Reduction of In-Stent Restenosis by Cholesteryl Ester

Transfer Protein Inhibition

Ben J. Wu, Yue Li, Kwok L. Ong, Yidan Sun, Sudichhya Shrestha, Liming Hou, Douglas Johns,
Philip J. Barter, Kerry-Anne Rye

Objective—Angioplasty and stent implantation, the most common treatment for atherosclerotic lesions, have a significant
failure rate because of restenosis. This study asks whether increasing plasma high-density lipoprotein (HDL) levels by
inhibiting cholesteryl ester transfer protein activity with the anacetrapib analog, des-fluoro-anacetrapib, prevents stent-
induced neointimal hyperplasia.
Approach and Results—New Zealand White rabbits received normal chow or chow supplemented with 0.14% (wt/wt) des-
fluoro-anacetrapib for 6 weeks. Iliac artery endothelial denudation and bare metal steel stent deployment were performed
after 2 weeks of des-fluoro-anacetrapib treatment. The animals were euthanized 4 weeks poststent deployment. Relative
to control, dietary supplementation with des-fluoro-anacetrapib reduced plasma cholesteryl ester transfer protein activity
and increased plasma apolipoprotein A-I and HDL cholesterol levels by 53±6.3% and 120±19%, respectively. Non-HDL
cholesterol levels were unaffected. Des-fluoro-anacetrapib treatment reduced the intimal area of the stented arteries by
43±5.6% (P<0.001), the media area was unchanged, and the arterial lumen area increased by 12±2.4% (P<0.05). Des-
fluoro-anacetrapib treatment inhibited vascular smooth muscle cell proliferation by 41±4.5% (P<0.001). Incubation of
isolated HDLs from des-fluoro-anacetrapib–treated animals with human aortic smooth muscle cells at apolipoprotein A-I
concentrations comparable to their plasma levels inhibited cell proliferation and migration. These effects were dependent
on scavenger receptor-B1, the adaptor protein PDZ domain-containing protein 1, and phosphatidylinositol-3-kinase/Akt
activation. HDLs from des-fluoro-anacetrapib–treated animals also inhibited proinflammatory cytokine-induced human
aortic smooth muscle cell proliferation and stent-induced vascular inflammation.
Conclusions—Inhibiting cholesteryl ester transfer protein activity in New Zealand White rabbits with iliac artery balloon injury and
stent deployment increases HDL levels, inhibits vascular smooth muscle cell proliferation, and reduces neointimal hyperplasia
in an scavenger receptor-B1, PDZ domain-containing protein 1– and phosphatidylinositol-3-kinase/Akt-dependent manner.
Visual Overview—An online visual overview is available for this article.   (Arterioscler Thromb Vasc Biol. 2017;37:
2333-2341. DOI: 10.1161/ATVBAHA.117.310051.)
Key Words: angioplasty ◼ apolipoprotein A-I ◼ cholesterol ester transfer protein ◼ iliac artery
◼ vascular smooth muscle cell proliferation

A ngioplasty and stent deployment, the most common pro-

cedures for treating atherosclerotic lesions, have a sig-
nificant failure rate because of restenosis of the culprit artery.1
Current restenosis rates with bare metal stents are 10% to 30%.2
Although the rate of restenosis is lower with drug-eluting stents
(≈3.5% after 1 year3), their use requires 12 months of dual anti-
platelet therapy to reduce the risk of stent-induced thrombosis.
This is associated with long-term safety concerns, especially
neoatherosclerosis and fracture-related adverse pathological
events.4,5 This complicates management of patients who require
further procedures during the first year after stent deployment.
Restenosis rates for bare metal and drug-eluting stents are also
increased in people with diabetes mellitus.2 There is thus an
unmet need for therapeutic interventions that prevent stent-
induced restenosis. The pathogenesis of in-stent restenosis is
multifactorial, with endothelial dysfunction,6 inflammatory
cell accumulation in blood vessels,7 intimal vascular smooth
muscle (VSMC) proliferation,8 thrombus formation,9 deposi-
tion of extracellular matrix,10 and oxidative modification of
proteins,11 all contributing to neointimal formation.
Plasma high-density lipoprotein cholesterol (HDL-C) lev-
els correlate inversely with the risk of having a cardiovascular
event.12 HDLs have several potentially cardioprotective proper-
ties, the best known of which relates to cholesterol removal from
macrophages in the artery wall in the first step of reverse cho-
lesterol transport.13,14 HDLs also inhibit vascular inflammation,15
suppress VSMC proliferation,16 promote endothelial repair,17
and enhance endothelial function.18 These cardioprotective
properties of HDLs suggest that therapies that increase endoge-
nous HDL-C levels may attenuate in-stent restenosis. Additional

Received on: August 2, 2017; final version accepted on: October 2, 2017.
From the School of Medical Sciences, The University of New South Wales Sydney, Australia (B.J.W., K.L.O., Y.S., S.S., L.H., P.J.B., K.-A.R.); Institute
of Pathophysiology and Immunology, Medical University of Graz, Austria (Y.S.); and Merck & Co., Inc, Kenilworth, NJ (D.J.).
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.117.310051/-/DC1.
Correspondence to Kerry-Anne Rye, PhD, or Ben J. Wu, PhD, School of Medical Sciences, Faculty of Medicine, University of New South Wales,
Sydney, NSW, Australia 2052. E-mail k.rye@unsw.edu.au or ben.wu@unsw.edu.au
© 2017 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.117.310051

2333
2334   Arterioscler Thromb Vasc Biol   December 2017
Nonstandard Abbreviations and Acronyms
ABC ATP-binding cassette transporter
ApoA-I apolipoprotein A-I
CETP cholesteryl ester transfer protein
HASMC human aortic smooth muscle cell
HDL-C high-density lipoprotein cholesterol
NZW New Zealand White
PDZK1 PDZ domain-containing protein 1
PI3K/Akt phosphatidylinositol-3-kinase/Akt
SR-B1 scavenger receptor-B1
TNF-α tumor necrosis factor-α
VSMC vascular smooth muscle cell
evidence that high HDL levels protect against in-stent reste-
nosis comes from peripheral artery disease and diabetes mel-
litus studies, where patients with in-stent restenosis have lower
plasma HDL-C levels than those without in-stent restenosis.19,20
A low HDL cholesterol efflux capacity at baseline also predicts
increased coronary restenosis risk in patients with stable angina
and stent implantation21 while treatment with reconstituted
HDLs inhibits in-stent stenosis in porcine coronary arteries.22
CETP (cholesteryl ester transfer protein) transfers choles-
teryl esters from HDLs to low-density lipoproteins and triglyc-
eride-rich lipoproteins.23 Inhibition of CETP activity increases
HDL-C and apoA-I (apolipoprotein A-I) levels and decreases
non-HDL cholesterol levels. We have reported that increasing
HDL-C levels with the CETP inhibitor des-fluoro-anacetrapib
enhances endothelial repair, improves endothelial function,
inhibits VSMC proliferation, and reduces intimal hyperplasia in
New Zealand White (NZW) rabbits with endothelial denudation
of the abdominal aorta.24 The peroxisome proliferator-activated
receptor-α agonist, fenofibrate, also inhibits CETP activity and
reduces intimal hyperplasia after coronary stenting.25
In this study, stent-induced neointimal hyperplasia was
assessed in des-fluoro-anacetrapib–treated NZW rabbits with
endothelial denudation of the iliac artery and stent deploy-
ment. Des-fluoro-anacetrapib is an active analog of anac-
etrapib with 1 less fluorine atom than the parent compound.24
Treatment with des-fluoro-anacetrapib increased plasma
HDL-C and apoA-I levels and inhibited stent-induced VSMC
proliferation and neointimal hyperplasia in these animals by a
mechanism that was dependent on scavenger receptor class B
type 1 (SR-B1), the adaptor PDZK1 (PDZ domain-containing
protein 1), and activation of the phosphatidylinositol-3-kinase
(PI3K)/Akt signal transduction pathway.
Materials and Methods
Materials and Methods are available in the online-only Data
Supplement.
Results
Treatment With Des-Fluoro-Anacetrapib Increases
HDL-C and ApoA-I Levels in NZW Rabbits
Dietary supplementation with 0.14% (wt/wt) des-fluoro-
anacetrapib reduced CETP activity by 85±9.8% in NZW rab-
bits relative to control, chow-fed animals (Figure IA in the
online-only Data Supplement; P<0.001). CETP inhibition
increased plasma apoA-I levels from 0.54±0.03 mg/mL for
the control animals to 0.83±0.04 mg/mL for the des-fluoro-
anacetrapib–treated animals (Figure IB in the online-only
Data Supplement; P<0.001). Plasma HDL-C levels increased
from 0.37±0.03 mmol/L in the control animals to 0.82±0.07
mmol/L in the des-fluoro-anacetrapib–treated animals (Figure
IC in the online-only Data Supplement; P<0.001). Resolution
of plasma lipoproteins by gel permeation chromatography
confirmed the increase in HDL-C levels. HDLs from the des-
fluoro-anacetrapib–treated animals eluted earlier than HDLs
from the control animals, which is consistent with an increase
in HDL particle size Figure ID in the online-only Data
Supplement). Treatment with des-fluoro-anacetrapib did not
affect non–HDL-C levels (Figure ID in the online-only Data
Supplement), and plasma triglyceride levels were unchanged
(2.01±0.34 mmol/L for control versus 1.56±0.36 mmol/L for
des-fluoro-anacetrapib–treated animals).
Treatment With Des-Fluoro-Anacetrapib
Inhibits Neointimal Hyperplasia in NZW
Rabbits With Balloon Injury and Stent
Deployment in the Iliac Artery
We have previously reported that des-fluoro-anacetrapib treat-
ment of NZW rabbits with balloon injury of the abdominal
aorta inhibits intimal hyperplasia and increases VSMC pro-
liferation.24 In the present study, endothelial denudation of the
iliac artery and bare metal stent deployment induced neointi-
mal formation (Figure 1) and VSMC proliferation in the con-
trol animals (Figure II in the online-only Data Supplement).
The neointimal area in the stented iliac arteries of the des-
fluoro-anacetrapib–treated rabbits was decreased by 43±5.6%
relative to control animals (Figure 1A and 1B; P<0.001) while
the media area was unaffected (Figure 1A and 1C). Des-
fluoro-anacetrapib treatment decreased the intima/media ratio
of the stented iliac arteries by 46±4.2% (Figure 2D) while the
lumen area increased by 12±2.4% (Figure 1E; P<0.001 and
P<0.05 compared with control, respectively). Treatment with
des-fluoro-anacetrapib also inhibited VSMC proliferation in
the stented iliac arteries (Figure IIA in the online-only Data
Supplement), with the number of PCNA+ (proliferating cell
nuclear antigen) cells decreasing by 41±4.5% relative to con-
trol (Figure IIB in the online-only Data Supplement; P<0.001).
HDLs From Des-Fluoro-Anacetrapib–
Treated NZW Rabbits Inhibit HASMC
Proliferation and Migration
To determine whether the decreased VSMC proliferation in
des-fluoro-anacetrapib–treated NZW rabbits was because of
improved HDL function, or due to increase in plasma HDL
levels, HDLs were isolated from the control- and des-fluoro-
anacetrapib–treated animals and incubated with human aortic
smooth muscle cells (HASMCs) at equivalent apoA-I concen-
trations and at apoA-I concentrations that replicated their on-
treatment plasma levels at the time of euthanasia.
Incubation of HASMCs with PBS or isolated HDLs from
control animals (Ctrl) at an apoA-I concentration comparable
to that in plasma (0.54 mg/mL) did not affect cell proliferation
 Wu et al   CETP Inhibition and In-Stent Restenosis   2335
Figure 1. Dietary supplementation with des-fluoro-anacetrapib
(dfAna) inhibits neointimal hyperplasia in rabbits in response to
iliac artery endothelial denudation and stent deployment. New
Zealand White rabbits (n=8/group) received chow (control) or
chow supplemented with 0.14% (wt/wt) dfAna for 6 wk. Endo-
thelial denudation of the iliac artery and bare metal steel deploy-
ment was performed after 2 wk of dfAna treatment. The animals
were euthanized 4 wk post-stent deployment. A, Verhoeff’s
hematoxylin-stained stented iliac artery cross-sections (bar=500
µm). Quantification of cross-sectional intimal area (B), media
area (C), intima-to-media ratio (D), and luminal area (E) of stented
iliac arteries. Data are expressed as mean±SEM, n=8, *P<0.05,
***P<0.001 vs control.
(Figure 2A). When the HASMCs were incubated with HDLs
from des-fluoro-anacetrapib–treated rabbits at an apoA-I con-
centration comparable to that in plasma (0.83 mg/mL, closed
bar), cell proliferation decreased by 40±11% relative to cells
incubated with PBS (open bar) and by 34±13% compared with
cells incubated with HDLs from control animals (Figure 2A,
closed bars; P<0.01 and P<0.05, respectively). To confirm that
HDLs isolated from des-fluoro-anacetrapib–treated rabbits
reduced HASMC proliferation more effectively than HDLs
from control rabbits, BrdU (5-bromo-2’-deoxyuridine)-pos-
itive cells in the HASMCs were quantified by flow cytom-
etry (Figure 2B). Incubation of HASMCs with HDLs from
des-fluoro-anacetrapib–treated rabbits decreased the number
of BrdU-positive cells by 36±7.2% relative to HDLs from
control animals (Ctrl-HDL, closed bars; P<0.01). There was
Figure 2. High-density lipoproteins (HDLs) from des-fluoro-
anacetrapib (dfAna)–treated New Zealand White (NZW) rabbits
decrease human aortic smooth muscle cell (HASMC) proliferation
and migration. HDLs were isolated from plasma of control and
dfAna-treated NZW rabbits after endothelial denudation of the
iliac artery and bare metal steel deployment as described in the
legend to Figure 1. HASMCs were incubated for 24 h with PBS or
isolated HDLs at final apolipoprotein A-I (apoA-I) concentrations
comparable to their plasma levels: 0.54 mg/mL for control (Ctrl)
and 0.83 mg/mL for 0.14% dfAna-treated rabbits. A, HASMC
proliferation. B, HASMC proliferation assessed as %BrdU
(5-bromo-2’-deoxyuridine)-positive cells. C, HASMC migration
was quantified as the number of cells that migrated past the
wound edge (black line). HASMC proliferation (D) and migration
(E) after incubation for 24 h with isolated HDLs from control (Ctrl)
and dfAna-treated rabbits at a final apoA-I concentration of 0.5
mg/mL. Data are expressed as mean±SEM, n=6 to 7, #P<0.05,
##P<0.01, ###P<0.001. hpf indicates high power field.
a trend toward reduced proliferation in the HASMCs that
were incubated with HDLs from the control animals relative
to HASMCs incubated with PBS (open bar), but this did not
reach statistical significance (Figure 2B).
In a scratch wound assay, HASMC migration was not
affected by incubation with PBS (open bar) or by incuba-
tion with HDLs from the control animals (Ctrl-HDL, closed
bar) at an apoA-I concentration equivalent to that in plasma
(Figure 2C). The number of cells that migrated across the
scratch wound when HASMCs were incubated with HDLs
from des-fluoro-anacetrapib–treated rabbits (des-fluoro-
anacetrapib–HDL) decreased by 46±3.2% compared with
HASMCs incubated with PBS (P<0.01; Figure 2C). Incubation
2336   Arterioscler Thromb Vasc Biol   December 2017

of HASMCs with HDLs from des-fluoro-anacetrapib–treated

rabbits reduced the number of cells that migrated across the
wound by 34±13% relative to HDLs from control rabbits
(Figure 2C, closed bars; P<0.05). Incubation of HASMCs
with HDLs from control and des-fluoro-anacetrapib–treated
rabbits at equivalent apoA-I concentrations did not affect cell
proliferation (Figure 2D) or migration (Figure 2E). Taken
together, these results indicate that des-fluoro-anacetrapib
treatment inhibits VSMC proliferation in balloon-injured,
stented NZW rabbit iliac arteries by increasing plasma HDL
levels, not by increasing HDL function.

HDLs From Des-Fluoro-Anacetrapib–Treated

NZW Rabbits Inhibit HASMC Proliferation
and Migration in an SR-B1-, PDZK1-, and
PI3K/Akt-Dependent Manner
As HDLs have been reported to inhibit smooth muscle cell
proliferation16 and migration26 in an SR-B1/PDZK1- and
PI3K/Akt-dependent manner,27,28 we next asked whether this
was also the case for HDLs from des-fluoro-anacetrapib–
treated rabbits.
Transfection of HASMCs with SR-B1 siRNA (small inter-
fering RNA) or PDZK1 siRNA decreased SR-B1 (Figure 3A)
and PDZK1 (Figure 3B) protein levels by 67±5.6% and
56±3.8%, respectively, compared with HASMCs transfected
with scrambled siRNA (siControl; P<0.01 for both). The trans-
fected cells were then incubated in the absence or presence of
HDLs from rabbits treated with des-fluoro-anacetrapib at an
apoA-I concentration identical to that in plasma at the time
of euthanasia (0.83 mg/mL). Incubation of nontransfected
HASMCs and cells transfected with scrambled siRNA in the
presence of PBS did not affect cell proliferation (Figure 3C) or
migration (Figure 3D). There was a trend toward decreased cell
proliferation (Figure 3E and 3G, closed bars) and migration
(Figure 3F and 3H, closed bars) in HASMCs transfected with
SR-B1 siRNA or PDZK1 siRNA, but this did not reach statis-
Figure 3. High-density lipoproteins (HDLs) from des-fluoro-
tical significance. Incubation of scrambled siRNA-transfected anacetrapib–treated New Zealand White (NZW) rabbits increase
HASMCs (siControl) with HDLs from des-fluoro-anacetra- human aortic smooth muscle cell (HASMC) proliferation and
pib–treated rabbits decreased cell proliferation by 29±3.2% migration in a scavenger receptor-B1 (SR-B1)- and PDZK1 (PDZ
(Figure 3E, open bars) and 43±8.4% (Figure 3G, open bars) domain-containing protein 1)-dependent manner. HASMCs were
transfected for 72 h with SR-B1 siRNA (small interfering RNA;
while cell migration decreased by 44±7.1% (Figure 3F, open A, E, F, closed bars) or for 24 h with PDZK1 siRNA (B, G, H,
bars) and 48±4.6% (Figure 3H, open bars; P<0.01 for all). closed bars) or scrambled siRNA (A–H, open bars). Nontrans-
Transfection of HASMCs with Akt siRNA decreased Akt fected HASMCs were incubated with PBS (C, D, closed bars).
The transfected HASMCs were incubated for 24 h in the absence
protein levels by 73±1.1% compared with cells transfected or presence of HDLs (final apolipoprotein A-I concentration
with scrambled siRNA (siControl; Figure 4A; P<0.001). 0.83 mg/mL) from NZW rabbits treated with 0.14% (wt/wt) des-
Incubation of Akt siRNA-transfected HASMCs in the absence fluoro-anacetrapib as described in the legend to Figure 1. A and
of HDLs did not affect cell proliferation (Figure 4B) or migra- B, Transfection efficiency monitored by Western blotting using
β-actin as a loading control. Data represent the mean±SEM (n=3).
tion (Figure 4C). Incubation of HASMCs transfected with *P<0.05 vs scrambled siRNA. C, E, G, HASMC proliferation. D, F,
scrambled siRNA (siControl) in the presence of HDLs from H, Number of HASMCs migrating past the scratch wound edge/
des-fluoro-anacetrapib–treated rabbits decreased the number high power field (hpf). Data are expressed as mean±SEM, n=6.
**P<0.01 vs siControl. #P<0.05, ##P<0.01.
of cells from 10±1.0×105 to 7.0±0.7×105 (Figure 4B, open
bars; P<0.05) while cell migration decreased from 190±19 to
117±11 cells/high power field (Figure 4C, open bars; P<0.01). in the presence of LY294002 alone did not affect cell pro-
HDLs did not inhibit cell proliferation (Figure 4B) or migra- liferation (Figure 4D, closed bar) or migration (Figure 4E,
tion (Figure 4C) in HASMCs transfected with Akt siRNA. closed bar). HASMC cell proliferation (Figure 4D, open
This was confirmed by pre-incubating HASMCs with the bars) and migration (Figure 4E, open bars) were decreased
PI3K/Akt inhibitor, LY294002 (LY) before incubation with by incubation with HDLs alone. Pre-incubation of HASMCs
HDLs from des-fluoro-anacetrapib–treated rabbits. Incubation with LY294002 before incubation with HDLs from
 Wu et al   CETP Inhibition and In-Stent Restenosis   2337
Figure 4. High-density lipoproteins (HDLs) from des-fluoro-anac-
etrapib–treated New Zealand White (NZW) rabbits increase human
aortic smooth muscle cell (HASMC) proliferation and migration in
a phosphatidylinositol-3-kinase/Akt-dependent manner. HASMCs
were transfected for 48 h with Akt siRNA (small interfering RNA;
A–C, closed bars) or scrambled siRNA (A–C, open bars). HASMCs
were also pre-incubated for 1 h without (D–E, open bars) or with
10 µmol/L LY294002 (LY; D–E, closed bars). HASMCs were incu-
bated for 24 h in the absence or presence of HDLs (final apolipo-
protein A-I concentration as described in the legend to Figure 1 is
0.83 mg/mL) from NZW rabbits treated with 0.14% (wt/wt) des-
fluoro-anacetrapib. A, Transfection efficiency was monitored by
Western blotting with anti-Akt antibodies using β-actin as a loading
control. Data represent the mean±SEM (n=4). *P<0.05 vs scram-
bled siRNA. B and D, HASMC proliferation. C and E, Number of
HCAECs (human coronary artery endothelial cells) migrating past
the scratch wound edge/high power field (hpf). Data are expressed
as mean±SEM, n=6. ***P<0.001 vs siControl. #P<0.05, ##P<0.01.
des-fluoro-anacetrapib–treated rabbits abolished the reduc-
tion in proliferation (Figure 4D, closed bars) and migration
(Figure 4E, closed bars).
To determine whether the HDL-mediated inhibition of
HASMC proliferation and migration was dependent on the
ATP-binding cassette transporters ABCA1 and ABCG1,
cells were transfected with ABCA1 siRNA, ABCG1 siRNA,
or scrambled siRNA (siControl). Transfection with ABCA1
and ABCG1 siRNA decreased ABCA1 (Figure IIIA in the
online-only Data Supplement) and ABCG1 (Figure IIIB in
the online-only Data Supplement) protein levels by 80±6.6%
and 72±3.3%, respectively (P<0.001 and P<0.01 compared
with cells transfected with scrambled siRNA, respectively).
Incubation of HASMCs transfected with ABCA1 siRNA or
ABCG1 siRNA in the absence of HDLs did not affect cell
proliferation (Figure IIIC and IIIE in the online-only Data
Supplement) or migration (Figure IIID and IIIF in the online-
only Data Supplement). When the HASMCs transfected with
scrambled siRNA (siControl) were incubated with HDLs from
des-fluoro-anacetrapib–treated rabbits, the number of cells
decreased from 7.9±1.0×105 to 5.3±0.3×105 (Figure IIIC in the
online-only Data Supplement, open bars; P<0.05) and from
11±0.7×105 to 6.8±0.5×105 (Figure IIIE in the online-only Data
Supplement, open bars; P<0.001). Cell migration decreased
from 121±21 to 63±11 cells/high power field (Figure IIID
in the online-only Data Supplement, open bars; P<0.05) and
from 210±28 to 128±15 cells/high power field (Figure IIIF in
the online-only Data Supplement, open bars; P<0.05) when the
HASMCs transfected with scrambled siRNA (siControl) were
incubated with HDLs from des-fluoro-anacetrapib–treated rab-
bits. When HASMCs transfected with ABCA1 siRNA (Figure
IIIC and IIID in the online-only Data Supplement, closed bars)
and ABCG1 siRNA (Figure IIIE and IIIF in the online-only
Data Supplement, closed bars) were incubated with HDLs
from des-fluoro-anacetrapib–treated rabbits, the reduction in
cell proliferation and migration was the same as in incubations
with scrambled siRNA-transfected cells. This indicates that
HDLs inhibit HASMC proliferation and migration by mecha-
nisms independent of ABCA1 and ABCG1.
HDLs From Des-Fluoro-Anacetrapib–
Treated NZW Rabbits Protect Against
TNF-α–Induced HASMCs Proliferation
Progression of neointimal hyperplasia after balloon angio-
plasty and stent placement has been attributed to vascular
inflammation.29 We have also reported that HDLs from nor-
mal human plasma protect against proinflammatory cytokine-
induced VSMC proliferation in vitro.16 To determine whether
this is also the case for HDLs from des-fluoro-anacetrapib–
treated NZW rabbits, HASMCs were pre-incubated with
HDLs from control and des-fluoro-anacetrapib–treated rab-
bits at apoA-I concentrations that replicated their on-treatment
plasma levels at euthanasia. The HASMCs were then incu-
bated in the presence and absence of tumor necrosis factor-α
(TNF-α). Relative to cells incubated in the absence of TNF-
α (Figure 5A, open bar), incubation with TNF-α increased
HASMC proliferation by 54±14% (Figure 5A; P<0.05). HDLs
from the control- and des-fluoro-anacetrapib–treated animals
decreased TNF-α–induced HASMC proliferation by 42±4.0%
and 53±2.3%, respectively (Figure 5A; P<0.001 versus TNF-
α only; P<0.05 HDL for control versus treated rabbits).
To determine whether the reduction in TNF-α–induced
HASMC proliferation was dependent on SR-B1, the incuba-
tions were repeated in HASMCs transfected with scrambled
siRNA (siControl) or SR-B1 siRNA. Incubation of the cells
transfected with SR-B1 siRNA in the absence of HDLs did
not affect TNF-α–induced cell proliferation (Figure 5B).
Incubation of scrambled siRNA-transfected HASMCs with
HDLs from des-fluoro-anacetrapib–treated rabbits decreased
2338   Arterioscler Thromb Vasc Biol   December 2017
Figure 5. High-density lipoproteins (HDLs) from des-fluoro-
anacetrapib (dfAna)–treated rabbits protect against tumor necrosis
factor-α (TNF-α)–induced smooth muscle cell proliferation in a
scavenger receptor-B1 (SR-B1)–dependent manner. Human aortic
smooth muscle cells (HASMCs) were incubated for 18 h in the
absence or presence of HDLs from control- and dfAna-treated ani-
mals as described in the legend to Figure 1. The cells were incu-
bated for 6 h in the absence (A, open bar) or presence (A, closed
bars) of TNF-α before assessment of cell proliferation. HASMCs
were transfected for 72 h with SR-B1 siRNA (small interfering RNA;
B, closed bars) or scrambled siRNA (B, open bars), incubated for
18 h in the absence or presence HDLs isolated from NZW rabbits
treated with 0.14% (wt/wt) dfAna, and then activated with TNF-
α for 6 h before assessment of HASMC proliferation. Data are
expressed as mean±SEM, n=6. #P<0.05, ##P<0.01, ###P<0.001.
TNF-α–induced cell proliferation from 12±0.7×105 to
5.7±0.4×105 (Figure 5B, open bars; P<0.01). The HDLs from
des-fluoro-anacetrapib–treated rabbits did not inhibit cell
proliferation when they were incubated with HASMCs trans-
fected with SR-B1 siRNA (Figure 5B, closed bar).
Treatment With Des-Fluoro-Anacetrapib
Inhibits Vascular Inflammation in NZW
Rabbits With Balloon Injury and Stent
Deployment in the Iliac Artery
Because extravasation of inflammatory cells into the artery
wall contributes to progression of neointimal hyperplasia after
balloon angioplasty and stent deployment,29 we asked whether
des-fluoro-anacetrapib treatment can also inhibit inflammatory
cell accumulation in balloon-injured and stented iliac arteries.
NZW rabbits received regular chow or chow supplemented
with 0.14% (wt/wt) des-fluoro-anacetrapib for 2 weeks before
balloon injury and stent deployment. The animals were eutha-
nized 24 hours poststent deployment.
Relative to control, plasma CETP activity in the des-
fluoro-anacetrapib–treated rabbits decreased by 90±17%
(Figure 6A; P<0.01) and plasma apoA-I and HDL-C lev-
els increased by 51±17% (Figure 6B; P<0.05) and 75±24%
(Figure 6C; P<0.05), respectively. The number of CD18+ cells
in the vessel wall was reduced by 85±9.8% in the des-fluoro-
anacetrapib–treated animals relative to control (Figure 6D;
P<0.001). This indicates that des-fluoro-anacetrapib treatment
inhibits acute vascular inflammatory responses after balloon
injury and stent deployment in the iliac artery of NZW rabbits.
Discussion
The effects of inhibiting CETP with the anacetrapib analogue,
des-fluoro-anacetrapib that were observed in this study are
most likely a consequence of increased HDL levels rather than
changes in non-HDL levels, that were minimal. This study
thus supports the notion that increasing HDL-C levels by
inhibiting CETP activity with des-fluoro-anacetrapib prevents
intimal hyperplasia and inhibits VSMC proliferation in NZW
rabbits with balloon injury of the iliac artery and stent deploy-
ment. We further demonstrate that HDLs isolated from these
animals decrease HASMC proliferation and migration in vitro
in an SR-B1/PDZK1- and PI3K/Akt-dependent manner, pro-
tect against inflammatory cytokine-induced HASMC prolif-
eration, and inhibit acute vascular inflammatory responses.
Because the amino acid sequence of rabbit SR-BI and its
human homologue, CLA-1, are 86% homologous, and the
proteins are functionally comparable,30 it is highly likely that
the results from the in vitro HASMC experiments also reflect
what occurs in smooth muscle cells in the NZW rabbit.
Restenosis is a common problem after stent implantation
in humans. Although drug-eluting stents have a lower reste-
nosis rate than bare metal stents, they are associated with a
higher rate of cardiac death and myocardial infarction because
of stent thrombosis.31 Even though use of new generation of
drug-eluting stents and long-term dual antiplatelet therapy
reduces stent thrombosis, neoatherosclerosis is increasingly
being recognized as a significant problem.31
CETP inhibition produces large, cholesteryl ester-
enriched HDL particles and increases plasma HDL levels in
rabbits and humans.32,33 CETP inhibition also inhibits athero-
sclerotic lesion development in rabbits,33–35 and anacetrapib
reduced major cardiovascular events in the recent REVEAL
trial (Randomized EValuation of the Effects of Anacetrapib
through Lipid modification).36 Cardiovascular events were,
by contrast, not reduced in earlier clinical outcome trials with
other CETP inhibitors.37–39
The present study indicates that increasing HDL levels by
inhibiting CETP activity with anacetrapib may reduce stent-
induced thrombosis and neoatherosclerosis. Because the cur-
rent results were obtained in normocholesterolemic rabbits,
additional investigations in cholesterol-fed animals are war-
ranted. Our results are, however, in line with a significant
reduction in revascularization in anacetrapib-treated subjects
in the REVEAL trial.36 We have further established that the
reduced neointimal hyperplasia in des-fluoro-anacetrapib–
treated rabbits can be attributed directly to the increase in
plasma HDL levels, rather than to an improvement in HDL
function for a given concentration of apoA-I. This provides
clear evidence that CETP inhibition does not generate dys-
functional HDLs, at least in terms of their ability to reduce
stent-induced neointimal hyperplasia. It will be of interest to
 Wu et al   CETP Inhibition and In-Stent Restenosis   2339

Figure 6. Des-fluoro-anacetrapib (dfAna) treatment inhibits inflammatory cell infiltration in New Zealand White (NZW) rabbits with endo-
thelial denudation and stent deployment of the iliac artery. NZW rabbits (n=5/group) received regular chow (control) or chow supple-
mented with 0.14% (wt/wt) dfAna for 2 wk before iliac artery endothelial denudation and stent deployment. The animals were euthanized
24 h poststent deployment. The stented arteries were resected, fixed in 4% (v/v) cold paraformaldehyde, and sectioned longitudinally
before stent removal. A, plasma CETP (cholesteryl ester transfer protein) activity. B, Plasma apolipoprotein A-I (apoA-I) levels. C, Plasma
high-density lipoprotein cholesterol (HDL-C) levels. D, Representative cross-sections of arteries immunostained for CD18 (bar=500 µm).
Data are expressed as mean±SEM, n=5, *P<0.05, **P<0.01, ***P<0.001 vs control.

determine if similar findings are apparent with other HDL-
raising strategies, such as reconstituted HDL infusions.
Consistent with previous reports, the present study estab-
lishes that HDLs from des-fluoro-anacetrapib–treated NZW
rabbits inhibit smooth muscle cell proliferation and migra-
tion in an SR-B1-, PDZK1-, and PI3K/Akt-dependent man-
ner.16,26–28 This is in line with evidence that several of the
cardioprotective and antidiabetic effects of HDLs are SR-B1
dependent. For example, HDL-induced endothelial nitric
oxide synthase activation,40 the promotion of endothelial cell
migration and re-endothelialization after endothelial injury,27
and increased glucose uptake by adipocytes and glycogen syn-
thesis in muscle41 are all SR-B1 dependent. Similarly, activa-
tion of downstream SR-B1 signaling pathways in endothelial
cells is dependent on binding of the C-terminal domain of
SR-B1 to PDZK1. Our findings suggest that this is also true
for HASMCs. However, a recent study has found that PDZK1
can also protect against neointima formation in ligated carotid
arteries in an SR-B1–independent manner.42 Whether this is
also the case for the inhibition of stent-induced neointimal
hyperplasia remains to be determined.
Stent implantation is associated with acute and chronic
inflammation in the vessel wall, both of which have the capac-
ity to contribute to neointimal proliferation.29 This is driven, at
least in part, by recruitment of inflammatory cells to the endo-
thelium and their subsequent migration into the vessel wall.
In this study, des-fluoro-anacetrapib treatment inhibited these
events in NZW rabbits with balloon injury and stent deploy-
ment in the iliac artery, with evidence that the benefit is attrib-
utable to the anti-inflammatory properties of HDLs.
In conclusion, this study establishes that increasing cir-
culating HDL levels by inhibiting CETP activity with des-
fluoro-anacetrapib protects against neointimal hyperplasia
in balloon-injured NZW rabbits with stent deployment sec-
ondary to inhibition of VSMC proliferation, migration, and
inflammation. It remains to be seen whether these mecha-
nisms are also responsible for reduced revascularization rates
in people treated with a CETP inhibitor.
2340   Arterioscler Thromb Vasc Biol   December 2017
Sources of Funding
This work was supported by Merck & Co., Inc and the National
Health and Medical Research Council (NHMRC) of Australia (grants
482800 and 1037903). K.L. Ong was supported by an NHMRC
Career Development Fellowship (1122854).
Disclosures
None.
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Highlights
• Increasing HDL levels by inhibiting CETP activity in NZW rabbits with des-fluoro-anacetrapib (1) reduces stent-induced neointimal hyperplasia,
(2) decreases vascular smooth muscle cell proliferation and migration, and (3) inhibits stent-induced acute vascular inflammatory responses.