Yoshitane Tsukamoto

Administration of CD34 + cells after stroke enhances neurogenesis via angiogenesis in a mouse model

Thrombo-occlusive cerebrovascular disease resulting in stroke and permanent neuronal loss is an important cause of morbidity and mortality. Because of the unique properties of cerebral vasculature and the limited reparative capability of neuronal tissue, it has been difficult to devise effective neuroprotective therapies in cerebral ischemia. Our results demonstrate that systemic administration of human cord blood-derived CD34(+) cells to immunocompromised mice subjected to stroke 48 hours earlier induces neovascularization in the ischemic zone and provides a favorable environment for neuronal regeneration. Endogenous neurogenesis, suppressed by an antiangiogenic agent, is accelerated as a result of enhanced migration of neuronal progenitor cells to the damaged area, followed by their maturation and functional recovery. Our data suggest an essential role for CD34(+) cells in promoting directly or indirectly an environment conducive to neovascularization of ischemic brain so that neuronal regeneration can proceed.

Prolonged Endoplasmic Reticulum Stress in Hypertrophic and Failing Heart After Aortic Constriction Possible Contribution of Endoplasmic Reticulum Stress to Cardiac Myocyte Apoptosis

Background—The endoplasmic reticulum (ER) is recognized as an organelle that participates in folding secretory and membrane proteins. The ER responds to stress by upregulating ER chaperones, but prolonged and/or excess ER stress leads to apoptosis. However, the potential role of ER stress in pathophysiological hearts remains unclear. Methods and Results—Mice were subjected to transverse aortic constriction (TAC) or sham operation. Echocardiographic analysis demonstrated that mice 1 and 4 weeks after TAC had cardiac hypertrophy and failure, respectively. Cardiac expression of ER chaperones was significantly increased 1 and 4 weeks after TAC, indicating that pressure overload by TAC induced prolonged ER stress. In addition, the number of terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL)–positive cells increased, and caspase-3 was cleaved in failing hearts. The antagonism of angiotensin II type 1 receptor prevented upregulation of ER chaperones and apoptosis in failing hearts. On the other hand, angiotensin II upregulated ER chaperones and induced apoptosis in cultured adult rat cardiac myocytes. We also investigated possible signaling pathways for ER-initiated apoptosis. The CHOP- (a transcription factor induced by ER stress), but not JNK- or caspase-12–, dependent pathway was activated in failing hearts by TAC. Pharmacological ER stress inducers upregulated ER chaperones and induced apoptosis in cultured cardiac myocytes. Finally, mRNA levels of ER chaperones were markedly increased in failing hearts of patients with elevated brain natriuretic peptide levels. Conclusions—These findings suggest that pressure overload by TAC induces prolonged ER stress, which may contribute to cardiac myocyte apoptosis during progression from cardiac hypertrophy to failure.

ORP150 protects against hypoxia/ischemia-induced neuronal death

Oxygen-regulated protein 150 kD (ORP150) is a novel endoplasmic-reticulum–associated chaperone induced by hypoxia/ischemia. Although ORP150 was sparingly upregulated in neurons from human brain undergoing ischemic stress, there was robust induction in astrocytes. Cultured neurons overexpressing ORP150 were resistant to hypoxemic stress, whereas astrocytes with inhibited ORP150 expression were more vulnerable. Mice with targeted neuronal overexpression of ORP150 had smaller strokes compared with controls. Neurons with increased ORP150 demonstrated suppressed caspase-3–like activity and enhanced brain-derived neurotrophic factor (BDNF) under hypoxia signaling. These data indicate that ORP150 is an integral participant in ischemic cytoprotective pathways.

Adrenomedullin Infusion Attenuates Myocardial Ischemia/Reperfusion Injury Through the Phosphatidylinositol 3-Kinase/Akt-Dependent Pathway

Background—Infusion of adrenomedullin (AM) has beneficial hemodynamic effects in patients with heart failure. However, the effect of AM on myocardial ischemia/reperfusion remains unknown. Methods and Results—Male Sprague-Dawley rats were exposed to a 30-minute period of ischemia induced by ligation of the left coronary artery. They were randomized to receive AM, AM plus wortmannin (a phosphatidylinositol 3-kinase [PI3K] inhibitor), or saline for 60 minutes after coronary ligation. Hemodynamics and infarct size were examined 24 hours after reperfusion. Myocardial apoptosis was also examined 6 hours after reperfusion. The effect of AM on Akt phosphorylation in cardiac tissues was examined by Western blotting. Intravenous administration of AM significantly reduced myocardial infarct size (28±4% to 16±1%, P <0.01), left ventricular end-diastolic pressure (19±2 to 8±2 mm Hg, P <0.05), and myocardial apoptotic death (19±2% to 9±4%, P <0.05). Western blot analysis showed that AM infusion accelerated Akt phosphorylation in cardiac tissues and that pretreatment with wortmannin significantly attenuated AM-induced Akt phosphorylation. Moreover, pretreatment with wortmannin abolished the beneficial effects of AM: a reduction of infarct size, a decrease in left ventricular end-diastolic pressure, and inhibition of myocardial apoptosis after ischemia/reperfusion. Conclusions—Short-term infusion of AM significantly attenuated myocardial ischemia/reperfusion injury. These cardioprotective effects are attributed mainly to antiapoptotic effects of AM via a PI3K/Akt-dependent pathway.

Rate of successful recording of blood flow signals in the middle cerebral artery using transcranial Doppler sonography.

Background and Purpose To assess the usefulness of transcranial Doppler sonography, we investigated the rate of blood flow signal recording failure in the middle cerebral artery in Japanese subjects. Furthermore, we studied the effect of increased emitted power on the rate of successful recording in some of the patients in whom recording failure had been detected at the standard transducer power of 100 mW/cm2. Methods To evaluate the rate of successful recording, we measured blood flow signals in 597 patients (age range, 16 to 89 years) for screening of cerebrovascular disease by using a 2-MHz range-gated, pulsed-wave Doppler instrument at the standard transducer power. In 18 elderly patients with recording failure at the standard power, we assessed the effect of increased emitted power of 400 mW/cm2 on flow signal recording. Results Blood flow signals were recorded in 920 (77.1%) of the 1194 middle cerebral arteries of the 597 patients studied. The rate of successful recording of bilateral middle cerebral artery flow signals (70.9%; 423 of 597 patients) decreased with age, especially in females (17.0% in women aged 70 years or older). In 12 of 18 elderly patients with recording failure at the standard power, blood flow signals could be detected at the increased emitted power of 400 mW/cm2. Conclusions The rate of successful recording of blood flow signals in Japanese subjects decreases with advancing age, especially in females. Increasing the emitted power markedly improves the successful recording rate.

Asymptomatic carotid lesions and silent cerebral infarction

Background and Purpose Few studies have investigated the relationships between asymptomatic carotid lesions and silent infarcts confirmed on magnetic resonance imaging. Methods A consecutive series of 117 subjects (average age, 62±9.4 years) who were free from neurological deficit but had at least one established risk factor for stroke were investigated by B-mode carotid ultrasonography and magnetic resonance imaging of the brain. Carotid lesions were evaluated by plaque score, maximum percent stenosis, and the existence of ulcerated lesions. The relations between the carotid lesions and the incidence, size, or localization of the brain lesions were investigated. Results The incidence of silent infarcts was 42% in all subjects and significantly increased with advancing age (P<.05). Most lesions were smaller than 1 cm in diameter and were usually localized in the subcortical white matter or the basal ganglia. The percentage of subjects with infarcts in- creased significantly as the plaque score increased (P<.05) or when subjects had high-grade stenosis (P<.05) or ulcerated lesions (P<.01). These relationships were also noted in each decade of age. A higher incidence of larger lesions (>1 cm) was found in the brain hemisphere ipsilateral to the carotid lesion, particularly in subjects with high-grade stenosis or ulcerated lesions (P<.01). Multivariate analysis indicated significant correlations with silent infarcts for age, hypertension, and plaque score. Conclusions Both the severity and characteristics of asymptomatic carotid lesions estimated by B-mode ultrasonography were closely related to the appearance of silent infarcts. These results demonstrate that noninvasive assessment of carotid lesions can be useful in predicting the existence of silent cerebral infarction even in patients free from neurological deficits.

Reactivity of cerebral blood flow to carbon dioxide in various types of ischemic cerebrovascular disease: evaluation by the transcranial Doppler method.

Background and Purpose The response of cerebral blood flow to changes in the arterial carbon dioxide partial pressure (i.e., carbon dioxide reactivity) has been evaluated as a parameter of cerebral perfusion reserve in patients with cerebrovascular disease. In this study, variations in this reactivity in various ischemic cerebrovascular diseases were evaluated by a newly established method, a transcranial Doppler technique. Methods Thirty-three patients with symptomatic cerebrovascular disease, 13 patients with asymptomatic cerebral infarction, and 25 age-matched normal control subjects were investigated. The symptomatic patients were divided into three groups; those with unilateral carotid artery obstruction, those with cortical infarction, and those with lacunar infarction. The carbon dioxide reactivity of each subject was determined by simultaneously measuring the mean spatial Doppler frequency in the middle cerebral artery and the end-tidal carbon dioxide partial pressure under normocapnia hypercapnic, and hypocapnic conditions. Results In the patients with carotid obstruction, the carbon dioxide reactivity of the hemisphere ipsilateral to the obstruction was more impaired than the reactivity of the symptomatic hemispheres in any other group, and was significantly less than in the contralateral asymptomatic hemisphere (p<0.01). In patients with cortical infarction, the carbon dioxide reactivity of the symptomatic hemisphere was significantly less than in normal control subjects (p<0.05) and was also less than that of the contralateral asymptomatic hemisphere (p<0.05). In patients with lacunar infarction, the carbon dioxide reactivity of both hemispheres was significantly less than that in normal controls (p<0.01), although there was no difference between the symptomatic and asymptomatic hemispheres. In patients with asymptomatic infarction, the carbon dioxide reactivity was also less than that in normal controls (p<0.01). Conclusions The carbon dioxide reactivity of cerebral blood flow measured by this transcranial Doppler technique may be useful for characterizing the hemodynamic changes that occur in various types of ischemic cerebrovascular disease.

Synovial sarcoma is a stem cell malignancy.

Synovial sarcoma (SS) is a malignant soft tissue tumor characterized by its unique t(X;18)(p11;q11) chromosomal translocation leading to the formation of the SS18‐SSX fusion gene. The resulting fusion protein product is considered to play as an aberrant transcription factor and transform target cells by perturbing their gene expression program. However, the cellular origin of SS is highly debated. We herein established two novel human SS cell lines, named Yamato‐SS and Aska‐SS, and investigated their biological properties. We found the self‐renewal ability of these cells to generate sarcospheres, to form tumors in serial xenotransplantation and reconstitute the tumor phenotypes without fractionation by any surface markers. Both SS cells as well as clinical tissue specimens from 15 patients expressed the marker genes‐associated stem cell identity, Oct3/4, Nanog, and Sox2. We also found that both SS cells displayed limited differentiation potentials for mesenchymal lineages into osteocytes and chondrocytes albeit with the expression of early mesenchymal and hematopoietic lineage genes. Upon SS18‐SSX silencing with sequence‐specific siRNAs, these SS cells exhibited morphological transition from spherical growth in suspension to adherent growth in monolayer, additional expression of later mesenchymal and hematopoietic lineage genes, and broader differentiation potentials into osteocytes, chondrocytes, adipocytes, and macrophages in appropriate differentiation cocktails. Collectively, these data suggest that a human multipotent mesenchymal stem cell can serve as a cell of origin for SS and SS is a stem cell malignancy resulting from dysregulation of self‐renewal and differentiation capacities driven by SS18‐SSX fusion protein. STEM CELLS 2010;28:1119–1131

Reactivity of cerebral blood flow to carbon dioxide in hypertensive patients: evaluation by the transcranial Doppler method.

OBJECTIVE To evaluate hypertensive cerebral involvement before cerebrovascular accidents. DESIGN Cerebral microvascular responses to changes in the arterial partial pressure of CO2 (pCO2; the CO2 reactivity) were compared among patients with different stages and severity of hypertensive disease. PATIENTS Fifty-eight patients with hypertension, 11 with borderline hypertension, 15 hypertensives with cerebral infarction and 58 normotensive controls were studied. METHODS The cerebrovascular CO2 reactivity was determined by measuring simultaneously the end-tidal pCO2 and the blood flow velocity in the middle cerebral artery using transcranial Doppler sonography under hypocapnic, normocapnic and hypercapnic conditions. RESULTS CO2 reactivity was impaired in the hypertensive patients compared with in the normotensive controls, but less so than in the symptomatic hemisphere of the hypertensive patients with cerebral infarction. The CO2 reactivity in the borderline hypertensive patients was greater than that in both the symptomatic and asymptomatic hemispheres of the hypertensive patients with cerebral infarction. In the subjects without cerebral infarction, two risk factors for cerebral atherosclerosis (age and hypertension) were negatively correlated with cerebrovascular CO2 reactivity. In the hypertensive patients age and the estimated duration of hypertension were negatively correlated with cerebrovascular CO2 reactivity. CO2 reactivity in the patients with hypertensive or arteriosclerotic retinopathy or ST-T changes on their electrocardiogram was impaired compared with that in the patients without such changes. CONCLUSIONS Hypertension affected the microvascular reactivity of the brain before the development of cerebrovascular accidents, and its effect varied dependently on the extent of involvement of other target organs.

ORP150/HSP12A protects renal tubular epithelium from ischemia-induced cell death

The 150 kDa oxygen‐regulated protein (ORP150) is an inducible endoplasmic reticulum (ER) chaperone with cytoprotective properties in settings of cell stress, such as ischemia/reperfusion (I/R). Renal tissue from patients with acute renal failure displayed strong induction of ORP150 in tubular epithelium. In a rodent model of renal I/R injury, ORP150 was expressed in both the ischemic and contralateral kidney, principally in the thick ascending loop of Henle (TAL) and distal tubules. Cultured renal epithelial cells exposed to hypoxic or hyperosmotic conditions displayed induction of ORP150. Renal tubular epithelial cells stably transfected with ORP150 sense or antisense cDNA displayed a strong correlation between ORP150 expression and vulnerability to hypoxic/osmotic stress; higher levels of ORP150 were protective, whereas lower levels increased susceptibility to cell death. Compared with nontransgenic controls, transgenic mice overexpressing ORP150 subjected to renal I/R displayed a blunted rise of serum creatinine and blood urea nitrogen, and enhanced survival of TAL, consistent with cytoprotection. In contrast, heterozygous ORP150+/− mice, with lower levels of ORP150, showed enhanced renal injury. These data are consistent with the possibility that ORP150 exerts cytoprotective effects in renal tubular epithelia subjected to I/R injury and suggest a key role for ER stress in the renal tubular response to acute renal failure.