progressive intracerebral accumulation of ?-amyloid (A?) peptides and neurofibrillary tangles. The level of proangiogenic growth
factors and inflammatory mediators with proangiogenic activity is known to be elevated in AD brains which has led to the supposition
shown to be antiangiogenic. To determine whether angiogenesis is compromised in the brains of two transgenic mouse models of AD
layer. Altogether our data suggest that the brain of transgenic mouse models of AD does not constitute a favorable environment to
Alzheimer’s disease (AD) is an ever-increasing health concern
among the aging population. While the cause of the disease is
uncertain, there are two major neuropathological hallmarks
present in the brains of AD patients: the extracellular senile
plaques containing a core of ?-amyloid (A?) peptide and the
intracellular neurofibrillary tangles made of hyperphosphory-
lated microtubule-associated protein tau. The progressive accu-
mulation of A? in the brain is believed to produce the clinical
phenotype of AD and, moreover, soluble A? rather than depos-
ited/fibrillar A? is associated with dementia (Selkoe, 2008). The
events that lead to the pathological accumulation of A? peptides
in AD are the subject of active investigations. There is evidence
that alterations in A? clearance across the blood brain barrier
(BBB) plays a major role in brain A? accumulation (Zlokovic et
al., 2000; Bell and Zlokovic, 2009). Other functional cerebrovas-
cular alterations have been observed in AD and in transgenic
mouse models of the disorder. In particular, cerebrovascular
blood flow (CBF) disturbances that reproduce some of the CBF
alteration observed in AD patients have been well characterized
in transgenic mouse models of AD overexpressing A? (Iadecola,
2004). Interestingly, dysregulation of serum response factor and
initiate a pathogenic cascade resulting in hypercontractility of
cerebral arterioles, CBF reduction (Chow et al., 2007) and de-
creased A? clearance across the BBB, consequently facilitating
the pathological accumulation of A? and the progression of AD
(Bell et al., 2009).
Clearly, increasing evidence points to vascular damage as an
early contributor to AD. Vascular pathologies synergistically ex-
which is reflected in that fact that AD patients with cerebrovas-
cular disease express the clinical symptoms of dementia with
fewer AD pathological changes (Petrovitch et al., 2000; Hoffman
cardiovascular risk factors increase the risk of AD (Skoog et al.,
1996). Numerous structural and functional cerebromicrovascu-
1997; Perry et al., 1998; Suter et al., 2002; Bouras et al., 2006;
Kitaguchi et al., 2007) and reduced expression of MEOX2 has been
observed in AD brain endothelial cells resulting in aberrant angio-
In AD brains, the levels of proangiogenic growth factors
(VEGF, bFGF, NGF) are elevated (Siedlak et al., 1991; Kalaria et
al., 1998; Tarkowski et al., 2002; Peng et al., 2004; Mashayekhi
and Salehin, 2006) suggesting that angiogenesis may be stimu-
lated. Angiogenesis is tightly regulated by the balance between
This work was supported by National Institutes of Health Grant R01A619250. We thank Diane and Robert
TheJournalofNeuroscience,August25,2010 • 30(34):11251–11258 • 11251
proangiogenic and antiangiogenic factors and it remains unclear
whether angiogenesis is actually stimulated or inhibited in AD
brains. Since the growth of solid tumors, especially gliomas,
which are highly vascularized, is dependent on angiogenesis we
evaluated the growth and vascularization of orthotopically im-
planted gliomas in transgenic mouse models of AD (Tg APPsw
and Tg PS1/APPsw) to determine whether tumorally induced
angiogenic processes may be altered in the brains of these
Transgenic mouse models of AD. Tg2576 (Tg APPsw) (Hsiao et al., 1996),
littermates (used as control of Tg APPsw and Tg PS1/APPsw) were ob-
tained by crossing heterozygous male Tg PS1/APPsw with wild-type fe-
male F1 B6/SJL purchased from The Jackson Laboratory. Animals have
under specific pathogen free condition in ventilated racks with sterile
bedding, water and irradiated food. All animal studies involving mice
were approved by the Institutional Animal Care and Use Committee of
precursor protein (?APP) containing the Swedish mutation (K670N/
M671L) under the control of a hamster prion promoter. Tg PS1/APPsw
were originally obtained from a cross between Tg APPsw and Tg PS1
(mutant M146L) and display elevated levels of A? compared with Tg
APPsw mice, resulting in an accelerated AD phenotype. Tg APPsw, Tg
PS1/APPsw and wild-type littermates used in this study were 32 weeks
old. At that age, Tg APPsw have elevated brain levels of A? but do not
have yet A? deposits (Hsiao et al., 1996) whereas Tg PS1/APPsw have
substantially higher levels of A? compared with Tg APPsw and start to
develop few parenchymal A? deposits forming senile plaque like struc-
tures (Holcomb et al., 1998).
Animal experimental procedures. An orthotopically implanted GL261
murine glioma model was chosen for its known invasive properties and
its well documented induction of neoangiogenesis (Zagzag et al., 2000).
GL261 were grown in DMEM containing 10% serum and 1% penicillin/
streptomycin/fungizone mixture. GL261 cells (105cells in 2 ?l) were
wild-type littermates (n ? 7) that were anesthetized with a mixture of
ketamine (80 mg/kg) and xylazine (10 mg/kg). Tumor cells were im-
planted in the right frontal lobe with a 25 gauge needle at a depth of 3
monitored daily for any neurological change, weight loss and for their
21 d following the implantation of tumor cells and their brains fixed in
4% paraformaldehyde for pathological evaluations.
Tumor volume measurements. Following 24 h of fixation in 4% para-
formaldehyde at 4°C, mouse brains were embedded in paraffin and sec-
tioned in 5 ?m sections with a microtome. Tumor volumes were
the tumor following hematoxylin/eosin staining using an image analysis
software (ImagePro Plus, Media Cybernetics) as previously described
(Wagemakers et al., 2009).
von Willebrand factor immunostaining. von Willebrand factor (VWF)
immunostaining was performed on deparaffinized sections that were
treated with 20 ?g/ml proteinase K for 15 min at room temperature.
Sections were blocked with the Protein Blok solution from Dako and
incubated overnight at 4°C with a 1:2000 dilution of a rabbit anti-VWF
al., 2008). Revelation was ensured using the Vectastain Elite ABC kit
(Vector Laboratories). To quantify the level of angiogenesis, the surface
area occupied by blood vessels (immune-positive for VWF) in brain
tumor sections was measured by image analysis (ImagePro Plus, Media
Cybernetics) in the most densely vascularized area of each tumor as
reported previously for orthotopically implanted gliomas (Kunkel et al.,
2001). A minimum of three microscopic fields at 400? magnification
were analyzed per histologic slide and three to five slides randomly dis-
tributed throughout the tumor mass were analyzed per tumor which
represents an adequate number to evaluate tumoral vascularization
(Kunkel et al., 2001). An average surface area of blood vessels was calcu-
lated for each tumor and expressed as a percentage area of the micro-
scopic fields analyzed.
Preparation of A? peptide. Human recombinant A?1-42 (purity
?95%) was purchased from rPeptide. The lyophilized peptide was dis-
minimize the formation of ?-sheet structures and promote ?-helical
secondary structure. The peptide was allowed to air dry in a chemical
fume hood for 1 h at room temperature, followed by further drying in a
Speed Vac (Thermo-Savant) for 30 min. The resulting clear film was
resuspended in 100% DMSO to a concentration of 1 mM, followed by
aliquoting and storage at ?80°C. Such preparations of HFIP-treated
equivalent monomeric concentration) in endothelial cell culture me-
dium following a 24 h incubation at 37°C (Paris et al., 2005). Within the
manuscript, the concentrations of A? reported do not account for the
an A?1-42 monomer (molecular weight ? 4514.1 Da).
Proliferation and toxicity assays. Gl261 proliferation was evaluated us-
a water-soluble salt of tetrazolium (WST) into formazan by mitochon-
drial dehydrogenase (Biovision). Briefly, GL261 at a low cellular density
(550 cells/mm2) in a 96-well plate were treated with a dose range of
containing 10% fetal calf serum and 1% penicillin/streptomycin/fungi-
dehydrogenase) cytotoxicity detection kit (Roche) and 10 ?l of WST
reagent was added to each cell culture well. The cell culture plate was
incubated at 37°C, 5% CO2for 1 h and the conversion of WST to
formazan quantified at 430 nm whereas LDH released was monitored at
was identically repeated, except that the GL261 culture medium was
supplemented with 100 ng/ml EGF (Cytoskeleton Inc). The effect of
freshly solubilized HFIP-treated human recombinant A?1-42 was also
culture plate following 24 h incubation using the LDH cytotoxicity de-
In vitro angiogenesis assay using cocultures of GL261 and human brain
microvascular endothelial cells. Capillary network assays were performed
as we previously published (Paris et al., 2004a,b, 2005; Patel et al., 2008).
Brain Microvascular Endothelial Cells (HBMEC) were obtained from
Sciencell and grown in EC medium supplemented with 5% serum, 1%
penicillin/streptomycin and 1% Endothelial Cell Growth Supplement
(Sciencell). Briefly, GL261 cells were plated onto 24-well plates and
grown in DMEM containing 10% serum and 1? penicillin/streptomy-
cin/fungizone mixture. Twenty-four hours after plating, confluent
GL261 cells were covered with 400 ?l of Matrigel (BD Bioscience). After
Matrigel polymerization at 37°C, 1 ml of DMEM medium containing
of the Matrigel layer was replaced with 500 ?l of EC medium containing
HBMEC (7.5 ? 104cells/ml) and treated with a dose range of HFIP-
treated (cf. above) human recombinant A?1-42 (2.5–10 ?M). Control
peptide. Following 24 h of incubation at 37°C and 5% CO2, capillary
network lengths were quantified by image analysis using the Image Pro
Plus software (Media Cybernetics). Capillary network formation exper-
iments were performed in triplicate and four random microscopic fields
(10? objective) per well were used to quantify the length of tube like
structures. Therefore, 12 microscopic fields were used for each culture
condition and results were expressed as an average capillary length ob-
served per 10? microscopic field in ?m.
11252 • J.Neurosci.,August25,2010 • 30(34):11251–11258Parisetal.•AngiogenesisandAlzheimer’sDisease
the cerebellum) from a 73-week-old Tg PS1/APPsw and wild-type litter-
mate were weighted and homogenized by sonication in ice-cold PBS
in the HBMEC culture medium (i.e., 1885 ?g of brain extract per milli-
liter of culture medium) surrounding HBMEC at the time of plating on
Matrigel. HBMEC were incubated for 24 h before evaluating capillary
morphogenesis by image analysis as indicated above.
A significant inhibition of tumor growth was observed in Tg
APPsw and Tg PS1/APPsw compared with their nontransgenic
littermates. Tumor volumes were reduced on average by ?60%
in Tg APPsw and by 50% in Tg PS1/APPsw compared with their
wild-type littermates (Fig. 1). Tumors in wild-type animals were
surrounded by numerous small satellite tumors compared with
suggesting a more invasive growth pattern of glioma tumors in
sity was significantly reduced by ?60% in Tg PS1/APPsw and by
?50% in Tg APPsw compared with their wild-type littermates
nially implanted in transgenic mouse models of AD.
density and observed that A? did not affect GL261 proliferation
a 24 h incubation period, under regular culture conditions (Fig.
and observed that A? did not induce cellular toxicity in GL261
cells (data not shown) showing that the inhibition of GL261 tu-
related to a direct cytotoxic effect of A? in tumor cells.
wild-type mice on in vitro angiogenesis
Brain homogenates from 73-week-old Tg PS1/APPsw and wild-
type littermate were diluted to 1% (volume to volume) in the
cent parenchyma representing the maximum cross-sectional area of the intracranial glioma
tive) depicting the presence of peritumoral satellites in wild-type mice suggestive of a more
TgAPPsw( p?0.005),TgPS1/APPsw( p?0.03)butnosignificantdifferencebetweenthe
0.04). Post hoc analyses show that vascularization is significantly reduced in tumor sections
from Tg APPsw ( p ? 0.03) and Tg PS1/APPsw ( p ? 0.02) compared with wild-type litter-
A, Representative pictures (60? objective) showing VWF immunostaining
Parisetal.•AngiogenesisandAlzheimer’sDiseaseJ.Neurosci.,August25,2010 • 30(34):11251–11258 • 11253
culture media surrounding HBMEC at the time of plating on
Matrigel. Following 24 h of culture, the formation of capillary
networks was analyzed by image analysis. A 55% reduction in
capillary network length was observed when Tg PS1/APPsw brain
homogenate was added to the culture medium of HBMEC com-
from Tg PS1/APPsw can impair the formation of capillary-like
networks in vitro (Fig. 4). No difference in LDH released in the
culture media surrounding HBMEC was observed between Tg
PS1/APPsw and wild-type brain homogenates treatments (data
is not directly cytotoxic to HBMEC.
by coculturing human brain microvascular cells (HBMEC) with
GL261 since angiogenesis is triggered by tumor expression of
various proangiogenic factors in gliomas (Kaur et al., 2004). We
observed that glioma GL261 cells stimulate the formation of
capillary-like networks by HBMEC (Fig. 5) and that freshly sol-
ubilized human recombinant A?1-42 dose-dependently inhibits
the formation of capillary-like structures stimulated by GL261
cells. In these cocultures, cellular contact between HBMEC and
GL261 cells was precluded by a layer of Matrigel suggesting that
diffusible factors produced by GL261 were acting on HBMEC to
stimulate the formation of capillary-like network and that A? is
able to inhibit the activity of these diffusible factors. LDH release
was assessed in the culture medium surrounding the capillary
networks and no increased LDH was observed following A?
treatment (Fig. 5) showing that the inhibition of angiogenesis
induced by A? was achieved without inducing direct cellular
toxicity to either GL261 cells or HBMEC.
diators with known angiogenic stimulatory activity are upregu-
lated in AD brains (Siedlak et al., 1991; Kalaria et al., 1998;
2006), which could suggest that excessive or aberrant angiogenic
angiogenic proteins such as VEGF and TIMP-1 in isolated AD mi-
ture is in a “proangiogenic state” in AD (Thirumangalakudi et al.,
2006). Others have suggested that anti-angiogenic therapies
could be beneficial since nonsteroidal anti-inflammatory drugs
(NSAIDs) and statins, which can inhibit angiogenesis, have been
GL261 cells ( p ? 0.001). ANOVA reveals no statistically significant main effect of A? dose
( p ? 0.788) showing that A?1-42 does not affect the proliferation of GL261 cells. B, The
culture medium. Slight toxicity of the positive control (celastrol) was observed ( p ? 0.01).
brain homogenate from wild-type and Tg PS1/APPsw mice. B, Histogram representing the
observed ( p ? 0.01) when HBMEC were cultured in the presence of Tg PS1/APPsw brain
11254 • J.Neurosci.,August25,2010 • 30(34):11251–11258 Parisetal.•AngiogenesisandAlzheimer’sDisease
associated with beneficial outcomes in AD patients (Vagnucci
and Li, 2003). However, recent clinical trials with statins or
NSAIDs have failed to provide evidence of protection in AD pa-
tients (Breitner et al., 2009; McGuinness et al., 2009). In AD
regions that immediately surround them, suggesting that amy-
loid plaques exclude capillaries or lead to their degeneration
(Kawai et al., 1990). Endothelial cell activation has been docu-
mented in an AD mouse model (APP23) as evidenced by in-
creased ?3-integrin expression restricted to ?-amyloid-positive
occur in this transgenic mouse model of AD.
Angiogenesis is a tightly regulated process in which the out-
and anti-angiogenic factors. Endostatin, a fragment of collagen
XVIII with potent antiangiogenic properties, has been shown to
accumulate in perivascular and cortical plaques of AD patients
multitude of in vitro and in vivo experimental models and antag-
giogenesis (Paris et al., 2004a,b, 2005;
Patel et al., 2008). Since VEGF and bFGF
are critical not only to initiate angiogene-
cerebrovasculature, our data suggest that
by antagonizing angiogenic growth fac-
cerebrovascular damage possibly leading
cated such findings (Cantara et al., 2004;
Donnini et al., 2006, 2010; Boscolo et al.,
2007; Drago et al., 2007; Hayashi et al.,
2009; Solito et al., 2009) and the hypothe-
sis that an aberrant angiogenic process
may contribute to AD dementia is now
emerging (Watson et al., 2005; Wu et al.,
2005; Zlokovic, 2005; Deane and Zlokovic,
To determine whether the overall en-
vironment is pro- or anti-angiogenic in
models of the AD brain, we examined the
growth of a tumor type that is critically
dependent upon angiogenesis. Specifi-
cally, we assessed the growth and vascu-
larization of an orthotopically implanted
glioma model in Tg APPsw and Tg PS1/
APPsw mice, two transgenic mouse mod-
ciated with a high degree of angiogenesis
(Plate and Risau, 1995). During tumori-
genesis, angiogenesis is triggered by tu-
mor expression of various proangiogenic
factors (Hanahan and Folkman, 1996)
which are induced by physiological stim-
uli such as hypoxia (Kaur et al., 2004).
Pathological evaluation of the tumor
samples shows that glioma growth is in-
hibited in the brains of Tg APPsw and Tg
PS1/APPsw compared with their wild-
type littermates. In addition, a decreased
VWF immunostaining in glioma tumors
from transgenic mouse models of AD was observed indicating a
decreased angiogenesis in glioma tumors. Although tumoral
angiogenesis is associated with rapid endothelial cell turnover
compared with healthy brain tissue, our data suggest that angio-
brain homogenate to HBMEC cultures on a Matrigel layer
impairs their differentiation into capillary networks further
suggesting that the brain environment of Tg PS1/APPsw mice
does not support angiogenesis.
Pathological levels of A? are clearly antiangiogenic both in
vitro and in vivo (Paris et al., 2004a,b; Donnini et al., 2010) and
appear to antagonize VEGF-R2 (the main receptor involved in
angiogenesis) in human brain microvascular endothelial cells
reduction of both soluble and insoluble A? is associated with
increased angiogenesis in Tg PS1/APP mice (Biscaro et al., 2009)
which supports the suggestion that A? is an angiogenic suppres-
sor in the brain. We therefore investigated the impact of A? on
glioma-induced angiogenesis using a coculture model using
(GL261).ANOVArevealedasignificantmaineffectofGL261cells( p?0.001)andofA?doses( p?0.001)oncapillarynetwork
cocultures ( p ? 0.001) showing that GL261 glioma cells stimulate capillary like network formations by HBMEC. In addition,
Effect of human recombinant A?1-42 on the angiogenesis stimulated by GL261 glioma cells. A, Representative
Parisetal.•AngiogenesisandAlzheimer’sDiseaseJ.Neurosci.,August25,2010 • 30(34):11251–11258 • 11255
GL261 cells and HBMEC. We found that GL261 glioma cells
stimulate capillary like network formation by HBMEC whereas
gross effect of A? even at high doses on GL261 glioma cell pro-
liferation and survival in vitro suggesting that the reduction of
tumor growth observed in transgenic mice overexpressing A? is
not related to a direct effect of A? on tumoral cell viability or
proliferation. In old Tg APPsw brains (21–23 months), the total
AD brain and is ?26,000 pmol/g (Kawarabayashi et al., 2001).
On average, the weight of an adult mouse brain is ?0.451 g for a
is uniformly distributed in the tissue, we can estimate the total
brain A? concentration to be ?106 ?g/ml in Tg APPsw mice
which is well within the range of the A? concentrations used in
our in vitro experiments showing that A? impairs the angiogen-
species responsible for the antiangiogenic activity of A? remains
unclear. We have determined previously that preparation of A?
peptides containing soluble A? oligomers are more potently an-
tiangiogenic than preparations that do not contain soluble A?
oligomers. In particular, we have shown that the mutant Dutch
its ability to form soluble A? oligomers more rapidly than wild-
type A? at low concentration (Paris et al., 2005).
Decreased capillary density has been documented in AD
brains (Hashimura et al., 1991; Kimura et al., 1991; Bue ´e et al.,
1997; Fischer et al., 1997; Bouras et al., 2006; Kitaguchi et al.,
2007) and in the brains of different AD mouse models (Paris et
al., 2004b; Lee et al., 2005; Kouznetsova et al., 2006; Meyer et
al., 2008; Takeuchi et al., 2008; Hayashi et al., 2009) despite in-
creased levels of angiogenic growth factors (Kalaria et al., 1998;
Tarkowski et al., 2002; Lopez-Lopez et al., 2007; Bu ¨rger et al.,
2009) suggesting that the angiogenic process may be aborted in
AD brains. We propose that A? may be one of the key factors
lar pathologies in AD by impairing vascular maintenance and
preventing the regeneration of the endothelium in response to
vascular injuries. The antiangiogenic activity of A? may explain
why AD patients appear particularly vulnerable to cerebrovascu-
lar infarcts (Snowdon et al., 1997). Capillary rarefaction appears
to contribute to the pathological and clinical presentation of AD
severity of dementia in AD (using the Clinical Dementia Rating
Scale) and with a higher burden of A? deposits and neurofibril-
lary tangles (Bouras et al., 2006).
Interestingly, donepezil, an acetylcholinesterase inhibitor fre-
quently prescribed to AD patients, has been shown to stimulate
brain angiogenesis in mice (Narimatsu et al., 2009). Overexpres-
brain of a mouse model of AD has been shown to promote a
significant recovery of vessel density in the hippocampus and to
reverse cognitive deficits induced by A? (Takeuchi et al., 2008).
factor I (Carro and Torres-Aleman, 2006; Lopez-Lopez et al.,
2007) or VEGF (Spuch et al., 2010) increase brain vascular den-
sity and improve cognition in transgenic mouse models of AD
Altogether our data show that the brains of transgenic mouse
models of AD overexpressing A? do not constitute an adequate
environment to sustain angiogenesis despite increased intracere-
Bu ¨rger et al., 2009). For this study, 32-week-old animals were
used. At that age, Tg APPsw mice do not present ?-amyloid
deposits whereas Tg PS1/APPsw mice have only a few A? depos-
in the absence of significant A? deposition is in agreement with
our previous observations showing that soluble forms of A? are
antiangiogenic whereas fibrillar/aggregated forms of A? are not
(Paris et al., 2005) and further suggest that angiogenesis inhibi-
tion may be an early event contributing to cerebrovascular dys-
functions in AD. Interestingly, there are reports of reduced
incidence of cancer in AD patients (Tirumalasetti et al., 1991;
2010) which may be the result of defective angiogenic functions
in these patients suggesting a clinical or potentially therapeutic
aspect to this effect.
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