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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3388-3393
Coexpression of Erythropoietin and Vascular Endothelial Growth
Factor in Nervous System Tumors Associated With von Hippel-Lindau
Tumor Suppressor Gene Loss of Function
By
Marion Krieg,
Hugo H. Marti, and
Karl
H. Plate
From the Department of Neuropathology, Freiburg
University Medical School, Freiburg; and the Department
of Molecular Cell Biology, Max-Planck Institute for
Physiological and Clinical Research, Bad Nauheim,
Germany.
 |
ABSTRACT |
Hemangioblastomas are highly vascular tumors of the central nervous
system that overexpress the hypoxia-inducible gene, vascular endothelial growth factor (VEGF), as a consequence of mutational inactivation of the von Hippel-Lindau tumor suppressor gene (VHL). Previous reports showed that hemangioblastomas can also express erythropoietin (Epo), which is also hypoxia-inducible. However, Epo
expression in hemangioblastomas was observed only in individual cases,
and the analyses were mainly based on indirect determination of
erythropoiesis-stimulating activity. Therefore, we analyzed a series of
11 hemangioblastomas for Epo, VEGF, and VHL expression by Northern blot
analysis and compared the results with normal brain and glioblastomas.
Surprisingly, we observed Epo mRNA expression in all hemangioblastoma
specimens analyzed, but in none of four glioblastomas. In contrast,
VEGF mRNA was expressed in all hemangioblastomas and all glioblastomas.
In situ hybridization revealed neoplastic stromal cells as Epo- and
VEGF-producing cells in hemangioblastomas. These results suggest that
in the nonhypoxic microenvironment of hemangioblastoma, Epo, similar to
VEGF, might be negatively regulated by the VHL gene product.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
ERYTHROPOIETIN (Epo) is a glycoprotein
hormone that acts as major regulator of erythropoiesis. The site of Epo
production switches during development from fetal liver to adult
kidney, with low-level expression remaining in adult
liver.1 It is well established that Epo gene expression is
controlled by oxygen tension. Hypoxic and anemic conditions result in
elevated Epo expression in mammals.2 Until recently,
erythroid precursor cells have been thought to be the exclusive target
site for Epo. However, recent reports suggest that Epo may also be
involved in neuronal functions. Epo gene expression was detected in
human, monkey, and murine brain by quantitative reverse
transcriptase-polymerase chain reaction,3 and Epo protein
was found in cerebrospinal fluid.4 Epo binding sites were
localized in defined areas of the mouse brain.5
Furthermore, in the brain of hypoxic rodents and primates, increased
Epo mRNA levels were observed.3,6
Epo production has also been reported in a variety of human tumors,
particularly renal cell carcinomas and cerebellar hemangioblastomas. Cerebellar hemangioblastomas are highly vascularized cystic tumors typically consisting of blood-filled capillaries separated by intervascular stromal cells. The histologic origin of stromal cells in
hemangioblastomas is not defined, but it has been shown that stromal
cells are the neoplastic cell type in hemangioblastomas.7,8 Current evidence suggests that hemangioblastoma formation is dependent on mutational inactivation of the von Hippel-Lindau tumor suppressor gene (VHL) because (1) hemangioblastomas are the most frequent manifestations of germline VHL mutations (eg, hereditary von
Hippel-Lindau disease9) and (2) sporadic nonhereditary
hemangioblastomas carry somatic mutations in VHL.10 More
recently, VHL has been shown to control tumor angiogenesis by negative
regulation of vascular endothelial growth factor (VEGF)
expression.11-13
VEGF is upregulated as much as 50-fold in certain types of brain
tumors, and exceptionally high levels have been found in the highly
vascularized hemangioblastomas and in glioblastomas.14,15 In hemangioblastomas, VEGF is expressed in almost all stromal cells,14,16 whereas in glioblastomas, VEGF expression is
restricted to tumor cells surrounding necrotic areas.17,18
Immunohistochemical studies and in situ hybridization demonstrated that
hypoxia is a microenvironmental stimulus for upregulation of VEGF in
glioblastomas.17,19 However, in hemangioblastomas, it is
unlikely that hypoxia is the major trigger of VEGF expression, since
these tumors are highly vascularized and show no signs of necrosis.
Interestingly, hypoxia-induced regulation of VEGF and Epo synthesis is
based on similar regulatory mechanisms involving transcriptional
activation and increased mRNA stability.20-22 Besides VEGF,
other hypoxia-inducible genes seem to be negatively regulated by the
VHL gene product11 and can thus be activated by a
hypoxia-independent mechanism. Although Epo has been suggested to be a
possible target gene for VHL regulation, extensive studies of Epo
expression in hemangioblastomas are missing. Therefore, we investigated
a series of capillary hemangioblastomas by Northern blot analysis for
expression of Epo. In comparison to normal cerebrum and normal
cerebellum, all analyzed hemangioblastomas showed upregulation of Epo
mRNA levels. In situ hybridization revealed coexpression of Epo with
VEGF in stromal cells, suggesting that Epo expression may be under
control of the VHL protein.
| |
MATERIALS AND METHODS |
Clinical data.
All clinical data were obtained from patient records at the
Neurocenter, Freiburg University Medical School, Freiburg, Germany.
Specimens.
All tumor specimens (11 hemangioblastomas and four glioblastomas) were
obtained from the brain tumor bank of the Department of Neuropathology,
Freiburg University Medical Center, at random. Tumor specimens were
immediately snap-frozen after removal and stored at  70°C. As a
control, normal cerebellum and normal cerebrum from two patients
without neurologic disease were collected postmortem and snap-frozen in
liquid nitrogen.
RNA extraction and Northern analysis.
Total RNA was isolated using the guanidinium thiocyanate
method.23 Aliquots of 20 µg total RNA were separated in
1% agarose gel containing 0.66 mol/L formaldehyde in 1× MOPS buffer
(40 mmol/L 3-(N-morpholino)propanesulfonic acid, 10 mmol/L
sodium acetate, and 1 mmol/L EDTA, pH 7.2) and transferred onto a nylon
membrane (Duralon, Stratagene, La Jolla, CA) by capillary
blotting in 20× SSC (1× SSC is 150 mmol/L NaCl plus 15 mmol/L
sodium citrate). For hybridizations, a human Epo cDNA (pe49f; kindly
provided by Dr C. Shoemaker, Boston, MA), a human VEGF cDNA (kindly
provided by Dr H. Weich, Braunschweig, Germany), and a human VHL cDNA
(kindly provided by Dr B. Zbar, Frederick, MD) were labeled with
32P-dCTP using the random priming labeling kit
(Stratagene).  -Actin cDNA was used for control hybridizations as
described previously.7
In situ hybridization.
The technique used for in situ hybridization was essentially as
described by Breier et al.24 RNA probes were generated by in vitro transcription of the plasmid pBShEPO3, a partial human Epo
cDNA clone. This plasmid was obtained by subcloning a 433-bp Kpn I-Stu I fragment spanning exon 2 to exon 5 of the
human Epo cDNA clone pe49f in BluescriptSK+. Single-strand antisense or sense RNA probes were generated using 100 µCi 35S-UTP and
T3 or T7 RNA polymerases as described by the manufacturer (Stratagene).
Snap-frozen hemangioblastomas embedded in tissue tec
(Sakura, Torrance, CA) were sectioned in a cryostat (Zeiss, Oberkochen,
Germany). Ten-micrometer sections, two on each slide, were
melted on 3-Aminopropyl-trimethoxysilan-coated (Fluka, Bucks, Switzerland) glass slides. Sections were incubated in 2×
SSC at 70°C, digested with pronase (40 µg/mL), fixed in 4%
paraformaldehyde, and acetylated with acetic anhydride diluted 1:400
with 0.1 mol/L triethanolamine. Hybridization was performed in buffer
containing 50% formamide, 10% dextran sulfate, 10 mmol/L Tris
hydrochloride (pH 7.5), 10 mmol/L sodium phosphate (pH 6.8), 2× SSC,
5 mmol/L EDTA, 150 µg/mL yeast tRNA, 0.1 mmol/L UTP, 1 mmol/L
ADPS, 1 mmol/L ATP S, 10 mmol/L dithiothreitol, 10 mmol/L
2-mercaptoethanol, and 2.5 to 5 × 104 cpm/mL
35S-labeled RNA probe overnight at 48°C. The slides were
washed in 2× SSC/50% formamide at 37°C for 4 hours, digested with
RNase (20 µg/mL) for 15 minutes, washed again with 2× SSC/50%
formamide overnight, and dehydrated in graded ethanol. They were then
coated with Kodak NTB-2 emulsion (Eastman Kodak, Rochester, NY) diluted 1:1 in water and exposed for 3 to 4 weeks. The slides were developed and counterstained with toluidine blue, air-dried, and mounted.
For identification of mast cells in hemangioblastomas, paraffin
sections were stained with toluidine blue according to standard protocols.
| |
RESULTS |
Clinical Data
We analyzed seven hemangioblastomas from six patients with clinically
confirmed VHL disease and four hemangioblastomas from four patients
without clinical evidence of VHL disease. Four glioblastomas from four
patients without evidence of VHL disease were used for comparison.
Preoperative hemoglobin and hematocrit levels were available in 9 of 10 hemangioblastoma patients and in all glioblastoma patients (Table
1). Hemoglobin and hematocrit levels in the
patients were within the normal range, with the exception of one
hemangioblastoma patient who presented with a slightly decreased
hemoglobin level. However, in general, hemoglobin and hematocrit levels
were higher in hemangioblastoma versus glioblastoma patients
(hemoglobin, 14.7 ± 1.5 v 13.0 ± 1.1 g/L; hematocrit,
43.7% ± 3.6% v 39.2% ± 2.5%, respectively).
Northern Analysis
Expression of VEGF mRNA.
We investigated some of the tumor and control specimens for expression
of the hypoxia-inducible angiogenesis factor VEGF (Fig 1). We observed a strong upregulation of
VEGF expression in all hemangioblastomas (lanes 12 to 17) and
glioblastomas (lanes 18 and 19) analyzed, consistent with our previous
findings and reports from other groups. Under the hybridization
conditions we used, no VEGF mRNA was detectable in normal cerebellum
and normal cerebrum (lanes 10 and 11).
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| Fig 1.
Northern blot analysis of total RNA from different human
brain tissues: lanes 1 and 10, normal cerebrum; 2 and 11, normal
cerebellum; 3-7 and 12-17, eleven different capillary
hemangioblastomas; 8, 9, 18, and 19, four different glioblastomas. The
membranes were hybridized with 32P-labeled human Epo cDNA
and partially reprobed with human VEGF and VHL probes. Hybridization
with -actin served as a loading control (ND, not determined).
|
|
Expression of Epo mRNA.
We analyzed 11 surgically removed hemangioblastomas, four surgically
removed glioblastomas, and four normal brain specimens obtained
postmortem for expression of Epo mRNA by Northern blot analysis using a
human Epo cDNA as the hybridization probe (Fig 1). Epo mRNA (size, 1.6 kb) could be detected in all hemangioblastomas tested, albeit at
different levels. High amounts of Epo mRNA were present in six of 11 hemangioblastomas (lanes 3, 7, 12, 13, 16, and 17), four samples
exhibited intermediate levels (lanes 4, 6, 14, and 15), and only one of
11 hemangioblastomas showed a weak signal (lane 5). In contrast, Epo
mRNA expression was not detectable either in normal brain tissues
(cerebellum, lanes 2 and 11; cerebrum, lanes 1 and 10) or in the four
different glioblastomas tested (lanes 8, 9, 18, and 19).
Expression of VHL mRNA.
In hemangioblastomas, VHL mRNA was detected in three of six specimens
(Fig 1, lanes 12, 13, and 15). The lack of VHL expression in three
hemangioblastomas might reflect VHL inactivation by transcriptional silencing due to gene hypermethylation, which has previously been described to occur in a proportion of VHL loss of function-associated tumors, including hemangioblastomas.25 In hemangioblastomas that expressed VHL mRNA, the protein is most likely
mutated.10 In all normal brain specimens, VHL mRNA was
expressed at similar levels. Glioblastomas also expressed VHL mRNA. We
assume that both normal brain and glioblastomas express wild-type VHL,
since glioblastomas are not associated with VHL disease and somatic mutations, to our knowledge, have not been described. Blots were rehybridized with a -actin probe as the loading control.
In Situ Hybridization
To determine the nature of Epo-producing cells in hemangioblastomas, we
performed in situ hybridization and compared the results with our
previous findings on VEGF mRNA in situ hybridization.7,16 A
strong hybridization signal was observed in stromal cells of hemangioblastomas (Fig 2B and E), whereas
endothelial cells remained negative. Compared with VEGF mRNA, which we
found to be highly expressed in almost all stromal cells, Epo mRNA
showed a weaker expression level in stromal cells. In addition, Epo
mRNA was not expressed in all stromal cells. However, by morphologic
analysis, we were unable to observe differences between stromal cells
that expressed Epo and those that did not. Since, by
immunohistochemistry, expression of Epo in hemangioblastomas has been
described in stromal cells and mast cells,26 we stained
tissue sections with toluidine blue. Within a given tumor, the number
of Epo mRNA-expressing cells significantly exceeded the number of mast
cells, suggesting that Epo mRNA-expressing cells are stromal cells.
Control hybridization using a sense Epo probe did not show significant
staining (Fig 2C and F). In glioblastoma cells, no Epo mRNA expression
was observed (not shown). In normal cerebellum specimens, a weak Epo
mRNA signal was observed in Purkinje cells (not shown).
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| Fig 2.
In situ hybridization for Epo mRNA in a human
hemangioblastoma using 35S-labeled antisense and sense cRNA
probes. Sections were counterstained with toluidine blue. Epo mRNA was
detected in intervascular stromal cells. A-C, 100× original
magnification; D-F, 400× original magnification; A and D, bright
field illumination; B and E, dark field illumination; C and F, sense
(control) hybridization and dark field illumination.
|
|
| |
DISCUSSION |
Previous reports on Epo expression in hemangioblastomas of the nervous
system were mainly based on the measurement of
erythropoiesis-stimulating activity in the fluid of cysts and tumor
extracts.27,28 Although hemangioblastomas are known for the
capability to induce erythrocytosis, data on mRNA and protein
expression in tumor tissues are sparse. Therefore, we examined 11 surgically removed and snap-frozen hemangioblastomas for expression of
Epo mRNA by Northern blot analysis. Surprisingly, Epo mRNA could be
detected in all hemangioblastomas tested, whereas Epo mRNA expression
was not present either in normal brain or in four different
glioblastomas. These findings suggest that expression of Epo is
intrinsic to all hemangioblastomas and might be of importance for
hemangioblastoma development and progression. In contrast to
hemangioblastomas, glioblastoma development and progression appear to
be independent from Epo production, since Epo mRNA could not be
detected. This observation is consistent with the finding that
glioblastomas are not known to induce erythrocytosis. To investigate
the mechanism responsible for selective Epo upregulation in
hemangioblastomas, we analyzed a portion of the tissue specimens for
expression of the hypoxia-inducible angiogenesis factor VEGF. By
Northern analysis, we observed a strong upregulation of VEGF expression
in all hemangioblastomas and glioblastomas analyzed, whereas in normal
cerebellum and normal cerebrum, no VEGF mRNA was detectable. These
findings are compatible with different regulatory mechanisms of VEGF
and Epo expression in hemangioblastomas and glioblastomas. Current
evidence suggests that VEGF expression in glioblastomas is driven by
hypoxia, leading to activation of VEGF gene transcription and an
increase in mRNA stability.19,22 Epo is also
hypoxia-inducible, but was not expressed in glioblastomas. Failure of
glioblastoma cells to produce Epo might reflect a highly cell-specific
expression pattern of Epo, inhibiting Epo expression in glioblastoma
cells even under hypoxic conditions, although in vitro normal
astrocytes have been shown to be a source of Epo.3,29 In
contrast to glioblastomas, hemangioblastomas are nonhypoxic tumors but
also express high levels of VEGF. The underlying mechanism of high VEGF
expression in hemangioblastomas has recently been identified, since
VEGF gene transcription and mRNA stability in VHL-deficient tumor cells
are increased in a hypoxia-independent manner.11-13 We
therefore examined VHL expression in all tissue specimens. We observed
similar amounts of VHL mRNA in normal brain and all glioblastomas by
Northern blot analysis. However, only three of six hemangioblastomas
expressed VHL mRNA. In addition, hemangioblastomas consistently contain
mutations in the VHL gene, whereas glioblastomas express wild-type VHL.
Given the common regulatory pathway of Epo and VEGF expression by
hypoxia21 and the finding that VHL inactivation leads to
upregulation of hypoxia-inducible genes, it is tempting to speculate
that VHL loss of function in hemangioblastomas leads to upregulation of
Epo. Indeed, we observed coexpression of VEGF and Epo in
hemangioblastoma stromal cells. Moreover, previous reports identified
stromal cells as VEGF-producing cells in
hemangioblastomas,14,16 which have also been shown to
selectively carry VHL mutations.8 These findings suggest that both VEGF and Epo are upregulated in hemangioblastoma stromal cells due to VHL loss of function. However, coexpression of VEGF mRNA
and Epo mRNA was not observed in all stromal cells, since VEGF-expressing cells outnumbered Epo-expressing cells. One possible explanation is that in hemangioblastomas, there are subsets of stromal
cells with different capacities to express Epo.
The precise role of Epo expression in hemangioblastomas remains
speculative. Tumor-induced upregulation of Epo mRNA can result in
secondary erythrocytosis, which was described in some tumors of the
central nervous system, for example, hemangioblastoma30 and
meningioma.31 In capillary hemangioblastomas, secondary erythrocytosis has been reported in up to 20% of
patients.32 None of our hemangioblastoma patients showed
erythrocytosis clinically. However, interestingly, hemoglobin and
hematocrit levels were higher in hemangioblastoma versus glioblastoma
patients. We assume that in hemangioblastomas, the blood-brain barrier
is leaky due to overexpression of VEGF, which induces vascular
permeability, and Epo is able to enter the systemic circulation.
However, the amount of Epo may not be sufficient to induce clinically
overt erythrocytosis, instead leading to an insignificant increase of hemoglobin and hematocrit levels. In addition to these systemic effects, Epo expression in hemangioblastomas may have functions that
are confined to the tumor microenvironment. In a detailed study of 26 hemangioblastomas, foci of extramedullary hematopoiesis characterized
by islands of normoblasts were detected in four tumors.33
These islands were found mostly within or adjacent to capillaries and
within areas of stromal cells. One possible function of Epo expression
in hemangioblastomas could be to act as a locally produced stimulus for
erythroid differentiation. In this context, it is noteworthy that
hemoglobin, a marker for erythroid differentiation, was found to be
present in neurons, where it may be involved in oxygen
homeostasis.34
There are also reports suggesting that Epo might play a role in
angiogenesis. Epo receptors have been shown to be expressed on rat
brain capillary endothelial cells35 and on human
endothelial cells, and Epo can induce cell proliferation and migration
of endothelial cells.36,37 Furthermore, Epo has been shown
to stimulate angiogenesis on rat aortic rings in vitro.38
However, since we have been unable to detect Epo receptor expression in hemangioblastomas by Northern analysis (not shown), evidence for a role
of Epo in tumor angiogenesis is still lacking.
An important question is whether Epo expression, similar to VEGF
expression, is under control of the VHL protein. Experiments to solve
this problem are hampered by the fact that VHL-deficient hemangioblastoma cell lines are not available and that most available VHL-deficient renal cell carcinoma cell lines lack Epo expression. However, by Northern blot analysis, human renal clear cell carcinomas were also shown to produce Epo39 and high amounts of
VEGF.40 Interestingly, renal clear cell carcinomas are also
known to be affected by mutations of the VHL gene and are associated
with hereditary VHL disease.41
In this study, we have shown strong Epo expression in hemangioblastomas
and identified stromal cells as Epo-producing cells. In concordance
with previous observations that stromal cells also express VEGF and
mutant VHL, these findings suggest that loss of function of the VHL
gene product may also upregulate Epo expression in hemangioblastomas.
| |
FOOTNOTES |
Submitted April 7, 1998;
accepted June 23, 1998.
Supported by Grant No. C4 from the Center for Clinical Research I,
Freiburg University Medical School, Grant No. PL 158-3/1 from the
Deutsche Forschungsgemeinschaft (K.H.P.), and in part by fellowships
from the Swiss National Science Foundation (Forschungskommission der
Universität Zürich) and the Schweizerische Stiftung
für Medizinisch-Biologische Stipendien (H.H.M.).
Address reprint requests to Karl H. Plate, MD,
Neurozentrum, Abteilung Neuropathologie, Breisacherstr. 64, 79106 Freiburg/Brsg; e-mail: plate{at}nz11.ukl.uni-freiburg.de.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr C. Shoemaker (Boston, MA) for providing human Epo cDNA, Dr
B. Zbar (Frederick, MD) for human VHL cDNA, Dr H. Weich (Braunschweig,
Germany) for human VEGF cDNA, and Richard Haas (Freiburg, Germany) for
technical assistance.
| |
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Abstract
Introduction
Abstract