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and Némeček
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Cerebral Angiogenesis Shows Nestin Expression
in Endothelial Cells
J
MOKRÝ
Department
Hradec
AND S
NÉMEČEK
of Histology
Králové,
Czech
and Embryology,
Charles
University,
Medical
Faculty,
Republic
A b s t r a c t . T h e class V I i n t e r m e d i a t e filament p r o t e i n nestin has b e e n generally consid­
ered as a specific m a r k e r for neural precursor cells or developing muscles In t h e p r e n a t a l
developing r a t central nervous s y s t e m (CNS) we localized i m m u n o r e a c t i v i t y for t h e n e s t i n
m b l o o d vessels A l t h o u g h t h e widespread n e s t i n expression m cerebral b l o o d vessels per­
sisted m early p o s t n a t a l periods, it was down-regulated in t h e a d u l t h o o d However, w h e n
t h e a d u l t r a t brains were subjected t o p r o c e d u r e s t h a t trigger neovascularization, e g
grafting fetal nervous tissue or C6 glioma, t h e a b u n d a n t i m m u n o r e a c t i v i t y was d e t e c t e d
m all newly formed vessels a n d adjacent host vasculature O u r results d e m o n s t r a t e t h a t
nestin expression m endothelial cells l m m g cerebral vessels a c c o m p a n i e s t h e process of
angiogenesis
K e y w o r d s : Angiogenesis — E n d o t h e l i u m — Nestin — I m m u n o h i s t o c h e m i s t r y — R a t
C o r r e s p o n d e n c e address J Mokrý, D e p a r t m e n t of Histology a n d Embryology, C h a r ­
les University, Medical Faculty, Šimkova 870, 500 01 Hradec Králové, Czech R e p u b l i c
E-mail mokryQlf hk c u m . cz
26
Gen Physiol Biophys (1999), 18, Suppl 1, Mokrý and Némeček
Introduction
Expression of the intermediate filament (IF) nestin in endothelial cells of the intact nervous
system and bram tumors has been noticed by several investigators (Dahlstrand et al 1992,
Tohyama et al 1992, Prisén et al 1995) Recently, we demonstrated that expression of
the nestin in vasculature was not restricted exclusively to cerebral blood vessels but was
ubiquitous The most abundant immunoreactivity in endothelial cells was observed during
embryonic development in both extra- and intraembryonic tissues (Mokrý and Némeček
1998) The aim of present study was to characterize events that are associated with
expression of the nestin in the cerebral vasculature
Materials and M e t h o d s
We screened paraffin-embedded sections of whole rat fetuses and brains of postnatal
Wistar rats (including pregnant rats) for the presence of IF nestin using anti-Rat-401
monoclonal antibody and peroxidase immunohistochemistry (labelled streptavidin-biotin
method) Rat-401 antibody was obtained from the Developmental Studies Hybridoma
Bank, maintained by the Department of Biological Sciences, University of Iowa, IA, USA
The bram tissue was obtained from animals of the following ages E14 (embryonic day
14, n = 3), E15 (n = 5), E16 (n = 2), E19 (n = 2), postanatal day 0 (PO, n = 2), P7
(n — 2), P14 (n = 2), three-month old adult (pregnant) rats (n = 6) In addition to
normal tissues, we also examined the expression of the nestin in sections of rat brains
that had been previously transplanted with fetal neural tissue or C6 glioma (n = 7)
Transplantation procedure has been thoroughly described elsewhere (Mokrý et al 1993
Mokrý and Némeček 1995)
Results
In the CNS of E14-E16 rat fetuses, immunohistochemistry using Rat-401 antibody re­
vealed the strongest positive signal in the neuroepithelium and radial fibres This overall
immunoreactivity for the nestin nearly masked lmmunostaming of blood vessels vascu­
larizing the neuroepithelium In blood vessels of choroid plexus pnmordia, nestin ex­
pression was identified in endothelial and perivascular cells In the prenatal (E19) and
neonatal (PO) rat bram, nestm-immunoreactivity ceased in postmitotic neural elements
of other areas Therefore, e g m the cortical parenchyma, blood vessels were responsible
for major immunoreactivity of the nestin Walls of the most vessels were ascertained by
nestm-immunostammg of varying intensity that was confined to perivascular lining and
endothelia In the CNS of P7 (postnatal day 7) rats, almost all cerebral vessels of differ­
ent calibres (inci vessels supplying the choroid plexus) became visible after peroxidase
immunodetection of the nestin (Fig 1) In P14 brains, endothelia of most capillaries ex­
pressed nestin, however, intensity of the lmmunostaming was lower when compared with
P7 brains and few vessels were devoid of any specific signal Completely different staining
pattern was specific for brains of adult pregnant rats The bram surface was lined with
nestin"1" endfeet of the marginal glia Similar positivity was obser\ed m astroglial perivas­
cular endfeet accompanying underlying blood vessels in the cortical molecular layer (I)
Deep cortical layers (II-VI) were devoid of any immunoreactivity for nestin Most endothe­
lial cells were nestm-negative, positive lmmunostaming being confined only to occasional
endothelial cells
G e n Physiol
Biophys
(1999), 1 8 , Suppl
F i g u r e 1. Immunohistochemical detection of
the nestm m the rat brain Nestin-immunoreactivity of neural cells ceased in the cortex of
P7 rats, but remained in blood vessels (Mag­
nification x60)
1, Mokrý
and
Némeček
27
Figure 2. Immunohistochemical localization
of the nestin in vasculature of neural grafts
Dilated lumma of blood vessels nourishing the
21d intracerebral transplant (TR) became
clearly apparent due to the expression of nes­
tin In the host parenchyma (H), only adja­
cent capillaries were apparent (Magnification
xllO)
O n e week after t r a n s p l a n t a t i o n of isogeneic embryonic neural tissue, t h e nestin was
identified m a c t i v a t e d astrocytes t h r o u g h o u t t h e host b r a m , of t h e positive a s t r o c y t e s only
elements adjacent t o a needle t r a c t area a d o p t e d morphology of reactive a s t r o c y t e s (RAs)
Overall strong expression of t h e nestm m astrocytic perivascular endfoot processes m a s k e d
l m m u n o s t a m i n g of o t h e r vascular cells T w o and t h r e e weeks postgraftmg, i m m u n o r e a c ­
tivity for nestin b e c a m e clearly visible m endothelia of capillaries nourishing i n t r a c e r e b r a l
t r a n s p l a n t due t o t h e ceased expression in astrocytes T h u s , anti-nestin i m m u n o h i s t o ­
chemistry enabled t o visualize capillaries as well as large vessels t h a t vascularized solid
grafts (Fig 2) T h i s reactivity for n e s t m was intense, it was mainly confined t o endothelia,
a l t h o u g h t h e positive perivascular s h e a t h of astrocytic endfeet was also p r e s e n t Nestin" 1 "
capillaries were also observed in t h e host p a r e n c h y m a m t h e close vicinity t o intracerebral
t r a n s p l a n t s In a n i m a l s t h a t were left t o survive for 1, 2 or 3 m o n t h s , only few nestin" 1 "
capillaries were found m peripheral p o r t i o n s of large intracerebral grafts I n intraventric­
ular grafts, intense i m m u n o r e a c t i v i t y was observed in blood vessels t h a t a p p o s e d t o a
wall of cerebral ventricles a n d b e c a m e vascularized In t h e s e solid i n t r a v e n t r i c u l a r grafts,
i m m u n o h i s t o c h e m i c a l detection of t h e nestin m endothelial cells allowed t o depict t h e i r
c o m p l e t e capillarization
In r a t brains i n o c u l a t e d mtracerebrally with C6 glioma cells 6 days previously, neo­
plastic cells filled m t h e whole needle-track area a n d b e g a n t o form a r a p i d l y increasing
28
Gen Physiol Biophys (1999), 18, Suppl 1, Mokrý and Némeček
tumour mass in the right hemisphere At the implantation site, blood vessels of different
calibres including large paralytic vessels became visualized with nestm immunostainmg
1
Perivascular spaces of these cerebral vessels were infiltrated with nestin" " C6 cells By
1
10 days a solid tumour composed of nestin" " C6 cells and capillaries was formed Antinestm immunohistochemistry visualized also peritumoral vessels which enabled perivas­
cular propagation of neoplastic cells (Fig 3) One month after the transplantation of C6
cells, tumor mass reached its largest size In areas rich in glioma cells, immunostainmg
for nestin revealed positive vascular endothelia surrounded by positive perivascular C6
cells or host RAs that were included m the tumour Perivascular processes of RAs lined
adjacent cerebral vessels and permitted to identify also blood vessels in the close vicinity
to tumor
Discussion
In E14-19 rat fetuses, we observed more distinct immunoreactivity of endothelial
cells for IF nestm in non neural tissues (Mokrý and Némeček 1998) than in the CNS
Nestin-positivity of CNS vessels became
quite clearly apparent in the first postna­
tal week when the nestin expression ceased
in surrounding neural cells As the num­
ber of cerebral vessels identified with antinestm immunohistochemistry declined in
the adulthood, it was apparent that ex­
pression of nestin gene in a vasculature
was temporally restricted The descrease of
nestin expression in the course of nervous
tissue maturation has been thoroughly de­
scribed in previous studies (Fredenksen
and McKay 1988, Lendahl et al 1990,
Tohyama et al 1992, Zimmerman et al
1994) In adult brains, immunohistochem­
istry utilizing Rat-401 monoclonal anti­
body as primary antibody labelled only
sporadic cerebral vessels reflecting the low
endothelial cell turnover
In the experimental part of our study,
Figure 3. Peroxidase immunohistochemistry
we investigated the nestm immunoreactiv­
identified the nestin expression in vascular cells
ity in adult rat brains that were mtracerein the periphery of lOd C6 glioma C6 cells
brally injected with cells that induce lo­
migrate along perivascular spaces of adjacent
cal neovascularization of the grafted tis­
blood vessels (arrowheads) in the recipient
brain tissue (Magnification x 130)
sue In the first group of experimental an­
imals, solid pieces of E14 fetal rat bram
were grafted into the bram parenchyma of adult animals Following transplantation, the
grafted tissue suffering from hypoxia obviously induces a release of signals that initiate the
process of vascularization This process consists of several steps which include ingrowth
of host capillaries into the transplant (angiogenesis) and their inoculation to pre-existing
and collapsed capillaries derived from the donor's brain tissue (Krum and Rosenstem
1987) Our finding of nestm expression in vessels in the close vicinity to the graft con­
firmed that also adjacent host cerebral vessels are really activated during angiogenesis
IM
\i
G e n Physiol
Biophys
(1999), 1 8 , Suppl 1, Mokrý
and Némeček
29
W i t h i n t h e graft, we observed t h e n e s t m i m m u n o r e a c t i v i t y in endothelia of all vessels
as well as in i m m a t u r e neural cells, i m m u n o r e a c t i v i t y confined t o v a s c u l a t u r e lasted for
long t i m e (more t h a n 3 m o n t h s ) whereas it rapidly ceased in neural cells t h a t m a t u r e d
After 1 m o n t h postgraftmg, all capillary endothelia in intraventricular grafts expressed
t h e n e s t m , whereas only peripheral blood vessels of intracerebral grafts r e m a i n e d n e s t m +
T h e second group of e x p e r i m e n t a l a n i m a l s was mtracerebrally injected w i t h t h e C6 glioma
cells We found t h a t in a t u m o r m a s s of a solid C6 glioma, endothelia of all blood vessels
1
nourishing t h e t u m o r expressed n e s t m T h e finding of nestm" " endothelial cells in h u m a n
a s t r o c y t o m a s a n d glioblastomas h a s been previously described by T o h y a m a et al (1992)
w h o used a n t i - n e s t m 129 polyclonal a n t i s e r u m t h a t binds t h e h u m a n nestin
Conclusions
O u r results d e m o n s t r a t e t h a t in t h e C N S , anti-nestin i m m u n o h i s t o c h e m i s t r y identifies
n o t only i m m a t u r e n e u r a l cells b u t also cerebral blood vessels W i d e s p r e a d vascular im­
m u n o r e a c t i v i t y for t h e n e s t m is r e s t r i c t e d t o p r e n a t a l a n d early p o s t n a t a l periods, whereas
it is greatly r e d u c e d in t h e e n d o t h e l i u m of blood vessels supplying t h e a d u l t i n t a c t brain
Neovascularization t h a t occurs e g in growing neural grafts or b r a m t u m o r s , is accom­
p a n i e d with specific expression of I F nestin in all endothelial cells O u r findings i n d i c a t e
t h a t t h e n e s t m expression m e n d o t h e l i u m of r a t cerebral vessels is closely associated w i t h
cerebral angiogenesis
A c k n o w l e d g e m e n t s . Authors are grateful to Mrs Hollerova and Hetešova for a technical assis
tance Supported by a grant No 304/97/1117 from the Grant Agency of Czech Republic
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