Takao Nakagawa

Secretion of matrix metalloproteinase-2 (72 kD gelatinase/type IV collagenase = gelatinase A) by malignant human glioma cell lines: implications for the growth and cellular invasion of the extracellular matrix

Human glioma cells (T98G and A172 cell lines) were cultured on various extracellular matrix (ECM) components including type 1, IV and V collagens, fibronectin, laminin, and reconstituted basement membrane (Matrigel), and the role of matrix metalloproteinases (MMPs) in their growth and invasion was examined. T98G glioma cells grew well on these ECM components and invaded the reconstituted basement membrane. In contrast, A172 glioma cells showed growth inhibition on collagen types IV and V and Matrigel without invasion of the Matrigel. Gelatin zymography and enzyme immunoassays demonstrated that T98G glioma cells, but not A172 cells, secrete a large amount of matrix metallproteinase-2 (MMP-2, 72 kD gelatinase/type IV collagenase = gelatinase A), and this was confirmed by immunoblotting and immunohistochemistry. Of the two different tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2), T98G cells produced only TIMP-1 during culture on Matrigel, whereas A172 cells secreted both. Although both human recombinant TIMP-1 and TIMP-2 stimulated T98G cell growth slightly on Matrigel, the in vitro invasiveness was significantly reduced by only recombinant TIMP-2. These results suggest that MMP-2 plays an important role in the ECM invasion of T98G human glioma cells in vitro.

Production of urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor-1 (PAI-1) in human brain tumours.

Summary We investigated the role of plasminogen activators (PAs) and their inhibitor (plasminogen activator inhibitor-1, PAI-1) in human brain tumours. The amounts of urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor-1 (PAI-1), and the activity of u-PA and t-PA were determined by enzyme-linked immunosorbent assay (ELISA), and u-PA and PAI-1 were immunolocalized using monoclonal antibodies in human brain tumours and normal brain tissues. The tissues were surgically removed from 64 patients; normal brain tissue (5 cases), low-grade glioma (4 cases), high-grade glioma (17 cases), metastatic tumour (9 cases), meningioma (benign 12 cases, malignant 6 cases), acoustic schwannoma (11 cases). u-PA activity and u-PA and PAI-1 antigen levels were significantly elevated in malignant brain tumours (malignant meningiomas, high-grade gliomas, and metastatic tumours) and acoustic schwannomas but very low in benign meningiomas, low-grade gliomas and normal brain. There was no difference in t-PA antigen levels among normal and malignant tissues, however levels of t-PA activity were markedly decreased in metastastic tumours. All malignant brain tumour tissues showed positive immunostaining for u-PA and PAI-1, however, some tumour cells showed negative intensity while others showed strong intensity for these antibodies. This contrasts to the homogeneous staining pattern found in acoustic schwannoma. These findings indicate that malignancy in human brain tumours is associated with elevated levels of u-PA and PAI-1 and that an imbalance between these proteins in a micro-enviroment contributes (ascribes) to tumour cell invasion.

Experimental and clinical study of detection of glioma at surgery using fluorescent imaging by a surgical microscope after fluorescein administration.

Total resection is the optimal treatment for malignant gliomas. However, an unexpected residual tumor mass is sometimes found on magnetic resonance imaging performed after an operation because of a macroscopically unclear margin of the tumor at surgery. This study was designed to evaluate the effectiveness of fluorescent imaging by a surgical microscope after fluorescein administration for the detection of gliomas at surgery. For this study, we produced two filters for the excitation and emission of fluorescein that can be easily fitted to and removed from a surgical microscope manually during the operation. For the experimental study, Wistar rat brains bearing C6 glioma were removed at appropriate intervals after intravenous administration of 10-20 mg kg-1 body weight of sodium fluorescein, and their surface and coronal section through the tumor were observed using a surgical microscope with the filters. In clinical cases, 1000 mg of sodium fluorescein was intravenously administered to five patients with glioma before tumor resection. In the experimental study, the C6 glioma itself and the edematous brain adjacent to the tumor (within 2-3 mm of the gross surface of the tumor) were well stained a brilliant yellowish green for a few hours. The normal brain was not stained. In clinical cases, the tumors were stained a brilliant yellowish green under fluorescent observation at surgery. The patients had no side effects. At all times the fluorescent observation could be quickly changed to ordinary observation by removing the filters from the surgical microscope. The tumor was also stained a faint yellow under ordinary nonfluorescent observation. Although this contributed to detection of the tumor, the fluorescent staining demarcated the tumor more clearly than nonfluorescent staining. These results suggest that this imaging technique by a surgical microscope with special filters at surgery may be practical and useful for detection of gliomas and warrants further clinical evaluations.

Neuronal loss and expression of neurotrophic factors in a model of rat chronic compressive spinal cord injury.

Study Design. An experimental animal study about neuronal loss and the expression of neurotrophic factors in the chronic compressive spinal cords. Objectives. To investigate neuronal loss and the expression of neurotrophic factors in the chronic compressive spinal cords of rats, and to evaluate effects of decompressive procedures for the neuronal loss. Summary of Background Data. Chronic compression of spinal cords induces the loss of motor neurons in the anterior horn. However, the precise mechanism of this neuronal loss is not still understood completely. Furthermore, it is uncertain whether decompressive procedures prevent this neuronal loss or not. Methods. A thin expanding polymer sheet was implanted microsurgically underneath T7 laminae of rats. After 6, 9, 12, and 15 weeks, the thoracic spinal cord was harvested and examined histopathologically. The expression of neurotrophic factors, including NGF, BDNF, NT-3, GDNF, CNTF, and VEGF, was analyzed using semiquantitative RT-PCR, enzyme immunoassay, and immunohistochemistry. Decompressive surgery was performed through the removal of T7 laminae and the compression materials 6, 9, and 12 weeks after starting compression. Three weeks later, respectively, the neuronal loss in the anterior horn was estimated. Results. The spinal cords were progressively flattened by the expanding of the implanted polymer sheet, and the number of motor neurons in the anterior horn decreased, especially from 6 to 9 weeks after starting compression. Semiquantitative RT-PCR analysis showed that the expression of NGF and BDNF mRNAs was decreased significantly in the spinal cords of 12-week compression group compared with the 6-week compression group and that NGF mRNA expression was up-regulated significantly in the 6-week compression group relative to the 6-week control group. Any changes of expression of other neurotrophic factors were not significant. Since BDNF, not NGF, has been known to be one of the powerful survival factors for spinal motoneurons, we investigated the levels of BDNF protein in the compressive spinal cords using enzyme immunoassay and immunohistochemistry. We demonstrated the level of BDNF protein in the compressive spinal cords was increased 6 weeks after compression but declined after 12 weeks. The decompressive procedure inthe 6 weeks after compression prevented neuronal loss, but the same procedure in the 9 or 12 weeks was ineffective. Conclusions. From the point of view of neuronal loss, decompressive surgery at an earlier stage, when compensatory mechanisms including the up-regulation of BDNF might be still effective, could provide better therapeutic results against chronic mechanical compressive spinal cord lesions.

Prognostic significance of the immunohistochemical expression of O6-methylguanine-DNA methyltransferase, P-glycoprotein, and multidrug resistance protein-1 in glioblastomas.

We studied the expression of O6‐methylguanine‐DNA methyltransferase (O6‐MGMT), P‐glycoprotein (Pgp), and multidrug resistance protein‐1 (MRP‐1) in 23 glioblastomas using RT‐PCR, methylation‐specific PCR, and immunohistochemistry, and analyzed their association with overall patient survival. Univariate analysis of collected data demonstrated that the expressions of O6‐MGMT and MRP‐1 detected by immunohistochemistry, in addition to the consistent factors, including preoperative Karnofsky performance scale (KPS), radical surgery, and tumor location and extension, were significant prognostic factors for the overall survival (OS) of patients with glioblastoma, who received nimustine (ACNU)‐based chemotherapy in association with surgery and radiotherapy. Among them, following multivariate analysis, preoperative KPS, radical surgery, tumor location, and the expression of O6‐MGMT remained as significant prognostic factors. These findings suggest that immunohistochemical analysis of O6‐MGMT in patients with glioblastoma can be a useful method to predict the effects of chemotherapy and identify alternative chemotherapeutic regimens for O6‐MGMT‐positive patients.

Astroblastoma: Immunohistochemical and ultrastructural study of distinctive epithelial and probable tanycytic differentiation

We report the clinicopathological findings of astroblastoma found in an 8‐year‐old girl who was subsequently treated for 11u2003years. The primary superficially circumscribed tumor was located in the frontoparietal lobe, while the recurrent and the second recurrent tumor were restricted to the same region 11u2003years later. The tumors obtained on these three occasions showed fundamentally the same histological, immunohistochemical and fine structural features. They exhibited astrocytic as well as ependymal tanycytic features with apparent epithelial cell lineage. The tumor cells showed typical features of astroblastoma comprising prominent perivascular pseudorosettes with remarkable vascular sclerosis. The immunohistochemical study revealed intensive positivity of GFAP, vimentin, epithelial membrane antigen (EMA), cytokeratin, connexin 26 and 32, desmocollin 1 and neuronal cadherin. The fine structure revealed divergent types of junctional complexes, some of which were connected with tonofilament bundles. Numerous microvilli protruded and basal lamina abutted on the tumor cell surface. We report these unique histological features, and stress that astroblastoma should be categorized as a specific type of neuroepithelial tumor.

Expression of vascular endothelial growth factor-A and mRNA stability factor HuR in human meningiomas

We studied the expression of vascular endothelial growth factor-A (VEGF-A) and mRNA stability factor HuR in 40 supratentorial meningiomas using RT-PCR, ELISA and immunohistochemistry, and analyzed their associations with the clinicopathological characteristics, including microvascular density (MVD), peritumoral brain edema (PTBE), histological subtypes and grades, and the performance of preoperative arterial embolization. Furthermore, we investigated the involvement of HuR in the upregulation of VEGF-A expression using primary meningioma cell cultures. The level of VEGF-A is elevated in meningiomas with PTBE and in higher grade meningiomas. Preoperative arterial embolization did not significantly increase the level of VEGF-A, but it did increase the expression of HuR in tumor tissues. HuR expression was correlated positively with VEGF-A expression in meningioma tissues. In inxa0vitro experiments, hypoxia induced the upregulation of VEGF-A expression and the cytoplasmic translocation of HuR protein in meningioma cells, and inhibition of the cytoplasmic translocation of HuR reduced the upregulation of VEGF-A expression in meningioma cells. These findings suggest that the expression of VEGF-A relates to the development of PTBE with meningiomas and the histological grade, and that HuR is involved in the upregulation of VEGF-A expression in human meningiomas.

Expression of vascular endothelial growth factor-A and mRNA stability factor HuR in human astrocytic tumors.

High‐grade astrocytic tumors, such as glioblastoma, possess rich vascular components, which are necessary for their growth. VEGF‐A is considered to be the major mediator of angiogenesis in malignant neoplasms including high‐grade astrocytic tumors. The upregulation of VEGF‐A expression in tumor cells is induced by two mechanisms: the transcriptional activation and the post‐transcriptional stabilization of VEGF‐A mRNA. While the former mechanism mediated by hypoxia inducible factor‐1 alpha (HIF‐1α) has been revealed, the latter mediated by mRNA stability factor HuR remains unclear in astrocytic tumors. In the present study, we investigated the expression of VEGF‐A and mRNA stability factor HuR in supratentorial astrocytic tumors of 27 adults using RT‐PCR, ELISA, and immunohistochemistry. Furthermore, we studied the involvement of HuR in the upregulation of VEGF‐A expression using malignant astrocytoma cell lines. In higher‐grade astrocytic tumors, the level of VEGF‐A and microvascular density were elevated, cytoplasmic expression of HuR, which potentially means the protection of VEGF‐A mRNA from degradation by ribonucleases, appeared, and they were correlated positively. In in vitro experiments, the inhibition of the cytoplasmic translocation of HuR protein by leptomycin B (LMB) reduced the upregulation of VEGF‐A expression in malignant astrocytic tumor cells under hypoxic conditions. These findings suggest that the expression of VEGF‐A and cytoplasmic translocation of HuR relates to the histological grade, and that HuR is involved in the upregulation of VEGF‐A expression, in human astrocytic tumors.

Glioma cells under hypoxic conditions block the brain microvascular endothelial cell death induced by serum starvation

Angiogenesis is one of essential components for the growth of neoplasms, including malignant gliomas. However, tumor vascularization is often poorly organized and marginally functional due to tumor strucutural abnormalities, inducing regional or temporal hypoxic conditions and nutritional shortages in tumor tissues. We investigated how during angiogenesis migrating endothelial cells survive in these hypoxic and reduced nutritional conditions. Human brain microvascular endothelial cells (HBMECs) underwent apoptosis and necrosis after serum withdrawal. This endothelial cell death was blocked by recombinant VEGF protein or the culture medium of U251 glioma cells exposed to hypoxia (H‐CM). Hypoxic treatment increased vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNF‐α) expression in U251 glioma cells. H‐CM activated nuclear factor‐κB (NFκB) protein and increased the gene expression of antiapoptotic factors including Bcl‐2, Bcl‐XL, survivin and X‐chromosome‐linked inhibitor of apoptosis protein (XIAP) in endothelial cells. The survival activity of H‐CM for endothelial cells was abolished by two kinds of VEGF inhibitors {Cyclopeptidic VEGF inhibitor and a VEGF receptor tyrosine kinase inhibitor (4‐[(4′‐chloro‐2′‐fluoro) phenylamino]‐6, 7‐dimethoxyquinazoline)} or NFκB inhibitors (ALLN and BAY 11–7082). These VEGF inhibitors did not block the activation of NFκB induced by H‐CM in endothelial cells. On the contrary, TNF‐α antagonist WP9QY enhanced the survival activity of H‐CM for endothelial cells and blocked NFκB activation induced by H‐CM under serum‐starved conditions. Taken together, our data suggest that both the secretion of VEGF from glioma cells and activation of NFκB in endothelial cells induced by TNF‐α are necessary for endothelial cell survival as they increase the expression of antiapoptotic genes in endothelial cells under conditions of serum starvation. These pathways may be one of the mechanisms by which angiogenesis is maintained in glioma tissues.

MR imaging and CT of pituitary abscess: Case report and review

Pituitary abscess is a rare disorder. However, preoperative diagnosis is important to prevent a cranial approach leading to severe meningitis. A case of a 55 year-old woman with pituitary abcess is reported. The patient was admitted with a several-week history of frontal headache and no signs of inflammation. Computed tomographic (CT) scan showed a slightly low-density suprasellar expanding mass lesion with an enhanced thin wall in the pituitary region. Magnetic resonance imaging showed a homogenous high-intensity signal relative to brain parenchyma on T1-weighted images with an enhanced thin wall and a homogenous low-intensity signal on T2-weighted images. This was histologically shown to be a pituitary abscess. Our case and review of the available literature suggest that pituitary abscess generally shows a homogenous low-density on CT scan and a homogenous low- to iso- and high-intensity signals or homogenous high- and low-intensity signals on T1- and T2-weighted images, respectively, with a cystic appearance and enhanced smooth wall.