growth arrest of neurites. The inhibitory effect on mRNA translation may thus be involved in the regulation of neurite outgrowth.

Animals↗
PubMed2006

A selective Sema3A inhibitor enhances regenerative responses and functional recovery of the injured spinal cord.

Axons in the adult mammalian central nervous system (CNS) exhibit little regeneration after injury. It has been suggested that several axonal growth inhibitors prevent CNS axonal regeneration. Recent research has demonstrated that semaphorin3A (Sema3A) is one of the major inhibitors of axonal regeneration. We identified a strong and selective inhibitor of Sema3A, SM-216289, from the fermentation broth of a fungal strain. To examine the effect of SM-216289 in vivo, we transected the spinal cord of adult rats and administered SM-216289 into the lesion site for 4 weeks. Rats treated with SM-216289 showed substantially enhanced regeneration and/or preservation of injured axons, robust Schwann cell-mediated myelination and axonal regeneration in the lesion site, appreciable decreases in apoptotic cell number and marked enhancement of angiogenesis, resulting in considerably better functional recovery. Thus, Sema3A is essential for the inhibition of axonal regeneration and other regenerative responses after spinal cord injury (SCI). These results support the possibility of using Sema3A inhibitors in the treatment of human SCI.

Animals↗
PubMed2006

The vasohibin family: a negative regulatory system of angiogenesis genetically programmed in endothelial cells.

Biological phenomena are under the precise control by the genome. For the regulation of angiogenesis, proangiogenic genes such as VEGFs and angiopoietins are highly conserved, act specifically on endothelial cells, and play a fundamental role. In this sense, nature should prepare specific antiangiogenic genes as well. However, this counterpart of genomic regulation of angiogenesis remains to be established. We recently isolated a novel endothelium-derived angiogenesis inhibitor and named it vasohibin. Vasohibin is dominantly expressed in endothelial cells, induced by the stimulation with VEGF or FGF-2, and selectively affects on endothelial cells and inhibits angiogenesis. Although the mechanism of how vasohibin inhibits angiogenesis remains to be elucidated, our discovery of vasohibin as an endothelium-derived VEGF-inducible angiogenesis inhibitor should shed light on the genomic basis of the negative regulation of angiogenesis.

Alternative Splicing↗
PubMed2006

Down syndrome candidate region 1 increases the stability of the IkappaBalpha protein: implications for its anti-inflammatory effects.

Down syndrome candidate region 1 (DSCR1), an endogenous inhibitor of calcineurin, inhibits the expression of genes involved in the inflammatory response. To elucidate the molecular basis of these anti-inflammatory effects, we analyzed the role of DSCR1 in the regulation of NF-kappaB transactivation using glioblastoma cells stably transfected with DSCR1.4 or its truncation mutants (DSCR1.4-(1-133) and DSCR1.4-(134-197)). Overexpression of DSCR1.4 significantly attenuated the induction of cyclooxygenase-2 (COX-2) expression by phorbol 12-myristate 13-acetate (PMA) via a calcineurin-independent mechanism. Experiments using inhibitors of the signaling molecules for NF-kappaB activation showed that NF-kappaB is responsible for the induction of COX-2. Full-length and truncated DSCR1.4 decreased the steady-state activity of NF-kappaB as well as PMA-induced activation of NF-kappaB, which correlated with attenuation of COX-2 induction. DSCR1.4 did not affect the PMA-stimulated phosphorylation or degradation kinetics of IkappaBalpha; however, DSCR1.4 significantly decreased the basal turnover rate of IkappaBalpha and consequently up-regulated its steady-state level. In the same context, knockdown of endogenous DSCR1.4 increased the turnover rate of IkappaBalpha as well as COX-2 induction. These results suggest that DSCR1 attenuates NF-kappaB-mediated transcriptional activation by stabilizing its inhibitory protein, IkappaBalpha.

Anti-Inflammatory Agents↗
PubMed2006

Vasohibin prevents arterial neointimal formation through angiogenesis inhibition.

Vasohibin is a VEGF-inducible angiogenesis inhibitor in vascular endothelium. Here we examined the presence of vasohibin in human arterial wall, and found it in endothelium of adventitial microvessels in atherosclerotic lesion. Adventitial angiogenesis is involved in the progression of neointimal formation. Even in the presence of endogenous angiogenesis inhibitors, pathological angiogenesis persists. However, the supplementation of exogenous angiogenesis inhibitors can prevent pathological angiogenesis. We evaluated the potential role of vasohibin in neointimal formation. Adenovirus-mediated human vasohibin gene transfer in mouse liver resulted in the release of vasohibin in plasma and exhibited anti-angiogenic effects at remote sites. This gene transfer inhibited adventitial angiogenesis, macrophage infiltration, and neointimal formation after cuff placement on mouse femoral artery. Vasohibin exhibited no direct effect on migration and proliferation of smooth muscle cells. Thus, vasohibin has an activity to prevent neointimal formation by inhibiting adventitial angiogenesis.

Adult↗
PubMed2006

Isolation and characterization of vasohibin-2 as a homologue of VEGF-inducible endothelium-derived angiogenesis inhibitor vasohibin.

OBJECTIVE: We recently isolated vasohibin, a novel vascular endothelial growth factor (VEGF)-inducible endothelium-derived angiogenesis inhibitor. Our aim is to find DNA sequences homologous to vasohibin and determine their expression profile. METHODS AND RESULTS: By the search of DNA sequences in the database, we found one homologous gene and designated it vasohibin-2. Overall amino acid sequence homology between the prototype vasohibin (vasohibin-1) and vasohibin-2 was >50%. Vasohibin-2 exhibited antiangiogenic activity. Vasohibin-2 expression in cultured endothelial cells was low and not inducible by the stimulation that induced vasohibin-1. However, the immunohistochemical analysis revealed that vasohibin-1 and -2 were diffusely expressed in endothelial cells in embryonic organs during mid-gestation. After that time point, vasohibin-1 and -2 became faint, but persisted to a certain extent in arterial endothelial cells from late gestation to neonate. Expression of vasohibin-1 and -2 could be augmented in vivo by local transfection with the VEGF gene in the embryonic brain or by cutaneous wounding in adult mice. CONCLUSIONS: These results suggest that vasohibin-2, in combination with vasohibin-1, forms a novel family of angiogenesis inhibitors.

Angiogenesis Inhibitors↗
PubMed2006

Multiple processing forms and their biological activities of a novel angiogenesis inhibitor vasohibin.

Vasohibin is a newly identified negative feedback regulator for angiogenesis. When expressed in cultured human endothelial cells, vasohibin polypeptides were detected in multiple distinct molecular weight forms, suggesting that some proteolytic events may occur within cells or the pericellular milieu. In order to identify the proteolysis sites, vasohibin cDNA mutants were generated to substitute some basic amino acids with alanine and then were transfected into endothelial cells. Western blots with anti-vasohibin monoclonal antibody following the transfection showed that there were at least two cleaving sites in the amino terminal region. Purified recombinant protein of the amino terminal truncated forms not only retained its inhibitory activity on angiogenesis in mouse corneal assay but also showed strong affinity to heparin. Moreover, deletion of some basic residues at the carboxyl terminal resulted in abrogation of both antiangiogenic and heparin-binding activities. Processing patterns and biological activities of the processed forms of this novel antiangiogenic factor are discussed.

Amino Acid Sequence↗
PubMed2006

Vasohibin is up-regulated by VEGF in the retina and suppresses VEGF receptor 2 and retinal neovascularization.

Vasohibin is a recently identified protein that is up-regulated in cultured vascular endothelial cells by vascular endothelial growth factor and fibroblast growth factor 2. It inhibits endothelial cell migration, proliferation, and tube formation, and suppresses angiogenesis in chick chorioallantoic membrane, after subcutaneous implantation of matrigel, and in a tumor xenograft model. This has led to the hypothesis that vasohibin functions as a negative feedback inhibitor of angiogenesis. In this study, we tested that hypothesis in a well-characterized model of retinal neovascularization. In ischemic retina, increased expression of VEGF was accompanied by elevation of vasohibin mRNA and blocking of the increase in vegf mRNA with vegf siRNA significantly attenuated the rise in vasohibin mRNA. In transgenic mice in which the rhodopsin promoter drives expression of VEGF in the retina, there was also a significant increase in vasohibin mRNA. In mice with ischemic retinopathy, there was increased expression of vasohibin in vascular endothelial cells, and vasohibin knockdown caused an increase in neovascularization. Conversely, intraocular injection of recombinant vasohibin or an adenoviral vector containing a vasohibin expression cassette strongly suppressed retinal neovascularization in mice with ischemic retinopathy. Knockdown of vasohibin mRNA in ischemic retina had no significant effect on vegf or vegf receptor 1 mRNA levels but caused a significant elevation in the level of vegf receptor 2 mRNA. These data support the hypothesis that vasohibin acts as a negative feedback regulator of neovascularization in the retina and suggest that suppression of VEGF receptor 2 may play some role in mediating its activity.

Animals↗
PubMed2006

Metronomic scheduling of a cyclic hexapeptide Ra-VII for anti-angiogenesis, tumor vessel maturation and anti-tumor activity.

RA-VII, a cyclic hexapeptide isolated from Rubiae radix, binds to actin, causing a conformational change in the actin molecule and inducing G2 arrest by inhibiting cytokinesis. Here we examined the effect of RA-VII, its water-soluble derivative, and related RA-III and RA-V on endothelial cells. Among the four compounds tested, RA-VII most potently inhibited angiogenesis-related properties of endothelial cells (i.e. migration and proliferation) in vitro. We confirmed the anti-angiogenic activity of RA-VII in vivo by using a mouse corneal model. We then applied RA-VII for the treatment of tumors in mice. Daily intraperitoneal injection of RA-VII (1.5 or 3 mg/kg/day) exhibited no toxic effect on the animals, but significantly and dose dependently inhibited the growth of Lewis lung carcinoma cells previously inoculated into the mice. Interestingly, although two doses of RA-VII decreased the tumor vascular area to a similar extent, a higher dose of RA-VII led to tumor vessel maturation together with a significant increase in tumor cell apoptosis. Also, RA-VII showed a cytotoxic effect on Lewis lung carcinoma cells. These results indicate that metronomic scheduling of RA-VII is efficient for cancer treatment. A careful dose setting of RA-VII is crucial to obtain therapeutic superiority, possibly through tumor vessel maturation and a better distribution of the compound in the tumor tissue.

Animals↗
PubMed2006

Puromycin insensitive leucyl-specific aminopeptidase (PILSAP) is required for the development of vascular as well as hematopoietic system in embryoid bodies.

We have shown that puromycin insensitive leucyl-specific aminopeptidase (PILSAP) is required for regulation of angiogenesis. However, it remains unclear whether PILSAP plays a role in endothelial cell (EC) differentiation. We examined the role of PILSAP by using an embryoid bodies (EBs) culture system. Fms-like tyrosine kinase-1 (Flk-1) showed two expression peaks on days 4 and 10 of culture. These two peaks represent populations of mesodermal precursors and mature ECs, respectively. Endothelial markers such as VE-cadherin, CD34, CD31 and Tie2 followed the first peak of Flk-1. Interestingly, the expression of PILSAP showed a pattern similar to that of Flk-1. ES cells transfected with mutant PILSAP (mtPILSAP) cDNA of a dominant negative activity organized less vascular structure and showed decreased levels of vascular lineage markers. The similar results were obtained in EBs treated with leucinethiol, a specific inhibitor of leucine aminopeptidase or siRNA for PILSAP. However, Flk-1 expression was unaffected on day 4. The expression of markers for hematopoietic lineage and muscle cells in mtPILSAP-EBs was also reduced. These results suggest that although PILSAP may not function in the initial generation of Flk-1 positive mesodermal precursors, it dose play a role in growth of vascular, hematopoietic, and muscular lineage population from those precursors.

Animals↗
PubMed2006

Effect of low-voltage electrical stimulation on angiogenic growth factors in ischaemic rat skeletal muscle.

1. Low-voltage electrical stimulation (LVES) in skeletal muscle at a level far below the threshold of muscle contraction has been reported to promote local angiogenesis. However, the mechanism underlying the promotion of local angiogenesis by LVES has not been fully elucidated. In the present study, we evaluated whether angiogenic factors, such as vascular endotherial growth factor (VEGF), hepatocyte growth factor (HGF) and fibroblast growth factor (FGF), as well as other disadvantageous factors, such as inflammation (interleukin (IL)-6) and hypoxia (hypoxia-inducible factor (HIF)-1alpha), contribute to the local angiogenesis produced by LVES. 2. We completely excised bilateral femoral arteries of male Sprague-Dawley rats. After the operation, electrodes were implanted onto the centre of the fascia of the bilateral tibialis anterior (TA) muscles, tunnelled subcutaneously and exteriorized at the level of the scapulae. The right TA muscles of rats were stimulated continuously at a stimulus frequency of 50 Hz, with a 0.1 V stimulus strength and no interval, for 5 days. The left TA muscles served as controls. 3. We found that both VEGF and HGF protein were significantly increased by LVES in stimulated muscles compared with control. The VEGF level of the LVES group was 89.10 +/- 17.19 ng/g, whereas that of the control group was 65.07 +/- 12.88 ng/g, as determined by ELISA (P < 0.05). The HGF level of the LVES and control groups was 8.52 +/- 1.96 and 5.80 +/- 2.14 ng/g, respectively (P < 0.05). In contrast, there was no difference in FGF, IL-6 and HIF-1alpha between the LVES and control groups. 4. These results suggest that LVES in a hindlimb ischaemia model of rats increases not only VEGF, but also HGF, production, which may be the main mechanism responsible for the angiogenesis produced by LVES.

Angiogenic Proteins↗
PubMed2005

Raf-1 is a binding partner of DSCR1.

Down syndrome critical region 1 (DSCR1) is recognized as an endogenous calcineurin inhibitor. DSCR1 is induced in endothelial cells and may play an important role in inflammation and angiogenesis. To address a novel function of DSCR1, we searched interacting partners of DSCR1. We performed pull-down analysis using DSCR1 as a bait and identified Raf-1 as a binding partner. The association of Raf-1 was confirmed by co-immunoprecipitation in GM7373 cells expressing green fluorescence protein tagged DSCR1. We determined two Raf-1 binding regions in DSCR1; one in the N-terminus and the other in the C-terminus regions. We further demonstrated that calpain cleaved DSCR1 and generated fragments with different binding affinity to Raf-1 or calcineurin. These results constitute the first demonstration of Raf-1 as a binding partner of DSCR1, and suggest a novel role of DSCR1.

Binding Sites↗
PubMed2005

Gene regulation of a novel angiogenesis inhibitor, vasohibin, in endothelial cells.

We recently reported that vasohibin is a negative feedback regulator of angiogenesis, and it is specifically expressed in endothelial cells. Here, we characterize the regulation of vasohibin expression. Two possible splicing variants were found, and the longer isoform was preferentially expressed. VEGF induced the expression of vasohibin, and this induction was abrogated by anti-VEGFR2 mAb but not by anti-VEGFR1 mAb. Pharmacological analysis revealed that the downstream targets of VEGFR2 were PKCs, especially PKCdelta. Actinomycin D did not alter the kinetics of vasohibin mRNA induction upon VEGF treatment, whereas cycloheximide completely abolished its induction. We tested the effect of various inflammatory cytokines on vasohibin expression. TNFalpha, IL1 and IFNgamma decreased VEGF-stimulated vasohibin expression. Actinomycin D did not alter the kinetics of vasohibin mRNA induction upon TNFalpha treatment. These results indicate that the expression of vasohibin in endothelial cells is regulated either positively or negatively by certain factors at the transcriptional level.

Angiogenesis Inhibitors↗
PubMed2004

Chronotopic organization of olfactory bulb axons in the lateral olfactory tract.

The arrangement of axons in a tract can have a specific effect on the organization of functional neuronal circuits. Here we describe olfactory bulb axons chronologically arranged in the lateral olfactory tract. Newly differentiated projection neurons over the whole olfactory bulb are similarly marked with transient expression of c-kit protein. Their axons are assembled together and project into the ventral superficial part of the tract, displacing the older axons. This special assembly of the axons explains the nontopographic relationships between the olfactory bulb and the lateral olfactory tract axons that have been described in previous studies and could possibly influence the subsequent selection of the olfactory target areas by these axons.

Animals↗
PubMed2004

Down syndrome candidate region 1,a downstream target of VEGF, participates in endothelial cell migration and angiogenesis.

Vascular endothelial growth factor (VEGF) is a principal stimulator of angiogenesis. However, the downstream targets of VEGF in endothelial cells (ECs) are not entirely clarified. Survey of downstream targets of VEGF in human ECs identified a number of genes, including Down syndrome candidate region 1 (DSCR1). Here, we confirmed the inducible expression of DSCR1 in ECs by Northern and Western blottings. Moreover, VEGF-stimulated induction of DSCR1 was blocked by anti-VEGF receptor-2 monoclonal antibody (mAb), or the specific calcineurin inhibitors cyclosporin A and FK506. The expression of DSCR1 in ECs of neovessels was further shown by immunohistochemical analysis. We therefore examined whether DSCR1 played any roles in angiogenesis. The specific downregulation of DSCR1 expression by antisense oligonucleotide (AS-ODN) inhibited VEGF-stimulated migration of ECs as well as angiogenesis in vivo. AS-ODN inhibited the spreading of ECs on vitronectin, as well as on the immobilized anti-alphavbeta3 mAb, but not on anti-alphavbeta5 mAb. Moreover, AS-ODN inhibited tyrosine phosphorylation of focal adhesion kinase when ECs were plated on a vitronectin-coated dish. Immunoprecipitation followed by Western blotting showed the coimmunoprecipitation of DSCR1 and integrin alphavbeta3. These results suggest that DSCR1 is involved in angiogenesis by regulating adhesion and migration of ECs via the interaction with integrin alphavbeta3.

Cell Movement↗
PubMed2004

Puromycin-insensitive leucyl-specific aminopeptidase (PILSAP) binds and catalyzes PDK1, allowing VEGF-stimulated activation of S6K for endothelial cell proliferation and angiogenesis.

Puromycin-insensitive leucyl-specific aminopeptidase (PILSAP) plays an important role in angiogenesis by regulating the proliferation and migration of endothelial cells (ECs). Here we characterize the mechanism by which PILSAP regulates the vascular endothelial growth factor (VEGF)-stimulated proliferation of ECs. The specific elimination of PILSAP expression or its enzymatic activity inhibited VEGF-stimulated G1/S transition in ECs. This G1 arrest correlated with reduced cyclin dependent kinase 4/6 (CDK4/6) activity and retinoblastoma (Rb) protein phosphorylation. Analyses of signaling molecules upstream of CDK4/6 revealed that S6 kinase (S6K) activation was affected by PILSAP, whereas that of phosphatidylinositol-3 kinase (PI3K), Akt, and extracellular signal-related kinase 1/2 (ERK1/2) was not. We further demonstrated that PILSAP bound phosphatidylinositol-dependent kinase 1 (PDK1) and removed 9 amino acids from its N-terminus, which allowed S6K to associate with PDK1 and PILSAP upon VEGF stimulation. We constructed mutant PILSAP, which lacked the aminopeptidase activity but bound PDK1. Mutant PILSAP abrogated S6K activation upon VEGF stimulation in a dominant-negative manner. An N-terminal truncated form of PDK1 abolished the dominant-negative effect of mutant PILSAP. Finally, the introduction of a mutated PILSAP gene in ECs inhibited angiogenesis and retarded tumor growth in vivo. These results indicate that PILSAP plays a crucial role in the cell cycle progression of ECs and angiogenesis via the binding and modification of PDK1.

3-Phosphoinositide-Dependent Protein Kinases↗