PubMed Health⌕ Search

Biomedical subjects

H Yonekura

Publications and source records attributed to H Yonekura.

At least 19 recordsLinked to original sources
PubMed2001

Hypoxia down-regulates endostatin production by human microvascular endothelial cells and pericytes.

Endostatin is a potent anti-angiogenic factor derived from the C-terminal region of collagen XVIII and is implicated in the regulation of physiological and pathological angiogenesis. In this study, reverse transcription-polymerase chain reaction analysis of poly(A+) RNA demonstrated the presence of mRNA for collagen XVIII in human endothelial cells (EC) and pericytes, the very constituents of microvessels wherein angiogenesis takes place. Enzyme immunoassay revealed that both cell types liberated endostatin into culture media and that the endostatin levels were decreased by hypoxia, the principal cause of angiogenesis. Northern and Western blot analyses revealed that while the collagen XVIII/endostatin mRNA levels were invariant between hypoxic and normoxic conditions, the collagen XVIII protein levels in EC and pericytes decreased by hypoxia. Further, exogenously administered intact endostatin was significantly decreased when it was incubated with hypoxic conditioned media of endothelial cells or pericytes, but not with normoxic media. The results suggest that the reduction of autocrine endostatin may take an active part in hypoxia-driven angiogenesis.

Angiogenesis Inhibitors↗
PubMed2001

Effects of high ELF magnetic fields on enzyme-catalyzed DNA and RNA synthesis in vitro and on a cell-free DNA mismatch repair.

Environmental electromagnetic fields have been implicated in human cancers. We examined whether high extremely low frequency (ELF) AC magnetic fields could affect DNA synthesis, transcription or repair, using in vitro model systems with defined sequences. The rate and fidelity of DNA polymerase catalyzed DNA synthesis, as well as of RNA polymerase catalyzed RNA synthesis, were not statistically significantly affected by 60 Hz 0.25-0.5 Tesla magnetic fields. The efficiency of mutS dependent mismatch repair with human cell extracts was also not affected by the magnetic field exposure. The results suggest that the core processes related to the transmission of genetic information are stable under high ELF magnetic fields.

Animals↗
PubMed2001

Antisense display: a new method for functional gene screen and its application to angiogenesis-related gene isolation.

We have developed an antisense-oriented method for functional gene screening, which we termed "antisense display." After evaluation in a cell-free system, we applied this method to a cellular system for isolating angiogenesis-related genes and came across a gene previously not described in humans. A screen of 10 mer antisense repertoire led to the identification of a unique 'angiostatic' antisense, and the gene corresponding to it was found to be preferentially expressed in vascular endothelial cells, coding for a putative type-2 membrane protein containing Rab-GAP/TBC and EF-hand-like domains. We named it vascular Rab-GAP/TBC domain-containing protein (VRP). The results obtained thus indicate that the antisense display method is useful for the isolation of functional genes responsible for phenotypes concerned.

Genetic Testing↗
PubMed2001

Development and prevention of advanced diabetic nephropathy in RAGE-overexpressing mice.

Vascular complications arising from multiple environmental and genetic factors are responsible for many of the disabilities and short life expectancy associated with diabetes mellitus. Here we provide the first direct in vivo evidence that interactions between advanced glycation end products (AGEs; nonenzymatically glycosylated protein derivatives formed during prolonged hyperglycemic exposure) and their receptor, RAGE, lead to diabetic vascular derangement. We created transgenic mice that overexpress human RAGE in vascular cells and crossbred them with another transgenic line that develops insulin-dependent diabetes shortly after birth. The resultant double transgenic mice exhibited increased hemoglobin A(1c) and serum AGE levels, as did the diabetic controls. The double transgenic mice demonstrated enlargement of the kidney, glomerular hypertrophy, increased albuminuria, mesangial expansion, advanced glomerulosclerosis, and increased serum creatinine compared with diabetic littermates lacking the RAGE transgene. To our knowledge, the development of this double transgenic mouse provides the first animal model that exhibits the renal changes seen in humans. Furthermore, the phenotypes of advanced diabetic nephropathy were prevented by administering an AGE inhibitor, (+/-)-2-isopropylidenehydrazono-4-oxo-thiazolidin-5-ylacetanilide (OPB-9195), thus establishing the AGE-RAGE system as a promising target for overcoming this aspect of diabetic pathogenesis.

Animals↗
PubMed2000

The receptor for advanced glycation end products is induced by the glycation products themselves and tumor necrosis factor-alpha through nuclear factor-kappa B, and by 17beta-estradiol through Sp-1 in human vascular endothelial cells.

The binding of advanced glycation end products (AGE) to the receptor for AGE (RAGE) is known to deteriorate various cell functions and is implicated in the pathogenesis of diabetic vascular complications. Here we show that AGE, tumor necrosis factor-alpha (TNF-alpha), and 17beta-estradiol (E(2)) up-regulated RAGE mRNA and protein levels in human microvascular endothelial cells and ECV304 cells, with the mRNA stability being essentially invariant. Transient transfection experiments with human RAGE promoter-luciferase chimeras revealed that the region from nucleotide number -751 to -629 and the region from -239 to -89 in the RAGE 5'-flanking sequence exhibited the AGE/TNF-alpha and E(2) responsiveness, respectively. Site-directed mutation of an nuclear factor-kappaB (NF-kappaB) site at -671 or of Sp-1 sites at -189 and -172 residing in those regions resulted in an abrogation of the AGE/TNF-alpha- or E(2)-mediated transcriptional activation. Electrophoretic mobility shift assays revealed that ECV304 cell nuclear extracts contained factors which retarded the NF-kappaB and Sp-1 elements, and that the DNA-protein complexes were supershifted by anti-p65/p50 NF-kappaB and anti-Sp-1/estrogen receptor alpha antibodies, respectively. These results suggest that AGE, TNF-alpha, and E(2) can activate the RAGE gene through NF-kappaB and Sp-1, causing enhanced AGE-RAGE interactions, which would lead to an exacerbation of diabetic microvasculopathy.

Blotting, Western↗
PubMed2000

Participation of syndecan 2 in the induction of stress fiber formation in cooperation with integrin alpha5beta1: structural characteristics of heparan sulfate chains with avidity to COOH-terminal heparin-binding domain of fibronectin.

The present study provides direct evidence that syndecan 2 participates selectively in the induction of stress fiber formation in cooperation with integrin alpha5beta1 through specific binding of its heparan sulfate side chains to the fibronectin substrate. Our previous study with Lewis lung carcinoma-derived P29 cells demonstrated that the cell surface heparan sulfate proteoglycan, which binds to fibronectin, is syndecan 2 (N. Itano et al., 1996, Biochem. J. 315, 925-930). We here report that in vitro treatment of the cells by antisense oligonucleotide for syndecan 2 resulted in a failure to form stress fibers on fibronectin substrate in association with specific suppression of its cell surface expression. Instead, localization of actin filaments in the cytoplasmic cortex occurred. A similar response of the cells was observed when the cells were treated to eliminate functions of cell surface heparan sulfates, including exogenous addition of heparin and pretreatment with anti-heparan sulfate antibody, F58-10E4, and with proteinase-free heparitinase I. Size- and structure-defined oligosaccharides prepared from heparin and chemically modified heparins were utilized as competitive inhibitors to examine the structural characteristics of the cell surface heparan sulfates involved in organization of the actin cytoskeleton. Their affinity chromatography on a column linked with a recombinant H-271 peptide containing a C-terminal heparin-binding domain of fibronectin demonstrated that 2-O-sulfated iduronates were essential for the binding. Inhibition studies revealed that a heparin-derived dodecasaccharide sample enriched with an IdoA(2OS)-GlcNS(6OS) disaccharide completely blocked binding of the syndecan 2 ectodomain to immobilized H-271 peptide. Finally, the dodecasaccharide sample was shown to inhibit stress fiber formation, triggered by adhesion of P29 cells to a CH-271 polypeptide consisting of both the RGD cell-binding and the C-terminal heparin-binding domains of fibronectin in a fused form. All these results consistently suggest that syndecan 2 proteoglycan interacts with the C-terminal heparin-binding domain of fibronectin at the highly sulfated cluster(s), such as [IdoA(2OS)-GlcNS(6OS)](6) present in its heparan sulfate chains, to result in the induction of stress fiber formation in cooperation with integrin alpha5beta1.

Actins↗
PubMed2000

Roles of the AGE-RAGE system in vascular injury in diabetes.

This study concerns whether advanced glycation endproducts (AGE) are related to microvascular derangement in diabetes, exemplified by pericyte loss and angiogenesis in retinopathy and by mesangial expansion in nephropathy. AGE caused a decrease in viable pericytes cultivated from bovine retina. On the other hand, AGE stimulated the growth and tube formation of human microvascular endothelial cells (EC), this being mediated by autocrine vascular endothelial growth factor. In AGE-exposed rat mesangial cells, type IV collagen synthesis was induced. Those AGE actions were dependent on a cell surface receptor for AGE (RAGE), because they were abolished by RAGE antisense or ribozyme. The AGE-RAGE system may thus participate in the development of diabetic microangiopathy. This proposition was supported by experiments with animal models; several indices characteristic of retinopathy were correlated with circulating AGE levels in OLETF rats. The predisposition to nephropathy was augmented in RAGE transgenic mice when they became diabetic.

Animals↗
PubMed1999

Placenta growth factor and vascular endothelial growth factor B and C expression in microvascular endothelial cells and pericytes. Implication in autocrine and paracrine regulation of angiogenesis.

We have shown previously that vascular endothelial growth factor (VEGF) synthesized by the cellular constituents of small vessels per se, viz. endothelial cells and pericytes, participates in the hypoxia-driven proliferation of both cell types (Nomura, M., Yamagishi, S., Harada, S., Hayashi, Y., Yamashima, T., Yamashita, J., Yamamoto, H. (1995) J. Biol. Chem. 270, 28316-28324; Yamagishi, S., Yonekura, H., Yamamoto, Y., Fujimori, H., Sakurai, S., Tanaka, N., and Yamamoto, H. (1999) Lab. Invest. 79, 501-509). In this study, we examined the expression of the recently isolated VEGF gene family members (placenta growth factor (PlGF), VEGF-B, and VEGF-C) in human dermal microvascular endothelial cells and bovine retinal pericytes cultured under various oxygen tensions. Quantitative reverse transcription-polymerase chain reaction analyses demonstrated that the two cell types possess not only VEGF (VEGF-A) mRNA, but also VEGF-B, VEGF-C, and PlGF mRNAs. Among them, only VEGF-A mRNA was induced under hypoxia. Competitive reverse transcription-polymerase chain reaction showed that, under normoxic conditions, the rank order of mRNA content in endothelial cells was PlGF > VEGF-B > VEGF-C > VEGF-A and that mRNA coding for PlGF was expressed at >100-fold higher levels than VEGF-A mRNA. In pericytes, the rank order was VEGF-C > VEGF-A > VEGF-B > PlGF, and approximately 7-fold higher levels of VEGF-C mRNA compared with VEGF-A mRNA were noted in this cell type. Furthermore, antisense inhibition of PlGF protein production lowered the endothelial cell synthesis of DNA under hypoxic conditions. The results suggest that these VEGF family members may also take active parts in angiogenesis.

Amino Acid Sequence↗
PubMed1999

Antisense display--a method for functional gene screening: evaluation in a cell-free system and isolation of angiogenesis-related genes.

Presented here is an antisense-oriented method for functional gene screening, which we propose naming 'antisense display'. In principle, it consists of four steps: (i) preparation of phosphorothioate antisense repertoires that would correspond to the Kozak's consensus sequence, (ii) subgroup screening to identify active antisense molecules that could cause changes in the cellular phenotypes concerned and (iii) RT-PCR cloning of cDNA with the 5[prime] sense complement and 3[prime] anchor primers and sequence determination, followed by (iv) functional assays of candidate genes. Cell-free translation in rabbit reticulocyte lysate revealed that 10mer or longer antisense effectively halted protein synthesis. This required the presence of RNase H, and was achieved without prior heat-denaturation of the RNA templates. Then, subpools of the 10mer repertoire were administered to human microvascular endothelial cells in culture, and screened for anti-angiogenic activities. A single species having the sequence 5[prime]-GGCTCATGGT-3[prime] consistently inhibited the endothelial cell growth under hypoxia. Through RT-PCR with the corresponding sense primer, we came across three candidate cDNAs. Experiments employing longer unique antisense reproduced marked growth inhibitions in two of the three cDNAs. One encoded a mitochondrial protein and the other, which encoded a putative type-2 membrane protein containing Rab-GAP/TBC and EF-hand like domains, was a gene previously undescribed in human. The results suggest that the antisense display method is potentially useful for isolating new genes towards elucidating their functions.

Amino Acid Sequence↗
PubMed1999

Advanced glycation endproducts accelerate calcification in microvascular pericytes.

Vascular calcification in advanced atherosclerosis is frequently associated with diabetes, and is a predictor of future cardiovascular events. To investigate the molecular mechanisms of vascular calcification, we examined whether advanced glycation endproducts (AGE) formed at an accelerated rate under diabetes induce the osteoblastic differentiation of pericytes, a mesenchymal progenitor. First, von Kossa staining demonstrated that AGE significantly increased the number of calcified nodules in a bovine pericyte culture. AGE were also found to induce calcium accumulation in the pericyte monolayer in time- and dose-dependent manners. Second, quantitative reverse transcription-polymerase chain reaction revealed that AGE increased the pericyte levels of mRNAs coding for alkaline phosphatase and osteopontin, the representative markers for early and late osteoblastic differentiation, respectively. Alkaline phosphatase activity was actually enhanced by AGE. The results suggest that AGE have the ability to induce the osteoblatic differentiation of pericytes, which would contribute to the development of vascular calcification in diabetes.

Alkaline Phosphatase↗
PubMed1999

Insulin stimulates the growth and tube formation of human microvascular endothelial cells through autocrine vascular endothelial growth factor.

Insulin treatment is known epidemiologically as an independent risk factor for the progression of diabetic retinopathy. However, how insulin exacerbates the retinopathy is not yet fully understood. In this study, we investigate the effects of insulin on the growth and tube formation of microvascular endothelial cells (EC). When human skin microvascular EC were grown under various concentrations of insulin, DNA synthesis as well as tube formation of EC was found to be significantly stimulated. We obtained evidence that it is mainly vascular endothelial growth factor (VEGF) that mediates the angiogenic activity of insulin as follows. (1) Insulin upregulates the level of mRNA coding for secretory forms of VEGF, while the expression of the two VEGF receptor genes, kinase insert domain-containing receptor (kdr) and fms-like tyrosine kinase1 (flt1), was essentially unchanged by exposure to insulin. (2) A monoclonal antibody against human VEGF can completely neutralize both the proliferation and the tube formation of EC induced by insulin. The angiogenic effects of insulin were additive with those of hypoxia, a principal factor that causes angiogenesis. Further, insulin significantly stimulated plasminogen activator inhibitor-1 activity in EC. The results thus suggest that insulin not only elicits angiogenesis through the induction of autocrine VEGF but also is a predisposing factor for thrombogenesis, which may give rise to focal ischemia that could superdrive angiogenesis, thereby leading to the exacerbation of diabetic retinopathy.

Autocrine Communication↗
PubMed1999

Vascular endothelial growth factor acts as a pericyte mitogen under hypoxic conditions.

Angiogenesis is the process by which new vascular networks are formed from preexisting capillaries. The small vessels are composed of two types of cells, namely endothelial cells (EC) and pericytes, with the former being encircled by the latter. We previously showed that hypoxia, the principal cause of angiogenesis, can induce the proliferation of pericytes as well as EC. In this report we present evidence that the hypoxic induction of pericyte growth can be ascribed at least in part to vascular endothelial growth factor (VEGF) produced by this very cell type. First, the finding that hypoxia can stimulate the proliferation of pericytes was confirmed by cultivating bovine retinal pericytes in a controlled-atmosphere culture chamber containing various concentrations of oxygen and then assaying pericyte synthesis of DNA. Second, Northern blot analysis revealed that pericyte levels of mRNA encoding VEGF increased as the atmospheric oxygen tension was decreased; this was accompanied by an increase in de novo synthesis of VEGF proteins. Third, pericytes were able to respond to exogenously added VEGF, resulting in a dose-dependent increase in viable cell numbers. Fourth, polyclonal antibodies against VEGF efficiently blocked the hypoxic induction of pericyte growth. Fifth, pericytes expressed the gene for fms-like tyrosine kinase 1 (flt1) as the predominant form of VEGF receptor, and tyrosine phosphorylation of this receptor protein was enhanced when pericytes were exposed to hypoxia, as it was when cells were exposed to VEGF. Sixth, the antisense DNA complement of flt1 mRNA abolished the hypoxia-induced stimulation of pericyte growth. Finally, exogenous VEGF stimulated the migration of pericytes in a dose-dependent manner. The results thus suggest that VEGF, which has been thought to be a specific mitogen for EC, also acts on neighboring pericytes, probably in both autocrine and paracrine manners, and that the hypoxia-induced overproduction of VEGF could promote not only EC sprouting but also the recruitment of pericytes, thereby contributing to the maturation of newly formed microvessels.

Animals↗
PubMed1999

Cornu Ammonis of the dog: a rudimentary CA2 field is only present in a small part of the dorsal division, and is absent in the ventral division of the cornu Ammonis.

The relationship between mossy fiber projections and cytoarchitecture was studied in the cornu Ammonis (CA) of three mongrel dogs and two Japanese white rabbits. Precise comparisons were accomplished by examining adjoining sections that were stained by two methods: 1) the Timm-Danscher method which stains for zinc, which is contained in high concentrations in the mossy fiber terminals, and 2) thionin staining of the Nissl substance, so that the mossy fiber projections and cytoarchitecture could be closely compared in each subdivision of the CA. There was a cytoarchitectonically transitional zone (TZ) between CA3 and CA1 in both dogs and rabbits. The TZ was composed of an intermingling of CA3-like large pyramidal cells and CA1-like small pyramidal cells. Across the dorso-ventral length of the rabbit CA, the mossy fiber projections emitted from the dentate granular cell layer extended as far as the distal border of CA3 toward the TZ but did not enter the TZ or CA1. The TZ of the rabbit was equivalent to CA2 of Lorente de Nó (1934), that was characterized by CA3-like large pyramidal cells which did not receive mossy fiber inputs. In the dorsal CA of the dog, the mossy fiber projections extended across the CA3/TZ border, as far as the most distal part of the TZ in one dog but only for a short distance in the other two dogs. That part of the TZ which did not receive mossy fiber projections corresponded with CA2. There were no mossy fiber projections to CA1. In the ventral CA of the dog, however, mossy fiber projections were more extensive than in the dorsal CA, extending across CA3/TZ as well as TZ/CA1 borders to enter the proximal part of CA1. These observations indicate that there is a distinct CA2 in the rabbit CA, and that a rudimentary CA2 is present only in a small part of the dorsal CA of the dog, whereas it is absent in the ventral CA.

Animals↗
PubMed1999

Lattice-like collagen fiber meshwork in the iris stroma of the cat: a possible mechanism to generate the tension directed towards the iris root which is required for pupillary dilatation in the sympathectomized eye.

NaOH digestion technique for collagen fiber dissection and scanning electron microscopy demonstrated a lattice-like meshwork in the anterior surface of the iris stroma of the cat. The mesh threads were made of collagen fibril bundles. In the constricted pupil, the meshes were square to rhomboid with the diagonals in the direction of the radius or circumference of the iris. In the dilated pupil, however, the meshes were strongly flattened rhomboid or ellipse with a longer diagnoal or axis in the circumferential direction. At the mesh corners facing the pupillary margin or the iris root, the collagen fibril bundles were strongly bent in the iris of the constricted pupil, while they were almost straight or slightly wavy in the iris of the dilated pupil. Accumulation of elasticity tension generated by this small distortion of the iris-mesh threads in the constricted pupil was considered to generate a tension directed towards the iris root, which is required for pupillary dilatation in the sympathectomized eye. On the posterior surface of the iris stroma, numerous thin pleats tightly woven with collagen fibrils traversed straightway through the radial length of the ciliary zone of the iris in both constricted and dilated pupils. The structural changes of these pleats in miosis and mydriasis were very small compared with the meshwork of the anterior aspect of the iris. Therefore, they were considered to work mainly as an iris skeleton.

Animals↗
PubMed1998

Transgenic mice overexpressing type 2 nitric-oxide synthase in pancreatic beta cells develop insulin-dependent diabetes without insulitis.

We generated transgenic mice carrying the mouse type 2 nitric-oxide synthase (NOS2) cDNA under the control of the insulin promoter. Western and immunohistochemical analyses revealed that NOS2 was expressed abundantly in transgenic islets but not in control islets. When islets were isolated and cultured, high levels of nitrite were released from the transgenic islets. In transgenic mice, the beta cell mass was markedly reduced without the infiltration of macrophages or lymphocytes, and extensive DNA strand breaks were detected in the islets by in situ nick translation. All the transgenic mice developed hypoinsulinemic diabetes by 4 weeks of age, and treatment with an inhibitor of NOS2, aminoguanidine (200 mg/kg body weight every 12 h), prevented or delayed the development of diabetes. The present study shows that the production of nitric oxide by beta cell NOS2 plays an essential role in the beta cell degeneration.

Animals↗
PubMed1998

Advanced glycation endproducts inhibit prostacyclin production and induce plasminogen activator inhibitor-1 in human microvascular endothelial cells.

Several thrombogenic abnormalities are associated with diabetes. To investigate the underlying molecular mechanisms, we examined the effects of advanced glycation endproducts (AGE), non-enzymatically glycated protein derivatives, on the production of prostacyclin (PGI2), an anti-thrombogenic prostanoid, and of plasminogen activator inhibitor-1 (PAI-1), a fast-acting serine protease inhibitor of fibrinolysis, in human microvascular endothelial cells (EC). Firstly, AGE-bovine serum albumin (BSA) but not non-glycated BSA, was found to considerably decrease the production of PGI2 to about two-thirds of the control value. Secondly, quantitative reverse transcription-polymerase chain reaction showed that AGE-BSA increased the EC levels of mRNA coding for PAI-1, this being associated with a concomitant increase in the immunoreactive PAI-1 contents and the anti-fibrinolytic activity. Thirdly, the effects of AGE on PGI2 and PAI-1 syntheses in EC were found to be mediated by a receptor for AGE (RAGE) because antisense DNA against RAGE mRNA could reverse the AGE effects. Further, it was found that AGE decreased the intracellular cyclic AMP concentrations in EC and that cyclic AMP agonists such as dibutyryl cyclic AMP, forskolin and PGI2 analogue reduced the AGE-stimulated PAI-1 production, suggesting the involvement of cyclic AMP in the AGE-signalling pathway. The results thus suggest that AGE have the ability to cause platelet aggregation and fibrin stabilization, resulting in a predisposition to thrombogenesis and thereby contributing to the development and progression of diabetic vascular complications.

Cyclic AMP↗
PubMed1997

Advanced glycation end products-driven angiogenesis in vitro. Induction of the growth and tube formation of human microvascular endothelial cells through autocrine vascular endothelial growth factor.

This study was undertaken to determine whether and how advanced glycation end products (AGE), senescent macroproteins accumulated in various tissues under hyperglycemic states, cause angiogenesis, the principal vascular derangement in diabetic microangiopathy. We first prepared AGE-bovine serum albumin (BSA) and anti-AGE antiserum using AGE-RNase A. Then AGE-BSA was administered to human skin microvascular endothelial cells in culture, and their growth was examined. The AGE-BSA, but not nonglycated BSA, was found to induce a statistically significant increase in the number of viable endothelial cells as well as their synthesis of DNA. The increase in DNA synthesis by AGE-BSA was abolished by anti-AGE antibodies. AGE-BSA also stimulated the tube formation of endothelial cells on Matrigel. We obtained the following evidence that it is vascular endothelial growth factor (VEGF) that mainly mediates the angiogenic activities of AGE. (1) Quantitative reverse transcription-polymerase chain reaction analysis of poly(A)+ RNA from microvascular endothelial cells revealed that AGE-BSA up-regulated the levels of mRNAs for the secretory forms of VEGF in time- and dose-dependent manners, while endothelial cell expression of the genes encoding the two VEGF receptors, kinase insert domain-containing receptor and fms-like tyrosine kinase 1, remained unchanged by the AGE treatment. Immunoprecipitation analysis revealed that AGE-BSA did increase de novo synthesis of VEGF. (2) Monoclonal antibody against human VEGF completely neutralized both the AGE-induced DNA synthesis and tube formation of the endothelial cells. The results suggest that AGE can elicit angiogenesis through the induction of autocrine vascular VEGF, thereby playing an active part in the development and progression of diabetic microangiopathies.

Cell Division↗