Hirokazu Sadahiro

Inhibition of Fatty Acid Synthase Decreases Expression of Stemness Markers in Glioma Stem Cells

Cellular metabolic changes, especially to lipid metabolism, have recently been recognized as a hallmark of various cancer cells. However, little is known about the significance of cellular lipid metabolism in the regulation of biological activity of glioma stem cells (GSCs). In this study, we examined the expression and role of fatty acid synthase (FASN), a key lipogenic enzyme, in GSCs. In the de novo lipid synthesis assay, GSCs exhibited higher lipogenesis than differentiated non-GSCs. Western blot and immunocytochemical analyses revealed that FASN is strongly expressed in multiple lines of patient-derived GSCs (G144 and Y10), but its expression was markedly reduced upon differentiation. When GSCs were treated with 20 μM cerulenin, a pharmacological inhibitor of FASN, their proliferation and migration were significantly suppressed and de novo lipogenesis decreased. Furthermore, following cerulenin treatment, expression of the GSC markers nestin, Sox2 and fatty acid binding protein (FABP7), markers of GCSs, decreased while that of glial fibrillary acidic protein (GFAP) expression increased. Taken together, our results indicate that FASN plays a pivotal role in the maintenance of GSC stemness, and FASN-mediated de novo lipid biosynthesis is closely associated with tumor growth and invasion in glioblastoma.

Fatty acid binding protein 7 as a marker of glioma stem cells.

Glioblastomas are the most aggressive brain tumors. Glioblastoma stem cells (GSCs) are thought to be responsible for the recurrence, chemoresistance, and poor prognosis of glioblastoma. Fatty acid binding protein 7 (FABP7), which is a cellular chaperone for a variety of omega‐3 fatty acids, is a known marker for neural stem cells. In this study, using a newly developed anti‐FABP7 antibody and patient‐derived GSC lines, we evaluated the expression of FABP7 in GSCs. Using immunocytochemistry, Western blotting, and qPCR analyses, FABP7 was found to be highly enriched in GSCs and its localization was found in cytosol and nuclei. FABP7 expression was significantly downregulated in differentiated GSCs induced by the addition of serum. In the glioma surgical specimens, FABP7 was highly expressed in the majority of glioblastoma. Double immunostaining for FABP7 and Sox2 showed that FABP7+Sox2+ tumor cells were significantly increased in glioblastoma (grade IV) compared with diffuse astrocytoma (grade II) and anaplastic astrocytoma (grade III). Our data introduces FABP7 as a marker for GSCs and further highlights its possible significance for glioma diagnosis and treatment.

Histological Characterization of the Tumorigenic "Peri-Necrotic Niche" Harboring Quiescent Stem-Like Tumor Cells in Glioblastoma.

Background Characterization of the niches for stem-like tumor cells is important to understand and control the behavior of glioblastomas. Cell-cycle quiescence might be a common mechanism underlying the long-term maintenance of stem-cell function in normal and neoplastic stem cells, and our previous study demonstrated that quiescence induced by hypoxia-inducible factor (HIF)-1α is associated with a high long-term repopulation capacity of hematopoietic stem cells. Based on this, we examined human astrocytoma tissues for HIF-1α-regulated quiescent stem-like tumor cells as a candidate for long-term tumorigenic cells and characterized their niche histologically. Methods Multi-color immunohistochemistry was used to visualize HIF-1α-expressing (HIF-1α+) quiescent stem-like tumor cells and their niche in astrocytoma (WHO grade II–IV) tissues. This niche was modeled using spheroids of cultured glioblastoma cells and its contribution to tumorigenicity was evaluated by sphere formation assay. Results A small subpopulation of HIF-1α+ quiescent stem-like tumor cells was found in glioblastomas but not in lower-grade astrocytomas. These cells were concentrated in the zone between large ischemic necroses and blood vessels and were closer to the necrotic tissues than to the blood vessels, which suggested that a moderately hypoxic microenvironment is their niche. We successfully modeled this niche containing cells of HIF-1α+ quiescent stem-like phenotype by incubating glioblastoma cell spheroids under an appropriately hypoxic condition, and the emergence of HIF-1α+ quiescent stem-like cells was shown to be associated with an enhanced sphere-forming activity. Conclusions These data suggest that the “peri-necrotic niche” harboring HIF-1α+ quiescent stem-like cells confers a higher tumorigenic potential on glioblastoma cells and therefore may be a therapeutic target to control the behavior of glioblastomas.

Pathological features of highly invasive glioma stem cells in a mouse xenograft model

Glioma stem cells (GSCs) may be a source of tumor progression and recurrence after multimodal therapy, because of their high invasive potential. The purpose of this study was to compare the invasive and migratory properties of GSCs and non-GSCs and examine the distribution of these cells in a mouse xenograft model. Three GSC lines, G144, Y02, and Y10, cultured from human glioblastoma, were used in the study. Matrigel-invasion assays of infiltration and time-lapse studies of migration were performed for comparison of the GSCs with the corresponding differentiated non-GSC lines. Cells were also transplanted into mouse brain and the different distribution of GSCs and non-GSCs was examined in the tumor xenograft model. All 3 GSC lines had greater invasion and migration ability than the corresponding non-GSCs. In vivo, GSCs infiltrated more widely than non-GSCs and reached the contralateral hemisphere via the corpus callosum in the early stage of tumorigenesis. GSCs also primarily penetrated the subventricular zone (SVZ). GSCs have high invasive potential and tend to be present in the outer tumor bulk and infiltrate the contralateral hemisphere via the corpus callosum, in addition to penetrating the SVZ.

Cognitive outcome differences on the side of carotid artery stenting

OBJECTIVE The right and left sides of the brain play different roles in cognition. Therefore, the side of treatment should be taken into consideration when evaluating cognitive outcome following revascularization. Thus, we designed a study to evaluate changes in right hemisphere cognitive function in patients undergoing right carotid artery stentings (CAS) and left hemispheric cognitive function in patients undergoing left CAS. In addition, we studied CAS-related changes in regional cerebral blood flow to determine potential correlations with changes in cognitive function. METHODS We performed a prospective assessment of 39 CAS patients, all of whom were right-handed. Patients with contralateral stenotic lesions were excluded. Twenty-one patients underwent CAS of the right internal carotid artery (Right CAS group) and 18 underwent CAS of the left internal carotid artery (Left CAS group). Neuropsychological testing was performed preoperatively and 6 months after endovascular treatment. Cerebral blood flow was determined by 123I-labeled N-isopropyl-p-iodoamphetamine single-photon emission computed tomography before and 6 months after CAS. RESULTS In the Right CAS group, postoperative performance intelligence quotient score (91.1±18.2) was significantly improved compared with the preoperative score (84.9±16.7; P<.001). In the Left CAS group, postoperative verbal intelligence quotient score (104.0±18.8) was significantly higher than that before endovascular treatment (97.9±15.8; P<.005). Postoperative regional cerebral blood flow was not significantly different from that before endovascular treatment in either group. However, regional cerebrovascular reactivity of the treated side showed significant improvement after treatment. CONCLUSIONS Amelioration of cognitive function may be dependent on the side of revascularization. Performance intelligence quotient improved after CAS in patients with severe carotid artery stenosis on the right side. Verbal intelligence quotient also improved on the left side after endovascular treatment. These effects seemed to involve improvement in regional cerebrovascular reactivity by CAS.

Comparison of lumbar drainage and external ventricular drainage for clearance of subarachnoid clots after Guglielmi detachable coil embolization for aneurysmal subarachnoid hemorrhage.

OBJECTIVE Subarachnoid clots play an important role in development of delayed vasospasm after subarachnoid hemorrhage (SAH). The purpose of this study was to compare clearance of subarachnoid clots using external ventricular drainage (EVD) or lumbar drainage (LD) after Guglielmi detachable coil (GDC) embolization for aneurysmal SAH. METHODS The subjects were 51 treated with GDC coil embolization for aneurysmal Fisher group 3 SAH within 72 h of ictus. Software-based volumetric quantification of the subarachnoid clots was performed on CT scans and the hemoglobin (Hb) level was measured in CSF drained from each catheter. RESULTS Clearance of subarachnoid clots was more rapid in patients treated with LD (n=34) compared to those treated with EVD (n=17). The Hb level in CSF was significantly higher in the LD group on Days 4-5 after onset of SAH (P<0.05), but was higher in the EVD group on Days 8-9. The incidence of symptomatic vasospasm did not differ between the two groups. The rate of occurrence of a new low density area on CT scans was higher in patients treated with EVD, but not significantly higher than the rate in the LD group. CONCLUSION GDC embolization followed by lumbar drainage accelerates the reduction of subarachnoid clots, but EVD may contribute to stasis of hemorrhage within subarachnoid spaces.

Intravenous thrombolysis with recombinant tissue plasminogen activator in a stroke patient treated with rivaroxaban.

As limited amounts of data are available regarding thrombolytic therapy for patients taking novel oral anticoagulants, thrombolytic therapy is not recommended in such cases. Here, we report an acute stroke patient taking rivaroxaban who received intravenous thrombolysis with recombinant tissue plasminogen activator (rt-PA). An 80-year-old man with a history of nonvalvular atrial fibrillation, who had been receiving 10 mg of rivaroxaban showed abrupt onset of aphasia and right hemiparesis. National Institutes of Health Stroke Scale score was 10. Onset of neurologic deficits occurred 4 hours after the last dose of rivaroxaban. Clinical data on admission were as follows: blood pressure, 170/90 mm Hg; prothrombin time (PT), 22.6 seconds (control, 12.9 seconds); international normalized ratio, 2.03; activated partial thromboplastin time, 46 seconds (normal, 23-32 seconds); and creatinine level, 1.11 mg/dL. Magnetic resonance angiography revealed occlusion of the superior trunk of the left middle cerebral artery. Intravenous infusion of .6 mg/kg of rt-PA (total dose, 36 mg) was performed 6 hours after the last rivaroxaban administration with informed consent. The neurologic deficit improved during infusion of rt-PA. Repeat brain computed tomography showed left frontal cortical infarction without hemorrhagic changes. In the case of rivaroxaban, it is difficult to accurately determine the drug activity. As the anticoagulant activity of rivaroxaban can be estimated from its pharmacokinetics and PT, it is clinically important to obtain accurate information about the timing of medication and blood sampling.

A novel trigger for cholesterol-dependent smooth muscle contraction mediated by the sphingosylphosphorylcholine-Rho-kinase pathway in the rat basilar artery: a mechanistic role for lipid rafts.

Hyperlipidemia is a risk factor for abnormal cerebrovascular events. Rafts are cholesterol-enriched membrane microdomains that influence signal transduction. We previously showed that Rho-kinase-mediated Ca2+ sensitization of vascular smooth muscle (VSM) induced by sphingosylphosphorylcholine (SPC) has a pivotal role in cerebral vasospasm. The goals of the study were to show SPC-Rho-kinase-mediated VSM contraction in vivo and to link this effect to cholesterol and rafts. The SPC-induced VSM contraction measured using a cranial window model was reversed by Y-27632, a Rho-kinase inhibitor, in rats fed a control diet. The extent of SPC-induced contraction correlated with serum total cholesterol. Total cholesterol levels in the internal carotid artery (ICA) were significantly higher in rats fed a cholesterol diet compared with a control diet or a β-cyclodextrin diet, which depletes VSM cholesterol. Western blotting and real-time PCR revealed increases in flotillin-1, a raft marker, and flotillin-1 mRNA in the ICA in rats fed a cholesterol diet, but not in rats fed the β-cyclodextrin diet. Depletion of cholesterol decreased rafts in VSM cells, and prevention of an increase in cholesterol by β-cyclodextrin inhibited SPC-induced contraction in a cranial window model. These results indicate that cholesterol potentiates SPC-Rho-kinase-mediated contractions of importance in cerebral vasospasm and are compatible with a role for rafts in this process.

Importance of Early Postoperative Body Temperature Management for Treatment of Subarachnoid Hemorrhage.

BACKGROUND The importance of acute-phase brain temperature management is widely accepted for prevention of exacerbation of brain damage by a high body temperature. METHODS In this study, we investigated the influence of body temperature in the early postoperative period on the outcomes of 62 patients with subarachnoid hemorrhage who were admitted to our department. Body temperature was measured from day 4 to day 14 after onset. The patients were divided into those treated with surgical clipping (clip group) and coil embolization (coil group), those graded I-III (mild) and IV-V (severe) based on the Hunt & Hess classification on admission, those with and without development of delayed cerebral ischemia (DCI), and those with favorable and poor outcomes. Body temperatures throughout the hospital stay were compared in each group. RESULTS There was no significant difference in body temperature between the clip and coil groups or between the mild and severe groups, but body temperature was significantly higher in patients with DCI compared to those without DCI, and in patients with a poor outcome compared to those with a favorable outcome. CONCLUSIONS Fever in the early postoperative period of subarachnoid hemorrhage is associated with development of DCI and a poor outcome.

Adverse Events after Unruptured Cerebral Aneurysm Treatment: A Single-center Experience with Clipping/Coil Embolization Combined Units

BACKGROUND Indications of clipping (Clip) or coil embolization (Coil) for unruptured cerebral aneurysms (uAN) was not elaborated because prediction of rupture and risk of treatment are difficult. This study aims to determine the risk-benefit analysis of treating uAN by a comprehensive and retrospective investigation of the adverse events and sequelae in patients treated by our Clip/Coil combined units. METHODS Clip and Coil were performed in 141 and 80 patients, respectively; Clip for middle cerebral artery AN and Coil for paraclinoid or basilar apex AN. Worsening of modified Rankin scale or mini-mental state examination was defined as major morbidity. Minor morbidity or transient morbidity was defined as other neurologic deficits. Mortality and these morbidities were considered as serious adverse events. Convulsion or events outside the brain were defined as mild adverse events. RESULTS Total mortality and major morbidity were low. Incidence of serious adverse events was not significantly different between the Clip and Coil (17 patients [12.1%] and 6 patients [7.5%]), but the number of total adverse events was significantly different (32 patients [22.7%] in Clip vs. 8 patients [10.0%] in Coil). Because mild morbidities were significantly more frequent in the Clip (20 patients [14.2%]) compared with the Coil (2 patients [2.5%]). Convulsion occurred in 11 (7.8%) patients in the Clip but none in the Coil. CONCLUSIONS Our combined unit decreased the occurrence of mortality/major morbidity; however, minor adverse effects were common, especially in the Clip group because of many intrinsic problems of Clip itself. This result suggests further consideration for the treatment modality for uAN.