市原 佐保子

We have investigated the effect of benzo[a]pyrene (B[a]P), a carcinogen of tobacco smoke and an agonist for the aryl hydrocarbon receptor (AHR), on hypoxia-induced angiogenesis. Ischemia was induced by femoral artery ligation in wild-type and AHR-null mice, and the animals were subjected to oral administration of B[a]P (125 mg/kg) once a week. Exposure to B[a]P up-regulated the expression of metallothionein in the ischemic hindlimb and markedly inhibited ischemia-induced angiogenesis in wild-type mice. The amounts of interleukin-6 and of vascular endothelial growth factor (VEGF) mRNA in the ischemic hindlimb of wild-type mice were reduced by exposure to B[a]P. These various effects of B[a]P were markedly attenuated in AHR-null mice. Our observations suggest that the loss of the inhibitory effect of B[a]P on ischemia-induced angiogenesis apparent in AHR-null mice may be attributable to maintenance of interleukin-6 expression and consequent promotion of angiogenesis through up-regulation of VEGF expression.

Despite recent advances in acute stroke therapy, stroke remains the leading cause of severe disability and the third leading cause of death, after heart disease and cancer, in Western countries and Japan. The identification of biomarkers of stroke risk is thus important both for risk prediction and for intervention to avert future events. Although genetic linkage analyses of families and sib-pairs as well as candidate gene and genome-wide association studies have implicated several loci and candidate genes in predisposition to ischemic or hemorrhagic stroke, the genes that contribute to genetic susceptibility to these conditions remain to be identified definitively. Given that vascular inflammation has been recognized as an important mechanism of atherosclerotic disease, proinflammatory genes may play pivotal roles in the pathogenesis of ischemic stroke. In this review, we summarize candidate genes that have been implicated in common forms of ischemic stroke by linkage analyses and association studies. We also review in more detail studies that have revealed an association of ischemic stroke with polymorphisms of proinflammatory genes of particular interest (LTA, IL6, and ALOX5AP) as well as with polymorphisms at chromosomal region 9p21.3, which has recently been identified as a susceptibility locus for coronary heart disease. Such studies may provide insight into the function of implicated genes as well as into the role of genetic factors in the development of ischemic stroke.

BACKGROUND AND PURPOSE: Although genetic epidemiologic studies have implicated several genetic variants as risk factors for ischemic or hemorrhagic stroke, the genetic determinants of these conditions remain largely unknown. We performed an association study to identify gene polymorphisms that confer susceptibility to atherothrombotic cerebral infarction, intracerebral hemorrhage, or subarachnoid hemorrhage. METHODS: The study population comprised 3432 unrelated Japanese individuals: 1362 stroke patients (822 with atherothrombotic cerebral infarction, 333 with intracerebral hemorrhage, and 207 with subarachnoid hemorrhage) and 2070 controls. The genotypes for 50 polymorphisms of 38 candidate genes were determined by a method that combines the polymerase chain reaction and sequence-specific oligonucleotide probes with suspension array technology. RESULTS: An initial chi(2) test (false discovery rate <0.05) and subsequent multivariable logistic-regression analysis with adjustment for conventional risk factors (P<0.05) revealed that the -14C-->T polymorphism (rs1800977) of ABCA1, the A-->C (rs3027898) and C-->T (Ser532Leu, rs1059703) polymorphisms of IRAK1, and the G-->C (Cys2229Ser) polymorphism (rs619203) of ROS1 were significantly associated with atherothrombotic cerebral infarction; that the -428G-->A polymorphism (rs710968) of LIMK1 was significantly associated with intracerebral hemorrhage; and that the 13989A-->G (Ile118Val) polymorphism (NC_000007.12) of CYP3A4 was significantly associated with subarachnoid hemorrhage. CONCLUSIONS: Genotypes for ABCA1, IRAK1, and ROS1 may prove useful for assessment of the genetic risk for atherothrombotic cerebral infarction, whereas those for LIMK1 and CYP3A4 may be similarly beneficial in assessment of the genetic risk for intracerebral hemorrhage and subarachnoid hemorrhage, respectively. Validation of these findings will require additional studies with independent subject panels.

The aim of the present study was to identify gene polymorphisms that confer susceptibility to obesity. A total of 5448 unrelated Japanese individuals from two independent populations were examined: subject panel A comprised 4252 individuals who visited participating hospitals; subject panel B comprised 1196 community-dwelling elderly individuals. The genotypes for 95 polymorphisms of 67 candidate genes were determined. The chi(2) test revealed that six polymorphisms were related (p<0.05) to the prevalence of obesity in subject panel A; after application of Bonferroni's correction, however, only the 2677G --> A/T polymorphism (rs2032582) of the ATP-binding cassette, subfamily B, member 1 gene (ABCB1) was significantly associated (p=0.0003) with obesity. Subsequent multivariable logistic regression analysis also revealed that the 2677G --> A/T polymorphism of ABCB1 was significantly associated with obesity. For validation of this association, the 2677G --> A/T polymorphism of ABCB1 was examined in subject panel B and again found to be significantly associated with obesity. Body mass index was significantly (p=0.01) greater for individuals with the variant T allele of this polymorphism than for those with the GG genotype in the combined subject panels A and B. Our results suggest that the ABCB1 genotype may prove informative for assessment of genetic risk for obesity in Japanese individuals.

Metabolic syndrome is a risk factor for cardiovascular disease. The aim of the present study was to identify genetic variants that confer susceptibility to atherothrombotic cerebral infarction among individuals with metabolic syndrome in order to allow prediction of genetic risk for this condition. The study population comprised 1284 unrelated Japanese individuals with metabolic syndrome, including 313 subjects with atherothrombotic cerebral infarction and 971 controls. The genotypes for 296 polymorphisms of 202 candidate genes were determined with a method that combines the polymerase chain reaction and sequence-specific oligonucleotide probes with suspension array technology. The Chi-square test, multivariable logistic regression analysis with adjustment for age, sex, body mass index, and the prevalence of hypertension, hypercholesterolemia, and diabetes mellitus, as well as a stepwise forward selection procedure revealed that the 2445G-->A (Ala54Thr) polymorphism (rs1799883) of FABP2, the -108/3G-->4G polymorphism of IPF1 (S82168), the A-->G (Thr94Ala) polymorphism (rs2241883) of FABP1, the G-->A (Asp2213Asn) polymorphism (rs529038) of ROS1, the -11377C-->G polymorphism (rs266729) of ADIPOQ, the 162A-->C polymorphism (rs4769055) of ALOX5AP, the -786T-->C polymorphism (rs2070744) of NOS3, and the 3279C-->T polymorphism (rs7291467) of LGALS2 were associated (P<0.05) with the prevalence of atherothrombotic cerebral infarction. Among these polymorphisms, the 2445G-->A (Ala54Thr) polymorphism of FABP2 was most significantly associated with this condition. Our results suggest that FABP2, IPF1, FABP1, ROS1, ADIPOQ, ALOX5AP, NOS3, and LGALS2 are susceptibility loci for atherothrombotic cerebral infarction among Japanese individuals with metabolic syndrome. Genotypes for these polymorphisms, especially for the 2445G-->A (Ala54Thr) polymorphism of FABP2, may prove informative for the prediction of genetic risk for atherothrombotic cerebral infarction among such individuals.

Although various environmental factors, such as a high-salt diet, a smoking habit, excessive alcohol intake, and physical inactivity, influence the development of hypertension, genetic variation also contributes to an individual's susceptibility to this condition. The purpose of the present study was to identify gene polymorphisms that confer susceptibility or resistance to hypertension, and thereby contribute to the prediction of the genetic risk for this condition. The study population comprised 2752 unrelated Japanese individuals (1370 men, 1382 women), including 1276 subjects with hypertension (774 men, 502 women) and 1476 controls (596 men, 880 women). The genotypes for 50 polymorphisms of 35 candidate genes were determined by a method that combines polymerase chain reaction and sequence-specific oligonucleotide probes with suspension array technology. Evaluation of genotype distributions by the Chi-square test and subsequent multivariable logistic regression analysis with adjustment for age, sex, body mass index, smoking status, and the prevalence of diabetes mellitus and hypercholesterolemia revealed that the -14C-->T polymorphism of ABCA1, the C-->G (Ser2229Cys) polymorphism of ROS1, the C-->T (Asn591Asn) polymorphism of LDLR, the 13989A-->G (Ile118Val) polymorphism of CYP3A4, the C-->G and A-->C polymorphisms of ADIPOR1, and the -519A-->G polymorphism of MMP1 were significantly (P<0.05) associated with the prevalence of hypertension. Systolic and diastolic blood pressure differed significantly among genotypes for the -14C-->T polymorphism of ABCA1 and the C-->G (Ser2229Cys) polymorphism of ROS1, with the variant T and G alleles, respectively, being related to increased blood pressure. These results suggest that polymorphisms of ABCA1 and ROS1 are determinants of blood pressure and the development of hypertension in Japanese individuals. Determination of genotypes for ABCA1 and ROS1 may thus prove informative for the prediction of the genetic risk for hypertension.

Myocardial infarction (MI) is an important clinical problem because of its large contribution to mortality. The main causal and treatable risk factors for MI include hypertension, hypercholesterolemia or dyslipidemia, diabetes mellitus, and smoking. In addition to these risk factors, recent studies have shown the importance of genetic factors and interactions between multiple genes and environmental factors. Disease prevention is an important strategy for reducing the overall burden of MI, with the identification of markers for disease risk being key both for risk prediction and for potential intervention to lower the chance of future events. Although genetic linkage analyses of families and sib-pairs as well as candidate gene and genome-wide association studies have implicated several loci and candidate genes in predisposition to coronary heart disease (CHD) or MI, the genes that contribute to genetic susceptibility to these conditions remain to be identified definitively. In this review, we summarize both candidate loci for CHD or MI identified by linkage analyses and candidate genes examined by association studies. We also review in more detail studies that have revealed the association with MI or CHD of polymorphisms in MTHFR, LPL, and APOE by the candidate gene approach and those in LTA and at chromosomal region 9p21.3 by genome-wide scans. Such studies may provide insight into the function of implicated genes as well as into the role of genetic factors in the development of CHD and MI. © 2008 Springer Science+Business Media B.V.

Previous experiments indicated that 1-bromopropane (1-BP), an alternative to chloroflurocarbons, is neurotoxic and inhibits spermiation in the testis. Here we investigated the reversibility of the toxic effects of 1-BP in rats. Male Wistar rats were divided into three equal groups of 24 each and exposed by inhalation to 0, 400 or 1000 ppm of 1-BP for 6 weeks (8 hrs/day, 7 days/week). Eight rats from each group were sacrificed at the end of 6 weeks exposure, and at 4 and 14 weeks after the end of exposure, to assess the recovery processes. We studied sperm count, motility, morphology and testicular histopathology, as well as blood pressure, skin temperature and hindlimb muscle strength. At the end of 6 weeks of exposure to 1000 ppm (0 week recovery), testicular weight, epididymal weight, sperm count and motility were low, morphologically abnormal sperm were increased and spermatogenic cells showed diffuse degeneration. These changes did not show full recovery at 14 weeks recovery, with the exception of the prostate and seminal vesicular weights, which recovered back to control values. At 400 ppm, increased retained spermatids at 0 week recovery returned to normal at 4 weeks recovery. Exposure to 1000 ppm produced sustained reduction of hindlimb muscle strength at 14 weeks recovery, whereas normalization of the skin temperature and blood pressure was noted after transient changes. Our study showed that the effect of 1-BP on spermatogenesis is dose-dependent; low exposure inhibited spermiation and hormone-dependent organ weight reduction and these changes were transient, while a higher dose of 1000 ppm 1-BP caused persistent depletion of spermatogenic cells.

Although several environmental factors, including a high-calorie diet and physical inactivity, influence the development of type 2 diabetes mellitus, genetic factors have been shown to contribute to individual susceptibility to this condition. The purpose of the present study was to identify gene polymorphisms that confer susceptibility or resistance to type 2 diabetes mellitus, and thereby to contribute to assessment of the genetic risk for this condition. The study population comprised 52.59 unrelated Japanese individuals (2980 men, 2279 women), including 1640 subjects with type 2 diabetes mellitus (1071 men, 569 women) and 3619 controls (1909 men, 1710 women). The genotypes for 94 polymorphisms of 67 genes were determined with a method that combines the polymerase chain reaction and sequence-specific oligonucleotide probes with suspension array technology. Evaluation of genotype distributions by the chi-square test revealed that the 13989A -&gt; G (I1e118Va1) polymorphism of the cytochrome P450, subfamily IIIA, polypeptide 4 gene (CYP3A4) was significantly (false discovery rate, 0.000009) associated with the prevalence of type 2 diabetes mellitus. Multivariable logistic regression analysis with adjustment for age and sex also revealed that the 13989A -&gt; G (Ile118Va1) polymorphism of CYP3A4 was significantly (P=0.00002) associated with the prevalence of type 2 diabetes mellitus, with the AG genotype being protective against this condition. Genotyping for CYP3A4 may thus prove informative for assessment of the genetic risk for type 2 diabetes mellitus.

The purpose of the present study was to identify genetic variants that confer susceptibility to dyslipidemia. A total of 5213 individuals from two independent populations were examined: Subject panel A comprised 3794 individuals who visited participating hospitals; subject panel B comprised 1419 community-dwelling elderly individuals. The genotypes for 100 polymorphisms of 65 candidate genes were determined. The chi(2) test and multivariable logistic regression analysis revealed that seven polymorphisms of APOA5, APOC3, APOA1, ACAT2, and LPL were significantly associated with hypertriglyceridemia, six polymorphisms of APOA5, LIPC, and CYP3A4 with low HDL-cholesterol, and three polymorphisms of APOE and CCR2 with high LDL-cholesterol in subject panel A. For validation of these associations, the same polymorphisms were examined in subject panel B. Six polymorphisms of APOA5, APOC3, APOA1, and LPL were again significantly associated with hypertriglyceridemia, three polymorphisms of APOA5 with low HDL-cholesterol, and two polymorphisms of APOE with high LDL-cholesterol. Serum triglyceride, HDL-cholesterol, and LDL-cholesterol concentrations differed significantly among genotypes of these corresponding polymorphisms in both subject panels. These results indicate that polymorphisms of APOA5, APOC3, APOA1, and LPL are determinants of hypertriglyceridemia and that those of APOA5 and APOE are determinants of low HDL-cholesterol and high LDL-cholesterol, respectively, in Japanese individuals.

Sepsis is characterized by various symptoms, signs and underlying pathophysiology. To investigate possible mechanisms underlying this diversity, we compared the cardiovascular effects of lipopolysaccharide (LPS) derived from Escherichia coli (E-LPS) with those of LPS from Pseudomonas aeruginosa (P-LPS) in rats. We also examined the possible roles of tumor necrosis factor-alpha (TNF-alpha) and oxidative stress in LPS-induced cardiovascular damage. E-LPS (10 mg/kg body weight) or P-LPS (2 mg/kg body weight) was administered intravenously to Wistar rats. Echocardiography was serially performed. E-LPS induced an increase in left ventricular fractional shortening that persisted for at least 6 h, whereas P-LPS elicited an initial increase and a subsequent decrease in this parameter. Histological analysis revealed that P-LPS induced interstitial edema, congestion, intramyocardial bleeding, myocardial necrosis, infiltration of inflammatory cells, and formation of fibrin thrombi in the heart, whereas no pathological changes were apparent in the hearts of rats treated with E-LPS. Furthermore, the plasma concentration of TNF-alpha in rats treated with P-LPS was greater than that in rats treated with E-LPS, but the glutathione redox ratio in the heart was not affected by either type of LPS. In conclusion, E-LPS and P-LPS induced distinct patterns of functional and structural responses in the cardiovascular systems of rats. These differential responses may be attributable in part to the difference in the associated increases in the plasma concentration of TNF-alpha. The cardiovascular effects of LPS thus depend on the causative organisms.

Cardiotoxicity is a treatment-limiting side effect of the anticancer drug doxorubicin (DOX). We have now investigated the roles of oxidative stress and signaling by the protein kinase Akt in DOX-induced cardiotoxicity as well as the effects on such toxicity both of fenofibrate, an agonist of peroxisome proliferator-activated receptor-alpha, and of polyethylene glycol-conjugated superoxide dismutase (PEG-SOD), an antioxidant. Mice injected intraperitoneally with DOX were treated for 4 days with fenofibrate or PEG-SOD. Fenofibrate and PEG-SOD each prevented the induction of cardiac dysfunction by DOX. Both drugs also inhibited the activation of the transcription factor NF-kappaB and increase in lipid peroxidation in the left ventricle induced by DOX, whereas only PEG-SOD inhibited the DOX-induced activation of Akt and Akt-regulated gene expression. These results suggest that fenofibrate and PEG-SOD prevented cardiac dysfunction induced by DOX through normalization of oxidative stress and redox-regulated NF-kappaB signaling.

OBJECTIVE: The aryl hydrocarbon receptor (AHR) is a transcription factor that binds to DNA as a heterodimer with the AHR nuclear translocator (ARNT) after interaction with ligands such as polycyclic and halogenated aromatic hydrocarbons found in tobacco smoke and the environment. We have investigated the interaction between AHR and hypoxia signaling pathways in regulation of angiogenesis with the use of a surgical model of ischemia. METHODS AND RESULTS: Ischemia was induced by femoral artery occlusion in wild-type and AHR-null mice. Ischemia-induced angiogenesis was markedly enhanced in AHR-null mice compared with that in wild-type animals. Ischemia-induced upregulation of the expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and ARNT as well as that of target genes for these transcription factors, such as that for vascular endothelial growth factor (VEGF), were also enhanced in AHR-null mice. Furthermore, the DNA binding activity of the HIF-1alpha-ARNT complex as well as the association of HIF-1alpha and ARNT with the VEGF gene promoter were increased by ischemia to a greater extent in AHR-null mice than in wild-type animals. CONCLUSIONS: Ablation of AHR resulted in enhancement of ischemia-induced angiogenesis. This effect was likely attributable in part to the associated enhancement of ischemia-induced VEGF expression, which in turn may be caused by an increased abundance and activity of the HIF-1alpha-ARNT heterodimer.

Objectives: The aim of the study was to identify gene polymorphisms that confer susceptibility to metabolic syndrome in order to allow reliable assessment of genetic risk for this condition. Methods and results: The study population comprised [788 unrelated Japanese individuals (1033 men, 755 women), including 1017 subjects with metabolic syndrome (634 men, 383 women) and 771 controls (399 men, 372 women). The genotypes for 158 polymorphisms of 133 candidate genes were determined with a method that combines the polymerase chain reaction and sequence-specific oligonucleotide probes with suspension array technology. Multivariable logistic regression analysis with adjustment for age, sex, and the prevalence of smoking revealed that the - 1131T -&gt; C polymorphism of the apolipoprotein A-V gene (APOA5) was significantly associated with the prevalence of metabolic syndrome, with the C allele representing a risk factor for this condition. A stepwise forward selection procedure demonstrated that APOA5 genotype (CC+ TC versus TT) significantly affected the prevalence of metabolic syndrome. The C allele of this polymorphism was associated with an increased serum concentration of triglycerides and a decreased concentration of HDL-cholesterol. Conclusions: Genotype for APOA5 may prove reliable for assessment of genetic risk for metabolic syndrome. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

Type 2 diabetes mellitus is a complex metabolic disorder in which endogenous sex hormones may contribute to sex-dependent etiologies. We hypothesized that genetic variants related to type 2 diabetes mellitus might differ between men and women. We thus performed a large-scale association study to identify gene polymorphisms associated with type 2 diabetes mellitus in men and women separately. The study population comprised 4854 unrelated Japanese individuals (2688 men, 2166 women), including 1490 subjects with type 2 diabetes mellitus (969 men, 521 women). The genotypes for 16 gene polymorphisms were determined with a method that combines the polymerase chain reaction and sequence-specific. oligonucleotide probes with suspension array technology. Multivariable logistic regression analysis with adjustment for age, body mass index, and smoking status revealed that the T-G (3' UTR) polymorphism of the thrombospondin 2 gene (THBS2), the -603A -&gt; G polymorphism of the coagulation factor III gene (F3), and the G,T (intron 2) polymorphism of the adipocyte, C1Q, and collagen domain containing (adiponectin) gene (ADIPOQ) were significantly associated with the prevalence of type 2 diabetes mellitus in men, and that the A-G (Arg160Gly) polymorphism of the paraoxonase 1 gene (PON1) was significantly associated with this condition in women. A stepwise forward selection procedure demonstrated that genotypes of THBS2, F3, and ADIPOQ were significant determinants of type 2 diabetes mellitus in men, and that genotype of PON1 significantly affected this condition in women. Genotyping of these polymorphisms may prove informative for assessment of the genetic component of type 2 diabetes mellitus for men and women separately.

Papillary breast lesions remain a source of diagnostic confusion because the full range of epithelial proliferations may arise within, or secondarily involve, papilloma. The expression of p63 and high-molecular-weight cytokeratins (HMWCK) was studied simultaneously in 33 papillary lesions including intraductal papilloma (IP, n = 10), atypical papilloma (AP, n = 8) and intraductal papillary carcinoma (IPC, n = 15) by double immunostaining. The myoepithelial cell nuclei were stained dark brown whereas the cytoplasms of usual ductal hyperplasia (UDH) and myoepithelium were stained purple. The myoepithelial layer was recognized as a dark brown dotted line at the epithelial stromal junction in all IP (10/10), most AP (7/8) and some IPC (7/15), suggesting that the retained myoepithelial layer in the papillary processes does not necessarily guarantee benignity. However, the malignant epithelial cells in AP and IPC were typically recognized as monotonous populations unstained with either chromogen. These monotonous cells contrasted with the proliferating cells of UDH in papilloma, which had intense purple cytoplasm in a mosaic-like fashion. The present data suggest that the double immunostaining with the two popular antibodies p63 and HMWCK is a useful tool for reproducible classification of papillary breast lesions. © 2007 The Authors.

The purpose of the present study was to assess the genetic risk for myocardial infarction (MI) in individuals with or without conventional coronary risk factors and thereby to contribute to the personalized prevention of MI in such individuals. The study population comprised 3483 unrelated Japanese individuals (1913 men, 1570 women). The 1192 subjects with MI (926 men, 266 women) and 2291 controls (987 men, 1304 women) either had or did not have conventional coronary risk factors, including hypertension, hypercholesterolemia, and diabetes mellitus. The genotypes for 164 polymorphisms of 137 candidate genes were determined by a method that combines the polymerase chain reaction and sequence-specific oligonucleotide probes with suspension array technology. Multivariable logistic regression analysis and a stepwise forward selection procedure revealed that nine different polymorphisms were significantly (P &lt; 0.005) associated with MI among individuals with or without hypertension, hypercholesterolemia, or diabetes mellitus: 1018C -&gt; T of GP1BA, -108/3G -&gt; 4G of IPF1, 677C -&gt; T of MTHFR, and G -&gt; A of UTS2 in hypertensive individuals; 2445G -&gt; A of FABP2, -108/3G -&gt; 4G of IPFI, 677C -&gt; T of MTHFR, -11,377C -&gt; G of ACDC, A -&gt; G of AKAP10, 11,496G -&gt; A of F7, and 46C -&gt; T of F12 in individuals without hypercholesterolemia; 2445G -&gt; A of FABP2 in diabetic individuals; and -108/3G -&gt; 4G of IPF1 in nondiabetic individuals. Polymorphisms associated with MI may thus differ among individuals with different conventional coronary risk factors. Stratification of subjects on the basis of such risk factors may thus be important in order to achieve personalized prevention of MI with the use of genetic information.

1. Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) manifest pleiotropic effects that may contribute to their therapeutic efficacy. However, the mechanism of the beneficial action of statins on cardiac hypertrophy and fibrosis remains unclear. We have now investigated this action of pitavastatin in Dahl salt-sensitive (DS) rats. 2. The DS rats progressively develop marked hypertension when fed a diet containing 8% NaCl from 7 weeks of age. These animals exhibited pronounced cardiac hypertrophy and fibrosis, as well as upregulation of fetal-type cardiac gene expression at 12 weeks of age, compared with DS rats fed a diet containing 0.3% NaCl. The abundance of mRNAs for collagen types I and III, angiotensin-converting enzyme, transforming growth factor-beta 1 and connective tissue growth factor was also increased in the heart of rats on the high-salt diet. 3. Treatment of rats on the high-salt diet with a non-antihypertensive dose of pitavastatin (0.3 or 1 mg/kg per day) from 7 to 12 weeks of age attenuated the development of cardiac hypertrophy and fibrosis, as well as inhibiting the upregulation of cardiac gene expression. Pitavastatin also blocked the translocation of RhoA to the membrane fraction of the left ventricle and RhoA activation, as well as the phosphorylation of the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK)-1 and ERK-2 and an increase in the DNA binding activity of serum response factor (SRF) in the heart induced by the high-salt diet. 4. These findings suggest that the effects of pitavastatin on load-induced cardiac hypertrophy and fibrosis are independent of its cholesterol-lowering action and may be mediated, at least in part, through inhibition of RhoA-ERK-SRF signalling.

Oxidative stress is an important susceptibility factor for dilated cardiomyopathy. We have investigated the effects of bisoprolol, a beta1-selective adrenoceptor blocker, on oxidative stress and the development of cardiac dysfunction in a model of dilated cardiomyopathy. Male TO-2 and control hamsters at 8 weeks of age were treated with bisoprolol (5 mg/kg per day) or vehicle for 4 weeks. Treatment with bisoprolol prevented the progression of cardiac dysfunction in TO-2 hamsters. This drug did not affect the increase in NADPH oxidase activity but prevented the reduction in activity and expression of mitochondrial manganese-dependent superoxide dismutase as well as the increases in the concentrations of interleukin-1beta and tumor necrosis factor-alpha in the left ventricle of TO-2 hamsters. Attenuation of the development of cardiac dysfunction by bisoprolol may thus result in part from normalization of the associated increases in the levels of oxidative stress and pro-inflammatory cytokines in the left ventricle.

The purpose of the present study was to identify gene polymorphisms for the reliable assessment of genetic factors for obesity. The study population comprised 3906 unrelated Japanese individuals (2286 men, 1620 women), including 1196 subjects (677 men, 519 women) with obesity (body mass index of &gt;= 25 kg/m(2)) and 2710 controls (1609 men, 1101 women). The genotypes for 147 polymorphisms of 124 candidate genes were determined with a method that combines the polymerases chain reaction and sequence-specific oligonucleotide probes with suspension array technology. Multivariable logistic regression analysis with adjustment for age, sex, and the prevalence of smoking revealed that the 30G -&gt; A polymorphism of GCK, the -240A -&gt; T polymorphism of ACE, and the -482C -&gt; T polymorphism of APOC3 were significantly (P &lt; 0.01) associated with the prevalence of obesity, and the -1989T -&gt; G polymorphism of ESR1 was almost significantly associated. A stepwise forward selection procedure demonstrated that ACE, GCK, and ESR1 genotypes significantly (P &lt; 0.01) and independently affected the prevalence of obesity. Combined genotype analysis for these three polymorphisms yielded a lowest odds ratio of 0.45 for the combined genotypes of AT or TT for ACE, GG for GCK, and GG for ESR1 in comparison with the combined genotypes of AA for ACE, GG for GCK, and TT or TG for ESR1. Genotypes for ACE, GCK, and ESR1 may prove reliable for the assessment of genetic factors for obesity. Determination of the combined genotypes for these genes may contribute to the personalized prevention of this condition.

The aim of the present study was to assess the genetic risk for atherothrombotic cerebral infarction (ACI) in men and women separately as well as in individuals with or without conventional risk factors for atherosclerosis and thereby to contribute to the personalized prevention of ACI. The study population comprised 2705 unrelated Japanese individuals (1244 men, 1461 women), including 636 subjects (372 men, 264 women) with ACI. Subjects with ACI and controls either had or did not have conventional risk factors for atherosclerosis, including hypertension, hypercholesterolemia, and diabetes mellitus. The genotypes for 202 polymorphisms of 152 candidate genes were determined by a method that combines polymerase chain reaction and sequence-specific oligonucleotide probes with suspension array technology. Multivariable logistic regression analysis and a stepwise forward selection procedure revealed that 11 different polymorphisms were significantly (P &lt; 0.005) associated with ACI in women or men or in individuals with or without hypertension, hypercholesterolemia, or diabetes mellitus: the 584C -&gt; T polymorphism of LIPG, 56656 -&gt; T of EDN1, and G -&gt; A of CCL11 in women; 677C -&gt; T of MTHFR, 1323C -&gt; T of ITGB2. 3932T -&gt; C of APOE, and -231A -&gt; G of EDNRA in men: -572 G -&gt; C of IL6 in hypertensive individuals; -4036 -&gt; A of CCL5 and G-A of COMT in individuals with hypercholesterolemia: and 3932T -&gt; C of APOE and A -&gt; G of TNFSF4 in diabetic individuals. Polymorphisms associated with ACI may thus differ between women and men as well as among individuals with different risk factors. Stratification of subjects on the basis of sex or conventional risk factors for atherosclerosis may therefore be important in order to achieve the personalized prevention of ACI with the use of genetic information.

Background: Although genetic epidemiologic studies have suggested that several genetic variants increase the risk for hypertension, the genes that underlie genetic susceptibility to this condition remain to be identified definitively. We have now performed a large-scale association study to identify gene polymorphisms for reliable assessment of the genetic component of hypertension. Methods: The study population comprised 4853 unrelated Japanese individuals, including 2818 subjects with hypertension (1677 men, 1141 women) and 2035 controls (1011 men, 1024 women). The genotypes for 150 polymorphisms of 128 candidate genes were determined with a method that combines the polymerase chain reaction and sequence-specific oligonucleotide probes with suspension array technology. Results: Multivariable logistic regression analysis with adjustment for age, sex, body mass index, and the prevalence of smoking revealed that four polymorphisms (16486 -&gt; A in ITGA2, -306 -&gt; A in GCK, A -&gt; G in SAH, and 1117C -&gt; A in PTGIS) were significantly (P &lt; .01) associated with hypertension. A stepwise forward selection procedure demonstrated that ITGA2, GCK, and PTGIS genotypes significantly affected the prevalence of hypertension. Combined genotype analysis of these polymorphisms yielded a lowest odds ratio of 0.47 for the genotypes of AA or AG for ITGA2, GA or AA for GCK, and CC for PTGIS, which were present in 1.1% and 2.0% of hypertensive and control individuals, respectively. Conclusions: These results suggest that the genotypes for ITGA2, GCK, and PTGIS may prove reliable for the assessment of the genetic component of hypertension. Determination of the combined genotypes for these genes may contribute to personalized prevention of this condition.

Objective-We performed an association study to identify gene polymorphisms for assessing the genetic risk of ischemic or hemorrhagic stroke. Methods and Results-The study population comprised 3151 unrelated Japanese individuals: 1141 stroke patients (636 with atherothrombotic cerebral infarction, 282 with intracerebral hemorrhage, and 223 with subarachnoid hemorrhage) and 2010 controls. The genotypes for 202 polymorphisms of 152 genes were determined by suspension array technology. Multivariable logistic regression analysis with adjustment for conventional risk factors revealed that the -572G -&gt; C polymorphism of the interleukin-6 (IL-6) gene (IL6) was significantly (P &lt; 0.001) associated with both atherothrombotic cerebral infarction and intracerebral hemorrhage and that the -55C -&gt; T polymorphism of the uncoupling protein 3 gene (UCP3), the -863C -&gt; A polymorphism of the tumor necrosis factor (TNF) gene (TNF), and the G-A (Gly243Asp) polymorphism of the polycystic kidney disease 1-like gene (PKD1-like) were significantly associated with subarachnoid hemorrhage. Conclusions-IL6 genotype may be useful in assessing the genetic risk for atherothrombotic cerebral infarction and intracerebral hemorrhage, and genotypes for UCP3, TNF, and PKD1-Iike may be similarly beneficial in assessment of the risk for subarachnoid hemorrhage. Validation of our findings will require additional studies with independent subject panels.

Peroxisome proliferator-activated receptor-alpha (PPAR-alpha) is a key regulator of lipid and glucose metabolism and is implicated in inflammation. We investigated the effects of the PPAR-alpha activator fenofibrate on, as well as the role of redox-regulated transcription factors, in the development of left ventricular (LV) hypertrophy and heart failure in Dahl salt-sensitive (DS) rats. DS rats were fed a high-salt diet and treated with either fenofibrate (30 or 50 mg/kg per day) or vehicle from 7 weeks of age. Fenofibrate inhibited the development of compensated hypertensive LV hypertrophy, attenuated the LV relaxation abnormality and systolic dysfunction, and improved the survival rate in DS rats. It also prevented a decrease in the ratio of reduced to oxidized glutathione and inhibited up-regulation of the DNA binding activities of the redox-regulated transcription factors NF-kappaB, AP-1, Egr-1, SP1, and Ets-1 induced in the left ventricle by the high-salt diet. Expression of target genes for these transcription factors, including those for adhesion molecules (VCAM-1, ICAM-1), cytokines (MCP-1), growth factors (TGF-beta, PDGF-B), and osteopontin, was also increased by the high-salt diet in a manner sensitive to treatment with fenofibrate. Furthermore, the infiltration of macrophages and T lymphocytes into the left ventricle and the increase in the plasma concentration of C-reactive protein were inhibited by fenofibrate. The PPAR-alpha activator fenofibrate thus attenuated the progression of heart failure and improved the survival rate in this rat model. These effects were associated with inhibition of the inflammatory response and of activation of redox-regulated transcription factors in the left ventricle.

The purpose of the present study was to identify gene polymorphisms for reliable assessment of genetic factors for type 2 diabetes mellitus. The study population comprised 4853 unrelated Japanese individuals (2688 men, 2165 women), including 1489 subjects with type 2 diabetes mellitus (969 men, 520 women) and 3364 controls (1719 men, 1645 women). The genotypes for 148 polymorphisms of 124 candidate genes were determined with a method that combines polymerase chain reaction and sequence-specific oligonucleotide probes with suspension array technology. Sixteen polymorphisms were related (p &lt; 0.05) to the prevalence of type 2 diabetes mellitus as determined by the chi-square test. Multivariable logistic regression analysis with adjustment for age, sex, and the prevalence of smoking revealed that, among these polymorphisms, the -603A -&gt; G polymorphism of the gene for coagulation factor III (F3) was significantly (p &lt; 0.001) associated with the prevalence of type 2 diabetes mellitus, with the -603G allele representing a risk factor for this condition. A stepwise forward selection procedure demonstrated that F3 genotype (GG versus AA + AG) significantly (p &lt; 0.00 1) and independently affected the prevalence of type 2 diabetes mellitus. Genotype for F3 may prove reliable for assessment of genetic factors for type 2 diabetes mellitus. Determination of the genotype for this gene may contribute to personalized prevention of this condition.

Although lifestyle and environmental factors influence the prevalence of myocardial infarction, genetic epidemiological studies have suggested that several genetic variants increase the risk for this condition. We have performed a large-scale association study to identify gene polymorphisms for reliable assessment of the genetic risk of myocardial infarction. The study population comprised 3,483 unrelated Japanese individuals (1,913 men; 1,570 women), including 1, 192 subjects with myocardial infarction and 2,291 controls. The genotypes for 164 polymorphisms of 137 candidate genes were determined with an oligonucleotide ligation assay based on analysis of fluorescent microspheres with suspension array technology. Multivariable logistic regression analysis with adjustment for age, sex, body mass index, and the prevalence of smoking, hypertension, diabetes mellitus, and hypercholesterolemia revealed that the 677C -&gt; T (Ala222Val) polymorphism of MTHFR, the 1595C -&gt; G (Ser447Stop) polymorphism of LPL, and the -108/3G -&gt; 4G polymorphism of IPF1 were significantly associated with the prevalence of myocardial infarction. A stepwise forward selection procedure demonstrated that IPF1, MTHFR, and LPL genotypes significantly affected the prevalence of myocardial infarction. Combined genotype analysis of these polymorphisms yielded a maximum odds ratio of 2.54 for the combined genotype of TT for MTHFR, CC for LPL, and 3G3G for IPF1. The genotypes for MTHFR, LPL, and IPF1 may prove reliable for assessment of genetic risk for myocardial infarction. Determination of the combined genotype for these genes may contribute to primary, personalized prevention of this condition.

1-Bromopropane (1-BP) exhibits neuroreproductive toxicities in adult rats and humans. Here, we determined the effects of exposure of rat dams to 1-BP during pregnancy and lactation on the growth and sexual maturation of their offspring. In Experiment 1, 40 rats were exposed to 0, 100, 400 and 800ppm 1-BP during pregnancy and lactation for 8h/day. Ten rats that were not placed in chambers throughout the experiment served to observe the effect of separation of dams from offspring. In Experiment 2, three groups of 10 pregnant rats each were exposed to fresh air in three chambers and 10 other rats were exposed to 800ppm 1-BP during pregnancy and lactation for 8h/day. After delivery, offspring of the exposed and non-exposed dams were swapped so that they were nursed by the opposite dams. In Experiment 1, the survival rate and body weight of offspring were lower than the non-exposed in 1-BP dose-dependent manner. In Experiment 2, the survival rate and body weight of offspring (Group A) nursed by exposed dams and those (Group B) of exposed dams were significantly lower than non-exposed groups. The body weight of Group A was lower than that of Group B, although the two groups showed a significant equal decrease in the survival rate. The number of dead offspring from Group A was significantly higher. Our results indicate that exposure to 1-BP during pregnancy and lactation has comparable effects on survival rate, but exposure during lactation has a more adverse effect on growth of offspring than that during pregnancy. Moreover, exposure during lactation is associated with reduced early survival of third generation (F2) rats.

Statin therapy may be associated with lower mortality in patients with heart failure, but the underlying mechanism of such an association is unknown. We have evaluated the effects of pitavastatin on cardiac function and survival in a rat model of hypertensive heart failure and investigated the molecular mechanism of the observed effects. Dahl salt-sensitive rats fed with high-salt diet from 7 weeks of age developed compensatory left ventricular hypertrophy at 12 weeks and heart failure at 19 weeks. Dahl salt-sensitive rats were treated with either vehicle or pitavastatin (0.3 mg/kg per day) from 7 or 12 weeks. Both early-onset and late-onset pitavastatin treatment reduced left ventricular fibrosis, improved cardiac function, and increased the survival rate apparent at 19 weeks. The increases in the expression levels of hypertrophic, profibrotic, and metalloproteinase genes as well as in gelatinase activities in the heart induced by the high-salt diet were suppressed by pitavastatin treatment. Furthermore, the level of cardiac endothelin-1 was increased in association with the development of heart failure in a manner sensitive to treatment with pitavastatin. Both early and late pitavastatin treatment thus improved cardiac function and survival, with modulation of extracellular matrix remodeling and endothelin-1 signaling possibly contributing to these beneficial effects.

Chronic elevation of plasma aldosterone contributes to heart failure. Mineralocorticoid receptor (MR) antagonism is cardioprotective in such a setting, but whether such protection occurs in the presence of low-aldosteroneconcentrations remains unclear. Weinvestigated whether MR blockade attenuates cardiac hypertrophy and failure in rats with salt-sensitive hypertension. Dahl salt-sensitive (DS) rats fed a high-salt diet from 7 weeks develop concentric left ventricular (LV) hypertrophy secondary to hypertension at 12 weeks followed by heart failure at 19 weeks (DS-CHF). DS rats on such a diet were treated with a non-antihypertensive dose of the selective MR antagonist eplerenone from 12 to 19 weeks. Renin activity and aldosterone concentration in plasma were decreased in DS-CHF rats compared with controls. LV hypertrophy and fibrosis, as well as macrophage infiltration around coronary vessels, were apparent in DS-CHF rats. The amounts of mRNAs for 11beta-hydroxysteroid dehydrogenase type 1, MR, monocyte chemoattractant protein 1, and osteopontin were increased in these hearts. Treatment of DS-CHF rats with eplerenone inhibited these changes in gene expression, as well as coronary va

Chronic elevation of plasma aldosterone contributes to heart failure. Mineralocorticoid receptor ( MR) antagonism is cardioprotective in such a setting, but whether such protection occurs in the presence of low-aldosterone concentrations remains unclear. We investigated whether MR blockade attenuates cardiac hypertrophy and failure in rats with salt-sensitive hypertension. Dahl salt-sensitive ( DS) rats fed a high-salt diet from 7 weeks develop concentric left ventricular ( LV) hypertrophy secondary to hypertension at 12 weeks followed by heart failure at 19 weeks ( DS-CHF). DS rats on such a diet were treated with a non-antihypertensive dose of the selective MR antagonist eplerenone from 12 to 19 weeks. Renin activity and aldosterone concentration in plasma were decreased in DS-CHF rats compared with controls. LV hypertrophy and fibrosis, as well as macrophage infiltration around coronary vessels, were apparent in DS-CHF rats. The amounts of mRNAs for 11 beta-hydroxysteroid dehydrogenase type 1, MR, monocyte chemoattractant protein 1, and osteopontin were increased in these hearts. Treatment of DS-CHF rats with eplerenone inhibited these changes in gene expression, as well as coronary vascular inflammation and heart failure. Eplerenone attenuated both the decrease in the ratio of reduced to oxidized glutathione and the increase in NADPH oxidase activity apparent in DS-CHF rat hearts. MR blockade with eplerenone thus resulted in attenuation of LV hypertrophy and failure, without an antihypertensive effect, in rats with low-aldosterone hypertension. The beneficial cardiac effects of eplerenone are likely attributable, at least in part, to attenuation of myocardial oxidative stress and coronary vascular inflammation induced by glucocorticoid-activated MRs.

OBJECTIVES: Oxidative stress is implicated in the pathogenesis of heart failure and affects the activity of matrix metalloproteinases (MMPs). We have now investigated the role of MMPs and their tissue inhibitors (TIMPs) in the transition from compensated left ventricular (LV) hypertrophy to heart failure as well as the effects of pravastatin on this transition in a rat model. METHODS: Dahl salt-sensitive rats were fed a high-salt (8% NaCl) diet and treated with pravastatin (50 or 100 mg/kg per day) or vehicle from 7 weeks of age. RESULTS: Pravastatin did not attenuate LV hypertrophy apparent at 12 or 18 weeks of age. However, the high dose of this drug markedly improved indices of diastolic function (early diastolic myocardial velocity) and systolic function (LV fractional shortening) at 18 weeks of age and increased the survival rate. It also prevented a decrease in the ratio of reduced to oxidized glutathione and an increase in NADPH oxidase activity in the left ventricle induced by the high-salt diet. The activities of MMP2 and MMP9 and the abundance of TIMP1 and TIMP2 in LV tissue were increased at 18 weeks of age, and pravastatin also prevented these changes. CONCLUSION: Although pravastatin did not attenuate LV hypertrophy, it prevented the transition from compensated hypertrophy to heart failure in this rat model. This effect of pravastatin may result from a reduction both in the level of oxidative stress and in MMP activity in the heart.

Mineralocorticoid receptor antagonism reduces mortality associated with heart failure by mechanisms that remain unclear. The effects of the mineralocorticoid receptor antagonist spironolactone on left ventricular (LV) function and chamber stiffness associated with myocardial fibrosis were investigated in mildly symptomatic patients with idiopathic dilated cardiomyopathy (DCM).

Background-Mineralocorticoid receptor antagonism reduces mortality associated with heart failure by mechanisms that remain unclear. The effects of the mineralocorticoid receptor antagonist spironolactone on left ventricular (LV) function and chamber stiffness associated with myocardial fibrosis were investigated in mildly symptomatic patients with idiopathic dilated cardiomyopathy (DCM). Methods and Results-Twenty-five DCM patients with a New York Heart Association functional class of I or II were examined before and after treatment with spironolactone for 12 months. LV pressures and volumes were measured simultaneously, and LV endomyocardial biopsy specimens were obtained. Serum concentrations of the carboxyl-terminal propeptide ( PIP) and carboxyl-terminal telopeptide (CITP) of collagen type I were measured. The patients were divided into 2 groups on the basis of the serum PIP/CITP ratio (&lt;= 35, group A, n = 12; &gt; 35, group B, n = 13), an index of myocardial collagen accumulation. LV diastolic chamber stiffness, the collagen volume fraction, and abundance of collagen type I and III mRNAs in biopsy tissue were greater and the LV early diastolic strain rate (tissue Doppler echocardiography) was smaller in group B than in group A at baseline. These differences and the difference in PIP/CITP were greatly reduced after treatment of patients in group B with spironolactone, with treatment having no effect on these parameters in group A. The collagen volume fraction was significantly correlated with PIP/CITP, LV early diastolic strain rate, and LV diastolic chamber stiffness for all patients before and after treatment with spironolactone. Conclusions-Spironolactone ameliorated LV diastolic dysfunction and reduced chamber stiffness in association with regression of myocardial fibrosis in mildly symptomatic patients with DCM. These effects appeared limited, however, to patients with increased myocardial collagen accumulation.

Long-term administration of vasodilators increases shear stress, which is thought to be important for vascular growth in the heart. Nicorandil, an activator of ATP-sensitive potassium channels with a nitrate-like action, is a potent vasodilator. We have now investigated the effects of nicorandil on vascular growth and gene expression in the failing heart of Dahl salt-sensitive (DS) hypertensive rats. DS rats fed a high-salt diet from 6 weeks of age develop concentric cardiac hypertrophy secondary to hypertension at 11 weeks, followed by heart failure at 18 weeks. DS rats on such a diet were treated with a nonantihypertensive oral dose of nicorandil (6 mg/kg per day) or vehicle from 11 to 18 weeks of age. Treatment of DS rats with nicorandil improved cardiac function and attenuated the development of heart failure. Myocardial capillary and arteriolar densities did not differ between vehicle-treated DS rats and age-matched controls. The abundance of mRNAs for endothelial NO synthase (eNOS), vascular endothelial growth factor (VEGF), the VEGF receptor Flt-1, and basic fibroblast growth factor (bFGF) in the myocardium was markedly reduced in vehicle-treated DS rats compared with contro

Long-term administration of vasodilators increases shear stress, which is thought to be important for vascular growth in the heart. Nicorandil, an activator of ATP-sensitive potassium channels with a nitrate-like action, is a potent vasodilator. We have now investigated the effects of nicorandil on vascular growth and gene expression in the failing heart of Dahl salt-sensitive (DS) hypertensive rats. DS rats fed a high-salt diet from 6 weeks of age develop concentric cardiac hypertrophy secondary to hypertension at I I weeks, followed by heart failure at IS weeks. DS rats on such a diet were treated with a nonantihypertensive oral dose of nicorandil (6 mg/kg per day) or vehicle from I I to 18 weeks of age. Treatment of DS rats with nicorandil improved cardiac function and attenuated the development of heart failure. Myocardial capillary and arteriolar densities did not differ between vehicle-treated DS rats and age-matched controls. The abundance of mRNAs for endothelial NO synthase (eNOS), vascular endothelial growth factor (VEGF), the VEGF receptor Flt-1, and basic fibroblast growth factor (bFGF) in the myocardium was markedly reduced in vehicle-treated DS rats compared with controls. Treatment of DS rats with nicorandil greatly increased capillary and arteriolar densities and inhibited the downregulation of eNOS, VEGF, fms-like tyrosin kinase-1. and bFGF gene expression. This, nicorandil stimulates coronary capillary and arteriolar growth and thereby likely suppresses the development of heart failure in DS rats. Nicorandil may prove beneficial for the treatment of hypertensive heart failure as well as of ischemic heart disease.

BACKGROUND: The relationship between enhanced myocardial oxidative stress and impaired beta-adrenergic signaling remains to be characterized during the development of dilated cardiomyopathy. METHODS AND RESULTS: Alterations in myocardial oxidative stress and beta-adrenergic signaling, as well as left ventricular (LV) functional and structural changes, were evaluated during the development of cardiomyopathy in TO-2 hamsters; F1B hamsters served as controls. LV dysfunction was first apparent at 8 weeks of age and deteriorated thereafter in the TO-2 hamsters. At 32 weeks, the animals exhibited heart failure with an increased plasma norepinephrine concentration. Cardiac myolysis, as demonstrated by elevated plasma concentration of cardiac troponin T, peaked at 8 weeks. The glutathione redox ratio revealed increased oxidative stress in the LV myocardium in TO-2 hamsters even at 4 weeks and became manifest after 8 weeks. The hearts of TO-2 hamsters had significantly reduced superoxide dismutase activity from 8 weeks onward compared with control hamsters. However, glutathione peroxidase activity was unchanged at any time point. The LV functional response to isoproterenol was markedly reduced at 8 weeks, without any apparent changes in the amount of beta-adrenergic signaling molecules, and it deteriorated thereafter. Adenylyl cyclase activity was significantly decreased, despite increased amounts of both G(s) alpha mRNA and protein, in the LV myocardium at 18 weeks. CONCLUSIONS: Myocardial oxidative stress is actually enhanced in the initial development of LV dysfunction. Both activation of myocardial oxidative stress and impairment of beta-adrenergic signaling become prominent at the stage of severe LV dysfunction. Myocardial oxidative stress may be involved in the development of beta-adrenergic desensitization.

Vascular inflammation plays an important role in the development of myocardial infarction (MI). Lymphotoxin alpha (LTA) is a cytokine with multiple functions in regulation of the immune system and inflammatory reactions. The aim of this study was to examine whether polymorphisms of the LTA gene are associated with the risk of MI in Japanese men and women. A case-control association study was performed for the 252A--&gt;G and 804C--&gt;A polymorphisms of the LTA gene and the prevalence of MI. The study population comprised 3,689 unrelated Japanese individuals (2,486 men, 1,203 women), including 1891 patients with MI (1,493 men, 398 women) and 1798 control subjects (993 men, 805 women). Among the control subjects 257 individuals (108 men, 149 women) who had none of the conventional risk factors for coronary artery disease (CAD) were defined as low-fisk controls. Genotypes for the two polymorphisms were determined with a fluorescence-based allele-specific DNA primer assay system. Among all study subjects the 252A--&gt;G and 804C--&gt;A polymorphisms exhibited linkage disequilibrium. No association of either polymorphism with MI was detected in men or in women in comparisons with total control or low-risk control subjects. However, each of the two polymorphisms was associated with the prevalence of type 2 diabetes mellitus both in men with MI and in those without MI in a recessive genetic model. No association was detected between the polymorphisms and other conventional risk factors for CAD. The LTA gene thus does not appear to be a susceptibility locus for MI in Japanese men or women, although it might affect susceptibility to type 2 diabetes in Japanese men.

Aims The aim of the study was to identify genes that confer susceptibility to coronary artery spasm and clarify the interaction between genetic and environmental factors in this condition. Methods and results The study population comprised 2188 Japanese individuals, including 593 subjects with coronary artery spasm (453 men, 140 women) and 1595 controls (762 men, 833 women). The genotypes for 35 polymorphisms of 29 candidate genes were determined with an allele-specific DNA primer-probe assay. Multivariable logistic regression analysis adjusted for age, body mass index, and the prevalence of smoking, hypertension, diabetes mellitus, hypercholesterotaemia, and hyperuricaemia revealed a significant association with coronary artery spasm of one polymorphism (242C--&gt;T in the NADH/NADPH oxidase p22 phox gene) in men and two polymorphisms (-1171/5A --&gt; 6A in the stromelysin-1 gene and -634C --&gt; G in the interleukin-6 gene) in women. A stepwise forward selection procedure revealed that smoking was the most important risk factor for coronary artery spasm and that the effects of these potymorphisms on this condition were statistically independent of smoking. Conclusion The NADH/NADPH oxidase p22 phox gene is a susceptibility Locus for coronary artery spasm in men, and the stromelysin-1 and interleukin-6 genes are susceptibility loci in women. (C) 2004 The European Society of Cardiology. Published by Elsevier Ltd. All rights reserved.

Plain old balloon angioplasty (POBA) is a useful therapeutic strategy especially for angioplasty of small coronary arteries. An association study was performed to identify genes that confer susceptibility to restenosis after POBA. The study population comprised 730 individuals (424 men, 306 women) who underwent successful POBA in at least one major coronary artery and were examined angiographically 6 months after the procedure. A total of 469 subjects (273 men, 196 women) exhibited no restenosis after POBA for any of the coronary lesions, whereas 261 subjects (151 men, 110 women) manifested restenosis for all lesions. The genotypes for 40 polymorphisms of 34 genes were determined with a fluorescence- or colorimetry-based allele-specific DNA primer-probe assay. Multivariate logistic regression analysis with adjustment for age, body mass index, and the prevalence of smoking, hypertension, diabetes mellitus, hypercholesterolemia, and hyperuricemia revealed that two polymorphisms (242C --&gt; T in the NADH/NADPH oxidase p22 phox (p22-PHOX) gene and 2136C --&gt; T in the thrombomodulin (THBD) gene) in men and two polymorphisms (584G --&gt; A in the paraoxonase 1 (PON1) gene and 2445G --&gt; A in the fatty acid-binding protein 2 (FABP2) gene) in women were significantly associated with restenosis after POBA. A stepwise forward selection procedure revealed that the effects of these polymorphisms on restenosis were statistically independent of conventional risk factors for coronary artery disease. Genotyping of these polymorphisms may prove informative for assessment of genetic risk for restenosis after POBA. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Given that a substantial proportion of individuals with coronary artery disease (CAD) also have type 2 diabetes, it is important to identify genes that confer susceptibility to CAD independently in subjects with type 2 diabetes and in those without this condition. A large-scale association study was performed to identify genes that confer susceptibility to CAD in either the absence or presence of type 2 diabetes. The study population comprised 5207 unrelated Japanese individuals, including 3085 subjects with CAD and 2122 controls. Among all subjects, 1704 individuals had type 2 diabetes and 3503 individuals did not have this condition. The genotypes for 33 polymorphisms of 27 candidate genes were determined with a fluorescence- or colorimetry-based allele-specific DNA primer-probe assay system. Multivariate logistic regression analysis with adjustment for age, body mass index, and the prevalence of smoking, hypertension, hypercholesterolemia, and hyperuricemia revealed that the following polymorphisms were significantly (P &lt; 0.005) associated with CAD: the 1019C &RARR; T of the connexin 37 gene for men with type 2 diabetes; the 2445G &RARR; A in the fatty acid-binding protein 2 gene for women with this condition; the -863C &RARR; A in the tumor necrosis factor-&alpha; gene, the -219G &RARR; T in the apolipoprotein E gene, the 1019C &RARR; T in the connexin 37 gene for men without type 2 diabetes; and the -482C &RARR; T in the apolipoprotein C-III gene for women without this condition. Genotyping of these polymorphisms may prove informative for assessment of the genetic risk for CAD in the absence or presence of type 2 diabetes. (C) 2004 Elsevier Inc. All rights reserved.

A substantial proportion of individuals with coronary artery disease (CAD) has concomitant hypercholesterolemia. A large-scale association study was performed to identify separately genes that confer susceptibility to CAD in the absence or presence of nonfamilial hypercholesterolemia. The study population comprised 5248 unrelated Japanese individuals, including 3085 subjects with CAD (2350 men, 735 women) and 2163 controls (1329 men. 834 women). Among all study subjects, 2541 individuals (1688 men, 853 women) had nonfamilial hypercholesterolemia, and 2707 individuals (1991 men, 716 women) did not have this condition. The genotypes for 33 polymorphisms of 27 candidate genes were determined with a fluorescence- or colorimetry-based allele-specific DNA primer-probe assay system. Multivariate logistic regression analysis with adjustment for age, body mass index, and the prevalence of smoking, hypertension, diabetes mellitus, and hyperuricemia revealed that three polymorphisms [994G --&gt; T (Va1279Phe) in the platelet-activating factor acetylhydrolase gene, 242C --&gt; T (His72Tyr) in the NADH/NADPH oxidase p22 phox gene, and 1100C --&gt; T in the apolipoprotein C-III gene] were significantly associated with CAD in men with hypercholesterolemia. Genotyping of these three polymorphisms may prove informative for prediction of the genetic risk for CAD in men with nonfamilial hypercholesterolemia. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

The anti-anginal drug nicorandil has been shown to inhibit apoptosis by activating mitochondrial ATP-sensitive potassium (K-ATP) channels. The possible contribution of the nitrate moiety of this drug to its anti-apoptotic effect has now been investigated in neonatal rat ventricular myocytes subjected to oxidative stress. Exposure of cultured myocytes to 100 mumol/l hydrogen peroxide (H2O2) increased the number of nuclei stained by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique as well as induced internucleosomal DNA fragmentation, loss of mitochondrial membrane potential, cytochrome c release into the cytosol, and activation of caspases-3 and -9, all of which are characteristics of apoptosis. Pretreatment of cells with nicorandil (100 mumol/l) inhibited these effects of H2O2. Both the mitochondrial K-ATP channel antagonist 5-hydroxydecanoate (5-HD) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, attenuated the anti-apoptotic effect of nicorandil in concentration-dependent manners. Coapplication of ODQ (10 mumol/l) and 5-HD (500 mumol/l) completely abolished nicorandil-induced cytoprotection. The effect of nicorandil was also reduced by an inhibitor of cGMP-dependent protein kinase (KT5823, 1 mumol/l). The nitric oxide donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP, 50 mumol/l) mimicked the protective effect of nicorandil in a manner sensitive to ODQ but not to 5-HD. A cell-permeable cGMP analog, 8-bromo-cGMP. also reduced H2O2-induced apoptosis. The inhibition of the H2O2-induced activation of caspase-3, but not that of caspase-9, by nicorandil in the presence of 5-HD or by SNAP was reversed by the addition of dithiothreitol to the enzyme assay. Nicorandil inhibits oxidative stress-induced apoptosis in cardiac myocytes through a nitric oxide/cGMP-dependent mechanism as well as by activating mitochondrial K-ATP channels. (C) 2003 Elsevier Ltd. All rights reserved.

Background: Although epidemiologic studies have suggested that several genetic variants increase the risk of myocardial infarction, large-scale association studies that examine many polymorphisms simultaneously are required to allow reliable prediction of the genetic risk of myocardial infarction. Methods: We used a fluorescence- or colorimetry-based allele-specific DNA-primer-probe assay system to determine the genotypes of 112 polymorphisms of 71 candidate genes in 2819 unrelated Japanese patients with myocardial infarction (2003 men and 816 women) and 2242 unrelated Japanese controls (1306 men and 936 women). Results: In an initial screening of the 112 polymorphisms for an association with myocardial infarction in 909 subjects, 19 polymorphisms were selected in men and 18 in women by means of logistic-regression analysis, after adjustment for age, body-mass index, and the prevalence of smoking, hypertension, diabetes mellitus, hypercholesterolemia, and hyperuricemia. In a large-scale study involving the selected polymorphisms and the remaining 4152 subjects, similar logistic-regression analysis revealed that the risk of myocardial infarction was significantly associated with the C1019T polymorphism in the connexin 37 gene (P&lt;0.001) in men and the 4G-668/5G polymorphism in the plasminogen-activator inhibitor type 1 gene (P&lt;0.001) and the 5A-1171/6A polymorphism in the stromelysin-1 gene (P&lt;0.001) in women. Conclusions: Determination of the genotypes of the connexin 37, plasminogen-activator inhibitor type 1, and stromelysin-1 genes may prove reliable in predicting the genetic risk of myocardial infarction and might thus contribute to the primary prevention of this condition.

Background-Accumulating evidence has suggested that the cardiac renin-angiotensin system is activated during the remodeling process after myocardial infarction (MI). Although 2 types of angiotensin II receptors (AT(1) and AT(2)) are upregulated in the infarcted tissue, the contribution of AT(2) to the subsequent fibrogenetic phase of wound healing is less certain. This study was conducted to evaluate the role of AT(2) in wound healing after MI using an in vivo intervention study in mice with MI. Methods and Results-We examined myocardial hypertrophy, cardiac fibrosis, and morphological evidence of fibrillar collagen accumulation at the infarcted and noninfarcted regions in male mice lacking the AT(2) receptor (Agtr2-/Y) and age-matched wild-type (WT) animals. Of the Agtr2-/Y mice, 63.6% died of cardiac rupture, whereas 23.5% of the WT mice died of the same cause within 1 week. The extent of fibrosis and that of collagen gene expression in Agtr2-/Y mice were significantly reduced compared with WT mice at 1 week after coronary ligation. Furthermore, MI resulted in a marked increase in the prostaglandin E-2 (PGE(2)) level at 4 days after surgery in Agtr2-/Y mice. In WT mice, the PGE(2) level was also elevated after MI but to a significantly lesser extent than in Agtr2-/Y mice. Conclusions-A chronic loss of AT(2) by gene targeting prevented the collagen deposition and caused cardiac rupture. The markedly elevated PGE(2) may be a mechanism that inhibits collagen synthesis in the infarcted region of Agtr2-/Y mice.

The possible role of calcineurin in the attenuation of cardiachypertrophy and fibrosis by blockade of the angiotensin II type 1 (AT1) receptor was investigated in Dahl salt-sensitive (DS) rats. The effect of the calcineurin inhibitor FK506 was also studied. DS rats progressively developed severe hypertension when fed a diet containing 8% NaCl from 7 weeks of age. In addition, marked cardiac hypertrophy and fibrosis were apparent and the activity of calcineurin and its mRNA expression in the myocardium was increased in these animals at 12 weeks in comparison with age-matched Dahl salt-resistant rats. The abundance of angiotensin-converting enzyme (ACE) and transforming growth factor (TGF)-beta1 mRNAs was also increased in the hearts of DS rats at 12 weeks. Treatment of DS rats with a non-antihypertensive dose of the selective AT1 receptor blocker candesartan (1 mg/kg per day) or FK506 (0.1 mg/kg per day) from 7 to 12 weeks attenuated both calcineurin activity and its mRNA expression in the heart, as well as the development of cardiac hypertrophy and fibrosis, without affecting cardiac function. Treatment with candesartan, but not FK506, prevented theupregulation of ACE and TGF-beta1

The possible role of calcineurin in the attenuation of cardiac hypertrophy and fibrosis by blockade of the angiotensin II type I (AT,) receptor was investigated in Dahl salt-sensitive (DS) rats. The effect of the calcineurin inhibitor FK506 was also studied. DS rats progressively developed severe hypertension when fed a diet containing 8% NaCl from 7 weeks of age. In addition, marked cardiac hypertrophy and fibrosis were apparent and the activity of calcineurin and its mRNA expression in the myocardium was increased in these animals at 12 weeks in comparison with age-matched Dahl salt-resistant rats. The abundance of angiotensin-converting enzyme (ACE) and transforming growth factor (TGF)-beta1 mRNAs was also increased in the hearts of DS rats at 12 weeks. Treatment of DS rats with a non-antihypertensive dose of the selective AT, receptor blocker candesartan (1 mg/kg per day) or FK506 (0.1 mg/kg per day) from 7 to 12 weeks attenuated both calcineurin activity and its mRNA expression in the heart, as well as the development of cardiac hypertrophy and fibrosis, without affecting cardiac function. Treatment with candesartan, but not FK506, prevented the upregulation of ACE and TGF-beta1 gene expression. Both candesartan and FK506 prevented the load-induced induction of fetal-type cardiac genes. These results demonstrate that AT, receptor blockade attenuates the development of cardiac hypertrophy and fibrosis as well as the activation of calcineurin, without an antihypertensive effect, in rats with salt-sensitive hypertension. Calcineurin may be downstream from TGF-beta1 in AT, receptor-mediated angiotensin II signaling in vivo.