- Email: [email protected]
Vascular endothelial growth factor in chronic subdural haematomas
Journal of Clinical Neuroscience (2001) 8(5), 411±415 & 2001 Harcourt Publishers Ltd DOI: 10.1054/jocn.2000.0951, available online at http://www.idealibrary.com on
Clinical study
Vascular endothelial growth factor in chronic subdural haematomas Tadahisa Shono1 MD PHD, Takanori Inamura1 MD PHD, Takato Morioka1 MD PHD, Ken-ichi Matsumoto1 MD, Satoshi O. Suzuki2 MD PHD, Kiyonobu Ikezaki1 MD PHD, Toru Iwaki2 MD PHD, Masashi Fukui1 MD PHD 1
Department of Neurosurgery, 2Neuropathology, Neurological Institute, Faculty of Medicine, Kyushu University, Fukuoka, Japan
Summary 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. & 2001 Harcourt Publishers Ltd Keywords chronic subdural haematoma, VEGF, bFGF, angiogenesis, vascular endothelial cell, macrophage
INTRODUCTION
MATERIALS AND METHODS
Chronic subdural haematoma (CSH) is one of the most common lesions encountered in neurosurgery. Nevertheless, the pathogenesis of CSH has not been fully elucidated. Morphologic studies of the haematoma membranes indicate that the outer membrane consists of loose collagen fibers, large capillaries (macrocapillaries), smooth muscle cells, dural border cells and inflammatory cells.1±3 Macrocapillaries in the outer membrane are considered to be important in the haematoma enlargement. The endothelial cells of macrocapillaries are abnormally permeable and fragile, and junctions between adjacent endothelial cells are loose, with gap junctions and absence of the basement membrane.4 Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is a potent, specific endothelial cell mitogen in vitro and induces angiogenesis in vivo.5,6 VEGF/ VPF has been found to be 1000 times more potent than histamine in inducing capillary permeability.7 Expression of VEGF correlates closely with neovascularisation in human brain tumours8 and with the degree of brain oedema surrounding meningiomas.9 No previous report has characterised expression of VEGF in CSH. To better understand the mechanisms underlying expansion of CSH, we quantified concentrations of VEGF as well as of another potent angiogenic factor, basic fibroblast growth factor (bFGF), in haematoma fluid and in serum from patients with CSH. We also examined the dura and outer membrane of CSH to determine the cell types producing VEGF in CSH by in situ hybridisation and immunohistochemistry.
Patients We studied 20 patients, 17 males and 3 females, with CSH. The age of the patients ranged from 24 to 90 years. All haematomas were unilateral, and there were no recurrences after primary burr hole surgery. Samples of haematoma fluid, blood, dura mater, and outer CSH membrane used for assay and were obtained during this operation at Kyushu University Hospital and affiliated hospitals beginning in 1998. Measurement of VEGF and bFGF Concentrations of VEGF and bFGF in haematoma fluid and in serum were measured with an enzyme-linked immunosorbent assay (ELISA) kit (Quantikine, R&D Systems, MN) according to the manufacturer's instructions. Immunohistochemistry The overlying dura and outer membrane of the CSH were resected during primary burr hole surgery. Resected specimens were fixed in 10% formalin solution, routinely processed, embedded in paraffin and sectioned at a thickness of 6 mm. Sections were stained immunohistochemically using an avidinbiotinylated peroxidase complex method with a mouse monoclonal antibody against the macrophage marker CD68 (KP-1; DAKO, Glostrup, Denmark), and then lightly counter-stained with haematoxylin. In situ hybridisation
Received 15 August 2000 Accepted 20 September 2000 Correspondence to: Takanori Inamura MD PhD, Department of Neurosurgery, Neurological Institute, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan. Tel.: 81-92-642-5524; Fax: 81-92-642-5526; E-mail: [email protected]
For in situ hybridisation, digoxigenin (DIG)-labeled sense and antisense RNA probes were synthesised with T7 RNA polymerase from a 210-base pair template cDNA using a DIG-RNA labeling kit (Boehringer Mannheim, Germany) according to the manufacturer's instructions. Prior to hybridisation, 6-mm-thick sections were treated with proteinase K 411
412 Shono et al.
(100 mg/ml) for 15 min at room temperature and postfixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 10 min. Thereafter, the sections were acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min and treated with 0.2 N HCl for 10 min to inactivate endogenous alkaline phosphatase. The pretreated sections were dehydrated, air-dried and hybridised overnight at 50 C with the DIGlabeled RNA probe in hybridisation buffer [50% formamide, 5X SSC (1X SSC 0.15 M NaCl and 0.015 M sodium citrate), 100 mg/ml tRNA and 0.05% heparin]. After hybridisation, sections was washed with 50% formamide/2X SSC for 30 min at 65 C and then treated with 50 mg/ml RNase A/TNE (10 mM Tris-HCl at pH 7.6, 500 mM NaCl, and 1 mM EDTA) for 30 min at 37 C. Sections were washed with 2X SSC for 20 min, followed by two washes with 0.2X SSC for 20 min each at 65 C. Hybridisation was detected immunohistochemically with monoclonal anti-DIG antibody (Boehringer Mannheim) and alkaline phosphatase-conjugated anti-mouse IgG antibody (Promega, Madison, WI), including visualisation with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT) (Promega). To compare distribution of macrophages with that of VEGF mRNA, immunohistochemical analysis and in situ hybridisation were performed sequentially using the same tissue section. Sections were incubated with a mouse monoclonal primary antibody against CD68 and subsequently with alkaline phosphatase-conjugated anti-mouse IgG antibody. Expression of CD68 was visualised with BCIP/NBT. After photomicrographs were obtained, the sections were washed with 99% ethanol overnight, completely removing the immunohistochemical reaction product. Following a brief rinse in tap water, in situ hybridisation was performed as described above.
Elevation of VEGF in CSH fluid In all 20 cases of CSH, concentrations of VEGF in the haematoma fluid were much higher than those of bFGF (Table 1). No correlation was detectable between VEGF concentrations in the haematoma and those of bFGF. Mean concentrations of
In sections stained with haematoxylin and eosin, many vessels and inflammatory cells were observed in the outer membrane (Fig. 2A). To determine cell types expressing VEGF mRNA, surgical specimens including dura and the outer membrane of CSH were analysed by in situ hybridisation. The cells surrounding vessels in the outer membrane were strongly stained when hybridised with the antisense probe (Fig. 2B), while cells in the dura showed little or no staining (not illustrated). Virtually no positive signal was seen in any cell in sections hybridised with the sense probe (Fig. 2C). When we immunohistochemically examined the distribution of macrophages, anti-CD68 antibody identified many infiltrating macrophages in the outer membrane, especially around vessels (Fig. 2D). To conclusively determine which cells expressed VEGF mRNA, immunohistochemical analysis and in situ hybridisation were performed sequentially in the same section. (Fig. 3). Most CD68-positive macrophages in the outer membrane expressed a large amount of VEGF mRNA (Fig. 3A and C). In addition, some vascular endothelial cells also showed hybridisation with the VEGF antisense probe (Fig. 3B and D). Other cells showed little or no staining for the transcript.
Angiogenesis occurs in various physiologic and pathologic conditions, including menstruation, ovulation, wound healing, inflammation and growth of solid tumours.10 The newly formed blood vessels appear to be more permeable and fragile than pre-existing vessels. To elucidate molecular aspects of the mechanisms causing expansion of CSH, we measured
Concentrations of VEGF and bFGF in serum and haematoma fluid from patients with chronic subdural haematoma
Age/Sex
57/F 84/M 90/M 78/F 68/M 86/F 63/M 59/M 71/M 83/M 51/M 71/M 85/M 58/M 70/M 75/M 24/M 77/M 87/M 69/M
Macrophages in the outer membrane are the main sources of VEGF in CSH
DISCUSSION
RESULTS
Table 1
VEGF were 355 pg/ml in serum and 10277 pg/ml in haematoma fluid (Fig. 1A); the mean concentration of VEGF in haematoma fluid was 28 times that in serum. On the other hand, mean bFGF concentrations were 4.74 pg/ml in serum and 3.04 pg/ml in haematoma fluid (Fig. 1B), with the concentration in the haematoma fluid being less than that in serum.
VEGF (pg/ml)
bFGF (pg/ml)
Serum
Haematoma
Serum
Haematoma
559 429 526 269 364 434 523 686 207 424 105 370 117 489 356 107 507 472 118 54
7589 12623 10609 8133 8887 14927 5510 8914 28556 7762 9113 5735 9536 7589 8053 8198 4000 18596 14782 6437
0 0 0 7.7 0.8 0 9.3 0 6.7 0 0 1.8 9.5 52.5 8.3 0 0 41.1 18.3 0
0 0.4 1.4 9.9 0.8 0 0 0 9.3 0.4 0.2 2.8 1.2 0 0 0 0 0 66.6 0
Journal of Clinical Neuroscience (2001) 8(5), 411±415
& 2001 Harcourt Publishers Ltd
VEGF and chronic subdural haematoma 413
Fig. 1 Protein levels in paired haematoma fluid and serum samples. Paired haematoma fluid and serum samples of patients with chronic subdural haematoma were analysed by ELISA for VEGF (A) and bFGF (B). Values represent the mean SE.
Fig. 2 Histologic, in situ hybridisation and immunostaining studies of the outer membrane of chronic subdural haematoma. (A) Haematoxylin and eosin staining (A). Many inflammatory cells and vessels are observed in the outer membrane (original magnification 200). In situ hybridisation of the outer membrane using a labeled antisense vascular endothelial growth factor probe (B) and a sense probe (C) (original magnification 200). Cells surrounding vessels in the outer membrane are strongly stained by the antisense probe, but not by the sense probe. Immunohistochemical staining for CD68 (D) demonstrates prominent macrophage infiltration in the outer membrane (original magnification 200).
concentrations of two representative angiogenic factors, VEGF and bFGF,11 in haematoma fluid and in serum from 20 patients with CSH. Concentrations of VEGF in haematomas were remarkably increased, while those of bFGF were not. Enhanced expression of VEGF is thought to be an important event in the formation of these haematomas. & 2001 Harcourt Publishers Ltd
Ito et al. have found that locally increased fibrinolysis, characterised by high concentrations of tissue-type plasminogen activator (tPA) and plasmin- -2-plasmin inhibitor complex, interferes with complete haemostasis and causes rebleeding into the central cavity of CSH.1,12,13 They also reported intense tPA immunoreactivity in the cytoplasm of Journal of Clinical Neuroscience (2001) 8(5), 411±415
414 Shono et al.
Fig. 3 Immunohistochemical staining for CD68 and in situ hybridisation for vascular endothelial growth factor (VEGF) in the same sequentially processed paraffin section of the outer membrane of a chronic subdural haematoma. After recording immunohistochemical observations (A and B), the sections were decolorized with 99% ethanol, and in situ hybridisation was performed in the same section (C and D). A and C show the same site in the outer membrane. Distributions of CD68-positive macrophages (A) and VEGF mRNA (C) generally are similar. B and D show the same site in the outer membrane. CD68-negative vascular endothelial cells (B) also have hybridised with the VEGF antisense probe (D, arrowheads; original magnification 400).
endothelial cells lining CSH sinusoids and capillaries.14 In the present study, the mean concentration of VEGF in haematomas exceeded 10 ng/ml and was 28 times the serum concentration. VEGF has been reported to stimulate growth of vascular endothelial cells in vitro at concentrations from 1 to 100 ng/ml.5,6 Furthermore, VEGF enhances plasminogen activator gene expression in vascular endothelial cells at the same concentration.5,15 The increased tPA activity found in CSH could result from stimulation of vascular endothelial cells by VEGF. VEGF is produced by various cell types, including endothelial cells, smooth muscle cells, fibroblasts, macrophages, neurons and glial cells in response to various environmental stimuli.10,11,16 In this study, we observed numerous CD68positive macrophages in the outer membrane. Macrophages play a key role in the angiogenesis cascade by producing a number of growth stimulators and inhibitors.16,17 The distribution of VEGF mRNA in the outer membranes of CSH was generally consistent with that of macrophages, indicating that infiltrating macrophages in the outer membrane are the main source of VEGF production in these haematomas. Vascular endothelial cells in the outer membrane also expressed VEGF mRNA. High-affinity receptors for VEGF, Flt-1 and Flk-1 have been demonstrated in vascular endothelial cells and some studies have identified VEGF as an autocrine growth factor for endothelial cells.16±20 Our data also support that VEGF produced by endothelial cells acts as an autocrine growth factor in these haematomas. Overexpression of VEGF in these cells, which can induce neovascularisation, excessive vascular permeability, and rebleeding is likely to be involved in the persistence and enlargement of CSH. Journal of Clinical Neuroscience (2001) 8(5), 411±415
CONCLUSION We observed marked elevation of VEGF in the fluid within CSH. Our results suggest that VEGF in the CSH is produced mainly by macrophages, and to a lesser extent by vascular endothelial cells, in the outer membrane of the haematoma. Enhanced production of VEGF by these cells is thought to be important in the pathogenesis of these haematomas. ACKNOWLEDGEMENT We thank Dr Yutaka Kimura (Kimura Hospital), Dr Tatsuyoshi Nishiyama (Amagi Central Hospital) and Dr Kazufumi Kamikaseda (Kaizuka Hospital) for providing some clinical samples. Excellent technical assistances were provided by Ms Mayumi Terasaki, Ms Fukuno Yaosaka, Ms Nami Kishikawa and Yoshie Hirosawa. This work was supported by a grant from Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (no. 9201, Tadahisa Shono MD PhD).
REFERENCES 1. 2. 3.
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Ito H, Yamamoto S, Komai T, Mizukoshi H. Role of local hyperfibrinolysis in the etiology of chronic subdural hematoma. J Neurosurg 1976; 45: 26±31. Kawano N, Suzuki K. Presence of smooth-muscle cells in the subdural neomembrane. J Neurosurg 1981; 54: 646±651. Takahashi Y, Ohkura A, Hirohata M,Tokutomi T, Shigemori M. Ultrastructure of obstructive tissue in malfunctioning ventricular catheters without infection. Neurol Med Chir (Tokyo) 1998; 38: 399±404. Yamashima T, Yamamoto S, Friede RL. The role of endothelial gap junctions in the enlargement of chronic subdural hematomas. J Neurosurg 1983; 59: 298±303.
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Bikfalvi A, Sauzeau C, Moukadiri H, Maclouf J, Busso N, Bryckaert M, Plouet J, Tobelem G. Interaction of vasculotropin/vascular endothelial cell growth factor with human umbilical vein endothelial cells: binding, internalization, degradation, and biological effects. J Cell Physiol 1991; 149: 50±59. 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: 1309±1312. 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: 983±985. Samoto K, Ikezaki K, Ono M, Shono T, Kohno K, Kuwano M, Fukui M. Expression of vascular endothelial growth factor and its possible relation with neovascularization in human brain tumors. Cancer Res 1995; 55: 1189±1193. 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: 1095±1101. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nature Med 1995; 1: 27±31. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996; 86: 353±364. Nomura S, Kashiwagi S, Ito H, Mimura Y, Nakamura K. Degradation of fibrinogen and fibrin by plasmin and nonplasmin proteases in the chronic subdural haematoma: evaluation by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and immunoblot. Electrophoresis 1993; 14: 1318±1321.
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Nomura S, Kashiwagi S, Fujisawa H, Ito H, Nakamura K. Characterization of local hyperfibrinolysis in chronic subdural haematomas by SDS-PAGE and immunoblot. J Neurosurg 1994; 81: 910±913. Fujisawa H, Ito H, Saito K, Ikeda K, Nitta H, Yamashita J. Immunohistochemical localization of tissue-type plasminogen activator in the lining wall of chronic subdural hematoma. Surg Neurol 1991; 35: 441±445. Pepper MS, Ferrara N, Orci L, Montesano R. Vascular endothelial growth factor (VEGF) induces plasminogen activators and plasminogen activator inhibitor-1 in microvascular endothelial cells. Biochem Biophys Res Commun 1991; 181: 902±906. Kovacs Z, Ikezaki K, Samoto K, Inamura T, Fukui M. VEGF and flt. Expression time kinetics in rat brain infarct. Stroke 1996; 27: 1865±1872. Yoshida S, Ono M, Shono T, Izumi H, Ishibashi T, Suzuki H, Kuwano M. Involvement of Interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis. Mol Cell Biol 1997; 17: 4015±4023. Millauer B, Wizigmann-Voos S, Schnurch H, Martinez R, Moller NP, Risau W, Ullrich A. High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis. Cell 1993; 72: 835±846. Quinn TP, Peters KG, De Vries C, Ferrara N, Williams LT Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium. Proc Natl Acad Sci USA 1993; 90: 7533±7537. Shibuya M, Yamaguchi S, Yamane A, Ikeda T, Tojo A, Matsushime H, Sato M. Nucloeotide sequence and expression of a novel human receptortype tyrosine kinase gene (flt) closely related to the fms family. Oncogene 1990; 5: 519±524.
Journal of Clinical Neuroscience (2001) 8(5), 411±415
Clinical study
Vascular endothelial growth factor in chronic subdural haematomas Tadahisa Shono1 MD PHD, Takanori Inamura1 MD PHD, Takato Morioka1 MD PHD, Ken-ichi Matsumoto1 MD, Satoshi O. Suzuki2 MD PHD, Kiyonobu Ikezaki1 MD PHD, Toru Iwaki2 MD PHD, Masashi Fukui1 MD PHD 1
Department of Neurosurgery, 2Neuropathology, Neurological Institute, Faculty of Medicine, Kyushu University, Fukuoka, Japan
Summary 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. & 2001 Harcourt Publishers Ltd Keywords chronic subdural haematoma, VEGF, bFGF, angiogenesis, vascular endothelial cell, macrophage
INTRODUCTION
MATERIALS AND METHODS
Chronic subdural haematoma (CSH) is one of the most common lesions encountered in neurosurgery. Nevertheless, the pathogenesis of CSH has not been fully elucidated. Morphologic studies of the haematoma membranes indicate that the outer membrane consists of loose collagen fibers, large capillaries (macrocapillaries), smooth muscle cells, dural border cells and inflammatory cells.1±3 Macrocapillaries in the outer membrane are considered to be important in the haematoma enlargement. The endothelial cells of macrocapillaries are abnormally permeable and fragile, and junctions between adjacent endothelial cells are loose, with gap junctions and absence of the basement membrane.4 Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is a potent, specific endothelial cell mitogen in vitro and induces angiogenesis in vivo.5,6 VEGF/ VPF has been found to be 1000 times more potent than histamine in inducing capillary permeability.7 Expression of VEGF correlates closely with neovascularisation in human brain tumours8 and with the degree of brain oedema surrounding meningiomas.9 No previous report has characterised expression of VEGF in CSH. To better understand the mechanisms underlying expansion of CSH, we quantified concentrations of VEGF as well as of another potent angiogenic factor, basic fibroblast growth factor (bFGF), in haematoma fluid and in serum from patients with CSH. We also examined the dura and outer membrane of CSH to determine the cell types producing VEGF in CSH by in situ hybridisation and immunohistochemistry.
Patients We studied 20 patients, 17 males and 3 females, with CSH. The age of the patients ranged from 24 to 90 years. All haematomas were unilateral, and there were no recurrences after primary burr hole surgery. Samples of haematoma fluid, blood, dura mater, and outer CSH membrane used for assay and were obtained during this operation at Kyushu University Hospital and affiliated hospitals beginning in 1998. Measurement of VEGF and bFGF Concentrations of VEGF and bFGF in haematoma fluid and in serum were measured with an enzyme-linked immunosorbent assay (ELISA) kit (Quantikine, R&D Systems, MN) according to the manufacturer's instructions. Immunohistochemistry The overlying dura and outer membrane of the CSH were resected during primary burr hole surgery. Resected specimens were fixed in 10% formalin solution, routinely processed, embedded in paraffin and sectioned at a thickness of 6 mm. Sections were stained immunohistochemically using an avidinbiotinylated peroxidase complex method with a mouse monoclonal antibody against the macrophage marker CD68 (KP-1; DAKO, Glostrup, Denmark), and then lightly counter-stained with haematoxylin. In situ hybridisation
Received 15 August 2000 Accepted 20 September 2000 Correspondence to: Takanori Inamura MD PhD, Department of Neurosurgery, Neurological Institute, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan. Tel.: 81-92-642-5524; Fax: 81-92-642-5526; E-mail: [email protected]
For in situ hybridisation, digoxigenin (DIG)-labeled sense and antisense RNA probes were synthesised with T7 RNA polymerase from a 210-base pair template cDNA using a DIG-RNA labeling kit (Boehringer Mannheim, Germany) according to the manufacturer's instructions. Prior to hybridisation, 6-mm-thick sections were treated with proteinase K 411
412 Shono et al.
(100 mg/ml) for 15 min at room temperature and postfixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 10 min. Thereafter, the sections were acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min and treated with 0.2 N HCl for 10 min to inactivate endogenous alkaline phosphatase. The pretreated sections were dehydrated, air-dried and hybridised overnight at 50 C with the DIGlabeled RNA probe in hybridisation buffer [50% formamide, 5X SSC (1X SSC 0.15 M NaCl and 0.015 M sodium citrate), 100 mg/ml tRNA and 0.05% heparin]. After hybridisation, sections was washed with 50% formamide/2X SSC for 30 min at 65 C and then treated with 50 mg/ml RNase A/TNE (10 mM Tris-HCl at pH 7.6, 500 mM NaCl, and 1 mM EDTA) for 30 min at 37 C. Sections were washed with 2X SSC for 20 min, followed by two washes with 0.2X SSC for 20 min each at 65 C. Hybridisation was detected immunohistochemically with monoclonal anti-DIG antibody (Boehringer Mannheim) and alkaline phosphatase-conjugated anti-mouse IgG antibody (Promega, Madison, WI), including visualisation with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT) (Promega). To compare distribution of macrophages with that of VEGF mRNA, immunohistochemical analysis and in situ hybridisation were performed sequentially using the same tissue section. Sections were incubated with a mouse monoclonal primary antibody against CD68 and subsequently with alkaline phosphatase-conjugated anti-mouse IgG antibody. Expression of CD68 was visualised with BCIP/NBT. After photomicrographs were obtained, the sections were washed with 99% ethanol overnight, completely removing the immunohistochemical reaction product. Following a brief rinse in tap water, in situ hybridisation was performed as described above.
Elevation of VEGF in CSH fluid In all 20 cases of CSH, concentrations of VEGF in the haematoma fluid were much higher than those of bFGF (Table 1). No correlation was detectable between VEGF concentrations in the haematoma and those of bFGF. Mean concentrations of
In sections stained with haematoxylin and eosin, many vessels and inflammatory cells were observed in the outer membrane (Fig. 2A). To determine cell types expressing VEGF mRNA, surgical specimens including dura and the outer membrane of CSH were analysed by in situ hybridisation. The cells surrounding vessels in the outer membrane were strongly stained when hybridised with the antisense probe (Fig. 2B), while cells in the dura showed little or no staining (not illustrated). Virtually no positive signal was seen in any cell in sections hybridised with the sense probe (Fig. 2C). When we immunohistochemically examined the distribution of macrophages, anti-CD68 antibody identified many infiltrating macrophages in the outer membrane, especially around vessels (Fig. 2D). To conclusively determine which cells expressed VEGF mRNA, immunohistochemical analysis and in situ hybridisation were performed sequentially in the same section. (Fig. 3). Most CD68-positive macrophages in the outer membrane expressed a large amount of VEGF mRNA (Fig. 3A and C). In addition, some vascular endothelial cells also showed hybridisation with the VEGF antisense probe (Fig. 3B and D). Other cells showed little or no staining for the transcript.
Angiogenesis occurs in various physiologic and pathologic conditions, including menstruation, ovulation, wound healing, inflammation and growth of solid tumours.10 The newly formed blood vessels appear to be more permeable and fragile than pre-existing vessels. To elucidate molecular aspects of the mechanisms causing expansion of CSH, we measured
Concentrations of VEGF and bFGF in serum and haematoma fluid from patients with chronic subdural haematoma
Age/Sex
57/F 84/M 90/M 78/F 68/M 86/F 63/M 59/M 71/M 83/M 51/M 71/M 85/M 58/M 70/M 75/M 24/M 77/M 87/M 69/M
Macrophages in the outer membrane are the main sources of VEGF in CSH
DISCUSSION
RESULTS
Table 1
VEGF were 355 pg/ml in serum and 10277 pg/ml in haematoma fluid (Fig. 1A); the mean concentration of VEGF in haematoma fluid was 28 times that in serum. On the other hand, mean bFGF concentrations were 4.74 pg/ml in serum and 3.04 pg/ml in haematoma fluid (Fig. 1B), with the concentration in the haematoma fluid being less than that in serum.
VEGF (pg/ml)
bFGF (pg/ml)
Serum
Haematoma
Serum
Haematoma
559 429 526 269 364 434 523 686 207 424 105 370 117 489 356 107 507 472 118 54
7589 12623 10609 8133 8887 14927 5510 8914 28556 7762 9113 5735 9536 7589 8053 8198 4000 18596 14782 6437
0 0 0 7.7 0.8 0 9.3 0 6.7 0 0 1.8 9.5 52.5 8.3 0 0 41.1 18.3 0
0 0.4 1.4 9.9 0.8 0 0 0 9.3 0.4 0.2 2.8 1.2 0 0 0 0 0 66.6 0
Journal of Clinical Neuroscience (2001) 8(5), 411±415
& 2001 Harcourt Publishers Ltd
VEGF and chronic subdural haematoma 413
Fig. 1 Protein levels in paired haematoma fluid and serum samples. Paired haematoma fluid and serum samples of patients with chronic subdural haematoma were analysed by ELISA for VEGF (A) and bFGF (B). Values represent the mean SE.
Fig. 2 Histologic, in situ hybridisation and immunostaining studies of the outer membrane of chronic subdural haematoma. (A) Haematoxylin and eosin staining (A). Many inflammatory cells and vessels are observed in the outer membrane (original magnification 200). In situ hybridisation of the outer membrane using a labeled antisense vascular endothelial growth factor probe (B) and a sense probe (C) (original magnification 200). Cells surrounding vessels in the outer membrane are strongly stained by the antisense probe, but not by the sense probe. Immunohistochemical staining for CD68 (D) demonstrates prominent macrophage infiltration in the outer membrane (original magnification 200).
concentrations of two representative angiogenic factors, VEGF and bFGF,11 in haematoma fluid and in serum from 20 patients with CSH. Concentrations of VEGF in haematomas were remarkably increased, while those of bFGF were not. Enhanced expression of VEGF is thought to be an important event in the formation of these haematomas. & 2001 Harcourt Publishers Ltd
Ito et al. have found that locally increased fibrinolysis, characterised by high concentrations of tissue-type plasminogen activator (tPA) and plasmin- -2-plasmin inhibitor complex, interferes with complete haemostasis and causes rebleeding into the central cavity of CSH.1,12,13 They also reported intense tPA immunoreactivity in the cytoplasm of Journal of Clinical Neuroscience (2001) 8(5), 411±415
414 Shono et al.
Fig. 3 Immunohistochemical staining for CD68 and in situ hybridisation for vascular endothelial growth factor (VEGF) in the same sequentially processed paraffin section of the outer membrane of a chronic subdural haematoma. After recording immunohistochemical observations (A and B), the sections were decolorized with 99% ethanol, and in situ hybridisation was performed in the same section (C and D). A and C show the same site in the outer membrane. Distributions of CD68-positive macrophages (A) and VEGF mRNA (C) generally are similar. B and D show the same site in the outer membrane. CD68-negative vascular endothelial cells (B) also have hybridised with the VEGF antisense probe (D, arrowheads; original magnification 400).
endothelial cells lining CSH sinusoids and capillaries.14 In the present study, the mean concentration of VEGF in haematomas exceeded 10 ng/ml and was 28 times the serum concentration. VEGF has been reported to stimulate growth of vascular endothelial cells in vitro at concentrations from 1 to 100 ng/ml.5,6 Furthermore, VEGF enhances plasminogen activator gene expression in vascular endothelial cells at the same concentration.5,15 The increased tPA activity found in CSH could result from stimulation of vascular endothelial cells by VEGF. VEGF is produced by various cell types, including endothelial cells, smooth muscle cells, fibroblasts, macrophages, neurons and glial cells in response to various environmental stimuli.10,11,16 In this study, we observed numerous CD68positive macrophages in the outer membrane. Macrophages play a key role in the angiogenesis cascade by producing a number of growth stimulators and inhibitors.16,17 The distribution of VEGF mRNA in the outer membranes of CSH was generally consistent with that of macrophages, indicating that infiltrating macrophages in the outer membrane are the main source of VEGF production in these haematomas. Vascular endothelial cells in the outer membrane also expressed VEGF mRNA. High-affinity receptors for VEGF, Flt-1 and Flk-1 have been demonstrated in vascular endothelial cells and some studies have identified VEGF as an autocrine growth factor for endothelial cells.16±20 Our data also support that VEGF produced by endothelial cells acts as an autocrine growth factor in these haematomas. Overexpression of VEGF in these cells, which can induce neovascularisation, excessive vascular permeability, and rebleeding is likely to be involved in the persistence and enlargement of CSH. Journal of Clinical Neuroscience (2001) 8(5), 411±415
CONCLUSION We observed marked elevation of VEGF in the fluid within CSH. Our results suggest that VEGF in the CSH is produced mainly by macrophages, and to a lesser extent by vascular endothelial cells, in the outer membrane of the haematoma. Enhanced production of VEGF by these cells is thought to be important in the pathogenesis of these haematomas. ACKNOWLEDGEMENT We thank Dr Yutaka Kimura (Kimura Hospital), Dr Tatsuyoshi Nishiyama (Amagi Central Hospital) and Dr Kazufumi Kamikaseda (Kaizuka Hospital) for providing some clinical samples. Excellent technical assistances were provided by Ms Mayumi Terasaki, Ms Fukuno Yaosaka, Ms Nami Kishikawa and Yoshie Hirosawa. This work was supported by a grant from Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (no. 9201, Tadahisa Shono MD PhD).
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Journal of Clinical Neuroscience (2001) 8(5), 411±415