Tamio Iwamoto

Molecular Variant of Angiotensinogen Gene Is Associated With Coronary Atherosclerosis

BACKGROUND A positive association was previously reported between angiotensin-converting enzyme (ACE) gene polymorphism and several cardiovascular diseases, such as myocardial infarction, left ventricular hypertrophy, and restenosis after percutaneous transluminal coronary angioplasty. Plasma ACE activity and carotid-wall thickening measured by ultrasonography were related, and it was postulated that long-term exposure to high levels of plasma ACE could be involved in structural changes of the arterial wall. In addition, angiotensinogen gene mutation was recently reported to be associated with essential hypertension and preeclampsia. There exists a possibility that the renin-angiotensin system plays an important role in the progress of cardiovascular diseases in humans. Therefore, we examined the association between the molecular variant of the angiotensin gene and coronary atherosclerosis. METHODS AND RESULTS This study included 82 patients who had coronary atherosclerosis and 160 control subjects; all study participants were Japanese. All patients with coronary atherosclerosis had at least one coronary artery with > 25% luminal diameter obstruction on average according to multiple coronary angiographic views. Angiotensinogen gene molecular variants were designated AA, Aa, and aa. The a allele indicated thymine-cytosine transition at nucleotide 704 in exon 2. Genomic DNA was extracted from peripheral blood leukocytes. Polymerase chain reaction was performed to amplify the concerned region of the angiotensinogen gene. After restriction enzyme digestion, it was possible to distinguish the molecular variant of the angiotensinogen gene. The frequencies of these genotypes were 7.3%, 26.8%, and 65.9% in the patients and 18.8%, 31.9%, and 49.3% in the control subjects for the AA, Aa, and aa alleles, respectively. There was an excess in the a allele among patients (P < .01). CONCLUSIONS We found a significant association between coronary atherosclerosis and a molecular variant of the angiotensin gene. The results suggested that the molecular variant of the angiotensinogen gene could be a new risk factor for coronary atherosclerosis.

Angiotensin I converting enzyme (ACE) gene polymorphism and essential hypertension in Japan. Ethnic difference of ACE genotype.

A polymorphism of the angiotensin I converting enzyme (ACE) gene has recently been reported and analysis of this polymorphism has indicated that it is associated with several cardiovascular diseases. However, the results are still controversial and such association has not yet been established conclusively. To determine whether the ACE gene may be responsible for essential hypertension in a Japanese population, we also compared the distribution of genotypes and the allele frequency of this polymorphism in our findings of a Japanese population with these features in other countries. Eighty-seven hypertensive patients with a family history of essential hypertension and 95 normotensive patients whose parents had no such history were enrolled in the study. Polymorphism of the ACE gene was determined by using the polymerase chain reaction. Homozygotes for this polymorphism had either a 490-bp band (II) or a 190-bp band (DD) and heterozygotes had both bands (ID). In hypertensive subjects, the numbers and frequency of the ACE genotypes were: II, 44 (0.51); ID, 26 (0.30); DD, 17 (0.19). In normotensive subjects these were: II, 35 (0.37); ID, 43 (0.45); DD, 17 (0.18). There were no significant differences between the two groups in derived allele frequencies (chi 2 = 1.41). The difference between the overall allelic frequency in Japan and that reported in several other countries was significant. We did not find any association between ACE gene polymorphism and essential hypertension in Japan. However, there were significant differences in derived allele frequencies between our findings in a Japanese population and those reported from Europe and Australia.

Adenosine A1 Receptor mRNA in Microdissected Rat Nephron Segments

Adenosine plays several roles in the kidney mediated by the specific receptors A1, A2, and possibly A3. We studied the localization of adenosine A1 receptor mRNA in rat nephron segments using reverse transcription and polymerase chain reaction (RT-PCR). The nephron segments of male Sprague-Dawley rats (6 to 8 weeks old) were microdissected. Total RNA was prepared by the acid-guanidinium-phenol-chloroform method and used in the following RT-PCR assay. Because the PCR primers spanned no intron, samples reacted in the absence of RT were used as controls for amplification of genomic DNA. The PCR products were size-fractionated by electrophoresis, visualized with ethidium bromide staining, and confirmed by Southern blot analysis. PCR products were detected in all of the nephron segments examined. No signals were detected in samples reacted in the absence of RT. Strong signals were detected in glomeruli, medullary collecting duct, cortical thick ascending limb, and medullary thick ascending limb, while weak signals were found in proximal convoluted and straight tubules. Previously, the presence of A1 receptors has been demonstrated in glomeruli, collecting duct, and thick ascending limb in the rat kidney by autoradiography and binding studies. In addition to these segments, we further detected A1 receptor mRNA in proximal convoluted and straight tubules. Thus, A1 receptor mRNA seems to be broadly expressed along the nephron.

Mechanism of cAMP regulation of renin gene transcription by proximal promoter.

Renin is produced mainly by the kidney, and cAMP is a main positive regulator of its synthesis. This study was undertaken to analyze the molecular mechanism of cAMP-mediated regulation of Ren-1C gene transcription by the proximal promoter. We first showed that the promoter region from -365 to +16 of the mouse renin gene (Ren-1C) mediated the cAMP-induced chloramphenicol acetyltransferase gene expression in embryonic kidney-derived 293 cells. Deletion analysis and heterologous promoter assay disclosed that the proximal promoter region from -75 to +16 was able to activate chloramphenicol acetyltransferase expression by cAMP, and indicated that the proximal promoter element from -75 to -47 (RP-2 element) overlapping the TATA-like region was able to confer cAMP responsiveness. Electrophoretic mobility shift assay and DNase I footprinting analysis demonstrated that novel nuclear factors in 293 cells interacted with the RP-2 element, and that cAMP increased the binding activity of these nuclear factors to the RP-2 element. Furthermore, we demonstrated that cAMP enhanced the binding of nuclear factors derived from juxtaglomerular cells, the main production site of renin in the kidney, to the RP-2 element in vivo. These results suggest that the RP-2 element plays an important role in the cAMP-mediated regulation of Ren-1C gene transcription through the proximal promoter.

Angiotensin-Converting Enzyme Gene Polymorphism Adds Risk for the Severity of Coronary Atherosclerosis in Smokers

To investigate the relation between the angiotensin-converting enzyme (ACE) gene polymorphism and acute coronary syndromes with respect to environmental factors, we analyzed the association of genotype with the coronary angiographic findings of patients with acute myocardial infarction or unstable angina pectoris, and we examined the linkage of each genotype with established risk factors for coronary artery disease. We determined the ACE genotype in 152 Japanese patients with acute coronary syndromes and 399 healthy individuals. The genotype distributions were not different between the two groups (P=.74, chi2 test). In the former group, coronary angiograms were evaluated by criteria based on the number of diseased vessels, the number of stenotic lesions (> or = 50%), and the relative abnormal arterial portion (extent index). Although the number of stenotic lesions was higher in patients with the DD genotype than in those with the ID or II genotype (P=.006), there were no differences in the number of diseased vessels or the extent index. When only smokers were analyzed, the number of diseased vessels (P=.032), number of stenotic lesions (P=.003), and extent index (P=.019) were all higher in patients with the DD genotype than in those with the ID or II genotype. In contrast, these differences in the respective parameters did not exist in nonsmokers. The results indicate smoking-associated effects of the ACE genotype on the severity of coronary atherosclerosis.

Molecular mechanism of adipogenic activation of the angiotensinogen gene.

Angiotensinogen gene expression is controlled in a tissue- and development-specific manner. Interestingly, the angiotensinogen gene is abundantly expressed in adipose tissues other than the liver, where it is mainly produced. We investigated the molecular mechanism of angiotensinogen gene expression in a 3T3-L1 preadipocyte-adipocyte system. Although angiotensinogen mRNA was barely detectable in preadipocytes, its levels increased significantly during differentiation. As a whole, the pattern of the change in transcriptional activity of the angiotensinogen promoter was similar to that of the angiotensinogen mRNA levels during adipogenic differentiation, indicating that the activation of the angiotensinogen promoter might be involved in the adipogenic differentiation-coupled gene expression. The proximal promoter region, from -96 to +22 of the transcriptional start site, was sufficient to confer adipogenic activation, and the proximal element from -96 to -52 of the transcriptional start site was necessary for this promoter stimulation. DNA-protein binding experiments showed that this proximal element specifically bound to a nuclear factor induced by adipogenic differentiation. These results suggest that the proximal promoter element from -96 to -52 plays a role in adipogenic activation of the angiotensinogen promoter.

AT1 RECEPTOR ANTAGONIST, TCV 116, DOES NOT PREVENT CARDIAC HYPERTROPHY IN SALT-LOADED DAHL SALT-SENSITIVE RATS

1. We investigated the effects of direct blockade of angiotensin II (AngII) by a potent, non‐peptide angiotensin II type 1 (AT1) receptor antagonist, TCV 116, on the development of cardiac hypertrophy in salt‐loaded Dahl salt‐sensitive rats.

Disruption of type 5 adenylyl cyclase negates the developmental increase in Gαolf expression in the striatum

The two stimulatory G protein α subunits, Gαs and Gαolf, activate adenylyl cyclase in a similar way. We examined whether type 5 adenylyl cyclase knockout, the major striatal isoform, can differentially and/or developmentally change the expression of these G proteins in the striatum. Gαs and Gαolf expressions at birth were unaffected in knockouts, which, however, demonstrated a blunted developmental increase in Gαolf, but not Gαs. Adenylyl cyclase activity was unaffected at birth, but subsequently became lower in knockouts. These findings suggest that type 5 adenylyl cyclase does not contribute to striatal cAMP signaling at birth. However, it may play an important role in developmental changes in the expression of Gαolf, but not Gαs.

DNA fingerprint analysis of Dahl-Iwai salt-sensitive rats (S) and salt-resistant rats (R)

Using DNA fingerprint analyses, extensive molecular heterogeneity has been found between spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY), suggesting a doubtful value of simply comparing these rats in the study of the pathogenesis of genetic hypertension. Therefore, we evaluated the genetic similarity between Dahl salt-sensitive and salt-resistant rats newly inbred by Dr.Iwai (Dahl-Iwai S and R rats), by use of DNA fingerprint analysis. Fingerprint patterns were generated by probing Hinf I- or Alu I-digested DNA with an oligonucleotide corresponded to the tandem repeat sequence of the human myoglobin 33.6 minisatellite. These fingerprint patterns were same within each strain. S and R rts shared 82 percent of the bands in Hinf I-digested DNA and 93 percent of those in Alu I-digested DNA. Although Dahl-Iwai S and R rats are more closely related than SHR and WKY rats, multiple genetic differences still exist between these Dahl strains.

Effect of dietary sodium on platelet alpha2-adrenoceptors in young normotensive men with or without a family history of hypertension

Objective: To study the effects of genetics on the response of platelet a2-adrenoceptors to a change in salt intake Methods: Biochemical measurements and radioligand binding assays in platelets were performed in 11 normotensive male university students with a family history of essential hypertension (FH ) and in 17 students without a family history of hypertension (FH− ). The 28 students were fed a high-sodium diet for 7 days and a low-sodium diet for 7 days Results: In FH + subjects the number of a2-adrenergic receptors on platelet membrane fractions increased significantly from the high-sodium diet to the low-sodium diet, even though plasma noradrenaline concentrations tended to increase with the low-sodium diet. There was no change in the number of a2-adrenoceptors in the FH —group. In both groups the radioligand binding affinity was decreased during a low-sodium period compared with in a high-sodium period Conclusion: In the FH+ subjects the change in platelet oc2-adrenoceptors associated with altered sodium status was similar to that seen in patients with salt-sensitive hypertension, suggesting that there is a genetic susceptibility to sodium