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J Kambayashi

Publications and source records attributed to J Kambayashi.

At least 37 records · Page 2Linked to original sources
PubMed1998

Expression of platelet-activating factor receptor transcript-2 is induced by shear stress in HUVEC.

Platelet-activating factor (PAF) is an important lipid involved in inflammation reaction and circulation regulation. The receptor for human PAF is synthesized from two spliced transcripts of the same gene. Our observation in the present study shows that HUVEC express transcript-1 only in the static condition. Shear stress induces the expression of transcript-2 in these cells but not transcript-1, resulting in increased PAF receptor expression as measured by FACS analysis. These results suggest that shear stress may increase the susceptibility of endothelial cells to PAF by inducing transcript-2 expression.

Alternative Splicing↗
PubMed1998

A role for local calcium gradients upon hypoxic injury in human umbilical vein endothelial cells (HUVEC).

Upon hypoxic injury, bleb formation is an early event of cell damage observed in a variety of cell types. Although a rise in cytosolic free Ca2+ ([Ca2+]i) has been considered to be involved in this process, the exact relationship between these phenomena remains ill-defined. In order to examine the relationship between bleb formation, and [Ca2+]i or nuclear free Ca2+ ([Ca2+]n), we analyzed [Ca2+]i and [Ca2+]n in HUVEC during hypoxic injury using confocal laser scanning microscopy. [Ca2+]i and [Ca2+]n were measured using Fluo-3, and cell viability and mitochondrial membrane potential were assessed by the exclusion of propidium iodide (PI) and rhodamine 123, respectively. After the initiation of hypoxia, [Ca2+]i and [Ca2+]n rose gradually up to 15 min reaching peak values of 447 +/- 62 and 516 +/- 105 nM, respectively, which was accompanied by a decrease in rhodamine 123 fluorescence and an increase in PI-stained cells. Bleb formation was observed after [Ca2+]i and [Ca2+]n had reached their peak values and the number of blebs increased thereafter. Confocal z-sectioning images revealed a localized increase in [Ca2+]i at the bleb forming site and this localized elevation in [Ca2+]i was observed before bleb formation in the corresponding area. In conclusion, bleb formation induced by hypoxic stress appears to involve Ca(2+)-dependent reactions that are linked to a regional elevation of [Ca2+]i.

Analog-Digital Conversion↗
PubMed1998

Upregulation of prostacyclin synthesis-related gene expression by shear stress in vascular endothelial cells.

Prostacyclin (prostaglandin I2, PGI2) has a variety of functions, including inhibition of smooth muscle cell proliferation, vasodilation, and antiplatelet aggregation. PGI2 production in endothelial cells has been reported to increase biphasically after shear loading, but the underlying mechanism is not well understood. To clarify the mechanism for the second phase of PGI2 upregulation, we examined the gene expression of the enzymes involved in PGI2 production in human umbilical vein endothelial cells (HUVECs) after shear-stress (24 dyne/cm2) loading. The production of 6-keto-PGF1alpha, a stable metabolite of PGI2, increased time-dependently under shear stress. The arachidonic acid liberation from membrane phospholipids in HUVECs after 12 hours of shear loading was increased significantly compared with the static condition. No change was observed for cytosolic phospholipase A2 expression, as detected by reverse transcription-polymerase chain reaction and Western blotting. Cyclooxygenase (COX)-1 mRNA increased after 1 hour of shear loading, and the increase lasted for 12 hours, the longest time tested, whereas COX-2 mRNA increased after 1 hour of shear loading and peaked at 6 hours. An increase of COX-1 expression was detected at 12 hours of shear loading by Western blotting. No expression of COX-2 was detected in the static control, but induced expression was observed at 6 hours after shear loading. PGI2 synthase was also found to be upregulated. These results suggest that the elevated PGI2 production by shear stress is mediated by increased arachidonic acid release and a combination of increased expression of COXs and PGI2 synthase.

6-Ketoprostaglandin F1 alpha↗
PubMed1998

Possible involvement of m-calpain in vascular smooth muscle cell proliferation.

Vascular smooth muscle cell (VSMC) proliferation still remains a poorly understood process, although it is believed to play a critical role in pathological states, including atherosclerosis and hypertension. Several reports have suggested that proteases may be directly involved in this process; however, it was still unclear which protease is responsible for VSMC proliferation. In this study, by use of a cell-permeable calpain inhibitor (calpeptin; benzyloxycarbonyl-Leu-nLeu-H), its analogue (benzyloxycarbonyl-Leu-Met-H), the cell-impermeable serine protease inhibitor leupeptin, and antisense oligonucleotide against m-calpain to inhibit proliferation of primarily cultured human VSMCs, we investigated whether calcium-activated neutral protease (calpain) is involved in VSMC proliferation. Calpeptin and its analogue, more specific for m-calpain, equally inhibited the proliferation of VSMCs in a dose-related manner, whereas a more limited antiproliferative effect was observed in leupeptin-treated VSMCs. Antisense oligonucleotide against m-calpain, but not scrambled antisense, dose-dependently inhibited m-calpain expression and proliferation of VSMCs. Maximal inhibition was an approximately 50% reduction of cell number and m-calpain antigen observed at 50 micromol/L of antisense oligonucleotide. Calpeptin or antisense oligonucleotide against m-calpain increased the expression of the endogenous calpain substrate pp125FAK (focal adhesion kinase), whereas the expression of the endogenous calpain inhibitor calpastatin was not affected. These results suggest that the proliferation of VSMCs requires protease activity, some of which is due to m-calpain.

Calcium-Binding Proteins↗
PubMed1997

Expanded polytetrafluoroethylene (ePTFE) vascular graft loses its thrombogenicity six months after implantation.

Although expanded polytetrafluoroethylene (ePTFE) has been believed to be an inert material for vascular prosthesis, it shows less tendency of graft maturation by means of endothelialization. The aim of this study is to evaluate long-term alteration in thrombogenicity of ePTFE grafts after implantation. Serial levels of thrombin-antithrombin III complex (TAT), D-dimer, C-reactive protein (CRP), and prothrombin time (PT) were examined in 77 patients following ePTFE Y-graft implantation for up to five years. TAT showed biphasic elevation after implantation; TAT increased from 16.4+/-8.6 ng/ml to 27.4+/-10.5 ng/ml at one day, decreased to 18.5+/-4.5 ng/ml at one week, and increased again to 25.3+/-8.5 ng/ml at two weeks. Elevated TAT gradually decreased after the second peak to reach a lower level than that before surgery after six months. There was no significant difference in TAT level after six months due to the difference in diagnosis or anti-thrombotic therapy. We suggest that ePTFE grafts lose their thrombogenicity six months after implantation, after which anti-thrombotic therapy might be unnecessary.

Adult↗
PubMed1997

The release mechanism of platelet-activating factor during shear-stress induced platelet aggregation.

We previously reported that 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor, PAF), released from activated platelets stimulated with thrombin plus collagen, is associated with platelet microparticles. In the present study, we found that PAF is concentrated and associated with microparticles released from high shear stress-induced activated platelets. The total amount of PAF released from 3 x 10(8) platelets under high shear stress (108 dyne/cm2) was 3.2 +/- 0.6 x 10(-15)mol (n = 5, mean +/- S.D.). Eighty percent of the PAF released from the platelets was recovered in the microparticle fraction after ultracentrifugation in the presence of albumin. Under high shear stress, PAF was not released from platelets within 3 minutes, although microparticles were released. In conclusion, microparticles released from activated platelets in the rheological condition of high shear stress are major carriers of PAF.

Animals↗
PubMed1997

Hypercholesterolemia as a risk factor for deep-vein thrombosis.

Our retrospective study has shown that hyperlipidemia is a novel etiologic factor in deep-vein thrombosis (DVT) and that most of the idiopathic DVT patients were hyperlipidemic (Thrombosis Research 79, 147-151, 1995). The aim of our current study is to analyze the interrelationship between hyperlipidemia and DVT by means of a case-control study. A series of lipid parameters were analyzed using serum from 109 patients with deep vein thrombosis (DVT). One hundred nine age- and sex-matched subjects served as controls. Diagnosis of hyperlipidemia was made if the serum cholesterol level was above 220 mg/dL or if the triglyceride level was above 150 mg/dL. Among several types of hyperlipidemia examined, the risk factor associated with the highest estimated odds ratio was carriage of hypercholesterolemia associated with hypertriglyceridemia (odds ratio 5.1) followed in order by hypercholesterolemia without hypertriglyceridemia (odds ratio 2.6) and hypertriglyceridemia without hypercholesterolemia (odds ratio 0.9). These findings support the hypothesis that hypercholesterolemia plays an important role in the pathogenesis of DVT.

Adult↗
PubMed1997

Identification of mu-, m-calpains and calpastatin and capture of mu-calpain activation in endothelial cells.

The presence of the calpain-calpastatin system in human umbilical vein endothelial cells (HUVEC) was investigated by means of ion exchange chromatography, Western blot analysis, and Northern blot analysis. On DEAE anion exchange chromatography, calpain and calpastatin activities were eluted at approximately 0.30 M and 0.15-0.25 M NaCl, respectively. For half-maximal activity, the protease required 800 microM Ca2+, comparable to the Ca2+ requirement of m-calpain. By Western blot analysis, the large subunit of mu-calpain (80 kDa) was found to be eluted with calpastatin (110 kDa). Both the large subunit of m-calpain (80 kDa) and calpastatin were detected in the respective active fractions. By Northern blot analysis, mRNAs for large subunits of mu- and m-calpains were detected in single bands, each corresponding to approximately 3.5 Kb. Calpastatin mRNA was observed in two bands corresponding to approximately 3.8 and 2.6 Kb. Furthermore, the activation of mu-calpain in HUVEC by a calcium ionophore was examined, using an antibody specifically recognizing an autolytic intermediate form of mu-calpain large subunit (78 kDa). Both talin and filamin of HUVEC were proteolyzed in a calcium-dependent manner, and the reactions were inhibited by calpeptin, a cell-permeable calpain specific inhibitor. Proteolysis of the cytoskeleton was preceded by the appearance of the autolytic intermediate form of mu-calpain, while the fully autolyzed postautolysis form of mu-calpain (76 kDa) remained below detectable levels at all time points examined. These results indicate that the calpain-calpastatin system is present in human endothelial cells and that mu-calpain may be involved in endothelial cell function mediated by Ca2+ via the limited proteolysis of various proteins.

Antibody Specificity↗
PubMed1997

Calpain activation in shear-induced platelet aggregation.

Fluid shear stress has been known to activate platelet reaction such as aggregation, but the exact mechanism of shear-induced platelet aggregation (SIPA) has not been fully understood. Calpain, an intracellular calcium-activated cysteine protease, is abundant in platelets and is considered to be activated and involved in the proteolytic processes during platelet activation. A possible activation of calpain in SIPA was investigated, employing a newly developed aggregometer and specific monoclonal antibodies to detect activation of calpain. When a shear stress gradient varying between 6 and 108 dyn/cm2 was applied to platelets, activation of mu-calpain was observed only in high-shear-stressed platelets, resulting in the proteolysis of talin. At 1 min after the onset of constant high shear stress of 108 dyn/cm2, mu-calpain activation and proteolysis of talin were detected and increased in a time-dependent manner. Constant shear stress more than 50 dyn/cm2, applied for 5 min, caused mu-calpain activation and proteolysis of talin, which were increased in a shear-force-dependent manner. Calpeptin, a calpain-specific peptide antagonist, caused the complete inhibition of both mu-calpain activation and proteolysis of talin, while SIPA profiles with calpeptin showed almost no change compared to those without calpeptin. These results suggest the possibility of calpain involvement in late phases of shear-induced platelet activation such as cytoskeletal reorganization.

Calpain↗
PubMed1997

Signal transduction system in epinephrine stimulated platelets; comparison between epinephrine sensitive and insensitive platelets.

We recently reported the high prevalence of impaired platelet responsiveness only to epinephrine in healthy Japanese. This abnormality was associated with a 50% decrease in the number of alpha 2-adrenergic receptors. Platelets from non-responders (NR) do not undergo secondary platelet aggregation even after exposure to 100 microM epinephrine, but they can potentiate the effect of ADP to provoke platelet aggregation. To further define the nature of the defect and to delineate controversial steps of epinephrine stimulated signal transduction, a signaling pathway of epinephrine was investigated in platelets from NR and R(normal responder to epinephrine). In a unique particle counting apparatus, epinephrine initially triggered the formation of small platelet aggregates composing of 10-1000 cells from both R and NR, but the aggregates became larger (4600 > cells) only in platelets from R. Thus, platelets from NR lack the ability to form larger aggregates. A similar defect was reproduced by treating normal platelets with aspirin. In the presence of fibrinogen, platelets from NR lacked phospholipase A2 activation, determined by arachidonic acid liberation in the presence of inhibitors to cyclooxygenase and lipoxygenase. In the absence of fibrinogen, aggregation and phospholipase A2 activation were not evident in R and NR. The surface expression of GPIIb/IIIa was markedly decreased in platelets from NR after stimulation by epinephrine, in comparison with those from R. The resting level and epinephrine stimulated increase in cAMP were not significantly different between NR and R. Incubating R platelets with a half saturating dose of yohimbine rendered them insensitive to epinephrine. These results indicated that the impaired platelet aggregation induced by epinephrine was due to the impaired surface exposure of glycoproteins GPIIbIIIa integral to the activation of phospholipase A2, which requires the full and normal occupancy of the alpha 2-adrenergic receptor by epinephrine.

Adenosine Triphosphate↗
PubMed1997

Sclerotherapy for varicose veins of the lower legs in patients with dysplasminogenemia.

Sclerotherapy combined with ligation has become a widely accepted treatment for varicose veins; however, it is associated with some risk of the serious complications of deep vein thrombosis (DVT). We investigated the incidence of thrombophilia in 164 consecutive patients undergoing treatment for varicose veins and determined the activities of antithrombin-III, protein C, and plasminogen. Of the 164 patients, 10 were diagnosed as having dysplasminogenemia (DPG), showing an incidence of 6.1%, in accordance with previous reports. DVT was not found to be caused by DPG in any patient, and no difference was found between patients with and those without DPG, suggesting that DPG is not a risk factor for varicose veins. We also investigated the activation of coagulation by measuring the thrombin-antithrombin III complex (TAT). The activation of coagulation after sclerotherapy was inhibited when ligation was performed 1 month prior to sclerotherapy, whereas it was increased when sclerotherapy and ligation were performed simultaneously. Of the 10 patients with DPG, 5 were treated uneventfully, and their TAT level increased to 4.0 micrograms/l, which was comparable to the level after sclerotherapy and ligation. These findings indicate that sclerotherapy can be performed safely in the majority of patients with DPG, and that the temporal separation of sclerotherapy and surgery is an alternative for these patients to prevent the activation of coagulation.

Adult↗
PubMed1997

Effect of low concentration of epinephrine on human platelet aggregation analyzed by particle counting method and confocal microscopy.

The effect of a physiologic concentration of epinephrine (Ep) on platelet activation was studied by using a novel method that simultaneously measures platelet aggregation by changes in light transmission and counts particles of various sizes by using light scattering. Detailed morphologic changes associated with activation process were studied by using confocal laser scanning microscopy (CLSM). A low concentration of Ep (20 nmol/L) corresponding to a high physiologic concentration triggered the formation of small platelet aggregates (diameter 7 to 30 microm) without any change in light transmission. The redistribution of filamentous actin (F-actin) and the expression of activated glycoprotein IIb-IIIa complex (GPIIb-IIIa), detected by PAC-1 binding, were also observed in the platelets comprising the small aggregates. Attempts were then made to detect changes in cytoplasmic ionized Ca2+ ((Ca2+)i) in individual platelets involved in the aggregate formation by CLSM with fluo-3, a Ca2+-indicating dye. Ep caused a weak (Ca2+)i increase in some individual platelets involved in the formation of small aggregates. This (Ca2+)i increase was associated with platelet aggregation, because no (Ca2+)i rise was detected in single platelets. Furthermore, platelets stimulated by Ep in the presence of RGDS or Ro 44-9883, a GPIIb-IIIa antagonist, did not form small aggregates or trigger a (Ca2+)i rise. Prior incubation with low concentrations of Ep (20, 100 nmol/L) enhanced the initial formation of small (diameter 7 to 30 microm), medium (diameter 30 to 50 microm), or large (diameter 50 to 70 microm) aggregates induced by a subthreshold concentration of adenosine diphosphate (0.25 micromol/L) as determined by the particle counting method. However, no apparent synergistic (Ca2+)i increase was observed in platelets involved in aggregate formation. From these observations the following conclusions have been reached. (1) A high physiologic concentration of Ep (20 nmol/L) is capable of triggering the formation of small aggregates, resulting in the redistribution of F-actin and the expression of activated GPIIb-IIIa complex. (2) An increase in (Ca2+)i is observed in platelets comprising the small aggregates. This increase is not related to the binding of Ep to its receptor but most likely is triggered by platelet-platelet association. (3) The characteristic potentiating effect of Ep is not due to the synergistic increase in (Ca2+)i.

Actins↗
PubMed1997

Middle ear inflation as a treatment for secretory otitis media in children.

Clinical evaluations of middle ear inflation for secretory otitis media (SOM) were performed with special emphasis on the influence of seasonal and aging factors. One hundred and forty-nine children between the ages of 3 and 9 years (227 ears) were all diagnosed as SOM by pneumatic-otoscopic findings, and type B tympanogram (TG) at 3 weeks or more after the onset of acute SOM or the initial observation of SOM. Middle ears were inflated by Politzer's method or by our modified method once or twice each week for 2 months. After inflation, TG displayed two different time sequences: one group changed to the A or C type immediately after inflating the ear one or more times, but usually returned gradually to the B type (TG-improved group): and the other group remained without any changes (TG-unchanged group) for the duration of this study. The healing rate in the TG-improved group was significantly higher than in the TG-unchanged group at the 2-month endpoint. The cure rate of SOM was significantly higher in spring than in autumn in the TG-unchanged group but not in the TG-improved group. There were almost no differences between the healing rates in the 3-5 and 6-9 year-old children. When a TG-unchanged ear is found in autumn during the 2-month inflation treatment, more careful and forcible treatments should be introduced later, especially to children between the ages of 3 and 9 years.

Child↗
PubMed1997

Evaluation of plasma (1-->3)-beta-D-glucan measurement by the kinetic turbidimetric Limulus test, for the clinical diagnosis of mycotic infections.

The present multicentre clinical study was conducted to assess the clinical utility of a new diagnostic method for deep mycosis in which (1-->3)-beta-D-glucan, a fungal cell wall component existing in plasma, was quantitatively measured by the kinetic turbidimetric Limulus test (WB003). Plasma (1-->3)-beta-D-glucan concentrations were 0.57 +/- 0.10 microgram/l in 92 healthy subjects and 0.62 +/- 0.32 microgram/l in 26 patients with non-mycotic diseases (disease control group). In comparison with these healthy subjects and patients with non-mycotic diseases, patients with mycosis had significantly higher plasma (1-->3)-beta-D-glucan concentrations: 19.63 +/- 73.28 micrograms/l in 12 patients with candidaemia, 11.28 +/- 21.42 micrograms/l in 7 patients with urinary Candida infection, 4.84 +/- 12.71 micrograms/l in 5 patients with pulmonary candidiasis, and 12.21 +/- 31.31 micrograms/l in 4 patients with invasive pulmonary aspergillosis. On the statistical analysis of these data, a cut-off value was set at 1.0 microgram/l. Using this cut-off value, 3 patients with pulmonary cryptococcosis and 4 patients (4/6) with pulmonary aspergilloma were all negative with low plasma (1-->3-beta-D-glucan levels. The test WB003 provided equivalent or higher efficiency of diagnosis of candidiasis and aspergillosis, in comparison with commercially available antigen detection kits, demonstrating its utility as a diagnostic reagent. It may also be useful in assessing therapeutic effectiveness when used periodically after treatment.

Adolescent↗
PubMed1996

Fluid shear stress induces actin polymerization in human neutrophils.

We have previously reported that a physiological range of shear stress induces neutrophil homotypic aggregation mediated by lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-3 (ICAM-3) interactions. To further characterized the homotypic aggregation, actin polymerization was investigated in neutrophils stimulated by shear stress in comparison with formyl-methionyl-leucyl-phenylalanine (fMLP). In fMLP-stimulated neutrophils, actin polymerization was localized in the pseudopods, and this reaction was not mediated by a cytosolic level of Ca2+. In contrast to fMLP stimulation, the actin polymerization induced by shear stress in a cone-plate viscometer was localized in cell-cell contact regions, and this polymerization required the increase of intracellular Ca2+. This shear stress-induced actin polymerization was not observed when neutrophils were pretreated with anti-LFA-1 or anti-ICAM-3 antibody. In conclusion, LFA-1 and ICAM-3 interaction mediated by the increase of [Ca2+]i generated the intercellular signal in order to accumulate F-actin in the cell-cell contact regions.

Actins↗
PubMed1996

Hemostasis activation during sclerotherapy of lower extremity varices.

The influence of compression sclerotherapy upon hemostasis activation was investigated in 41 consecutive patients with lower extremity varices by serial measurement of thrombin-antithrombin III complexes (TAT), D-dimer, fibrinogen and C-reactive protein (CRP). Blood sampling was carried out before operation and on the 7th and 28th post-operative day in patients randomly assigned to either the control group (n = 18), in which high ligation of sapheno-femoral junction and local excision of varices were performed, or the sclerotherapy group (n = 23) in which the comparable surgical intervention and compression sclerotherapy using hypertonic saline were performed simultaneously. In both groups, the TAT, D-dimer and fibrinogen concentrations at day 7 were significantly elevated compared to the value before operation while CRP showed no significant change during the observation period. In the sclerotherapy group, higher incidence of superficial thrombosis was observed and the TAT concentration at day 7 was significantly higher than that in the control group (p < 0.01), and the TAT at day 28 was still significantly elevated compared to the pre-operative level (p < 0.05). However, no relationship between TAT and D-dimer concentrations and the extent of superficial thrombosis was observed. We conclude that compression sclerotherapy for lower extremity varices causes latent activation of coagulation system and can be a risk factor for venous thromboembolism.

Antithrombin III↗
PubMed1996

LFA-1/ICAM-3 mediates neutrophil homotypic aggregation under fluid shear stress.

We found that human neutrophils undergo homotypic aggregation by loading the physiological range of fluid shear stress (12-30 dynes/cm2). Under the fluid shear stress, an increase of intracellular Ca2+ concentration of neutrophils was observed. This increase of intracellular Ca2+ concentration was caused by Ca2+ influx, and the blockage of the flux by NiCl2 suppressed the neutrophil homotypic aggregation. Furthermore, this neutrophil aggregation under fluid shear stress was completely inhibited by pretreatment with antibody against LFA-1 or ICAM-3. These results suggested that NiCl2-sensitive Ca2+ channel played an important role in LFA-1/ICAM-3-mediated neutrophil homotypic aggregation under fluid shear stress.

Antigens, CD↗