Yoshitaka Ohashi

Hypotension and reduced nitric oxide-elicited vasorelaxation in transgenic mice overexpressing endothelial nitric oxide synthase.

Nitric oxide (NO), constitutively produced by endothelial nitric oxide synthase (eNOS), plays a major role in the regulation of blood pressure and vascular tone. We generated transgenic mice overexpressing bovine eNOS in the vascular wall using murine preproendothelin-1 promoter. In transgenic lineages with three to eight transgene copies, bovine eNOS-specific mRNA, protein expression in the particulate fractions, and calcium-dependent NOS activity were confirmed by RNase protection assay, immunoblotting, and L-arginine/citrulline conversion. Immunohistochemical studies revealed that eNOS protein was predominantly localized in the endothelial cells of aorta, heart, and lung. Blood pressure was significantly lower in eNOS-overexpressing mice than in control littermates. In the transgenic aorta, basal NO release (estimated by Nomega-nitro-L-arginine-induced facilitation of the contraction by prostaglandin F2alpha) and basal cGMP levels (measured by enzyme immunoassay) were significantly increased. In contrast, relaxations of transgenic aorta in response to acetylcholine and sodium nitroprusside were significantly attenuated, and the reduced vascular reactivity was associated with reduced response of cGMP elevation to these agents as compared with control aortas. Thus, our novel mouse model of chronic eNOS overexpression demonstrates that, in addition to the essential role of eNOS in blood pressure regulation, tonic NO release by eNOS in the endothelium induces the reduced vascular reactivity to NO-mediated vasodilators, providing several insights into the pathogenesis of nitrate tolerance.

Interaction of Oxidative Stress and Inflammatory Response in Coronary Plaque Instability: Important Role of C-Reactive Protein

Objective—C-reactive protein (CRP), a predictor of cardiovascular events, localizes in atherosclerotic arteries and exerts proinflammatory effects on vascular cells. Reactive oxygen species (ROS) have been implicated in atherogenesis and plaque instability. Methods and Results—Expressional pattern of CRP in directional coronary atherectomy specimens from 39 patients was examined. Characteristics of histological plaque instability and higher levels of serum CRP and fibrinogen were associated with the CRP immunoreactivity. In situ hybridization revealed the presence of CRP mRNA in coronary vasculature. Furthermore, the expression of CRP mRNA and protein was detected in cultured human coronary artery smooth muscle cells (CASMCs) by reverse transcriptase–polymerase chain reaction and Western blotting. In addition, CRP was frequently colocalized with p22phox, an essential component of NADH/NADPH oxidase, which is an important source of ROS in vasculature. Moreover, the incubation of cultured CASMCs with CRP resulted in the enhanced p22phox protein expression and in the generation of intracellular ROS. Conclusions—The expression of CRP in coronary arteries was associated with histological and clinical features of vulnerable plaque, and it had a prooxidative effect on cultured CASMCs, suggesting that it might play a crucial role in plaque instability and in the pathogenesis of acute coronary syndrome via its prooxidative effect.

Superoxide Generation in Directional Coronary Atherectomy Specimens of Patients With Angina Pectoris: Important Role of NAD(P)H Oxidase

Objective—NADH/NADPH oxidase is an important source of reactive oxygen species (ROS) in the vasculature. Recently, we demonstrated that p22phox, an essential component of this oxidase, was expressed in human coronary arteries and that its expression was enhanced with the progression of atherosclerosis. The present study was undertaken to investigate its functional importance in the pathogenesis of coronary artery disease. For this aim, the expression of p22phox, the distribution of oxidized low density lipoprotein (LDL), and the generation of ROS in directional coronary atherectomy (DCA) specimens were examined. Methods and Results—DCA specimens were obtained from patients with stable or unstable angina pectoris. The distribution of p22phox and of oxidized LDL was examined by immunohistochemistry. The generation of superoxide in DCA specimens was assessed by the dihydroethidium method and lucigenin-enhanced chemiluminescence. ROS were closely associated with the distribution of p22phox and oxidized LDL. Not only inflammatory cells but also smooth muscle cells and fibroblasts generated ROS. There was a correlation between ROS and the expression of p22phox or oxidized LDL. The generation of ROS was significantly higher in unstable angina pectoris compared with stable angina pectoris. Conclusions—ROS generated by p22phox-based NADH/NADPH oxidase likely mediate the oxidative modification of LDL and might play a major role in pathogenesis of atherosclerotic coronary artery disease.

Resistance to Endotoxin Shock in Transgenic Mice Overexpressing Endothelial Nitric Oxide Synthase

BACKGROUND Nitric oxide (NO) plays a central role in the pathogenesis of septic shock. However, the role of the NO produced by endothelial NO synthase (eNOS) in septic shock is still unclear. We examined the effect of chronic eNOS overexpression and the role of eNOS-derived NO in lipopolysaccharide (LPS)-induced septic shock using eNOS transgenic (Tg) mice. METHODS AND RESULTS LPS was intraperitoneally injected into Tg and control mice. No differences existed in the peak plasma nitrate and nitrate levels induced by LPS between the 2 genotypes. In LPS-treated control mice, blood pressure progressively declined and reached 60% of basal levels (from 97+/-3 to 59+/-3 mm Hg) 24 hours after LPS injection. In contrast, the blood pressure of LPS-treated Tg mice fell only 15% from basal levels (from 84+/-4 to 71+/-4 mm Hg) after the first 6 hours and, thereafter, it remained at this level. LPS-induced increases in the expression of the mRNA of both vascular cell adhesion molecule-1 and intracellular adhesion molecule-1 in the lungs were significantly lower in Tg mice than in control mice. LPS-induced pulmonary leukocyte infiltration and increases in lung water content were also significantly attenuated in Tg mice. Histological examination revealed that lung injury after LPS injection was milder in Tg mice. Furthermore, Tg mice exhibited enhanced survival from LPS-induced septic shock compared with control mice. CONCLUSIONS Chronic eNOS overexpression in the endothelium of mice resulted in resistance to LPS-induced hypotension, lung injury, and death. These effects are associated with the reduced vascular reactivity to NO and the reduced anti-inflammatory effects of NO.

Possible Role of Brain-Derived Neurotrophic Factor in the Pathogenesis of Coronary Artery Disease

Background— The neurotrophin (NT) family, including nerve growth factor NT-3 and brain-derived neurotrophic factor (BDNF), has a critical role in the survival, growth, maintenance, and death of central and peripheral neurons. NTs and their receptors are expressed in atherosclerotic lesions; however, their significance in cardiovascular disease remains unclear. Methods and Results— To clarify the role of NTs in the pathogenesis of coronary artery disease, NT plasma levels in the aorta, coronary sinus, and peripheral veins of patients with unstable angina (n=38), stable effort angina (n=45), and non–coronary artery disease (n=24) were examined. In addition, regional expression of BDNF in coronary arteries was examined in autopsy cases and patients with angina pectoris by directional coronary atherectomy. The difference in BDNF levels, but not NT-3, between the coronary sinus and aorta was significantly greater in the unstable angina group compared with the stable effort angina and non–coronary artery disease groups. Immunohistochemical investigations demonstrated BDNF expression in the atheromatous intima and adventitia in atherosclerotic coronary arteries. BDNF expression was enhanced in macrophages and smooth muscle cells in atherosclerotic coronary arteries. Stimulation with recombinant BDNF significantly enhanced NAD(P)H oxidase activity and the generation of reactive oxygen species in cultured human coronary artery smooth muscle cells. Conclusions— BDNF has an important role in atherogenesis and plaque instability via the activation of NAD(P)H oxidase.

Reduced Hypoxic Pulmonary Vascular Remodeling by Nitric Oxide From the Endothelium

We examined whether overproduction of endogenous nitric oxide (NO) can prevent hypoxia-induced pulmonary hypertension and vascular remodeling by using endothelial NO-overexpressing (eNOS-Tg) mice. Male eNOS-Tg mice and their littermates (wild-type, WT) were maintained in normoxic or 10% hypoxic condition for 3 weeks. In normoxia, eNOS protein levels, Ca2+-dependent NOS activity, and cGMP levels in the lung of eNOS-Tg mice were higher than those of WT mice. Activity of eNOS and cGMP production in the lung did not change significantly by hypoxic exposure in either genotype. Chronic hypoxia did not induce iNOS expression nor increase its activity in either genotype. Plasma and lung endothelin-1 levels were increased by chronic hypoxia, but these levels were not significantly different between the 2 genotypes. In hemodynamic analysis, right ventricular systolic pressure (RVSP) in eNOS-Tg mice was similar to that in WT mice in normoxia. Chronic hypoxia increased RVSP and induced right ventricular hypertrophy in both genotypes; however, the degrees of these increases were significantly smaller in eNOS-Tg mice. Histological examination revealed that hypoxic mice showed medial wall thickening in pulmonary arteries. However, the increase of the wall thickening in small arteries (diameter <80 &mgr;m) by chronic hypoxia was inhibited in eNOS-Tg mice. Furthermore, muscularization of small arterioles was significantly attenuated in eNOS-Tg mice. Thus, we demonstrated directly that overproduction of eNOS-derived NO can inhibit not only the increase in RVSP associated with pulmonary hypertension but also remodeling of the pulmonary vasculature and right ventricular hypertrophy induced by chronic hypoxia.

Mechanisms of Reduced Nitric Oxide/cGMP–Mediated Vasorelaxation in Transgenic Mice Overexpressing Endothelial Nitric Oxide Synthase

NO, constitutively produced by endothelial NO synthase (eNOS), plays a key regulatory role in vascular wall homeostasis. We generated transgenic (Tg) mice overexpressing eNOS in the endothelium and reported the presence of reduced NO-elicited relaxation. The purpose of this study was to clarify mechanisms of the reduced response to NO-mediated vasodilators in eNOS-Tg mice. Thoracic aortas of Tg and control mice were surgically isolated for vasomotor studies. Relaxations to acetylcholine and sodium nitroprusside were significantly reduced in Tg vessels compared with control vessels. Relaxations to atrial natriuretic peptide and 8-bromo-cGMP were also significantly reduced in Tg vessels. Reduced relaxations to these agents were restored by chronic N(G)-nitro-L-arginine methyl ester treatment. Basal cGMP levels of aortas were higher in Tg mice than in control mice, whereas soluble guanylate cyclase (sGC) activity in Tg vessels was approximately 50% of the activity in control vessels. Moreover, cGMP-dependent protein kinase (PKG) protein levels and PKG enzyme activity were decreased in Tg vessels. These observations indicate that chronic overexpression of eNOS in the endothelium resulted in resistance to the NO/cGMP-mediated vasodilators and that at least 2 distinct mechanisms might be involved: one is reduced sGC activity, and the other is a decrease in PKG protein levels. We reported for the first time that increased NO release from the endothelium reduces sGC and PKG activity in mice. These data may provide a new insight into the mechanisms of nitrate tolerance and cross tolerance to nitrovasodilators.

Endothelial NO Synthase Overexpression Inhibits Lesion Formation in Mouse Model of Vascular Remodeling

Abstract —NO produced by endothelial NO synthase (eNOS) plays important roles in the regulation of vascular tone and structure. The purpose of this study was to clarify the role of eNOS-derived NO on vascular remodeling by use of eNOS-transgenic (eNOS-Tg) mice. The common carotid artery was ligated just proximal to the carotid bifurcation. Four weeks later, the proximal carotid artery of the ligation site was histologically examined. In this vascular remodeling model, the endothelium remains uninjured, but neointimal and medial thickening occurs in combination with a reduction in vascular diameter at the proximal portion of the ligation. At 4 weeks after ligation, the respective neointimal and medial areas in wild-type mice were 17 200±1100 and 24 300±1500 &mgr;m2, whereas both were reduced to 8000±1900 (P <0.01) and 18 400±700 &mgr;m2 (P <0.01) in eNOS-Tg mice (n=8). Total vascular area was not different between the 2 genotypes. NG-Nitro-l-arginine methyl ester treatment increased neointimal and medial areas to the same extent in both genotypes. Leukocyte infiltration was observed in the luminal side of the vessel, but the number of infiltrating cells was significantly attenuated in eNOS-Tg mice compared with wild-type mice. This reduction of leukocyte infiltration in eNOS-Tg mice was associated with reduced expressions of intracellular adhesion molecule-1 and vascular cellular adhesion molecule-1 on the endothelium. In conclusion, chronic eNOS overexpression in the endothelium reduced leukocyte infiltration and inhibited neointimal formation and medial thickening. Our data provide the evidence for the regulatory role of NO from the endothelium on vascular structure integrity.

“Arteries Within the Artery” After Kawasaki Disease A Lotus Root Appearance by Intravascular Ultrasound

A 26-year-old man underwent cardiac catheterization because of abnormal electrocardiography (QS in leads V1 to V3) and thallium stress scintigraphy (a fixed defect in the anteroseptal wall). The patient had a history of suspected Kawasaki disease with sudden cardiac arrest at the age of 1 year. Coronary angiography showed no significant …

Effect of Culprit-Lesion Remodeling Versus Plaque Rupture on Three-Year Outcome in Patients With Acute Coronary Syndrome

To investigate intravascular ultrasound predictors of long-term clinical outcome in patients with acute coronary syndrome, 94 patients with a first acute coronary syndrome with both preintervention intravascular ultrasound imaging and long-term follow-up were enrolled in this study. Remodeling index was defined as external elastic membrane cross-sectional area at the target lesion divided by that at the proximal reference. Arterial remodeling was defined as either positive (PR: remodeling index >1.05) or intermediate/negative remodeling (remodeling index < or =1.05). Clinical events were death, myocardial infarction, and target-lesion revascularization. Patients were followed up for a mean of 3 years. PR was observed in 50 (53%), and intermediate/negative remodeling, in 44 (47%). During the 3-year follow-up, there were 20 target-lesion revascularization events and 5 deaths (2 cardiac and 3 noncardiac), but no myocardial infarctions. Patients with PR showed significantly lower major adverse cardiac event (MACE; death, myocardial infarction, and target-lesion revascularization)-free survival (log-rank p = 0.03). However, patients with plaque rupture showed a nonsignificant trend toward lower MACE-free survival (p = 0.13), but there were no significant differences in MACE-free survival between those with single versus multiple plaque ruptures. Using multivariate logistic regression analysis, only culprit lesion PR was an independent predictor of MACEs (p = 0.04). In conclusion, culprit-lesion remodeling rather than the presence or absence of culprit-lesion plaque rupture was a strong predictor of long-term (3-year) clinical outcome in patients with acute coronary syndrome.