Normal Brain Tissue Specimens Research Articles

Significantly higher expression of high-mobility group AT hook protein 2 (HMGA2) in the border zone of glioblastoma.

High-mobility group AT-hook protein 2 (HMGA2) is a gene regulatory protein that is correlated with metastatic potential and poor prognosis. It has been shown that HMGA2 is overexpressed in various tumors such as lung cancer or pancreatic cancer. The invasive character and highly aggressive structure of glioblastoma let us to investigate HMGA2 expression in the border zone of the tumor more closely. We compared HMGA2 expression between glioblastoma and normal brain tissue. In addition, we analyzed and compared HMGA2 expression in the border and center zones of tumors. Correlation tests between HMGA expression and clinical parameters such as MGMT-status and survival were performed. Samples from 23 patients with WHO grade 4 glioblastomas were analyzed for HMGA2 expression using quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry (IHC) and correlated with clinical parameters. The areas from the tumor center and border were analyzed separately. Two normal brain tissue specimens were used as the controls. Our results confirm that HMGA2 is higher expressed in glioblastoma compared to healthy brain tissue (qPCR, P=0.013; IHC, P=0.04). Moreover, immunohistochemistry revealed significantly higher HMGA2 expression in the border zone of the tumor than in the tumor center zone (P=0.012). Survival analysis revealed a tendency for shorter survival when HMGA2 was highly expressed in the border zone. The results reveal an overexpression of HMGA2 in the border zone of glioblastomas; thus, the expression cluster of HMGA2 seems to be heterogenous and thorough borough surgical resection of the vital and aggressive border cells might be important to inhibit the invasive character of the tumor.

Knockdown of lncRNA HOXD-AS2 Improves the Prognosis of Glioma Patients by Inhibiting the Proliferation and Migration of Glioma Cells.

Increasing studies reported that long noncoding RNAs are involved in regulating glioma progression. However, the specific roles and mechanisms of lncRNAs in glioma remain unclear. Here, we sought to explore the functions of HOXD-AS2 in glioma progression. Gene expressions of lncRNAs in 5 normal brain tissue specimens and 5 glioblastoma tissue specimens were detected by gene expression profile chip technology. Bioinformatic analysis was performed to see whether differential expression of lncRNAs played any significant role in glioma occurrence and progression. The relationship between HOXD-AS2 level and clinical prognosis of the patients was analyzed. HOXD-AS2 was specifically interfered with by siRNA technology to observe its effects on U251 cell growth, proliferation, apoptosis, and invasion. The expression level of HOXD-AS2 gene in glioma was significantly higher than that in the normal brain tissue, which was related to the tumor grade. The level of HOXD-AS2 gene in patients with high-grade glioma was higher than that in patients with low-grade glioma. High expression of HOXD-AS2 gene was a risk factor for poor prognosis of glioma patients. Knocking down the expression of HOXD-AS2 in glioma cell line U251 arrested the cell cycle and reduced the cell proliferation. Furthermore, it could significantly reduce the migration ability of the cells but had no significant effect on the invasion. HOXD-AS2 is an oncogenic lncRNA associated with the poor prognosis of glioma. Knockdown of HOXD-AS2 may reduce the growth of glioma, which may provide a new avenue for treatment.

ZEB1 induces N-cadherin expression in human glioblastoma and may alter patient survival.

The present study investigated the expression of epithelial-mesenchymal transition (EMT)-related factors zinc finger E-box-binding homeobox 1 (ZEB1), cadherin-1 (CDH1), cadherin-2 (CDH2) and the cell cycle modulating kinase cyclin-dependent kinase 1 (CDK1) in human glioblastoma (GBM) compared to normal brain tissue, as well as whether the levels of expression were associated with the overall and progression-free survival of the GBM patients. In 44 GBM and five normal brain tissue specimens, the expression levels of ZEB1, CDH1, CDH2 and CDK1 were evaluated by real-time PCR and immunostaining, and the results were correlated with clinical data. The expression levels of all investigated genes as detected by immunostaining were significantly higher in the GBM when compared to the normal brain tissues. There was no influence on survival. A linear correlation between ZEB1 and CDH2 and CDK1 expression was observed in GBM. Moreover, ZEB1 was involved in EMT (e.g., signaling in human GBM) and high ZEB1 levels were linked to an aberrant cell cycle processing, marked by CDK1 overexpression.

Tenascin-C expression contributes to pediatric brainstem glioma tumor phenotype and represents a novel biomarker of disease

Diffuse intrinsic pontine glioma (DIPG), an infiltrative, high grade glioma (HGG) affecting young children, has the highest mortality rate of all pediatric cancers. Despite treatment, average survival is less than twelve months, and five-year survival under 5%. We previously detected increased expression of Tenascin-C (TNC) protein in DIPG cerebrospinal fluid and tumor tissue relative to normal specimens. TNC is an extracellular matrix (ECM) glycoprotein that mediates cell-matrix interactions, guides migrating neurons during normal brain development and is thought to maintain the periventricular stem cell niche in the developing brain. Tumor TNC expression is reported in adult glioma and other cancers. However, the pattern and effects of TNC expression in DIPG has not been previously explored. Here, we characterize TNC expression in patient derived pediatric supratentorial HGG (n = 3) and DIPG (n = 6) cell lines, as well as pediatric glioma tumor (n = 50) and normal brain tissue specimens (n = 3). We found tumor specific TNC gene and protein overexpression that directly correlated with higher tumor grade (WHO III and IV, p = 0.05), H3K27 M mutation (p = 0.012), shorter progression free survival (p = 0.034), and poorer overall survival (0.041) in association with these factors. TNC knockdown via lentiviral shRNA transfection of HGG (n = 1) and DIPG (n = 3) cell lines resulted in decreased cell proliferation, migration, and invasion in vitro (p < 0.01), while TNC cDNA transfection resulted in increased cell migration, invasion and proliferation (p < 0.01) as well as altered cell morphology in H3K27 M mutant DIPG lines. Whole transcriptome sequencing analysis (RNA-Seq) on DIPG (n = 3) and HGG (n = 2) cell lines after TNC cDNA, shRNA, and empty vector control transfection revealed the effects of TNC expression level on global gene expression profiles. Together, our findings reveal TNC expression in DIPG in association with H3K27 M mutation and VEGF signaling, and suggest that TNC may contribute to DIPG tumor phenotype, and serve as a clinically detectable biomarker for DIPG.

Expression of long non-coding RNA MAGI2‑AS3 in human gliomas and its prognostic significance.

It has been confirmed that the dysregulation of long noncoding RNAs (lncRNAs) is associated with various diseases, especially cancer. LncRNA MAGI2 antisense RNA 3 (MAGI2-AS3) has been reported to be involved in the progression of bladder cancer and breast cancer. In this study, we aimed to explore its expression and clinical significance in glioma. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) assays were performed to detect the expression levels of MAGI2-AS3 in fresh glioma and matched adjacent normal brain tissue specimens, which were collected from 178 patients. Then the association between MAGI2-AS3 expression and clinical-pathological parameters were further evaluated using the Chi-square test. The overall survival (OS) was analyzed by the log-rank test, and the survival curves were plotted according to Kaplan-Meier. Univariate and multivariate analyses were performed to analyze the prognostic significance of MAGI2-AS3 expression. We found that the relative expression level of MAGI2-AS3 in glioma tissues was significantly lower than those in adjacent normal brain tissues (p<0.01). Lower MAGI2-AS3 expression was observed to be positively correlated with the World Health Organization (WHO) grade (p=0.031) and KPS score (p=0.003) in glioma patients. The Kaplan-Meier analysis indicated that patients with low MAGI2-AS3 expression levels tended to have worse overall survival than those with high levels of MAGI2-AS3 expression (p=0.0042). In the multivariate analysis, we further observed that MAGI2-AS3 expression in glioma tissues was an independent prognostic factor for overall survival (HR=3.098, 95% CI: 1.289-4.118, p=0.014). MAGI2-AS3 expression represents a significant favorable prognostic factor for patients with glioma.

MicroRNA-16 inhibits the proliferation, migration and invasion of glioma cells by targeting Sal-like protein4.

MicroRNAs (miRNAs or miRs), a class of non-coding RNAs 18-25nucleotides in length, act as key regulators in the development and malignant progression of various human cancers by modulating the expression of their target genes. Recently, miR‑16 has been demonstrated to be play a role in glioma. However, the regulatory mechanisms of miR‑16 in glioma growth and metastasis remain largely unclear. In the present study, qRT-PCR revealed that miR‑16 was significantly downregulated in 23glioma tissue specimens compared to 7normal brain tissue specimens. Moreover, its levels were markedly lower in the glioma samples at stagesT2-T4 compared to those at stageT1. The overexpression of miR-16 significantly suppressed the proliferation, migration and invasion of U251 and U87glioma cells. Luciferase reporter assay identified Sal-like protein4(SALL4) as a target gene of miR‑16, and its protein levels were found to be decreased in miR‑16-overexpressing U251 and U87cells. Furthermore, the overexpression of SALL4 significantly reversed the suppressive effects of miR‑16 on the proliferation, migration and invasion of U251 and U87 cells, suggesting that miR‑16 playsa tumor suppressor role in glioma by inhibiting cell proliferation and invasion through the targeting of SALL4. Finally, we found that SALL4 was significantly upregulated in glioma tissues compared to normal brain tissues, and its levels were markedly higher in the glioma tissues at stagesT2-T4 compared to those at stageT1. In addition, the expression levels of SALL4 inversely correlated with the miR‑16 levels in glioma tissues, suggesting that the downregulation of miR‑16 contributes to the upregulation of SALL4 in glioma. On the whole, the findings of this study indicate a role for the miR‑16/SALL4 axis in glioma. Our data may also provide a potential therapeutic target for the treatment of glioma.

Differential expression of insulin-like growth factor binding protein- 3 in the different grades of glioma and its correlation with malignancy of glial tumors

Objective To explore the differential expression of insulin- like growth factor- binding protein- 3(IGFBP- 3)in normal brain tissue and different grades of gliomas,and its corralation with the differentiation and malignance of gliomas. Methods By real- time quantitative polymerase chain reaction (Real- time PCR)and immunohistochemical staining methods, the expression of IGFBP- 3 was detected in 11 cases of grade-Ⅱglioma tissue samples, 14 cases of grade-Ⅲglioma tissue specimens, 9 cases of grade-Ⅳgrade glioma tissue samples,and 16 specimens of normal brain tissue. Results The average optical density values of IGFBP- 3 in normal brain tissue specimens and the specimens of grade-Ⅱ,-Ⅲ,-Ⅳgliomas were 1.082 2±0.334 7, 1.535 0±0.226 7, 4.383 1±0.521 8, and 5.094 1±0.652 5 respectively.Single factor analysis of variance(One- way ANOVA)and LSD, SNK- t analysis comparison showed there was significant difference in the expression of IGFBP- 3 between human gliomas and normal brain tissue(P< 0.05), and between grade-Ⅱand grade-Ⅲor grade-Ⅳgliomas(P< 0.05).By immunohistochemical staining methods,the positive expression rate of IGFBP- 3 was 20.0% in normal brain tissue specimens, which was significantly lower than in grade-Ⅱ(33.3%),-Ⅲ(57.1%), and-Ⅳ(80.0%)glimas(P<0.05 for all).There was no significant difference among different grades of gliomas. Conclusion The expression of IGFBP- 3 was significantly reduced in gliomas,and closely related with disease progression. Key words: Glioma; Insulin- like growth factor binding protein- 3; Promote apoptosis

Expression differences of N-acetyl amino galactosyl transferase-14 in the different levels of malignant glioma and the correlation of malignant degree with glial tumors

Objective To explore the expression differences of N-acetyl amino galactosyl transferase-14 (GalNAc-T14) in normal brain tissue and different levels of malignant gliomas and to observe the relationship between the expression of GalNAc-T14 with the differentiation and malignant degree of gliomas.Methods By real-time quantitative polymerase chain reaction detecting system (QPCR) and immunohistochemical staining methods,the expression of GalNAc-T14 was detected in 11 cases of Ⅱ grade glioma tissue samples,14 cases of Ⅲ grade glioma tissue specimens,9 cases of Ⅳ grade glioma tissue samples and 16 specimens of normal brain tissue.Results The average optical density values of GalNAc-T14 in normal brain tissue specimens and the specimens of Ⅱ,Ⅲ and Ⅳ grade glioma cells were 0.956 4 ±0.079 1,0.525 6 ±0.064 7,0.298 9 ±0.080 8,and0.151 4 ±0.062 4 respectively by QPCR.Single factor analysis of variance (One-way ANOVA) and LSD,SNK-t analysis comparison showed the expression of GalNAc-T14 in human gliomas was significantly different from that in the normal brain tissue (P ＜ 0.05).The same significance was also found between grade Ⅱ and grade Ⅲ or grade Ⅳ (P ＜ 0.05).Immunohistochemical staining method revealed that the positive rate of the GalNAc-T14 expression was 68.7％ in normal brain tissue specimens and 63.6％,42.9％ and 33.3％ in glioma tissues of grade Ⅱ,Ⅲ and Ⅳ,and there was significant difference between glioma group and normal brain tissue groups (P ＜ 0.05).Different levels of brain glioma cells showed no obvious consistency.Conclusion The expression of GalNAc-T14 was significantly reduced in malignant glioma cells,and was closely related with disease progression. Key words: Glioma; N-acetyl amino galactosyl transferase-14; Apoptosis; O-glycosylation

Expression of erythropoietin receptor and its relation with neovascularization in gliomas

Objective To investigate the expression of erythropoietin receptor (EPOR) in human gliomas and its relationship with histopathological degree and neovascularization.Methods Immunohistochemistry of the tissue microarray was used to detect the expression of EPOR and CD105 in gliomas.Tissue samples included 152 gliomas of different WHO grades and 20 normal brain tissue specimens.Twenty-one gliomas and 6 normal brain tissue specimens were detected by using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR).Results The expression of EPOR was significantly higher in glioma specimens than in normal brain tissues (x2 =7.7038,P ＜ 0.01),and EPOR expression was positively correlated with the stage of the tumor (rx =0.3151,P ＜ 0.01).CD105 counts of EPOR (艹,(艹)) were greater than those of EPOR(－,+) (P ＜0.05).There was no significant difference in the EPOR mRNA expression in glioma group and normal brain group.Conclusion Tbe expression of EPOR in human glioma exhibits a positive correlation with histopathological degree and neovascularization.There is no up-regulation of EPOR mRNA. Key words: Glioma; Erythropoietin receptors; CD105 ; Tissue arrayer

Identification of primary tumors of brain metastases by Raman imaging and support vector machines

Vibrational spectroscopic imaging techniques are new tools for visualizing chemical components in tissue without staining. The spectroscopic signature can be used as a molecular fingerprint of pathological tissues. Fourier transform infrared imaging which is more common than Raman imaging so far has already been applied to identify the primary tumor of brain metastases. The current study introduces a two level discrimination model for Raman microspectroscopic images to distinguish normal brain, necrosis and tumor tissue, and subsequently to determine the primary tumor. 22 Specimens of normal brain tissue and brain metastasis of bladder carcinoma, lung carcinoma, mamma carcinoma, colon carcinoma, prostate carcinoma and renal cell carcinoma were snap frozen, and thin tissue sections were prepared. Raman microscopic images were collected with 785nm laser excitation at 10μm step size. Cluster analysis, vertex component analysis and principal component analysis were applied for data preprocessing. Then, data of 17 specimens were used to train the discrimination model based on support vector machines with radial basis functions kernel. The training data were discriminated with accuracy better than 99%. Finally, the discrimination model correctly predicted independent specimens. The results were superior to discrimination by partial least squares discriminant analysis and support vector machines with linear basis function kernel that were applied for comparison.

Identification of MicroRNAs as Potential Prognostic Markers in Ependymoma

IntroductionWe have examined expression of microRNAs (miRNAs) in ependymomas to identify molecular markers of value for clinical management. miRNAs are non-coding RNAs that can block mRNA translation and affect mRNA stability. Changes in the expression of miRNAs have been correlated with many human cancers.Materials and MethodsWe have utilized TaqMan Low Density Arrays to evaluate the expression of 365 miRNAs in ependymomas and normal brain tissue. We first demonstrated the similarity of expression profiles of paired frozen tissue (FT) and paraffin-embedded specimens (FFPE). We compared the miRNA expression profiles of 34 FFPE ependymoma samples with 8 microdissected normal brain tissue specimens enriched for ependymal cells. miRNA expression profiles were then correlated with tumor location, histology and other clinicopathological features.ResultsWe have identified miRNAs that are over-expressed in ependymomas, such as miR-135a and miR-17-5p, and down-regulated, such as miR-383 and miR-485-5p. We have also uncovered associations between expression of specific miRNAs which portend a worse prognosis. For example, we have identified a cluster of miRNAs on human chromosome 14q32 that is associated with time to relapse. We also found that miR-203 is an independent marker for relapse compared to the parameters that are currently used. Additionally, we have identified three miRNAs (let-7d, miR-596 and miR-367) that strongly correlate to overall survival.ConclusionWe have identified miRNAs that are differentially expressed in ependymomas compared with normal ependymal tissue. We have also uncovered significant associations of miRNAs with clinical behavior. This is the first report of clinically relevant miRNAs in ependymomas.

The relationship between expression of PIAS3 and malignancy of human brain meningioma

Objective To explore the relationship between PIAS3 expression and the degree of malignancy of meningiomas.Methods 72 cases of different degree of malignant meningioma and 13 cases of surgical specimens of normal brain tissue were extracted the total RNA,and detected the levels of PIAS3 in different degree of meningioma and normal brain tissue through comparative control study by using real-time fluorescence quantitative PCR.Results PIAS3 was low expressed in normal brain tissue,however,high expressed in the human brain meningioma with significant differences(P＜0.01).There were also significant differences found in expression level of PIAS3 between meningiomas grade I and Ⅱ,or Ⅲ level(P＜0.05),but the differences between meningiomas grade Ⅱ and Ⅲ were not obvious(P＞0.05).And PIAS3 expression was not related with the depth of infiltration in invasive meningiomas(P＞0.05).Conclusion PIAS3 was overexpression in meningiomas,and was be closely related to the occurrence and development of meningiomas. Key words: Meningeal; Protein inhibitors of activated STAT

The methylation and mRNA expression of SLC5A8 and TMS1/ASC genes in human glioma

To study the methylation status of the SLC5A8 and TMS1/ASC genes, candidate tumor-inhibiting genes closely related to the central nervous system, in the promoter regions, the mRNA expression of these 2 genes, and their correlation with the clinical characteristics in human glioma. The methylation status of SLC5A8 and TMS1/ASC genes in the promoter regions was studied by methylation specific PCR (MSP) in the specimens of primary astrocytoma from 88 patients, 55 males and 33 females, aged 12 - 81, and 10 specimens of normal brain tissue, all obtained during operation, and in the human glioma cells of the lines U251 and SHG-44. The mRNA expression levels of SLC5A8 and TMS1/ASC genes in 30 specimens of primary glioma and 10 specimens of normal brain tissue were determined by conventional RT-PCR and real-time PCR. 5-Aza-2'-deoxycytidine (5-Aza-CdR), a demethylating agent, was added into the culture fluid of the U251 and SHG-44 cells, and then real-time PCR was used to the methylation status and mRNA expression levels of the SLC5A8 and TMS1/ASC genes. MSP showed that the SLC5A8 promoter region was hypermethylated in 62 of the 88 specimens of astrocytoma (70.45%) and the TMS1/ASC promoter region was hypermethylated in 51 of the88 specimens of astrocytoma (57.95%). But no methylation of SLC5A8 and TMS1/ASC promoter was detected in the 10 specimens of normal brain tissue. The mRNA expression of SLC5A8 gene and the mRNA expression of TMS1/ASC gene in the specimens of astrocytoma of different pathological grades were all significantly decreased compared to the specimens of normal brain tissue (all P < 0.05). The mRNA expression of SLC5A8 gene was not significantly related to the age and sex, however, the mRNA expression of TMS1/ASC was significantly higher in the age group > 60 than in other age groups (all P < 0.05). Both U251 and SHG-44 glioma cells showed methylation of SLC5A8 and TMS1/ASC genes and after the treatment of 5-Aza-CdR both genes showed reactivated mRNA expression. Hypermethylation of SLC5A8 and TMS1/ASC genes in the promoter regions may play an important role in the down-regulation of their mRNA levels in glioma. The methylation frequency and mRNA levels of SLC5A8 or TMS1/ASC genes are closely related to the malignant development of glioma.

Microdosimetric analysis for high LET radiation

For short range high linear energy transfer (LET) radiation therapy the biological effects are strongly affected by the heterogeneity of the specific energy (z) distribution delivered to tumour cells. Three-dimensional (3-D) dosimetry information at the cellular level is required for this study. An ideal approach would be the reconstruction of the cell and the radiation source microdistribution from sequential autoradiographic sections, which is, however, not a practical solution. In this paper, a novel microdosimetry analysis method, which obtains the specific energy (z) distribution directly from the morphological information in individual autoradiographic sections, is applied to human glioblastoma multifore (GBM) and normal brain tissue specimens in boron neutron capture therapy. The results are consistent with Monte Carlo simulation and demonstrate a uniform radiation source distribution in both GBM and normal brain tissues. We also hypothesise a biophysical model based on specific energy for survival analysis. The specific energy distributions to cell nuclei were calculated with a uniform radiation source distribution. By combining this microdosimetric analysis with measured cell survival data at the low dose region, a cell survival curve at high doses is predicted, which is consistent with the commonly used simple exponential curve model for high LET radiation.

Overexpression of Heparin-Binding Growth-Associated Molecule in Malignant Glioma Cells

The highly invasive and angiogenic characteristics of malignant gliomas depend on the production of growth factors and angiogenic factors. Heparin-binding growth-associated molecule (HB-GAM) is a secreted growth factor that is mitogenic for endothelial cells. To examine the expression profile of HB-GAM in malignant glioma cells, messenger ribonucleic acid (mRNA) expression was analyzed in 10 malignant glioma cell lines, two glioblastoma tissue specimens, and two normal brain tissue specimens by the reverse transcription-polymerase chain reaction. HB-GAM mRNA was expressed in all specimens including normal brain tissue specimens. Western blot analysis revealed that HB-GAM protein contents in glioma cell lines and glioblastoma tissues were 1.8 to 6.3 times higher than those in normal brain tissues. The effect of neutralizing anti-platelet-derived growth factor (PDGF) antibody was also examined on the production of HB-GAM in malignant glioma cells, since malignant glioma cells secrete PDGF that upregulates HB-GAM expression. Treatment of U251 and T98G glioblastoma cells with the anti-PDGF antibody did not affect the HB-GAM production. These results suggest that HB-GAM is overexpressed in malignant glioma cells and is involved in tumor growth.

Proteolytic Cleavage of the CD44 Adhesion Molecule in Multiple Human Tumors

Cell surface adhesion molecules are crucial for the development and/or pathogenesis of various diseases including cancer. CD44 has received much interest as a major adhesion molecule that is involved in tumor progression. We have previously demonstrated that the ectodomain of CD44 undergoes proteolytic cleavage by membrane-associated metalloproteases in various tumor cell lines. The remaining membrane-bound CD44 cleavage product can be detected using antibodies against the cytoplasmic domain of CD44 (anti-CD44cyto antibody). However, the cleavage of CD44 in primary human tumors has not been investigated. Using Western blots with anti-CD44cyto antibody to assay human tumor tissues, we show that the CD44 cleavage product can be detected in 58% (42 of 72) of gliomas but not in normal brain. Enhanced CD44 cleavage was also found in 67% (28 of 42) of breast carcinomas, 45% (5 of 11) of non-small cell lung carcinomas, 90% (9 of 10) of colon carcinomas, and 25% (3 of 12) of ovarian carcinomas. Tumors expressing a CD44 splice variant showed a significantly higher incidence of enhanced CD44 cleavage. The wide prevalence of CD44 cleavage suggests that it plays an important role in the pathogenesis of human tumors.

Endothelial nitric oxide synthase expression in tumor vasculature is correlated with malignancy in human supratentorial astrocytic tumors.

Endothelial nitric oxide synthase (eNOS) may play an important role in the regulation of tumor blood flow and vascular permeability. However, there have been no reports describing alterations of eNOS expression in relation to malignant progression in human astrocytic tumors. We immunohistochemically studied the relationship between eNOS expression in tumor vasculature and malignancy in supratentorial astrocytic tumors. Tissue samples were obtained from 12 patients with low-grade astrocytomas, 10 with anaplastic astrocytomas, and 17 with glioblastomas. Normal brain tissue samples were obtained from four patients with other brain diseases. Immunohistochemical staining was performed using the avidin-biotin complex method, with polyclonal anti-eNOS antibody, and the levels of eNOS expression in endothelial cells were evaluated as slight, moderate, or intense on the basis of eNOS immunoreactivity. The proliferative potential was assessed as the MIB-1 staining index for tumor cells. The expression of eNOS was slight in all specimens of normal brain tissue, slight in 7 and moderate in 5 specimens of low-grade astrocytoma, slight in 2, moderate in 6, and intense in 2 specimens of anaplastic astrocytoma, and moderate in 5 and intense in 12 specimens of glioblastoma. The MIB-1 staining index (mean+/-standard deviation) was 0.2+/-0.2% for normal specimens, 1.8+/-0.6% for low-grade astrocytomas, 9.6+/-6.9% for anaplastic astrocytomas, and 18.5+/-7.7% for glioblastomas. The MIB-1 staining indices for slight, moderate, and intense eNOS expression were 2.0+/-2.3%, 10.8+/-9.8%, and 16.9+/-7.7%, respectively. Expression of eNOS in tumor vessels was significantly correlated with histological grade and proliferative potential. These findings suggest that astrocytic tumor vessels possess higher activity for nitric oxide production than do normal vessels.