Risk Factors for Coronary Atherosclerosis in Recent Japanese Population Study: Hisayama Study Based on Pathological Assessment

Yutaka Nakashima
Graduate School of Medical Sciences Kyushu University, Fukuoka, Japan

Autopsy data from patients in a long-term observational study identified age, hypertension, hyperlipidemia, obesity, and glucose intolerance to be closely related to coronary atherosclerosis in the Japanese. Although cerebrovascular diseases have decreased in Japan, from treatment of hypertension, coronary artery disease (CAD) is increasing from the Westernization of the Japanese lifestyle.

The 40-year-old Hisayama study investigated recent risk factors for coronary atherosclerosis in the Japanese using a population-based consecutive autopsy series in people from the city of Hisayama, Japan, near Fukuoka, and developed a method for the global assessment of coronary atherosclerosis.

In this series, autopsy was performed on 233 Hisayama residents who had a medical exam in 1988 (80.9% participation) and died during the 8-year follow-up period (79.1% participation). This series is comprised of 125 men, aged 44-94 years with a mean age of 72 years, and 108 women, aged 46-96 years with a mean age of 74 years. Risk factors studied were: systolic blood pressure (SBP), diastolic blood pressure, body mass index, waist-to-hip ratio, serum total cholesterol, HDL cholesterol, hemoglobin A1c, smoking habit, daily alcohol intake.

The 14 sites examined in the coronary arteries, selected on the basis of the AHA reporting system to ensure thorough study of the atherosclerosis, are shown in Figure 1. The degree of atherosclerosis is determined based on histological changes and stenotic ratio, determined as illustrated in Figure 2. Vessels are photographed on autopsy and computer-corrected into circles for measurements to determine the stenotic ratio. The two types of atherosclerosis on histology are pre-atheroma, an accumulation of foam cells with adhesion of lipids in the extracellular regions, and atheroma, necrotic tissue covered by a fibrous cap (Figure 3). One characteristic of the atheroma in Japanese is severe fibrotic changes, with limited lipid deposition. The grading system used in the global assessment of coronary atherosclerosis is illustrated in Figure 4.

The prevalence of coronary atherosclerosis by grade and age is shown in Figure 5. Women less than 60 years had milder atherosclerosis, but after 60 years, the prevalence was similar for women and men. The incidence of ischemic heart disease was rare before menopause, but increased, along with its morbidity, after menopause.

Risk factors for coronary atherosclerosis in men were SBP, waist-to-hip ratio body mass index, serum total cholesterol, and hemoglobin A1c. In women the risk factors were SBP, waist-to-hip ratio body mass index, and HDL cholesterol, on age-adjusted regression analysis. Smoking and alcohol intake were not significant risk factors for coronary atherosclerosis. Stepwise multiple regression analysis revealed risk factors for men were age, SBP, serum total cholesterol, and hemoglobin A1c, and for women were age, SBP, and waist-to-hip ratio.

Comparison of the Effects of Beta-Blockers and Calcium Antagonists on Cardiovascular Events after Acute Myocardial Infarction in Japanese

Hisao Ogawa
Kumamoto University School of Medicine, Kumamoto, Japan

The higher incidence of coronary spasm in Japanese compared to Caucasians is established. About 40% of patients with angina pectoris and 60% of patients with organic angina in Japan have coronary spasm. Sasayama and colleagues have shown a greater incidence of coronary spasm after acute myocardial infarction (AMI) in Japanese compared to Caucasians.

The Japanese Beta-blockers Calcium Antagonists Myocardial Infarction (JBMCI) study compared the effects of ß-blockers and calcium channel blockers (CCB) on cardiovascular (CV) events after AMI, in a multicenter, open-label, controlled, randomized trial. The benefit of ß-blockers in AMI has been established, but that of calcium antagonists has not. Yasue and co-workers showed that CCB effectively suppress coronary spasm, particularly during exercise, but ß-blockers aggravate the attacks in some AMI patients. In the Japanese Antiplatelet Myocardial Infarction Study (JAMIS) 2 study by Yasue, about 80% of patients has a CCB and 5% a ß-blocker.

The design of the JBCMI study is outlined in Figure 1. From 1998 to 2000, 1,090 patients were randomized to ß-blockers or CCB (545 in each group). The primary endpoints were CV death including sudden death; nonfatal reinfarction, unstable angina requiring readmission, heart failure requiring readmission, and nonfatal ischemic stroke.

The ß-blockers administered were carvedilol in 310 patients, bisoprolol 138 patients, atenolol 59 patients, and metoprolol 38 patients. CCB were amlodipine in 469 patients, nifedipine 61 patients, manidipine 14 patients, and nisolopine 1 patient. The mean age was 64 years and time from onset to admission was 14-17 hours. Baseline characteristics are outlined in Figure 2.

During the mean follow-up of 455 days, the incidence of total CV events, defined as CV death, reinfarction, uncontrolled unstable angina, and nonfatal ischemic stroke, was higher in the ß-blocker group compared to the CCB group (101 vs 78 events, p=0.0298) (Figure 3). The incidence of heart failure was significantly higher in the ß-blocker group than in the CCB group (23 vs 6, p=0.0011) (Figure 4). Coronary spasm was higher in the ß-blocker group than in the CCB group (7 vs 1, p=0.0271). Because patients with coronary spasm are readmitted to the hospital for refractory chest pain, this number may exclude the cases of mild coronary spasm responding to drug therapy, said Hisao Ogawa of Kumamoto University School of Medicine, a study investigator. The onset of coronary spasm was delayed with CCB but not with ß-blocker (Figure 5).

In summary, the incidence of coronary spasm was 40.2% in 2195 consecutive patients with angina pectoris in this multicenter study in Japan. CCB may be more useful than ß-blockers for AMI in Japanese patients, especially for the prevention of heart failure and coronary spasm. However, CV events other than heart failure, including cardiac death and refractory angina, were the same in the two groups. ß-blockers are beneficial in patients with left ventricular dysfunction after AMI. Thus, in patients with AMI, ß-blockers should be carefully administered with small initial doses with more gradual dose titration. In the absence of heart failure, a usual starting dose may be initiated.

Importance of Rho-Kinase in the Pathogenesis of Arteriosclerotic Vascular Disease in Japanese

Hiroaki Shimokawa
Kyushu University Graduate School of Medicine, Fukuoka, Japan

The Rho/Rho-kinase pathway is involved in the development of cardiovascular (CV) diseases (Figure 1). Research by Hiroaki Shimokawa and colleagues at Kyushu University Graduate School of Medicine shows that Rho-kinase may be involved in spasm-related diseases of the vessel and the development of atherosclerosis.

Coronary inflammation changes at the adventitia is seen in persons who die from coronary spasm. In the porcine model given microbeads with the inflammatory cytokine IL-1ß, this group showed serotonin-induced enhanced coronary artery spasm. This porcine model was used to study the changes in the Rho-kinase pathway, in which the expression of Rho mRNA is enhanced. Rho-kinase has 3 domains, catalytic, coiled-coil, and PH, with a Rho-binding (RB) domain at the coiled-coil domain. Rho-kinase activity is blocked by overexpression of the RB-PH domains.

Fasudil, approved for coronary spasm, is metabolized by the liver into hydroxyfasudil, which is highly selective for blocking Rho-kinase. In the porcine model with IL-1ß, hydroxyfasudil suppressed serotonin-induced coronary spasm in a dose-dependent manner, but not coronary spasm in normal coronary arteries.

The myosin binding subunit of myosin phosphatase is phosphorylated and inactivated by Rho-kinase, showing a Rho-kinase activity. The Rho-kinase inhibitors, fasudil and Y-27632 suppressed the enhanced Rho-kinase activity in the porcine model of coronary spasm/arteriosclerosis. The close correlation between the Rho-kinase and coronary spasm supports the involvement of Rho-kinase in the pathogenesis of coronary spasm.

The mechanisms for coronary spasm include suppression of myosin phosphotase and resultant enhancement of myosin phosphorylation (Figure 2). Interaction with actins induces vascular spasm. Rho-kinase expression is increased in inflammatory atherosclerotic lesions

In humans, fasudil suppresses acetylcholine-induced focal spasm, but not normal coronary artery constrictions. Fasudil also suppresses the incidence of diffuse multivessel spasm in patients with vasospastic angina.

Pokkuri disease, or sudden nocturnal death, occurs in persons with no previous sign of disease. A comparison of the lipid profiles in persons who died from Pokkuri disease and from accidents showed that the triglyceride level and particularly remnant lipoprotein triglyceride level (RLP-TG) are very high in Pokkuri disease. Pokkuri disease and coronary spasm are more frequent in the early morning and in Asian males. RLP-TG might be associated with both.

A comparison of Pokkuri-derived RLP fractions and bound fractions showed that serotonin caused a severe contraction only at the coronary segments treated with RLP fraction. This was significantly suppressed with the pre-administration of the Rho-kinase inhibitor, hydroxyfasudil, in the porcine model. Rho-kinase expression is significantly enhanced in the RLP fractions compared to the bound fractions from patients with Pokkuri disease. A high level of RLP may induce fatal coronary spasm, stated Shimokawa.

Atherosclerosis and Rho-kinase

Rho-kinase down regulates eNOS, BMP-2, and osteocalcin, which are said to suppress atherosclerosis. In contrast, Rho-kinase upregulates MCP-1, PAI-1, tissue factor, TGF-ß1, collagen I, IL-6, Rho-kinase, NAD(P)H, MIF, and IFN-.

In a balloon injury porcine model, pre-administration of Rho-kinase prevented atherosclerotic changes. MCP-1 induces atherosclerotic lesions, but fasudil suppresses this action. Also, some studies have shown that Rho-kinase inhibitors may prevent atherosclerosis after heart and liver transplantation.

Long-term inhibition of Rho-kinase can eliminate the constrictive remodeling of the porcine coronary artery. Constrictive remodeling occurs at the inflammatory coronary lesions, but the transfection of dominant-negative Rho-kinase regresses the remodeling, as shown by increased luminal cross-sectional area on angiography.

Pulmonary hypertension and Rho-kinase

Although the pathophysiology of pulmonary hypertension is unclear, Rho-kinase is thought to be involved with the inflammatory and proliferative changes of the pulmonary arteries that may lead to proliferation and hypertrophy, adhesion and migration of the inflammatory cells, injury and thrombosis of endothelium, and hypercontraction of vascular smooth muscle cells.

In the pulmonary hypertensive rat model, severe pulmonary hypertension was established 3 weeks after injection of monocrotaline (MCT). When untreated, 80% of the rats died within 8 weeks. Survival is improved with long-term oral treatment with fasudil. Further, fasudil improved survival even after pulmonary hypertension was established. The Rho-kinase inhibitor improves pulmonary vascular lesions and right ventricular hypertrophy in pulmonary hypertension. Fasudil, but not nitric oxide or nifedipine, reduced vascular resistance in patients with severe pulmonary hypertension (Figure 3).

In summary, Rho-kinase is a therapeutic target, whose inhibitors have been shown to be effective for cerebral vasospasm, coronary artery spasm, hypertension, pulmonary hypertension, and sudden death (Figure 4). Atherosclerotic diseases, such as angina, myocardial infarction, restenosis, coronary artery dysfunction after transplantation may be treated by targeting Rho-kinase.

G (-670) A Polymorphism in the Promoter Region of the Fas Gene is a Risk Factor for Myocardial Infarction

Keisuke Fukuo
Osaka University Medical School, Osaka, Japan

Fas is a member of the TNF- receptor family. Binding to the Fas ligand activates apoptosis-related enzymes such as caspase. Although Fas is expressed in most cell types, Fas ligand (FasL) is expressed only in natural killer cells, activated T cells, macrophages, and other immune and inflammatory cells. When the FasL is cut off from the membrane by metalloproteinase, it becomes soluble (sFas), which may be involved in the induction of the inflammatory reactions and other apoptotic activity, explained Keisuke Fukuo, Osaka University Medical School.

The role of the Fas/FasL system in atherogenesis is being elucidated by work from this group and others. Atherogenic factors such as oxidative stress induces endothelial cell apoptosis via Fas-mediated pathways. Fas-mediated vascular smooth muscle cell apoptosis may exaggerate plaque vulnerability via induction of inflammation. FasL expressed in endothelial cells may regulate the accumulation of inflammatory cells into the vessel wall. Soluble fasL (SfasL) released from endothelial cells may protect against hypoxia-induced endothelial cell apoptosis. Plasma sFasL levels are increased in patients with heart failure and acute coronary syndrome. Fas-mediated apoptosis may serve as a negative feed back mechanism for the elimination of activated macrophages in the vessel wall.

Infiltration of inflammatory cells into endothelium causes plaque instability and is related to the development of cardiovascular (CV) events. The mechanisms for the accumulation of macrophages in the vessel wall may include MCP-1, which may be related to the migration or chemotaxis, proliferation, and apoptosis. This group showed in mice studies that activated macrophages cause apoptosis in control mice, but not in Fas-lacking mouse. The macrophage accumulation in the perivascular space is greater in the Fas-lacking mice than in controls.

Work by this group and others has shown the following. The atherogenic diet induced vasculitis, lipid accumulation, and a higher incidence of myocardial infarction (MI) in Fas-lacking mice compared to controls. Mutations in the Fas gene were associated with autoimmune disease. Importantly, patients with SLE and other autoimmune disease are associated with a higher CV mortality. Fas signaling is required in the compensation of pressure overload, and its absence leads to rapid ventricular dilation, failure and increased mortality, without compensatory hypertrophy. Thus, abnormality of the Fas gene may be a genetic risk factor for CV disease.

Fas gene polymorphism and MI

This group conducted a study to assess whether a polymorphism in the promoter region of the Fas gene is associated with an increased risk for MI in Japanese subjects. Two polymorphisms have been identified in the promoter region of the Fas gene. The single base change from GA to GG was the focus of this presentation.

The TaqMan (the fluorogenic 5’ nuclease polymerase change reaction)-PCR method was used. Six hundred sixteen patients were studied (AMI positive 154, AMI negative 462), with a mean age of 63 years (82% male in both groups). Smoking (61% vs 38%), hypertension (52% vs 40%), diabetes (36%vs 8%), and hyperlipidemia (48% vs 22%) were greater in the AMI positive than the AMI negative patients.

No age or sex difference in the 3 groups based on the polymorphism. However, the incidence of MI was higher in the AA group compared to the GA and GG groups (35%, 23%, 19%, respectively). An A allele is associated with an odds ratio of 2.62 for an MI, and it is an independent risk factor for MI, based on logistic regression analysis.

JAK and STAT seem to be stimulated in the system regulating Fas expression. STAT3 is reported to have a negative effect on FAS expression. The GAS region is the dominant binding region. A GAS sequence is present in patients with an A allele, but not those with a G allele. In A allele patients, Fas expression is suppressed by GAS sequence-stimulation of STAT3. This does not occur in G allele patients.

Leukemia inhibitory factor (LIF), an activator of STAT3, suppressed Fas expression in the AA genotype but not in the GG genotype in an experiment by this group. Further work showed that LIF suppressed the promoter activity in Cos 7 cells transfected with the construct containing AA but not GG genotype. Hence, the difference in genotype seems to be related to the difference in transcription activity.

In patients with the AA genotype, there are 3 defects in the GAS sequence. Cytokine stimulation of STAT3 suppresses Fas expression, leading to Fas-mediated apoptosis.

Thus, the mechanism to eliminate inflammatory cells from the endothelium is weakened or suppressed, causing unstable plaque formation. The lack of a GAS sequence in the GG phenotype means no stimulation of STAT3 and suppression of Fas expression, allowing inflammatory cells to be eliminated from the endothelium, making the plaque more stable (Figure 1).

In conclusion, the A allele of the GA polymorphism in the promoter region of the Fas gene may be a new genetic risk factor for MI via downregulation of Fas expression.