1521-0103/344/1/41–49$25.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/jpet.112.199257
J Pharmacol Exp Ther 344:41–49, January 2013
Anti-Inflammatory Functions of Apolipoprotein A-I and
High-Density Lipoprotein Are Preserved in Trimeric
Apolipoprotein A-I
Andrew J. Murphy, Anh Hoang, Andrea Aprico, Dmitri Sviridov, and Jaye Chin-Dusting
Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia
Received August 11, 2012; accepted October 2, 2012
Introduction
Cardiovascular disease remains a leading cause of death
worldwide. The cardioprotective properties of high-density
lipoprotein (HDL) are well established (Murphy et al., 2009b),
with plasma levels negatively correlating with cardiovascular
events (Gordon et al., 1977). The ability of HDL to modulate
a number of different cellular processes ranging from regulating hematopoiesis (Murphy et al., 2011a) to inhibiting
activation of circulating cells (Murphy et al., 2008, 2011b) to
having a major role in the reverse cholesterol transport
system has made HDL an attractive potential therapy for
cardiovascular disease (Murphy et al., 2010, 2012).
There are a number of therapeutic approaches to increase
plasma HDL levels. Although the premature termination of
the AIM-HIGH (Boden et al., 2011) and dal-OUTCOMES
(Schwartz et al., 2009) studies for futility would appear to
dispute the beneficial effect of HDL-raising therapies, it is
possible that more dramatic and direct elevations of HDL levels
with a high cholesterol efflux capacity are required to produce
clinically meaningful results. One such method to achieve this
would be to administer cholesterol-poor reconstituted HDL
(rHDL) particles, which have been demonstrated in a number of
short-term clinical studies to deliver beneficial outcomes
(Murphy et al., 2009a). A study in patients with type II diabetes
showed that a single infusion of native rHDL improves glucose
metabolism and reduces a number of inflammatory parameters
(Drew et al., 2009; Patel et al., 2009). We have also reported
dx.doi.org/10.1124/jpet.112.199257.
similar rate as native apoA-I particles in both ABCA1-dependent
and -independent pathways. Trimeric particles inhibited ICAM-1
and VCAM-1 expression and the ability of the endothelium to
capture monocytes under shear flow. Monocyte activation, CD11bdependent adhesion, and monocyte recruitment under shear flow
conditions were perturbed by the trimeric particles. Our data
suggest that trimeric rHDL particles can be constructed without any
loss of function, preserving the anti-inflammatory effects of HDL
that are key to its in vivo actions.
that a single infusion of rHDL into patients with peripheral
vascular disease can decrease the inflammatory profile and
remodel atherosclerotic plaques, attenuating vascular inflammation and decreasing plaque macrophage content, ultimately
resulting in a more stable lesion (Shaw et al., 2008). In
addition, a single infusion of rHDL can inhibit hematopoietic
stem cell proliferation, mobilization, and monocytosis in atherosclerotic and leukemic mouse models (Westerterp et al.,
2012; Murphy et al., 2011a), suggesting it could be used to
treat myeloproliferative disorders. Finally, repeated infusions
of rHDL have been shown not only to remodel but also regress
atherosclerotic plaques in patients with acute coronary syndrome (Nicholls et al., 2006; Nissen et al., 2003; Tardif et al.,
2007).
Recently, Ohnsorg et al. (2011) published on the reversed
cholesterol transport capabilities of trimeric apoA-I, a recombinant high molecular mass variant of apoA-I, engineered
by fusing three apoA-I molecules for the purpose of decreasing the rapid clearance properties of apoA-I, hence increasing plasma half-life (Graversen et al., 2008). In the current
study, we explored the functional properties, including the antiinflammatory capabilities, of the trimeric form of recombinant
apoA-I (T-apoA-I) and trimeric-rHDL (T-rHDL) particles with
native apoA-I and rHDL, respectively.
Materials and Methods
Blood Collection. Blood was collected from healthy consenting
(approved by the Human Ethics Committee of the Alfred Hospital)
volunteers by venipuncture and drawn into syringes containing
sodium citrate.
ABBREVIATIONS: apoA-I, human apoA-I; apoA-IE, Escherichia coli apoA-I; rHDL, human ApoA-I/DMPC; rHDLE, E. coli apoA-I/DMPC; T-apoA-I,
trimeric-apoA-I; T-rHDL, trimeric-apoA-I/DMPC.
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ABSTRACT
Raising high-density lipoprotein (HDL) levels is proposed as an
attractive target to treat cardiovascular disease. However, a
number of clinical studies examining the effect of HDL-raising
therapies have been prematurely halted due to futility. Therefore
there is a need for alternative therapies. Infusion of reconstituted
HDL (rHDL) particles is still considered as a viable approach to
increasing HDL levels. In this study we have profiled the antiinflammatory effects of a trimeric-HDL particle. We show that
trimeric apoA-I and rHDL particles promote cholesterol efflux to a
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Murphy et al.
12-well plates and incubated in media with or without the various
cholesterol acceptors (1 mg/ml) for 16 h prior to stimulation with TNF-a
(0.1 ng/ml) for 5 h at 37°C. Cells were then washed with cold PBS and
incubated with fluorescein isothiocyanate (FITC) conjugated antibodies
to either ICAM-1 (Abs Serotec, clone 15.2) or VCAM-1 (AbD Serotec,
Kidlington, United Kingdom, clone 1.G11B1) for 1 h at 4°C on a shaker.
The cells were then washed twice with PBS (Ca/Mg free), and trypsin
was added to remove the cells from the wells. After the cells began to
lift, trypsin-neutralizing solution was added and the cells were
transferred into flow cytometry tubes containing 4% paraformaldehyde.
Samples were compensated for by using the isotype matched negative
control (FITC-anti-mouse IgG, AbD Serotec, clone W3/25). ICAM-1 and
VCAM-1 expression was measured by flow cytometry using FACS
Calibur (BD Biosciences; San Jose, CA). Analysis was conducted using
the Cell Quest Pro software. Results were expressed as percentage of
the unstimulated control (100%).
Monocyte Isolation. Resting human monocytes were isolated
by density centrifugation with Lymphoprep (Axis Shield, Dundee,
Scotland) followed by Dynal negative monocyte isolation kit (Invitrogen) as described previously (Murphy et al., 2008). Monocytes
were resuspended in PBS and cell number was determined on
an automated hematology analyzer (KX-21N; Sysmex, Mundelein,
IL).
CD11b Expression-Flow Cytometry. Monocytes were stimulated with either 1 mmol/l PMA or 1 mg/ml LPS (Sigma, Sydney, NSW,
Australia) in the presence or absence HDL (50 mg/ml) or apoA-I (40
mg/ml) and incubated with the FITC conjugated Ab the active epitope
of CD11b (eBiosciences, San Diego, CA, Clone CBRM1/5) for 15 min at
37°C, unless otherwise stated. Cells were then fixed with 4%
paraformaldehyde. The samples were run, data were analyzed, and
results were expressed as in the HCAECs studies.
Perfusion Studies. Perfusion studies were conducted using the
parallel plate flow-chamber (GlycotechGaithersburg, MD) as previously
described (Murphy et al., 2008). HCAEC were cultured in 6-well plates
to form a confluent monolayer with HCAEC Media (Cell Applications).
Prior to perfusion, HCAECs were washed twice with PBS. Isolated
monocytes (1 106/ml) in PBS were perfused over HCAECs under
negative pressure (PHD 2000, Harvard Apparatus, Holliston, MA), at
a shear rate of 150 s21 (1.1 dyn/cm2) for 5 min, with an additional
washout period of 5 min with PBS. To study the inhibitory effect of the
rHDL molecules on monocyte-dependent adhesion, monocytes were
pretreated with 1 mmol/l of PMA 6 50 mg/ml rHDL for 15 min at 37°C
prior to perfusion. To study the effect of the rHDL on endothelialdependent adhesion, HCAECs were incubated for 16 h with rHDL (1
mg/ml) or PBS (control) prior to stimulation with TNF-a for 5 h.
Monocyte-endothelial cell interactions were visualized using phase
microscopy (20 lens, Olympus, Mount Waverley, VIC, Australia)
captured digitally (MDC-1004, Imperx, Boca Raton, FL) at 30 frames/s
with XCAP software v2.2 (Epix, Buffalo Grove, IL) and analyzed offline
using Image Pro-Plus v5.1 (Media Cybernetics, Bethesda, MD). Time
0 was defined by the first adhering monocyte. Fields (274 mm 275 mm)
were recorded for 10 s at 0.5 min, 1 min (1 visual field each/time point
was analyzed), 2.5 min, 5 min, and 10 min (5 visual fields each/time
point were analyzed) for offline analysis.
Static Adhesion Assay. Monocyte adhesion to immobilized
fibrinogen was performed as previously described (Murphy et al.,
2008). Monocytes (1 106/ml) were allowed to adhere for 15 min at
37°C with the appropriate treatment. Monocyte adhesion was
quantified by phase microscopy (40), counting five random fields
for each slide. Treatments were carried out in triplicate. Results are
expressed as the average amount of cells per field.
Statistical Analysis. Values are presented as the mean 6 S.E.M.
or percentage of control 6 S.E.M. All results were analyzed for
statistical significance using one-way analysis of variance followed by Bonferroni post hoc test, with the exception of perfusion
studies, which were analyzed using a two-way analysis of variance
followed by Bonferroni post hoc test. Significance was accepted at
P , 0.05.
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Trimeric apoA-I, rHDL Particles, and Liposomes. Recombinant human apoA-I (apoA-IE) and trimeric apoA-I and rHDL
preparations were kindly provided by Hoffmann-La Roche, Basel,
Switzerland. ApoA-I was constructed as previously described by
Ohnsorg et al. (2011). Reconstituted HDL (rHDL) and dimyristoylphosphatidylcholine (DMPC) liposomes were prepared as previously
described (Murphy et al., 2008). HDL was isolated from plasma using
sequential ultracentrifugation (density 1.085 to 1.21 g/ml), and
protein content was measured. All acceptors were used at the
concentrations stated in each figure as calculated by protein for
HDL particles or concentration of phospholipid for liposomes.
Particle Characterization. To measure the Stokes diameter, the
respective apoA-I and rHDL particles were subjected to native 6%
PAGE and Stokes diameter was measured as previously described
(Hoang et al., 2012). To determine the number of apoA-I particles per
rHDL particle, crosslinking studies were performed as previously
described (Sviridov et al., 2000). Briefly, 10 mg of the respective
particles was crosslinked for 30 min at room temperature in dithiobis
(succinimidylpro-pionate) (DSP; 200 mM); the reaction was then
stopped by adding 50 mM of Tris-HCl for 15 min. Samples were then
run on a 4–20% SDS-PAGE under denaturing conditions.
Cholesterol Efflux. Cholesterol efflux assays were performed as
previously described (Ohnsorg et al., 2011). THP-1 was differentiated
into macrophages using phorbol 12-myristate 13-acetate (PMA; 100
ng/ml) and loaded with acetylated low-density lipoprotein and 3Hcholesterol. Cells were then washed and incubated with or without
a liver X receptor (LXR) agonist (TO901317 1mM) for 18 h. Cells were
then incubated with various concentrations of the apoA-I molecules
for 4 h. Supernatant was collected and filtered centrifuged to remove
cellular debris. and 100 ml was taken for counting on the scintillation
counter (Perkin Elmer, MA). The remaining cells were washed, lyzed
by the addition of 5% Nonidet P-40, and an aliquot was taken for
counting. Fractional efflux was calculated as the ratio of radioactivity
measured in the supernatant to the sum of the radioactivity measured
in both the cell lysate and supernatant.
Endothelial Cells. Human coronary aortic endothelial cells
(HCAEC; Cell Applications) were cultured in 6-well or 12-well plates
to form a confluent monolayer with HCAEC Media (Cell Applications,
San Diego, CA). The cells were either used for perfusion studies (6
well) or for examining adhesion molecule expression (12 well). Freshly
isolated human umbilical vein endothelia cells (HUVECs) were
seeded onto cell culture coverslips in a 24-well plate and cultured to
form a confluent monolayer.
Endothelial Adhesion Molecule Expression–Immunohistochemistry. HUVECs were grown on glass coverslips to form a
confluent monolayer. Media were replaced with or without T-rHDL (1
mg/ml) or DMPC liposomes and incubated for 16 h, after which the
cells were stimulated with tumor necrosis factor (TNF)-a (0.1 ng/ml).
The cells were then fixed with 2% paraformaldehyde for 10 min and
washed with phosphate-buffered saline (PBS). Endogenous peroxidase
activity was blocked using peroxidase block (EnVision-kit, Dako
Cytomation, Melbourne, VIC, Australia) for 5 min at room temperature
(RT) followed by washing in Tris-buffered saline (TBS). The samples
were then blocked with goat serum (1:10 in TBS) for 20 min at RT. The
primary antibody to VCAM-1 (Dako Cytomation) (1:85 in TBS) was
allowed to bind for 1 h at RT followed by washing with TBS. Detection
of the primary antibody was achieved by adding a horseradish
peroxidase-goat anti-mouse IgG for 30 min at RT followed by
washing with PBS. The substrate-chromogen was then applied, and
the sample was counterstained with Mayer’s hematoxylin nuclear
staining solution (Bio-Optica, Milano, Italy). The samples were
rinsed with dH2O before been dehydrated in ethanol and cleared in
xylene. The coverslips were then mounted onto glass slides using
Histomount (Invitrogen, Mulgrave, VIC, Australia). Images were
captured using at 20 magnification, and the percentage of positive
stained cells was quantified using imageJ.
Endothelial Adhesion Molecule Expression-Flow Cytometry.
HUVECs or HCAECs were cultured as a confluent monolayer in
Anti-Inflammatory Properties of Trimeric apoA-I
Results
Fig. 1. Characterization of T-apoA-I. (A) particle size was determined by
running the respective apoA-I and rHDL particles using a 6% native
PAGE. (B) respective particles were run under denaturing conditions on
a 4–20% SDS-PAGE to determine the number of apoA-I molecules.
endothelial adhesion molecules. Incubating endothelial cells
with TNF-a promotes to a dramatic increase in the number of
VCAM-1-positive cells. Analysis of immunohistochemistry
micrographs showed that T-rHDL particles could indeed
inhibit VCAM-1 expression on endothelial cells stimulated
with TNF-a (Fig. 3, A and E). T-rHDL particles significantly
inhibited VCAM-1 expression to a greater extent than DMPC
liposomes, and thus subsequent experiments focused on the
rHDL particles (Fig. 3A). Flow cytometry was used to compare
the anti-inflammatory ability of T-rHDL and rHDL particles.
Both particles equally well reduced VCAM-1 expression (Fig.
3F). Similar results were obtained when comparing the ability
of native and trimeric particles to inhibit ICAM-1 expression
(Fig. 3G). These findings were confirmed in HCAECs. The
rHDL molecules suppressed both VCAM-1 and ICAM-1
expression (Fig. 3, H and I). Incubating HCAECs with lipidfree apoA-IE or DMPC liposomes failed to significantly inhibit
adhesion molecule expression (Fig. 3, H and I).
Trimeric apoA-I Inhibits Monocyte Activation. Both
HDL and apoA-I have recently been reported as having potent
anti-inflammatory effects on monocytes as determined by measuring the conformational change of the adhesion molecule
CD11b (Murphy et al., 2008). The efficacy of T-apoA-I or TrHDL to inhibit monocyte activation was assessed. Stimulating monocytes with PMA caused a significant increase in
CD11b activation. This activation of CD11b was significantly
inhibited with only 5 mg/ml, with a nadir at 20 mg/ml for each
of the respective apoA-I molecules (Fig. 4A). Additionally,
there was no difference in the efficacy of each molecule, and
the trimeric molecules were equally as efficient as native
apoA-IE. The ability of apoA-IE, T-apoA-I, or T-rHDL to
inhibit monocyte activation stimulated with a receptordependent agonist LPS was also examined. Again, each of
the apoA-I molecules inhibited CD11b activation to a similar
extent (Fig. 4B).
Effect of rHDL Particles on Endothelial-Dependent
Shear Flow Cell Adhesion. A shear flow cell adhesion
model was employed to examine whether rHDL and T-rHDL
were able to inhibit endothelial-dependent monocyte adhesion. Total adhesion, stationary adhesion, and rolling flux
were assessed. Incubating endothelial cells with TNF-a prior
to the perfusion of resting monocytes resulted in a significant
increase in monocyte adhesion (Fig. 5, A and D–G, respectively). Preincubating endothelial cells with rHDL prior
to stimulation with TNF-a resulted in a significant attenuation in total monocyte adhesion after 10 min of perfusion
compared with endothelial cells stimulated with TNF-a alone
(Fig. 5, A, H, and I). Preincubating endothelial cells with TrHDL before stimulation resulted in a significant decrease in
monocyte adhesion at the 5-min time point (Fig. 5, A, J, and
K). T-rHDL was also able to reduce significantly stationary
monocyte adhesion after 5 min of perfusion compared with
TNF-a-treated cells. This was a more potent inhibition
compared with rHDL, where a significant reduction in
stationary adhesion was not observed until 10 min of
perfusion (Fig. 5B). The number of monocytes rolling along
the endothelium was significantly higher when the endothelial cells were pretreated with rHDL; there also was
a tendency for a higher number of rolling monocytes in the
T-rHDL group, suggesting that pretreatment with rHDL
inhibited the ability of the endothelial cells to capture and
arrest the monocytes (Fig. 5C).
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Characterization of rHDL Particles. rHDL particles
based on apoA-IE and T-apoA-I were characterized using nondenaturing electrophoresis in native 6% gels and in crosslinking experiments (Fig. 1). The predominant population of
rHDL particles assembled with apoA-IE had a Stokes diameter of 9.2 nm (Fig. 1A). T-apoA-I formed a number of
particles, with the predominant particle having a Stokes
diameter of 9.5 nm. Analysis of crosslinking demonstrated
that rHDL particles formed from apoA-IE contained two
molecules of apoA-I, whereas T-apoA-I formed particles that
contained predominantly three molecules of T-apoA-I per
rHDL particle (Fig. 1B). The level of heterogeneity of T-HDL
was unexpectedly high for a particle with the same number of
apolipoprotein molecules per particle. This may be a reflection
of variability in lipid and protein packing in T-HDL resulting
in a mixture of differently sized particles.
Trimeric apoA-I Facilitates Cholesterol Efflux Via
ABC Transporters. The ability for T-apoA-I particles to
promote cholesterol efflux was examined using THP-1derived macrophages either unstimulated or treated with an
LXR agonist (T01901317). Native apoA-IE and T-apoA-I
facilitated cholesterol efflux comparably over all concentrations tested in unstimulated cells (Fig. 2A). Moreover, the
efficiency of these molecules to remove cholesterol was equal
to purified native human apoA-I. As expected, T-rHDL
removed cholesterol more efficiently than the nonlipidated
acceptors. A significant difference in cholesterol efflux by TrHDL was observed at 6.6 mg/ml. Similar results were
observed in THP-1 macrophages activated with an LXR
agonist. As expected, the overall level of cholesterol efflux was
increased after LXR activation to all acceptors. Again the
level of efflux was equal between native apoA-I and T-apoA-I
over all concentrations examined, and the level of efflux was
also equal to purified human apoA-I (Fig. 2B). Incubating
macrophages with T-rHDL led to a significant increase in
cholesterol efflux at 20 mg/ml.
Trimeric rHDL Inhibits Endothelial Adhesion Molecules. HDL has previously been shown to inhibit the
expression of adhesion molecules on HUVECs. Immunohistochemistry and flow cytometry were employed to determine
if T-rHDL particles could also inhibit the expression of
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min, continuing to the end of the perfusion period (10 min)
(Fig. 6, B and E). Both T-apoA-I and T-rHDL also inhibited
monocyte adhesion to HCAECs under shear flow after 5 min
of perfusion, respectively (Fig. 6, B, F, and G). Furthermore,
all apoA-I molecules returned adhesion levels to near basal
conditions at 10 min of perfusion.
Discussion
Effect of rHDL Particles on Monocyte-Dependent
Shear Flow Cell Adhesion. To determine if the inhibition
of CD11b activation by T-apoA-I particles translates into
a decrease in monocyte inflammatory function, we aimed to
examine the effect of these molecules on monocyte adhesion.
Initially we examined CD11b-dependent monocyte adhesion
using the extracellular matrix protein fibrinogen as the
substrate (Gresham et al., 1989). Monocytes stimulated with
PMA displayed a significant increase in adhesion to the
fibrinogen matrix compared with control, correlating with the
increase in CD11b activation. Incubation of PMA-stimulated
monocytes with either of the apoA-I molecules significantly
inhibited CD11b-dependent monocyte adhesion (Fig. 6A).
Again there was no difference in the degree of reduction in
monocyte adhesion between each of the respective apoA-I
molecules. Next we examined the anti-inflammatory effects of
the apoA-I molecules on monocyte adhesion to endothelial
cells under shear flow conditions. Stimulating monocytes with
PMA prior to perfusion over HCAECs lead to a significant
increase in monocyte adhesion compared with control. Incubating PMA-stimulated monocytes with apoA-I prior to
perfusion lead to a significant attenuation in adhesion after 5
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Fig. 2. T-apoA-I and T-rHDL mediate cholesterol efflux through SR-BI
and ABC transporters. THP-1 macrophages were loaded with (A) Ac-LDL
and in (B) activated with an LXR agonist before cholesterol efflux to the
various acceptors was assessed over a dose response. T-rHDL versus TapoA-I, *P , 0.05, ***P , 0.01, n = 3.
Increasing HDL levels to reduce risk of cardiovascular
disease is a strategy of choice for many research groups and
industries. However, safely and effectively increasing functional cardioprotective HDL in disease has proven challenging. Previous studies suggest that oligomers of apoA-I have
increased plasma half-life (Graversen et al., 2008; Pedersen
et al., 2009). With this in mind, we explored the possibility
that a stable trimer of apoA-I may be more effective than
native apoA-I or rHDL. In the current study we describe a
trimeric-apoA-I molecule that retains the anti-inflammatory
properties of native apoA-I; this is the first report assessing
the anti-inflammatory properties of apoA-I and/or rHDL containing three apoA-I molecules.
The ability of the trimeric particles to facilitate cholesterol
efflux via the major reverse cholesterol transport pathways
was first assessed. In acetylated-low-density lipoproteinloaded THP-1 macrophages cholesterol efflux to the lipidpoor apoA-I molecules was similar and also comparable with
native human apoA-I. T-rHDL facilitated significantly more
efflux than lipid poor apoA-I, a finding that is likely due to the
interaction with other cellular transporters and not just
ABCA1 (Favari et al., 2009; Lorenzi et al., 2008). In LXRactivated THP-1 macrophages ABC transporter-mediated
cholesterol efflux was again similar between the different
apoA-I molecules, whereas the T-rHDL was only more
effective at higher concentrations. Although lipid-poor apoAI molecules are removing cholesterol via an ABCA1dependent pathway, it has been shown previously that rHDL
particles with three or more apoA-I molecules remove the
majority of cholesterol via an ABCG1-dependent pathway in
LXR-activated macrophages (Favari et al., 2009). Interestingly, although ABCA1 can efflux to rHDL particles, this
appears to be only to small particles containing two apoA-I
molecules with a low phospholipid concentration and at
a significantly lower efficiency than lipid-free apoA-I (Favari
et al., 2009). A previous study that employed the trimerization
domain of human tetranectin fused to apoA-I to create
a trimeric apoA-I molecule describe enhanced cholesterol
efflux over native apoA-I at higher concentrations only
(Graversen et al., 2008). However, both forms of apoA-I
display equal binding efficiencies to ABCA1-expressing cells
(Graversen et al., 2008).
The ability of the apoA-I molecules to inhibit endothelial
inflammation was then examined. It has been previously
shown that lipid-free apoA-I has a limited role in regulating
endothelial adhesion molecules (Mcgrath et al., 2009), a finding
we confirm in the current study using HCAECs. Consistent
with the literature, we observed a significant inhibition of
VCAM-1 expression with DMPC liposomes alone; however, this
inhibition was dramatically enhanced by T-rHDL particles. We
also observed via flow cytometry that both rHDL and T-rHDL
were equal at inhibiting TNF-a2induced VCAM-1 and ICAM-1
expression. This inhibition of endothelial adhesion molecule
Anti-Inflammatory Properties of Trimeric apoA-I
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Fig. 3. T-rHDL inhibits endothelial cell adhesion molecules. (A–E) HUVECs were preincubated with either saline, 1 mg/ml of T-rHDL, or DMPC
liposomes for 16 h prior to incubation with 0.1 ng/ml of TNF-a. VCAM-1 expression was detected via immunohistochemistry. **P , 0.01, ***P , 0.001
versus TNF-a, #P , 0.05 versus DMPC. Microscope images control (B), TNF-a (C), DMPC liposomes 1 TNF-a (D), T-rHDL 1 TNF-a (E). 20 objective.
By using the above incubation conditions, VCAM-1 (F) and ICAM-1 (G) expression was measured in HUVECs by flow cytometry using FITC conjugated
antibodies to the respective receptors. Results are expressed as percentage of control. *P , 0.05, **P , 0.01 versus TNF-a, n = 6. Flow cytometry was also
employed to determine VCAM-1 (H) or CAM-1 (I) on HCAECs preincubated with the various cholesterol acceptors. *P , 0.05, **P , 0.01 versus TNF-a,
n = 5.
expression by the rHDL particles is likely to be via an SR-BIdependent pathway as previously described (Kimura et al.,
2006; Mcgrath et al., 2009; Murphy et al., 2006; Murphy and
Woollard, 2010), showing the diversity of these rHDL particles
to interact with a number of HDL receptors. The functional
significance of the inhibition of endothelial inflammation was
observed in shear flow cell adhesion studies. Preincubating
endothelial cell monolayers with either form of rHDL prior to
TNF-a stimulation and subsequent perfusion of monocytes
resulted in a significant reduction in total and stationary
adhesion. There was an increase in rolling adhesion with rHDL
treatment, which is more than likely due to the reduction in
ICAM-1 not allowing the monocyte to firmly adhere via receptors
including CD11b (Diamond et al., 1991; Springer, 1994).
In a previous study we described a potent anti-inflammatory
role for both apoA-I and HDL in attenuating monocyte
activation (Murphy et al., 2008). All forms of apoA-I and
T-rHDL appeared to have equal effects in inhibiting monocyte
activation to both receptor and nonreceptor agonists. This
finding translated into a decrease in CD11b-dependent
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Fig. 4. T-apoA-I and T-rHDL inhibits monocyte activation. (A) isolated human monocytes were incubated with PMA (1 mmol/l) with or without the
various apoA-I molecules in increasing doses (2.5–40 mg/ml). CD11b activation was assessed via flow cytometry and results were expressed as
a percentage of control. *apoA-I compared with PMA, #T-apoA-I compared with PMA, and ^T-rHDL compared with PMA, n = 5. *P , 0.05 versus PMA
(0mg/ml), ^^, ##, **P , 0.01 versus PMA (0mg/ml), ^^^, ###, ***P , 0.001 versus PMA (0mg/ml). (B) monocytes were stimulated with LPS (1 mg/ml) in the
presence or absence of the cholesterol acceptors (40 mg/ml). Flow cytometry was used to determine the CD11b activation. *P , 0.05, **P , 0.01, n = 5.
adhesion as assessed by monocyte binding to fibrinogen
(Gresham et al., 1989). By using shear flow cell adhesion
experiments we were again able to demonstrate a functional
effect of the apoA-I molecules. All molecules inhibited monocyte adhesion to the endothelial monolayer to a similar extent.
Importantly, these trimeric particles did not appear to have
any superphysiologic effects, suggesting they should be
equally as safe as native particles in vivo.
In conclusion, we describe a trimeric apoA-I molecule that
can form a rHDL particle comprised of three apoA-I molecules
that retains the most important cardioprotective effects of
native apoA-I and HDL. The trimeric complex can efficiently
facilitate cholesterol efflux from lipid-loaded macrophages.
T-apoA-I and T-rHDL are also able to modulate leukocyte
recruitment to the endothelium by inhibiting endothelial
and monocyte activation. Although further investigation
of T-apoA-I in vivo is required, the data presented here
suggest an alternative therapeutic approach to the sustained
increase of apoA-I/HDL to treat cardiovascular-related
diseases.
Anti-Inflammatory Properties of Trimeric apoA-I
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Fig. 5. T-rHDL inhibits endothelial-mediated monocyte recruitment under shear flow. HCAECs were preincubated with rHDLs before stimulation with
TNF-a and subsequent perfusion of purified monocytes. (A) total adhesion, (B) stationary adhesionm and (C) rolling flux. T-rHDL inhibited total and
stationary adhesion after 5 min, whereas rHDLE after 10 min compared with TNF-a. T-rHDL resulted in a significant increase in rolling flux after 5 min
compared with TNF-a. Phase-contrast images at 5 and 10 min, respectively, control (D and E), TNF-a (F and G), rHDLE (H and I), and T-rHDL (J and K).
*P , 0.05 TNF-a1T-rHDL versus TNF-a, **P , 0.01 TNF-a1T-rHDL versus TNF-a, ***P , 0.001 TNF-a1T-rHDL versus TNF-a. #P , 0.05 TNF-a1
rHDLE versus TNF-a, ###P , 0.001 TNF-a1rHDLE versus TNF-a.
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Fig. 6. T-apoA-I and T-rHDL inhibit CD11b-dependent monocyte adhesion and recruitment under shear flow. (A) isolated human monocytes were
stimulated with PMA with or without the various cholesterol acceptors (40 mg/ml) and allowed to adhere to fibrinogen-coated glass coverslips. Cell
adhesion was determined by counting five random fields from each slide (40 objective). **P , 0.001, n = 3. (B) isolated monocytes were pretreated as
above prior to perfusion over HCAECs. Number of adhered monocytes was counted and plotted as average number of cells per field 6 S.E.M. (C–G)
phase-contrast images of adherent monocytes to HCAECs after 5 min of shear flow. *P , 0.05 versus apoA-IE vs PMA, **P , 0.01 versus apoA-IE versus
PMA and ***P , 0.001 versus apoA-IE vs PMA. #P , 0.05 versus T-apoA-I versus PMA, ##P , 0.01 versus T-apoA-I versus PMA, ^P , 0.05 versus TrHDL versus PMA, ^^P , 0.01 versus T-rHDL versus PMA. (C) unstimulated, (D) PMA, (E) apoA-IE 1 PMA, (F) T-apoA-I 1 PMA, and (G) T-rHDL 1
PMA. 20 objective, bar = 56 mm. **P , 0.01, ***P , 0.001, n = 4.
Acknowledgments
The authors acknowledge the kind contribution of Hoffmann La
Roche for generous donation of the compounds studied. In particular,
the authors acknowledge the discussions and assistance received
from Michel Pech, Juergen Fingerle, and Dorothee Kling from Roche.
Conducted experiments: Murphy, Hoang, Aprico.
Performed data analysis: Murphy, Hoang, Aprico.
Wrote or contributed to the writing of the manuscript: Murphy,
Sviridov, Chin-Dusting.
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