Nobuo Nyui

Endothelial Nitric Oxide Synthase Gene Polymorphism and Acute Myocardial Infarction

Recently a point mutation of guanine to thymine at nucleotide position 1917 in the endothelial nitric oxide synthase (eNOS) gene has been reported to be associated with coronary artery spasm. In addition, a significant association of the 4a/b polymorphism in intron 4 of the eNOS gene with coronary artery disease has been reported. However, the implications of these polymorphisms with respect to acute myocardial infarction (AMI) remain to be established. We conducted a case-control study of 226 patients with AMI and 357 healthy gender- and age-matched control subjects. In the former group, coronary angiograms were evaluated according to angiographic criteria based on the number of diseased vessels (>/=75%) and the number of stenotic lesions (>/=50%). Homozygosity for the Glu-Asp298 polymorphism existed in 5 of 226 patients with AMI (2.2%) but not in any of the 357 control subjects (P=.0085). However, when we evaluated the coronary angiograms of 226 case patients, there was no difference in the number of diseased vessels or the number of stenotic lesions between the patients with this homozygote and those without it. By contrast, there was no evidence of a significant increase in the risk of AMI or the severity of coronary atherosclerosis among individuals with the a/a genotype of the eNOS4a/b polymorphism. Our results imply that patients who are homozygous for the Glu-Asp298 polymorphism may be genetically predisposed to AMI; however, this mutation apparently is not related to the severity of coronary atherosclerosis. Further studies are needed to confirm our results and characterize the molecular mechanisms by which eNOS is involved in susceptibility to AMI.

Plasma Angiotensinogen Concentrations in Obese Patients

A close relationship between obesity and hypertension has been recognized, and plasma angiotensinogen concentrations (p-AGT) have been reported to correlate with blood pressure (BP). However, little is known about AGT in obese patients with hypertension. To define the role of AGT in obese hypertension, we measured p-AGT in obese patients. The subjects were 42 obese patients diagnosed on the basis of a body mass index (BMI) of more than 25 kg/m2, and 21 sex- and age-matched nonobese patients, whose BMI was less than 25 kg/m2. The hypertensive patients had not previously received antihypertensive drugs. P-AGT (P < .05) and mean BP (P < .0001) was increased in the obese patients as compared with the nonobese patients. Positive correlations were observed between BMI and p-AGT, mean BP and p-AGT, and BMI and mean BP (all P < .05). However, after adjustment for blood pressure, p-AGT was not different between groups, and after adjustment a positive correlation remained only between BMI and mean BP. These results suggested the possible involvement of increased p-AGT in hypertension in obese patients, although this may be a secondary change to hypertension or obesity.

Activation of angiotensinogen gene in cardiac myocytes by angiotensin II and mechanical stretch

Circulating and cardiac renin-angiotensin systems (RAS) play important roles in the development of cardiac hypertrophy. Mechanical stretch of cardiac myocytes induces secretion of ANG II and evokes hypertrophic responses. Angiotensinogen is a unique substrate of the RAS. This study was performed to examine the regulation of the angiotensinogen gene in cardiac myocytes in response to ANG II and stretch. ANG II and stretch significantly increased the levels of angiotensinogen mRNA in cardiac myocytes. Actinomycin D completely inhibited ANG II- and stretch-mediated increases in angiotensinogen mRNA. Although CV-11974 abolished ANG II-mediated increases in mRNA level and promoter activity of the angiotensinogen gene, the inhibition of stretch-mediated activation by CV-11974 was significant but not complete. These results indicate that ANG II activates transcription of the angiotensinogen gene exclusively via ANG II type 1-receptor pathway and that stretch activates such transcription mainly via the same pathway in cardiac myocytes. Furthermore, factors other than ANG II may also be involved in stretch-mediated activation of the angiotensinogen gene in cardiac myocytes.Circulating and cardiac renin-angiotensin systems (RAS) play important roles in the development of cardiac hypertrophy. Mechanical stretch of cardiac myocytes induces secretion of ANG II and evokes hypertrophic responses. Angiotensinogen is a unique substrate of the RAS. This study was performed to examine the regulation of the angiotensinogen gene in cardiac myocytes in response to ANG II and stretch. ANG II and stretch significantly increased the levels of angiotensinogen mRNA in cardiac myocytes. Actinomycin D completely inhibited ANG II- and stretch-mediated increases in angiotensinogen mRNA. Although CV-11974 abolished ANG II-mediated increases in mRNA level and promoter activity of the angiotensinogen gene, the inhibition of stretch-mediated activation by CV-11974 was significant but not complete. These results indicate that ANG II activates transcription of the angiotensinogen gene exclusively via ANG II type 1-receptor pathway and that stretch activates such transcription mainly via the same pathway in cardiac myocytes. Furthermore, factors other than ANG II may also be involved in stretch-mediated activation of the angiotensinogen gene in cardiac myocytes.

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.

Tissue Angiotensinogen Gene Expression Induced by Lipopolysaccharide in Hypertensive Rats

There is now convincing evidence that various tissues express their own tissue renin-angiotensin system, which may be regulated independently of the systemic renin-angiotensin system. However, little information is available on the regulation of the tissue renin-angiotensin system. We investigated the regulation of tissue angiotensinogen gene expression with respect to the development of hypertension. We measured basal and lipopolysaccharide-stimulated plasma angiotensinogen concentrations by radioimmunoassay and examined the expression of tissue angiotensinogen by Northern blot analysis in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) at 4 and 13 weeks of age. Basal plasma angiotensinogen concentration in SHR was comparable to that in WKY at 4 weeks of age and was significantly higher than that in WKY at 13 weeks of age. Lipopolysaccharide induced a significant increase in plasma angiotensinogen concentration in both WKY and SHR at 4 and 13 weeks of age. At 4 weeks of age, the basal levels of angiotensinogen mRNA in the liver, fat, adrenal, and aorta were higher in WKY than in SHR. At 13 weeks of age, the basal levels of angiotensinogen mRNA in the fat, adrenal, aorta, spleen, and kidney were higher in WKY than in SHR, while that in the liver did not differ significantly between the two strains. At 4 weeks of age, pretreatment with lipopolysaccharide increased the angiotensinogen mRNA levels in the liver, fat, adrenal, and aorta in both WKY and SHR. At 13 weeks of age, pretreatment with lipopolysaccharide increased the angiotensinogen mRNA levels in the liver, aorta, and adrenal; decreased those in the spleen; and had no effect in the kidney in both WKY and SHR. Interestingly, lipopolysaccharide increased the angiotensinogen mRNA level in fat only in SHR, with no effect in WKY, at 13 weeks of age. Lipopolysaccharide stimulated tumor necrosis factor-a mRNA expression in fat of WKY and SHR, and the increase in tumor necrosis factor-alpha mRNA level in SHR was significantly greater than that in WKY. Therefore, the increased tumor necrosis factor-alpha mRNA expression may be involved in the increased lipopolysaccharide-induced expression of angiotensinogen gene in fat of SHR at 13 weeks of age. These data suggest that the transcriptional and probably posttranscriptional regulation of angiotensinogen mRNA differs between SHR and WKY, that the regulation of angiotensinogen gene expression is tissue-specific, and that the altered expression of the angiotensinogen gene may be involved in the development of hypertension.

Endocrinological abnormalities in angiotensinogen-gene knockout mice: studies of hormonal responses to dietary salt loading.

Objective Physiological roles of the renin-angiotensin system in maintaining blood pressure and sodium-water balance in angiotensinogen gene-knockout mice were evaluated with special reference to endogenous pressor substances. Methods Angiotensinogen-gene knockout mice and control mice were fed a 0.3 or 4% NaCl diet for 2 weeks. Systolic blood pressure and urinary excretions of electrolytes, creatinine, aldosterone, adrenaline, noradrenaline, dopamine and vasopressin were measured. Results About 60% of our angiotensinogen-gene knockout mice did not survive until weaning. These mice presented with hypotension and polyuria. Urinary excretion of aldosterone from such mice was significantly lower (not detected) than that from control mice (2.0 ± 0.3 pg/mg creatinine). In contrast, urinary excretion of vasopressin from angiotensinogen-gene knockout mice (0.7 ± 0.1 ng/mg creatinine) was greater than that from control mice (0.3 ± 0.1 ng/mg creatinine), and those of adrenaline and of noradrenaline were similar for knockout and control mice. After salt loading (a 4% NaCl diet), angiotensinogen-gene knockout mice exhibited a significant increase in systolic blood pressure (from 68.3 ± 2.9 to 95.9 ± 5.9 mmHg), significant decreases in urinary excretions of adrenaline (from 65 ± 8 to 40 ± 7 pg/mg creatinine) and noradrenaline (from 467 ± 48 to 281 ± 41 pg/mg creatinine) and no change in excretion of vasopressin compared with such mice fed a 0.3% NaCl diet. Conclusion The present results with angiotensinogen gene knockout mice confirm that the renin-angiotensin system plays fundamental roles in maintaining the blood pressure and sodium-water balance. Because the vasopressin and catecholaminergic systems may be altered by lack of angiotensin in angiotensinogen-gene knockout mice, these systems perhaps are not able to restore blood pressure and sodium-water depletion to normal levels in these mice. J Hypertens 16:285–289

Effect of Genetic Deficiency of Angiotensinogen on the Renin-Angiotensin System

This study examined expression of renin-angiotensin system (RAS) component mRNAs in angiotensinogen gene knockout (Atg-/-) mice. Wild-type (Atg+/+) and Atg-/- mice were fed a normal-salt (0.3% NaCl) or high-salt (4% NaCl) diet for 2 weeks. Angiotensinogen, renin, angiotensin-converting enzyme (ACE), angiotensin II type la receptor (AT1A), and angiotensin II type 2 receptor (AT2) mRNA levels were measured by Northern blot analysis. In Atg+/+ mice, activities of circulating RAS and renal angiotensinogen mRNA level were decreased by salt loading, whereas levels of renal and cardiac ACE; renal, brain, and cardiac AT1A; and brain and cardiac AT2 mRNA were increased by salt loading. Although activities of circulating RAS were not detected in Atg-/- mice, salt loading increased blood pressure in Atg-/- mice. In Atg-/- mice, renal renin mRNA level was decreased by salt loading; in contrast, salt loading increased renal AT1A and cardiac AT2 mRNA levels in Atg-/- mice, and these activated levels in Atg-/- mice were higher than those in Atg+/+ mice fed the high-salt diet. Thus, expression of each component of the RAS is regulated in a tissue-specific manner that is distinct from other components of systemic and local RAS and that appears to be mediated by a mechanism other than changes in the circulating or tissue levels of angiotensin peptides.

Expression of renin-angiotensin system and extracellular matrix genes in cardiovascular cells and its regulation through AT1 receptor

Angiotensinogen (AGT) is a unique substrate of the renin-angiotensin system and fibronectin (FN) is an important component of the extracellular matrix. These play critical roles in the pathophysiological changes including cardiovascular remodeling and hypertrophy in response to hypertension. This study was performed to examine the regulation of AGT and FN gene in cardiac myocytes (CMs) and vascular smooth muscle cells (VSMCs) in response to mechanical stretch. Mechanical stretch significantly increased the AGT mRNA expression in CMs, while these stimuli did not affect FN mRNA levels. On the other hand, Mechanical stretch upregulated FN mRNA levels in VSMCs, whereas no increase in AGT mRNA levels was observed in response to stretch stimuli. An angiotensin II type 1 (AT1) receptor antagonist (CV11974) significantly decreased these stretch-mediated increases in mRNA level and promoter activity of the AGT and FN gene, whereas angiotensin II type 2 (AT2) receptor antagonist (PD123319) did not affect the induction. These results indicate that mechanical stretch activates transcription of the AGT and FN gene mainly via AT1 receptor-pathway in CMs and VSMCs. Furthermore, mechanisms regulating AGT and FN gene seem to be different between CMs and VSMCs.

Relationship between hepatic angiotensinogen mRNA expression and plasma angiotensinogen in patients with chronic hepatitis.

Recent association and linkage studies suggested that angiotensinogen may play an important role in the pathogenasis of essential hypertension. However, there is little information in human concerning a relationship between plasma angiotensinogen levels and the angiotensinogen mRNA expression in the liver, which is the main production site of angiotensinogen. Therefore, the aim of this study was to examine whether hepatic angiotensinogen gene expression determines the level of circulating angiotensinogen and the activity of the renin-angiotensin system in humans. The subjects were 36 patients with chronic hepatitis. Blood was collected from each patients for estimation of plasma renin activity, plasma angiotensinogen and angiotensin II concentrations and several parameters of liver function. In addition, total RNA was isolated from liver biopsy specimens, which were then used to measure angiotensinogen mRNA with Northern blot analysis. Levels of angiotensinogen mRNA were detected easily in the liver biopsy specimens in all of the patients. Hepatic angiotensinogen mRNA levels were positively correlated with plasma angiotensinogen levels (r=0.41, P=0.013). In contrast, hepatic angiotensinogen mRNA levels did not show any significant relationship with plasma renin activity, plasma angiotensin II concentration, histological subgroup of hepatitis, histological activity index and parameters of liver function tests. The present study demonstrated, for the first time, that hepatic angiotensinogen mRNA levels correlated with plasma angiotensinogen concentration in humans.

Increased Cardiac Angiotensin II Receptors in Angiotensinogen-Deficient Mice

Two subtypes of angiotensin II (Ang II) receptors, type 1 (AT1-R) and type 2 (AT2-R), have been identified in the heart. However, little is known about the regulation of cardiac AT1-R and AT2-R by Ang II in vivo. Thus, we examined cardiac AT1-R and AT2-R in angiotensinogen-deficient (Atg-/-) mice that are hypotensive and lack circulating Ang II. Cardiac Ang II receptors (Ang II-R) were assessed by radioligand binding with 125I-[Sar1,Ile8]-Ang II in plasma membrane fractions. AT1-R and AT2-R were distinguished using their specific antagonists CV-11974 and PD123319, respectively. Total densities of Ang II-R and AT1-R density were significantly greater in the Atg-/- mice than Atg+/+ mice (31.1+/-2.8 versus 18.8+/-2.1, 28.7+/-3.0 versus 16.9+/-2.3 fmol/mg protein, P<.01, respectively), and AT2-R showed a slight but not significant increase in Atg-/- mice relative to Atg+/+ control animals. Kd values were not different between the two groups. In contrast to binding experiments, levels of Ang II type 1a receptor (AT1a-R) and AT2-R mRNA did not differ between Atg-/- and Atg+/+ mice. These results suggest that lack of Ang II may upregulate AT1-R through translational and/or posttranslational mechanisms in Atg-/- mice.