Expression of Vascular Endothelial Growth Factor in Human Meningiomas and Peritumoral Brain Areas

Transcription

1 344 Available online at Annals of Clinical & Laboratory Science, vol. 38, no. 4, 2008 Expression of Vascular Endothelial Growth Factor in Human Meningiomas and Peritumoral Brain Areas Ya-Suo Ding, Han-Dong Wang, Ke Tang, Zhi-Gang Hu, Wei Jin, and Wei Yan Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, China Abstract. Vascular endothelial growth factor (VEGF) is a regulator of angiogenesis, vasculogenesis, and vascular permeability. Recent reports suggest that VEGF may play a critical role in formation of peritumoral brain edema (PTBE) associated with meningiomas. While VEGF expression has been shown in meningiomas, studies have not focused on VEGF in the adjacent peritumoral brain regions. The present study examined the protein and gene expression of VEGF in human meningiomas and peritumoral brain areas. Biopsies were obtained from 37 patients. Immunohistochemical staining and immunoblotting were performed to detect the expression of VEGF protein. Reverse-transcriptase polymerase chain reaction (RT- PCR) was used to analyze the presence and quantity of VEGF mrna. The extent of PTBE was estimated as an edema index (EI) based on preoperative magnetic resonance imaging. In meningiomas, western blot and RT-PCR results were congruent and the expression of both protein and mrna had a significant correlation with EI. However, in peritumoral areas, western blot results were not consistent with the RT- PCR results. Protein results showed high correlation with EI, but mrna was almost undetectable. In VEGF-positive cases, a decreasing gradient of VEGF protein expression was observed with increasing distance from tumors. These data suggest that peritumoral tissue does not produce VEGF and that VEGF protein levels in peritumoral tissues have a high correlation with EI. We conclude that VEGF macromolecules are secreted by the tumor tissue and enter peritumoral normal brain tissue to induce edema. Keywords: meningiomas, vascular endothelial growth factor, peritumoral brain edema Introduction Peritumoral brain edema (PTBE) is one of the most serious complications in the management of intracranial meningiomas and plays an important role in the pathophysiology and clinical profiles of meningiomas [1]. The severity of PTBE may limit operative exposure and increase the difficulty and complexity of intraoperative procedures. The development of PTBE occurs in approximately 60% of meningiomas [8,36]. Nevertheless, the pathophysiological mechanism remains unclear. Various causative factors have been discussed, including patient age [21], gender [21], tumor size Address correspondence to Dr. Han-Dong Wang, Department of Neurosurgery, Jinling Hospital, 305 East Zhongshan Road, Nanjing , P. R. China; tel ; fax ; hdwang1@gmail.com. [5,7-9,25,29,30,47], tumor location [7-9,15,25,29, 30], histological differentiation [7,18,19,25,29,30, 33], arterial blood supply to the tumor [6,10,15,25, 38], intratumoral venous congestion [41], relation to hormone receptors [2,33], brain ischemia caused by tumor compression [42], and stasis induced by compromised venous drainage resulting from tumor mass effects [5,13,22]. VEGF has been implicated in many studies as a critical factor in the formation of PTBE associated with meningiomas [20,26,34,35,39,40]. VEGF is a specific mitogen of endothelial cells in vitro [27,28] and a potent promoter of angiogenesis in vivo [46]. Furthermore, VEGF has an effect on vascular permeability that is similar to vascular permeability factor (VPF) [27,28]. VEGF is a homodimeric glycoprotein of kda. Molecular cloning of the cdna) has revealed 4 types of VEGF in /08/ $ by the Association of Clinical Scientists, Inc.

2 VEGF in meningiomas and peritumoral brain areas 345 human cells. These forms result from alternative splicing of mrna [23,44] and have been termed VEGF121, VEGF165, VEGF189, and VEGF206. The best characterized and most common form is VEGF165, which is a secretable and heparinbinding isoform [17]. Although VEGF has been hypothesized to be secreted by the tumor tissues and to enter peritumoral normal brain tissue [47], no studies have been performed to test whether it is expressed in the adjacent peritumoral brain region. This study examines the pattern of VEGF expression in meningioma tissue and peritumoral brain areas. Materials and Methods Sample collection. Thirty-seven patients with 40 intracranial supratentorial meningiomas were recruited for the study. They underwent surgery at our department between July 2005 and June The study was approved by the Ethical Committee of the Faculty of Medicine of Jinling Hospital. After obtaining the patients informed consent, we resected the tumors and removed some peritumoral brain tissue. Patients consisted of 13 males and 24 females (mean age, 56.5 yr; range, 25 to 75 yr). Magnetic resonance imaging (MRI) was performed in all cases. The presence of PTBE was determined and quantified based on the MRI images. Using intraoperative navigation, tissue samples were collected during surgery from two sites: (a) tumor tissue, from the main bulk of the tumor tissue; and (b) peritumoral brain tissue, from the brain tissue adjacent to the tumor and involved in edema. The sample size was about 5 mm 3. Normal brain white matter was obtained from five patients with intractable epilepsy treated by partial temporal lobectomy after obtaining the patients informed consent. Tissue samples were fixed in 10% formalin and embedded in paraffin for histological examinations. For western blotting and RT-PCR, materials were snap-frozen in liquid nitrogen and kept at 80 C until use. All tumors were reviewed independently by two neuropathologists and diagnosed according to the revised WHO classification of brain tumors. The cases included 14 transitional, 11 meningothelial, 6 fibrous, 2 microcystic, 1 psammomatous, 1 angiomatous, 1 secretory, 3 atypical, and 1 malignant (anaplastic) meningioma. Tumors were further classified by location: 17 convex, 9 parasagittal, 7 falx, 3 middle fossa, 2 sphenoid ridge, and 2 frontal base. Evaluation of magnetic resonance imaging. Tumor and edema volumes were approximated from MRI scans, similar to methods of other authors [6,12,25,41]. Tumor volume was estimated on T1-weighted scans using gadolinium-diethylenetriamine penta-acetic acid (0.1 mmol/kg) enhancement. PTBE was evaluated on T2-weighted or fluid-attenuated inversion recovery scans. Maximum perpendicular diameters (radius: a,b) were measured from the axial images and the extent in the coronal direction (radius: c) was estimated from the number of axial images displaying the structure multiplied by slice thickness. The total volume of the tumor (V tumor ) and the volume of the hyperintensity area on T2-weighted images (V T2-high ) were then approximated using the formula for a spheroid (V=4 π abc/3). The relation of the tumor to PTBE volume (edema index [EI]) was defined using the formula EI = (V T2-high - V tumor )/V tumor. The formula yielded a value of 0 when no brain edema was found. The grading of edema severity according to EI was performed as follows: grade 0, no edema or negligible edema (EI <0.1); grade 1, mild edema (0.1 <EI <1.0); grade 2, moderate edema (1.0 <EI <2.0); grade 3, severe edema (EI >2) [32]. Immunocytochemistry. Tissue was fixed with 10% neutral buffered formalin and embedded in paraffin. Tissue sections (4 µm) were used for immunohistochemical staining, which was performed with an anti-vegf polyclonal antibody (diluted 1:150, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The sections were incubated with the diluted antibody overnight at 4 C, washed, and blocked with 1.6% H 2 O 2 in phosphate-buffered saline (PBS) for 10 min. After washing with PBS, each section was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (diluted 1:500, Santa Cruz Biotechnology) for 60 min at room temperature. Diaminobenzidine (DAB) was used as the chromogen and counterstaining was done with hematoxylin. Negative controls consisted of PBS instead of primary antibodies. Evaluation of the immunohistochemical staining was carried out independently by two observers. Specimen sections and controls were rated on an arbitrary 1-to-4-point scale based on staining intensity, which was considered to be VEGF immunoreactivity. The ratings were arbitrarily designated as follows: no staining = 1, mild staining = 2, moderate staining = 3, and intense staining = 4. Staining intensity scores were the mean values assigned by observers. Western blot analysis. The frozen tumor and brain tissue was mechanically lysed in 20 mm Tris, ph 7.6, which contained 0.2% SDS, 1% Triton X-100, 1% deoxycholate, 1 mm phenylmethylsulphonyl fluoride (PMSF), and 0.11 IU/ml aprotinin (all from Sigma-Aldrich, St. Louis, MO, USA). Lysates were centrifuged at 12,000 g for 20 min at 4 C. The protein concentration was estimated by the Bradford method using a protein assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The samples (60 µg per lane) were separated by 8% SDS-PAGE and electro-transferred onto a polyvinylidene-difluoride (PVDF) membrane (Bio-Rad Lab, Hercules, CA, USA). The membrane was blocked with 5% skim milk for 2 hr at room temperature and incubated overnight at 4 C with primary antibodies directed against the VEGF protein in PBS+Tween 20 (PBST) at a dilution of 1:200; β-actin (diluted 1:8,000 in PBST, Sigma-Aldrich) was used as a loading control. After the membrane was washed 6 times for 10 min each in PBST, it was incubated in the appropriate HRP-conjugated secondary antibody (diluted 1:400 in PBST) for 2 hr. Blotted protein bands were visualized by enhanced chemiluminescence (ECL) western blot detection reagents (Amersham, Arlington Heights, IL, USA) and were

3 346 Annals of Clinical & Laboratory Science, vol. 38, no. 4, 2008 exposed to X-ray film. Developed films were digitized using an Epson Perfection 2480 scanner (Seiko Corp, Nagano, Japan). Optical densities were obtained using Glyko Bandscan software (Glyko, Novato, CA, USA) and VEGF expression levels were normalized to β-actin. All experiments were repeated at least 3 times. VEGF mrna expression determined by RT-PCR. Total RNA was extracted with TriPure Reagent (Roche Diagnostics Corp., Indianapolis, IN, USA) according to the manufacturer s instructions. The cdna synthesis from the isolated RNA was performed using a reverse transcriptional system. Briefly, 4 µg of total RNA was reversely transcribed using 0.5 µg oligo(dt)15 and incubated with 15 U Avian Myeloblastosis Virus Reverse Transcriptase (AMV RT) (all from Promega, Madison, WI, USA). The cdna was amplified by PCR using specific oligonucleotide primers (Table 1). The amplified fragments were detected by agarose gel electrophoresis and visualized by ethidium bromide staining. The intensity of the bands was quantified using Glyko Bandscan software. The positive control, β-actin mrna, was detected in all samples; VEGF/ β-actin product ratios were calculated and used as an index of VEGF mrna expression. All experiments were repeated at least 3 times. Statistical Analysis. Results are expressed as mean ± SE unless otherwise noted. Student s t-test was used for comparisons of VEGF/β-actin ratio between the PTBE-negligible group and the PTBE-severe group. The relationship between VEGF/βactin ratio and EI was evaluated using Pearson s correlation coefficient. Calculations were performed using a statistical analysis system (SPSS-System). Differences were considered significant when p was <0.05. Results Relationship between intratumoral VEGF expression and PTBE. For assessment of the localization of VEGF in meningiomas, an immunohistochemical study of VEGF was performed. We observed that positive VEGF reactivity occurred mainly in the cytoplasm of tumor cells (Fig. 1A) and in the endothelium of tumor vessels (Fig. 1B). To quantify the expression of VEGF, western blots were performed to assess VEGF protein levels in the tumors. Two obvious protein bands were detected in all PTBE-positive tumor samples. The major form migrated at approximately 23 kda, and a minor band at 20 kda (Fig. 2B). For all cases, immunohistochemical and immunoblotting results were congruent. Samples that were negative immunohistochemically did not show any detectable band by western blot analysis, although western blots showed a low level of VEGF protein in the PTBE-negligible group (Fig. 2B,C). To quantify the expression of VEGF, the intensity of the two bands was summed. Compared to PTBEnegligible meningiomas, tumors with severe PTBE revealed higher VEGF expression (VEGF relative intensity = 0.08 vs VEGF relative intensity = 0.53; p <0.01). Furthermore, EI correlated significantly Table 1. PCR primer sequences. Primers Primer sequences Product Annealing Cycles size (bp) temperature ( C) VEGF Sense: 5 -TCGGGCCTCCGAAACCATGA Anti-sense: 5 -CCTGGTGAGAGATCTGGTTC b-actin Sense: 5 -TACTGCCATCCAATCGAGACC Anti-sense: 5 -AATGCTTTCTCCGCTCTG-3 Table 2. Protein and mrna ratio of VEGF in different brain regions (mean ± SE). Intratumoral tissues Peritumoral tissues Normal brain tissues Mean protein ratio (VEGF/b-actin) 0.38± ± ±0.02 Mean mrna ratio (VEGF/b-actin) 0.48± ± ±0.01

4 VEGF in meningiomas and peritumoral brain areas 347 Fig. 1. Immunohistochemical staining for VEGF. (A) In tumor tissue, positive VEGF reactivity was mainly in the cytoplasm of tumor cells. (B) In tumor tissue, positive VEGF reactivity was also in the endothelium of tumor vessels. (C) In peritumoral brain tissue, VEGF reactivity was scattered in the interstitial tissue. with the intensity of VEGF protein expression (r = 0.892, p <0.01). Fig. 3B shows VEGF mrna expression in meningiomas. Two obvious mrna bands were also detected. The major fragment was 648 bp; the second major band was 516 bp (Fig. 3B). In all cases, RT-PCR and immunohistochemical results were consistent. RT-PCR analysis showed that VEGF mrna was expressed at a low level in the PTBE-negligible group, whereas VEGF mrna was expressed at a high level in the PTBE-severe group. To quantitatively compare the expression of VEGF, the intensity of the two bands was also summed. There was a significant difference between the severe group and the negligible group (VEGF Ratio = 0.07 vs VEGF Ratio = 0.68; p <0.01). EI correlated significantly with the intensity of VEGF mrna (r = 0.875, p <0.01). Relationship between peritumoral VEGF expression and PTBE. To assess of the localization and expression of VEGF in peritumoral brain tissues, an immunohistochemical study for VEGF was performed. The positive VEGF reactivity was mainly in the interstitial tissue (Fig. 1C). Western blotting was performed to quantify VEGF protein levels in peritumoral brain tissues. Two clear protein bands were detected. The major form migrated at approximately 23 kda, and a second major band was found 20 kda (Fig. 2A). For all cases, the immunohistochemical and immunoblotting results were congruent. Western blot analysis showed a low level of VEGF protein in the PTBE-negligible group (Fig. 2A, C). Compared to the PTBE-negligible group, the severe PTBE group revealed significantly higher VEGF expression (VEGF relative intensity = 0.04 vs VEGF relative intensity = 0.35; p <0.01). Moreover, the EI correlated significantly with the intensity of VEGF protein expression (r = 0.912, p <0.01). Fig. 3A shows VEGF mrna expression in peritumoral brain tissues. VEGF mrna was practically undetectable. Fig. 3C shows that there was no significant difference between the PTBE-negligible group and the severe PTBE group. VEGF expression in intratumoral, peritumoral. and normal tissues. To highlight the relationship of VEGF expression in intratumoral, peritumoral, and normal tissues, we calculated the mean protein ratio and mean mrna ratio in 3 different regions

5 348 Annals of Clinical & Laboratory Science, vol. 38, no. 4, 2008 Fig. 2. (A) Representative autoradiogram of VEGF expression in peritumoral brain tissues. Two clear protein bands were detected. The major form migrated at approximately 23 kda, and the second major band at 20 kda. Lane 1: negligible; Lanes 2, 3, and 4: mild, moderate, and severe groups, respectively. (B) Representative autoradiogram of VEGF expression in meningiomas. Two obvious protein bands were detected in all positive tumor samples. The major form migrated at approximately 23 kda, and the minor band at 20 kda. Lane 1: negligible; Lanes 2, 3, and 4: mild, moderate, and severe groups, respectively. (C) Quantitative analysis of western blot results for VEGF. Open bars represent peritumoral groups; solid bars, intratumoral groups. Compared to PTBE-negligible meningiomas, peritumoral group and intratumoral group with severe PTBE revealed a significantly higher VEGF expression. **P<0.01 vs intratumoral group with negligible PTBE; ##P<0.01 vs peritumoral group with negligible PTBE. Bars represent the mean ± SE. To quantitatively compare the expression of VEGF, the intensity of the two bands was summed. Fig. 3. (A) Representative RT-PCR for the expression of VEGF mrna in the peritumoral brain tissues. VEGF mrna was virtually undetectable. Lanes 1-4: negligible, mild, moderate, and severe, respectively. b-actin (430 bp) was used as a control for RNA integrity. (B) Representative RT-PCR for the expression of VEGF mrna in the meningiomas. Lanes 1-4: negligible, mild, moderate, and severe, respectively. (C) Quantitative analysis of the level of VEGF mrna. Open bars represent peritumoral groups; solid bars, intratumoral groups. In intratumoral groups, there was a statistically significant difference between the severe group and the negligible group. **P<0.01 vs intratumoral group with negligible PTBE. However, there was no statistically significant difference between any two peritumoral groups. Bars represent the mean ± SE. To quantitatively compare the expression of VEGF, the intensity of the two bands was summed.. for VEGF-positive cases (Table 2). Twenty-eight meningiomas expressed VEGF protein, but only 21 peritumoral brain tissues had detectable VEGF protein. A decreasing gradient of VEGF protein expression was observed with increasing distance from tumors. Consistent with the protein expression, 28 meningioma tumor tissues were detected with VEGF mrna. However, VEGF mrna was almost undetectable in peritumoral brain tissues. This value was similar to the value found in normal brain tissue (Table 2). Discussion The most important findings of this study are that (1) in VEGF-positive cases, VEGF protein expression decreases with increasing distance away from tumors; (2) VEGF mrna could only be detected in tumors; and (3) in the peritumoral areas, VEGF protein levels showed high correlation with EI. Meningiomas grow extra-axially and are separated from the brain parenchyma by the

6 VEGF in meningiomas and peritumoral brain areas 349 arachnoid, the subarachnoid space, and the pia mater. Part of the blood brain barrier, the arachnoid, is impermeable to fluids. In contrast, the pia mater shows high permeability to water and electrolytes, but is far less permeable to macromolecules, such as proteins in edema fluid [18]. In addition, the cerebral cortex, because of its intricately interwoven cellular processes, poses a structural hindrance that is almost impossible for vasogenic edema to overcome. Thus, the reasons for the formation of PTBE are complicated. Various groups have proposed hypotheses to explain the generation of edema, including ischemic processes [42], venous congestion due to tumor compression [5,13,22], a secretory excretory phenomenon [20,26,34,35,39,40], and a hydrodynamic process [3]. Moreover, controversy persists about the importance of tumor size [5,7-9,25,29,30,47], location [7-9,15,25,29,30], and histology [7,18,19,25,29,30,33] for the genesis of edema. There is no consensus regarding PTBE pathogenesis. However, VEGF has been implicated as a critical factor in the formation of PTBE [20,26, 34,35,39,40] based on the following findings: First, it is widely accepted that brain edema accompanying meningiomas arises from a vasogenic rather than a cytotoxic origin. Electron microscopy has revealed similarities between meningioma-associated edema and experimentally induced vasogenic edema [18]. Second, it is widely accepted that VEGF is a potent inducer of tumor angiogenesis and vascular permeability. VEGF is 1,000-fold more potent than histamine in inducing capillary permeability [11]. Third, Dvorak et al [16] have found that VEGF is synthesized in tumor cells and accumulates in the endothelium of tumor vessels. Meningiomas with strong VEGF staining demonstrated a significantly higher edema incidence and edema index than VEGF-negative cases [4,20]. Other authors noted increased VEGF mrna-expression in edema-associated meningiomas compared to cases without edema [26]. Fourth, a previously published study demonstrated strong correlation between the adherence of the tumor to the surrounding brain tissue and the occurrence of edema [24]. Another study found a qualitative and quantitative correlation between pial blush and PTBE in meningiomas [6]. Paek et al [12] found that MMP-2 and MMP-9, which are well-known proteolytic enzymes that break down the basal membrane and connective tissue, were strongly correlated with the presence of PTBE in meningiomas. These studies showed disruption of the arachnoid and close spatial relationship between the tumor surface and adjacent brain parenchyma. Based on the above facts, it is thought that VEGF is synthesized in meningioma tumor cells. It is also thought that, through disruptions in the arachnoid, edema-inducing substances and macromolecules enter the peritumoral normal brain tissue and promote vascular permeability, giving impetus to edemagenesis in meningiomas. Additional questions must be answered to support this hypothesis: (1) Do VEGF proteins exist in peritumoral brain tissues, and does peritumoral VEGF have a relationship with EI? (2) If so, it must be determined whether VEGF is produced by peritumoral brain tissues induced by ischemia and hypoxia. The present study examined the protein and mrna expression of VEGF in human meningioma and peritumoral brain areas, which helps to answer these questions. Our study shows that protein and gene expressions are congruent in the meningiomas and that both have a significant correlation with EI. In peritumoral areas, western blot results showed correlation with EI. However, mrna was almost undetectable. In VEGF-positive cases, a decreasing gradient of VEGF expression was observed with increasing distance from tumors. This may explain why 28 meningiomas showed VEGF expression but only 21 had peritumoral edema. We found that VEGF mrna could only be detected in tumors. Moreover, VEGF has two major endothelial cell-specific, high-affinity receptors, flt-1 [14,31] and KDR [31,43]. Cerebral vessels of adults do not express VEGF receptors [45]. Further studies are needed to evaluate the expression of flt-1 and KDR in peritumoral brain tissues. If studies confirm these results, treatment with anti-vegf antibodies could influence the extent of peritumoral brain edema. Other molecules, such as aquaporin-4 [48], may cooperate with VEGF in the genesis of brain edema. This should be tested by further research.

7 350 Annals of Clinical & Laboratory Science, vol. 38, no. 4, 2008 In conclusion, the present study demonstrates that peritumoral tissue does not produce VEGF and that the VEGF protein intensity in peritumoral tissues has a high correlation with EI. Thus, we conclude that VEGF macromolecules are secreted by tumor tissue and enter normal peritumoral tissue to induce edemagenesis in meningiomas. Acknowledgements We thank Dr. Bo Wu for technical assistance. References 1. Abe T, Black PM, Ojemann RG, Hedley-White E. Cerebral edema in intracranial meningiomas: evidence for local and diffuse patterns and factors associated with its occurrence. Surg Neurol 1994;42: Benzel EC, Gelder FB. Correlation between sex hormone binding and peritumoral edema in intracranial meningiomas. Neurosurgery 1988;23: Bitzer M, Nagele T, Geist-Barth B, Klose U, Gronewaller E, Morgalla M, Heiss E, Voigt K. Role of hydrodynamic processes in the pathogenesis of peritumoral brain edema in meningiomas. J Neurosurg 2000;93: Bitzer M, Opitz H, Popp J, Morgalla M, Gruber A, Heiss E, Voigt K. Angiogenesis and brain oedema in intracranial meningiomas: influence of vascular endothelial growth factor. Acta Neurochir 1998;140: Bitzer M, Topka H, Morgalla M, Friese S, Wockel L, Voigt K. Tumor-related venous obstruction and development of peritumoral brain edema in meningiomas. Neurosurgery 1998;42: Bitzer M, Wockel L, Luft AR, Wakhloo AK, Petersen D, Opitz H, Sievert T, Ernemann U, Voigt K. The importance of pial blood supply to the development of peritumoral brain edema in meningiomas. J Neurosurg 1997;87: Bitzer M, Wockel L, Morgalla M, Keller C, Friese S, Heiss E, Meyermann R, Grote E, Voigt K. Peritumoural brain oedema in intracranial meningiomas: influence of tumour size, location and histology. Acta Neurochir 1997;139: Bradac GB, Ferszt R, Bender A, Schorner W. Peritumoral edema in meningiomas. A radiological and histological study. Neuroradiology 1986;28: Brandis A, Mirzai S, Tatagiba M, Walter GF, Samii M, Ostertag H. Immunohistochemical detection of female sex hormone receptors in meningiomas: correlation with clinical and histological features. Neurosurgery 1993;33: Challa VR, Moody DM, Marshall RB, Kelly DL Jr. The vascular component in meningiomas associated with severe cerebral edema. Neurosurgery 1980;7: Connolly DT. Vascular permeability factor: a unique regulator of blood vessel function. J Cell Biochem 1991; 47: Constantini S, Tamir J, Gomori MJ, Shohami E. Tumor prostaglandin levels correlate with edema around supratentorial meningiomas. Neurosurgery 1993;33: Czernicki Z, Grochowski W, Uchman G, Tychmanowicz K, Razumowski AE. Occlusion of the superior sagittal sinus caused by meningioma, intracranial volumepressure relations and brain edema. Neurol Neurochir Pol 1991;25: de Vries C, Escobedo JA, Ueno H, Houck K, Ferrara N, Williams LT. The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science 1992;255: de Vries J, Wakhloo AK. Cerebral oedema associated with WHO-I, WHO-II, and WHO-III-meningiomas: correlation of clinical, computed tomographic, operative and histological findings. Acta Neurochir 1993;125: Dvorak HF, Sioussat TM, Brown LF, Berse B, Nagy JA, Sotrel A, Manseau EJ, Van de Water L, Senger DR. Distribution of vascular permeability factor (vascular endothelial growth factor) in tumors: concentration in tumor blood vessels. J Exp Med 1991;174: Fiebich BL, Jager B, Schollmann C, Weindel K, Wilting J, Kochs G, Marme D, Hug H, Weich HA. Synthesis and assembly of functionally active human vascular endothelial growth factor homodimers in insect cells. Eur J Biochem 1993;211: Gilbert JJ, Paulseth JE, Coates RK, Malott D. Cerebral edema associated with meningiomas. Neurosurgery 1983;12: Go KG, Wilmink JT, Molenaar WM. Peritumoral brain edema associated with meningiomas. Neurosurgery 1988;23: Goldman CK, Bharara S, Palmer CA, Vitek J, Tsai JC, Weiss HL, Gillespie GY. Brain edema in meningiomas is associated with increased vascular endothelial growth factor expression. Neurosurgery 1997;40: Gurkanlar D, Er U, Sanli M, Ozkan M, Sekerci Z. Peritumoral brain edema in intracranial meningiomas. J Clin Neurosci 2005;12: Hiyama H, Kubo O, Tajika Y, Tohyama T, Takakura K. Meningiomas associated with peritumoural venous stasis: three types on cerebral angiogram. Acta Neurochir 1994;129: Houck KA, Ferrara N, Winer J, Cachianes G, Li B, Leung DW. The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA. Mol Endocrinol 1991;5: Ide M, Jimbo M, Yamamoto M, Umebara Y, Hagiwara S, Kubo O. MIB-1 staining index and peritumoral brain edema of meningiomas. Cancer 1996;78: Inamura T, Nishio S, Takeshita I, Fujiwara S, Fukui M. Peritumoral brain edema in meningiomas influence of

8 VEGF in meningiomas and peritumoral brain areas 351 vascular supply on its development. Neurosurgery 1992; 31: Kalkanis SN, Carroll RS, Zhang J, Zamani AA, Black PM. Correlation of vascular endothelial growth factor messenger RNA expression with peritumoral vasogenic cerebral edema in meningiomas. J Neurosurg 1996; 85: Keck PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Feder J, Connolly DT. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science 1989; 246: Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 1989;246: Lobato RD, Alday R, Gomez PA, Rivas JJ, Dominguez J, Cabrera A, Madero S, Ayerbe J. Brain oedema in patients with intracranial meningioma. Correlation between clinical, radiological, and histological factors and the presence and intensity of oedema. Acta Neurochir 1996;138: Maiuri F, Gangemi M, Cirillo S, Delehaye L, Gallicchio B, Carandente M, Giamundo A. Cerebral edema associated with meningiomas. Surg Neurol 1987;27: Otsuka S, Tamiya T, Ono Y, Michiue H, Kurozumi K, Daido S, Kambara H, Date I, Ohmoto T. The relationship between peritumoral brain edema and the expression of vascular endothelial growth factor and its receptors in intracranial meningiomas. J Neurooncol 2004;70: Paek SH, Kim CY, Kim YY, Park IA, Kim MS, Kim DG, Jung HW. Correlation of clinical and biological parameters with peritumoral edema in meningioma. J Neurooncol 2002;60: Philippon J, Foncin JF, Grob R, Srour A, Poisson M, Pertuiset BF. Cerebral edema associated with meningiomas: possible role of a secretory-excretory phenomenon. Neurosurgery 1984;14: Provias J, Claffey K, delaguila L, Lau N, Feldkamp M, Guha A. Meningiomas: role of vascular endothelial growth factor/vascular permeability factor in angiogenesis and peritumoral edema. Neurosurgery 1997;40: Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvorak HF. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 1983;219: Sigel RM, Messina AV. Computed tomography; the anatomic basis of the zone of diminished density surrounding meningiomas. Am J Roentgenol 1976;127: Sternberger LA, Hardy PH Jr, Cuculis JJ, Meyer HG. The unlabeled antibody enzyme method of immunohistochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 1970;18: Stevens JM, Ruiz JS, Kendall BE. Observations on peritumoral oedema in meningioma. Part II: Mechanisms of oedema production. Neuroradiology 1983;25: Strugar J, Rothbart D, Harrington W, Criscuolo GR. Vascular permeability factor in brain metastases: correlation with vasogenic brain edema and tumor angiogenesis. J Neurosurg 1994;81: Strugar JG, Criscuolo GR, Rothbart D, Harrington WN. Vascular endothelial growth/permeability factor expression in human glioma specimens: correlation with vasogenic brain edema and tumor-associated cysts. J Neurosurg 1995;83: Tanaka M, Imhof HG, Schucknecht B, Kollias S, Yonekawa Y, Valavanis A. Correlation between the efferent venous drainage of the tumor and peritumoral edema in intracranial meningiomas: superselective angiographic analysis of 25 cases. J Neurosurg 2006; 104: Tatagiba M, Mirzai S, Samii M. Peritumoral blood flow in intracranial meningiomas. Neurosurgery 1991;28: Terman BI, Carrion ME, Kovacs E, Rasmussen BA, Eddy RL, Shows TB. Identification of a new endothelial cell growth factor receptor tyrosine kinase. Oncogene 1991;6: Tischer E, Mitchell R, Hartman T, Silva M, Gospodarowicz D, Fiddes JC, Abraham JA. The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem 1991;266: Weindel K, Moringlane JR, Marme D, Weich HA. Detection and quantification of vascular endothelial growth factor/vascular permeability factor in brain tumor tissue and cyst fluid: the key to angiogenesis? Neurosurgery 1994;35: Wilting J, Christ B, Weich HA. The effects of growth factors on the day 13 chorioallantoic membrane (CAM): a study of VEGF165 and PDGF-BB. Anat Embryol 1992;186: Yoshioka H, Hama S, Taniguchi E, Sugiyama K, Arita K, Kurisu K. Peritumoral brain edema associated with meningioma: influence of vascular endothelial growth factor expression and vascular blood supply. Cancer 1999;85: Tan WL, Wong JH, Liew D, Ng IH. Aquaporin-4 is correlated with peri-tumoural oedema in meningiomas. Ann Acad Med Singapore 2004;33(5 Suppl):S87-S89..