Hiromi Tasaki

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.

Vasculoprotective roles of neuronal nitric oxide synthase

Nitric oxide (NO) has multiple important actions that contribute to the maintenance of vascular homeostasis. NO is synthesized by three different isoforms of NO synthase (NOS), all of which have been reported to be expressed in human atherosclerotic vascular lesions. Although the regulatory roles of endothelial NOS (eNOS) and inducible NOS (iNOS) on the development of atherosclerosis have been described, little is known about the role of neuronal NOS (nNOS). Here, we show that nNOS also exerts important vasculoprotective effects in vivo. In a carotid artery ligation model, nNOS gene‐deficient (nNOS‐KO) mice exhibited accelerated neointimal formation and constrictive vascular remodeling caused by blood flow disruption. In a rat balloon injury model, the selective inhibition of nNOS activity potently enhanced vasoconstrictor responses to a variety of calcium‐mobilizing stimuli, suppressed tissue cGMP concentrations, a marker of vascular NO production, and exacerbated neointimal formation. In both models, nNOS was absent before injury and was up‐regulated only after the injury, and was predominantly expressed in the neointima and medial smooth muscle cells. These results provide the first direct evidence that nNOS plays important roles in suppressing arteriosclerotic vascular lesion formation in vivo.

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.

Urotensin II-induced activation of extracellular signal-regulated kinase in cultured vascular smooth muscle cells: Involvement of cell adhesion-mediated integrin signaling

Urotensin II (UII), a cyclic dodecapeptide, is a potent mammalian vasoconstrictive substance recently shown to induce proliferation of vascular smooth muscle cells (VSMCs). However, little is known about mechanisms involved in UII-induced mitogenic response such as cell proliferation. To investigate the intracellular signaling pathways involved in this process, we examined the effects of UII on activation of extracellular signal-regulated kinase (ERK) and focal adhesion kinase (FAK) in VSMCs. UII stimulated in time- and dose-dependent manners the phosphorylation level of ERK. In contrast, UII failed to alter the phosphorylation level of FAK. Although angiotensin II-induced ERK phosphorylation was noted even in suspended cells, UII failed to induce an increase in ERK phosphorylation in such cells. On the other hand, UII induced an increase in the phosphorylation level of ERK, but not FAK, in cells adherent to fibronectin. Furthermore, UII-induced proliferation of VSMCs was inhibited by ERK kinase inhibitor PD98059. Our results suggested that activation of integrin-mediated signaling pathways play a critical role in UII-induced phosphorylation of ERK, leading to proliferation of VSMCs, which does not involved increased phosphorylation of FAK.

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.

Efficacy and Safety of Alirocumab in Japanese Patients With Heterozygous Familial Hypercholesterolemia or at High Cardiovascular Risk With Hypercholesterolemia Not Adequately Controlled With Statins – ODYSSEY JAPAN Randomized Controlled Trial –

BACKGROUND The ODYSSEY Japan study was designed to demonstrate the reduction in low-density lipoprotein cholesterol (LDL-C) by alirocumab as add-on to existing lipid-lowering therapy in Japanese patients with heterozygous familial hypercholesterolemia (heFH) or non-FH at high cardiovascular risk who require additional pharmacological management to achieve their LDL-C treatment goal (<2.6 or <3.1 mmol/L, depending on risk category). METHODSANDRESULTS This randomized, double-blind, parallel-group, 52-week study was conducted in Japan. Patients (n=216) with heFH, non-FH at high cardiovascular risk with coronary disease, or classified as category III were enrolled. The prespecified safety analysis was done after the last patient completed 52 weeks. Patients were randomized (2:1, alirocumab:placebo) with stratification for heFH to s.c. alirocumab (75 mg every 2 weeks [Q2 W] with increase to 150 mg if week 8 LDL-C ≥2.6/3.1 mmol/L) or placebo for 52 weeks plus stable statin therapy. At week 24, mean±SE change in LDL-C from baseline was -62.5±1.3% in the alirocumab group and 1.6±1.8% in the placebo group (difference, -64.1±2.2%; P<0.0001); the reduction was sustained to week 52 (alirocumab, -62.5±1.4%; placebo, -3.6±1.9%). No patterns were evident between treatment groups for adverse events at 52 weeks. CONCLUSIONS In high-risk Japanese patients with hypercholesterolemia on stable statin therapy, alirocumab markedly reduced LDL-C vs. placebo and was well tolerated over 52 weeks. (Circ J 2016; 80: 1980-1987).

Comparison of serum lipid values in variant angina pectoris and fixed coronary artery disease with normal subjects

Lipid and apolipoprotein (apo) levels in patients with variant angina were examined and compared with patients with coronary artery disease (CAD) and normal subjects (control). Cholesterol and triglyceride levels in plasma, lipoprotein fractions and several apolipoproteins were measured in 108 men (90 of whom had undergone coronary angiography): 22 had variant angina, 56 had fixed CAD (effort angina and old myocardial infarction) and 30 were normal subjects. Patients with variant angina showed more severe atherosclerotic lesions than the control group, but less severe lesions than the patients with fixed CAD. In comparison with lipid and apolipoprotein, high density lipoprotein cholesterol, apo AI and apo AII decreased significantly in control, variant angina and fixed CAD groups, respectively. Additionally, stepwise discriminant analysis revealed that apo AI was the best discriminator among the 3 groups or between variant angina or fixed CAD and the control group. Variant angina and fixed CAD patients could be discriminated from the control subjects by an apo AI level of 135 and 126 mg/dl, with 71% (p less than 0.025) and 73% (p less than 0.005) accuracy, respectively. By these criteria 77% of the patients with variant angina and 73% of the patients with fixed CAD were precisely discriminated. Discrimination between variant angina and fixed CAD patients, however, was not practical, even if the best discriminator was used. Thus, the apo AI level is useful in discriminating patients with variant angina and fixed CAD from normal subjects. Therefore, symptomatic patients with low apo AI levels should be aggressively examined.