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Biomedical subjects

U Ikeda

Publications and source records attributed to U Ikeda.

At least 145 records · Page 8Linked to original sources
PubMed1997

Gene transfer into vascular cells using adeno-associated virus (AAV) vectors.

OBJECTIVES: Recombinant viral vectors based on the nonpathogenic parvovirus, adeno-associated virus (AAV), have a number of attractive features for gene therapy, including the ability to transduce non-dividing cells and its long-term transgene expression. In this study, an AAV vector containing bacterial beta-galactosidase gene (lacZ) was used to transduce cultured rat vascular smooth muscle cells (VSMC) in vitro and rat thoracic aortas ex vivo. METHODS: VSMC were transduced with AAV-lacZ at multiplicities of infection (MOI) ranging from 5.0 x 10(5) to 1.0 x 10(7). Expression of beta-galactosidase (beta-gal) in VSMC was evaluated by X-gal staining and a beta-gal ELISA method. Excised rat aortas were incubated with medium containing AAV-lacZ. Expression of beta-gal in the aortic segments was evaluated by X-gal staining. RESULTS: With increasing MOI, up to 50% of cultured VSMC were positive by X-gal staining and the beta-gal expression increased up to 15 ng/mg protein. The expression gradually decreased during the culture but was detectable for at least 1 month. In the ex vivo study, AAV vectors transduced endothelial and adventitial cells in rat aortic segments, while no expression was seen in medial VSMC. CONCLUSIONS: AAV vectors can efficiently transduce rat VSMC in vitro. AAV-mediated ex vivo gene transfer into the normal aorta resulted in efficient gene transfer into endothelial and adventitial cells but not into medial VSMC. These findings suggest that AAV-based vectors are promising for use in cardiovascular gene therapy.

Aorta, Thoracic↗
PubMed1997

Tranilast inhibits contraction and Ca2+ movement of porcine coronary arteries.

In a recent clinical study, tranilast, an anti-allergic agent, was shown to reduce the rate of coronary restenosis after percutaneous transluminal coronary angioplasty, although the mechanism of this effect is unclear. The present study was undertaken to investigate the effects of tranilast on contraction and Ca2+ movement of the coronary arteries. We characterized the effects of tranilast on isometric force and aequorin-estimated intracellular Ca2+ concentrations ([Ca2+]i) of porcine coronary artery strips. Tranilast concentration-dependently (10-500 microM) inhibited histamine (3 x 10(-5) M)-induced contraction of the coronary arteries. A similar tendency was observed in the response to high K+ (30 mM) stimulation. Histamine caused phasic and tonic increases in [Ca2+]i, and high K+ caused a tonic increase in [Ca2+]i of smooth muscle, both of which were significantly suppressed in the presence of tranilast. These results suggest that tranilast inhibits the contraction of coronary arteries by inhibiting both Ca2+ influx from extracellular environment and Ca2+ release from intracellular Ca2+ stores, which might be related to its preventive effect on restenosis after coronary angioplasty.

Animals↗
PubMed1997

Endothelial-derived nitric oxide preserves anticoagulant heparan sulfate expression in cultured porcine aortic endothelial cells.

Nitric oxide (NO) has been shown to inhibit platelet adhesion and aggregation, but there are no reports on its interaction with the coagulation system. We investigated the effect of the L-arginine analogues, N-nitro-L-arginine (LNA), N(G)-nitro-L-arginine methyl ester (L-NAME), and N(G)-monomethyl-L arginine (L-NMMA), competitive inhibitors of NO production, on endothelial-surface heparan sulfate. Addition of LNA to porcine aortic endothelial cells reduced 125I-labeled antithrombin III binding to the cell surface heparan sulfate in a dose- and time-dependent fashion. Significant inhibition was observed with 1 mM LNA, and the maximal suppression (-50% of control) occurred at 10 mM LNA after 12 h. L-NAME (1 mM) and L-NMMA (1 mM) also significantly inhibited the antithrombin III binding. The iron chelator desferrioxamine significantly prevented the reduction of antithrombin III binding to LNA-treated cells. We further investigated the effect of L-NAME on intracellular oxidative stress of endothelial cells using a hydroperoxide-sensitive fluorochrome, carboxy-dichloro-dihydrofluorescein diacetate bisacetoxymethyl ester probe, and revealed that inhibition of NO synthesis by L-NAME led to a marked increase in intracellular oxidative stress. These results demonstrated that the prolonged inhibition of NO synthesis in porcine aortic endothelial cells decreases the expression of anticoagulant heparan sulfate on endothelial cells through the increase in intracellular oxidative stress, perhaps comprising another mechanism by which NO affects the coagulation system in the vasculature.

Animals↗
PubMed1997

Diurnal blood pressure rhythm in hypertensives with parental history of stroke.

To investigate whether the lack of nocturnal decline of blood pressure (nondipper) is a primary cause of stroke or a secondary abnormality due to stoke, we examined the relation between the blood pressure variation and parental history of stroke in 110 hypertensive patients. In nondippers (n = 54), the frequency of positive parental history of stroke was significantly higher than in dippers (n = 56) (53.7% v 33.9%, chi2 = 4.37, P = .0366). We observed a significant increase in the incidence of positive parental history of stroke in nondippers, suggesting that some genetic factors may regulate blood pressure profiles before stroke develops.

Aged↗
PubMed1997

Angiotensin converting enzyme gene polymorphism in essential hypertension based on ambulatory blood pressure monitoring.

The association of the angiotensin converting enzyme (ACE) gene polymorphism with essential hypertension is still controversial. We studied its polymorphism in 41 patients with hypertension based on ambulatory blood pressure (ABP) and 34 subjects with normal blood pressure. The ACE genotype was not significantly different between hypertensive and normotensive subjects. Casual blood pressure levels, 24 h, and daytime and nighttime ABP levels did not differ among the ACE genotype in patients with hypertension. In conclusion, the ACE genotype is not associated with essential hypertension based on ABP monitoring.

Alleles↗
PubMed1997

Activation of human monocytes for enhanced production of interleukin 8 during transendothelial migration in vitro.

Interleukin-8 (IL-8) is a chemokine for polymorphonuclear leukocytes (PMNs) and lymphocytes, which promotes the extravasation of these inflammatory cells. In this study, we investigated IL-8 synthesis induced by the adhesive interaction between monocytes and endothelial cells during transmigration and the capacity of transmigrated monocytes to produce IL-8. Cocultured human monocytes and human umbilical vein endothelial cell (HUVEC) monolayers induced the synergistic production of IL-8, compared with cultures of either monocytes or HUVEC monolayers alone. Coculture-induced IL-8 production almost doubled after HUVECs were stimulated with IL-1 beta. The induced IL-8 mRNA expression was consistent with the protein data, indicating the de novo synthesis of IL-8 by the coculture. Monoclonal antibodies (mAbs) against IL-8 inhibited the transendothelial chemotactic activity of the supernatants for PMNs by 55%. Immunohistochemistry revealed that both adherent and transmigrated monocytes and unstimulated HUVECs expressed IL-8 protein, whereas nonadherent monocytes did little. Transmigrated monocytes spontaneously secreted a 3.8-fold greater amount of IL-8 than the initial monocytes. Coculture-induced IL-8 production was inhibited about 30% by polyclonal Abs against IL-alpha, IL-1 beta, or tumor necrosis factor alpha, while it was not affected by mAbs against intercellular adhesion molecule 1 or vascular cell adhesion molecule 1. The results suggested that adhesive interaction during the transmigration of monocytes through HUVEC monolayers activates both cell types to produce IL-8 and that transmigrated monocytes are capable of producing ample IL-8.

Antibodies, Monoclonal↗
PubMed1997

Factor VII R353Q polymorphism and lacunar stroke in Japanese hypertensive patients and normotensive controls.

Elevated plasma factor VII (FVII) levels are reported to be associated with cardiovascular disease in both Caucasians and Japanese. Recent reports indicate that individuals with the FVII 353Q allele have decreased plasma FVII levels. Thus, we investigated the association between lacunar stroke and the FVII R353Q polymorphism in 137 hypertensive patients with silent or overt lacunar stroke (stroke group), 83 non-stroke hypertensives without any lacunae detected by magnetic resonance imaging (non-stroke group), and 97 normotensive control subjects matched for age, sex, and smoking status recruited at an annual health examination (normotensive control group). The frequency of the FVII 353Q allele was 0.057 in the normotensive control group, 0.051 in the non-stroke group and 0.061 in the stroke group. These frequencies, as well as genotype distribution, were not significantly different from each other, even when we subclassified the ischemic group into silent (n = 54) and clinically overt (n = 64) lacunar stroke subgroups. These results suggest that the FVII 353Q allele is not an important genetic determinant for cerebrovascular disease in Japanese individuals.

Adult↗
PubMed1997

Thyroid hormone stimulates Na(+)-Ca2+ exchanger expression in rat cardiac myocytes.

We investigated whether thyroid hormone directly affects Na(+)-Ca2+ exchanger expression in cardiac myocytes. Cultured neonatal rat cardiocytes were prepared from 1-day-old Sprague-Dawley rats. Intracellular Na+ concentration ([Na+]i) in cardiocytes was measured by using the Na(+)-sensitive dye sodium-binding benzofran isophthalate (SBFI). Na(+)-Ca2+ exchanger messenger RNA (mRNA) and protein expression were assayed by Northern and Western blotting, respectively. Triiodothyronine (T3; 10(-8) M) showed no effect on [Na+]i in cardiocytes, whereas ouabain (100 microM) caused a significant increase in [Na+]i from 11.3 +/- 5.0 to 21.8 +/- 5.0 mM. Exposure of cardiocytes to ouabain caused a rapid increase in Na(+)-Ca2+ exchanger mRNA accumulation, with a maximal twofold elevation at 12 h. The ouabain-induced Na(+)-Ca2+ exchanger mRNA accumulation was still observed in the Ca(2+)-free culture medium. On the other hand, exposure of cardiocytes to T3 induced a gradual increase in Na+ exchanger mRNA accumulation, with a maximal threefold increase at 24 h. Even in Na(+)-free medium, T3 still induced a twofold increase in Na(+)-Ca2+ exchanger mRNA accumulation in cardiocytes. Exposure of cardiocytes to T3 for 24-48 h also caused a marked increase in Na(+)-Ca2+ exchanger protein accumulation. In conclusion, thyroid hormone directly increases cardiac Na(+)-Ca2+ exchanger expression, independent of alterations in Na+ mobilization. These findings suggest also that thyroid hormone and Na+ regulate Na(+)-Ca2+ exchanger gene expression through distinct molecular regulatory pathways.

Animals↗
PubMed1997

Adenosine stimulates nitric oxide synthesis in rat cardiac myocytes.

We investigated the effects of adenosine on nitric oxide (NO) synthesis by measuring the production of nitrite, a stable metabolite of NO, in cultured neonatal rat cardiac myocytes. Incubation of cultures with interleukin-1 beta (10 ng/ml) for 24 h caused a significant increase in nitrite production. The interleukin-1 beta-induced nitrite production by cardiac myocytes was significantly increased by adenosine or its stable analog 2-chloroadenosine in a dose-dependent manner (10(-7)-10(-4) M). The adenosine A2-receptor antagonist KF-17837 (10(-6) M), but not the A1-receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine (10(-6) M), significantly inhibited 2-chloroadenosine-mediated nitrite production. The 2-chloroadenosine-induced nitrite production by interleukin-1 beta-stimulated cells was accompanied by inducible NO synthase mRNA and protein accumulation. In the presence of N6, 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (cAMP) (10(-3) M) or isoproterenol (10(-5) M), interleukin-1 beta-induced nitrite accumulation was further increased, but the effect of 2-chloroadenosine was not additive or synergistic. The protein kinase C inhibitor calphostin C did not inhibit the effect of 2-chloroadenosine. These results indicate that adenosine acts on A2 receptors and augments NO synthesis in interleukin-1 beta-stimulated cardiac myocytes, at least partially through a cAMP-dependent pathway.

2-Chloroadenosine↗
PubMed1997

Endothelin-1 inhibits nitric oxide synthesis in vascular smooth muscle cells.

We investigated the effects of endothelin-1 on nitric oxide synthesis in vascular smooth muscle cells. We measured the production of nitrite, a stable metabolite of nitric oxide, and the expression of inducible nitric oxide synthase mRNA and protein in cultured rat vascular smooth muscle cells. Incubation of the cultures with interleukin-1 beta (10 ng/mL) for 24 hours caused a significant increase in nitrite production. Endothelin-1 significantly decreased the interleukin-1 beta-induced nitrite production by vascular smooth muscle cells in a dose-dependent manner (10(-11) to 10(-8) mol/L). Incubation with interleukin-1 beta for 24 hours induced expression of inducible nitric oxide synthase mRNA and protein in vascular smooth muscle cells, whereas endothelin-1 showed a suppressive effect on their expressions. Addition of the endothelin type A receptor antagonist BQ-485, but not the endothelin type B receptor antagonist BQ-788, dose-dependently inhibited the effect of endothelin-1. After protein kinase C activity was functionally depleted by treatment of cells with phorbol 12-myristate 13-acetate for 24 hours, the effect of endothelin-1 was abolished. These results indicate that endothelin-1 acts on endothelin type A receptors and inhibits nitric oxide synthesis in interleukin-1 beta-stimulated vascular smooth muscle cells at least partially through a protein kinase C-dependent pathway.

Animals↗
PubMed1997

Arginine vasopressin increases nitric oxide synthesis in cytokine-stimulated rat cardiac myocytes.

We investigated the effects of arginine vasopressin (AVP) on nitric oxide (NO) synthase activity in cardiac myocytes by measuring the production of nitrite, a stable metabolite of NO, and the expression of inducible NO synthase (iNOS) mRNA and protein. Incubation of cultured neonatal rat cardiac myocytes for 24 hours with interleukin-1beta (IL-1beta) caused a significant increase in NO production. Both AVP and V1a receptor agonist [Phe2,Ile3,Orn8]vasopressin augmented NO synthesis in IL-1beta-stimulated, but not in unstimulated myocytes, in a dose-dependent manner. The V1a receptor antagonist [d(CH2)[5]1,O-Me-Tyr2,Arg8]vasopressin completely inhibited the effect of AVP. The AVP-induced NO production by IL-1beta-stimulated cells was accompanied by increased iNOS mRNA and protein accumulation. AVP caused a significant increase in cytosolic free Ca2+ levels of cardiac myocytes, whereas it showed no effect on cytosolic cAMP levels. After protein kinase C activity was functionally depleted by treating cells with phorbol 12-myristate 13-acetate for 24 hours, AVP did not augment IL-1beta-induced NO production. The effect of AVP was also inhibited in the presence of the protein kinase C inhibitor calphostin C. The addition of AVP increased protein kinase C activity in cardiac myocytes, and its effect was significantly inhibited in the presence of calphostin C. These results support the hypothesis that the heart may be a target organ for AVP and that AVP modulates IL-1beta-induced iNOS expression in myocytes through the V1a receptor, which is mediated at least partially via activation of protein kinase C.

Animals↗
PubMed1997

Autonomic nervous system dysfunction in elderly hypertensive patients with abnormal diurnal blood pressure variation: relation to silent cerebrovascular disease.

To investigate the relationships among diurnal blood pressure (BP) variations and autonomic nervous system dysfunction, we assessed heart rate variability (HRV) using power spectral analysis of the 24-hour RR interval in 51 asymptomatic elderly hypertensive patients with various patterns of nocturnal BP fall. The extreme-dippers with marked nocturnal BP fall (n=16) had lower asleep low-frequency power (LF)/high-frequency power (HF) ratios (a relative index of sympathetic nervous system activity), while the nondippers without nocturnal BP fall (n=18) had lower awake LF/HF ratios and asleep/awake ratio for HF (an index of parasympathetic nervous activity), when compared with dippers with appropriate nocturnal BP fall (n=17). The incidence of multiple lacunar infarction detected by brain magnetic resonance imaging was 56% in the extreme-dippers and 38% in the nondippers, and both were markedly higher than that (6.3%) in the dippers (both P<.01). There was no significant relationship between the BP level and any HRV parameter for either the daytime or nighttime period. The asleep/awake ratio for systolic BP was significantly correlated with the asleep/awake ratio for HF (r= -.363, P<.01) and with the asleep/awake ratio for the LF/HF ratio (r=.540, P<.001), regardless of whether multiple lacunar infarction was present. In conclusion, the autonomic nervous system activity is not related to high BP level per se, rather its diurnal variation is more important as a determinant of the diurnal BP patterns, regardless of the presence or absence of cerebrovascular disease.

Aged↗
PubMed1997

Arginine vasopressin inhibits nitric oxide synthesis in cytokine-stimulated vascular smooth muscle cells.

The purpose of this study was to investigate the effects of arginine vasopressin (AVP) on nitric oxide (NO) synthesis in vascular smooth muscle cells (VSMCs). We measured the production of nitrite, a stable metabolite of NO, and the expression of inducible NO synthase (iNOS) mRNA in cultured rat VSMCs. Incubation of VSMCs for 24 h with interleukin-1 beta (IL-1 beta) caused a significant increase in NO production. Both AVP and the V1a receptor agonist [Phe2, Ile3, Orn8]vasopressin inhibited NO synthesis in IL-1 beta-stimulated cells, but not in unstimulated cells, in a dose-dependent manner. The V1a receptor antagonist [d(CH2)5(1), O-Me-Tyr2, Arg8]vasopressin completely inhibited the effect of AVP. Incubation with IL-1 beta for 24 h induced the expression of iNOS mRNA in VSMCs, while AVP suppressed its expression. After functional depletion of protein kinase C activity by treating cells with phorbol 12-myristate 13-acetate for 24 h, AVP did not inhibit IL-1 beta-induced NO production. The effect of AVP was also inhibited in the presence of the protein kinase C inhibitor calphostin C in a dose-dependent manner. These results indicate that AVP inhibits IL-1 beta-induced iNOS expression in VSMCs through the V1a receptor, which is mediated at least partially via activation of protein kinase C.

Animals↗
PubMed1996

Circadian variation in incidence of cardioembolism.

Cardioembolism is most likely to occur after waking in the morning. The identification of periods of high risk for cardioembolism may have important therapeutic implications, such as matching drug effects with vulnerability.

Adult↗
PubMed1996

Adrenomedullin augments inducible nitric oxide synthase expression in cytokine-stimulated cardiac myocytes.

BACKGROUND: Plasma levels of adrenomedullin are increased in patients with congestive heart failure, but there has been no report concerning the effects of adrenomedullin on the heart. We investigated the effects of adrenomedullin on NO synthase activity in cardiac myocytes. METHODS AND RESULTS: We measured the production of nitrite, a stable metabolite of NO, in cultured neonatal rat cardiac myocytes with the Griess reagent. Inducible NO synthase mRNA and protein expression were assayed by Northern and Western blotting, respectively. Incubation of the cultures with interleukin-1 beta (10 ng/mL) for 24 hours caused a significant increase in nitrite accumulation. Adrenomedullin significantly augmented nitrite production by interleukin-1 beta-stimulated but not by unstimulated cardiac myocytes in a dose-dependent manner (10(-10) to 10(-6) mol/L). The adrenomedullin-induced nitrite production by interleukin-1 beta-stimulated cells was accompanied by increased inducible NO synthase mRNA and protein expression. In the presence of dibutyryl cAMP, the interleukin-1 beta-induced nitrite accumulation was increased further, but the stimulatory effect of adrenomedullin on nitrite production was abolished. Adrenomedullin dose-dependently increased intracellular cAMP levels in cardiac myocytes. Addition of the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP[8-37] to the culture dose-dependently inhibited both cAMP and NO generation stimulated by adrenomedullin. CONCLUSIONS: These results indicate that adrenomedullin acts on cardiac myocytes and augments NO synthesis in these cells under cytokine-stimulated conditions, at least partially through a cAMP-dependent pathway.

Adrenomedullin↗
PubMed1996

Effect of cilostazol, a cAMP phosphodiesterase inhibitor, on nitric oxide production by vascular smooth muscle cells.

We investigated the effects of cilostazol, a cAMP phosphodiesterase inhibitor, on nitric oxide (NO) synthesis in cultured rat vascular smooth muscle cells. Incubation of the cultures with interleukin-1 beta (10 ng/ml) for 24 h caused a significant increase in the accumulation of nitrite, a stable metabolite of NO. Although cilostazol itself showed no effect on nitrite accumulation, it stimulated interleukin-1 beta-induced nitrite accumulation in a concentration-dependent manner (10(-8)-10(-5) M). This effect of cilostazol was completely abolished in the presence of NG-monomethyl-L-arginine, actinomycin D or dexamethasone. The cilostazol-induced nitrite production was accompanied by increased inducible NO synthase protein expression. In the presence of dibutyryl-cAMP, interleukin-1 beta-induced nitrite accumulation was further increased, but the stimulatory effect of cilostazol on nitrite accumulation was blunted. The effect of cilostazol was also abolished in the presence of Rp-8-bromoadenosine-3',5-cyclic monophosphorothioate, a competitive inhibitor of protein kinase A. Addition of cilostazol to the cultures significantly increased intracellular cAMP levels of vascular smooth muscle cells. These results indicate that cilostazol increases NO synthesis in interleukin-1 beta-stimulated smooth muscle cells, at least partially through a cAMP-dependent pathway.

Animals↗
PubMed1996

Human monocyte-endothelial cell interaction induces synthesis of granulocyte-macrophage colony-stimulating factor.

BACKGROUND: Adhesion of monocytes to the endothelium is an initial step in the early stages of atherosclerosis and inflammation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates a range of functional activities of monocytes, including regulation of monocyte adhesion and induction of cytokine production. We investigated in this study whether CM-CSF synthesis was induced by the direct cell-to-cell interaction between human monocytes and human umbilical vein endothelial cells (ECs). METHODS AND RESULTS: The expressions of GM-CSF mRNA and protein were analyzed by Northern blotting and ELISA, respectively. Coculture of monocytes and ECs induced the high levels of GM-CSF mRNA expression, whereas culture of ECs or monocytes alone or coculture of neutrophils with ECs induced no GM-CSF mRNA expression. A large amount of GM-CSF was secreted into the supernatant upon coculture of monocytes with ECs. The supernatant from the coculture markedly stimulated 02- release in neutrophils, and this effect was significantly inhibited by anti-GM-CSF antibody (Ab). Immunohistochemistry and in situ hybridization revealed that GM-CSF protein and mRNA were clearly detectable in both ECs and monocytes adhered to ECs but not in nonadherent monocytes. The GM-CSF production by the coculture was markedly inhibited by genistein and partially inhibited by Abs against interleukin-1 and tumor necrosis factor-alpha. CONCLUSIONS: The present results indicate that GM-CSF is produced by direct interaction between monocytes and ECs and suggest that GM-CSF produced locally by monocyte-EC adhesive interaction plays an important role in the pathogenesis of atherosclerosis and inflammation by modulating monocyte/macrophage functions in vivo.

Adult↗