Enhanced Ab1–40Production in Endothelial Cells
Stimulated with Fibrillar Ab1–42
Jayakumar Rajadas4*, Wenchao Sun4, Hai Li4, Mohammed Inayathullah4, Damiano Cereghetti4,
Aaron Tan5, Valeria de Mello Coelho2, Francis J. Chrest3, John W. Kusiak3, Wanli Wei Smith3,
Dennis Taub2, Joseph C. Wu6,7, Joseph M. Rifkind1*
1Molecular Dynamics Section, National Institute on Aging, Baltimore, Maryland, United States of America, 2Laboratory of Immunology, National Institute on Aging,
Baltimore, Maryland, United States of America, 3Research Resources Branch, National Institute on Aging, Baltimore, Maryland, United States of America, 4Biomaterials
and Advanced Drug Delivery Laboratory, Stanford University School of Medicine, Stanford, California, United States of America, 5Centre for Nanotechnology and
Regenerative Medicine, UCL Division of Surgery and Interventional Science, UCL Medical School, University College London, United Kingdom, 6Department of Medicine,
Stanford University School of Medicine, Stanford, California, United States of America, 7Institute for Stem Cell Biology and Regenerative Medicine, Stanford University
School of Medicine, Stanford, California, United States of America
Amyloid accumulation in the brain of Alzheimer’s patients results from altered processing of the 39- to 43-amino acid
amyloid b protein (Ab). The mechanisms for the elevated amyloid (Ab1–42) are considered to be over-expression of the
amyloid precursor protein (APP), enhanced cleavage of APP to Ab, and decreased clearance of Ab from the central nervous
system (CNS). We report herein studies of Ab stimulated effects on endothelial cells. We observe an interesting and as yet
unprecedented feedback effect involving Ab1–42fibril-induced synthesis of APP by Western blot analysis in the endothelial
cell line Hep-1. We further observe an increase in the expression of Ab1–40by flow cytometry and fluorescence microscopy.
This phenomenon is reproducible for cultures grown both in the presence and absence of serum. In the former case, flow
cytometry reveals that Ab1–40accumulation is less pronounced than under serum-free conditions. Immunofluorescence
staining further corroborates these observations. Cellular responses to fibrillar Ab1–42 treatment involving eNOS
upregulation and increased autophagy are also reported.
Citation: Rajadas J, Sun W, Li H, Inayathullah M, Cereghetti D, et al. (2013) Enhanced Ab1–40Production in Endothelial Cells Stimulated with Fibrillar Ab1–42. PLoS
ONE 8(3): e58194. doi:10.1371/journal.pone.0058194
Editor: Hemachandra Reddy, Oregon Health & Science University, United States of America
Received August 14, 2012; Accepted February 4, 2013; Published March 7, 2013
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This research was in part supported by the Intramural Research Program the National Institute of Health, National Institute on Aging and National
Institute of Health HL095571. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (J. Rajadas); email@example.com (J. Rifkind)
Alzheimer’s disease (AD) is characterized by neuronal de-
generation and accumulation of senile plaques, which are
composed of amyloid-b (Ab) peptides predominantly consisting
of 40 and 42 amino acids [1,2]. The excessive accumulation of Ab
peptides in AD may be due to enhanced endoproteolytic cleavage
of membrane bound amyloid precursor protein (APP), over-
expression of APP and/or decreased clearance of Ab from the
central nervous system (CNS) [3–5].
Postmortem analyses of AD subjects reveal that amyloid plaques
in the brain suffuse vascular cells in addition to the parenchymal.
The implications of this vascular infiltration for AD has been less
well studied than the parenchymal Ab, but has generated
considerable interest with studies that b-amyloid fibrils accumulate
in small arteries, arterioles and capillaries of the brain [6–8].
Cerebrovascular amyloid toxicity generally manifests itself in the
breach of the blood-brain-barrier and enhanced inflammation in
the cerebrovasculature [9,10].
The mechanism for the onset of pathological vascular changes
has yet to be elucidated . Two mechanisms that have been
proposed involve: (1) The production of excess superoxide by
amyloid-b induced oxidative stress [12,13] and (2) the formation of
amyloid aggregates whose resistance to protease degradation turns
them into cellular ‘‘tombstones’’ that impair blood flow and
cellular function [14,15].
The oxidative stress mechanism was used to explain an in vitro
investigation where the interaction with Ab fibrils resulted in the
endothelial lining of the rat aorta undergoing rapid damage
leading to exposure of smooth muscle cells and connective tissue
. In agreement with these observations, it has been shown that
antioxidant treatment and superoxide dismutase (SOD) treatment
can reduce damage of endothelial cells caused by amyloid-
The ‘‘tombstone’’ mechanism is consistent with biochemical
and biophysical studies of synthetic Ab peptides indicating that the
more toxic Ab peptides ending at residue 42 aggregate more
rapidly than peptides of 39 or 40 amino acids [19–22]. This
feature of Ab1–42 makes it less susceptible to proteolytic
degradation [23–25]. Further support for this mechanism comes
from studies on the APP mutation found in HCHWA-Dutch type,
which results in the production of Ab with enhanced tendency to
aggregate relative to that of wild type Ab. Fibril formation in this
mutation is limited to the cerebrovasculature and amyloidosis
leads to cerebral hemorrhage [26,27].
PLOS ONE | www.plosone.org1March 2013 | Volume 8 | Issue 3 | e58194
APP synthesis and processing to Ab normally takes place only to
a limited extent in endothelial cells . It has, however been
shown that amyloids can alter the expression pattern of specific
proteins. Thus, the accumulation of Ab1–42in lysosomes down
regulates the catabolism of APP resulting in enhanced production
of amyloidogenic fragments . Production of individual iso-
forms of Ab induced by the same isoform has been shown in
smooth muscle cells . On the basis of these studies we
investigated the possibility that amyloid fibrils can induce synthesis
of more APP and amyloid of its other isoforms. Such a process
would provide a synergistic mechanism whereby amyloid fibrils in
circulation potentiate damage to the blood brain barrier endo-
thelium. For this purpose we used synthetic, preformed Ab1–42
fibrils and established the resultant accumulation of APP and Ab1–
Materials and Methods
Media for cell culture was obtained from Invitrogen (Carlsbad,
CA). Synthetic Ab1–40, Ab1–42and monoclonal antibody against
Ab1–40were purchased from Biosource International (Camarillo,
CA). Fluorescein and phycoerythrin labeled streptavidin were
purchased from PharMingen (Becton Dickinson, San Jose, CA.).
Preparation of Amyloid Fibrils
Synthetic Ab1–42 was disaggregated by pretreatment with
trifluoroacetic acid (TFA) followed by treatment with trifluor-
oethanol (TFE) three times to remove traces of TFA. After each
step, solvents were evaporated to form a film. Ab1–42fibrils were
formed from disaggregated Ab1–42 by following a known pro-
cedure . Briefly, Ab1–42(250 mM) was dissolved in phosphate
buffered saline, (PBS), pH 7.5, and polymerized in Eppendorf
tubes (1.5 ml), at 37uC for 2 days. Newly formed fibrils were then
separated from monomeric peptides by centrifugation at 4uC for
90 min at 15,000 rpm, using a Sorvall RC-5B centrifuge. The
fibril pellet was re-suspended in PBS and used for in vitro studies
Cell Culture and Flow Cytometry
Human endothelial (Hep-1) cell line is a kind gift from Dr. John
Kusiak at the National Institute on Aging. This is an immortal
human endothelial cell line derived from an adenocarcinoma of
the liver . Cells were grown in DMEM medium containing
10% fetal bovine serum (FBS Prime, Biofluids), 16MEM non-
essential amino acids, 2 mM L-glutamine, 100 units/ml penicillin,
and 100 mg/ml streptomycin in a humidified atmosphere at 37uC
in the presence of 5% CO2. 16106cells were incubated with
varying concentrations of Ab1–42fibrils in the presence or absence
of FBS for 12 hrs. Cells were then harvested, washed with PBS,
fixed in 2% glutaraldehyde, and probed with biotinylated
monoclonal antibodies against Ab1–40. This antibody detects low
molecular weight Ab1–40, but not fibrils. Cells were counter-
labeled with 20 mg/ml phycoerythrin-streptavidin for 15 to
30 min. Labeled cells were analyzed on a FACScan flow
cytometer (Becton Dickinson Immunocytometry Systems, San
Jose CA) equipped with a 15 mW argon laser. Phycoerythrin
labeled cells were excited with 488 nm light from the argon laser
and emitted fluorescence was collected using the FL-2 (585/42)
bands pass filter. A total of 20,000 events were analyzed for each
sample. The amount of Ab1–40present in the endothelial cells was
determined by mean fluorescence values. Light scatter signals were
also collected in the forward and side scatter detectors. Experi-
ments were repeated three times. The mean and standard error of
the mean values were calculated.
Cells grown on glass cover slips were incubated at 37uC for
12 hrs, and then washed with PBS. Cells with and without Ab
treatment were subsequently fixed in 2% glutaraldehyde for
10 min and washed with 100 mM glycine in PBS containing 2%
bovine serum albumin. After washing, samples were incubated for
1 hr with a biotin-conjugated anti-Ab1–40antibody (1:500 dilution)
at room temperature, washed with PBS and stained with FITC-
streptavidin conjugate. Immunofluorescence images were cap-
tured with an Axiovert S100 microscope (Zeiss, Munich,
Germany). Images were recorded at the same exposure time
using a digital SPOT camera (Diagnostic Instruments, Sterling
Cells were lysed in 300 ml of 20 mM HEPES, pH 7.4,
supplemented with 2 mM EGTA, 50 mM b-glycerol phosphate,
1% Triton X-100, 10% glycerol, 1 mM dithiothreitol (DTT),
1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 10 mg/
ml aprotinin, 1 mM Na3VO4, and 5 mM NaF. The resulting
lysates were resolved on 4–15% SDS Tris-glycine gels (30 mg/
lane) and transferred onto polyvinylidene difluoride membranes
(Millipore, Bedford, MA). The membranes were blocked in TBST
(10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20)
containing 5% non-fat milk and then probed with the following
primary antibodies: monoclonal mouse anti-human APP (8E5
Elan Pharmaceuticals; 1:500 dilution), monoclonal mouse anti-
human endothelial nitric oxide synthase (eNOS) (BD Biosciences;
1:2500 dilution), monoclonal mouse anti-human nNOS (BD
Biosciences; 1:2500 dilution), rabbit polyclonal anti-caveolin 1
antibody (ab2910 abcam; 1:2000 dilution) and rabbit polyclonal
anti-LC3B antibody (ab48394 abcam; 1:1000 dilution). Proteins
were detected with HRP conjugated secondary antibodies and
enhanced chemiluminescence (ECL) reagents (NEN Life Science,
Boston, MA). The blot was quantitated by densitometric analysis
and the difference between treated and control groups were
analyzed by unpaired t test (tails=2) using Excel software.
APP Upregulation by Fibrillar Ab1–42
Ab1–42fibrils were prepared from synthetic Ab1–42peptides and
morphologically characterized by TEM (Fig. 1A). Western blotting
was used to determine whether Ab1–42fibrils can increase synthesis
of APP. For this purpose human endothelial cells (Hep-1) were
incubated with Ab1–42fibrils for 12 hrs and then harvested in
a lysis buffer for Western blotting. As shown in Fig. 1B and C, the
amount of APP was increased in cells incubated with Ab1–42in
a dose dependent manner.
Ab1–40Production in Amyloidic Condition
The association of the increased APP synthesis with an increase
in Ab was investigated in the same cell model. It was possible to
discriminate between newly synthesized amyloids and the Ab1–42
used to treat Hep-1 cells by using antibodies that are specific for
Ab1–40, which only detects low molecular weight Ab1–40, but not
fibrils. The latter sequence accounts for about 90% of newly
processed amyloid but no cross reactivity between Ab1–42and
Ab1–40was observed, in agreement with previous reports .
The flow cytometry histograms, of cultures grown with and
without FBS (Fig. 2A and B, respectively), show the amyloid-
Stimulation of Ab Synthesis in Endothelial Cells
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induced accumulation of Ab1–40, which is further accentuated by
the stress associated with the absence of serum. In both cases, it is
clear that Ab1–42fibrils increase Ab1–40synthesis in a concentration
dependent manner as shown by the plots of the mean fluorescence
in Fig. 3. The concentration dependence further highlights the
effect of stress associated with the absence of serum. Thus, in the
presence of serum a gradual raise in fluorescence takes place over
the range of amyloid concentrations tested (Fig. 3A), while more
dramatic changes occur in the absence of serum (Fig. 3B) with the
relative fluorescence increasing from 250 to 800 at the lowest
concentration tested (1 mM). At higher concentrations only minor
additional increases in fluorescence are detected.
Because the antibody used only detects low molecular weight
amyloid, the observed Ab1–40cannot be derived from cleavage of
added Ab1–42fibrils. To confirm that a cleaved fibril cannot be
detected, Hep-1 endothelial cells were treated with 20 mM
protofibrillar Ab1–40(10 hrs aged peptide) and analyzed by flow
cytometry. Only a negligible increase in Ab1–40was observed and
the fluorescence histogram was similar to that of control (data not
Flow cytometry provides scattering data in addition to the
fluorescence. The intensity light scattered at small angles (0.5–2.0u)
from the incident laser beam (referred to as forward scattering) is
proportional to the cell size. The side scattering at larger angles is
a measure of structural changes such as granulated structure on
the membrane or cytoplasm that increase the scattering.
Examination of changes in scattering properties of Ab1–42treated
cells revealed no difference in the forward scattering consistent
indicating no change in cell size. On the other hand, changes in
the granulated structure were apparent as evidenced by an
increase in the intensity of side scattering (Fig. 4), which was even
more pronounced in the absence of serum.
Morphological changes as well as increased production of Ab1–
40in Ab1–42treated cells were further corroborated by light and
Figure 1. Fibrillar Ab1–42induced APP upregulation in Hep-1
cells. Morphology of fibrillar Ab1–42was confirmed by TEM (A).Cells
were untreated or treated with 2 mM, 10 mM, 20 mM Ab1–42for 24 hrs.
Cell lysates were subjected to Western blot analysis using 8E5 antibody
(B). The blot was quantitated by densitometric analysis (C). The fold
increase of APP production compared to untreated control cells were
shown as mean 6 SD (n=4). *, P,0.05; **, P,0.01 compared to control.
Figure 2. Detection of fibrillar Ab1–42 induced Ab1–40 pro-
duction by flow cytometry. For the analysis of Ab1–40synthesis in
amyloidic condition, Hep-1 cells were treated with fibrillar Ab1–42in the
presence (A) or absence (B) of serum, stained with anti Ab1–40
antibodies (biotinylated) and streptavidin conjugated phycoerythrin.
Histograms are shown for the control with no added amyloid (ctrl) and
for cells incubated with various concentrations of Ab1–42.as indicated in
Stimulation of Ab Synthesis in Endothelial Cells
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confocal microscopy (Fig. 5). As found by flow cytometry, no effect
was observed by microscopy when the endothelial cells were
treated with Ab1–40instead of Ab1–42(Fig. 5A and B). In contrast,
shape changes in some of the cells as well as numerous intracellular
and superficial granular patches (tentatively attributed to big
amyloid aggregates) were clearly visible by light microscopy when
cells were treated with Ab1–42fibrils (Fig. 5C and D).
Treated and non-treated endothelial cells were stained with
antibodies against Ab1–40and observed under confocal microsco-
py (Fig. 5E, F, G, H). A fluorescence signal was only detected in
those cells that were exposed to Ab1–42(Fig. 5G and H).
Ab1–42Treatment Affects eNOS Expression and
To further study the cellular response to Ab1–42treatment, we
examined the protein levels of the nitric oxide synthases (NOS)
which catalyze the production of NO, a known modulator of CNS
vascular and neuronal function. After the cells were treated with 5
and 10 mM fibrillar Ab1–42, Western blot of different NOS
isoforms showed a mild but statistically significant increase of
endothelial NOS (eNOS) and no change in neuronal NOS
(nNOS) level (Fig. 6A and B). Inducible NOS (iNOS) was not
detected with or without treatment (data not shown). Since
caveolin-1 is shown to interact with eNOS within endothelial
plasmalemmal caveolae and affect eNOS function , we also
examined caveolin-1 protein level but found no change (Fig. 6A
and B). Since autophagy is induced under conditions of cell stress
and shown to be involved in APP processing , we studied the
state of autophagy after Ab1–42 treatment. Increased ratio of
LC3B-II/I (Fig. 6A and C), a reliable marker of autophagosomes,
indicated activation of autophagy.
Major attention has been devoted to the role played by
neuronal cells in AD development. Most of the amyloid found in
AD subjects is produced in the brain and contained in plaques
with a high content of highly aggregated amyloids. However,
cognitive function does not generally correlate with the level of
cerebral plaques. It was only in recent years that the contribution
of the cerebrovascular system has been appreciated [37–39]. In
this respect, it should be noted that a large proportion of AD
patients have cerebral amyloid angiopathy (CAA) involving
vascular amyloidosis within the cerebral circulation. It has in fact
been proposed that vascular pathology and the resultant impair-
ment of oxygen delivery to the brain may play a primary role in
the development of AD . In this study, we have delineated
a new role for the cerebrovascular system involving Ab1–42fibril
induced APP synthesis in the human endothelial cell line Hep-1.
We first demonstrate that incubation of endothelial cells with
fibrillar Ab1–42results in elevated levels of the APP (Fig. 1). Using
an antibody specific for Ab1–40, we also report an increase in the
concentration of Ab1–40peptides. This finding is indicated both by
flow cytometry (Fig. 2 and 3) and confocal microscopy (Fig. 5E, F,
G, H). The increased levels of APP together with increased Ab1–40
cannot be explained just by altered processing of APP and requires
increased synthesis of APP by the endothelial cells.
Such an effect would seem to require that the Ab1–42fibrils are
endocytosed by cells. Evidence for amyloid uptake and transcytosis
in endothelial cells was provided more than a decade ago .
Among the potentially physiologically relevant amyloid receptors
that can be involved in amyloid uptake, the RAGE (receptor for
advanced glycation end products) [42–44] was shown to bind Ab1–
40even in the fibrillar form with a high affinity [45,46].
Support, from our data, for a contribution of stress to the
observed increase in APP and Ab comes from the augmentation of
the effects under serum-deprived conditions . An analogous
stimulation of amyloid production has been reported  in
serum-deprived human primary neuron cultures.
Altered cellular function can be triggered by the aggregation
state of the amyloids in addition to stress. Our observation that
only Ab1–42fibrils but not Ab1–40fibrils induce cellular changes
implies that this phenomenon is triggered by the highly aggregated
fibrillar form that is favored by the Ab peptide ending at residue
42 . Additional studies will be required to explain how the
cellular changes produced by stress and/or amyloid aggregation
induce APP synthesis and its processing. Two processes induced by
exogenous Ab fibrils are likely to be involved: inflammatory
response and oxidative stress. As for increased APP synthesis, we
have previously shown that cellular uptake of fibrillar Ab induces
interleukin-1a (IL-1a) expression . The link between IL-1a
and APP can be found in an earlier study which showed an IL-1a
Figure 3. The mean fluorescence values obtained from the flow
cytometry histogram were plotted against incubated Ab1–42
concentration in the presence of serum (A) and under serum
deprived condition (B).
Stimulation of Ab Synthesis in Endothelial Cells
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mediated upregulation of APP at the translational level .
Interestingly, the APP mRNA level was also unchanged after the
Hep-1 cells were treated with Ab1–42(Rajadas unpublished data)
indicating the increase of APP could also be translational in our
study. As for increased Ab production, there is ample evidence
that in neuronal cells, Ab accumulation and oxidative stress, each
accelerating the other, generate a vicious circle of more Ab
production and oxidation . Based on our observation, it is
possible that the similar process is also taking place in endothelial
cells. This notion is supported by an Ab immunotherapy study
Figure 4. The morphological changes of Hep-1 cells incubated with Ab1–42in the presence and absence of serum analyzed by
scattering data. The effect of Ab1–42concentration (A, D: 0 mM; B, E: 5 mM; C, F: 10 mM) in both forward and side scatter values is given in the dot
Stimulation of Ab Synthesis in Endothelial Cells
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which indicated that Ab depositions in the brain parenchyma and
blood vessels occur independently .
Once inside the cell, the amyloid can affect many other cellular
responses. One of the cellular responses we observed is the
upregulation of eNOS (Fig. 6) which is consistent with a previous
study showing a Ca2+dependent induction of eNOS by hydrogen
peroxide in endothelial cells. This upregulation has been
attributed to a compensatory mechanism for increased superoxide
formation [54,55]. Elevated eNOS expression has been observed
in cardiovascular and other vascular pathologies, wherein in-
creased levels of ROS have been detected . Another cellular
response we observed is increased autophagy (Fig. 6). Since
autophagy is involved in APP processing as a protective mecha-
nism , this response is expected and also explains the increased
Ab1–40production. The intricate relations between oxidative stress
and autophagy and its implication in AD vascular pathogenesis
awaits further elucidation.
The increased cellular granulation indicated both by changes in
side-scattering in the flow cytometry experiment (Fig. 4) as well as
by microscopy (Fig. 5) seem to indicate damage to the cells. Thus,
coupled with the altered cellular function, and the new synthesis of
APP and its metabolism, amyloid toxicity results in damage to the
In conclusion, our observation of increased APP and amyloid in
ECs incubated with Ab1–42fibrils establishes, for the first time, that
the exposure of endothelial cells to Ab1–42fibrils results in an
Figure 5. Ab1–40expression monitored by immunofluorescence microscopy. Endothelial cells were incubated with different concentrations
of fibrils for 12 hrs and processed as described in the materials and method. Phase contrast (left panels) and fluorescence images (right panel) were
taken showing the same field.
Stimulation of Ab Synthesis in Endothelial Cells
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elevated amyloid load in endothelial cells. This process provides
a new potential pathway for amyloidosis with these newly formed
amyloids in the endothelial cells able to contribute to both vascular
amyloidosis and/or parenchyma amyloidosis.
We thank Eliza Anna Ruben and Zhen Cheng for help in editing the
Conceived and designed the experiments: J. Rajadas, W. Sun, J. Rifkind.
Performed the experiments: J. Rajadas W. Sun HL MI VMC FJC W.
Smith. Analyzed the data: J. Rajadas W. Sun HL MI DC. Contributed
reagents/materials/analysis tools: AT JWK DT JCW J. Rifkind. Wrote the
paper: J. Rajadas W. Sun J. Rifkind.
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Stimulation of Ab Synthesis in Endothelial Cells
PLOS ONE | www.plosone.org8March 2013 | Volume 8 | Issue 3 | e58194