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Biomedical subjects

R Nagai

Publications and source records attributed to R Nagai.

At least 127 records · Page 7Linked to original sources
PubMed2000

In vivo klotho gene delivery protects against endothelial dysfunction in multiple risk factor syndrome.

The klotho gene, originally identified by insertional mutagenesis in mice, suppresses multiple aging phenotypes (e.g., arteriosclerosis, pulmonary emphysema, osteoporosis, infertility, and short life span). We have previously shown that mice heterozygous for a defect in the klotho gene upon parabiosis with wild-type mice show improved endothelial function, suggesting that the klotho gene product protects against endothelial dysfunction. In the present study, using the Otsuka Long-Evans Tokushima Fatty (OLETF) rat which demonstrates multiple atherogenic risk factors (e.g., hypertension, obesity, severe hyperglycemia, and hypertriglyceridemia) and is thus considered an experimental animal model of atherosclerotic disease, we show that adenovirus-mediated klotho gene delivery can (1) ameliorate vascular endothelial dysfunction, (2) increase nitric oxide production, (3) reduce elevated blood pressure, and (4) prevent medial hypertrophy and perivascular fibrosis. Based on these findings, klotho gene delivery improves endothelial dysfunction through a pathway involving nitric oxide, and is involved in modulating vascular function (e.g., hypertension and vascular remodeling). Our findings establish the basis for the therapeutic potential of klotho gene delivery in atherosclerotic disease.

Adenoviridae↗
PubMed2000

Characterization of S818L mutation in HERG C-terminus in LQT2. Modification of activation-deactivation gating properties.

We examined the mechanism(s) for HERG channel dysfunction in an S818L mutation in the HERG C-terminus using the heterologous expression system in Xenopus oocytes. Injection of S818L cRNA alone did not produce expressed currents. Coinjection of an equal amount of S818L cRNA with wild-type (WT) cRNA into oocytes did not exhibit apparent dominant-negative suppression. However, coinjection of excess amounts of S818L cRNAs with WT cRNA into oocytes decreased HERG current amplitudes and shifted the voltage dependence of activation to negative potentials, accelerated its activation and deactivation. The data suggest that S818L alone cannot form functional channels, whereas S818L subunits can, at least in part, coassemble with WT subunits to form heterotetrameric functional channels, and imply that the HERG C-terminus may contain a domain involving the activation-deactivation process of the channel. These findings may provide new insights into the structure-function relationships of the HERG C-terminus.

Amino Acid Substitution↗
PubMed2000

Restricted usage of T-cell receptor Vgamma-Vdelta genes and expression of costimulatory molecules in Takayasu's arteritis.

To further investigate the immunological mechanisms involved in Takayasu's arteritis, we analyzed the T-cell receptor (TCR) Vgamma and Vdelta gene usage by infiltrating gammadelta T-cells and the expression of costimulatory molecules B7-1, B7-2, CD40, CD27 ligand (CD27L), CD30L, OX40L in the arterial tissue of a patient with Takayasu's arteritis. We found that the repertoires of TCR Vgamma as well as Vdelta gene transcripts of the infiltrating cells were restricted as compared with those of peripheral blood lymphocytes from a patient with Takayasu's arteritis. This strongly suggests that gammadelta T-cells as well as alphabeta T-cells, as we previously reported, were specifically involved in the pathogenesis of Takayasu's arteritis. We also found that B7-1, B7-2, CD40, CD27L, CD30L, and OX40L were expressed in the arterial tissue, suggesting the roles for these costimulatory molecules in T-cell-mediated vascular injury in Takayasu's arteritis. Our findings strongly support the involvement of T-cell-mediated immunological mechanisms in the pathogenesis of Takayasu's arteritis.

Antibodies, Monoclonal↗
PubMed2000

Application of hydrostatic pressure facilitates ex vivo adenovirus gene transfer into rabbit aortas.

We investigated whether application of hydrostatic pressure facilitates ex vivo adenovirus gene transfer into rabbit aortas. Assay of beta-galactosidase activity was performed in the aortas subjected to adenovirus encoding LacZ gene (Ad-LacZ) transfer. Application of hydrostatic pressure at 8 atmospheres during Ad-LacZ gene transfer (10(10) pfu/mL, 10 min) to aortic segments resulted in an approximately 4.5-fold increase in transgene efficiency. X-Gal staining showed predominant beta-galactosidase activity in the endothelial and the adventitial cells in the aorta subjected to pressure-supported Ad-LacZ gene transfer. Then we examined the effect of heme oxygenase-1 (HO-1), which catabolizes heme to carbon monoxide and biliverdin, by transferring adenovirus encoding HO-1 (Ad-HO-1) into rabbit aortas using this pressurization system. The Ad-HO-1-infected aortic segment showed a significantly decreased contractile response to phenylephrine compared to the Ad-LacZ-infected aortic segment. Pressure-supported adenovirus gene transfer may increase the feasibility of exploiting intraoperative adenovirus-mediated gene transfer.

Acetylcholine↗
PubMed2000

Lecithinized copper, zinc-superoxide dismutase ameliorates prolonged hypoxia-induced injury of cardiomyocytes.

Recent studies have suggested that prolonged hypoxia results in increased production of reactive oxygen species in cardiomyocytes, which leads to apoptosis of these cells. We previously showed that lecithinized recombinant human copper, zinc-superoxide dismutase (rhSOD) showed increased bioavailability through greater membrane affinity and a longer half-life than unmodified SOD. The purpose of this study was to investigate whether lecithinized SOD plays a protective role against hypoxic injury in cardiomyocytes. Cultured rat cardiomyocytes incubated with lecithinized SOD (100 U/ml), unmodified SOD (100 U/ml), or vehicle alone were subjected to hypoxia for up to 72 h. Lecithinized SOD, but not unmodified SOD, was successfully delivered intracellularly, which was verified by Western blot and confocal laser-scanning microscopy. Treatment of cells with lecithinized SOD significantly suppressed hypoxia-induced cell damage. Since lecithinized SOD also suppressed hypoxia-induced DNA fragmentation, the improved cell survival provided by lecithinized SOD is thought to be mediated by its antiapoptotic effect. In summary, lecithinization resulted in a facilitated rhSOD delivery into cultured cardiomyocytes, which reduced mortality of cardiomyocytes exposed to prolonged hypoxia.

Animals↗
PubMed2000

Oral administration of branched chain amino acids improves virus-induced glucose intolerance in mice.

We investigated the therapeutic effect of branched chain amino acids (BCAA) on mice with glucose intolerance induced by encephalomyocarditis virus (EMCV). Male DBA/2 mice were divided into three groups: treated with BCAA, (such as valine, leucine, and isoleucine), untreated, and control. BCAA-treated and -untreated groups were inoculated intraperitoneally with the NDK25 variant of EMCV at 200 plaque-forming units per mouse. The BCAA-treated group was administered orally 0.9 g/kg/day of each BCAA from the day after viral inoculation. The control group neither received virus inoculation nor was treated with BCAA. One week after inoculation, oral glucose tolerance tests (OGTT) were performed. After the glucose loading at 1.5 g/kg of body weight, blood glucose levels in the untreated group were 92.0+/-10.0 mg/dl at baseline, 224.6+/-10.9 mg/dl at 30 min, and 169.4+/-21.4 mg/dl at 60 min, which were significantly (P<0.05) higher than those in the control group (62. 7+/-3.6 mg/dl, 167.2+/-16.4, and 83.8+/-6.0 mg/dl, respectively). Blood glucose levels in the BCAA-treated group were 54.5+/-3.7 mg/dl at baseline, 145.2+/-8.7 mg/dl at 30 min, and 128.7+/-18.3 mg/dl at 60 min after the glucose loading, which were not significantly higher than those in the control group. Immunoreactive insulin levels at 30 min after the glucose loading were lower in the untreated group than in the control group at 1 week after virus inoculation. Histological investigations showed that the grade of insulitis in the pancreas of mice of the BCAA-treated group was lower than that of the mice of the untreated group. These results suggest that oral administration of BCAA is able to improve glucose intolerance induced by EMCV.

Administration, Oral↗
PubMed2000

Transcriptional regulation of human cardiac homeobox gene CSX1.

Cardiac homeobox gene Csx/Nkx-2.5 is essential for normal heart development and morphogenesis and is the earliest marker for cardiogenesis. To elucidate the regulatory mechanisms of Csx/Nkx-2.5 expression, we have isolated and characterized the upstream regulatory region of human Csx/Nkx-2.5 (CSX1). Transfection of the reporter gene containing a 965-bp CSX1 5' flanking region indicated that this region confers cardiomyocyte-predominant expression of CSX1. Deletion and mutational analyses revealed two positive cis-regulatory elements in this region that are essential for CSX1 expression in cardiomyocytes. Electrophoretic mobility shift assay revealed that nuclear proteins prepared from cardiac myocytes bound to these elements in a sequence-specific manner. The identification of cis-regulatory sequences of the Csx/Nkx-2.5 gene will facilitate further analysis for the upstream regulatory factors that control the expression of Csx/Nkx-2.5 and the process of vertebrate heart development.

Animals↗
PubMed2000

Ca2+/calmodulin-dependent kinase II and calcineurin play critical roles in endothelin-1-induced cardiomyocyte hypertrophy.

Endothelin-1 (ET-1) induces cardiac hypertrophy. Because Ca(2+) is a major second messenger of ET-1, the role of Ca(2+) in ET-1-induced hypertrophic responses in cultured cardiac myocytes of neonatal rats was examined. ET-1 activated the promoter of the beta-type myosin heavy chain gene (beta-MHC) (-354 to +34 base pairs) by about 4-fold. This activation was inhibited by chelation of Ca(2+) and the blocking of protein kinase C activity. Similarly, the beta-MHC promoter was activated by Ca(2+) ionophores and a protein kinase C activator. beta-MHC promoter activation induced by ET-1 was suppressed by pretreatment with the calmodulin inhibitor, W7, the Ca(2+)/calmodulin-dependent kinase II (CaMKII) inhibitor, KN62, and the calcineurin inhibitor, cyclosporin A. beta-MHC promoter activation by ET-1 was also attenuated by overexpression of dominant-negative mutants of CaMKII and calcineurin. ET-1 increased the activity of CaMKII and calcineurin in cardiac myocytes. Pretreatment with KN62 and cyclosporin A strongly suppressed ET-1-induced increases in [(3)H]phenylalanine uptake and in cell size. These results suggest that Ca(2+) plays a critical role in ET-1-induced cardiomyocyte hypertrophy by activating CaMKII- and calcineurin-dependent pathways.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2000

Hypotension and resistance to lipopolysaccharide-induced shock in transgenic mice overexpressing adrenomedullin in their vasculature.

BACKGROUND: Adrenomedullin (AM) is a vasodilating peptide involved in the regulation of circulatory homeostasis and in the pathophysiology of certain cardiovascular diseases. To determine the extent to which chronic AM overproduction affects circulatory physiology under normal and pathological conditions, we used a preproendothelin-1 promoter to establish transgenic mouse lines overexpressing AM in their vasculature. METHODS AND RESULTS: Transgenic mice overexpressing AM mainly in vascular endothelial and smooth muscle cells exhibited significantly lower blood pressure (BP) and higher plasma cGMP levels than their wild-type littermates. Blockade of NO synthase with N(G)-monomethyl-L-arginine elevated BP to a greater degree in AM transgenic mice, offsetting the BP difference between the 2 groups. Despite their lower basal BP, administration of bacterial lipopolysaccharide elicited smaller declines in BP and less severe organ damage in AM transgenic mice than in wild-type mice. Furthermore, the 24-hour survival rate after induction of lipopolysaccharide shock was significantly higher in the transgenic mice. CONCLUSIONS: A chronic increase in vascular AM production reduces BP at least in part via an NO-dependent pathway. In addition, smaller responses to LPS in transgenic mice suggest that AM is protective against the circulatory collapse, organ damage, and mortality characteristic of endotoxic shock.

Adrenomedullin↗
PubMed2000

Up-regulation of natriuretic peptides in the ventricle of Csx/Nkx2-5 transgenic mice.

A cardiac homeobox-containing gene Csx/Nkx2-5, which is essential for cardiac development, is abundantly expressed in the adult heart as well as in the heart primordia. Targeted disruption of this gene results in embryonic lethality due to abnormal heart morphogenesis. To elucidate the role of Csx/Nkx2-5 in the adult heart, we generated transgenic mice which overexpress human Csx/Nkx2-5. The transgene was expressed abundantly in the heart and the skeletal muscle. mRNA levels of several cardiac genes including natriuretic peptides, CARP, MLC2v, and endogenous Csx/Nkx2-5 were increased in the ventricle of the transgenic mice. Electron microscopic analysis revealed that the ventricular myocardium of the transgenic mice had many secretory granules, which disappeared after administration of vasopressin. These results suggest that Csx/Nkx2-5 regulates many cardiac genes and induces formation of secretory granules in the adult ventricle.

Animals↗
PubMed2000

Pressurization facilitates adenovirus-mediated gene transfer into vein graft.

We investigated whether application of non-distending hydrostatic pressure facilitates gene transfer into vein grafts. An external jugular vein was placed in a chamber with 100 microl adenovirus solution at a titer of 10(10) pfu/ml and was pressurized to up to 8 atm above ambient pressure for 10 min. Histochemical analysis demonstrated a positive transgene expression in all layers of the vessel wall. Gene transfer with 8 atm pressurization resulted in an approximately 50 times higher transgene expression than that without pressurization. Under 8 atm pressurization, the efficiency of gene transfer reached a plateau at 7.5 min. The application of hydrostatic pressure may improve the effectiveness of intraoperative genetic engineering of vein grafts.

Adenoviridae↗
PubMed2000

Lipopolysaccharide augments expression and secretion of vascular endothelial growth factor in rat ventricular myocytes.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is highly expressed in the myocardium under various stimuli including hypoxia and ischemia. On the other hand, lipopolysaccharide (LPS) causes systemic inflammatory response syndrome (SIRS), which consists of systemic pathophysiological changes related to vascular hyperpermeability. To test the hypothesis that VEGF is one of the important mediators of SIRS, we examined effects of LPS on the VEGF expression and secretion in cultured neonatal rat ventricular myocytes. LPS (10 microg/ml) rapidly (within 1 h) augmented the levels of VEGF mRNA in these cells. Pharmacological inhibition of nucleic factor-kappaB or tyrosine kinases did not affect the LPS-induced augmentation of VEGF mRNA expression, while these treatments markedly suppressed the up-regulation of inducible nitric oxide synthase (iNOS) expression by LPS. The VEGF concentrations in the conditioned media were also significantly increased by the LPS treatment of 6 h. In conclusion, LPS augments VEGF expression and secretion in rat ventricular myocytes, suggesting that VEGF may be involved in pathogenesis of SIRS. LPS may induce VEGF mRNA through the signaling pathways that are distinct from those responsible for the iNOS induction.

Animals↗
PubMed2000

Expression of genes encoding vascular endothelial growth factor and its Flk-1 receptor in the chick embryonic heart.

Vascular endothelial growth factor (VEGF) is known to play an essential role in embryonic vascular development. The heart is one of the main organs that produce VEGF, but it is still unknown how expression of VEGF gene is regulated in embryonic cardiac myocytes. Thus, we cloned cDNAs encoding VEGF and its receptor (a KDR/flk-1 or Quek1 homologue) from cultured 10-day-old chick embryonic ventricular myocytes (CEVM). Reverse transcription-polymerase chain reaction revealed that the chick VEGF mRNAs consisted of at least four different species corresponding to the isoforms of 190, 166, 146 and 122 amino acids. In the embryonic heart and CEVM, the isoforms of 166 and 122 amino acids were dominant. Northern blot analysis detected an abundance of VEGF mRNA in both the embryonic heart and CEVM, even at the basal state. The levels of VEGF mRNA in CEVM were significantly augmented by forskolin (100 microM), or phorbol 12-myristate, 13-acetate (200 nM) in a time-dependent manner in CEVM. In contrast, the basal levels of VEGF mRNA were attenuated by genistein (100 microM), but not by H89 (100 microM) or bisindolylmaleimide (75 microM). Northern blot analysis also detected the chick flk-1 mRNA in abundance in the embryonic heart, and to a much lesser extent in CEVM. The expression levels of VEGF and flk-1 mRNA species were continuously high in the 6, 8 and 10-day-old chick embryonic hearts. In the 10-day-old embryonic hearts, in situ hybridization confirmed that mRNA encoding VEGF was mainly expressed in ventricular myocytes. In contrast, the flk-1 mRNA was detected in the microvascular endothelial cells, and to a lesser extent in the ventricular myocytes. These data suggest that VEGF is produced in embryonic ventricular myocytes, even at the basal state, and that the levels of VEGF mRNA may be differently regulated by various protein kinases. VEGF produced by the chick embryonic heart may play important roles in embryonic cardiovascular development by acting on surrounding endothelial cells and, possibly, on ventricular myocytes themselves.

Amino Acid Sequence↗
PubMed2000

Doxorubicin represses CARP gene transcription through the generation of oxidative stress in neonatal rat cardiac myocytes: possible role of serine/threonine kinase-dependent pathways.

Doxorubicin (Dox), an anthracyclin antineoplastic agent, causes dilated cardiomyopathy. CARP has been identified as a nuclear protein whose mRNA levels are exquisitely sensitive to Dox. In this study we investigated the molecular mechanisms underlying the repression of CARP expression by Dox in cultured neonatal rat cardiac myocytes. Dox (1 micromol/l)-mediated decrease in CARP mRNA levels was strongly correlated with BNP but not with ANP mRNA levels. Hydrogen peroxide scavenger catalase (1 mg/ml) but not hydroxyl radical scavengers dimethylthiourea (10 mmol/l) or mannitol (10 mmol/l) blunted the Dox-mediated decrease in CARP and BNP expression. Superoxide dismutase inhibitor diethyldithiocarbamic acid (10 mmol/l), which inhibits the generation of hydrogen peroxide from superoxide metabolism, attenuated the repression. PD98059 (MEK1 inhibitor, 50 micromol/l), SB203580 (p38 MAP kinase inhibitor, 10 micromol/l), calphostin C (protein kinase C (PKC) inhibitor, 1 micromol/l), non-selective protein tyrosine kinase inhibitors genistein (50 micromol/l) or herbimycin A (1 micromol/l) failed to abrogate the downregulation of CARP and BNP expression by Dox. In contrast, H7 (30 micromol/l), a potent inhibitor of serine/threonine kinase, significantly blocked Dox-mediated downregulation of CARP and BNP expression. Transient transfection of a series of 5'-deletion and site-specific mutation constructs revealed that M-CAT element located at -37 of the human CARP promoter mediates Dox-induced repression of CARP promoter activity. These results suggest that a genetic response to Dox is mediated through the generation of hydrogen peroxide, which is selectively linked to the activation of H7-sensitive serine/threonine kinase distinct from PKC and well characterized mitogen-activated protein (MAP) kinases (ERK and p38MAP kinase). Furthermore, our data implicated M-CAT element as a Dox-response element within the CARP promoter in cardiac myocytes.

Animals↗
PubMed2000

Induction of VEGF gene transcription by IL-1 beta is mediated through stress-activated MAP kinases and Sp1 sites in cardiac myocytes.

Interleukin-1 beta (IL-1 beta) is a multipotent cytokine participating in a variety of cardiovascular diseases. In this study, we examined the effects of IL-1 beta on the expression of vascular endothelial cell growth factor (VEGF) and pursued the molecular mechanisms underlying this effect. Treatment of cultured neonatal rat cardiac myocytes with IL-1 beta increased the levels of VEGF mRNA in a time- and a concentration-dependent manner. These effects were completely abolished by SB203580 and SB202190 (p38 MAPK inhibitors) but not by PD98059 (MEK1 inhibitor), calphostin C (protein kinase C inhibitor), or genistein (tyrosine kinase inhibitor). While IL-1 beta phosphorylated c-Jun N-terminus protein kinase (JNK) rapidly and transiently, the effect of IL-1 beta on p38 mitogen-activated protein kinase (MAPK) was gradual and persistent. Transient transfection assays showed that IL-1 beta increases the transcription from the VEGF promoter. A series of 5;-deletion and site-specific mutation analyses indicated that IL-1 beta as well as overexpression of p38 MAPK and JNK activate VEGF promoter activity through two G+C-rich sequences located at -73 and -62. Electrophoretic mobility shift and supershift assays showed Sp1 and Sp3 proteins specifically bind to the G+C-rich sequences. The half-life of VEGF mRNA was significantly increased in cells treated with IL-1 beta. Together, these results indicate that IL-1 beta induces VEGF gene expression at both transcriptional and post-transcriptional levels, and IL-1 beta evokes p38 MAPK and JNK signalings, which in turn stimulate the transcription of the VEGF gene through Sp1-binding sites. These findings suggest the role of IL-1 beta as a cytokine inducing VEGF in cardiac myocytes, and imply that activation of stress-activated MAP kinases regulate Sp1 sites-dependent transcription.

Animals↗
PubMed2000

A mouse model of vascular injury that induces rapid onset of medial cell apoptosis followed by reproducible neointimal hyperplasia.

Genetically modified mice serve as a powerful tool to determine the role of specific molecules in a wide variety of biological phenomena including vascular remodeling. Several models of arterial injury have been proposed to analyze transgenic/knock-out mice, but many questions have been raised about their reproducibility and physiological significance. Here, we report a new mouse model of vascular injury that resembles balloon-angioplasty. A straight spring wire was inserted into the femoral artery via arterioctomy in a small muscular branch. The wire was left in place for one minute to denude and dilate the artery. After the wire was removed, the muscular branch was tied off and the blood flow of the femoral artery was restored. The lumen was enlarged with rapid onset of medial cell apoptosis. While the circumference of the external elastic lamina remained enlarged, the lumen was gradually narrowed by neointimal hyperplasia composed of smooth muscle cells. At 4 weeks, a concentric and homogeneous neointimal lesion was formed reproducibly in the region where the wire had been inserted. Similar exuberant hyperplasia could be induced in all strains examined (C57BL/6J, C3H/HeJ, BALB/c, and 129/SVj). This model may be widely used to study the molecular mechanism of post-angioplasty restenosis at the genetic level.

Angioplasty, Balloon↗