Masako Togo

Heme Oxygenase-1 Gene Promoter Polymorphism Is Associated With Coronary Artery Disease in Japanese Patients With Coronary Risk Factors

Objective—Heme oxygenase (HO) is important in the defense against oxidative stress and as a factor in an antiatherogenic mechanism. Compared with long (GT)n repeats, short (GT)n repeats in the human HO-1 gene promoter were shown to have higher transcriptional activity in response to oxidative stress. There is a strong link between oxidative stress and the pathogenesis of coronary artery disease (CAD). Methods and Results—We screened the allelic frequencies of (GT)n repeats in the HO-1 gene promoter in 577 patients who underwent coronary angiography. Because the distribution of numbers of (GT)n repeats was bimodal, we divided the alleles into 2 subclasses: class S included shorter (<27) repeats, and class L included longer (≥27) repeats. Multivariate logistic regression models including standard coronary risk factors revealed that the genotypes were significantly related to CAD status in hypercholesterolemic, diabetic patients or in smokers. In this study, the patients with shorter GT repeats were less likely to have CAD. Conclusions—Length polymorphism in the HO-1 gene promoter is related to CAD susceptibility in Japanese people who also have coronary risk factors such as hypercholesterolemia, diabetes, and smoking. HO-1 may play an antiatherogenic role in Japanese patients with these coronary risk factors.

Association Between Insulin Resistance and Carotid Arteriosclerosis in Subjects With Normal Fasting Glucose and Normal Glucose Tolerance

Objective—We examined the possible association between insulin resistance and carotid arteriosclerosis in subjects who had both normal fasting glucose and normal glucose tolerance after intake of a glucose load. Methods and Results—Our subjects were individuals who underwent general health screening at our institute, which included carotid ultrasound and oral glucose tolerance testing. Of the 1238 subjects enrolled in our study, 738 (60%) were classified as normal, defined as a normal fasting glucose level and normal glucose tolerance, and 334 (27%) and 166 (13%) were classified as borderline and diabetic, respectively, according to the criteria of the Japan Diabetes Society. The homeostasis model assessment of insulin resistance (HOMA-IR) was used as the index to measure insulin resistance. In normal-type subjects, univariate analysis showed that insulin resistance, but not insulin secretion, was associated with the presence of carotid plaque. Multivariate analysis showed that HOMA-IR was positively associated with carotid plaque in normal-type subjects, with an odds ratio of 1.19 (95% confidence interval, 1.00 to 1.41;P <0.05). Conclusions—These data suggest the possibility that the presence of higher insulin resistance could be a risk factor for carotid arteriosclerosis in subjects with normal fasting glucose and normal glucose tolerance.

Isoform-Dependent Cholesterol Efflux From Macrophages by Apolipoprotein E Is Modulated by Cell Surface Proteoglycans

Objective—Apolipoprotein E (apoE) mediates cellular cholesterol efflux and plays a crucial role in the inhibition of atherogenesis. We investigated whether there is an isoform-specific difference in its function for cholesterol efflux from cholesterol-loaded RAW264.7 cells, a murine macrophage cell line that lacks endogenous apoE expression. Methods and Results—When human apoE was expressed in RAW264.7 cells, apoE2 reduced cellular total cholesterol (TC) and esterified cholesterol (EC) levels significantly, whereas apoE3 and apoE4 had no effect. However, treatment of cells with 4-methylumbelliferyl-7-&bgr;-d-xyloside (&bgr;-DX) resulted in all 3 isoforms’ reducing cellular TC and EC contents significantly. We also investigated the effect of exogenously derived apoE on cholesterol efflux by utilizing the medium harvested from HeLa cells expressing apoE. ApoE2 and E3 reduced both cellular TC and EC contents significantly, whereas apoE4 did not. However, treatment of the cells with &bgr;-DX resulted in all 3 exogenously derived apoE isoforms’ reducing TC and EC contents significantly. The binding ability of apoE to heparan sulfate proteoglycans examined by heparinase I treatment revealed less binding ability of apoE2 compared with that of apoE3 or apoE4. Conclusions—The present study clarified the differential cellular cholesterol-modulating effect of apoE isoforms in macrophages, which would be due to the difference in their binding to proteoglycans.

ROLES OF MYOSIN LIGHT-CHAIN KINASE IN PLATELET SHAPE CHANGE AND AGGREGATION

We examined the roles of myosin light-chain kinase in platelet responses to ADP using wortmannin, which almost completely inhibited myosin light-chain kinase at 3-6 microM. This concentration of wortmannin did not affect ADP-induced changes in the shape of the platelets, but it markedly inhibited aggregation in platelet-rich plasma and washed platelets. ML-9, another inhibitor of myosin light chain kinase, elicited similar effects on the platelet responses to wortmannin. Electron microscopic studies showed that there was no wortmannin effect on the ADP-induced spheration of discoid platelets, pseudopod formation, or granule centralization. Wortmannin at concentrations which prevented myosin light-chain kinase also inhibited platelet aggregation induced by ADP in the presence of U46619, an analogue of thromboxane A2, which is a prerequisite for ADP-induced irreversible aggregation. Although wortmannin partially inhibited protein kinase C, the protein kinase C inhibitor Ro-31-7549 (5 microM) prevented neither ADP- or ADP/U46619-induced changes in the shape of the platelets nor aggregation. These results suggest that myosin light-chain kinase activation is a prerequisite for ADP-induced platelet aggregation, but not for changes in their shape.

Protein kinase C-dependent and -independent mechanisms of dense granule exocytosis by human platelets

We examined the mechanisms of ATP release by human platelets using Ro-31-7549, a specific inhibitor of protein kinase C. Ro-31-7549 almost completely inhibited TPA-induced platelet aggregation and ATP release at 5-10 microM in washed platelets and in platelet-rich plasma. However, it suppressed thrombin- and U46619-induced ATP release by only 48% and 21%, respectively, and had little effect on aggregation in washed platelet suspensions containing serum or in platelet-rich plasma. The addition of GRGDS to prevent aggregation inhibited this residual thrombin-induced release by 53% and the residual U46619 release by 100% in the presence of Ro-31-7549. In washed platelet suspensions free of serum or plasma, Ro-31-7549 almost completely inhibited the ATP release and partially suppressed the aggregation induced by these agonists. These results suggested that there are protein kinase C-dependent and -independent mechanisms for ATP release by human platelets and that activation of the latter mechanism may depend on aggregation and plasma factors.

Identification of a novel mutation for phytosterolemia. Genetic analyses of 2 cases.

BACKGROUND Phytosterolemia is one of the genetic disorders causing hypercholesterolemia and atherosclerosis together with the accumulation of plant sterol in plasma and tissues. The mutations in ABCG5 and ABCG8 genes, encoding sterolin-1 and -2, respectively, are responsible for phytosterolemia. METHODS We performed genetic analyses on 2 Japanese phytosterolemia patients. RESULTS We identified 2 mutations in the ABCG5 gene in these patients. The first patient was homozygous for a novel mutation, which was a 19-base pair tandem repeat insertion in exon 7, leading to a premature termination at codon 288. The second patient was a compound heterozygote; one of the mutations was the same as that found in the first patient, while the other mutation was a C to T substitution in exon 10, resulting in a premature termination at codon 446 (R446X). No other mutation was found in the ABCG5 and ABCG8 genes. CONCLUSIONS This result was concordant with previous observations that found most Asian phytosterolemia patients possessed mutations in the ABCG5 gene, and the site of the novel mutation was completely different from these previous reports, necessitating the extensive analyses for phytosterolemia.

Ca2+ entry pathways activated by the tumor promoter thapsigargin in human platelets

Thapsigargin-activated Ca2+ entry into platelets was examined in the presence of S-145, a thromboxane A2 receptor antagonist, to inhibit indirect effects by endogenously formed prostaglandin H2/thromboxane A2. With external Ca2+ present, 0.2 microM thapsigargin caused a prompt increase in intracellular Ca2+ concentration ([Ca2+]i) followed by a gradual increase. Pretreatment with 6 microM wortmannin, a specific inhibitor of myosin light chain kinase, partly inhibited the increase in [Ca2+]i. In Ca(2+)-free EGTA buffer, thapsigargin induced a smaller increase in [Ca2+]i, and subsequent addition of Ca2+ to the buffer caused a further prompt increase in [Ca2+]i, demonstrating external Ca2+ entry. Wortmannin only partly inhibited this entry of external Ca2+. The wortmannin-insensitive Ca2+ entry pathway remained open for more than 6 min in Ca(2+)-free buffer. On the other hand, when receptor agonists such as thrombin and U46619 were substituted for thapsigargin, activation of the wortmannin-insensitive Ca2+ entry was transient (Hashimoto et al., J. Biol. Chem (1992) 267, 17078-17081). In the presence of S-145 and wortmannin, thapsigargin stimulated phosphorylation of neither the 20-kDa myosin light chain nor the 47-kDa protein, a substrate of protein kinase C. These results suggest that thapsigargin induces external Ca2+ entry by two mechanisms: (1) a mechanism involving myosin light chain kinase; (2) a mechanism, not activated by receptor agonists, that is independent of the major protein kinases of platelets.

Relationship between the Serum Level of Leptin and Life-Style Habits in Japanese Men

Aims/Methods: To investigate whether smoking affects the serum level of leptin, 708 male workers aged 25–65 years old were cross-sectionally surveyed. Results: Multiple regression analysis indicated that among the various parameters examined, the level of leptin was positively associated with the body mass index and the levels of insulin, total cholesterol and uric acid, and was inversely associated with physical activity and the level of creatinine. The partial correlation coefficient of leptin was highest against the body mass index (r = 0.40), followed by insulin (0.29) and physical activity (–0.14), after adjustment for other leptin-related variables. However, no association was observed between the level of leptin and smoking (0.05), alcohol consumption (0.09) or age (0.09). Conclusions: The findings suggest that among life-style habits, physical activity, but not smoking or alcohol consumption, significantly affects the serum level of leptin in Japanese men.

Protein kinase C plays a key role in the cross-talk between intracellular signalings via prostanoid receptors in a megakaryoblastic cell line, MEG-01s

In a previous study, we characterized prostanoid and thrombin receptors expressed on a megakaryoblastic cell line, MEG-01s (Blood 78, 2328-2336, 1991). In this study, we examines the mechanism of cross-talk between intracellular Ca2+ ([Ca2+]i) and cAMP signalings through prostanoid and thrombin receptors. Addition of a thromboxane (TX)A2 mimetic (U46619 or STA2) or thrombin stimulated the formation of inositol phosphates and dose-dependently augmented a prostaglandin (PG)I2 mimetic (iloprost)- or forskolin-induced cAMP formation. 12-O-tetradecanoylphorbol-13-acetate (TPA) and ionomycin, to lesser extent, also augmented iloprost-induced cAMP formation. The enhancing effect of U46619 or TPA on cAMP formation was inhibited by prolonged pretreatment of the cells with TPA (2.5 microM, 24 h), but not with calmodulin-antagonists; W-7, W-5, or KN-62. The elevation of [Ca2+]i induced by thrombin, STA2 or PGE2 was significantly suppressed by pretreatment of the cells with TPA (100 nM) as well as cAMP mimetics such as dibutyryl cAMP (5 mM), forskolin (5 microM) and iloprost (1 microM). These results suggest the key role of PKC on the cross-talk between [Ca2+]i and cAMP signalings through prostanoid and thrombin receptors; PKC, which is activated with TXA2 or thrombin, concomitantly suppress further [Ca2+]i elevation and enhances the PGI2 receptor-mediated cAMP formation, which, in turn, suppress [Ca2+]i elevation.

Genetic analysis of phytosterolaemia.

Two women with multiple xanthomas, intermittent arthritis and thrombocytopenia were diagnosed as phytosterolaemia, an autosomal-recessive lipid storage disease, based on their increased serum concentrations of β-sitosterol, campesterol and sitostanol. The gene responsible for this disease is located within a distance of 18 cM between microsatellite markers of D2S1788 and D2S1352 at chromosome 2p21. We genotyped the patients and their family members with 16 microsatellite markers around this locus. The results from the homozygosity mapping of one family suggested that the gene was located within the distance of 12.6 cM between D2S2328 and D2S1352. We have shortened the genetic distance by 5.4 cM.