Translational Sciences

Cholesteryl Ester Transfer Protein Inhibition Enhances Endothelial Repair and Improves Endothelial Function in the RabbitSignificance

Ben J. Wu, Sudichhya Shrestha, Kwok L. Ong, Douglas Johns, Liming Hou, Philip J. Barter, Kerry-Anne Rye
https://doi.org/10.1161/ATVBAHA.114.304747
Arteriosclerosis, Thrombosis, and Vascular Biology. 2015;35:628-636
Originally published January 29, 2015

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Abstract

Objective—High-density lipoproteins (HDLs) can potentially protect against atherosclerosis by multiple mechanisms, including enhancement of endothelial repair and improvement of endothelial function. This study asks if increasing HDL levels by inhibiting cholesteryl ester transfer protein activity with the anacetrapib analog, des-fluoro-anacetrapib, enhances endothelial repair and improves endothelial function in New Zealand White rabbits with balloon injury of the abdominal aorta.

Approach and Results—New Zealand White rabbits received chow or chow supplemented with 0.07% or 0.14% (wt/wt) des-fluoro-anacetrapib for 8 weeks. Endothelial denudation of the abdominal aorta was carried out after 2 weeks. The animals were euthanized 6 weeks postinjury. Treatment with 0.07% and 0.14% des-fluoro-anacetrapib reduced cholesteryl ester transfer protein activity by 81±4.9% and 92±12%, increased plasma apolipoprotein A–I levels by 1.4±0.1-fold and 1.5±0.1-fold, increased plasma HDL-cholesterol levels by 1.8±0.2-fold and 1.9±0.1-fold, reduced intimal hyperplasia by 37±11% and 51±10%, and inhibited vascular cell proliferation by 25±6.1% and 35±6.7%, respectively. Re-endothelialization of the injured aorta increased from 43±6.7% (control) to 69±6.6% and 76±7.7% in the 0.07% and 0.14% des-fluoro-anacetrapib-treated animals, respectively. Aortic ring relaxation and guanosine 3′,5′-cyclic monophosphate production in response to acetylcholine were also improved. Incubation of HDLs from the des-fluoro-anacetrapib-treated animals with human coronary artery endothelial cells increased cell proliferation and migration relative to control. These effects were abolished by knockdown of scavenger receptor-B1 and PDZ domain-containing protein 1 and by pharmacological inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt.

Conclusions—Increasing HDL levels by inhibiting cholesteryl ester transfer protein reduces intimal thickening and regenerates functional endothelium in damaged New Zealand White rabbit aortas in an scavenger receptor-B1-dependent and phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt-dependent manner.

Introduction

Population studies have consistently shown that plasma high-density lipoprotein cholesterol (HDL-C) levels correlate inversely with the risk of having a cardiovascular event.1 HDLs have several potentially cardioprotective properties, the best known of which relates to the removal of cholesterol from macrophages in the artery wall in the first step of reverse cholesterol transport.2 HDLs also inhibit vascular inflammation,3 reduce oxidative stress in macrophages,4 prevent oxidation of low-density lipoproteins (LDLs),5 reduce thrombosis,6 promote endothelial repair,7 enhance endothelial function,8 increase angiogenesis,9 and improve pancreatic β-cell function.10

We have previously reported that discoidal reconstituted HDLs comprising apolipoprotein (apo) A–I, the main HDL apolipoprotein, complexed with phospholipid, (A–I)rHDLs, inhibit adhesion molecule expression in cytokine-activated human umbilical vein endothelial cells and human coronary artery endothelial cells (HCAECs).11–13 We have also reported that intravenous infusions of lipid-free apoA–I and (A–I)rHDLs inhibit acute vascular inflammation in normocholesterolemic New Zealand White (NZW) rabbits3,14,15 and reduce atherosclerosis in cholesterol-fed NZW rabbits.16 This suggests that therapies that increase endogenous HDL levels may attenuate atherosclerotic lesion development and progression.

Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters from HDLs to LDLs and triglyceride-rich lipoproteins.17 Inhibition of CETP activity increases HDL-C and apoA–I levels and has been proposed as a strategy for reducing cardiovascular events. Genetic studies have also indicated that CETP inhibition is potentially cardioprotective.18–20 Furthermore, increasing HDL-C levels with the CETP inhibitors torcetrapib and JTT-705 inhibits atherosclerotic lesion development in rabbits, which have naturally high levels of CETP activity,21–24 but has so far not reduced cardiovascular events in humans.25,26

As endothelial injury is a key early event in atherosclerotic lesion development, and we have reported previously that increasing circulating HDL levels with (A–I)rHDL infusions enhances the repair of damaged endothelium in mice,7 we ask in this study if increasing HDL levels by inhibiting CETP generates functional endothelium in NZW rabbits with balloon injury of the abdominal aorta. This question has been addressed using des-fluoro-anacetrapib, an analog of the CETP inhibitor, anacetrapib (Figure I in the online-only Data Supplement), to treat NZW rabbits with endothelial denudation of the abdominal aorta. The results establish that des-fluoro-anacetrapib treatment increases HDL-C and apoA–I levels, enhances endothelial repair, and improves endothelial function in these animals. We also show that this outcome is dependent on scavenger receptor-B1 (SR-B1) and activation of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt signaling pathway.

Materials and Methods

Materials and Methods are available in the online-only Data Supplement.

Results

Des-fluoro-anacetrapib Treatment Increases HDL Size and HDL-C Levels in NZW Rabbits

Three groups of NZW rabbits (n=6/group) were used for the study. The control animals were maintained on normal chow for 8 weeks. The remaining animals consumed chow supplemented with 0.07% or 0.14% (wt/wt) des-fluoro-anacetrapib for 8 weeks. All the animals were subjected to endothelial denudation of the abdominal aorta 2 weeks after study commencement and were euthanized at 6 weeks postinjury. At euthanasia, the plasma concentration in the animals treated with 0.07% or 0.14% (wt/wt) des-fluoro-anacetrapib was 0.34±0.13 μmol/L and 0.65±0.34 μmol/L, respectively.

At euthanasia, plasma CETP activity (expressed as the % cholesteryl esters transferred from HDL3 to LDLs) in the animals treated with 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib was 2.9±0.8% and 1.1±1.8%, respectively, compared with 15.7±1.7% in the control animals (P<0.05; Table). Plasma total cholesterol levels increased from 0.56±0.16 mmol/L for the control animals to 0.98±0.12 and 1.01±0.13 mmol/L for the 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib-treated animals, respectively (P<0.05). HDL total cholesterol levels increased from 0.49±0.03 mmol/L (control) to 0.75±0.11 and 0.78±0.05 mmol/L, and HDL unesterified cholesterol levels increased from 0.11±0.01 mmol/L in the control animals to 0.17±0.02 and 0.19±0.03 mmol/L in the 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib-treated animals (P<0.05). HDL cholesteryl ester levels increased from 0.38±0.03 mmol/L (control) to 0.58±0.08 and 0.60±0.07 mmol/L, respectively, in the animals treated with 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib (P<0.05). Treatment with 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib also increased HDL-phospholipid levels from 0.81±0.02 mmol/L (control) to 0.97±0.07 and 1.03±0.08 mmol/L (P<0.05) while apoA–I levels increased from 0.43±0.06 mg/mL to 0.58±0.05 and 0.61±0.05 mg/mL (Table).

View this table:
Table.

Effect of Des-fluoro-anacetrapib on CETP Activity and Plasma Lipid and apoA–I Levels in New Zealand White Rabbits

Gel permeation chromatography of plasma samples from the control and des-fluoro-anacetrapib-treated animals established that HDL total cholesterol levels were increased and the HDLs eluted earlier in the des-fluoro-anacetrapib-treated animals compared with the control animals (Figure 1A). This indicates that des-fluoro-anacetrapib treatment increases HDL particle size and HDL-C levels. The increase in HDL size was confirmed by subjecting plasma to 2D gel electrophoresis and immunoblotting for apoA–I (Figure 1B). The apoA–I-containing particles in the control and des-fluoro-anacetrapib-treated animals migrated to an α-position when subjected to agarose gel electrophoresis. The absence of pre-β-migrating particles in these samples is consistent with what we have reported previously.27 Populations of large, α-migrating HDL particles were apparent in the des-fluoro-anacetrapib-treated, but not in the control, animals.

Figure 1.
Figure 1.

Des-fluoro-anacetrapib treatment increases high-density lipoprotein (HDL) size and HDL-cholesterol levels in New Zealand White (NZW) rabbits. NZW rabbits (n=6/group) received chow (control) or chow supplemented with 0.07% or 0.14% (wt/wt) des-fluoro-anacetrapib for 8 weeks. Endothelial denudation of the abdominal aorta was carried out with a balloon catheter after 2 weeks of des-fluoro-anacetrapib treatment. The animals were euthanized 6 weeks after aortic balloon injury and blood was collected. A, Plasma (200 μL) was loaded onto 2 Superdex 200 columns connected in series. Very-low-density lipoproteins (VLDLs), low-density lipoproteins (LDLs), and HDLs were resolved at a flow rate of 0.3 mL/min. Fractions were collected at 1-minute intervals and total cholesterol levels were measured. Each data point represents the mean value from 6 animals (n=6). B, Plasma was subjected to 2D gel electrophoresis and immunoblotted for apolipoprotein A–I.

Des-fluoro-anacetrapib Treatment Inhibits Intimal Hyperplasia in NZW Rabbits in Response to Aortic Balloon Injury

We have reported previously that balloon injury of the abdominal aorta induces intimal hyperplasia and increases vascular smooth muscle cell proliferation.28 When balloon-injured NZW rabbits were treated with 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib, intimal hyperplasia decreased by 35±5.9% and 56±8.2%, respectively, compared with control (Figure 2A and 2B; P<0.05 for both). Aortic media area was not affected (Figure 2C). The aortic intima/media ratio decreased by 37±11% and 51±10% in the animals treated with 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib, respectively (Figure 2D; P<0.05 for both). Treatment with 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib also inhibited cell proliferation within the injured aortas, with the number of PCNA+ (proliferating cell nuclear antigen) cells decreasing by 25±6.1% and 35±6.7% (wt/wt), respectively, relative to control (Figure 2E; P<0.05 for both).

Figure 2.
Figure 2.

Des-fluoro-anacetrapib treatment inhibits intimal hyperplasia in New Zealand White (NZW) rabbits with aortic balloon injury. NZW rabbits were subjected to endothelial denudation of the abdominal aorta as described in the legend of Figure 1. A, Representative Verhoeff’s hematoxylin-stained aortic cross-sections. B–D, Quantification of intimal and media areas and the intima/media ratio in aortic cross-sections. E, Cell proliferation in aortic cross-sections assessed as the % proliferating cell nuclear antigen (PCNA+) cells relative to the total cells. Data are expressed as mean±SEM (n=6; *P<0.05 vs control [Ctrl]).

Des-fluoro-anacetrapib Treatment Promotes Functional Re-Endothelialization of Balloon-Injured NZW Rabbit Aortas

Re-endothelialization is a key repair process in response to arterial injury.28 At 6 weeks postinjury, the damaged aortic surface in the balloon-injured animals was partially covered by CD31+ endothelial cells that had regenerated from branch orifices (Figure 3A). In the control animals, 43±6.7% of the endothelial surface was CD31+ compared with 69±6.6% and 76±7.7% for the 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib-treated animals, respectively (Figure 3B; P<0.05 for both).

Figure 3.
Figure 3.

Des-fluoro-anacetrapib treatment promotes functional re-endothelialization of balloon-injured New Zealand White (NZW) rabbit aortas. NZW rabbits were subjected to endothelial denudation of the abdominal aorta as described in the legend of Figure 1. A, Representative aortic cross-sections with a branch orifice showing CD31+ cells covering the injured aortic surface (red arrows). B, Re-endothelialization of aortic cross-sections assessed as the %CD31+ cells. C, Endothelial-dependent relaxation of precontracted aortic rings in response to acetylcholine (ACh). D, Endothelial-independent relaxation of precontracted aortic rings in response to sodium nitroprusside (SNP). E, cGMP content of aortic rings in response to ACh. Data are expressed as mean±SEM (n=6; *P<0.05 vs control [Ctrl]).

The increased re-endothelialization in the des-fluoro-anacetrapib-treated animals was associated with improved endothelial function. Treatment with 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib enhanced maximal endothelium-dependent relaxation in response to acetylcholine in preconstricted aortic rings by 1.8±0.3-fold and 2.2±0.5-fold, respectively, relative to control (Figure 3C; P<0.05 for both). Assessment of endothelium-independent relaxation with sodium nitroprusside was, by contrast, comparable for the control and des-fluoro-anacetrapib-treated animals (Figure 3D). Aortic guanosine 3′,5′-cyclic monophosphate levels in response to acetylcholine, as a measure of nitric oxide synthase activity, increased from 151±18 pmol/g wet wt (control) to 215±20 and 261±34 pmol/g wet wt in the 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib-treated animals, respectively (Figure 3E; P<0.05 for both).

We have reported previously that endothelial progenitor cells (EPCs) contribute to endothelial repair in NZW rabbits, with a maximal response being apparent at 4 days post balloon injury.29 We have also reported that (A–I)rHDL infusions increase EPC (stem cell antigen-1+-cells) recruitment to damaged endothelium in mice.7 To determine if des-fluoro-anacetrapib treatment enhances re-endothelialization of balloon-injured NZW rabbit aortas by recruiting EPCs to areas of damage, 3 additional groups of rabbits (n=6/group) were maintained on chow (control) or chow supplemented with 0.07% or 0.14% (wt/wt) des-fluoro-anacetrapib for 18 days. Endothelial denudation of the abdominal aorta was carried out after 14 days of dietary des-fluoro-anacetrapib supplementation, and the animals were euthanized 4 days later.

At euthanasia, circulating fetal liver kinase 1/stem cell antigen-1+-EPCs (Figure IIA in the online-only Data Supplement) and the number of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine-labeled acetylated LDL/fluorescein isothiocyanate-lectin/4′,6-diamidino-2-phenylindole+ cells (Figure IIB in the online-only Data Supplement) in cultured monocytes from the treated and control animals were comparable. Recruitment of stem cell antigen-1+-cells to the damaged endothelium was minimal in the control animals and not enhanced in the des-fluoro-anacetrapib-treated animals (Figure IIC in the online-only Data Supplement).

When taken together, these results indicate that treatment with des-fluoro-anacetrapib increases the regeneration of functional endothelium in NZW rabbits in response to aortic balloon injury by enhancing the proliferation and migration of endothelial cells from branching blood vessels adjacent to the site of vascular injury.

HDLs From Des-fluoro-anacetrapib-Treated NZW Rabbits Increase HCAEC Proliferation and Migration

To determine if the enhanced re-endothelialization in des-fluoro-anacetrapib-treated, balloon-injured NZW rabbits was due to improved HDL particle function or an increase in plasma HDL levels, HDLs were isolated from control- and des-fluoro-anacetrapib-treated animals and incubated with HCAECs at identical apoA–I concentrations or at apoA–I concentrations that reflected their on-treatment plasma levels at euthanasia.

The effect of des-fluoro-anacetrapib treatment on HCAEC proliferation was assessed by trypan blue exclusion. Incubation of HCAECs with HDLs from control and des-fluoro-anacetrapib-treated rabbits at concentrations comparable to their plasma apoA–I levels (0.43 mg/mL for the control animals, 0.58 mg/mL for the animals treated with 0.07% [wt/wt] des-fluoro-anacetrapib, and 0.61 mg/mL for the animals that received 0.14% [wt/wt] des-fluoro anacetrapib) increased the number of viable HCAECs from 14±1.8×104 cells (control) to 20±1.8×104 and 25±3.0×104 cells, respectively (Figure 4A; P<0.05 for both). Cell proliferation was further verified using the xCELLigence system. The cell index, which is a quantitative measure of the number of cells in each well, was 0.59±0.06 for the control samples and 0.87±0.04 and 0.89±0.03 for the animals that received 0.07% and 0.14% (wt/wt) des-fluoro anacetrapib, respectively (P<0.05 for both; Figure III in the online-only Data Supplement). When identical incubation conditions were used to assess endothelial cell migration with the scratch-wound assay, migration of HCAECs across the wound increased by 43±13% in the animals treated with 0.07% (wt/wt) des-fluoro-anacetrapib and by 73±21% in the 0.14% (wt/wt) des-fluoro-anacetrapib-treated animals (Figure 4B).

Figure 4.
Figure 4.

High-density lipoproteins (HDLs) from des-fluoro-anacetrapib-treated New Zealand White (NZW) rabbits increase human coronary artery endothelial cell (HCAEC) proliferation and migration. HCAECs were incubated for 24 hours with ultracentrifugally isolated HDLs at final apolipoprotein A–I concentrations comparable to the plasma levels: 0.43 mg/mL for control (Ctrl), 0.58 mg/mL for the animals treated with 0.07% (wt/wt) des-fluoro-anacetrapib, and 0.61 mg/mL for the animals treated with 0.14% (wt/wt) des-fluoro-anacetrapib. A, HCAEC proliferation assessed by trypan blue exclusion. B, A scratch wound was introduced into the HCAECs with a cell scraper (black line). The HCAECs were then incubated with HDLs for 24 hours. Migration was quantified as the number of cells migrating past the wound edge (black line). Data are expressed as mean±SEM of 3 independent experiments. *P<0.05 vs Ctrl.

When HDLs from the des-fluoro-anacetrapib-treated, balloon-injured NZW rabbits were incubated with HCAECs at equivalent apoA–I concentrations, cell proliferation (Figure IVA in the online-only Data Supplement) and migration (Figure IVB in the online-only Data Supplement) was comparable to that of the control animals. This indicates that des-fluoro-anacetrapib treatment enhances endothelial repair in balloon-injured NZW rabbits by increasing plasma HDL levels and not by increasing HDL particle function.

HDLs From Des-fluoro-anacetrapib-Treated NZW Rabbits Increase HCAEC Proliferation and Migration in an SR-B1-, PDZ Domain-Containing Protein 1-, and PI3K/Akt-Dependent Manner

As HDLs promote endothelial cell migration by activating the PI3K/Akt signal transduction pathway in a scavenger receptor class B-type I (SR-BI)- and PDZ1-dependent manner,30,31 we also asked if this pathway explained why HDLs from des-fluoro-anacetrapib-treated, balloon-injured NZW rabbits increased endothelial cell proliferation and migration.

Transfection of HCAECs with SR-B1 small interfering RNA (siRNA), PDZ domain-containing protein 1 (PDZK1) siRNA, or scrambled siRNA (siControl) decreased SR-B1 and PDZK1 protein levels by 73±9.5% and 85±7.1%, respectively (Figure VA and VB in the online-only Data Supplement; P<0.05 for both). The transfected cells were incubated in the absence or presence of HDLs from rabbits treated for 8 weeks with 0.14% (wt/wt) des-fluoro-anacetrapib. The concentration of apoA–I in the incubations (0.61 mg/mL) was identical to the plasma apoA–I level at euthanasia.

Incubation of the transfected HCAECs in the absence of HDLs did not affect endothelial cell proliferation (Figure 5A and 5C) or migration (Figure 5B and 5D). Incubation of scrambled siRNA-transfected HCAECs (siControl) with HDLs from the des-fluoro-anacetrapib-treated rabbits increased the number of endothelial cells from 11±1.5×104 to 25±2.7×104 (Figure 5A and 5C, open bars), while endothelial cell migration increased from 21±2.7 to 61±7.6 cells/area (Figure 5B and 5D, open bars; P<0.05 for all). The capacity of the HDLs to enhance endothelial cell proliferation and migration in HCAECs transfected with SR-B1 siRNA was inhibited by 46±7.2% (Figure 5A, closed bar) and 50±6.3% (Figure 5B, closed bar), respectively (P<0.05 for both). Similarly, incubation with HDLs inhibited proliferation and migration by 47±11% (Figure 5C, closed bar) and 59±7.8% (Figure 5D, closed bar), respectively (P<0.05 for both), in HCAECs transfected with PDZK1 siRNA.

Figure 5.
Figure 5.

High-density lipoproteins (HDLs) from des-fluoro-anacetrapib-treated New Zealand White (NZW) rabbits increase human coronary artery endothelial cell (HCAEC) proliferation and migration in a scavenger receptor-B1 (SR-B1)-, PDZ domain-containing protein 1 (PDZK1)-, and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt-dependent manner. HCAECs were transfected for 48 hours with SR-B1 small interfering RNA (siRNA; A and B, closed bars), for 24 hours with PDZK1 siRNA (C and D, closed bars), or with scrambled siRNA for the appropriate time (A–D, open bars). HCAECs were also preincubated for 1 hour without (E and F, open bars) or with 10 μmol/L LY294002 (LY; E and F, closed bars) and then incubated for 24 hours in the absence or presence of ultracentrifugally isolated HDLs (final apolipoprotein A–I concentration, 0.61 mg/mL) from NZW rabbits treated for 8 weeks with 0.14% (wt/wt) des-fluoro-anacetrapib. A, C, and E, HCAEC proliferation assessed by trypan blue exclusion. B, D, and F, HCAEC migration determined as the number of cells migrating past the scratch wound edge. Data are expressed as mean±SEM of 3 independent experiments. #P<0.05.

Involvement of the PI3K/Akt pathway in HCAEC proliferation and migration was also assessed. Incubation of nontransfected HCAECs with HDLs increased cell proliferation by 2.2±0.2-fold (from 12±2.1×104 cells to 26±1.8×104 cells; Figure 5E, open bars) and migration from 20±6.5 to 59±4.8 cells/area (Figure 5F, open bars), respectively (P<0.05 for both). Preincubation of the HCAECs with the PI3K/Akt inhibitor, LY294002, prior to incubation with HDLs, reduced endothelial cell proliferation and migration by 32±5.5% (Figure 5E, closed bar) and 43±10% (Figure 5F, closed bar), respectively (P<0.05 for both versus control).

Activation of PI3K/Akt by HDLs from des-fluoro-anacetrapib-treated rabbits was also investigated. HCAECs were incubated for ≤6 hours with HDLs from rabbits treated for 8 weeks with 0.14% (wt/wt) des-fluoro-anacetrapib. The concentration of apoA–I in these incubations was identical to the plasma apoA–I level at the time of euthanasia. Relative to cells incubated in the absence of HDLs, Akt phosphorylation (p-Akt) increased 6.7±0.8-fold (P<0.05), 4.6±0.7-fold (P<0.05), 2.7±0.5-fold (P<0.05), and 1.6±0.5-fold (ns) in HCAECs incubated with HDLs for 1, 2, 4, and 6 hours, respectively (Figure VIA in the online-only Data Supplement).

To ascertain if activation of PI3K/Akt by HDLs was dependent on SR-B1, HCAECs were transfected with SR-B1 siRNA or scrambled siRNA (siControl), then incubated for 1 hour in the absence or presence of HDLs from rabbits treated for 8 weeks with 0.14% (wt/wt) des-fluoro-anacetrapib. The concentration of apoA–I in the incubations was identical to the plasma apoA–I level at the time of euthanasia. Under these conditions, p-Akt protein levels increased 5.2±0.5-fold (P<0.05) in the scrambled siRNA-transfected HCAECs (Figure VIB in the online-only Data Supplement, open bars), whereas p-Akt levels were reduced by 45±7.7% in the SR-B1 siRNA-transfected HCAECs (P<0.05; Figure VIB in the online-only Data Supplement, closed bar).

Collectively, these results indicate that HDLs from rabbits treated with 0.14% (wt/wt) des-fluoro-anacetrapib promote endothelial cell proliferation and migration by activating the PI3K/Akt signal transduction pathway in an SR-B1-dependent manner.

To determine if the HDL-mediated enhancement of HCAEC proliferation and migration was dependent on the ATP-binding cassette transporters, ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1), cells were transfected with ABCA1 siRNA, ABCG1 siRNA, or scrambled siRNA (siControl). ABCA1 and ABCG1 protein levels were reduced by 80±7.5% and 74±4.2%, respectively, in the transfected cells (Figure VC and VD in the online-only Data Supplement; P<0.05 for both). Incubation of the ABCA1 siRNA- and ABCG1 siRNA-transfected cells with or without HDLs from des-fluoro-anacetrapib-treated rabbits at a final apoA–I concentration of 0.61 mg/mL did not affect endothelial cell proliferation (Figure VIIA in the online-only Data Supplement) or migration (Figure VIIB in the online-only Data Supplement). This indicates that HDLs enhance endothelial cell proliferation and migration independent of ABCA1 and ABCG1.

Overall, these results indicate that HDLs from des-fluoro-anacetrapib-treated rabbits enhance endothelial cell proliferation and migration via an SR-B1/PDZK1/PI3K/Akt-dependent pathway.

Discussion

This study establishes that increasing HDL and apoA–I levels by inhibiting CETP activity with des-fluoro-anacetrapib protects against intimal hyperplasia (Figure 2) and promotes functional re-endothelialization (Figure 3) in normocholesterolemic NZW rabbits with endothelial denudation of the abdominal aorta. We further demonstrate that HDLs isolated from des-fluoro-anacetrapib-treated NZW rabbits increase endothelial cell proliferation and migration in vitro in an SR-B1/PDZK1- and PI3K/Akt-dependent manner (Figures 4 and 5).

The results showing that treatment of NZW rabbits with des-fluoro-anacetrapib increases HDL and apoA–I levels, and HDL particle size is in agreement with what has been reported for humans32 and NZW rabbits24 treated with the CETP inhibitor, torcetrapib. One of the key questions arising from these observations is whether the cardiovascular benefit of increasing circulating HDL levels by inhibiting CETP activity improves HDL particle function or whether it is related to the increase in circulating HDL levels. The present results indicate that the reduced intimal hyperplasia, enhanced re-endothelialization, and improved vascular reactivity that was observed in the des-fluoro-anacetrapib-treated, balloon-injured NZW rabbits are due to the increase in HDL levels and not due to an improvement of HDL particle function.

HDLs have been shown to enhance endothelial cell migration in a nitric oxide–dependent manner in processes that involve SR-B1 as well as Rac GTPase, Src kinases, phosphatidylinositol 3-kinase, and p44/42 mitogen-activated protein kinase activation.30 HDLs also increase endothelial cell proliferation via a cell surface F(1)-ATPase.33 By contrast, HDLs reduce myeloid cell proliferation by increasing cholesterol efflux via the ATP-binding cassette transporters, ABCA1 and ABCG1.34 These opposing effects of HDLs on endothelial cell and myeloid cell proliferation highlight the functional diversity of the HDL fraction.

One of the most interesting outcomes of the current study is that the HDL-mediated increase in endothelial cell proliferation and migration in the des-fluoro-anacetrapib-treated animals was dependent on SR-B1, but not ABCA1 or ABCG1. Several of the cardioprotective and antidiabetic effects of HDLs are mediated by binding to SR-B1. For example, HDL-induced endothelial nitric oxide synthase activation,35 the promotion of endothelial cell migration and re-endothelialization after endothelial injury,30 and increased glucose uptake by adipocytes and glycogen synthesis in muscle36 all involve HDL-SR-B1 interactions. The ability of HDLs to improve endothelial cell migration has also been reported to involve PI3K/Akt signal transduction, SR-B1, and PDZK1.30,31

Circulating EPCs play an important role in vascular endothelial cell repair29 and are related to plasma HDL levels.37 We have shown previously that EPCs contribute to endothelial repair in NZW rabbits29 and that (A–I)rHDL infusions increase EPC recruitment to damaged endothelium in mice.7 However, EPCs did not contribute to the functional aortic re-endothelialization in balloon-injured, des-fluoro-anacetrapib-treated NZW rabbits in the present study (Figure II in the online-only Data Supplement). This may be because large HDL particles impair EPC function,38,39 and moderate to high concentrations of HDLs enhance EPC senescence.40 This suggests that increasing HDL particle size by inhibiting CETP activity may selectively attenuate EPC function without altering the ability of individual HDL particles to increase the proliferation and migration of pre-existing endothelial cells.

A potential limitation of this study was the use of normocholesterolemic NZW rabbits that have low plasma LDL levels. Although it may have been more physiologically relevant to use cholesterol-fed rabbits, which have elevated LDL levels, this would have confounded the results because des-fluoro-anacetrapib treatment increases HDL levels and decreases LDL levels. As such it would not have been possible to attribute the outcome of the study unequivocally to an increase in plasma HDL levels. This issue was circumvented by treating normocholesterolemic NZW rabbits with des-fluoro-anacetrapib, so that the reduction in LDL levels was minor relative to the increase in HDL levels.

In conclusion, this study establishes that treatment with des-fluoro-anacetrapib protects against intimal hyperplasia and promotes functional re-endothelialization in NZW rabbits with endothelial denudation of the abdominal aorta. It remains to be seen if these beneficial effects are also apparent in humans and whether they translate into a reduction in cardiovascular events in people treated with CETP inhibitors.

Sources of Funding

This work was supported by Merck & Co Inc (IISP39674) and the National Health and Medical Research Council of Australia (grants 482800 and 1037903).

Disclosures

D. Johns is an employee of Merck & Co. The other authors report no conflicts.

Footnotes

  • The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.114.304747/-/DC1.

  • Nonstandard Abbreviations and Acronyms
    ABCA1
    ATP-binding cassette transporter A1
    ABCG1
    ATP-binding cassette transporter G1
    apoA–I
    apolipoprotein A–I
    CETP
    cholesteryl ester transfer protein
    EPCs
    endothelial progenitor cells
    HCAECs
    human coronary artery endothelial cells
    HDL
    high-density lipoprotein
    HDL-C
    high-density lipoprotein cholesterol
    LDL
    low-density lipoprotein
    NZW
    New Zealand White
    PDZK1
    PDZ domain-containing protein 1
    PI3K
    phosphatidylinositol-4,5-bisphosphate 3-kinase
    rHDL
    reconstituted HDL
    siRNA
    small interfering RNA
    SR-B1
    scavenger receptor-B1

  • Received October 1, 2014.
  • Accepted January 20, 2015.

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Significance

This study establishes that elevating plasma high-density lipoprotein and apolipoprotein A–I levels by inhibiting cholesteryl ester transfer protein activity in New Zealand White rabbits enhances endothelial repair by increasing endothelial cell proliferation and the migration of endothelial cells to areas of damage. Inhibition of cholesteryl ester transfer protein also improves endothelial function and reduces intimal hyperplasia. These findings establish that cholesteryl ester transfer protein inhibition improves several of the key cardioprotective functions of high-density lipoproteins. Further studies are required to ascertain if these beneficial effects of cholesteryl ester transfer protein inhibition are also apparent in humans.

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  • Cholesteryl Ester Transfer Protein Inhibition Enhances Endothelial Repair and Improves Endothelial Function in the RabbitSignificance
    Ben J. Wu, Sudichhya Shrestha, Kwok L. Ong, Douglas Johns, Liming Hou, Philip J. Barter and Kerry-Anne Rye
    Arteriosclerosis, Thrombosis, and Vascular Biology. 2015;35:628-636, originally published January 29, 2015
    https://doi.org/10.1161/ATVBAHA.114.304747
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