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Regular Articles |
From the Department of Neuropathology,*
Freiburg
University Medical School, Freiburg, Germany, and the Department of
Molecular Cell Biology,
Max-Planck-Institute
for Physiological and Clinical Research, Bad Nauheim, Germany
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several studies3 suggest that solid tumor growth to a clinically relevant size depends on adequate blood supply. Solid tumors recruit blood vessels from the neighboring tissue by angiogenesis, eg, the sprouting of capillaries from pre-existing vessels that migrate into the tumor and form a new vascular network. To stimulate angiogenesis, tumors secrete growth factors that act on endothelial cells. It is thought that the resulting neovasculature supports tumor expansion and metastasis.
According to gene targeting studies in mice, vascular endothelial growth factor (VEGF) and its tyrosine kinase receptors VEGF-R1 (flt-1) and VEGF-R2 (flk-1/KDR) are major regulators of angiogenesis and vasculogenesis in the developing embryo.4 Mice deficient for VEGF die at embryonic day 8.5–9.0 by impairment of both vasculogenesis and angiogenesis.5,6 Disruption of the VEGF-R2 gene interferes with endothelial cell differentiation and causes embryonic death on embryonic day 8.5.7 Disruption of the VEGF-R1 gene permits differentiation of endothelial cells but results in phenotypically abnormal, disorganized blood vessels leading to death of embryos at embryonic day 9.0.8 In addition to embryonic angiogenesis, VEGF and its receptors are also key regulators of tumor angiogenesis. VEGF mRNA is highly up-regulated in hypoxic tumor cells in human and rat glioblastomas, while VEGF receptors are up-regulated in tumor endothelial cells.9-13 A role for the VEGF/VEGF receptor system in tumor angiogenesis has been proven by several experimental approaches using anti-VEGF antibodies,14,15 VEGF-antisense cDNA,16,17 dominant-negative VEGF-R2 mutants,18-20 anti-VEGF-R2 antibodies,21 and soluble VEGF-R2.22 Using different animal models, all approaches resulted in significant inhibition of tumor angiogenesis.
Tie1 and Tie2 (tek) are members of the only other known endothelial cell-specific receptor tyrosine kinase family.23-25 Like VEGF and its receptors, Tie1 and Tie2 are essential for the formation of embryonic vasculature. Transgenic mice deficient for Tie1 or Tie2 showed vascular defects such as edema and localized hemorrhage (Tie1 knockout),26,27 dilated blood vessels, absence of capillary sprouts, and an abnormal vascular network (Tie2 knockout),27,28 resulting in embryonic or early postnatal death. In contrast to VEGF signaling, which is essential for endothelial cell differentiation, Tie1 and Tie2 seem to be important for vascular remodeling and sprouting.28 In contrast to the established role of Tie1 and Tie2 in embryonic angiogenesis and vessel maturation, their role in blood vessel growth associated with tumor development remains unclear. It has been shown that Tie1 is up-regulated in the vascular endothelium of metastatic melanomas,29 brain tumors,30 and mammary carcinomas.31 The potential importance of Tie2 in pathological vascular growth was suggested by inhibition studies using soluble extracellular domains of Tie2.32
A ligand for the Tie1 receptor has not yet been found but the newly discovered ligand for the Tie2 receptor, Angiopoietin-1 (Ang-1), has recently been shown to be essential for normal vascular development in the mouse.33,34 Its naturally occurring antagonist Angiopoietin-2 (Ang-2) binds with similar affinity to Tie2 but, unlike Ang-1, which induced receptor phosphorylation on ligand binding, Ang-2 blocked Tie2 activity.35 The absence of Ang-1 caused severe vascular abnormalities in the developing mouse embryo including a less complex vascular network with fewer branches and dilated vessels.34 Transgenic overexpression of Ang-2 revealed malformation of the vascular network and widespread vessel discontinuities in the embryo, defects similar to those seen in mice lacking Ang-1 or Tie2.35 Based on these studies, it has been proposed that the angiopoietin/Tie2 receptor system plays a role in the interaction of endothelial cells with smooth muscle cells/pericytes (SMC/PC).36 Pericytes have been shown to inhibit endothelial cell proliferation in vitro and are thought to be an important regulator of blood vessel growth in vivo.37
To define a putative role for the Tie2/angiopoietin system in glioblastoma angiogenesis, we examined expression and cellular distribution of Ang-1 and Ang-2 mRNA by Northern blot analysis and in situ hybridization, and of Tie2 by immunohistochemistry. We observed cell type-specific up-regulation of Tie2 and its ligands Ang-1 and Ang-2 during tumor progression in a pattern compatible with a role in tumor-induced angiogenesis.
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Materials and Methods |
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Fourteen glioma specimens (one pilocytic astrocytoma WHO grade I, four astrocytomas WHO grade II, two anaplastic astrocytomas WHO grade III, and seven glioblastomas WHO grade IV) were freshly received from the neurosurgical theater, directly frozen in liquid nitrogen, and stored at -80°C before use. Normal brain and cerebellar tissue from two patients without neurological disease were also snap-frozen. The frozen tissue was cryosectioned for immunohistochemistry and in situ hybridization. Part of the material was used for extraction of total RNA.
For in situ hybridization frozen sections (10 µm thick) from TissueTek (Miles, Elkhart, IN) embedded tumor tissue were melted on silan-covered (TESPA, 3-aminopropyl-triethoxy-silane) glass slides. Two serial sections were put on each slide and used for antisense and sense (control) hybridization. Sections were fixed in 4% paraformaldehyde and dehydrated in a series of 30%, 60%, 80%, 95%, and 100% ethanol. After air-drying slides were stored at -80°C until used for in situ hybridization.
For immunohistochemistry cryostat sections (10 µm) were air-dried and acetone-fixed. Sections were stored at -20°C before use.
Immunohistochemistry
Slides were washed in phosphate-buffered saline (PBS) and
incubated for 30 minutes in 5% bovine serum albumin, Fraction V
(Sigma, Deisenhofen, Germany) in PBS. Sections were then
incubated with primary antibody for 1 hour at room temperature. Primary
antibodies used were monoclonal antibodies against human CD31 (dilution
1:40, Dako, Geostrup, Demnark), against 
smooth muscle actin
(dilution 1:300, Sigma), and against human Tie238
(10 µg/ml, gift of Dr. Kevin Peters, Durham, NC), and polyclonal
antibodies against human von Willebrand factor (1:1000, Sigma). To
demonstrate specificity of Tie2 immunohistochemistry, 10µg/ml of
antibody were preincubated with 230 µg/ml recombinant extracellular
tie2-Fc35
(gift of Dr. G. D. Yancopoulos,
Tarrytown, NY) for 15 minutes at room temperature. After washing with
PBS, 0.1% Triton-X-100 and blocking with 20% normal goat serum
(Dianova, Hamburg, Germany) in PBS for 30 minutes, slides were
incubated with secondary antibody for 1 hour at room temperature
(biotinylated goat anti-mouse IgG, 1:300 or biotinylated goat
anti-rabbit IgG, 1:300). For blocking of endogenous peroxidase activity
sections were incubated in 0.3%
H2O2 in methanol for 30
minutes at 4°C. Finally, sections were incubated in a
streptavidin-peroxidase-complex (Vectastain, Vector, Burlingame,
CA) according to the manufacturer's protocol. Color detection
of immunoreactivity was achieved using AEC (3-amino-9-ethyl-carbazole,
Sigma, 0.26 mg/ml in acetate buffer, pH 5.2) and 0.03%
H2O2 as a substrate for
peroxidase. Color reaction time was 10–30 minutes at room temperature.
Isolation of Total RNA and Northern Blotting
Total cytoplasmic RNA was isolated from 100 mg of tissue by the guanidinium-isothiocyanate method.39 Aliquots of 10–12 µg RNA were electrophoresed on a 1.4% agarose gel containing 5.5% formaldehyde in 1x MOPS buffer (20 mmol/L 3-(n-morpholino) propane sulfonic acid, pH 7.0, 8 mmol/L sodium acetate, 1 mmol/L EDTA) and transferred to a nylon membrane (Duralon, Stratagene, Heidelberg, Germany) in 20x standard saline citrate (SSC) buffer. RNA was UV-crosslinked to the membrane (120 mJ/cm2) and filters were hybridized with [32P]dCTP-labeled cDNA probe (Prime-It II random primer labeling kit, Stratagene). Human Ang-cDNA templates described in the next paragraph were used for random priming. Hybridization in QuikHyb hybridization solution (Stratagene) plus 200 mg/ml salmon sperm DNA was for 1 hour at 68°C. Two stringency washes in 2x SSC, 0.1% sodium dodecyl sulfate (SDS) at 60°C for 10 minutes were followed by a final wash in 0.1x SSC, 0.1% SDS at 60°C for 5 minutes. Blots were exposed to Kodak X-OMAT-XAR-2 films with an intensifying screen at -70°C for 2–10 days.
RNA Probe Generation by in Vitro Transcription
A 570-bp SpeI-EcoRI fragment from human Ang-1 cDNA subcloned into XbaI/EcoRI site of pBluescript KS+ containing 70 bp of 5'-untranslated region and 500 bp of coding region ending at amino acid 166 was used to generate nonradioactive antisense and sense RNA probes by in vitro transcription. After linearization of the plasmid, single-stranded runoff transcripts were synthesized from the T7/T3 polymerase promoters using Digoxigenin-labeled uridine triphosphate as a substrate according to the manufacturer's instructions (Digoxigenin RNA labeling kit, Boehringer, Mannheim, Germany). RNA probes for human Ang-2 were transcribed from a 640-bp EcoRI-HindIII fragment from human Ang-2 cDNA containing 360 bp of 5'-untranslated region and 280 bp of coding region ending at amino acid 99 subcloned into EcoRI/HindIII site of pBluescript KS+. After determination of concentration and labeling efficiency, Digoxigenin labeled probes were stored at -20°C or used at once for in situ hybridization analysis. Sense probes served as a control.
Nonradioactive in Situ Hybridization
Prehybridization treatment consisted of 0.2 mol/L HCl for 5 minutes followed by digestion with Proteinase K (10 µg/ml) for 10 minutes at room temperature. After postfixation with 4% paraformaldehyde sections were acetylated with 0.1 mol/L triethanolamine and 0.25% acetic anhydride for 10 minutes at room temperature. Prehybridization solution containing 4x SSC, 50% deionized formamide, 2% SDS, 5x Denhardt's solution, 10% dextran sulfate and 0.5 mg/ml of yeast tRNA was applied for 1.5 hours at 48°C. RNA sense/antisense probe concentration for hybridization was 0.1–0.5 ng/µl of initial transcript. Tissue sections were incubated in a humidified chamber under glass coverslips at 48°C overnight. Posthybridization stringency washes at 48°C included 2x SSC for 30 minutes, 2x SSC plus 0.1% SDS for 5 minutes, 0.1x SSC plus 0.1% SDS for 15 minutes. Each wash was carried out twice. After RNase A treatment (2.5 µg/ml in 2x SSC) for 5 minutes at 37°C, hybridized probes were detected by anti-DIG antibody conjugated to alkaline phosphatase (Boehringer) diluted 1:500 for 1 hour at room temperature. Nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate solution was used as color substrate in 0.1 mol/L Tris/Cl, 0.1 mol/L NaCl, pH 9.5. Color reaction times ranged from 14 to 20 hours, after which slides were rinsed in TE (10 mmol/L Tris/Cl, 1 mmol/L EDTA, pH 8.0) and mounted in glycerol.
In Situ Hybridization/Immunohistochemistry Double Labeling Analysis
To identify Ang-2 mRNA expressing cells, a double labeling technique combining in situ hybridization and immunohistochemistry was applied. Slides were subjected to the in situ hybridization procedure described above and then directly transferred into PBS. Subsequent immunohistochemistry with anti-van Willebrand factor and anti-smooth muscle actin antibodies was performed following the above protocol but omitting the hydrogen peroxide/methanol blocking step.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. In all tissue specimens, Tie2
expression was confined to endothelial cells and neither normal
neuroectodermal cells nor glioma cells expressed Tie2. Smooth muscle
actin was strongly expressed in periendothelial cells (probably smooth
muscle cells and pericytes) but not in endothelial cells or tumor
cells. Small capillaries were associated with single SMA-positive
cells, but larger tumor vessels showed an abundance of SMA-positive
cells when compared to vessels of similar size in normal
brain.
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. Whereas in normal brain and low-grade
gliomas no or faint signals were observed, Ang-1 mRNA expression was
clearly detectable in high-grade gliomas. Like Ang-1, Ang-2 mRNA was
not observed in normal brain but its expression was seen in
glioblastoma specimens, albeit at a lower level.
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In glioblastoma, Ang-1 was expressed in almost all tumor cells. Areas
of more intense staining alternated with areas of less intense or very
weak staining. No focal up-regulation of angiopoietin-1, which has been
found to be characteristic for hypoxia-induced VEGF expression in
glioblastomas, was observed (Figure 3c)
. Ang-2 expression in normal
brain was observed in neurons such as cerebellar Purkinje's cells. No
expression was seen in vascular cells (Figure 3e)
. In glioblastoma,
Ang-2 transcripts were not observed in tumor cells but found associated
with tumor vessels (Figure 3g)
. However, Ang-2 transcripts were
restricted to a subset of blood vessels in a given section. Smaller
vessels and capillaries were stained for Ang-2 mRNA (Figure 3i)
,
whereas larger vessels showed only weak staining or were completely
negative (Figure 3j)
.
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. We
therefore conclude that Ang-2 is expressed in endothelial cells of
glioblastoma vessels. vWF stained endothelial cells of both smaller
capillaries and large vessels. Colocalization of Ang-2 and vWF protein
was observed only in endothelial cells of smaller vessels, whereas
endothelial cells of large vessels only expressed vWF (Figure 4
c).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the potential role of Ang-1 and Ang-2 in glioma vascularization, we analyzed their expression in normal brain and in low- and high-grade gliomas. We observed cell type-specific up-regulation of the mRNA for both angiopoietins during glioma progression. Ang-1 was expressed only weakly in low-grade gliomas, but showed strong expression in high-grade gliomas. In glioblastomas, Ang-1 mRNA was expressed in almost all tumor cells. No focal up-regulation in certain tumor areas, typically observed for VEGF mRNA in perinecrotic palisading cells, was found for Ang-1 in the tumor sections analyzed. This finding is consistent with the observation that unlike VEGF mRNA, Ang-1 mRNA is not up-regulated by hypoxia.40 Ang-2 was expressed in vascular cells of high-grade gliomas, but not in vessels of low-grade gliomas and normal brain. Although vascular expression of Ang-2 has been described previously, the exact cell type has not been defined. From previous experiments in E12.5 mice,34 it is known that Ang-2 transcripts are associated with vascular structures such as the smooth muscle cell layer of the dorsal aorta and major aortic branches. In fetal liver Ang-2 mRNA was located at or close to the lumen of hepatic vessels in so-called endothelial-like cells. Ang-2 mRNA, however, was observed in some but not all hepatic vessels. Because it was difficult to determine whether in our glioblastoma sections Ang-2 mRNA was expressed in endothelial or periendothelial cells, we applied a double-labeling technique consisting of nonradioactive in situ hybridization to detect Ang-2 mRNA combined with immunohistochemical detection of endothelial (vWF) and smooth muscle/pericyte proteins (SMA) on the same tissue section. With this technique tumor endothelial cells were unequivocally identified as Ang-2 mRNA-expressing cells. Interestingly, the overlap of Ang-2 mRNA and vWF protein was confined to smaller vessels, whereas in larger vessels no Ang-2 expression could be observed in endothelial cells.
We suspect that endothelial Ang-2 expression characterizes angiogenic glioblastoma blood vessels. Because Ang-1 is thought to be important for stabilizing the vessel wall, local Ang-2 expression might promote smooth muscle cell/pericyte dropoff, which is thought to be a requirement for rendering and maintaining endothelial cells accessible to angiogenic inducers. In glioblastomas, this angiogenic signal most likely is VEGF, which has been shown to be necessary for glioma angiogenesis.14,16-18 Absence of Ang-2 in larger glioblastoma vessels was negatively correlated with the number of SMC/PC because endothelial cells in larger vessels were associated with many more SMA-expressing cells than those in smaller vessels. It has recently been proposed that the Angiopoietin/Tie2 system might regulate the interaction of endothelial cells with surrounding mesenchymal cells.41 This model is based on ultrastructural analysis in Ang-1 (-/-) mouse embryos that revealed a defect in smooth muscle cell and pericyte precursor cell recruitment, resulting in an abnormal vasculature.34 Because a similar phenotype was observed in Ang-2-overexpressing mice,35 it was suggested that a physiological role of Ang-1 could be to recruit periendothelial cells and support the physical association of endothelial cells with these cells. According to this model, inhibition of Ang-1 by Ang-2 drives angiogenesis in the presence of angiogenesis inducers such as VEGF by loosening contacts between endothelial and periendothelial cells.41 Although this hypothesis remains speculative, our findings in human gliomas favor this model.
Glioblastomas are highly angiogenic tumors that overexpress VEGF and platelet-derived growth factor-B (PDGF-B) chain.9,10,12,42 Whereas VEGF is a specific mitogen for vascular endothelial cells, PDGF-B stimulates the proliferation of mesenchymal cells, including smooth muscle cells and pericytes.43 Glioblastoma blood vessels typically consist of endothelial cells covered by multiple layers of SMC/PC.44,45 Whereas endothelial cells express PDGF-B and VEGF-R1 and-R2, SMC/PC express PDGF-Rß.1,11,42,46 It is therefore likely that in glioblastomas endothelial cell proliferation is driven by tumor-derived VEGF, whereas SMC/PC proliferation is driven by endothelium-derived (and probably also tumor-derived) PDGF-B. Our observation that endothelial Ang-2 expression in glioblastomas is limited to small vessels with few SMC/PC, whereas larger vessels with many SMC/PC did not express Ang-2, supports the hypothesis that Ang-2 promotes angiogenesis in situ. Down-regulation of Ang-2 in quiescent endothelial cells may permit physical interaction of endothelial cells with SMC/PC, leading to inhibition of endothelial cell proliferation and maturation of the vascular wall. This hypothesis is supported by angiopoietin expression observed in normal brain because in quiescent brain vessels Ang-1 but not Ang-2 was expressed. Thus, whereas in normal blood vessel Ang-1 expression exceeds Ang-2 expression, the opposite is true in tumor blood vessels. Ang-2 up-regulation in endothelial cells is therefore associated with tumor angiogenesis and may even be a prerequisite for the induction of tumor angiogenesis. In addition to its putative function in vascular remodeling, Ang-1 has been found to induce vascular sprouting in vitro.47 Thus, it also appears possible that Ang-1 acts synergistically with Ang-2 to promote glioblastoma angiogenesis by inducing vascular sprouting.
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Acknowledgements |
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Footnotes |
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Supported by grant number W44/94/Ri 2 from the Deutsche Krebshilfe.
Accepted for publication August 31, 1998.
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 U. Fiedle |
