Setsuro Ibayashi

Nox4 as the Major Catalytic Component of an Endothelial NAD(P)H Oxidase

Background—Recent evidence has suggested that reactive oxygen species are important signaling molecules in vascular cells and play a pivotal role in the development of vascular diseases. The activity of NAD(P)H oxidase has been identified as the major source of reactive oxygen species in vascular endothelial cells. However, the precise molecular structure and the mechanism of activation of the oxidase have remained poorly understood. Methods and Results—Here, we investigated the molecular identities and the superoxide-producing activity of endothelial NAD(P)H oxidase. We found that Nox4, a homologue of gp91phox/Nox2, was abundantly expressed in endothelial cells. The expression of Nox4 in endothelial cells markedly exceeded that of other Nox proteins, including gp91phox/Nox2, and was affected by cell growth. Using electron spin resonance and chemiluminescence, we measured the superoxide production and found that the endothelial membranes had an NAD(P)H-dependent superoxide-producing activity comparable to that of the neutrophil membranes, whereas the activity was not enhanced by the 2 recombinant proteins p47phox and p67phox, in contrast to that of the neutrophil membranes. Downregulation of Nox4 by an antisense oligonucleotide reduced superoxide production in endothelial cells in vivo and in vitro. Conclusions—These findings suggest that Nox4 may function as the major catalytic component of an endothelial NAD(P)H oxidase.

Incidence and Risk Factors for Subtypes of Cerebral Infarction in a General Population: The Hisayama Study

Background and Purpose We estimated the incidence of first-ever cerebral infarction in regard to its subtypes and analyzed their risk factors separately in a community-based prospective cohort study in Japan. Methods Stroke-free subjects (n=1621) aged ≥40 years were followed up for 32 years from 1961. During this period, 298 cerebral infarctions occurred and were divided into 167 lacunar, 62 atherothrombotic, 56 cardioembolic, and 13 undetermined subtypes of infarction on the basis of clinical information including brain imaging and autopsy findings. Results The age-adjusted incidence of lacunar infarction (3.8 per 1000 person-years for men and 2.0 for women) was higher than that of atherothrombotic infarction (1.2, 0.7) and cardioembolic infarction (1.3, 0.5) in both sexes. Time-dependent Cox’s proportional hazard analysis revealed systolic blood pressure as well as age to be independent risk factors for all subtypes of cerebral infarction except for cardioembolic infarction in men. Additionally, ST depression on ECG, glucose intolerance, and smoking in men and left ventricular hypertrophy on ECG and body mass index in women remained significant risk factors for lacunar infarction. ST depression was also significantly related to events of atherothrombotic infarction in women. The risk of atrial fibrillation for cardioembolic infarction was outstandingly high in both sexes, and left ventricular hypertrophy and lower total cholesterol were additional risk factors for cardioembolic infarction in women. Conclusions In this Japanese population, lacunar infarction was the most common subtype of cerebral infarction and had a greater variety of risk factors, including not only hypertension but also ECG abnormalities, diabetes, obesity, and smoking, than did atherothrombotic infarction or cardioembolic infarction.

A nonsynonymous SNP in PRKCH (protein kinase C η) increases the risk of cerebral infarction

Cerebral infarction is the most common type of stroke and often causes long-term disability. To investigate the genetic contribution to cerebral infarction, we conducted a case-control study using 52,608 gene-based tag SNPs selected from the JSNP database. Here we report that a nonsynonymous SNP in a member of protein kinase C (PKC) family, PRKCH, was significantly associated with lacunar infarction in two independent Japanese samples (P = 5.1 × 10−7, crude odds ratio of 1.40). This SNP is likely to affect PKC activity. Furthermore, a 14-year follow-up cohort study in Hisayama (Fukuoka, Japan) supported involvement of this SNP in the development of cerebral infarction (P = 0.03, age- and sex-adjusted hazard ratio of 2.83). We also found that PKCη was expressed mainly in vascular endothelial cells and foamy macrophages in human atherosclerotic lesions, and its expression increased as the lesion type progressed. Our results support a role for PRKCH in the pathogenesis of cerebral infarction.

Antiplatelet therapy contributes to acute deterioration of intracerebral hemorrhage.

Objective: The purpose of this study was to examine the effect of antiplatelet therapy on the initial severity and the acute outcome of intracerebral hemorrhage (ICH). Methods: The authors reviewed records of 251 consecutive patients hospitalized in their cerebrovascular center within 24 hours after onset of ICH. Results: Fifty-seven patients (23%) had development of ICH during oral antiplatelet therapy. The major indication for antiplatelet therapy was the prevention of stroke recurrence (63%). As compared with patients without antiplatelet therapy, those who received antiplatelet therapy more frequently were aged 70 years or older (60% vs 35%; p < 0.001), had previous symptomatic ischemic stroke (54% vs 7%; p < 0.0001), had diabetes mellitus (26% vs 15%; p < 0.05), and had heart disease (32% vs 8%; p < 0.0001). Antiplatelet therapy was predictive of an increase in the hematoma volume by more than 40% on the second hospital day (hematoma enlargement, odds ratio [OR] 7.67, 95% CI 1.62 to 36.4) and the need for emergent surgical evacuation of the hematoma (OR 3.10, 95% CI 1.18 to 8.15). Antiplatelet therapy was an independent predictor for the occurrence of any of hematoma enlargement, emergent death, or evacuation surgery, which suggests that clinical deterioration occurs into the second hospital day (OR 7.45, 95% CI 2.46 to 22.5). Conclusions: Antiplatelet therapy seems to contribute to the acute clinical deterioration of intracerebral hemorrhage.

Postischemic Gene Transfer of Interleukin-10 Protects Against Both Focal and Global Brain Ischemia

Background—Gene therapy may be a promising approach for treatment of brain ischemia, although the efficiency of postischemic gene therapy is not established. Our goal in this study was to examine the effects of gene transfer of interleukin-10 (IL-10), an antiinflammatory cytokine, after induction of brain ischemia. Methods and Results—Brain ischemia was produced by either photochemical occlusion of distal middle cerebral artery for focal ischemia or bilateral carotid occlusion for global ischemia in spontaneously hypertensive rats. Adenoviral vectors encoding human IL-10 (AdIL10) or &bgr;-galactosidase (control) were injected into the lateral ventricle 90 or 60 minutes after focal or global ischemia. Five days after ischemia, IL-10, IL-1&bgr;, or tissue necrosis factor-&agr; in the cerebrospinal fluid, infarct volume, infiltrations of leukocytes/macrophages in the infarct area, or hippocampal neuronal damages were determined. The transduced IL-10 was released to the cerebrospinal fluid from the ventricular wall and increased to 7623±2965 pg/mL 5 days after AdIL10 transfection. Cerebral blood flow during ischemia was not different between treatments in either focal or global ischemia. Brain infarction of the AdIL10 group was significantly smaller and infiltrations of leukocytes and macrophages were fewer in the IL-10 treatment than control. Hippocampal neurons after global ischemia were more preserved, and the terminal deoxynucleotidyl transferase–mediated dUTP-biotin in situ nick end labeling–positive cells were diminished by the IL-10 gene transfer with attenuated IL-1&bgr; and augmented tissue necrosis factor-&agr;. Conclusions—Postischemic gene transfer of IL-10 into the lateral ventricle attenuated brain infarction and hippocampal damages, suggesting the promise for treatment of brain ischemia.

LDL Cholesterol and the Development of Stroke Subtypes and Coronary Heart Disease in a General Japanese Population The Hisayama Study

Background and Purpose— Although the relation between serum LDL cholesterol level and coronary heart disease (CHD) is well established, its relation with stroke subtypes is less clear. Methods— A total of 2351 inhabitants age ≥40 years in a Japanese community were followed up for 19 years. Results— During follow-up, 271 subjects developed stroke and 144 developed CHD. Whereas the age- and sex-adjusted incidences of CHD significantly increased with increasing LDL cholesterol levels (P for trend <0.001), the associations between LDL cholesterol level and the incidences of ischemic or hemorrhagic stroke were not significant. The age- and sex-adjusted incidences of atherothrombotic infarctions (ATIs) and lacunar infarctions (LIs) significantly increased with increasing LDL cholesterol level (P for trend=0.03 for ATIs and=0.02 for LIs), but no such association was observed for cardioembolic infarction. After multivariate adjustment, the positive associations of LDL cholesterol level with the risks of ATI and CHD remained significant (P for trend=0.02 for ATIs and=0.03 for CHD), whereas the association with LIs was not significant. The risk of ATI significantly increased in the fourth quartile of LDL cholesterol compared with the first quartile (multivariate-adjusted hazard ratio=2.84; 95% CI, 1.17 to 6.93). The multivariate-adjusted risks for developing nonembolic infarction (ATIs and LIs) and CHD were significantly elevated in the groups with elevated LDL cholesterol values with and without the metabolic syndrome. Conclusions— Our findings suggest that an elevated LDL cholesterol level is a significant risk factor for developing ATI as well as CHD, and these associations are independent of the metabolic syndrome.

Free radical scavenger, edaravone, in stroke with internal carotid artery occlusion

BACKGROUND Edaravone has potent free radical quenching and antioxidant actions. The agent has been recently in commercial use for acute ischemic stroke patients. In this study, we investigated the therapeutic effect of edaravone on severe carotid-territorial stroke. METHODS Stroke patients with internal carotid artery occlusion and baseline NIH Stroke Scale Score > or =15 were treated for 14 days with drip intravenous infusion of edaravone (n=30) and were compared with a historical control cohort of similar patients (n=31). Glycerol was also administered to all patients in both groups. RESULTS Infarct volume (P<0.02) and midline shift (P<0.02) on CT performed on day 2 of the patients treated with edaravone were smaller than those without edaravone. For patients with edaravone, infarct volume (P<0.0001) and midline shift (P<0.0001) on days 5-7 were greater than those on day 2. Hemorrhagic transformation of infarcts on day 2 was less severe in patients with than without edaravone (P<0.03). Within 14 days after the onset of stroke, 6 patients with edaravone (20%) and 14 without edaravone (45%) died directly of stroke (P<0.03). Among all patients, only two treated with edaravone were independent without any assistance 8 weeks after the onset. CONCLUSIONS Edaravone was associated with delayed evolution of infarcts and edema in patients with severe carotid-territorial stroke and decreased mortality during the acute stage. The agent, however, failed to prevent evolution of infarcts and edema on later days, and did not significantly improve functional outcome among the surviving patients.

NAD(P)H Oxidases in Rat Basilar Arterial Endothelial Cells

Background and Purpose— Reactive oxygen species (ROS) may play a critical role in the regulation of vascular tone and development of vascular diseases, such as stroke. NAD(P)H oxidase is a major source of ROS in vascular cells, including endothelial cells. It has been considered that Nox2 and Nox4 are exclusively expressed among Nox homologues in the endothelial cells of noncerebral blood vessels. However, the precise molecular identity of the NAD(P)H oxidase in the endothelial cells of the cerebral arteries is not fully understood. We examined the expression of Nox homologues and their activation mechanism in the endothelial cells of the cerebral arteries. Methods— We isolated and cultured basilar artery endothelial cells (BAECs) of Sprague-Dawley rats. Expression of NAD(P)H oxidase was examined by reverse-transcription-polymerase chain reaction (RT-PCR) and immunohistological staining. Results— RT-PCR disclosed abundant expression of Nox4 with marginal Nox2 in BAEC. In addition, Nox1 was expressed highly both at mRNA and protein levels in BAECs. Immunohistological staining also showed the prominent expression of Nox1 in the endothelial cells of the basilar artery. With respect to the cytosolic components of NAD(P)H oxidases, BAECs expressed p67phox and, to a lesser extent, p47phox, Noxo1, and Noxa1. Both NADH and NADPH induced superoxide production of the BAEC membranes. The phagocyte-type cytosolic components, p47phox and p67phox, significantly enhanced the NADH-induced superoxide production of the BAEC membranes, whereas the components failed to increase the NADPH-induced superoxide production. Conclusions— Nox1 is highly expressed in the endothelial cells of the cerebral arteries along with Nox2 and Nox4, and the endothelial NAD(P)H oxidase of the cerebral arteries may have a unique activation mechanism by the phagocyte-type cytosolic components.

Leukoaraiosis and dementia in hypertensive patients.

Background and Purpose: Although our previous study demonstrated that dementia of the Binswanger type may be a disconnection dementia caused by leukoaraiosis, some hypertensive patients with marked leukoaraiosis do not develop dementia. The goal of the present study is to elucidate the pathophysiology of nondemented hypertensive patients with leukoaraiosis. Methods: We performed clinical and neuroradiological studies, including positron emission tomography, in eight hypertensive patients with leukoaraiosis. Results: Four patients were demented, and two among the other four who were not demented at the first examination developed dementia during the follow-up period. Digital subtraction angiography of the cervical and intracranial arteries demonstrated stenotic lesions in only one patient. Cerebral blood flow and oxygen metabolism in patients with dementia were markedly reduced in the white matter (59–67% of control values). In contrast, cerebral blood flow in the white matter of patients without dementia was reduced less markedly (74% of control), oxygen extraction fraction in the white matter was significantly increased (130% of control), and oxygen metabolism remained at almost-normal levels not only in the white matter but also in the cortical area. Conclusions: Hypertension-caused arteriosclerotic changes of the long penetrating medullary arteries may cause misery perfusion and later ischemic damage in the periventricular white matter. Preserved oxygen metabolism in hypertensive patients with leukoaraiosis may represent the early stage of vascular dementia of the Binswanger type.

Ovariectomy Exacerbates and Estrogen Replacement Attenuates Photothrombotic Focal Ischemic Brain Injury in Rats

BACKGROUND AND PURPOSE We previously reported the infarct volumes in female spontaneously hypertensive rats (SHR) to be significantly smaller than those in male SHR. The purpose of the present study was to determine whether estrogen is responsible for the sex difference in ischemic vulnerability in SHR. METHODS In experiment 1, 1 week (short-term) or 4 weeks (long-term) after the ovariectomy (OVX), female SHR (5 months old) were randomly subjected to photothrombotic occlusion of the middle cerebral artery, and the infarct volumes were determined. In experiment 2, the rats were randomly assigned to 3 groups (ie, the sham-ovariectomized, ovariectomized, and estrogen replacement groups). In the replacement group, estradiol valerate (200 microgram/kg) was subcutaneously injected once a week after the OVX. Four weeks after the OVX or sham-OVX, all rats were subjected to middle cerebral artery occlusion. Changes in regional cerebral blood flow were determined by laser-Doppler flowmetry. RESULTS In experiment 1, the infarct volume produced 1 week after the OVX was not different from that of the sham-ovariectomized group. In contrast, the infarct volume produced 4 weeks after the OVX was significantly larger than that of the sham-ovariectomized group (82.4+/-11.6 versus 54.5+/-16.0 mm(3), P=0.0058). In experiment 2, estradiol replacement after the OVX was observed to attenuate the infarct volume compared with the ovariectomized group (55.6+/-18.8 versus 78.5+/-21.0 mm(3), P=0.0321). The degrees of regional cerebral blood flow reduction did not differ among the sham-ovariectomized, ovariectomized, and estrogen replacement groups. CONCLUSIONS Chronic estrogen depletion was thus found to increase the infarct size, which was attenuated by estradiol replacement. These findings indicate that estrogen contributes to the sex difference in ischemic vulnerability and that endogenous estrogen also has a neuroprotective effect against ischemic brain damage.