Amyloid-b Inhibits No-cGMP Signaling in a CD36- and
Thomas W. Miller1, Jeff S. Isenberg1,2, Hubert B. Shih1,3, Yichen Wang1, David D. Roberts1*
1Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America,
2Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America, 3Howard Hughes Medical Institute–National
Institutes of Health Research Scholars Program, Bethesda, Maryland, United States of America
Amyloid-b interacts with two cell surface receptors, CD36 and CD47, through which the matricellular protein
thrombospondin-1 inhibits soluble guanylate cyclase activation. Here we examine whether amyloid-b shares this inhibitory
activity. Amyloid-b inhibited both drug and nitric oxide-mediated activation of soluble guanylate cyclase in several cell
types. Known cGMP-dependent functional responses to nitric oxide in platelets and vascular smooth muscle cells were
correspondingly inhibited by amyloid-b. Functional interaction of amyloid-b with the scavenger receptor CD36 was
indicated by inhibition of free fatty acid uptake via this receptor. Both soluble oligomer and fibrillar forms of amyloid-b were
active. In contrast, amyloid-b did not compete with the known ligand SIRPa for binding to CD47. However, both receptors
were necessary for amyloid-b to inhibit cGMP accumulation. These data suggest that amyloid-b interaction with CD36
induces a CD47-dependent signal that inhibits soluble guanylate cyclase activation. Combined with the pleiotropic effects
of inhibiting free fatty acid transport via CD36, these data provides a molecular mechanism through which amyloid-b can
contribute to the nitric oxide signaling deficiencies associated with Alzheimer’s disease.
Citation: Miller TW, Isenberg JS, Shih HB, Wang Y, Roberts DD (2010) Amyloid-b Inhibits No-cGMP Signaling in a CD36- and CD47-Dependent Manner. PLoS
ONE 5(12): e15686. doi:10.1371/journal.pone.0015686
Editor: Sergio T. Ferreira, Federal University of Rio de Janeiro, Brazil
Received August 9, 2010; Accepted November 21, 2010; Published December 22, 2010
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public
domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: This work was supported by the Intramural Research Program of the National Institutes of Health (NIH), National Cancer Institute, Center for Cancer
Research (D.D.R.), NIH grant CA128616 (J.S.I.) and by the Howard Hughes Medical Institute-NIH Research Scholars Program (H.B.S.). 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
The pathogenesis of Alzheimer’s disease is closely associated
with the accumulation of amyloid-b (Ab) peptides, which
eventually form neuronal deposits known as senile plaques on
the outside surface of the neurons  and lead to neuron death.
Ab is a peptide of 37–43 amino acids in length that originates by
proteolytic cleavage from the amyloid precursor protein, which is
a neuronal transmembrane protein that contributes to innate
antimicrobial immunity and has unknown function in the CNS
. Binding of Ab to the plasma membrane is thought to be a
critical step in development of Alzheimer’s disease , and the
formation of Ab plaques is a primary trigger of neuron
degeneration . However, our molecular understanding of how
Ab contributes to the pathogenesis of Alzheimer’s disease remains
Nitric oxide (NO) is a cell signaling molecule that plays an
important role in regulating vascular, immune, and neurological
processes. For example, both hippocampal and cortical long-term
potentiation, a physiological correlate of synaptic plasticity thought
to underlie learning and memory, involve NO signaling cascades
[8,9]. NO can originate from exogenous sources and diffuse across
the cell membrane, or it can be synthesized from L-arginine within
the cell by nitric oxide synthases (NOS). NO activates soluble
guanylate cyclase (sGC) to produce cGMP , which activates
cGMP-dependent kinase (cGK), a major cellular receptor of
cGMP . cGK then catalyzes the phosphorylation of its
substrates, which initiate various cellular responses such as smooth
muscle relaxation, delayed platelet aggregation, intestinal secre-
tion, and long term potentiation [12,13,14,15].
NO in the brain can be produced either by inducible NOS
(iNOS/NOS2) in microglia and astrocytes, or by constitutive NOS
in neurons and endothelial cells (nNOS/NOS1 and eNOS/
NOS3). A large body of evidence suggests that the NO produced
by neuronal and endothelial constitutive NOS is responsible for
neuroprotection during Ab-induced cell death, while NO
production in the case of iNOS activation plays a neurotoxic role
due to the inflammatory response caused by the over generation of
other reactive nitrogen species from NO (see review ). A
decrease in neuronal NOS and an increase in hippocampal iNOS
have been demonstrated in aged rats , thus suggesting the dual
roles of NO. In mice, the higher level of constitutive NO produced
by iNOS protects beta-amyloid transgenic mice from developing
most typical human symptoms of Alzheimer’s disease . When
crossed into an iNOS-null background these mice displayed
extensive tau pathology associated with regions of dense
microvascular amyloid deposition.
The protective role of NO in Alzheimer’s disease pathogenesis
has been linked to NO/sGC/cGMP/cGK signaling cascades.
Treatment with NO donors and cGMP analogues suppresses cell
death , and increasing intracellular cGMP levels prevents
inflammatory responses in brain cells . Moreover, the use of
the NO donors, sGC stimulators, and cGMP-analogs reverses
learning and memory impairment through cGK activation, in part
PLoS ONE | www.plosone.org1December 2010 | Volume 5 | Issue 12 | e15686
by reestablishing the enhancement of the transcription factor
cAMP-responsive element-binding protein (CREB), which is
phosphorylated during long term potentiation .
However, an accumulation of Ab inhibits the NO signaling
pathway and therefore may suppress the protective effects of
endogenous NO in the brain. Chronic administration of fibrillar
Ab decreases the expression of sGC in cultured rat astrocytes,
desensitizing them to treatment with sodium nitroprusside .
Acute Ab administration blocks NO-induced vasoactivity in rats
[23,24] and inhibits NO-stimulated phosphorylation of CREB
. The molecular mechanisms behind the down regulation of
NO signaling by acute Ab exposure remain a mystery, and are the
focus of this paper. Understanding these mechanisms can
potentially provide the basis for a novel therapeutic application
of drugs aimed at limiting the adverse effects of Ab.
The well-studied inhibitor of NO-cGMP signaling thrombos-
pondin-1 (TSP1) shares several features with Ab that suggested a
common mechanism to inhibit NO signaling. Among the known
cell surface TSP1 receptors, three have been proposed to also
interact with Ab: CD36, CD47, and a6b1 integrin [25,26].
Although direct binding of Ab to each of these receptors has not
been established, the ability of Ab to stimulate interleukin-1b
release and reactive oxygen species production in microglial cells
was inhibited by antibodies to each receptor . Because CD36
and CD47 are each known to associate with some b1integrins, the
authors proposed that Ab binds to a complex of these 3 receptors.
Microglial phagocytosis of Ab fibrils was subsequently shown to be
inhibited by antagonists of the same receptor . A GST-CD36
fusion protein and a CD47-binding peptide inhibited Ab-
stimulated Vav1 phosphorylation in THP-1 monocytes .
Furthermore, the ability of Ab to stimulate histamine release by
mast cells was inhibited by antibodies against CD47 and b1
integrin subunit and by a modified TSP1 peptide that binds to
CD47 . Contrary to these receptors recognizing Ab as a
complex, phagocytosis of Ab was enhanced by conditions that
increased CD36 but decreased CD47 recruitment to lipid raft
domains of microglial cells .
TSP1 inhibits NO signaling in vascular cells by binding to
CD47 to decrease sGC activity and cGMP levels [25,31,32]. TSP1
binding to CD36 can also inhibit this pathway, but only in cells
that also express CD47 . To test whether Ab suppresses sGC
activity and thus inhibits NO signal transduction by the same
mechanism as TSP1, we used intracellular cGMP production as
an indicator of sGC activity after stimulation by NO. We
demonstrate that inhibition of the NO-cGMP signaling pathway
by Ab requires CD47 and CD36 but may not involve a direct
interaction of Ab with CD47. Rather, CD47 signaling may be
perturbed downstream of Ab interacting with CD36.
Ab interacts directly with CD36 but not with CD47
Ab(1–42) has 3 predominate conformations, monomeric
peptide, soluble oligomers, and fibrillar, that depend on the
amount of time it is left in solution to aggregate. Controversy still
exists as to which of these mediate Ab pathology, and both the
soluble and fibrillar forms are inhibitors of vascular responses
[24,33,34,35]. Considering this ambiguity, we compared the
ability of fibrillar and soluble peptide forms to interact with CD36
based on their ability to inhibit the fatty acid translocase activity of
CD36 [36,37]. Both forms inhibited [3H]-myristic acid uptake by
both glial and vascular cells in a dose-dependent manner (Fig. 1A–
C). While their responses were equipotent in human aortic VSMC
and HUVEC (Fig. 1A, B), the soluble peptide was more potent in
microglial cells (Fig. 1C). Based on these results, we used the
soluble peptide for the remainder of the studies.
Previous antibody and peptide inhibition studies implicated
CD47 in Ab signaling but did not determine whether Ab binds
directly to CD47 [26,27,28,29]. The prototypical ligand for CD47
is the extracellular domain of its counter-receptor SIRPa . We
have previously shown that binding of the CD47 ligand TSP1 can
be measured by displacement of labeled SIRPa-Fc fusion protein
. We employed the same assay to determine whether Ab binds
directly to CD47 but observed no significant inhibition of SIRPa-
Fc binding at Ab concentrations up to 10 mM (Fig. 1D). There-
fore, the role of CD47 in mediating the inhibitory activity of Ab
may be indirect, as previously shown for peptide ligands of CD36
that also do not bind to CD47 .
Although we cannot exclude the possibility that Ab indirectly
inhibits the translocase activity of CD36, these data support a
direct interaction between Ab and the scavenger receptor CD36
but not with CD47.
Soluble Ab inhibits NO and BAY 41-2272 stimulated sGC
TSP1 engagement of CD47 or CD36 inhibits activation of sGC
in several cell types [25,32,40,41]. To evaluate the effect of Ab on
sGC activation, we tested whether Ab directly blocks NO-
stimulated cGMP production. Treatment of BAEC with 10 mM
of Ab led to a slight decrease in the basal levels of cGMP (Fig. 2A).
As expected, addition of the fast-releasing NO donor DEA/NO
(10 mM) caused a significant increase of cGMP production. Cells
that were treated with Ab followed by DEA/NO exhibited lower
levels of cGMP than those given only the NO donor, suggesting
that Ab signaling directly blocks sGC activation. The cGMP data
represented in Figure 1 were obtained without inclusion of a
phosphodiesterase inhibitor such as 3-isobutyl-1-methylxanthine
or sildenifil. Thus, the effect of Ab on cGMP flux could result
either from inactivation of sGC or stimulation of phosphodiester-
ase activity. However, Ab also inhibited cGMP accumulation in
the presence of 3-isobutyl-1-methylxanthine (IBMX, Fig 2B)
establishing that Ab signaling regulates sGC activation.
We recently reported that TSP1 also inhibits drug-induced
activation of sGC . Treatment of Jurkat cells with 10 mM of
the synthetic sGC activator BAY 41–2272 led to an increase in
cGMP production similar to that observed after treatment with
DEA/NO (Fig. 2C). The increase intracellular cGMP induced by
the BAY compound was inhibited by the addition of 10 mM of Ab
peptide. Therefore, Ab suppresses both NO-induced and synthetic
sGC stimulator-mediated sGC activation, reducing cGMP pro-
duction and inhibiting NO signaling.
The inhibitory effect of Ab on sGC activation extends to both
Jurkat human T lymphoma cells (Fig. 2D) and porcine VSMC
(Fig. 2E). A dose response in porcine VSMC revealed an IC50
value of about 5 mM Ab, while the Jurkat T cells were more
sensitive, having an IC50of less than 100 nM. A dose of 10 mM Ab
was used in subsequent experiments as it caused a greater than
50% inhibition in all three cell types.
TSP1 inhibits NO/cGMP-stimulated VSMC adhesion on
collagen , and we used this adhesion as a functional assay of
the effect of Ab on cGMP production. Paralleling its inhibition of
cGMP production in primary porcine VSMCs (Fig. 2E), Ab dose
dependently inhibited NO (10 mM DEA/NO) stimulated adhesion
of VSMC to collagen coated wells (Fig. 2F). 10 mM Ab inhibited
NO-stimulated VSMC adhesion by 40612%.
Thrombin-induced platelet aggregation is potently delayed by
NO-cGMP signaling . Under standard high sheer conditions,
thrombin induced aggregation of washed human platelets was
Amyloid-b Inhibits sGC Activation
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delayed by the addition of 10 nM DEA/NO (Fig. 2G). This delay
was reversed by pre-treating the platelets with 10 mM Ab prior to
the addition of 10 nM DEA/NO. The same effect of Ab was also
observed when platelet aggregation was assessed under low sheer
conditions (data not shown). Thus, consistent with the inhibition of
NO and sGC activator induced stimulation of cGMP production,
Ab inhibits at least two functional outcomes of cGMP signaling in
Ab inhibition of NO signaling requires CD36
CD36 has been implicated as a receptor/mediator for other
targets of Ab signaling [26,28]. Data in Figures 1A–C suggest that
Ab directly binds to CD36 based on its inhibiting cellular uptake
of the CD36 ligand myristic acid, but we cannot exclude that Ab
inhibits myristate update indirectly by binding to another CD36-
associated protein. Previous results from our lab have shown that
binding of recombinant type 1 repeats of TSP1, a synthetic
peptide derived from this domain of TSP1, or a related
peptidomimetic to CD36 is sufficient to inhibit both myristate
uptake and NO-cGMP signaling [25,32]. To examine whether Ab
inhibition of cGMP signaling requires CD36, BAEC and Jurkat
cells were pretreated with a splice-blocking CD36 morpholino
oligonucleotide. In both cell types this knockdown resulted in
decreased Ab inhibition of NO induced cGMP accumulation
(Fig. 3A, B). Nearly complete reversal of Ab inhibition occurred
when CD36 expression was suppressed in HUVEC. Therefore,
unlike the inhibitory activity of TSP1, CD36 expression is
necessary for Ab to inhibit sGC activation in these cells.
Ab inhibition of NO signaling also requires CD47
Although ligation of CD36 is sufficient to inhibit cGMP
signaling, this inhibition is lost in cells lacking CD47 .
Therefore, CD47 is necessary for CD36-mediated inhibition of
cGMP signaling. In contrast, TSP1 inhibition of cGMP signaling
via of its high affinity binding to CD47 does not require CD36
. This suggests that modulation of cGMP signaling by CD36
ligands is mediated by cross-talk with CD47. Consistent with the
BAEC data described above, wild-type murine vascular cells
expressing CD47 and treated with 10 mM DEA/NO showed a 4-
fold increase in intracellular cGMP production that was
inhibited by the addition of Ab (Fig. 4A). In contrast, Ab failed
to suppress the increase in the cGMP production induced by
Figure 1. Fibrillar and soluble Ab inhibit cellular myristate uptake but not SIRPa binding to CD47. [3H]-Myristic acid uptake after 5 min
into human aortic VSMC (A), HUVEC (B), and microglial cells (C) was determined in the presence of the indicated concentrations of Ab (soluble or
fibrillar) after lysis by liquid scintillation counting. D125I-SIRPa-Fc binding to Jurkat T-cells was measured in the presence of soluble Ab (0.1-10 mM) or
a CD47-specific function-blocking antibody (B6H12) for 1 hour at 25uC.
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Figure 2. Ab inhibits NO-induced cGMP synthesis and function. A BAEC were pretreated with 10 mM Ab followed by 10 mM DEA/NO.
B HUVEC were pretreated with 10 mM Ab followed by 90 mM IBMX and 10 mM DEA/NO. C Jurkat cells were pretreated with 10 mM Ab followed by an
NO-independent sGC activator (BAY 41-2272). D Jurkat cells were pretreated with Ab (0.1–100 mM) followed by 1 mM DEA/NO. E Porcine VSMC were
pretreated with Ab (0.1–10 mM) followed by 10 mM DEA/NO. Following treatment, cells were lysed and assayed for cGMP production. n=3, * denotes
P,0.05. F Porcine VSMC adhesion to collagen coated wells was assessed in the presence of Ab (0.01–10 mM) for 1 hour. G Thrombin(0.2 U)-induced
aggregation of washed human platelets was assessed in the absence (control) or presence of DEA/NO (0.01 mM) and with a 5 min pretreatment of Ab
(10 mM) followed by DEA/NO.
Amyloid-b Inhibits sGC Activation
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DEA/NO in murine CD472/2 lung endothelial cells. Con-
versely, suppressing CD47 expression in Jurkat T-cells using a
CD47-specific morpholino abolished the effect of Ab on NO-
stimulated cGMP accumulation (Fig. 4B). Thus, two indepen-
dent approaches confirm that CD47 is necessary for Ab to
suppress NO signaling.
Ab inhibition of NO signaling is TSP1 independent
TSP1 is known to inhibit NO signaling by suppressing sGC
activity through CD47, in the same manner through which we
observed Ab to inhibit NO signaling. To rule out the possibility
that the observed Ab inhibitory activity is due to or influenced by
mobilization of endogenous TSP1, we used TSP1 null cells to
evaluate Ab inhibition of NO signaling. As shown in Figure 5, in
wild type primary murine lung endothelial cells bearing TSP1, the
addition of DEA/NO increased intracellular cGMP production,
while treatment with Ab inhibited DEA/NO stimulated cGMP
production, as expected. In the corresponding TSP1 null cells, a
similar pattern was observed in which Ab inhibited an increase in
cGMP levels caused by DEA/NO and thus inhibited sGC activity.
Figure 3. Ab inhibition of NO signaling is dependent on CD36. A Jurkat cells or B BAEC were incubated with 10 mM CD36 antisense
morpholino or 10 mM 5-mis-splice CD36 control morpholino for 48 hrs. Following CD36 knockdown, cells were pretreated with 10 mM Ab followed by
10 mM DEA/NO. Following treatment, cell were lysed and assayed for cGMP production. n=3, * denotes P,0.05.
Amyloid-b Inhibits sGC Activation
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Therefore, Ab inhibition of NO signaling is not an artifact of
modulating endogenous TSP1.
Although previous studies have suggested that Ab is involved in
the inhibition of NO-induced processes such as hippocampal long
term potentiation , vascular tone , and CREB phosphor-
ylation (18), the molecular mechanism through which inhibition of
NO signaling occurs was not defined. Identifying how Ab is able to
suppress the NO signaling pathway is important in the
development of therapeutics aimed at slowing Alzheimer’s disease
pathogenesis because enhancing neuronal NO has the potential to
protect against neurodegeneration. Here, we provide evidence that
Ab inhibits the NO signaling pathways through its interactions
with CD36, which causes a CD47-dependent decrease in sGC
activity and cGMP production. This inhibition was reproduced in
VSMC, endothelial cells, and T cells and prevents both NO- and
drug-mediated activation of sGC. We also showed that Ab can
inhibit uptake of free fatty acids via CD36, which was previously
established to regulate NO synthesis in vascular cells [37,44].
T cells suggests that pathological accumulation of Ab can play a key
role in limiting the NO signaling pathway (7). Previously, various
downstream targets of NO have been shown to be inhibited by Ab
(18, 19). Here, we provide a link between Ab and NO signaling by
showing that all the downstream inhibitory responses could result
that Ab offsets increases in cGMP levels caused by both NO donors
and synthetic sGC activators, indicating that Ab can inhibit sGC
independent of its NO-binding heme prosthetic group. This is
important because others have shown that sGC can be inhibited by
oxidizing the Fe2+in this heme.
Our results further show that decreased sGC activity and NO
signaling caused by Ab are dependent on the presence of CD47.
Previously, Ab fibrils were proposed to attach to microglial cells by
Figure 4. Ab inhibition of NO signaling is dependent on CD47. A Wild type or CD472/2 primary murine lung endothelial cells were
pretreated with 10 mM Ab followed by 10 mM DEA/NO. B Wild type Jurkat cells were incubated with 10 mM CD47 antisense morpholino or 10 mM of a
5 base mismatched CD47 control morpholino for 48 hrs. Following CD47 knockdown, cells were pretreated with 10 mM Ab followed by 1 mM DEA/
NO. Following treatment, cell were lysed and assayed for cGMP production. n=3, * denotes P,0.05.
Amyloid-b Inhibits sGC Activation
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interacting with a cell surface receptor complex that includes
CD47, a6b1 integrin, and CD36 . While the evidence for
functional involvement of CD36 in Ab signaling was strong, the
evidence for CD47 binding was only inferred based on sensitivity
to 4N1K, a thrombospondin-1-based peptide with both CD47-
dependent and CD47-independent activities [45,46]. The ability
of Ab to directly interact with integrins is also controversial. An
Arg-His-Asp sequence in Ab was shown to be recognized by a5b1
integrin and mediate its uptake and degradation . However,
another study concluded that integrin antagonists increase uptake
of Ab and increase its neurotoxicity in brain tissue . Beta-2
integrins have also been identified as Ab receptors , and a2b1
and avb1were implicated in fibrillar amyloid deposition .
Therefore, it is unlikely that a6b1is a specific integrin receptor for
Ab that could account for its inhibition of NO signaling.
We propose a revised version of the Ab binding model
presented by Bamberger et al. Ab inhibition of cGMP-dependent
signaling requires CD47, but Ab binding may depend more on the
interaction with the scavenger receptor CD36, which is well
known for its promiscuous binding to a variety of extracellular
ligands (reviewed in ). Binding of Ab to CD36 or to an
associated molecule generates an inhibitory signal that is probably
transduced via CD47 (Figure 6). This model is consistent with the
requirement for both CD36 and CD47 and the inability of Ab to
displace SIRPa, a specific CD47 ligand. CD47 also plays a role in
Ab inhibition of kinase-based signal transduction cascades 
and Vav1 activation , but like inhibition of sGC activation,
these responses may be independent of direct binding between Ab
Ab regulation of NO signaling through CD47 differs slightly
from TSP1 inhibition of NO signaling in vascular cells . CD47
is a high affinity receptor for TSP1, and this binding inhibits NO
signaling by suppression of sGC activation and a decrease in
cGMP levels (20, 22). In contrast, Ab does not interact directly
with CD47. TSP1 can also bind to CD36, but the affinity of this
interaction is lower. Thus, CD47 is the dominant signaling
receptor for physiological concentrations of TSP1. Ab appears to
not bind to CD47, but CD47 is necessary for Ab’s inhibition of
sGC activation. The details of how CD47 contributes to CD36-
mediated Ab signaling to decrease sGC activation remain to be
Recognizing that Ab inhibits NO signaling by suppressing sGC
activity through CD36 and CD47 may clarify Ab’s role in
Alzheimer’s disease pathogenesis and could lead to novel
therapeutics aimed at limiting the adverse effects of Ab in the
brain. As mentioned previously, NO can protect against neuron
degeneration and inflammation, but its signaling is inhibited by Ab
at the level of sGC activation. Thus, elevating the steady state NO
levels would be more effective in combination with an agent
targeting CD47 to relieve the block at sGC. Realizing that this
inhibition occurs through CD47 and CD36, therapeutic ap-
proaches could be developed to block Ab signaling through these
receptors to rescue the NO signaling pathway.
In addition to providing a mechanistic basis for the recognized
deficiencies in NO/cGMP signaling associated with Alzheimer’s
disease pathogenesis, our data may have implications for
peripheral vascular disease in light of the documented Ab
accumulation in diseased peripheral as well as cerebral blood
vessels. Consistent with the inhibitory activities of Ab we describe
using large vessel endothelial and VSMC, Ab was previously
shown to inhibit vascular reactivity in isolated rabbit and rat aorta
[33,52]. Furthermore, the secretion of Ab by activated platelets
during aging and hypercholesterolemia may expose the peripheral
vasculature to this NO antagonist and account for part of the NO
insufficiency and insensitivity of these diseases [53,54].
Mutations resulting in CD36 deficiency occur in humans and
some strains of spontaneously hypertensive rats. Humans with
CD36 deficiency exhibit hyperlipidemia, increased remnant
lipoproteins, impaired glucose metabolism based upon insulin
resistance, and mild hypertension . Based on our observation
that Ab impairs the fatty acid translocase activity of CD36, the
accumulation of vascular-associated Ab in Alzheimer’s disease
could cause a local functional CD36 deficiency by blocking lipid
and, potentially, oxidized lipoprotein uptake via this receptor. In
addition to perturbing vascular NO signaling via CD36, this direct
effect of Ab on CD36 translocase function merits further study.
Materials and Methods
Cells and reagents
Bovine aortic endothelial cells (BAECs) prepared from fresh
aortic segments and human umbilical vein endothelial cells
(HUVEC) obtained from Lonza (Walkersville, MD) were cultured
Figure 5. Ab inhibition of NO signaling is not dependent on
TSP1. A Wild type or TSP12/2 primary murine lung endothelial cells
were pretreated with 10 mM Ab followed by 10 mM DEA/NO. Following
treatment, cell were lysed and assayed for cGMP production. n=3,
* denotes P,0.05.
Figure 6. Proposed model of Ab inhibition of cGMP production.
Ab binds directly to CD36 to inhibit uptake of free fatty acids. In the
presence of CD47, CD36 engagement transduces an inhibitory signal to
sGC, limiting its activation and production of cGMP.
Amyloid-b Inhibits sGC Activation
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in endothelial growth medium (EGM; Lonza, Walkersville, MD)
in 5% CO2at 37uC, and were used at passages 3–8. Jurkat human
T-lymphoma cells were cultured in RPMI 1640 (Invitrogen)
with 10% FBS, glutamine, and penicillin/streptomycin. Cells were
kept at a density of between 16105and 56105and used between
passages 7 and 20. Primary mouse endothelial cells were obtained
from the lungs of wild-type, TSP1-null, and CD47-null C57BL/6
mice after sacrifice, and grown in EGM as above. BV2
immortalized murine microglial cells (a gift of Dr. Sandra
Rempel, Hermelin Brain Tumor Center, Henry Ford Health
System, Detroit, MI) were maintained in DMEM (Invitrogen) with
10% FBS, glutamine, and penicillin/streptomycin. Porcine
vascular smooth muscle cells (white hairless Yucatan miniature
pig, as described in ) and human aortic vascular smooth
muscle cells (VSMC, Lonza) were cultured in SMGM2 (Lonza;
Walkersville, MD) and were used at passages 3–8. DEA/NO was
provided by Dr. Larry Keefer (NCI, Frederick, MD). BAY 41–
2272 and IBMX (3-isobutyl-1-methylxanthine) were obtained
from Calbiochem (La Jolla, CA). Ab(1–42) peptides were
purchased from Anaspec (San Jose, CA). Fibrillar Ab was made
by incubating peptides in sterile distilled water for 1 week at 37uC.
Extracellular domain of SIRPa fused to a modified human Fc
domain was prepared and labeled with125I as previously described
[39,60]. Anti-CD47 antibody B6H12 was purchased from Abcam
(Cambridge, UK). Care and handling of animals was in
accordance with and approved by Animal Care and Use
Committees of the National Cancer Institute (Protocol LP-012).
Intracellular cGMP assay
BAEC, porcine VSMC, or mouse primary cells were plated in
6-well plates. The cells were grown in serum containing media
until reaching 80% confluence, at which time they were serum
starved overnight in endothelial basal medium (EBM; Lonza) or
smooth muscle basal medium (SMBM; Lonza) with 0.1% bovine
serum albumin (BSA; Sigma-Aldrich, St Louis, MO). Jurkat cells
were assayed at 56105cells per condition in 0.5 ml basal media
(RPMI, glutamine, penicillin/streptomycin, 0.01% BSA). The
relevant cells were pre-incubated for 15 min with the indicated
concentrations of Ab and then treated with DEA/NO for 2 min at
room temperature. The cells were then lysed, and total
intracellular cGMP levels were measured via immunoassay using
a cGMP kit (GE/Amersham Healthcare, Amersham, United
Kingdom) according to the manufacturer’s instructions. A mBCA
protein assay (Thermo Scientific, Rockford, IL) was performed to
determine the protein concentration for each of the samples.
cGMP levels were normalized based on the amount of protein
Experiments using BAY 41–2272 followed the same above
procedure, except 10 mM of BAY 41–2272 in DMSO was used
instead of DEA/NO. In the experiments involving BAY 41–2272,
dimethyl sulfoxide (DMSO; Sigma) was also added to control wells.
[3H]-myristate uptake assay
The [3H]myristic acid uptake assays were performed using 80–
90% confluent HUVEC, human microglial, and human aortic
VSMC cells (56105cells/well) in 24-well culture plates (Nunc,
Denmark). Trace amounts of [3H]myristic acid (5 mCi/ml,
0.9 mM) mixed with 9.1 mM nonradioactive myristic acid were
dissolved in a FAF BSA solution at a myristic acid/BSA molar
ratio of 1:2. Cells were incubated in medium with treatment agents
for the indicated time intervals at 37uC. The uptake was stopped
by removal of the solution followed by the addition of chilled 0.9%
NaCl with 0.5% BSA. The stop solution was discharged, and the
cells were washed again with stop solution. Cells were lysed by
adding 0.2 M NaOH (200 ml/well) and incubated for 2 h at 37uC.
On completion of solubilization, 0.2 M HCl in 1.5 m Tris-HCl
(200 ml) was added to each well. Radioactivity was determined in
10 ml of Ecoscint A (National Diagnostics, Atlanta, GA) using a
1900CA liquid scintillation counter (Packard Instrument Co.).
125I-SIRPa-Fc binding assay
16106Jurkat T-cells in PBS with cations and 0.1% BSA were
incubated with 0.4 ug/ml125I-SIRPa and the indicated concen-
trations of Ab peptide or CD47-specific monoclonal antibody
(B6H12) shaking at 25uC for 1hour. Cells were separated from
unbound125I-SIRPa by centrifugation through silicone oil (nyosil
M25, Nye Co, New Bedford, MA). Cell-bound radioactivity was
quantified using a PerkinElmer Life Sciences gamma counter.
Data are represented as percent of total counts (no cells), n=3.
VSMC adhesion assay
Cell adhesion was carried out in 96-well plates (Nunc,
Denmark). After precoating wells with type I collagen (3 mg/ml),
porcine VSMC were plated at a density of 16104cells/well in
SMBM containing 0.1% BSA and treatment agents and incubated
in 5% CO2for 1 h. Wells were washed with PBS, and the cells
were fixed with 1% glutaraldehyde for 10 min, washed, and
stained with 1% crystal violet for 20 min. Excess stain was rinsed
away, the cells were extracted with 10% acetic acid, and the plates
were read at 570 nm.
Preparation of human platelets
Platelets were obtained obtained as byproducts from healthy
volunteers through the NIH department of transfusion medicine
blood bank. ‘‘Byproducts’’ of transfusion donations are provided
anonymously, without any identifying code or number. The link
between the donor and the product is irreversibly destroyed by the
DTM. As such, distribution of these products is exempt from the
need for IRB approval. Platelets were pelleted from platelet-rich
plasma by centrifugation for 10 min at 200 g. They were then
resuspended in acid citrate dextrose (ACD; 85 mM citric acid,
65 mM sodium citrate, 100 mM glucose, pH 5.1) at a ratio of 1:10
at room temperature. Platelets were pelleted again and resus-
pended in 10 ml of Tyrode buffer (137 mM NaCl, 3 mM KCl,
12 mM NaHCO3, 0.3 mM NaHPO4, 2 mM CaCl2, 1 mM
MgCl2, 5.5 mM glucose, 5 mM N-2-hydroxyethylpiperazine- N-
2-ethanesulphonic acid (HEPES), 3.5 mg/ml BSA, pH 7.4). The
final platelet number was adjusted to 6.56105platelets/ml in a
cuvette containing 500 ml of Tyrode buffer.
Platelet aggregation assay
Aggregation of human platelets under high shear conditions was
assessed using a standard optical aggregometer (Lumi- Dual
Aggregometer; Chrono-Log, Havertown, PA, USA) at 37uC and
1200 rpm in a volume of 500 mL buffer with a final platelet
concentration of 6.56105platelets/ml over a 5 min interval.
Preincubation with Ab (10 mM) was for 5 min prior to addition of
DEA/NO (0.01 mM) or vehicle control, which were incubated
5 min prior to the initiation of aggregation with thrombin (0.2 U).
All assays were repeated at least in triplicate and some are
normalized to percents of control in order to account for the
differences in cell count and conditions between trials. The results
were expressed as means 6 SD or shown as representative data.
Statistical significance was determined by the Student t test. A P-
value less than 0.05 was regarded as statistically significant.
Amyloid-b Inhibits sGC Activation
PLoS ONE | www.plosone.org8December 2010 | Volume 5 | Issue 12 | e15686
We thank L. Keefer for providing reagents.
Conceived and designed the experiments: TWM JSI HBS YW DDR.
Performed the experiments: TWM JSI HBS YW. Analyzed the data:
TWM JSI HBS YW DDR. Wrote the paper: TWM JSI HBS YW DDR.
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PLoS ONE | www.plosone.org10 December 2010 | Volume 5 | Issue 12 | e15686