Hideo Ariyoshi

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.

Ischemia–Reperfusion Injury of the Liver with Special Reference to Calcium-Dependent Mechanisms

Abstract Ischemia–reperfusion (I/R) injury is a complex process involving numerous intracellular signaling pathways, mediators, cells, and pathophysiological disturbances; its prevention during liver surgery is of utmost importance. In this review, we divide hepatic I/R injury into two phases, intracellular and extracellular, for a better understanding of the processes involved. Ca2+ and Ca2+-dependent reactions play an important role as a trigger in the former phase, while the subsequent generation of bioactive substances plays a predominant role in the latter phase. These findings indicate that a combination of different therapeutic approaches against Ca2+-dependent steps may help prevent I/R injury of the liver.

Prevalence of impaired responsiveness to epinephrine in platelets among Japanese

The responsiveness of platelets only to epinephrine was markedly impaired in 23/140 (16%) healthy Japanese. The impaired responsiveness was not altered by changes in time and environment. Circulating level of catecholamines did not affect the responsiveness of platelets to epinephrine. A pilot family study indicated a possible familial nature of the defect. 3H methyl-yohimbine binding studies indicated that this defect was due to the decreased number of alpha 2 adrenergic receptor. Despite the defect, the potentiating effect of epinephrine on platelet aggregation stimulated by a low dose of ADP was normal. This abnormality is not apparently associated with any bleeding disorders and the clinical implication is unknown at present. It is, however, essential to acknowledge the prevalence of such defect in pursuing research on platelets stimulated by epinephrine.

Shear stress down‐regulates gene transcription and production of adrenomedullin in human aortic endothelial cells

Vascular endothelial cells are potent modulators of vascular tone in response to shear stress. Levels of vasoactive peptides such as adrenomedullin (AM), endothelin‐1 (ET‐1), C‐type natriuretic peptide (CNP), and nitric oxide (NO) are affected by fluid shear stress. AM, a potent vasodilator and suppressor of smooth muscle cell proliferation, contains the shear stress responsive element (SSRE) “GAGACC” in its promoter region. To examine the role of AM in the shear stress response, cultured human aortic endothelial cells (HAoECs) were exposed to fluid shear stresses of 12 and 24 dynes/cm2 in a cone‐plate shear stress loading apparatus for various time periods, and the levels of AM gene expression and peptide secretion from HAoECs were measured by Northern blotting analysis and radioimmunoassay (RIA), respectively. Both AM gene transcription and AM peptide levels were down‐regulated by fluid shear stress in a time‐ and magnitude‐dependent manner. Our results demonstrate that the normal level of arterial shear stress down‐regulates AM expression in HAoECs, suggesting that AM participates in the modulation of vascular tone by fluid shear stress. J. Cell. Biochem. 71:109–115, 1998.

Proteasome and its novel endogeneous activator in human platelets

Proteasome, a high molecular weight multicatalytic protease, was purified from the cytosolic fraction of human platelets for the first time. The biochemical properties of the enzyme including substrate specificity, optimal pH and effects of various inhibitors were almost identical with those of other cells. During the purification with a Heparin-Sepharose chromatography, a novel endogenous activator of the protease was identified and was partially purified. The activator enhanced both chymotrypsin or trypsin like activities of the proteasome in a dose related manner and was inactivated by heating at 56 degrees C for 30 min. This newly identified activator may serve as an important regulator or cofactor of intracellular activities of the proteasome.

Cancer Cells Cause Vascular Endothelial Cell (vEC) Retraction via 12(S)HETE Secretion; The Possible Role of Cancer Cell Derived Microparticle

BackgroundCancer cell mediated vascular endothelial cell (vEC) retraction plays a pivotal role in cancer metastasis. The aim of this study is to clarify the biochemical character of vEC retraction factor derived from human breast cancer cell line, MCF-7.Methods and ResultsIn order to estimate vEC retracting activity, transwell chamber assay system was employed. We first tested the effects of trypsin digestion as well as lipid extraction of culture medium (CM). Trypsin digestion of CM resulted in approximately 40% loss of vEC retracting activity and lipid extraction of CM by Brigh and Dyer methods recovered approximately 60% of vEC retracting activity, suggesting that approximately 60% of vEC retracting activity in MCF-7 derived CM is due to lipid. Although Nordihydroguaiaretic acid (NDGA), the specific lipoxygenase inhibitor, suppressed vEC retracting activity in CM, Acetyl salicylic acid (ASA), a specific cyclooxygenase inhibitor, did not affect the activity, suggesting that lipid exerting vEC retracting activity in CM belongs to lipoxygenase mediated arachidonate metabolites. Thin layer chromatography clearly demonstrated that Rf value of lipid vEC retracting factor in CM is identical to 12HETE. Authentic 12(S)HETE, but not 12(R)HETE, showed vEC retracting activity. After the ultracentrifugation of CM, most lipid vEC retracting activity was recovered from the pellet fraction, and flow cytometric analysis using specific antibody against 12(S)HETE clearly showed the association of 12(S)HETE with small particle in CM.ConclusionThese findings suggested the principal involvement of 12(S)HETE in cancer cell derived microparticles in cancer cell mediated vEC retraction.

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.

Common C677T Polymorphism in the Methylenetetrahydrofolate Reductase Gene Increases the Risk for Deep Vein Thrombosis in Patients with Predisposition of Thrombophilia

The alanine/valine (A/V) gene polymorphism of 5, 10-methylenetetrahydrofolate reductase (MTHFR), one of the key enzymes catalyzing remethylation of homocysteine, has been reported and the VV genotype is associated with increased plasma homocysteine levels as a result of the reduced activity and increased thermolability of this enzyme. Although previous studies have suggested that the VV genotype is a risk factor for arterial occlusive disease, whether the VV genotype is a risk factor for venous thrombosis is still controversial. Here we screened 72 Japanese patients with deep venous thrombosis (DVT) and 85 controls for this mutation, and we measured plasma levels of homocysteine to determine whether the thermolabile variant with the VV genotype is a risk factor for DVT in a Japanese population. Of the 72 patients with DVT, 10 (13.9%) were found to be homozygous for the VV genotype, and in 6 (7.0%) of 85, control individuals and the difference was not significant (odds ratio=2.12, 95% CI=0.73-6.16, p=0.19). When we divided the DVT patients into subgroups, with and without predisposition of thrombophilia, including deficiencies of proteins C and S, plasminogen, and lupus anticoagulant, the prevalence of the VV genotype in DVT patients with predisposition was significantly higher than that of the normal controls (odds ratio=5.99, 95% CI=1. 56-22.96, p=0.01). However, the prevalence of the VV genotype in DVT patients without predisposition was not significantly different from that of the normal controls (odds ratio=1.20, 95% CI=0.32-4.47, p=0. 75). The plasma homocysteine levels in patients with DVT (11.6+/-5.2 nmol/ml) was not significantly different from that of the control subjects (11.6+/-3.7 nmol/ml). Individuals with the VV genotype showed higher plasma homocysteine levels (15.4+/-6.9 nmol/ml) than did individuals with the AV genotype (11.2+/-3.7 nmol/ml, p=0.009) or in individuals with the AA genotype (11.1+/-4.2 nmol/ml, p=0.004). Serum folate and vitamin B12 levels were not correlated with the plasma homocysteine levels. In conclusion, even though homozygosity for the VV genotype of the MTHFR gene was associated with higher plasma homocysteine levels, we found no association between plasma levels of homocysteine and DVT or between the genotype of the MTHFR gene and the DVT incidence. However, we found that the VV genotype of the MTHFR gene is a risk factor for DVT only when combined with the predisposition of thrombophilia.

Separate analysis of nuclear and cytosolic Ca2+ concentrations in human umbilical vein endothelial cells

Ca2+ concentration inside human umbilical vein endothelial cells was studied separately in cytosol and nucleus by a confocal laser scanning microscopy using fluo‐3. The in vivo calibration curve for cytosol and nucleus showed good linearity between fluorescence intensity and Ca2+ concentration in cytosol ([Ca2+]i) and nuclei ([Ca2+]n). After calibration, [Ca2+]n was constantly higher than [Ca2+]i before and after the chelation of extracellular Ca2+ suggesting an active Ca2+ accumulation system on nuclear membrane. [Ca2+]n was also constantly higher than [Ca2+]i after the stimulation of thrombin (0.05 U/ml), FCS (10%), and thapsigargin (Tsg, 1μM). The temporal change of [Ca2+]n and [Ca2+]i was identical, and [Ca2+]i gradient towards the nucleus and peripheral or central [Ca2+]n rise was observed after these stimulations. From these results, [Ca2+]n is not only regulated by the active Ca2+ accumulation system on nuclear membrane at rest but also the generation of Inositol‐triphosphate. FCS caused heterogeneous [Ca2+]n or [Ca2+]i rise from cell to cell; single spike or oscillatory change of [Ca2+]n and [Ca2+]i was observed in about 56% of cells, which were completely abolished by the chelation of extracellular Ca2+, suggesting that FCS stimulated [Ca2+]n and [Ca2+]i rise solely depending on Ca2+ influx from extracellular medium. The higher concentration of [Ca2+]n and heterogeneous [Ca2+]n rise may have important roles in nuclear‐specific cellular responses.

Simultaneous digital imaging analysis of cytosolic calcium and morphological change in platelets activated by surface contact

The dynamic change of cytoplasmic Ca2+ concentration ([Ca2+]i) and morphological change were investigated simultaneously by confocal laser scanning microscopy using fluo‐3 and by differential interference contrast optics in platelets activated by contact with the following types of surfaces: native glass and glass treated with poly‐L‐lysine (PLL), fibrinogen (Fg), or von Willebrand factor (vWF). The initial [Ca2+]i values just after the surface contact were comparable (approximately 100 nM) among platelets deposited on the four surface types. On the PLL‐surface, no morphological change or [Ca2+]i elevation was observed. Glass‐, Fg‐, and vWF‐surface adhered platelets showed pseudopod formation and spreading associated with the inhomogeneous [Ca2+]i rise. The platelets on the Fg‐surface were the most active in terms of [Ca2+]i rise and morphological change. During pseudopod formation, the mean [Ca2+]i value was maximal and localized high [Ca2+]i zones were observed inside pseudopods, as well as in the center of the platelets. After spreading, high [Ca2+]i zones still remained in the center of the cell. This new technique enabled simultaneous observation of [Ca2+]i and cell shape and we clearly demonstrated a close relationship between [Ca2+]i and morphological alterations.