Full text of DOI:10.1038/labinvest.3780428

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LABORATORY INVESTIGATION
Vol. 82, No. 3, p. 345, 2002
Copyright © 2002 by The United States and Canadian Academy of Pathology, Inc.
Printed in U.S.A.
Nestin as a Marker for Proliferative Endothelium
in Gliomas
Ken-ichi Sugawara, Hideyuki Kurihara, Mitsuko Negishi, Nobuhito Saito,
Yoichi Nakazato, Tomio Sasaki, and Toshiyuki Takeuchi
Department of Molecular Medicine (KS, MN, TT), Institute for Molecular and Cellular Regulation, Gunma University,
and Departments of Neurosurgery (KS, HK, NS, TS) and Pathology (YN), Gunma University School of Medicine,
Maebashi 371-8512, Japan
SUMMARY: Nestin is one of the intermediate filaments abundantly produced in the developing central nervous system and
somites in the embryonic stage. Nestin is also reportedly detected in gliomas/glioblastomas. We retested nestin expression in
brain tumors having a range of malignancy grades using immunostaining. The intensity of nestin immunostaining roughly
paralleled the malignancy grade of the gliomas. However, many tumors were negative for nestin immunostaining, while nestin
immunostaining was invariably detected in tumor endothelium regardless of glioma malignancy grades or brain tumor types. We
suspected that angiogenic epithelial cells may express nestin, and we found that nestin was highly positive in bovine aortic
endothelial cells in static culture. However, nestin expression decreased when the endothelial cells underwent laminar shear
stress flow, under which endothelial cells exhibit differentiated features and a decreased rate of growth. Because nestin is highly
expressed in growing endothelial cells, we examined its expression in hemangioblastomas because hemangioblasts are thought
to be a precursor for angiogenic epithelial cells. As expected, nestin immunostained strongly in all four samples of
hemangioblastomas. We suggest that nestin is not only a marker for neuroepithelial stem cells and glioma cells but also for tumor
endothelial cells during rapid growth. (Lab Invest 2002, 82:345–351).
estin is one of the intermediate filaments, to-
gether with vimentin and glial fibrillary acidic
protein (GFAP), and is detected abundantly in neuro-
epithelial stem/progenitor cells in the growing central
nervous system of embryonal rats and humans
nestin has also been detected in human gliomas and
glioblastomas (Dahlstrand et al, 1992). Nestin immu-
nostaining has frequently been observed in highly
malignant gliomas, especially glioblastomas, as
compared with the less malignant forms such as
pilocytic astrocytomas. In contrast, nestin is rarely
detected by immunostaining in nonneoplastic brain
tissues, occurring sometimes faintly in vascular en-
dothelial cells.
Nestin mRNA is approximately 6.2 kilobases long,
and its gene contains three introns. Interestingly,
neuroepithelium-specific nestin expression is driven
by the second intron of the nestin gene, whereas
muscle precursor-specific expression is driven by the
first intron (Lothian and Lendahl, 1997; Zimmerman et
al, 1994). We previously examined nestin expression
in seven human glioma/glioblastoma-derived culture
cell lines (Kurihara et al, 2000). The level of expression
varied from high (U251, KG-1C) to nondetectable
N
(
Lendahl et al, 1990; Messam et al, 2000; Tohyama
et al, 1992, 1993). Nestin forms intermediate fila-
ment bundles, perhaps with vimentin, by copoly-
merization in neuroepithelial cells (Eliasson et al,
1
999; Rutka et al, 1999). Nestin mRNA is expressed
highly in the cerebrum of the developing rat embryo
at embryonic day 15 (E15), declines toward postna-
tal day 12 (P12), and disappears from P18 to the
adult stage (Lendahl et al, 1990). Using nestin
transgene-promoted ␤-galactosidase expression
analysis in mice, LacZ activity has been detected in
the neuroepithelium and somites shortly after neural
tube closure (E9) (Zimmerman et al, 1994). The LacZ
staining becomes stronger in the proliferative ven-
tricular zones of the mouse embryonic striatum and
cerebral cortex at E14.5 and E16.5 and decreases in
expression in the adult cortex, becoming restricted
to a population of ependymal cells. Substantial
(NP-2, LN-Z308, T98G) according to Northern blot
analysis. The expression levels did not parallel the
growth rates of the cell lines, although the degree of
malignancy generally reflects tumor doubling time in
vivo. The neuronal cell-specific regulator, consisting of
the second intron before the 5' upstream region of the
gene, drove LacZ expression in parallel with the extent
of mRNA expression in each cell line (Kurihara et al,
Received December 12, 2001.
This work is supported by grants-in-aid from the Ministry of Education,
Science, Sports, and Culture.
Address reprint requests to: Dr. Toshiyuki Takeuchi, Department of Mo-
lecular Medicine, Institute for Molecular and Cellular Regulation, Gunma
University, Showa-machi, Maebashi 371–8512, Japan. E-mail:
tstake@showa.gunma-u.ac.jp
2000). This variability in nestin expression levels in the
glioma/glioblastoma cell lines caused us to reevaluate
nestin expression in human glioma/glioblastomas
from low to high malignancy grades.
Laboratory Investigation • March 2002 • Volume 82 • Number 3 345

Sugawara et al
For this purpose, we used nestin-specific antiserum
and found that vascular endothelial cells contained in
glioma tissues express nestin constantly, whether
nestin is highly expressed or not in the glial tumor
cells. Thus, nestin is a marker protein not only for
immature neuroepithelial and glioma cells but also for
endothelial cells in active proliferation.
Results
Nestin Immunostaining for Brain Tumors
The antibody to nestin was raised in rabbits by inject-
ing the synthetic oligopeptide covering the C-terminal
17 amino acids of the human nestin sequence. This
antibody reacted with proteins of 210 to 240 kd from
U251 cell extracts by Western blotting, as reported
previously (Messam et al, 2000; Tohyama et al, 1992).
The immunoblot resembled doublets, perhaps be-
cause of the difference in carbohydrate modification,
as reported previously (Messam et al, 2000). The
immunoblot disappeared when the synthetic oli-
gopeptide was added to the U251 cell lysate, indicat-
ing that the immunoblot represents the nestin protein
(
Fig. 1a).
We further tested the cross-reactivity of this anti-
body with other intermediate filaments including vi-
mentin, GFAP, keratins, and desmin (Fig. 1b). The
antibody to human vimentin showed a single band at
a little beyond the 50-kd marker position with both
U251 and HeLa cell extracts. The antibody to GFAP
displayed an approximately 50-kd band with U251 cell
extract, but not with HeLa cell extract. For detecting
keratins, we used pooled mouse monoclonal antibod-
ies, anti-cytokeratin AE1/AE3, which recognize a
broad subfamily of acidic and basic keratins. The
anti-cytokeratin AE1/AE3 recognized an approxi-
mately 50-kd protein from HeLa cell extract, but faintly
from U251 cell extract. The antibody to human desmin
did not display bands with either U251 or HeLa cell
extract. Nonimmune rabbit serum did not show any
artifactual bands with either U251 or HeLa cell extract.
Thus, the antibody to nestin displayed large-sized
bands corresponding to nestin molecular-sized pro-
teins reported previously (Messam et al, 2000; To-
hyama et al, 1992) but did not cross-react with other
intermediate filaments (Fig. 1b).
Figure 1.
A, Western blot of nestin. Cell lysates of U251 human glioma cells were run on
7
.5% polyacrylamide gel. After blotting to a nylon membrane, the membrane
was immunostained with anti-nestin antibody (1:7500 dilution). Molecular size
(kd) is shown to the left of the gel. Left lane, lysate only; right lane, lysate
mixed with the synthetic oligopeptide covering C-terminal nestin sequence. B,
Western blot of other intermediate filaments, vimentin, glial fibrillary acidic
protein (GFAP), keratins, and desmin. Cell lysates of U251 human glioma cells
and HeLa human cervix epithelioid cells were used for analysis. Antibodies
were used according to the manufacturer’s instructions. Six Western blots are
shown from left to right, with the antibody to nestin, vimentin, GFAP, keratins,
and desmin, and with nonimmune rabbit serum.
was limited to proliferative endothelium, as shown in
Figure 3c (glioblastoma, grade IV) and in Figure 3d
(anaplastic oligodendroglioma, grade III). In the low-
We then immunostained 71 brain tumor samples
with this antibody. Table 1 is a summary of the
immunostaining. Normal brain cortex tissues were not
immunostained with this antibody (Fig. 2, a and b),
although a few vascular endothelial cells showed
occasional faint staining, as described previously
grade gliomas, the staining was weak to negligible in a
considerable number of tumors, but distinct staining
was noted along the endothelium in the tumor, as
shown in Figure 4, a and b (oligodendroglioma, grade
II). This tendency was more marked in other types of
brain tumors (Schwannoma, Fig. 4c; and meningioma,
Fig. 4d), whose epithelium immunostained strongly
positive for nestin while the tumor cells did not stain at
all. Thus, the tumor endothelial cells expressed nestin
without regard to the WHO grade for malignancy.
(
Dahlstrand et al, 1992). In the glioblastomas (WHO
grade IV), the typical nestin staining was fibrillar dis-
tribution along the processes of the tumor cells, as
shown in Figure 3a. The staining intensity was classi-
fied as 4ϩ in this tumor. Nestin staining was also
evident as a button-like cluster in the cytoplasm of
round-shaped tumor cells, as shown in Figure 3b
Nestin Expression in Replicating Endothelial Cells
(
anaplastic oligoastrocytoma, staining intensity is 3ϩ).
Because tumor epithelium is characterized by neoan-
giogenic growth (Bergers et al, 2000; Croix et al,
In some grade III and grade IV gliomas, the staining
3
46 Laboratory Investigation • March 2002 • Volume 82 • Number 3

Nestin as a Growing Endothelium Marker
Table 1. Summary of Nestin Immunostaining in Brain Tumors
Intensity of nestin staining
Tumor cells
Endothelial cells
Histology
n
0
1ϩ
2ϩ
3ϩ
4ϩ
0
1ϩ
2ϩ
Glioma
WHO grade I
Pilocytic astrocytoma
Subependymoma
Ganglioglioma
4
1
1
2
0
1
1
0
0
1
0
0
0
1
0
0
0
0
0
0
0
2
0
1
2
1
0
WHO grade II
Astrocytoma
Oligodendroglioma
Ependymoma
Oligoastrocytoma
WHO grade III
3
1
4
3
2
0
0
0
0
0
0
0
1
0
3
1
0
1
1
2
0
0
0
0
0
0
0
0
2
0
4
1
1
1
0
2
Anaplastic astrocytoma
Anaplastic oligodendroglioma
Anaplastic ependymoma
Anaplastic oligoastrocytoma
WHO grade IV
7
7
2
2
1
1
0
0
1
1
0
0
4
1
0
0
0
4
1
1
1
0
1
1
1
0
0
0
4
1
1
2
2
6
1
0
Glioblastoma
22
2
1
4
8
7
0
12
10
Others
Hemangioblastoma
Medulloblastoma
Atypical teratoid/rhabdoid tumor
Meningioma
4
2
1
3
2
2
71
*
2
1
3
2
0
17
*
0
0
0
0
0
4
*
0
0
0
0
1
16
*
0
0
0
0
1
20
*
0
0
0
0
0
10
0
0
0
0
0
0
1
0
1
0
2
0
0
33
4
1
1
1
2
2
37
Atypical meningioma
Schwannoma
Total
n, sample number.
In hemangioblastomas, tumor cells are precursors for endothelial cells. To evaluate the nestin immunostaining intensity, we classified hemangioblastoma cells
*
as endothelial cells, but not as tumor cells. Pathologic sections were scanned at low magnification to search for strong nestin-stained areas. Any field containing a
large nestin-stained area was then observed with a 10ϫ objective (ϫ100 magnification) for classification of the staining intensity.
Definition of nestin immunostaining intensity:
Tumor cells: % of stained cells over total cell population in one field under ϫ100 magnification. 0, no staining; 1ϩ, less than 1% of total tumor cell population
positive; 2ϩ, 1–5% of total tumor cell population positive; 3ϩ, 5–30% of total cell population positive; 4ϩ, over 30% of total cell population positive.
Endothelial cells: positive cells found in one microscopic field. 0, no staining; 1ϩ, positive cells found by over ϫ200 magnification; 2ϩ, positive cells found by
ϫ100 magnification.
2
000), we suspected that nestin expression is specific
hemangioblastomas because hemangioblasts are
thought to be a precursor for both hematopoietic cells
and angioblasts (Eichmann et al, 1997). To examine
this issue, we tested four human hemangioblastomas
(Table 1). An endothelial cell marker, von Willebrand
factor, positively immunostained along endothelium
and microcapillary vessels in the hemangioblastoma
for proliferating endothelial cells. To examine endothe-
lial nestin expression, we used bovine aortic endothe-
lial cells (BAECs). Nestin was expressed strongly in
BAECs in a static culture by both Northern blot anal-
ysis and immunostaining (Fig. 5, left lane, and Fig. 6a).
Endothelial cells proliferate by cell division in static
culture, whereas proliferation decreases under physi-
ologic laminar flow (approximately 15 dyn/cm ) (Malek
et al, 1999). To examine whether the nestin expression
is proliferation dependent, BAECs were subjected to a
shear stress flow of 15 dyn/cm for 12 hours. Expres-
sion of nestin mRNA diminished significantly with
shear stress flow by Northern blot analysis (Fig. 5,
right lane). Furthermore, we confirmed
dependent decrease of nestin expression by immuno-
staining (Fig. 6b).
(Fig. 7b) (Böhling et al, 2000). Nestin also immuno-
2
stained mostly in microcapillary vessels in the heman-
gioblastoma (Fig. 7c). However, typical endothelium
consisting of thin cytoplasm with a convex-shaped
nucleus was not positive for nestin staining (red ar-
rows in Fig. 7c). Because the appearance of endothe-
lium is similar to normal vessels, this type of endothe-
lium may have been well-differentiated and lost nestin
expression. Nestin-positive cells consisting of heman-
gioblastomas may reflect genuine transformed he-
mangioblasts. Thus, nestin is a marker protein not
only for neuroepithelial stem cells and glioma cells but
also for hemangioblasts and proliferating endothelial
cells.
2
a flow-
Nestin Expression in Hemangioblastomas
Because nestin was expressed in proliferating endo-
thelial cells, we suspected that it is expressed even in
Laboratory Investigation • March 2002 • Volume 82 • Number 3 347

Sugawara et al
Figure 4.
Nestin immunostaining of WHO low-grade malignancy gliomas and other types
of brain tumors. Nestin staining is not found in tumor cells but is instead
limited to the proliferative endothelium. a, Grade II oligodendroglioma. The
staining intensity of the endothelium is 2ϩ. b, High magnification of a. c,
Schwannoma. The staining intensity is 2ϩ. d, Meningioma. The staining
intensity is 2ϩ. Scales, 200 ␮m (a) and 50 ␮m (b to d).
Figure 2.
Nestin immunostaining of control human brain tissues. a, human cortex. Scale,
2
00 ␮m. b, close-up of a. Scale, 50 ␮m.
Figure 5.
Expression of nestin mRNA in shear stress-loaded endothelial cells. Upper
panel, nestin mRNA blot: left lane, RNA from bovine aortic endothelial cells
(BAECs) in static culture; right lane, RNA from shear stress-loaded BAECs for
1
2 hours. 28S indicates ribosomal RNA size. Lower panel, glyceraldehyde-3-
phosphate dehydrogenase mRNA blot as a control: right and left lanes are the
same as those in the upper panel. 18S indicates ribosomal RNA size.
Figure 3.
Nestin immunostaining of WHO high-grade malignancy gliomas. a, Grade IV
glioblastoma. The intensity of the nestin staining is 4ϩ. b, Grade III anaplastic
oligoastrocytoma. The intensity is 3ϩ. c, Grade IV glioblastoma. The staining
is limited to the proliferative endothelium. The staining intensity of the
endothelium is 2ϩ. d, Grade III anaplastic oligodendroglioma. The staining is
also limited to the proliferative endothelium. The staining intensity of the
endothelium is 2ϩ. Scales, 50 ␮m (a to c) and 200 ␮m (d).
Discussion
The present study demonstrated that the neuroepithe-
lial stem cell–specific marker nestin filament is ex-
pressed distinctly in the vascular endothelium draining
brain tumors. The nestin immunostaining was distinct
in tumor endothelium but faint to negligible in brain
tumor cells (Fig. 3, c and d, and Fig. 4, a to d).
Proliferating endothelial cells during active angiogen-
esis for tumor expansion appear to express the nestin
filament. This expression may not be specific for
tumor-associated angiogenesis because bovine en-
dothelial cells in culture express nestin to a great
Figure 6.
Nestin immunostaining in shear stress-loaded endothelial cells. a, BAECs in
2
static culture. b, BAECs under shear stress load (15 dyn/cm ) for 12 hours.
Scales, 50 ␮m (a and b).
escence and an atheroprotective gene expression,
whereas low or turbulent shear stress flow stimulates
the expression of proinflammatory and atherogenic
genes (Gimbrone et al, 1997; Malek et al, 1999). When
the physiologic level of shear stress flow was loaded
to endothelial cells, nestin gene expression decreased
extent. Physiologic level of laminar shear stress flow
2
(
approximately 15 dyn/cm ) induces endothelial qui-
3
48 Laboratory Investigation • March 2002 • Volume 82 • Number 3

Nestin as a Growing Endothelium Marker
branes, resulting in active angiogenesis (Dang and
Semenza, 1999). Endothelial cells express HIF-like
factor (HLF, or EPAS1), which is also induced by
hypoxia and is degraded by the pVHL degradation
system (Ema et al, 1997; Krieg et al, 2000). HIF-1 and
HLF are known to act on its consensus enhancer
element 5'-CACGTG-3'. We looked for this element on
the first and second introns of the nestin gene using a
computer search and found at least one candidate site
on the first intron. It will be important to examine
whether this candidate site is functional for the HIF/
HLF-dependent expression of the nestin gene in tu-
mor endothelium.
Although a number of angiogenesis-related genes
are reported in colorectal cancer endothelium, the
nestin gene is not included in the list (Croix et al,
Figure 7.
Staining of hemangioblastomas. a, Hematoxylin and eosin staining. b, Immu-
nostaining for von Willebrand factor. c, Immunostaining for nestin. Red
arrows, nonstained endothelium. Scales, 50 ␮m (a to c). HE ϭ hematoxylin
and eosin; vWF ϭ von Willebrand factor.
2000). Angiogenesis-related genes in brain tumor en-
dothelium may be different from those in colorectal
endothelium. It is noteworthy that strong nestin ex-
pression is found in brain tumor endothelium even if
no nestin expression is found in the brain tumor cells.
We suggest that nestin serves as an excellent endo-
thelium marker for brain tumors such as gliomas,
hemangioblastomas, Schwannomas, medulloblasto-
mas, and meningiomas.
remarkably, suggesting that the nestin expression is
specific for proliferating endothelial cells. Thus, nestin
serves as a marker for proliferating endothelial cells,
especially for angiogenic endothelium draining brain
tumor tissues.
Nestin was initially found in growing neuroepithelial
stem cells and gliomas and has been recognized as an
oncofetal marker protein for gliomas (Dahlstrand et al,
Materials and Methods
Preparation and Characterization of Nestin Antibody
1992; Lendahl et al, 1990). When nestin expression
was further investigated in detail by nestin promoter/
intron-driven LacZ expression in mice, the expression
was found to be limited to neuroepithelial stem cells
and somites in developing mouse embryos (Zimmer-
man et al, 1994). This neuroepithelial stem cell–spe-
cific expression was shown to be driven by the second
intron of the nestin gene, whereas the somite-specific
expression seemed to be promoted by the first intron.
Although nestin immunostaining has been reported to
be weak in a few vascular endothelial cells in normal
brain tissues in rats and humans (Dahlstrand et al,
The antibody to human nestin was generated in rabbits
using the synthetic oligopeptide covering the C-terminal
17 amino acids: 1602-KFTQREGDRESWSSGED-1618
(Messam et al, 2000). A cysteine residue was added to
the N-terminal of this oligopeptide for covalent conjuga-
tion with the carrier protein keyhole limpet hemocyanin.
The oligopeptide-protein conjugate was emulsified with
Freund’s complete adjuvant and injected into two New
Zealand White rabbits. After several booster injections,
the rabbits developed antibodies against the oligopeptide.
The specificity of the antibody was examined by
Western blot analysis using cell extracts from a human
glioblastoma cell line U251 and from a human cervix
epithelioid cell line HeLa. For testing cross-reactivity
of the nestin antibody with other intermediate fila-
ments, we used four antibodies recognizing human
vimentin, human GFAP, human keratins, and human
desmin. Antibody to GFAP was made previously by
one of the coauthors, Dr. Nakazato (Nakazato et al,
1992; Tohyama et al, 1992, 1993), the endothelium-
specific expression of the nestin gene remains to be
investigated. Because both somite and endothelium
originate from the embryonal mesoderm, it should be
investigated whether endothelium-specific nestin ex-
pression is driven by the first intron.
The extensive nestin immunostaining in hemangio-
blastomas is of interest because dysfunction of the
tumor suppressor gene von Hippel-Lindau (pVHL)
predisposes towards the development of central ner-
vous system hemangioblastomas (Böhling et al, 2000).
pVHL functions as a component of the proteasome
complex for degrading hypoxia-inducible factors (HIF)
1982). Other antibodies were purchased from DAKO,
Glostrup, Denmark.
Morphologic Studies
(
Maxwell et al, 1999). Although HIF-1 is easily induc-
We used 71 human brain tumor samples including 57
gliomas and 14 other brain tumors. The gliomas in-
cluded 6 WHO grade I tumors, 11 grade II tumors, 18
grade III tumors, and 22 grade IV tumors, as listed in
Table 1. Other brain tumors included four hemangio-
blastomas, two medulloblastomas, one atypical tera-
toid/rhabdoid tumor, three meningiomas, two atypical
meningiomas, and two Schwannomas. All 71 brain
tumors were resected at the Department of Neurosur-
ible by hypoxia, HIF-1 is readily degraded by ubiquiti-
nation in a pVHL-dependent fashion in normal vascu-
lar stromal cells (Ohh et al, 2000). However,
dysfunction of pVHL by genetic defects results in an
increase in HIF-1 because of lack of its degradation
system. Increased HIF-1 molecules induce vascular
endothelial growth factor, which in turn activates its
receptor Flk-1 and Flt-1 on endothelial plasma mem-
Laboratory Investigation • March 2002 • Volume 82 • Number 3 349

Sugawara et al
gery, Gunma University School of Medicine. The diag-
noses were established by routine pathologic exami-
nations according to the revised WHO classification at
the Department of Pathology, Gunma University
School of Medicine.
The human brain and tumor tissues and BAECs
were fixed in 4% paraformaldehyde in 0.1 M phos-
phate buffer, pH 7.4, for 24 hours (tissues) or 1 hour
deoxy-CTP by a random priming procedure. A
glyceraldehyde-3-phosphate dehydrogenase probe
was used as a control.
For Western blotting, U251 human glioblastoma
cells were solubilized for cell lysates in lysis buffer (70
mM Tris-HCl, pH 6.8, 11.2% glycerol, 3% SDS, 0.01%
bromophenol blue, 5% 2-mercaptoethanol). The cell
lysates were then subjected to electrophoresis on a
7.5% polyacrylamide gel under a reducing condition,
then blotted onto a nitrocellulose membrane for prob-
ing with rabbit anti-human nestin antiserum at a dilu-
tion of 1:7500. Nestin blots were detected utilizing an
ECL detection system (Amersham, Buckinghamshire,
United Kingdom).
(
culture cells). Small pieces of the tissue sample were
embedded in optimal cutting temperature compound
for microtome sectioning. BAECs were treated with 50
mM NH CL in PBS to quench any free aldehyde, then
4
made permeable by 0.1% saponin and 0.4% bovine
serum albumin before the primary antibody incuba-
tion. The tissue sections or BAECs were first incu-
bated with the primary antibody to nestin at a dilution
of 1:5000. For brain and tumor tissues, an LSAB2/HRP
staining kit (DAKO) was used as the secondary anti-
body reaction system. The procedure consists of a
secondary antibody reaction followed by an enzyme
Acknowledgements
We thank Ms. Eiko Hamana for her secretarial
assistance and Ms. Mari Hosoi for her technical
assistance.
reaction with
a
horseradish peroxidase-labeled
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Northern and Western Blot Analyses
4
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