Glomeruloid vascular structures in glioblastoma multiforme: an ...

J Neurosurg 85:1078–1084, 1996

Glomeruloid vascular structures in glioblastoma multiforme: an immunohistochemical and ultrastructural study AMYN M. ROJIANI, M.D., PH.D., AND KATERINA DOROVINI-ZIS, M.D. Department of Pathology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida; and Department of Pathology and Laboratory Medicine, Section of Neuropathology, Vancouver Hospital and Health Sciences Center, University of British Columbia, Vancouver, British Columbia, Canada U Microvascular proliferation and glomeruloid vascular structures are important histopathological features of glioblastoma multiforme (GBM). The nature of cells participating in the formation of these structures remains unclear and is the subject of this study. To define these cells better, immunohistochemical markers directed against Factor VIII–related antigen (FVIIIR:Ag), alpha smooth-muscle actin (a-SMA), and the lectin Ulex europaeus agglutinin type I (UEA-I) were used. Cells lining the vascular channels and a large number of proliferating abluminal cells participating in glomeruloid vascular structure formation showed positive cytoplasmic staining for FVIIIR:Ag and UEA-I. Abluminal and luminal cells were variably labeled for a-SMA. Ultrastructurally, complex aggregates of focally anastomosing capillaries with narrow lumina composed the glomeruloid vascular structure. Endothelial cells were hyperplastic, varied in size and shape, overlapped focally, and contained numerous cytoplasmic filaments. Tight junctions bound together adjacent and overlapping endothelial cells. Weibel–Palade bodies, usually absent from brain microvessels, were present in increased numbers in the newly formed capillaries. Each capillary loop was surrounded by basal lamina encompassing a discontinuous layer of pericytes. This study indicates that glomeruloid vascular structures in GBM are complex aggregates of newly formed microchannels lined with hyperplastic endothelial cells that have an altered morphological phenotype and that these microchannels are supported by basal lamina and pericytes and are devoid of astrocytic end-feet.

KEY WORDS • glomeruloid vascular structure • glioblastoma multiforme • immunohistochemistry • electron microscopic study

NE of the histological hallmarks of high-grade gliomas is florid proliferation of small blood vessels within the main mass and in the advancing front of the tumor. This neovascularization is often associated with the formation of complex glomeruloid structures that are a diagnostic criterion for glioblastoma multiforme (GBM). Previous electron microscopic studies have shown that the morphology of the small blood vessels within malignant gliomas is significantly different from that of the normal brain microvessels from which they originate.15,23,26,36 Thus, immature capillaries with slitlike lumina devoid of pericapillary glial processes prevail within the main tumor.37 Endothelial cells lining these abnormal vessels show evidence of hyperplasia, increased numbers of Weibel–Palade bodies,13 and focal distentions in the interendothelial junctions that may play a major role in the blood-brain barrier defect and increased vascular permeability in GBM.6 The exact nature of the cells participating in the formation of these elaborate vascular structures in GBM has not been fully elucidated. Although cells lining the narrow lumina appear to be partly of endothelial origin on the basis of light microscopic and immunohistochemical studies, other nonendothelial cell

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types have been implicated as primary contributors to the formation of the glomeruloid structures. These include pericytes, fibroblasts,25 and smooth muscle cells.10 In the present study the morphology and cellular composition of the glomeruloid structures in GBM were investigated using a combined immunohistochemical and ultrastructural approach. The results indicate that these complex vascular structures represent closely packed newly formed capillary buds, which are lined by hyperplastic endothelial cells that are partly invested by pericytes and devoid of astrocytic end-feet. Materials and Methods Biopsy or surgical resection specimens from 24 cases of adult supratentorial GBM treated at the Vancouver Hospital and Health Sciences Center between January 1988 and December 1989 were examined. Twelve cases showing marked vascular proliferation with formation of glomeruloid vascular structures and in which adequate tissue specimens embedded in paraffin and plastic were available were selected for the study. For routine light microscopy all specimens were fixed in 10% phosphate-buffered formalin and sections were stained with hematoxylin and eosin.

J. Neurosurg. / Volume 85 / December, 1996

Glomeruloid vascular structures in glioblastoma in nonimmune serum (1:20) for 10 minutes and then incubated with a 1:100 dilution of rabbit antiserum to FVIIIR:Ag or a 1:8000 dilution of mouse anti-a-SMA MAb for 60 minutes at room temperature. For the demonstration of UEA-I, sections were first incubated with a 1:400 dilution of UEA-I lectin for 2 hours at room temperature and then with a 1:100 dilution of rabbit antiserum to UEA-I for 60 minutes. Sections were rinsed with Tris buffer and incubated with a 1:200 dilution of HRP-conjugated goat anti–rabbit (for FVIIIR:Ag and UEA-I) or goat anti–mouse (for a-SMA) immunoglobulin G for 90 minutes at room temperature. After washing in Tris buffer, the sections were incubated with 3-amino9-ethyl carbazol for 15 minutes, counterstained with hematoxylin and mounted with aqua-mount. Double-immunolabeling for glial fibrillary acidic protein and UEA-I or a-SMA was performed in some tumors, and 3,39-diaminobenzidine was used as the second chromogen. Controls consisted of sections incubated with normal rabbit or mouse serum and positive controls. Staining of endothelial cells with UEA-I was inhibited by preincubation of the lectin in 0.2 M a-L-fucose solution for 30 minutes. In addition, sections of vessels in normal cortex, as well as proliferating vessels at the edge of a recent infarct, were examined and served as controls. Twelve cases were examined by immunohistochemical methods. One to three blocks were studied from each case and a total of 46 optical fields (up to three 3 10 optical fields per block) containing prominent vascular proliferations were assessed semiquantitatively. Individual cells were distinguished on the basis of their nuclei. The degree of immunolabeling of cells lining definable vascular lumina was graded on a 1+ to 3+ scale for each field. Sections were scored as 3+ if all cells lining the lumina were positive, 2+ if some cells lining the lumina did not stain and 1+ if the majority of lining cells were negative. Cells peripheral to or unassociated with visible lumina were similarly assessed and graded as 1+ to 3+ with regard to the degree of immunolabeling.

FIG. 1. Photomicrographs showing glomeruloid vascular structures in glioblastoma multiforme. A: Proliferating endothelial cells form compact masses encompassing small, narrow vascular channels. A microvessel at one edge of the glomeruloid vascular structure (arrowhead) may represent the vessel of origin of this structure. The microvascular aggregates are clearly delineated from the surrounding neoplastic astrocytes. H & E. B and C: Endothelial cells lining slit-like lumina (some indicated by arrows), as well as cells not clearly associated with apparent vascular lumina (arrowheads point to some positive cells) exhibit positive staining for Factor VIII–related antigen (FVIIIR:Ag; B) and Ulex europaeus agglutinin type I (UEA-I; C). Consistent with the semiquantitative score, a greater number of luminal (arrows) than abluminal (arrowheads) cells are positive for FVIIIR:Ag and UEA-I. Immunoperoxidase staining for FVIIIR:Ag and UEA-I. D: Photomicrograph showing a small number of cells, mostly luminal (arrows), positively stained for alpha smooth-muscle actin (a-SMA) in this glomeruloid structure. Staining is in the form of delicate granular perinuclear deposits. Immunoperoxidase staining for a-SMA. Bars = 10 mm.

Electron Microscopy Specimens were fixed in 2.5% glutaraldehyde overnight, washed in 0.1 M cacodylate buffer, postfixed with 1% osmium tetroxide in veronal acetate buffer for 90 minutes, block stained in 4% uranyl acetate for 15 minutes, dehydrated through a graded ethanol series and embedded in epoxy resin. One micrometer-thick toluidine bluestained sections were screened for areas of vascular proliferation and ultrathin sections were then examined with an electron microscope. Sources of Supplies Rabbit antisera to FVIIIR:Ag and UEA-I lectin were obtained from Dakopatts Inc., Santa Barbara, CA. Mouse MAb to a-SMA and a-L-fucose were obtained from Sigma Chemical Co., St. Louis, MO. Horseradish peroxidase–conjugated goat anti–rabbit and goat anti–mouse secondary antibodies were obtained from Jackson Immunoresearch Laboratories, West Grove, PA. The UEA-I lectin was obtained from Vector Laboratories, Mississauga, Ontario, Canada. Aqua-mount was obtained from Lerner Laboratories, New Haven, CT, and Efepoxy resin from E. F. Fullam, Inc., Latham, NY.

Results Immunohistochemical Studies

Light Microscopy

Antibodies. Rabbit antisera to Factor VIII–related antigen (FVIIIR:Ag), Ulex europaeus agglutinin type I (UEA-I) lectin mouse monoclonal antibody (MAb) to alpha smooth-muscle actin (aSMA), and horseradish peroxidase (HRP)–conjugated goat anti– rabbit and goat anti–mouse secondary antibodies were used. Immunoperoxidase Technique. For the immunohistochemical demonstration of FVIIIR:Ag, UEA-I, and a-SMA, the indirect immunoperoxidase technique was performed. Paraffin sections cut 3 mm thick were deparaffinized and the endogenous peroxidase activity was blocked with 0.6% hydrogen peroxide in methanol for 45 minutes. After washing in Tris buffer, sections were immersed

Glomeruloid vascular structures were well delineated from the surrounding tumor cells (Fig. 1). Unlike normal brain capillaries that are lined by one or two flattened endothelial cells (Fig. 2), glomeruloid vascular structures were composed of masses of closely associated cells that enclosed several small and often slitlike vascular channels (Fig. 1A). Usually one to three dilated thin-walled blood vessels were observed in the center or at one edge of the glomeruloid vascular structures, closely surrounded by the proliferating microvessels, and they seemed to provide


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FIG. 2. Photomicrograph showing normal brain capillaries (arrows) with thin walls lined with a single layer of attenuated endothelium that is strongly positive for Factor VIII–related antigen. H & E, bar = 10 mm.

the mother vessel from which endothelial sprouts originated. Immunoperoxidase staining for FVIIIR:Ag showed intense cytoplasmic staining of luminal and to a considerable extent of immediately subjacent (peripheral or abluminal) cells forming the glomeruloid vascular structures (Fig. 1B). Similarly, cells lining the vascular channels within the glomeruloid vascular structures displayed strongly positive staining for UEA-I lectin. Several, but not all, peripheral cells also demonstrated the ability to bind the lectin (Fig. 1C). The intensity of staining was greatest in the luminal surface of cells lining the narrow lumina. Staining for a-SMA demonstrated a small number of positive cytoplasmic profiles among the luminal and peripheral cells (Fig. 1D). However, cells lining the narrow lumina were not as uniformly stained as with FVIIIR:Ag and UEA-I. Sections of normal brain used as controls showed positive staining for a-SMA of smoothmuscle cells around large vessels. The results of semiquantitative assessment of the staining pattern of luminal and peripheral cells with the three markers are illustrated in Fig. 3. As described in Materials and Methods, the results represent the total score of 46 optic fields graded on a 1+ to 3+ scale. Thus, with both FVIIIR:Ag and UEA-I, all cells lining identifiable lumina stained positive, generating a semiquantitative score of 138, whereas fewer luminal cells (109) stained positively with a-SMA. Interestingly, the number of peripheral cells exhibiting positive reaction for a-SMA was smaller compared to the number of positive luminal cells (62: 109). On the other hand, a much larger number of peripheral cells were immunopositive for FVIIIR:Ag and UEAI (semiquantitative score: 95 and 84, respectively) than for a-SMA. Electron Microscopy

All glomeruloid vascular structures examined displayed a similar ultrastructural appearance, being composed of closely associated microvessels with the morphology of capillaries. The number and size of capillaries varied among different glomeruloid vascular structures. Usually more than three vascular channels comprised a single 1080

FIG. 3. Bar graph showing semiquantitative assessment of the immunoperoxidase staining pattern of luminal and peripheral cells for Factor VIII–related antigen, Ulex europaeus agglutinin type I, and alpha smooth-muscle actin. Bars represent numbers of positive luminal and peripheral cells in 46 optical fields with areas of prominent vascular proliferation.

glomeruloid vascular structure. One or two capillaries in a given glomeruloid vascular structure with a lumen large enough to accommodate one or two red or white blood cells were adjacent to capillary buds with narrow slitlike lumina. The lumina were irregular, too small to be patent to blood flow and commonly filled with amorphous material, endothelial cell processes, and cellular debris (Fig. 4). Not infrequently, the lumina of adjacent capillary buds were interconnected. Endothelial cells lining the capillaries and vascular sprouts appeared hyperplastic and varied considerably from the endothelium of normal brain capillaries. Thus, whereas the normal capillary lumen is lined by one or two flattened endothelial cells that are uniform in appearance, surrounded by basal lamina within which the pericytes rest (Fig. 5), in glomeruloid vascular structures several endothelial cells of remarkably different sizes and shapes were aligned around a small lumen. Tall cuboidal or rounded cells protruding into the lumen lay next to small or attenuated flattened cells with long, thin cytoplasm (Fig. 6). Occasionally an endothelial cell overlapped a neighboring cell by extending a process over and establishing contacts with the underlying cell. Even less common was the observation that, after making proper contact through tight junction formation with an adjacent cell, an endothelial cell would extend a long process beneath and along the abluminal surface of one or two neighboring endothelial cells, and would then become reincorporated into the same capillary wall and make contact with another cell. The luminal surface of the cytoplasm was highly irregular, with numerous villous processes protruding into the lumen (Fig. 6). Adjacent endothelial cells were bound together by junctional complexes, focally sealing long interendothelial contacts and having the ultrastructural appearance of tight junctions (Figs. 4 and 7). These were also observed along the juxtaposed surfaces of overlapping cells (Fig. 4). Focal distentions of interjunctional spaces were sometimes observed between adjacent cells. The endothelial cell cytoplasm contained numerous miJ. Neurosurg. / Volume 85 / December, 1996

Glomeruloid vascular structures in glioblastoma

FIG. 4. Electron micrograph showing three adjacent endothelial cells forming a capillary sprout with a slitlike lumen (L). A tight junction (arrow) is present between adjacent endothelial cells. The largest cell overlaps a process of another endothelial cell (arrowhead) and the two are focally bound together by tight junctions. A pericyte (p) partly surrounds the capillary bud, from which it is separated by basal lamina. Bar = 1 mm.

crofilaments and intermediate filaments that filled and distended the cytoplasm of some cells as the predominant feature. Although microfilaments were distributed haphazardly throughout the cytoplasm, they were often more prominent in the part facing the lumen. The number of plain and coated cytoplasmic vesicles and mitochondria did not appear to be increased, although no attempt was made to quantitate these organelles. Weibel–Palade bodies were observed in variable numbers in most but not all endothelial cells and their numbers were considerably increased in some cells (Fig. 8). This variability in the numbers of Weibel–Palade bodies was evident even within adjacent cells forming the wall of a single capillary loop. Basal lamina of variable thickness and electron density surrounded the newly formed microvessels. Astrocytic end-feet that form a continuous sheath around normal cerebral capillaries were completely absent from capillaries of glomeruloid vascular structures (Figs. 4 and 6). Embedded within the basal lamina were pericytes, which formed a discontinuous layer around each capillary (Figs. 4 and 6). Pericytes displayed characteristic subplasmalemmal densities, a paucity of cytoplasmic organelles, dense bodies, and intermediate filaments. The number of endothelial cells lining the capillaries of glomeruloid vascular structures exceeded that of surrounding pericytes (Fig. 6). The basal lamina separating pericytes from tumor cells was often thick and duplicated and focally continuous with collagen fibers. Basal lamina and pericytes were the only tissue components separating adjacent capillary loops. No other cell types were identified within any of the glomeruloid vascular structures examined. Although processes of tumor cells were seen abutting on the thickened perivascular basement membrane, the glomeruloid J. Neurosurg. / Volume 85 / December, 1996

FIG. 5. Electron micrograph showing two endothelial cells bound together by tight junctions (arrows) and forming a thin cell layer around the lumen of this normal brain capillary. The cells are devoid of Weibel–Palade bodies and have prominent mitochondria and inconspicuous cytoplasmic filaments. Embedded within the basement membrane (asterisk) are processes of pericytes (arrowheads). Bar = 1 mm.

vascular structures were often separated from the surrounding neoplastic cells by expanded acellular spaces (Fig. 6). Discussion The results of our studies indicate that the complex glomeruloid vascular structures that develop as a consequence of florid microvascular proliferation in GBM are composed of several closely associated capillaries and endothelial sprouts surrounded by variably thickened basement membrane within which a limited number of pericapillary pericytes is embedded. Smooth-muscle cells

FIG. 6. Electron micrograph showing endothelial cells (E) lining two adjacent interconnecting capillary loops and differing greatly in size, shape, and density of cytoplasmic organelles. Hyperplastic cells protrude into and compromise the lumen, which barely accommodates a polymorphonuclear leukocyte (PMN). Pericytes (p), separated from endothelial cells by basal lamina, surround the capillaries and interpose themselves between adjacent loops. Bar = 10 mm.

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FIG. 7. Electron micrograph showing tight junctions with pentalaminar configuration that are present between adjacent capillary endothelial cells of glomeruloid vascular structures (arrowheads, inset). Bar = 0.1 mm.

and neoplastic astrocytes do not participate in the formation of glomeruloid vascular structures. The capillaries forming the glomeruloid vascular structure are made up of hypertrophic endothelial cells that protrude into and restrict the diameter of the vascular lumen. This is in contrast to the normal cerebral capillary wall, which is lined by thin endothelium with dense cytoplasm encompassing a large blood-filled lumen. Endothelial sprouts with slitlike lumina have been described as forming primitive capillaries in the developing brain of human embryos1 and in the rat cerebral cortex during the first 10 postnatal days.5 Although these primordial vessels consist of one to six endothelial cells in a given crosssection, the number of cells lining the capillary walls in glomeruloid vascular structures is often much greater. Unlike normal cerebral endothelium, cells lining capillary loops in glomeruloid vascular structures exhibit great variability in size and shape, which partly contributes to the formation of irregular, tortuous, narrow lumina. These are often not patent to blood flow and because of their size they cannot be visualized by light microscopy. Therefore, cells positive for FVIIIR:Ag and UEA-I, designated as “abluminal” on light microscopy in the present study or “adventitial” by others,25 are indeed endothelial cells lining winding slitlike lumina of closely associated vascular loops. Another factor contributing to the positive staining of abluminal cells for endothelial markers is the focal overlapping of endothelial cells in the wall of microvessels in glomeruloid vascular structures. Such overlapping has been reported infrequently in developing capillaries of the human fetal cerebrum.1 The results of this study demonstrate that ultrastructural characterization of the glomeruloid vascular structure is required for identification of endothelial cells that may otherwise be misinterpreted as abluminal cells (pericytes and/or smooth-muscle cells) because of the poor resolution of light microscopy immunohistochemistry. The cytoplasm of endothelial cells in glomeruloid vascular structures, unlike normal resting endothelium, contains an unusually large number of randomly arranged filaments. Although the exact role of intermediate filaments in cell function remains uncertain, actin microfilaments are considered important in adherence of cells to the subendothelial substratum, maintaining close contact between adjacent cells, and in cell migration.9 Interendothelial junctions with the ultrastructural appearance of tight junctions were routinely observed be1082

FIG. 8. Electron micrograph showing a cell containing large numbers of Weibel–Palade bodies. Weibel–Palade bodies are a prominent feature of most endothelial cells participating in the formation of glomeruloid vascular structures, although their density varies greatly even among endothelial cells of the same capillary. L = lumen. Bar = 0.5 mm.

tween adjacent endothelial cells in glomeruloid vascular structures. They were less numerous than in normal brain endothelium and often appeared to focally seal only part of the long intercellular contact between two apposed hypertrophic cells. In the absence of tracer studies, the functional impermeability of these junctions remains unknown. Weibel–Palade bodies were frequently observed in the hyperplastic endothelium in glomeruloid vascular structure. These FVIII:Ag-storing organelles are extremely rare or absent in normal human cerebral capillaries14,15 and in human brain microvessel endothelial cells in primary culture.7 Their presence in large numbers in endothelial cells lining microvessels in certain childhood brain tumors has been interpreted as indicating active capillary growth.20 The conspicuous presence of Weibel–Palade bodies in the endothelium of glomeruloid vascular structure is in agreement with the above findings and contrasts sharply with their absence in normal brain microvessels. Their numbers did not correspond with the size of individual cells and varied even among cells lining a single vascular loop, from none to 23 per cell. The mechanism(s) leading to increase of Weibel–Palade bodies in glomeruloid vascular structures and their functional significance remain to be elucidated. Each capillary loop in a given glomeruloid vascular structure was surrounded by a variably dense basal lamina enveloping cells with the morphological features of pericytes. These pericapillary cells appeared to keep pace with the newly formed capillary loops in that they were always observed as a single discontinuous layer around individual capillaries, separated adjacent microvessels, and were always outnumbered by the overlying endothelial cells. This relationship between endothelium and surrounding pericytes in glomeruloid vascular structures is of interest in view of the recent concepts regarding the effects of pericytes on endothelial cell growth. Thus, loss of pericytes from the retinal microvessels of diabetic J. Neurosurg. / Volume 85 / December, 1996

Glomeruloid vascular structures in glioblastoma patients appears to facilitate subsequent neovascularization.21,34 In addition, studies of pericyte–endothelial interactions in coculture systems have demonstrated that pericytes specifically inhibit the growth of endothelial cells in a reversible fashion.27 This growth inhibition is dependent on contact between the two cell types27 and is mediated by transforming growth factor-b (TGFb) secreted by pericytes.28 The lack of pericytic proliferation in glomeruloid vascular structures might therefore create a permissive environment for active endothelial proliferation and new vessel growth. Positive staining of cells comprising the hyperplastic vascular structures in glioblastoma for FVIIIR:Ag and to a lesser extent a-SMA has been reported previously.32 In the present study, smooth-muscle cells were not encountered among the cell populations of glomeruloid vascular structures. A similar lack of inclusion of cells other than endothelial cells and pericytes in the wall of the newly formed vessels has been reported in an in vivo experimental model of tumor angiogenesis in rats.2 Our findings in this respect differ from recent observations based on immunoperoxidase staining of glomeruloid vascular structures in glioblastoma38,39 and metastatic brain tumors39 in which cells positive for a-SMA and a marker for “activated” pericytes (HMW-MAA) were the predominant cell type. This discrepancy, however, may well reflect differing interpretations of the immunohistochemical staining results. Because cells with the typical ultrastructural appearance of smooth muscle were not observed in any of the glomeruloid vascular structures ultrastructurally, it is quite possible that cells previously interpreted as smooth muscle cells are indeed a-SMA– positive pericytes. This is not surprising, because pericytes contain both nonmuscle and smooth-muscle actin.11,33 Whereas endothelial cells do not normally express a-SMA, in vitro growth in the absence of endothelial cell growth supplement and heparin induces murine and porcine cerebral capillary endothelium to express a-SMA and to resemble smooth-muscle cells morphologically.35 Furthermore, recent studies have shown that TGFb1 induces differentiation of aortic endothelial cells into a smooth-muscle–like phenotype expressing both FVIIIR:Ag and a-SMA or a-SMA only.3 Because the presence of TGFb1 has been documented in human glioblastomas,4 in particular in the extracellular space of glomeruloid vascular structure,17 it is possible that expression of a-SMA by endothelial cells participating in glomeruloid vascular structures is induced via a similar mechanism. On the basis of these findings, therefore, expression of a-SMA by phenotypically altered endothelial cells in glomeruloid vascular structures cannot be excluded, and, along with the adjacent pericytes, could account for the variable presence of a-SMA–positive cells in glomeruloid vascular structures. The specific factors that mediate endothelial cell proliferation and neovascularization in GBM have not been fully elucidated. Production of angiogenic factors by human brain tumors, mainly astrocytomas and glioblastomas, has been strongly suggested by studies showing that supernatants from human glioma cell cultures promote in vitro proliferation of human umbilical vein endothelial cells.16,19,24 Several endothelial growth factors have been characterized, including platelet-derived growth facJ. Neurosurg. / Volume 85 / December, 1996

tor (PDGF),18 fibroblast growth factors (FGFs),8 and vascular endothelial growth factor (VEGF).22 The PDGF has been found in tumor cells in human malignant gliomas as well as in tumor endothelial cells that also express its receptor, thus suggesting an autocrine stimulation by PDGF via its receptor.12 Localization of FGF has also been reported in a variety of primary and metastatic brain tumors and in endothelial cells in these tumors.29 Recent studies indicate that VEGF may be an important tumor angiogenesis factor in high-grade human astrocytomas and experimental rat gliomas, because VEGF messenger RNA was found to be markedly upregulated in tumor cells but not in normal brain. This upregulation was associated with strong expression of VEGF receptor messenger RNA specifically in endothelial cells in these tumors.30,31 The relative significance of these and possibly other growth factors in the proliferation of endothelial cells in glomeruloid vascular structures remains to be investigated further. Acknowledgments The authors gratefully acknowledge the expert technical assistance with immunohistochemical analysis provided by B. Dupuis and S. Borget. References 1. Allsopp G, Gamble HJ: Light and electron microscopic observations on the development of the blood vascular system of the human brain. J Anat 128:461–477, 1979 2. Antonelli-Orlidge A, Saunders KB, Smith SR, et al: An activated form of transforming growth factor b is produced by cocultures of endothelial cells and pericytes. Proc Natl Acad Sci USA 86:4544–4548, 1989 3. Arciniegas E, Sutton AB, Allen TD, et al: Transforming growth factor beta 1 promotes the differentiation of endothelial cells into smooth muscle-like cells in vitro. J Cell Sci 103:521–529, 1992 4. Bodmer S, Strommer K, Frei K, et al: Immunosuppression and transforming growth factor-b in glioblastoma. Preferential production of transforming growth factor-b2. J Immunol 143: 3222–3229, 1989 5. Caley DW, Maxwell DS: Development of the blood vessels and extracellular spaces during postnatal maturation of rat cerebral cortex. J Comp Neurol 138:31–48, 1970 6. Coomber BL, Stewart PA, Hayakawa K, et al: Quantitative morphology of human glioblastoma multiforme microvessels: structural basis of blood-brain barrier defect. J Neurooncol 5: 299–307, 1987 7. Dorovini-Zis K, Huynh HK: Ultrastructural localization of factor VIII-related antigen in cultured human brain microvessel endothelial cells. J Histochem Cytochem 40:689–696, 1992 8. Gospodarowicz D, Ferrara N, Schweigerer L, et al: Structural characterization and biological functions of fibroblast growth factor. Endocrin Rev 8:95–114, 1987 9. Gotlieb AI, Wong MKK: Current concepts on the role of the endothelial cytoskeleton in endothelial integrity, repair, and dysfunction, in Ryan US (ed): Endothelial Cells. Boca Raton, FL: CRC Press, 1988, Vol 2, pp 81–101 10. Haddad SF, Moore SA, Schelper RL, et al: Vascular smooth muscle hyperplasia underlies the formation of glomeruloid vascular structures of glioblastoma multiforme. J Neuropathol Exp Neurol 51:488–492, 1992 11. Herman IM, D’Amore PA: Microvascular pericytes contain muscle and nonmuscle actin. J Cell Biol 101:43–52, 1985 12. Hermansson M, Nistér M, Betsholtz C, et al: Endothelial cell hyperplasia in human glioblastoma: coexpression of mRNA for

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Manuscript received September 29, 1995. Accepted in final form June 26, 1996. This study was supported by grants from the Medical Research Council of Canada (MT–12209) and the British Columbia Health Care Research Foundation. Address reprint requests to: Katerina Dorovini-Zis, M.D., Department of Pathology, Section of Neuropathology, Vancouver Hospital and Health Sciences Center, 855 West 12th Avenue, Vancouver, British Columbia, V5Z 1M9, Canada.
