Tadahisa Shono

Involvement of interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis.

Tumor necrosis factor alpha (TNF-alpha) is a macrophage/monocyte-derived polypeptide which modulates the expression of various genes in vascular endothelial cells and induces angiogenesis. However, the underlying mechanism by which TNF-alpha mediates angiogenesis is not completely understood. In this study, we assessed whether TNF-alpha-induced angiogenesis is mediated through TNF-alpha itself or indirectly through other TNF-alpha-induced angiogenesis-promoting factors. Cellular mRNA levels of interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and their receptors were increased after the treatment of human microvascular endothelial cells with TNF-alpha (100 U/ml). TNF-alpha-dependent tubular morphogenesis in vascular endothelial cells was inhibited by the administration of anti-IL-8, anti-VEGF, and anti-bFGF antibodies, and coadministration of all three antibodies almost completely abrogated tubular formation. Moreover, treatment with Sp1, NF-kappaB, and c-Jun antisense oligonucleotides inhibited TNF-alpha-dependent tubular morphogenesis by microvascular endothelial cells. Administration of a NF-kappaB antisense oligonucleotide almost completely inhibited TNF-alpha-dependent IL-8 production and partially abrogated TNF-alpha-dependent VEGF production, and an Sp1 antisense sequence partially inhibited TNF-alpha-dependent production of VEGF. A c-Jun antisense oligonucleotide significantly inhibited TNF-alpha-dependent bFGF production but did not affect the production of IL-8 and VEGF. Administration of an anti-IL-8 or anti-VEGF antibody also blocked TNF-alpha-induced neovascularization in the rabbit cornea in vivo. Thus, angiogenesis by TNF-alpha appears to be modulated through various angiogenic factors, both in vitro and in vivo, and this pathway is controlled through paracrine and/or autocrine mechanisms.

Involvement of the transcription factor NF-kappaB in tubular morphogenesis of human microvascular endothelial cells by oxidative stress.

Oxygen radicals are induced under various pathologic conditions associated with neovascularization. Oxygen radicals modulate angiogenesis in cultured human microvascular endothelial cells by an unknown mechanism. Treatment of human microvascular endothelial cells for 15 min with 0.1 to 0.5 mM hydrogen peroxide (H2O2) or 100 U of tumor necrosis factor alpha per ml induced tubular morphogenesis in type I collagen gels. Gel shift assays with nuclear extracts demonstrated that H2O2 increases the binding activities of two transcription factors, NF-kappaB and AP-1, but not of Spl. Tumor necrosis factor alpha increased the binding activities of all three factors. A supershift assay with specific antibodies against JunB, JunD, and c-Jun (Jun family) showed that the antibody against c-Jun supershifted the AP-1 complex after H2O2 treatment. Coadministration of the antisense sequence of NF-kappaB inhibited H2O2-dependent tubular morphogenesis, and the antisense c-Jun oligonucleotide caused partial inhibition. The angiogenic factor responsible for H2O2-induced tubular morphogenesis was examined. Cellular mRNA levels of vascular endothelial growth factor and interleukin-8 (IL-8), but not those of transforming growth factor alpha, were increased after treatment with 0.5 mM H2O2. Coadministration of anti-IL-8 antibody inhibited tubular morphogenesis enhanced by H2O2, and IL-8 itself also enhanced the formation of tube-like structures. Treatment with antisense NF-kappaB oligonucleotide completely blocked H2O2-dependent IL-8 production by endothelial cells. The tubular morphogenesis of vascular endothelial cells after treatment with oxidative stimuli and its possible association with NF-kappaB and IL-8, is examined.

MiRNA-196 Is Upregulated in Glioblastoma But Not in Anaplastic Astrocytoma and Has Prognostic Significance

Purpose: MicroRNAs (miRNA) are short noncoding RNAs that can play critical roles in diverse biological processes. They are implicated in tumorigenesis and function both as tumor suppressors and as oncogenes. The clinical significance of miRNA expression profiles in malignant gliomas remains unclear. Experimental Design: In this study, we examined the expression levels of 365 mature human miRNAs in 12 malignant gliomas, including 8 glioblastomas and 4 anaplastic astrocytomas, using TaqMan real-time quantitative PCR arrays. A validation study was done to corroborate a subset of the results, including expression levels of miR-196a, -196b, -21, and -15b, by analyzing 92 malignant gliomas by conventional real-time PCR. We modeled the relationship between the expression levels of these miRNAs and the survival rate of 39 glioblastoma patients by Kaplan-Meier method and multivariate analysis. Results: Expression profiles in glioblastomas and anaplastic astrocytomas suggested that 16 miRNAs were candidate markers associated with the malignant progression of gliomas. Among them, miR-196a showed the most significant difference (P = 0.0038), with miR-196b also having a high significance (P = 0.0371). Both miRNAs showed increased expression levels in glioblastomas relative to both anaplastic astrocytomas and normal brains in the validation study. Furthermore, patients with high miR-196 expression levels showed significantly poorer survival by the Kaplan-Meier method (P = 0.0073). Multivariate analysis showed that miR-196 expression levels were an independent predictor of overall survival in all 39 glioblastoma patients (P = 0.021; hazard ratio, 2.81). Conclusions: Our results suggest that miR-196 may play a role in the malignant progression of gliomas and may be a prognostic predictor in glioblastomas. Clin Cancer Res; 16(16); 4289–97. ©2010 AACR.

Perfusion Imaging of Brain Tumors Using Arterial Spin-Labeling : Correlation with Histopathologic Vascular Density

BACKGROUND AND PURPOSE: We investigated the relationship between tumor blood-flow measurement based on perfusion imaging by arterial spin-labeling (ASL-PI) and histopathologic findings in brain tumors. MATERIALS AND METHODS: We used ASL-PI to examine 35 patients with brain tumors, including 11 gliomas, 9 meningiomas, 9 schwannomas, 1 diffuse large B-cell lymphoma, 4 hemangioblastomas, and 1 metastatic brain tumor. As an index of tumor perfusion, the relative signal intensity (SI) of each tumor (%Signal intensity) was determined as a percentage of the maximal SI within the tumor per averaged SI within normal cerebral gray matter on ASL-PI. Relative vascular attenuation (%Vessel) was determined as the total microvessel area per the entire tissue area on CD-34–immunostained histopathologic specimens. MIB1 indices of gliomas were also calculated. The differences in %Signal intensity among different histopathologic types and between high- and low-grade gliomas were compared. In addition, the correlations between %Signal intensity and %Vessel or MIB1 index were evaluated in gliomas. RESULTS: Statistically significant differences in %Signal intensity were observed between hemangioblastomas versus gliomas (P < .005), meningiomas (P < .05), and schwannomas (P < .005). Among gliomas, %Signal intensity was significantly higher for high-grade than for low-grade tumors (P < .05). Correlation analyses revealed significant positive correlations between %Signal intensity and %Vessel in 35 patients, including all 6 histopathologic types (rs = 0.782, P < .00005) and in gliomas (rs = 0.773, P < .05). In addition, in gliomas, %Signal intensity and MIB1 index were significantly positively correlated (rs = 0.700, P < .05). CONCLUSION: ASL-PI may predict histopathologic vascular densities of brain tumors and may be useful in distinguishing between high- and low-grade gliomas and in differentiating hemangioblastomas from other brain tumors.

Enhanced expression of NADPH oxidase Nox4 in human gliomas and its roles in cell proliferation and survival

Reactive oxygen species (ROS) have been attracting attention as mediators of various cell‐signaling pathways. Nox‐family NADPH oxidases have proven to be a major source of ROS production in various cell types and have crucial roles in various physiological and pathological processes. In this study, we show that Nox4, a member of Nox family, is prominently expressed in various neuroepithelial tumors by reverse transcription‐polymerase chain reaction (RT‐PCR) and immunohistochemical studies. We quantified Nox4 mRNA expression by real‐time PCR in tumor specimens from 58 patients with astrocytomas and found that the expression levels of Nox4 mRNA in glioblastomas (WHO grade IV) were significantly higher than those in other astrocytomas (WHO grade II and III). In addition, we show that specific knockdown of Nox4 expression by RNA interference results in cell‐growth inhibition and enhances induction of apoptosis by chemotherapeutic agents, such as cisplatin, in cultured glioma cell lines. Based on these observations, enhanced expression of Nox4 appears to be involved in cell proliferation and survival in glioma cells.

Results of a long-term follow-up after neuroendoscopic biopsy procedure and third ventriculostomy in patients with intracranial germinomas

OBJECT The authors report the results of long-term follow-ups in 12 patients with intracranial germinomas who underwent neuroendoscopic procedures before chemotherapy and radiotherapy, and discuss the usefulness and safety of these procedures. METHODS Between January 1996 and December 2005 at Kyushu University Hospital, 12 patients with intracranial germinomas underwent neuroendoscopic biopsy procedures involving a flexible fiberscope. Eight patients simultaneously underwent endoscopic third ventriculostomy (ETV) for existing obstructive hydrocephalus. All patients received chemotherapy and radiotherapy postoperatively, according to the regimen promulgated by the Japanese Pediatric Brain Tumor Study Group. The patients were followed for an average of 78.6 months (range 15-134 months), and a retrospective study was conducted. RESULTS Germinomas were histologically verified in all patients. No postoperative deaths or permanent morbidity was related to the neuroendoscopic procedures. No other cerebrospinal fluid diversion, such as that achieved with a ventriculoperitoneal shunt, was needed for the management of hydrocephalus. A complete response to postoperative chemotherapy and radiotherapy was achieved in all cases. Only one patient had a recurrent lesion in the spinal cord 6 years after the initial treatment; however, this patient had undergone only the neuroendoscopic biopsy procedure without ETV. CONCLUSIONS Neuroendoscopic procedures can permit a precise histological diagnosis of intracranial germinomas and are safe and effective in the management of hydrocephalus associated with these tumors. The risk of tumor dissemination due to the neuroendoscopic procedures appears to be minimal when the appropriate chemotherapy and radiotherapy are provided postoperatively.

Vascular endothelial growth factor in chronic subdural haematomas.

OBJECTIVE To elucidate molecular aspects of the mechanisms of expansion of chronic subdural haematomas (CSH), we examined the expression of two representative angiogenic factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in CSH. METHODS We quantified VEGF and bFGF in haematoma fluid and serum of 20 patients with CSH using an enzyme-linked immunosorbent assay. Mean concentrations of VEGF in the haematoma fluid (10277 pg/ml) and in serum, (355 pg/ml) were much greater than those of bFGF (haematoma, 3.04 pg/ml; serum, 4.74 pg/ml). Surgical specimens, including dura and the outer membrane of the CSH were analysed by in situ hybridisation to detect VEGF mRNA. Macrophages and vascular endothelial cells in the outer membrane over expressed VEGF mRNA. CONCLUSIONS Enhanced production of VEGF by macrophages and vascular endothelial cells in the outer membrane is thought to be pathogenetically important in CSH.

Dual Pathways to Tubular Morphogenesis of Vascular Endothelial Cells by Human Glioma Cells : Vascular Endothelial Growth Factor/Basic Fibroblast Growth Factor and Interleukin-8

In this study, we examined whether human glioma cells are angiogenic in a model using human microvascular endothelial cells, and also which factor is responsible for the glioma‐dependent angiogenesis. Tubular morphogenesis in type I collagen gel by human microvascular endothelial cells was stimulated in the presence of 10 and 100 ng/ml of vascular endothelial growth factor (VEGF), 10 ng/ml basic fihroblast growth factor (bFGF) and 10 ng/ml of interleukin‐8 (IL‐8). Tube formation of the microvascular endothelial cells was assayed in the glioma cell lines IN157 and IN301, co‐cultured using the double chamber method. IN301 cells had much higher levels of VEGF, bFGF and transforming growth factor‐(mRNA than IN157 cells, whereas the two had similar levels of transforming growth factor‐Alfa mRNA. By contrast, IN157 cells had much higher levels of IL‐8 mRNA than IN301 cells. IN301‐dependent tubular morphogenesis was inhibited by anti‐VEGF or anti‐bFGF antibody, and the inhibition was almost complete when anti‐VEGF and anti‐bFGF antibodies were present. On the other hand, IN157‐dependent tubular morphogenesis was inhibited by anti‐IL‐8 antibody, but not by anti‐VEGF or anti‐bFGF antibodies. These findings demonstrated dual paracrine controls of tumor angiogenesis by human glioma cells. One is mediated through VEGF and/or bFGF, and the other, through IL‐8.

Effect of tumor removal on tinnitus in patients with vestibular schwannoma.

OBJECT Tinnitus is one of the most common symptoms in patients with vestibular schwannomas (VSs), but the effect of surgery on this symptom has not been fully evaluated. The aim of this study was to define the effect on tinnitus of tumor removal, cochlear nerve resection, and useful hearing preservation in patients with VSs. METHODS The authors retrospectively analyzed the status of tinnitus before and after surgery in 242 patients with unilateral VSs who underwent surgery via the retrosigmoid lateral suboccipital approach. RESULTS Of 242 patients, 171 (70.7%) complained of tinnitus before surgery; the symptom disappeared in 25.2%, improved in 33.3%, remained unchanged in 31.6%, and worsened in 9.9% of these cases after tumor removal. In the 171 patients with preoperative tinnitus, the cochlear nerve was resected in 85 (49.7%) and preserved in 86 (50.3%), but there was no significant difference in the incidence of postoperative tinnitus between these 2 groups (p = 0.293). In the 71 patients without preoperative tinnitus, the symptom developed postoperatively in 6 cases (8.5%). Among those without preoperative tinnitus, the cochlear nerve was resected in 45 cases (63.4%) and tinnitus appeared postoperatively in 3 (6.7%). The authors also analyzed the association between postoperative tinnitus and useful hearing preservation, but could not find any statistically significant association between the 2 factors (p = 0.153). CONCLUSIONS Tumor removal via the retrosigmoid lateral suboccipital approach may provide some chance for improvement of tinnitus in patients with VSs; however, neither cochlear nerve resection nor useful hearing preservation affects the postoperative development of tinnitus.

Expression of VEGF and its receptor genes in intracranial schwannomas

Vascular endothelial growth factor (VEGF) is considered to be a major regulator of angiogenesis in various brain tumors. In this study, we determined the expression levels of VEGF, and vascular endothelial growth factor receptor (VEGFR)-1 and -2 mRNA in 46 intracranial schwannomas by quantitative real-time PCR, and correlated these with various clinical factors or other molecular markers. We found that these tumors expressed significant amounts of VEGF mRNA in comparison with other brain tumors, including malignant gliomas and meningiomas. In addition, we performed immunohistochemical studies for VEGF and VEGFR-1, and confirmed that these tumors prominently express these proteins. The expression levels of VEGF and VEGFR-1 mRNA in recurrent tumors were higher than those in primary tumors. When we divided patients into two groups according to VEGF mRNA expression in the tumor, there was no significant difference in patient age, gender, or cranial nerves of origin between groups; however, the tumor volume tended to be larger in the high VEGF group than in the low VEGF group. The levels of VEGFR-1 mRNA and neurofibromatosis-2 mRNA in the high VEGF group were significantly greater than those in the low VEGF group. Levels of VEGFR-2 mRNA and DNA topoisomerase IIα mRNA, and the MIB-1 labeling index in the high VEGF group were slightly higher than those in the low VEGF group; however, the difference was not statistically significant. Based on these observations, the significance of VEGF and its receptor genes in intracranial schwannomas is discussed.