Neuropathology and Applied Neurobiology 1991,17, 11-16
U1tr astructural localization of Fact or VIII-related antigen in endothelial proliferations of malignant gliomas" A. MIGHELI, A. ATTANASIO, C. M O C E L L I N I A N D D. S C H I F F E R 2nd Department of Neurology, University of Turin, Turin, Italy
MIGHELI A., ATTANASIO A., MOCELLINI C. & SCHIFFERD. (1991) Neuropathology and Applied Neurobiology 17,ll-16 Ultrastructurallocalization of Factor VIII-related antigen in endothelial proliferations of malignantgliomas The ultrastructural localization of Factor VIII-related antigen (FVIII/RAg) in the endothelial cells of malignant gliomas was studied with immunogold labelling on thin sections of LR Whiteembedded tissue. FVIII/RAg was localized in Weibel-Palade bodies (WPBs) as well as in cytoplasmic vacuoles and vesicles, and in extracellular spaces. Stained WPBs were found both in cells surrounding vascular channels and in several proliferating cells of solid buds and glomeruli. Our results confirm previous ultrastructural observations on non-neural tissues that FVIII/RAg is produced by endothelial cells and stored in WPBs. The presence of FVIII/RAgstained WPBs in capillary buds and tufts indicates that at least some of the proliferating cells are endothelial in origin rather than pericytes. Thus, the contribution of endothelial cells to new vessel formation seems to be more substantial than previously reported. Keywords: FVIII/RAg, Weibel-Palade bodies, gliomas, immunogold labelling
INTRODUCTION Factor VIII-related antigen (FVIII/RAg) is a complex glycoprotein which has procoagulant activity and is involved in platelet plug formation (Sakariassen, Bolhuis & Sixma, 1979). In vitro studies have demonstrated its synthesis by endothelial cells (Jaffe, Hoyer & Nachman, 1973); this finding has been supported by ultrastructural investigations (Piovella et al., 1978; Rand et al., 1980; Jeanneau & Sultan, 1982). For this reason, FVIII/RAg is commonly used as an endothelial cell marker in normal and pathological conditions (Mukai, Rosai & Burgdorf, 1980; Sehested & Hou-Hensen, 1981). Immunoelectron microscopy studies have shown that Weibel-Palade bodies (Weibel & Palade, 1964) (WPBs) are the major site of storage of the protein in endothelial cells (Wagner, Olmsted & Marder, 1982; Warhol & Sweet, 1984). Recently, a report has appeared on the ultrastructural distribution of FVIII/RAg in brain tumours, including gliomas (Miyagami et a/., 1987), using immunoperoxidase-DAB; WPBs were not mentioned. In the present paper we have used an immunogold labelling technique to investigate the ultrastructural localization of FVIII/RAg in vascular proliferations of gliomas. Correspondenceto: Dr Antonio Migheli, 2nd Department of Neurology, University of Turin,via Cherasco IS, 10126
Turin,Italy. *Supportedby Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan, Italy.
12
A . Migheli et al.
Figure 1. FVIII/RAg-gold labelled Weibel-Palade bodies. a, Elongated profiles. b, Rounded profiles. a, x 24 000. b, x 36 000.
MATERIALS AND METHODS Surgical samples of six malignant gliomas (anaplastic astrocytomas, glioblastomas) were cut into small blocks and placed in the fixative for 3 h. The following fixatives were used: (a) 4% paraformaldehyde+0.2% glutaraldehyde in 0.1 M phosphate buffer (PB); (b) 4% paraformaldehyde in 0.1 M PB; (c) 2.5% glutaraldehyde in 0.1 M PB. After fixation, blocks of tissue were rinsed in PB, dehydrated in graded alcohols and embedded in the hydrophilic resin LR White (Newman, Jasani &Williams, 1983). Thin sections were collected on Formvar coated grids, and treated as follows: (a) normal goat serum diluted 1/10 in Tris buffer saline (TBS) for 30 min; (b) rabbit anti-FVIII/RAg serum diluted 1/800 in TBS for 45 min; (c) goat anti-rabbit IgGs coupled with 20 nm gold (BioCell) diluted 1/50in TBS for 45 min. Grids were counterstained with uranyl acetate and lead citrate. As controls, the primary antibody was replaced by either non-immune serum or by buffer. RESULTS The presence of FVIII/R4g immunoreactivity was investigated in the cells forming various types of vessel proliferations, ranging from simple endothelial hyperplasias to more composite formations, such as glomeruloid tufts and solid buds. Gold decoration was found in the lumina and in the extracellular spaces. In endothelial cells lining vessel lumina, intracellular labelled deposits were composed of three types: (a) small, elongated or roundish dense bodies, corresponding to WPBs (Figure 1); (b) vesicles; and (c) large vacuoles, containing filamentous or amorphous material. Large vacuoles were especially
FVIII in gliomas
13
Figure 2. a, Typical solid capillary bud. x 8300. b, Detail of area indicated in a, showing FVIII/RAg labelling in a rounded dense body. x 54 100.
abundant on the luminal side, where they might appear partially fused with the plasma membrane. On the side facing the basal membrane, fusion of vesicles with the plasmalemma and extrusion of their content were seen. Solid buds were identified as structures composed of groups of cells which either lacked a clear-cut lumen, or had slit-like lumina that did not contain blood; sometimes, cavitation of the cytoplasm was seen. Cells in the solid capillary buds contained variable amounts,ofFVIII/RAgdecorated WPBs, while large vacuoles were absent (Figure 2). In glomeruli, stained WPBs could be found in cells surrounding vascular channels, as well as in a few cells which were not in a periluminal position (Figure 3). Gold labelling was similar with the various fixation schedules; in particular, fixation in glutaraldehyde alone gave comparable results in terms of number of gold granules as paraformaldehyde, but the morphology was obviously improved. DISCUSSION In the present study we have used an immunogold labelling technique to investigate the expression of FVIII/RAg by endothelial cells of malignant gliomas. First of all, some technical considerations must be taken into account. Previous data on the ultrastructural localization of FVIIIiRAg by means of immunogold labelling were obtained in Lowicryl-embedded tissue, using a low-temperature embedding technique (Warhol & Sweet, 1984). We have shown here
14
A . Migheliet al.
Figure 3. FVIII/FUg staining in glomeruli. Multiple labelled Weibel-Paladebodies are seen in an endothelialcell which is not in contact with or surroundinga lumen. a, x 14 000. b, Detail of area indicated in a. x 35 200.
that post-embedding staining in LR White-embedded tissue is equally suitable. Furthermore, enhanced preservation of the morphology could be achieved by using fixation in glutaraldehyde, which did not seem to significantly affect the antigenicity of FVIII/RAg. Previous findings obtained with peroxidase-DAB showed FVIII/RAg localization in the endoplasmic reticulum, in extracellular spaces and in the lumina of endothelial proliferations in gliomas (Miyagami et al., 1987). By gold labelling, we have demonstrated that FVIII/RAg is contained in roundish or elongated dense bodies, which can be identified as Weibel-Palade bodies (WPBs).This observation is in agreement with previous ultrastructural studies on nonneural tissues, which have shown FVIII/RAg-stained WPBs in cultured endothelial cells from umbilical vein (Wagner et al., 1982; Hormia, Lehto & Virtanen, 1984), and, in vivo, in endothelial cells of muscle and breast tissues, and in a capillary haemangioma (Warhol & Sweet, 1984). WPBs are rarely seen in normal brain (Herrlinger et al., 1974); by contrast, they are frequently found in the endothelial cells of brain tumours (Hirano, 1974). Their number seems to correlate with brain tumour growth, so that they have been considered as markers for actively growing capillaries (Kumar et al., 1980). The demonstration of stained WPBs, which are storage sites of FVIII/RAg before its release into the plasma (Warhol & Sweet, 1984), indicates that endothelial cells of vascular proliferations are able to synthesize FVIII/RAg, and not merely to adsorb it from the plasma as previously suggested (Miyagami et al., 1987). On the other hand, the large stained vacuoles at the luminal side reflect the absorptive properties of the endothelium; in fact, they were only found in lumina-lining cells. As for the labelled vesicles in the proximity of basal membranes, this finding suggests an active excretion by the endothelial cell.
FVIII in gliomas
15
Our observations have led to a reappraisal of the problem of angogenesis in malignant gliomas. The origin of the cells forming vascular proliferations in malignant gliomas is a controversial issue. Immunoperoxidase studies on FVIII/RAg localization in the light (Weller et al., 1981; McComb etal., 1982) and electron microscope (Miyagami et al., 1987) showed that FVIII/RAg staining was limited to endothelial cells lining lumina, while the other cells of vascular proliferations were reportedly negative. On the basis of their ultrastructural appearances, a pericytic origin was proposed for these cells (Weller et al., 1981). In other reports, a transition in the expression of FVIIIiRAg was found in the light microscope between hyperplastic endothelial cells and the other cells forming vascular proliferations of glioblastomas (Schiffer et al., 1984,1989).These observations suggested that at least some of the cells forming the stromal component of malignant gliomas were of endothelial nature. The present demonstration of FVIII/RAg-stained WPBs in several proliferating cells of solid buds and glomeruli indicates that: (a) these cells are endothelial in origin; (b) contrary to previous reports (Guarda et al., 1982), they may synthesize FVIII/RAg even if not stimulated by the blood; and (c)endothelial cells are not strictly located around vascular channels, but may be also found in places where a lumen is not evident ultrastructurally. Therefore, their contribution to new vessel formation seems to be more substantial than previously reported. Further immunoelectron microscopy studies are required to investigate the localization of a-smooth muscle actin, a marker of pericytes (Skalli et al., 1986), and the distribution of endothelial and pericytic cells in vessel proliferations, by means of double immunogold labelling techniques.
REFERENCES Guarda L.A., Ordonez N.G., Smith J.L. & Haussen G. (1982) Immunoperoxidaselocalization of Factor VIII/RAg in angiosarcomas. Archives of Pathology and Laboratory Medicine 106,s 15-5 16 Herrlinger H., Anzil A.P., Blindnger K. & Kronski D. (1974) Endothelial microtubular bodies in human brain capillaries and venules. Journal of Anatomy 118,205-209 Hirano A. (1974) Fine structural alterations of small vessels in the nervous system. In PathoLogy of Cerebral Microcirculation, Ed. J. Cerv&-Navarro, pp. 203-21 7. De Gruyter, Berlin Hormia M., Lehto V.-P. & Virtanen I. (1984) Intracellular localization of factor VIII-related antigen and fibronectin in cultured human endothelial cells: evidence for divergent routes of intracellular translocation. European Journal of Cell Biology 33,217-228 Jaffe E.A., Hoyer, L.W. & Nachman R.L. (1973) Synthesis of antihemophilic factor antigen by cultured human endothelid cells. Journal of Clinical Investigation 52,2757-2764 Jeanneau C. & Sultan Y. (1982) Localization of Factor VIII/von Willebrand factor antigen by immunoelectron microscopy in human endothelid cells using Fab fragments coupled to peroxidase. The Journal of Histochemistry and Cytochemistry 30,109 1-1096
Kumar P., Kumar S., Marsden H.B., Lynch P.G. & Earnshaw E. (1980) Weibel-Palade bodies in endothelid cells as a marker for angiogenesisin brain tumors. Cancer Research 40,2010-2019 McComb R.D., Jones T.R., Pizzo S.V. & Bigner D.D. (1982) Immunohistochemical detection of Factor VIII/von Willebrand factor in hyperplastic endothelial cells in glioblastoma multiforme and mixed glioma/sarcoma. Journal of Neuropathology and Experimental Neurology 41,479-489
Miyagami M., Smith B.H., McKeever P.E., Chronvall B.M., Greenwood M.A. & Kornblith P.L.(1987) Immunocytochemical localization of factor VIII-related antigen in tumors of the central nervous system. Journal of Neuro-Oncology 4,269-285
Mukai K., Rosai J. & Burgd0rfW.H.C. (1980) Localization of factor VIII-related antigen in vascular endothelid cells using an immunoperoxidasemethod. The American Journal of Surgical Pa!hoIogy 4,273-276 Newman R., Jasani B. & Williams E.D. (1983) A simple post-embedding system for the rapid demonstration of tissue antigens under the electron microscope. Histochemical Journal 15,543-555 Piovella F., Nalli G., Malamani G.D., Majolino I., Frassoni F., Sitar G.M., Ruggeri A., Dell’Orbo C. & Ascari E. (1978) The ultrastructural localization of Factor VIII-antigen in human platelets, megakaryocytes and endothelialcells utilizing a ferritin-labelled antibody. British Journal of Haematology 39,209-213
16
A . Migheli et al.
Rand J.H., Sussman I.I., Gordon R.E., Chu S.V. &Solomon V. (1980) Localization of Factor-VIII-related antigen in human vascular subendothelium. Blood 55,752-756 Sakariassen K.S..Bolhuis P.A. & Sixma J.J. (1979) Human blood platelet adhesion to artery subendothelium is mediated by Factor VIII-von Willeband factor bound to subendothelium. Nature (London) 279,63-38 Schiffer D., Giordana M.T., Mauro A. & Migheli A. (1984) GFAP, F VIII/RAg, laminin and fibronectin in gliosarcomas: an immunohistochemical study. Acta Neuropathologica (Berlin) 63, 108-1 16 Schiffer D., Chio’ A,, Giordana M.T., Mauro A., Migheli A. & Vigliani M.C. (1989) The vascular response to tumor infiltration in malignant gliomas. Morphometric and reconstruction study. Acta Neuropathologica (Berlin) 77, 369-378 Sehested M. & Hou-Jensen K. (198 1) Factor VIII related antigen as an endothelial cell marker in benign and malignant diseases. Virchows Archiv A [Pathological Anatomy and Histology] 391,217-224 Skalli 0..Ropraz P., Trzeciak A,, Benzonana G.,Gillessen D. & Gabbiani G. (1986) A monoclonal antibody against asmooth muscle actin: a new probe for smooth muscle differentiation. The Journalof CeN Biology 103,2787-2796 Wagner D.D., Olmsted J.B. & Marder V.J. (1982) Immunolocalization of von Willebrand protein in Weibel-Palade bodies of human endothelial cells. The Journal of Cell Biology 95,355-360 Warhol M.J. & Sweet J.M. (1984) The ultrastructural localization of von Willebrand factor in endothelial cells. American Journal of Pathology 117,3 1&3 15 Weibel E.R. & Palade G.E. (1964) New cytoplasmic components in arterial endothelia. The Journal of Cell Biology 23, 101-112 Weller R.O., Davis B.E., Wilson P.O.G. & Mitchell J. (1981) Capillary proliferation in cerebral infarction, gliomas, angioblastic meningiomas, and hemangioblastomas. In Cerebral Microcirculation and Metabolism, eds J. Cerv6s-Navarro & E. Fritschka, pp. 41-48. Raven Press, New York
Received I May I990 Accepted after revision 7 July 1990
U1tr astructural localization of Fact or VIII-related antigen in endothelial proliferations of malignant gliomas" A. MIGHELI, A. ATTANASIO, C. M O C E L L I N I A N D D. S C H I F F E R 2nd Department of Neurology, University of Turin, Turin, Italy
MIGHELI A., ATTANASIO A., MOCELLINI C. & SCHIFFERD. (1991) Neuropathology and Applied Neurobiology 17,ll-16 Ultrastructurallocalization of Factor VIII-related antigen in endothelial proliferations of malignantgliomas The ultrastructural localization of Factor VIII-related antigen (FVIII/RAg) in the endothelial cells of malignant gliomas was studied with immunogold labelling on thin sections of LR Whiteembedded tissue. FVIII/RAg was localized in Weibel-Palade bodies (WPBs) as well as in cytoplasmic vacuoles and vesicles, and in extracellular spaces. Stained WPBs were found both in cells surrounding vascular channels and in several proliferating cells of solid buds and glomeruli. Our results confirm previous ultrastructural observations on non-neural tissues that FVIII/RAg is produced by endothelial cells and stored in WPBs. The presence of FVIII/RAgstained WPBs in capillary buds and tufts indicates that at least some of the proliferating cells are endothelial in origin rather than pericytes. Thus, the contribution of endothelial cells to new vessel formation seems to be more substantial than previously reported. Keywords: FVIII/RAg, Weibel-Palade bodies, gliomas, immunogold labelling
INTRODUCTION Factor VIII-related antigen (FVIII/RAg) is a complex glycoprotein which has procoagulant activity and is involved in platelet plug formation (Sakariassen, Bolhuis & Sixma, 1979). In vitro studies have demonstrated its synthesis by endothelial cells (Jaffe, Hoyer & Nachman, 1973); this finding has been supported by ultrastructural investigations (Piovella et al., 1978; Rand et al., 1980; Jeanneau & Sultan, 1982). For this reason, FVIII/RAg is commonly used as an endothelial cell marker in normal and pathological conditions (Mukai, Rosai & Burgdorf, 1980; Sehested & Hou-Hensen, 1981). Immunoelectron microscopy studies have shown that Weibel-Palade bodies (Weibel & Palade, 1964) (WPBs) are the major site of storage of the protein in endothelial cells (Wagner, Olmsted & Marder, 1982; Warhol & Sweet, 1984). Recently, a report has appeared on the ultrastructural distribution of FVIII/RAg in brain tumours, including gliomas (Miyagami et a/., 1987), using immunoperoxidase-DAB; WPBs were not mentioned. In the present paper we have used an immunogold labelling technique to investigate the ultrastructural localization of FVIII/RAg in vascular proliferations of gliomas. Correspondenceto: Dr Antonio Migheli, 2nd Department of Neurology, University of Turin,via Cherasco IS, 10126
Turin,Italy. *Supportedby Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan, Italy.
12
A . Migheli et al.
Figure 1. FVIII/RAg-gold labelled Weibel-Palade bodies. a, Elongated profiles. b, Rounded profiles. a, x 24 000. b, x 36 000.
MATERIALS AND METHODS Surgical samples of six malignant gliomas (anaplastic astrocytomas, glioblastomas) were cut into small blocks and placed in the fixative for 3 h. The following fixatives were used: (a) 4% paraformaldehyde+0.2% glutaraldehyde in 0.1 M phosphate buffer (PB); (b) 4% paraformaldehyde in 0.1 M PB; (c) 2.5% glutaraldehyde in 0.1 M PB. After fixation, blocks of tissue were rinsed in PB, dehydrated in graded alcohols and embedded in the hydrophilic resin LR White (Newman, Jasani &Williams, 1983). Thin sections were collected on Formvar coated grids, and treated as follows: (a) normal goat serum diluted 1/10 in Tris buffer saline (TBS) for 30 min; (b) rabbit anti-FVIII/RAg serum diluted 1/800 in TBS for 45 min; (c) goat anti-rabbit IgGs coupled with 20 nm gold (BioCell) diluted 1/50in TBS for 45 min. Grids were counterstained with uranyl acetate and lead citrate. As controls, the primary antibody was replaced by either non-immune serum or by buffer. RESULTS The presence of FVIII/R4g immunoreactivity was investigated in the cells forming various types of vessel proliferations, ranging from simple endothelial hyperplasias to more composite formations, such as glomeruloid tufts and solid buds. Gold decoration was found in the lumina and in the extracellular spaces. In endothelial cells lining vessel lumina, intracellular labelled deposits were composed of three types: (a) small, elongated or roundish dense bodies, corresponding to WPBs (Figure 1); (b) vesicles; and (c) large vacuoles, containing filamentous or amorphous material. Large vacuoles were especially
FVIII in gliomas
13
Figure 2. a, Typical solid capillary bud. x 8300. b, Detail of area indicated in a, showing FVIII/RAg labelling in a rounded dense body. x 54 100.
abundant on the luminal side, where they might appear partially fused with the plasma membrane. On the side facing the basal membrane, fusion of vesicles with the plasmalemma and extrusion of their content were seen. Solid buds were identified as structures composed of groups of cells which either lacked a clear-cut lumen, or had slit-like lumina that did not contain blood; sometimes, cavitation of the cytoplasm was seen. Cells in the solid capillary buds contained variable amounts,ofFVIII/RAgdecorated WPBs, while large vacuoles were absent (Figure 2). In glomeruli, stained WPBs could be found in cells surrounding vascular channels, as well as in a few cells which were not in a periluminal position (Figure 3). Gold labelling was similar with the various fixation schedules; in particular, fixation in glutaraldehyde alone gave comparable results in terms of number of gold granules as paraformaldehyde, but the morphology was obviously improved. DISCUSSION In the present study we have used an immunogold labelling technique to investigate the expression of FVIII/RAg by endothelial cells of malignant gliomas. First of all, some technical considerations must be taken into account. Previous data on the ultrastructural localization of FVIIIiRAg by means of immunogold labelling were obtained in Lowicryl-embedded tissue, using a low-temperature embedding technique (Warhol & Sweet, 1984). We have shown here
14
A . Migheliet al.
Figure 3. FVIII/FUg staining in glomeruli. Multiple labelled Weibel-Paladebodies are seen in an endothelialcell which is not in contact with or surroundinga lumen. a, x 14 000. b, Detail of area indicated in a. x 35 200.
that post-embedding staining in LR White-embedded tissue is equally suitable. Furthermore, enhanced preservation of the morphology could be achieved by using fixation in glutaraldehyde, which did not seem to significantly affect the antigenicity of FVIII/RAg. Previous findings obtained with peroxidase-DAB showed FVIII/RAg localization in the endoplasmic reticulum, in extracellular spaces and in the lumina of endothelial proliferations in gliomas (Miyagami et al., 1987). By gold labelling, we have demonstrated that FVIII/RAg is contained in roundish or elongated dense bodies, which can be identified as Weibel-Palade bodies (WPBs).This observation is in agreement with previous ultrastructural studies on nonneural tissues, which have shown FVIII/RAg-stained WPBs in cultured endothelial cells from umbilical vein (Wagner et al., 1982; Hormia, Lehto & Virtanen, 1984), and, in vivo, in endothelial cells of muscle and breast tissues, and in a capillary haemangioma (Warhol & Sweet, 1984). WPBs are rarely seen in normal brain (Herrlinger et al., 1974); by contrast, they are frequently found in the endothelial cells of brain tumours (Hirano, 1974). Their number seems to correlate with brain tumour growth, so that they have been considered as markers for actively growing capillaries (Kumar et al., 1980). The demonstration of stained WPBs, which are storage sites of FVIII/RAg before its release into the plasma (Warhol & Sweet, 1984), indicates that endothelial cells of vascular proliferations are able to synthesize FVIII/RAg, and not merely to adsorb it from the plasma as previously suggested (Miyagami et al., 1987). On the other hand, the large stained vacuoles at the luminal side reflect the absorptive properties of the endothelium; in fact, they were only found in lumina-lining cells. As for the labelled vesicles in the proximity of basal membranes, this finding suggests an active excretion by the endothelial cell.
FVIII in gliomas
15
Our observations have led to a reappraisal of the problem of angogenesis in malignant gliomas. The origin of the cells forming vascular proliferations in malignant gliomas is a controversial issue. Immunoperoxidase studies on FVIII/RAg localization in the light (Weller et al., 1981; McComb etal., 1982) and electron microscope (Miyagami et al., 1987) showed that FVIII/RAg staining was limited to endothelial cells lining lumina, while the other cells of vascular proliferations were reportedly negative. On the basis of their ultrastructural appearances, a pericytic origin was proposed for these cells (Weller et al., 1981). In other reports, a transition in the expression of FVIIIiRAg was found in the light microscope between hyperplastic endothelial cells and the other cells forming vascular proliferations of glioblastomas (Schiffer et al., 1984,1989).These observations suggested that at least some of the cells forming the stromal component of malignant gliomas were of endothelial nature. The present demonstration of FVIII/RAg-stained WPBs in several proliferating cells of solid buds and glomeruli indicates that: (a) these cells are endothelial in origin; (b) contrary to previous reports (Guarda et al., 1982), they may synthesize FVIII/RAg even if not stimulated by the blood; and (c)endothelial cells are not strictly located around vascular channels, but may be also found in places where a lumen is not evident ultrastructurally. Therefore, their contribution to new vessel formation seems to be more substantial than previously reported. Further immunoelectron microscopy studies are required to investigate the localization of a-smooth muscle actin, a marker of pericytes (Skalli et al., 1986), and the distribution of endothelial and pericytic cells in vessel proliferations, by means of double immunogold labelling techniques.
REFERENCES Guarda L.A., Ordonez N.G., Smith J.L. & Haussen G. (1982) Immunoperoxidaselocalization of Factor VIII/RAg in angiosarcomas. Archives of Pathology and Laboratory Medicine 106,s 15-5 16 Herrlinger H., Anzil A.P., Blindnger K. & Kronski D. (1974) Endothelial microtubular bodies in human brain capillaries and venules. Journal of Anatomy 118,205-209 Hirano A. (1974) Fine structural alterations of small vessels in the nervous system. In PathoLogy of Cerebral Microcirculation, Ed. J. Cerv&-Navarro, pp. 203-21 7. De Gruyter, Berlin Hormia M., Lehto V.-P. & Virtanen I. (1984) Intracellular localization of factor VIII-related antigen and fibronectin in cultured human endothelial cells: evidence for divergent routes of intracellular translocation. European Journal of Cell Biology 33,217-228 Jaffe E.A., Hoyer, L.W. & Nachman R.L. (1973) Synthesis of antihemophilic factor antigen by cultured human endothelid cells. Journal of Clinical Investigation 52,2757-2764 Jeanneau C. & Sultan Y. (1982) Localization of Factor VIII/von Willebrand factor antigen by immunoelectron microscopy in human endothelid cells using Fab fragments coupled to peroxidase. The Journal of Histochemistry and Cytochemistry 30,109 1-1096
Kumar P., Kumar S., Marsden H.B., Lynch P.G. & Earnshaw E. (1980) Weibel-Palade bodies in endothelid cells as a marker for angiogenesisin brain tumors. Cancer Research 40,2010-2019 McComb R.D., Jones T.R., Pizzo S.V. & Bigner D.D. (1982) Immunohistochemical detection of Factor VIII/von Willebrand factor in hyperplastic endothelial cells in glioblastoma multiforme and mixed glioma/sarcoma. Journal of Neuropathology and Experimental Neurology 41,479-489
Miyagami M., Smith B.H., McKeever P.E., Chronvall B.M., Greenwood M.A. & Kornblith P.L.(1987) Immunocytochemical localization of factor VIII-related antigen in tumors of the central nervous system. Journal of Neuro-Oncology 4,269-285
Mukai K., Rosai J. & Burgd0rfW.H.C. (1980) Localization of factor VIII-related antigen in vascular endothelid cells using an immunoperoxidasemethod. The American Journal of Surgical Pa!hoIogy 4,273-276 Newman R., Jasani B. & Williams E.D. (1983) A simple post-embedding system for the rapid demonstration of tissue antigens under the electron microscope. Histochemical Journal 15,543-555 Piovella F., Nalli G., Malamani G.D., Majolino I., Frassoni F., Sitar G.M., Ruggeri A., Dell’Orbo C. & Ascari E. (1978) The ultrastructural localization of Factor VIII-antigen in human platelets, megakaryocytes and endothelialcells utilizing a ferritin-labelled antibody. British Journal of Haematology 39,209-213
16
A . Migheli et al.
Rand J.H., Sussman I.I., Gordon R.E., Chu S.V. &Solomon V. (1980) Localization of Factor-VIII-related antigen in human vascular subendothelium. Blood 55,752-756 Sakariassen K.S..Bolhuis P.A. & Sixma J.J. (1979) Human blood platelet adhesion to artery subendothelium is mediated by Factor VIII-von Willeband factor bound to subendothelium. Nature (London) 279,63-38 Schiffer D., Giordana M.T., Mauro A. & Migheli A. (1984) GFAP, F VIII/RAg, laminin and fibronectin in gliosarcomas: an immunohistochemical study. Acta Neuropathologica (Berlin) 63, 108-1 16 Schiffer D., Chio’ A,, Giordana M.T., Mauro A., Migheli A. & Vigliani M.C. (1989) The vascular response to tumor infiltration in malignant gliomas. Morphometric and reconstruction study. Acta Neuropathologica (Berlin) 77, 369-378 Sehested M. & Hou-Jensen K. (198 1) Factor VIII related antigen as an endothelial cell marker in benign and malignant diseases. Virchows Archiv A [Pathological Anatomy and Histology] 391,217-224 Skalli 0..Ropraz P., Trzeciak A,, Benzonana G.,Gillessen D. & Gabbiani G. (1986) A monoclonal antibody against asmooth muscle actin: a new probe for smooth muscle differentiation. The Journalof CeN Biology 103,2787-2796 Wagner D.D., Olmsted J.B. & Marder V.J. (1982) Immunolocalization of von Willebrand protein in Weibel-Palade bodies of human endothelial cells. The Journal of Cell Biology 95,355-360 Warhol M.J. & Sweet J.M. (1984) The ultrastructural localization of von Willebrand factor in endothelial cells. American Journal of Pathology 117,3 1&3 15 Weibel E.R. & Palade G.E. (1964) New cytoplasmic components in arterial endothelia. The Journal of Cell Biology 23, 101-112 Weller R.O., Davis B.E., Wilson P.O.G. & Mitchell J. (1981) Capillary proliferation in cerebral infarction, gliomas, angioblastic meningiomas, and hemangioblastomas. In Cerebral Microcirculation and Metabolism, eds J. Cerv6s-Navarro & E. Fritschka, pp. 41-48. Raven Press, New York
Received I May I990 Accepted after revision 7 July 1990
