Sei Nakata

Asymmetric Dimethylarginine Produces Vascular Lesions in Endothelial Nitric Oxide Synthase–Deficient Mice: Involvement of Renin-Angiotensin System and Oxidative Stress

Objective—Asymmetric dimethylarginine (ADMA) is widely believed to be an endogenous nitric oxide synthase (eNOS) inhibitor. However, in this study, we examined our hypothesis that the long-term vascular effects of ADMA are not mediated by inhibition of endothelial NO synthesis. Methods and Results—ADMA was infused in wild-type and eNOS-knockout (KO) mice by osmotic minipump for 4 weeks. In wild-type mice, long-term treatment with ADMA caused significant coronary microvascular lesions. Importantly, in eNOS-KO mice, treatment with ADMA also caused an extent of coronary microvascular lesions that was comparable to that in wild-type mice. These vascular effects of ADMA were not prevented by supplementation of l-arginine, and vascular NO production was not reduced by ADMA treatment. Treatment with ADMA caused upregulation of angiotensin-converting enzyme (ACE) and an increase in superoxide production that were comparable in both strains and that were abolished by simultaneous treatment with temocapril (ACE inhibitor) or olmesartan (AT1 receptor antagonist), which simultaneously suppressed vascular lesion formation. Conclusions—These results provide the first direct evidence that the long-term vascular effects of ADMA are not solely mediated by simple inhibition of endothelial NO synthesis. Direct upregulation of ACE and increased oxidative stress through AT1 receptor appear to be involved in the long-term vascular effects of ADMA in vivo.

Asymmetric Dimethylarginine Causes Arteriosclerotic Lesions in Endothelial Nitric Oxide Synthase-Deficient Mice. Involvement of Renin-Angiotensin System and Oxidative Stress

Objective—Asymmetric dimethylarginine (ADMA) is widely believed to be an endogenous nitric oxide synthase (eNOS) inhibitor. However, in this study, we examined our hypothesis that the long-term vascular effects of ADMA are not mediated by inhibition of endothelial NO synthesis. Methods and Results—ADMA was infused in wild-type and eNOS-knockout (KO) mice by osmotic minipump for 4 weeks. In wild-type mice, long-term treatment with ADMA caused significant coronary microvascular lesions. Importantly, in eNOS-KO mice, treatment with ADMA also caused an extent of coronary microvascular lesions that was comparable to that in wild-type mice. These vascular effects of ADMA were not prevented by supplementation of l-arginine, and vascular NO production was not reduced by ADMA treatment. Treatment with ADMA caused upregulation of angiotensin-converting enzyme (ACE) and an increase in superoxide production that were comparable in both strains and that were abolished by simultaneous treatment with temocapril (ACE inhibitor) or olmesartan (AT1 receptor antagonist), which simultaneously suppressed vascular lesion formation. Conclusions—These results provide the first direct evidence that the long-term vascular effects of ADMA are not solely mediated by simple inhibition of endothelial NO synthesis. Direct upregulation of ACE and increased oxidative stress through AT1 receptor appear to be involved in the long-term vascular effects of ADMA in vivo.

Spontaneous Myocardial Infarction in Mice Lacking All Nitric Oxide Synthase Isoforms

Background— The roles of nitric oxide (NO) in the cardiovascular system have been investigated extensively in pharmacological studies with NO synthase (NOS) inhibitors and in studies with NOS isoform–deficient mice. However, because of the nonspecificity of the NOS inhibitors and the compensatory interactions among NOS isoforms (nNOS, iNOS, and eNOS), the ultimate roles of endogenous NO derived from the entire NOS system are still poorly understood. In this study, we examined this point in mice deficient in all 3 NOS isoforms (triply n/i/eNOS−/− mice) that we have recently developed. Methods and Results— The triply n/i/eNOS−/− mice, but not singly eNOS−/− mice, exhibited markedly reduced survival, possibly due to spontaneous myocardial infarction accompanied by severe coronary arteriosclerotic lesions. Furthermore, the triply n/i/eNOS−/− mice manifested phenotypes that resembled metabolic syndrome in humans, including visceral obesity, hypertension, hypertriglyceridemia, and impaired glucose tolerance. Importantly, activation of the renin-angiotensin system was noted in the triply n/i/eNOS−/− mice, and long-term oral treatment with an angiotensin II type 1 receptor blocker significantly suppressed coronary arteriosclerotic lesion formation and the occurrence of spontaneous myocardial infarction and improved the prognosis of those mice, along with ameliorating the metabolic abnormalities. Conclusions— These results provide the first direct evidence that genetic disruption of the whole NOS system causes spontaneous myocardial infarction associated with multiple cardiovascular risk factors of metabolic origin in mice in vivo through the angiotensin II type 1 receptor pathway, demonstrating the critical role of the endogenous NOS system in maintaining cardiovascular and metabolic homeostasis.

Statin Treatment Upregulates Vascular Neuronal Nitric Oxide Synthase Through Akt/NF-κB Pathway

Objective—Three-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are known to enhance vascular expression of endothelial (eNOS) and inducible nitric oxide synthase (iNOS). In this study, we examined whether statins also upregulate vascular expression of neuronal NOS (nNOS). Methods and Results—In cultured rat aortic smooth muscle cells, treatment with atorvastatin significantly increased nNOS expression, associated with activation of Akt and NF-&kgr;B. Inhibition of Akt by dominant-negative Akt suppressed atorvastatin-induced nNOS expression as well as Akt and NF-&kgr;B activation. Inhibition of NF-&kgr;B by dominant-negative I&kgr;B also attenuated atorvastatin-induced nNOS expression and NF-&kgr;B activation, but not Akt activation. We further examined whether atorvastatin also enhances nNOS expression in isolated mouse aorta, and if so, how much nNOS-derived NO accounts for atorvastatin-induced NOx production. In isolated aortas of wild-type mice, atorvastatin significantly increased all three NOS isoform expression and NOx production. In isolated aortas of doubly i/eNOS−/−, n/eNOS−/−, and n/iNOS−/− mice, which express only nNOS, iNOS, and eNOS, respectively, atorvastatin-induced NOx production was approximately 25%, 25%, and 50% to that of wild-type mice, respectively, suggesting that nNOS accounts for 25% of the atorvastatin-mediated NOx production. Conclusions—These results indicate that atorvastatin upregulates vascular nNOS through Akt/NF-&kgr;B pathway, demonstrating a novel nNOS-mediated vascular effect of the statin.

Vascular neuronal NO synthase is selectively upregulated by platelet-derived growth factor: involvement of the MEK/ERK pathway.

Objective—We demonstrated recently that neuronal NO synthase (NOS) is expressed in arteriosclerotic lesions and exerts important vasculoprotective effects in vivo. In this study, we examined the molecular mechanism(s) for vascular neuronal NOS (nNOS) expression. Methods and Results—In cultured rat aortic smooth muscle cells, treatment with platelet-derived growth factor (PDGF) selectively upregulated nNOS expression but not inducible NOS (iNOS) or endothelial NOS (eNOS) expression. Treatment with PDGF also significantly caused activation of mitogen-activated protein kinase (MAPK) family, including extracellular signal-regulated kinase (ERK), p38MAPK, and c-Jun N-terminal kinase (JNK). ERK kinase (MAPK kinase [MEK]) inhibitors inhibited PDGF-induced nNOS expression, whereas a p38MAPK inhibitor or JNK inhibitor was without effects. Importantly, gene transfer of MEK per se elicited nNOS induction, and gene transfer of dominant-negative MEK abolished PDGF-induced nNOS expression. In isolated aortas of wild-type, eNOS−/−, and iNOS−/− mice, but not in those of nNOS−/− mice, treatment with PDGF significantly enhanced nNOS expression and nitrite plus nitrate production, both of which were again attenuated by a MEK inhibitor. Conclusions—These results provide the first evidence that vascular nNOS expression is upregulated selectively in response to PDGF through the MEK/ERK pathway. Upregulated nNOS may play an important compensatory role under arteriosclerotic/inflammatory conditions associated with eNOS dysfunction to maintain vascular homeostasis.

Vascular Neuronal NO Synthase Is Selectively Upregulated by Platelet-Derived Growth Factor. Involvement of the Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Pathway

Objective—We demonstrated recently that neuronal NO synthase (NOS) is expressed in arteriosclerotic lesions and exerts important vasculoprotective effects in vivo. In this study, we examined the molecular mechanism(s) for vascular neuronal NOS (nNOS) expression. Methods and Results—In cultured rat aortic smooth muscle cells, treatment with platelet-derived growth factor (PDGF) selectively upregulated nNOS expression but not inducible NOS (iNOS) or endothelial NOS (eNOS) expression. Treatment with PDGF also significantly caused activation of mitogen-activated protein kinase (MAPK) family, including extracellular signal-regulated kinase (ERK), p38MAPK, and c-Jun N-terminal kinase (JNK). ERK kinase (MAPK kinase [MEK]) inhibitors inhibited PDGF-induced nNOS expression, whereas a p38MAPK inhibitor or JNK inhibitor was without effects. Importantly, gene transfer of MEK per se elicited nNOS induction, and gene transfer of dominant-negative MEK abolished PDGF-induced nNOS expression. In isolated aortas of wild-type, eNOS−/−, and iNOS−/− mice, but not in those of nNOS−/− mice, treatment with PDGF significantly enhanced nNOS expression and nitrite plus nitrate production, both of which were again attenuated by a MEK inhibitor. Conclusions—These results provide the first evidence that vascular nNOS expression is upregulated selectively in response to PDGF through the MEK/ERK pathway. Upregulated nNOS may play an important compensatory role under arteriosclerotic/inflammatory conditions associated with eNOS dysfunction to maintain vascular homeostasis.

Upregulation of Vascular Extracellular Superoxide Dismutase in Patients With Acute Coronary Syndromes

Objective—We examined the vascular expression levels of extracellular superoxide dismutase (EC-SOD), a major antioxidant enzyme in the cardiovascular system, in patients with acute coronary syndromes. Methods and Results—Twenty-one consecutive patients with acute myocardial infarction (AMI), 14 patients with unstable angina, 11 patients with stable angina, and 20 control subjects were studied. The levels of vascular EC-SOD expression were assessed by the difference in plasma EC-SOD concentrations before and after intravenous heparan injection. In the patients with AMI, vascular EC-SOD expression (ng/mL) was significantly higher on day 1 after the onset of AMI (148±10) as compared with the control subjects (116±6, P <0.05). The vascular EC-SOD expression returned to the normal range on day 7 (104±8), and that level persisted thereafter. The vascular EC-SOD expression was also significantly higher in the patients with unstable angina (160±13) than in those with stable angina (122±10) or in the controls (116±6) (P <0.05 each). Moreover, in the patients with AMI, higher levels of vascular EC-SOD expression on day 1 were significantly associated with smaller myocardial infarct size (P <0.05). Conclusions—This is the first clinical demonstration showing that vascular EC-SOD may be upregulated in acute coronary syndromes in humans in vivo. EC-SOD may play an important protective role against increased oxidative stress during acute ischemic coronary events.

Severe dyslipidaemia, atherosclerosis, and sudden cardiac death in mice lacking all NO synthases fed a high-fat diet

AIMS The precise role of the nitric oxide synthase (NOS) system in lipid metabolism remains to be elucidated. We addressed this point in mice that we have recently developed and that lack all three NOS isoforms. METHODS AND RESULTS Wild-type (WT), singly, doubly, and triply NOS(-/-) mice were fed either a regular or high-cholesterol diet for 3-5 months. The high-cholesterol diet significantly increased serum low-density lipoprotein (LDL) cholesterol levels in all the genotypes when compared with the regular diet. Importantly, when compared with the WT genotype, the serum LDL cholesterol levels in the high-cholesterol diet were significantly and markedly elevated only in the triply NOS(-/-) genotype, but not in any singly or doubly NOS(-/-) genotypes, and this was associated with remarkable atherosclerosis and sudden cardiac death, which occurred mainly in the 4-5 months after the high-cholesterol diet. Finally, hepatic LDL receptor expression was markedly reduced only in the triply NOS(-/-) genotype, accounting for the diet-induced dyslipidaemia in the genotype. CONCLUSION These results provide the first direct evidence that complete disruption of all NOS genes causes severe dyslipidaemia, atherosclerosis, and sudden cardiac death in response to a high-fat diet in mice in vivo through the down-regulation of the hepatic LDL receptor, demonstrating the critical role of the whole endogenous NOS system in maintaining lipid homeostasis.

Histamine Upregulates the Expression of Inducible Nitric Oxide Synthase in Human Intimal Smooth Muscle Cells via Histamine H1 Receptor and NF-κB Signaling Pathway

Objective—Histamine increases endothelial nitric oxide (NO) production as an endothelium-dependent vasodilator, which acts as a vasoconstrictor in atherosclerotic coronary arteries. To investigate the relation between histamine and NO production in intimal smooth muscle cells (SMCs), we studied the effect of histamine on inducible NO synthase (iNOS) expression in the SMCs. Methods and Results—In cultured human intimal SMCs, histamine increased NO production, iNOS expression, and NF-&kgr;B nuclear translocation, which were inhibited by histamine H1 blocker and NF-&kgr;B inhibitor. Luciferase assay using −8.3 kb upstream of human iNOS promoter region and electrophoretic mobility shift assay suggested that a NF-&kgr;B motif located at −3922 to −3914 would be necessary for histamine-inducible promoter activity. In addition, H1 blocker, NF-&kgr;B inhibitor, and dominant negative I&kgr;Bα or I&kgr;B kinase β downregulated the histamine-induced iNOS promoter activity. In the human aorta, histamine content was estimated to be 310±66 pmol/mg protein in the atherosclerotic intima, while that was to be 43±22 pmol/mg protein in the media (P<0.001). Conclusions—Histamine stimulates intimal SMCs to increase iNOS expression via H1 receptors and NF-&kgr;B signaling pathway. Histamine could be one of NO-regulating factors, by inducing iNOS expression in intimal SMCs, and may be related to atherogenesis.

Association of seasonal variation in the prevalence of metabolic syndrome with insulin resistance

The aim of this study was to examine the hypothesis that seasonal variation in the prevalence of metabolic syndrome (MetS) is associated with increased insulin resistance. Among 840 Japanese male workers who were evaluated using the homeostasis model assessment of insulin resistance (HOMA-IR) in June (summer) 2010, we prospectively studied a total of 758 subjects (40–65 years of age) who underwent an assessment in December (winter) 2010. MetS was defined according to the criteria proposed by the International Diabetes Federation (IDF) and the Japanese Society of Internal Medicine (JSIM). The median level of HOMA-IR in the study subjects was 0.84 (interquartile range: 0.60–1.19). The prevalence rates of IDF- and JSIM-MetS significantly increased from 12.4 and 9.6% in the summer to 16.6 and 13.3% in the winter, respectively (each P<0.05). Our data suggest that these increases are mainly due to increases in blood pressure (BP) and glucose during the winter assessment. The prevalence rates of IDF-MetS in the first, second, third and fourth quartiles of HOMA-IR were 1.1, 5.8, 14.3 and 29.1% in the summer and 3.1, 10.6, 21.9, and 31.3% in the winter, respectively. Similar results were obtained when using the JSIM criteria. In the third quartile, the frequency of elevated BP increased from 42.4% in the summer to 61.2% in the winter (P<0.05), and these values were mainly correlated with significant variations in IDF- and JSIM-MetS prevalence rates. This study demonstrates that seasonal variation in MetS prevalence is associated with mildly to moderately increased insulin resistance in middle-aged Japanese men.