Bone Marrow–Derived Endothelial Progenitor Cells
Participate in Cerebral Neovascularization After Focal
Cerebral Ischemia in the Adult Mouse
Zheng Gang Zhang, Li Zhang, Quan Jiang, Michael Chopp
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Abstract—We investigated whether circulating endothelial progenitor cells contribute to neovascularization after stroke.
Donor bone marrow cells obtained from transgenic mice constitutively expressing ␤-galactosidase transcriptionally
regulated by an endothelial-specific promoter, Tie2, were injected into adult mice. Focal cerebral ischemia was induced
by embolic middle cerebral artery (MCA) occlusion and changes of cerebral blood flow (CBF) were measured by
perfusion-weighted magnetic resonance imaging (MRI). Laser scanning confocal microscopy (LSCM), immunohistochemistry and X-gal staining were performed. Perfusion-weighted MRI demonstrated increases in CBF around the
boundary of an infarct area 1 month after ischemia. Morphological and 3-dimensional image analyses revealed enlarged
and thin-walled blood vessels with sprouting or intussusception at the boundary of the ischemic lesion, which closely
corresponded to elevated CBF areas detected on perfusion-weighted MRI, indicating the presence of neovascularization.
X-gal and double immunostaining demonstrated that Tie2-lacZ–positive cells incorporated into sites of neovascularization at the border of the infarct, and these cells exhibited an endothelial antigenic marker (von Willebrand factor).
In addition, bone marrow recipient mice without ischemia showed incorporation of Tie2-lacZ– expressing cells into
vessels of the choroid plexus. These data suggest that formation of new blood vessels in the adult brain after stroke is
not restricted to angiogenesis but also involves vasculogenesis and that circulating endothelial progenitor cells from
bone marrow contribute to the vascular substructure of the choroid plexus. (Circ Res. 2002;90:284-288.)
Key Words: bone marrow 䡲 endothelial progenitor cells 䡲 neovascularization 䡲 cerebral ischemia 䡲 Tie2
I
thelial cells are linked by complex tight junctions that form
the blood brain barrier (BBB).5,6 In the adult brain, proliferation of the cerebral endothelial cells ceases, and the turnover
rate of endothelial cells is approximately 3 years.8,9
Studies from human and experimental stroke indicate that
neovascularization is present in the adult brain after ischemia.10,11 However, development of new blood vessels in
ischemic adult brain is incompletely understood, and it
remains unknown whether newly formed vessels are induced
by proliferation of preexisting vascular endothelial cells or by
recruitment of circulating endothelial progenitor cells to the
brain after stroke. In the present study, in ischemic adult
mouse brain, we measured bone marrow cells obtained from
transgenic mice constitutively expressing ␤-galactosidase
(lacZ) transcriptionally regulated by an endothelial-specific
promoter, Tie2.12
n the early embryogenesis, the vascular system develops
from vasculogenesis in which angioblasts differentiate into
endothelial cells to form a primitive capillary network,
whereas angiogenesis, the sprouting of capillaries from preexisting blood vessels, is involved in the late stage of
embryogenesis and in the adult.1 Angioblast-like circulating
endothelial progenitor cells are present in the peripheral
blood and have been isolated from adult animals.2,3 Injection
of circulating endothelial progenitor cells into animals with
hindlimb or myocardial ischemia results in incorporation of
circulating endothelial progenitor cells into neovasculature at
the site of ischemia, suggesting that circulating endothelial
progenitor cells contribute to formation of new blood vessels
in the adult tissue.4
The endothelial cells of cerebral capillaries differ functionally and morphologically from those of noncerebral capillaries.5,6 During embryonic development, the cerebral vascular
system originates from the perineural plexus when vascular
sprouts invade the proliferating neuroectoderm, indicating
that the cerebral vascular system is primarily developed by
angiogenesis and not by vasculogenesis.7 The cerebral endo-
Materials and Methods
All experimental procedures have been approved by the Care of
Experimental Animals Committee of Henry Ford Hospital.
Original received May 2, 2001; resubmission received September 26, 2001; revised resubmission received December 19, 2001; accepted December 19,
2001.
From the Department of Neurology (Z.G.Z., L.Z., Q.J., M.C.), Henry Ford Health Sciences Center, Detroit, Mich; and the Department of Physics
(M.C.), Oakland University, Rochester, Mich.
Correspondence to Michael Chopp, PhD, Henry Ford Hospital, Department of Neurology, 2799 West Grand Blvd, Detroit, MI 48202. E-mail
[email protected]
© 2002 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/hh0302.104460
284
Zhang et al
Animal Model
Focal embolic cerebral ischemia was induced in male FVB mice (26
to 30 g, n⫽24) as previously reported with some modifications.13
Briefly, the right middle cerebral artery (MCA) was occluded by
placing a single 8-mm length intact, fibrin-rich, 24-hours-old, homologous clot at the origin of the MCA via an 8-mm length of
modified PE-50 catheter. The rationale for selecting a relatively short
clot is that a long (10 mm) clot induces the ipsilateral cortical and
subcortical lesions supplied by the right MCA, and mice with this
type of large ischemic lesion usually do not survive for a week,
whereas a short clot induces primarily a subcortical lesion and
animals can survive for months.14 This model of embolic stroke is
the most relevant one to human stroke.15
Bone Marrow Transplantation
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Using a syringe with 1 mL phosphate-buffered saline (PBS), fresh
bone marrow was harvested aseptically by flushing tibias and femurs
from age-matched (3 months) transgenic mice constitutively expressing ␤-galactosidase under transcriptional regulation of a Tie2 promoter12 (FVB/N-TgN, TIE2LacZ, The Jackson Laboratory, Bar
Harbor, Maine). The Tie2 receptor is expressed in endothelial
lineage cells that participate in neovascularization.12 Bone marrow
was then mechanically dissociated until a single cell suspension was
achieved. Bone marrow cells (2⫻106) were intravenously injected
into a recipient mouse via a tail vein. At 4 weeks after bone marrow
transplantation, by which time the donor bone marrow cells can be
detected in the recipient bone marrow cells,16 recipient mice were
subjected to embolic MCA occlusion and euthanized at 2, 30, and 60
days after MCA occlusion.
Bromodeoxyuridine Labeling
Bromodeoxyuridine (BrdU, Sigma Chemical), the thymidine analog
that is incorporated into the DNA of dividing cells during S-phase,
was used for mitotic labeling. BrdU (50 mg/kg) was intraperitoneally
injected daily for 7 consecutive days into ischemic mice starting 7
days after MCA occlusion. These mice (n⫽4) were euthanized 14
days after ischemia.
Magnetic Resonance Imaging Measurements
To dynamically measure changes of cerebral blood flow (CBF),
perfusion-weighted magnetic resonance imaging (MRI) was performed on mice before and 1 month after MCA occlusion using a 7.0
T magnet (Magnex Scientific) equipped with actively shielded
gradients and a Surrey Medical Imaging Systems console.17 MR
images were recorded using a birdcage RF coil, as previously
described.17 This technique is based on the selective inversion of
blood water protons at the level of the carotid arteries prior to 1H
MRI measurement in the brain.17 Two images were obtained for
perfusion measurement with parameters: repetition time (TR)⫽1.055
seconds; echo time (TE)⫽30 ms, 64⫻64 image matrix, 2-mm slice
thickness, and a 40-mm field of view (FOV). The duration of the
inversion pulse was 1 second and 0.3 kHz in amplitude. In addition,
the apparent diffusion coefficient of water (ADCw) was measured.17
CBF values were calculated as following: A region of interest (ROI)
was chosen by tracing the ipsilateral hyperemia area on the CBF map
1 month after onset of ischemia. CBF values of homologous area in
the contralateral hemisphere were measured. Then, values of CBF
corresponding to the ROI on the CBF map obtained 1 day after
ischemia were calculated.
Quantitative Measurements of Vascular Perimeters
Each vWF immunostained coronal section was digitized under a
20⫻ or 40⫻ objective (Olympus B⫻40) for measurement of
perimeters of vWF immunoreactive vessels18 using a 3-CCD color
video camera (Sony DXC-970 MD) interfaced with MCID image
analysis system (Imaging Research).
Three-Dimensional Image Acquisition
To examine neovascularization in ischemic brain, fluorescein isothiocyanate (FITC) dextran (2⫻106 molecular weight, Sigma; 0.1
Endothelial Progenitor Cells in Ischemic Brain
285
mL of 50 mg/mL) was administered intravenously to the ischemic
mice subjected to 30 days of MCA occlusion. FITC-dextran remains
dissolved and free in plasma.18 This dye circulated for 1 minute, after
which the anesthetized animals were euthanized by decapitation. The
brains were rapidly removed from the severed heads and placed in
4% of paraformaldehyde at 4°C for 48 hours. Coronal sections (100
␮m) were cut on a vibratome. The vibratome sections were analyzed
with a Bio-Rad MRC 1024 (argon and krypton) laser-scanning
confocal imaging system mounted onto a Zeiss microscope (BioRad), as previously described.18
LacZ Staining and Immunohistochemistry
Mice were perfused with heparinized saline. Brains were embedded
in Tissue-Tek OCT compound (Miles, Inc), frozen in
2-methylbutane (Fisher Scientific), and cooled on dry ice. Coronal
brain sections (8 to 40 ␮m thick) were cut on a cryostat and
thaw-mounted onto gelatin coated slides. LacZ staining was performed using a ␤-galactosidase reporter gene staining kit according
to manufacturer’s protocol (Sigma). Briefly, after postfixation,
coronal sections were incubated in X-gal solution for 2 days at 37°C
and then counterstained with light eosin. To identify cell types of
Tie2-lacZ– expressing cells, double immunofluorescence labeling for
von Willebrand factor (vWF), a marker for endothelial cells,19 or
microtubule-associated protein-2 (MAP2), a marker for neurons,18 or
glial fibrillary acidic protein (GFAP), a marker for astrocytes, and
␤-galactosidase was performed. A monoclonal antibody (mAb)
against vWF (DAKO, Carpinteria, Calif), a mAb against MAP2
(clone AP20, Boehringer Mannheim Biochemicals, Indianapolis,
Ind), a polyclonal antibody against GFAP (DAKO), and a polyclonal
antibody against ␤-galactosidase (Chemicon) were used at a titer of
1:20, 1:50, 1:200, and 1:200, respectively. Coronal sections were
incubated with the antibody against vWF, MAP2, or GFAP for 3
days at 4°C, and sections were then incubated with the anti-mouse or
anti-rabbit immunoglobulin antibody conjugated to Cy3 (Vector,
Burlingame, Calif). These sections were incubated with the antibody
against ␤-galactosidase for 3 days at 4°C and then with the
anti-rabbit immunoglobulin antibody conjugated to FITC. Single
immunostaining for vWF was performed for morphological analysis
of vessels. For BrdU immunostaining, DNA was first denatured by
incubating brain sections (6 ␮m) in 50% formamide 2X SSC at 65°C
for 2 hours and then in 2N HCl at 37°C for 30 minutes. Sections were
then rinsed with tris buffer and treated with 1% of H2O2 to block
endogenous peroxidase. Sections were incubated with a mouse
monoclonal antibody (mAb) against BrdU (1:1000, Boehringer
Mannheim) overnight and incubated with biotinylated secondary
antibody (1:200, Vector) for 1 hour. Control experiments consisted
of staining brain coronal tissue sections as outlined above but
omitted the primary antibodies.
Results
To determine dynamic changes of CBF after ischemia,
perfusion-weighted MRI measurements were performed on
the bone marrow–transplanted mice 2 hours and 1 month
after MCA occlusion. Substantial reduction of CBF in the
right subcortex was detected in all mice (n⫽8) (Figure 1A),
indicating the presence of an ischemic lesion. However, an
elevated CBF was detected around the boundary of the
ischemic lesion 1 month after ischemia (Figure 1B). Quantitative analysis of CBF revealed 74% of CBF reduction in
ischemic lesion compared with levels of CBF in the contralateral homologous tissue at 2 hours after MCA occlusion
(n⫽8). In contrast, 86% of CBF increase was detected in the
ischemic boundary at 1 month after ischemia (n⫽8). Laser
scanning confocal microscopy (LSCM) images of coronal
sections that matched MRI sections from the same animal
showed enlarged FITC-dextran–perfused cerebral vessels in
the lesion boundary areas (Figure 1C, arrow), which closely
286
Circulation Research
February 22, 2002
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Figure 1. Images of perfusion-weighted MRI, LSCM, and immunohistochemistry. Perfusion-weighted MRIs show reduction of
CBF in the subcortical area at 2 hours (A) and elevated CBF at
boundary of ischemia at 1 month after MCA occlusion (B,
arrow). A coronal section obtained from LSCM corresponding to
perfusion-weighted MRI shows the presence of enlarged FITCperfused vessels around infarct (C, arrow). Coronal sections immunostained by a vWF antibody show enlarged and thin-walled
vessels around infarct (D), and these vessels exhibited sprouting
(E, an arrow) and intusussception (F, arrows). A composite
image obtained from 15 LSCM sections (1 ␮m/section) shows
sprouting vessels (G, arrows). BrdU immunoreactive endothelial
cells were observed on lumen of enlarged vessels (H, arrows).
Bar in D⫽50 ␮m, bar in F⫽10 ␮m for E and F, and bars in G
and H⫽10 ␮m. Quantitative data (I) show vascular perimeters in
the ischemic boundary (I, ipsilateral) and the contralateral
homologous areas (I, contralateral) from the 3 mice. Vascular
perimeters are significantly increased in the ischemic boundary
zone compared with perimeters in homologous contralateral
tissue.
corresponded to the elevated CBF areas detected on
perfusion-weighted MRI (Figure 1B, arrow). Morphological
analysis of vWF stained vessels revealed enlarged and thinwalled blood vessels at the boundary of the ischemic lesion
(Figure 1D) and some of them exhibited sprouting (Figure
1E, arrow) or intussusception (Figure 1F, arrows). Sprouting
vessels were also detected in 3-dimensional images (Figure
1G, arrows), indicating neovascularization. Few BrdU immunoreactive cells were detected in vessels in the contralateral
hemisphere. In contrast, BrdU immunoreactive cells were
present on lumen of small and enlarged vessels (Figure 1H,
arrows) around the ischemic lesion 14 days after ischemia.
Quantitative measurements of vascular perimeter show that
perimeters of blood vessels in the ipsilateral hemisphere
(n⫽6) were significantly (P⬍0.05) larger than perimeters in
the contralateral homologous areas (Figure 1I).
Nonischemic mice with bone marrow transplantation exhibited Tie2-lacZ– expressing cells in the choroid plexus of
the both hemispheres (Figure 2A), and higher magnification
showed that blue color was localized to the inner layer of
Figure 2. Localization of Tie2-lacZ– expressing cells in the brain
and bone marrow. Tie2-lacZ– expressing cells were localized to
the choroid plexus in nonischemic mice 2 months after bone
marrow transplantation (A and B, blue color, section thickness
40 ␮m, B corresponding to the box in A). Mice without bone
marrow transplantation did not show any blue color (C). Tie2lacZ– expressing cells were detected at the border of infarct (D,
arrows, section thickness 40 ␮m) and localized to vessels (E,
arrows, section thickness 8 ␮m). Single immunostaining for lacZ
shows lacZ immunoreactive endothelial cells in the lumen surface of the vessel (F, arrows) and an arrowhead indicates a lacZ
immuno-negative endothelial cell (F). Double immunostaining for
vWF and lacZ on FITC-dextran–perfused tissue shows that vWF
immunoreactivity (H and J, blue) surrounded a FITC-dextran–
perfused vessel (G and J, green) and was colocalized to lacZ
immunoreactivity (I and J, red, arrows). Tie2-lacZ– expressing
cells were not detected in nonischemic parenchyma (K, section
thickness 40 ␮m). Tie2-lacZ– expressing cells were detected in
the recipient bone marrow 2 months after transplantation (L,
arrows). Bar in A⫽200 ␮m, bar in B⫽50 ␮m, bar in D⫽20 ␮m
for D, K, and L, bar in E⫽10 ␮m, bar in F⫽25 ␮m, and bar in
J⫽10 ␮m for G to J.
choroid plexus (Figure 2B), suggesting these are endothelial
cells rather than epithelial cells. Tie2-lacZ– expressing cells
were not detected on parenchymal cerebral vessels in these
mice (Figure 2A). Nonischemic mice without bone marrow
transplantation did not show any Tie2-lacZ– expressing cells
as assessed by blue color (Figure 2C). However, ischemic
mice receiving bone marrow transplantation exhibited Tie2lacZ–positive cells at the border of the infarct 1 month after
ischemia (Figure 2D), and higher magnification showed that
these cells were localized to cerebral microvessels (Figure
2E). LacZ immunostaining revealed lacZ immunoreactive
cells in the lumen surface of the vessel (Figure 2F). Double
immunofluorescent staining on FITC-dextran–perfused brain
tissue showed a vessel perfused with FITC-dextran (Figure
2G and 2J, green) and colocalization of vWF immunoreactivity (Figure 2H and 2J, blue) with lacZ immunoreactivity
(Figure 2I and 2J, red, arrows) on luminal surface, suggesting
that these cells are endothelial cells. Tie2-lacZ positive cells
were not detected outside of ischemic lesion (Figure 2K).
Tie2-lacZ–positive cells were also founded in recipient bone
marrow cells 2 months after transplantation (Figure 2L,
Zhang et al
arrows). Tie2-lacZ bone marrow–recipient mice killed at 48
hours after ischemia did not exhibit any blue color in the
ischemic lesion. LacZ immunoreactive cells did not exhibit
GFAP or MAP2 immunoreactivity (data not shown).
Discussion
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The present study provides evidence that circulating endothelial progenitor cells from bone marrow participate in neovascularization processes in the adult brain of mice after ischemia. In addition, circulating endothelial progenitor cells
contribute to the microvascular structure of the choroid
plexus.
Enlarged thin-walled vessels are termed “mother” vessels
and have been found in the pathological angiogenesis.20
Mother vessels can develop into small vessels by sprouting,
by invaginating, or by forming transluminal endothelial
bridges to structure smaller caliber daughter vessels and
glomeruloid bodies.20 In parallel, we observed that enlarged
and thin-walled blood vessels localized to the ischemic
boundary and that some of these vessels sprout or show
intussusception at the boundary of the ischemic lesion. These
data indicate neovascularization occurs in the adult ischemic
brain.11,20 Furthermore, colocalization of increased CBF with
neovascularization implies that these newly formed vessels
function. This is consistent with previous reports that functional imaging of stroke patients shows increased cerebral
blood flow and metabolism in tissue surrounding focal brain
infarcts.21 Our observation of a near absence of BrdU
immunoreactive cells in vessels of the contralateral hemisphere is consistent with that proliferation of the cerebral
endothelial cells ceases,8,9 whereas increases in proliferated
endothelial cells in the ischemic vessels may reflect angiogenesis. Formation of new vessels may arise from the
proliferation and migration of endothelial cells from the
adjacent tissue and from circulating endothelial progenitor
cells.
In the present study, using bone marrow transplantation
from transgenic mice in which constitutive lacZ expression
was regulated by an endothelial-specific promoter, Tie2, we
found that bone marrow recipient mice incorporated Tie2lacZ– expressing cells into the site of neovascularization
around infarct areas after ischemia. Double immunostaining
analysis shows that Tie2-lacZ immunoreactive cells had an
endothelial antigenic marker, indicating that marrow-derived
cells may differentiate into endothelial cells and contribute to
neovascularization. These data suggest that formation of new
blood vessels in the adult brain after stroke is not restricted to
angiogenesis but also involves vasculogenesis. Our findings
are consistent with the concept that vasculogenesis contributes to neovascularization in the adult muscle and heart.4
Interestingly, nonischemic mice with bone marrow transplantation showed incorporation of Tie2-lacZ– expressing
cells into the choroid plexus. The choroid plexus is composed
of a single layer of epithelial cells surrounding a central core
containing capillaries.22 It is believed that endothelial cells in
the choroid plexus do not proliferate.5 Our observations of
presence of Tie2-lacZ– expressing cells in the inner side of
the choroid plexus indicate that these cells are endothelial
cells on capillaries. Using male bone marrow cells, others
Endothelial Progenitor Cells in Ischemic Brain
287
have reported the presence of male bone marrow– derived
cells in the choroid plexus of female mice.23 Taken together,
these data suggest that circulating endothelial progenitor cells
contribute to the microvascular structure of the choroid
plexus. Recent studies suggest that active vascular recruitment and endothelial cells provide cues mediating neurogenesis in the brain.24,25 Neurogenesis takes place in the subventricular zone and the dentate gyrus throughout of the rodent
life.26,27 Observation of presence of circulating endothelial
progenitor cells in the choroid plexus suggest that these cells
may support neurogenesis in the subventricular zone and the
dentate gyrus by secreting growth factors associated with
neurogenesis, such as vascular endothelial growth factor or
brain-derived neurotrophic factor.24,25
Cells derived from bone marrow can migrate into the
brains of adult mice and differentiate into astrocytes, microglia, and neurons, indicating that bone marrow– derived
progenitor cells are reservoirs of normal tissues.4,23,28,29 An
absence of neuronal and glial markers for Tie2-lacZ– expressing cells in the brain of any transplant recipients suggests that
a subclass of bone marrow– derived progenitor cells may
contribute to differentiation of endothelial cells. In fact,
circulating endothelial progenitor cells have been isolated.2
Exogenous angioblasts might be used to augment compromised vascularization in ischemic brain. Our observations of
incorporation of circulating endothelial progenitor cells into
sites of neovascularization suggest that recruitment of circulating endothelial progenitor cells induced by focal cerebral
ischemia may be regulated by cytokines, soluble receptors,
and adhesive molecules released from the ischemic lesion.
Maximizing the contribution of circulating endothelial progenitor cells to new vessel formation by exogenous angioblasts or by augmentation of endogenous endothelial progenitor cells may provide another avenue for treatment of stroke.
Acknowledgments
This work was supported by National Institute of Neurological
Disorders and Stroke (NINDS) grants PO1NS23393, RO1NS33627,
and RO1NS38292 and National Heart, Lung, and Blood Institute
(NHLBI) grant RO1HL64766. The authors wish to thank Cecylia
Powers, Cynthier Roberts, Anton Goussev, and Qingjiang Li for
technical assistance.
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Bone Marrow-Derived Endothelial Progenitor Cells Participate in Cerebral
Neovascularization After Focal Cerebral Ischemia in the Adult Mouse
Zheng Gang Zhang, Li Zhang, Quan Jiang and Michael Chopp
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Circ Res. 2002;90:284-288; originally published online January 3, 2002;
doi: 10.1161/hh0302.104460
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