A novel apoA-I mimetic peptide suppresses atherosclerosis by promoting physiological HDL function in apoE?/? mice

Gou Sanhu (Orcid ID: 0000-0003-3231-7427)

A novel apoA-I mimetic peptide suppresses atherosclerosis by promoting physiological

HDL function in apoE⁻^/⁻mice

Running title: Novel apoA-I mimetic peptide suppresses atherosclerosis in mice

1

1, 2

1

Sanhu Gou , Li Wang , Chao Zhong , Xinyue Chen , Xu Ouyang , Beibei Li ,

3

1

Guangjun Bao , Hui Liu , Yun Zhang , Jingman Ni

¹Institute of Pharmaceutics, School of Pharmacy, Lanzhou University, Lanzhou P.R. China

²Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic

Medical Sciences, Lanzhou University, Lanzhou P.R. China

³Institute of Biochemistry and Molecular Biology, School of Life Sciences, Lanzhou

Correspondence

Jingman Ni, PhD, Professor, Institute of Pharmaceutics, School of Pharmacy, Lanzhou

University, 222 Tianshui South Road, Lanzhou, 730000, P.R. China.

Email: nijm@lzu.edu.cn

This article has been accepted for publication and undergone full peer review but has not been

through the copyediting, typesetting, pagination and proofreading process which may lead to

differences between this version and the Version of Record. Please cite this article as doi:

10.1111/bph.15213

Funding information

National Natural Science Foundation of China, Grant/Award Number: 81273440, 81773564.

Word count (excluding figure legends and references): 7037

Abstract:

Background and Purpose: Apolipoprotein A-I (apoA-I) mimetic peptides (AMPs) are short

peptides that can mimic the physiological effects of apoA-I, including the suppression of

atherosclerosis by reversely transporting peripheral cholesterol to the liver. As the

hydrophobicity of apoA-I is considered important for its lipid transport, novel AMPs were

designed and synthesized in this study by gradually increasing the hydrophobicity of the

parent peptide and their anti-atherosclerotic effects were tested.

Experimental Approach: Seventeen new AMPs (P1–P17) with incrementally increased

hydrophobicity were designed and synthesized by replacing the amino acids 221–240 of

apoA-I (VLESFKVSFLSALEEYTKKL). Their effects on cholesterol efflux were evaluated.

Their cytotoxicity and hemolytic activity were also measured. The in vitro mechanism of the

action of the new peptides were explored. Adult apolipoprotein E⁻^/⁻mice were used to

evaluate the anti-atherosclerotic activity of the best candidate, and the mechanistic basis of its

anti-atherosclerotic effects were explored.

Key Results: Seventeen new AMPs (P1–P17) were synthesized and their cholesterol efflux

activity and cytotoxicity were closely related to their hydrophobicity. P12

(FLEKLKELLEHLKELLTKLL), as the best candidate, most strongly promoted cholesterol

efflux among the non-toxic peptides (P1–P12). With its phospholipid affinity, P12 can

effortlessly facilitate cholesterol transport through ATP-binding cassette transporter A1. In

vivo, P12 exhibited prominent anti-atherosclerotic activity via coupling with HDL.

Conclusions and Implications: P12 featured proper hydrophobicity, which ensured its

efficient binding with cytomembrane phospholipids, cholesterol, and HDL and provided a

basis for its ability to reversely transport cholesterol and treat atherosclerosis.

Keywords: Atherosclerosis, apolipoprotein A-I mimetic peptide, hydrophobicity, lipoprotein,

cholesterol

Abbreviations: ABCA1, ATP-binding cassette transporter A1; ApoA-I, apolipoprotein A-I; AMP,

ApoA-I mimetic peptide; C-TPP, 5-(4-carboxyphenyl)-10, 15, 20-triphenylporphyrin; DMPC,

Dimyristoyl phosphatidylcholine; RCT, reverse cholesterol transport. TC, total cholesterol; TG,

triglyceride.

1

. Introduction

Reverse cholesterol transport (RCT) has received increasing attention as an anti-

atherosclerotic strategy in recent decades (Lewis & Rader, 2005; Ohashi, Mu, Wang, Yao &

Chen, 2005). However, despite intense efforts, drugs specifically designed to stimulate RCT

are barely available in the market (Reddy, Navab, Anantharamaiah & Fogelman, 2014). In

vivo, HDL undergoes RCT, thereby removing parts of cholesterol from peripheral cells

(Fisher, Feig, Hewing, Hazen & Smith, 2012). In the formation of HDL, lipid-free

apolipoprotein A-I (apoA-I) is synthesized and secreted from the liver, after which it binds

phospholipids through interactions with ATP-binding cassette transporter protein A1

(ABCA1) and forms nascent disc-shaped HDL (preβ-HDL) particles. Preβ-HDL particles

constantly encapsulate peripheral intracellular cholesterol ester into its hydrophobic core to

generate mature spherical HDL (α-HDL) and have stronger cholesterol carrying capacity than

mature α-HDL particles. Eventually, cholesterol ester is removed from HDL and transported

into hepatocytes via scavenger receptor BI (SR-BI) or transferred to LDL by cholesterol

ester-transfer protein. Thus, cholesterol is mobilized and transferred from the peripheral

tissues to the liver throughout the RCT process (Khera & Rader, 2010).

ApoA-I, a structural and functional protein of HDL, is the key protein that drives the RCT

process. (Mei & Atkinson, 2015). ApoA-I consists of 243 amino acids and is divided into 10

amphipathic α-helical fragments by proline (Leman, Maryanoff & Ghadiri, 2014). These 10

fragments share common structural features (Ingenito et al., 2010; Islam et al., 2018): 1)

Their secondary structure is an amphiphilic α-helix that continuously possesses hydrophobic

and hydrophilic surfaces at proper. 2) The connection points of amphiphilic surfaces are

positively charged amino acid residues, such as Lys, Arg, and His. 3) The middle of the

hydrophilic surface contains negatively charged amino acid residues, such as Asp and Glu.

These characteristics are closely related to its activity. ApoA-I has high lipid affinity and

drastically promotes peripheral cell cholesterol efflux. Unfortunately, the use of apoA-I as a

drug has been restricted by its high production cost and lack of oral bioavailability.

Fortunately, apoA-I mimetic peptides (AMPs) may represent alternative treatment options.

Therefore, based on their sequence characteristics, many AMPs with short sequences have

been derived. The class A and amphipathic α-helix peptide, 18A

(DWLKAFYDKVAEKLKEAF) was designed to mimic apoA-I, which has hydrophobic

amino acids (W, L, A, F, Y, V) on the hydrophobic face and hydrophilic amino acids (D, E, K,

R) on the hydrophilic face of its α-helix, as well as a positively charged amino acid (K) at the

hydrophobic/hydrophilic interface and a negatively charged amino acid (D or E) at the center

of the hydrophilic face. 18A was found to associate strongly with liposomes and displace

apoA-I from native HDL and apolipoprotein E (apoE) from native VLDL (Getz & Reardon,

2

011). 5A (DWLKAFYDKVAEKLKEAF-P-DWAKAAYDKAAEKAKEAA), a proline-

linked dimer of 18A and its derivative, more strongly promoted cholesterol efflux than 18A.

This dimer readily complexed with phospholipids and formed an HDL-like structure (Amar

et al., 2010). 4F (Ac-DWFKAFYDKVAEKFKEAF-NH

the leucine of 18A at sites 3 and 14 with phenylalanine, is the most representative AMP. L-4F

L-amino acid) and D-4F (enantiomer of L-4F) were revealed to have good cholesterol efflux

2

), which was produced by replacing

(

activity in vitro and in vivo. In clinical research, oral and intraperitoneal 4F reduced evolving

atherosclerotic lesions, plaque lipids, and macrophage activity in vein grafts, but the drug did

not affect established lesions in the aortic sinus. In addition, its dosage and cost of production

were major issues in the project (Navab et al., 2002; Navab et al., 2011; Osei-Hwedieh, Amar,

Sviridov & Remaley, 2011). These AMPs feature the aforementioned three common

sequence-structure characteristics of the 10 fragments of apoA-I.

Regarding the anti-atherosclerotic mechanism of AMPs, after binding to HDL, the

peptides promote RCT from arteriosclerotic plaques to the liver. AMPs must have a strong

affinity with phospholipids and cholesterol in HDL or cells to transport cholesterol

(Stoekenbroek, Stroes & Hovingh, 2015). Cholesterol efflux is a rate-limited step in the

process of RCT. The factors affecting cholesterol efflux induced by AMPs are

hydrophobicity, charge, and secondary structure (Leman, Maryanoff & Ghadiri, 2014).

Among them, hydrophobicity is the most important and fundamental factor, that can both

affect the solubility and absorption of the peptide, and directly determine its lipid affinity and

secondary structure (Sviridov et al., 2013). Sufficient hydrophobicity of AMPs can promote

binding between AMPs and ABCA1 for membrane phospholipid efflux to form preβ-HDL

and induce cellular cholesterol efflux (D’Souza et al., 2010). In short, hydrophobicity has a

considerable influence on cholesterol efflux induced by AMPs, highlighting the need for

detailed studies of this variable.

Chroni and colleagues found that amino acids 220–231 of apoA-I comprise the binding

domain of ABCA1 (Chroni et al., 2003). The tenth helix of apoA-I (containing amino acids

2

20–231) is the least conserved but most indispensable sequence (Palgunachari et al., 1996;

Panagotopulos, Witting, Horace, Hui, Maiorano & Davidson, 2002). Truncation mutants of

apoA-I lacking the tenth helix do not promote cholesterol efflux (Panagotopulos, Witting,

Horace, Hui, Maiorano & Davidson, 2002). In the present study, based on apoA-I221–240

which comprises the active fragment of the apoA-I C-terminal (Wool, Reardon & Getz,

,

2

014), 17 new AMPs were designed (P1–P17) and synthesized by gradually increasing their

hydrophobicity through amino acid replacement. The relationships of hydrophobicity with the

secondary structures, anti-atherosclerotic activity, and toxicity of the new mimetic peptides

were evaluated. Of these, P12, a lipid-free 20-amino acid AMP featuring an amphiphilic α-

helix structure and the best biological function of apoA-I, was revealed to have the greatest

effects on cholesterol efflux. The anti-atherosclerotic mechanisms and effect of the candidate

AMP were elucidated in apoE⁻^/⁻mice.

2

. Methods

.1 Peptide and fluorescent probe synthesis

ApoA-I221–240, 17 novel AMPs (P1–P17), L-4F, and 5-(4-carboxyphenyl)-10, 15, 20-

triphenylporphyrin (C-TPP)-labeled P12 were synthesized using Wang resin and N-9-

fluorenyl methoxycarbonyl (Fmoc) based on solid-phase peptide synthesis. The peptide

extension reaction conditions were as follows: 3-fold excess of 2-(1H-benzotriazol-1-yl)-1, 1,

3

, 3-tetramethyluronium hexafluorophosphate (HBTU)/hydroxybenzotriazole (HOBt) as the

coupling reagents, N-dimethylformamide as a solvent, 6-fold excess of

diisopropylethylamine, and 3-fold excess of Fmoc protecting group amino acids. The

cleavage of the peptide from the resin was performed using a reagent composed of 95%

trifluoroacetic acid, 2.5% triisopropylsilane, and 2.5% deionized water (3 h, room

temperature). The peptide was precipitated in diethyl ether (1:1, volume ratio).

C-TPP and C-TPP-P12 were generated as described previously (Ushasri et al., 2014).

Briefly, 4-carboxybenzaldehyde and benzaldehyde were mixed with propionic acid. The

reaction mixture was heated up to 120°C, and pyrrole was added dropwise. The obtained

solid material was subjected to silica gel column chromatography and eluted with

CH

2

Cl

2

/CH OH to obtain pure C-TPP. Then 1 equivalent of P12 that was not cleaved from

3

the resin, 10 equivalents of C-TPP, 10 equivalents of HBTU and 10, equivalents of HOBt

were dissolved in dimethylformamide separately and mixed. This mixture was added to the

resin, and 10 equivalents of diisopropylethylamine were added to start the coupling reaction.

After 24 h, the resin was dried, and the cleavage of peptide from the resin was achieved by

treatment using a solution containing trifluoroacetic acid at room temperature for 10 h

(Figure S1 and S2).

The obtained crude peptides were purified by reversed-phase-HPLC (Waters, MA, USA)

on a C18 column (19 mm × 250 mm). The hydrophobicity and purity of the peptides were

analyzed using reversed-phase-HPLC on a C18 column (4.6 mm × 250 mm). All peptides

prepared were more than 95% pure. Their molecular masses were confirmed via electrospray

ionization MS (MaXis 4G, Bruker, USA).

2

.2 The design strategy of AMP

As shown in Table 1 and Figure 1, 17 novel AMPs were designed and synthesized using

apoA-I221–240 (VLESFKVSFLSALEEYTKKL) as the parent peptide. To ensure alignment to

the amphiphilic surface of apoA-I221–240, L , S , F , V , S , F

of apoA-I221–240 were replaced with V , K , A , E , A , A

respectively. Then, the amino acids on the hydrophilic surface of the new peptides were

2

4

5

7

8

9

, L10, S11, L13, E15, Y16, and K19

2

4

5

7

8

9

, E10, H11, K13, A15, A16, and A19,

confirmed, The template of the novel AMPs was

EKX KEX 20 (X represents hydrophobic amino acids), and

X

1

X

2

5

8

9

X EHX12KEX15X16TKX19X

the hydrophobicity of P1–P17 was gradually increased by replacing the hydrophobic amino

acid with Ala, Leu, Val, Phe or Trp. Hydrophobicity and the hydrophobic moment were

calculated using HeliQuest online software.

2

.3 Circular dichroism (CD) measurements

To study the relationship between the secondary structures and cholesterol efflux activity

of the new peptides, the CD spectra of the peptides were measured at 25°C on a J-810

spectrometer (Jasco, Japan) using a quartz cell with a 1.0 mm path length. Recording was

–1

performed over the range of 190–260 nm with a scanning speed of 50 nm·min , band width

of 1 nm, and response time of 1 s. The peptide solutions were prepared at a final

concentration of 50 μM in deionized water (mimicking an aqueous environment) and 50%

trifluoroethanol (TFE, mimicking the hydrophobic environment of the biological membrane).

The acquired CD spectra were then converted to the mean residue ellipticity using the

following equation:

θ

M

= (θobs × 1000)/(c × l × n),

2

–1

where θ is the residue ellipticity (deg·cm ·dmol ), θobs is the observed ellipticity

M

corrected for the buffer at a given wavelength (mdeg), c is the peptide concentration (mM), l

is the path length (mm), and n is the number of amino acids.

2

.4 DMPC vesicle solubilization assay

Dimyristoyl phosphatidylcholine (DMPC) vesicles (1 mg·mL ) were prepared by

−

1

resuspending DMPC in 75°C PBS (pH= 7.4) and vortexed. Then, 5 μL of 1mM AMP in PBS

–

1

(

pH= 7.4) was added to 96-well plates, followed by 95 μL of 1 mg·mL DMPC vesicles in

PBS. The working concentration of the peptide was 50 μM. Changes in light scattering upon

peptide addition were monitored at 23.9°C every 15 s for 90 min at 430 nm, with shaking in a

multimode plate reader. Each sample had five parallel duplicates and the test was repeated in

five to six independent experiments.

2

.5 Cell culture

RAW264.7 cells (RRID:CVCL_0493) were cultured in DMEM supplemented with 10%

FBS, and THP-1 cells (RRID:CVCL_0006) were cultured in RPMI-1640 medium

supplemented with 10% FBS. Cells were maintained at 37°C in 5% CO

.6 Determination of cellular cholesterol efflux

The cholesterol efflux activity of the new peptides was determined using a previously

described method with modifications (Avdulov, Chochina, Igbavboa & Wood, 2000; Zhang et

2

.

2

4

–1

4

–1)

al., 2017). Briefly, RAW264.7 cells (4 × 10 ·well ) or THP-1 (1 × 10 ·well were labeled

–

1

with 22-NBD-cholesterol (2.5 mg·L ) for 24 h in serum-free medium, and then cellular

cholesterol efflux was examined in the presence of 0, 1, 2, 5, 10, 20, 50, 100 or 200 μM

AMPs for 12 h in serum-free medium. Cells were lysed with 2% Triton X-100. The

fluorescence intensity (FI, Ex = 469 nm, Em = 538 nm) of 22-NBD-cholesterol in medium

and cell lysates was monitored using a multimode reader. The cholesterol efflux rate was

calculated as (FImedium)/(FImedium + FIcell) × 100%. Each sample had five parallel duplicates,

and the test was repeated in five to six independent experiments.

For the further determination of cholesterol efflux using flow cytometry, RAW264.7 and

–

1

THP-1 cells were labeled with 22-NBD-cholesterol (2.5 mg·L ) for 24 h in 24-well plates,

and cellular cholesterol efflux was induced by incubation with apoA-I221–240 (10 μM), L-4F

(

10 μM), or P12 (1, 10 and 20 μM) for 12 h. The uptake and efflux of 22-NBD-cholesterol

Ex = 488 nm, Em = 550 nm) in cells were determined using a flow cytometer (LSRFortessa,

(

BD). Each sample had five parallel duplicates, and the test was repeated in five to six

independent experiments.

Finally, ABCA1-dependent cellular cholesterol efflux stimulated by P12 was determined

using two methods:

In the first method (Shimizu et al., 2015; Uehara et al., 2013), RAW264.7 or THP-1 cells

–

1

were labeled with 22-NBD-cholesterol (2.5 mg·L ) for 12 h in serum-free medium. Cells

were washed with PBS (pH= 7.4). To promote the ABCA1 expression, the labeled cells were

treated with 9-cis-retinoic acid (retinoid X receptor ligand) and TO-901317 (liver X receptor

agonist) for 10 h in serum-free medium (each 5 μM). To suppress ABCA1 function, cells

were treated with 20 μM probucol for 2 h at 37°C. Intracellular cholesterol efflux was

induced by treatment with P12 (1 and 10 μM) for 4 h, and L-4F was used as a positive

control. FI of 22-NBD-cholesterol in medium and cell lysates was monitored using a

multimode reader.

In the second method (Carballo-Jane et al., 2010), RAW264.7 or THP-1 cells were

–

1

labeled with 22-NBD-cholesterol (2.5 mg·L ) for 24 h in serum-free medium, and then

cellular cholesterol efflux was induced by exposure to P12 (0, 1, 5, 10 and 20 μM) while

providing energy for ABCA1 activity using cAMP (0.3 mM) for 12 h in serum-free medium.

L-4F was used as a positive control. FI of 22-NBD-cholesterol in medium and cell lysates

was monitored using a multimode reader.

2

.7 Cytotoxicity assay and hemolytic activity

RAW264.7 cells were seeded on 96-well plates at a density of 1×10 ·well . After

4

–1

permitting adherence for 6 h, 100 μL of AMP (0, 1, 2, 5, 10, 20, 50, 100, or 200 μM) in

serum-free medium was added to the plates for 12 h, after which 10 μL of MTT (50 mg·mL^–¹

in waters) was added. After 4 h, the formazan in the cells was dissolved in DMSO. The

absorbance was measured using a multimode reader at 490 nm. Each sample had five parallel

duplicates, and the test was repeated in five to six independent experiments.

The venous blood of C57BL/6J mice (male, 20 ± 2 g) was collected from the venous

plexus of the fundus and centrifuged at 1000 × g for 10 min. The plasma supernatant was

removed, and 8% erythrocyte suspension was prepared with PBS (pH= 7.4). Then, 100 μL of

8

% erythrocyte suspension and 100 μL of AMPs (0, 1, 2, 5, 10, 20, 50, 100, or 200 μM) in

PBS were added to the 96-well plates, and 2% Triton X-100 was used as a positive control.

The plates were incubated at 37°C for 1 h and then centrifuged at 12,000 × g for 15 min.

Plasma supernatants (150 μL) were transferred to new 96-well plates. The absorbance was

measured using a multimode reader at 490 nm. Each sample had five parallel duplicates, and

the test was repeated in five to six independent experiments.

2

.8 Localization of P12 and 22-NBD-cholesterol in macrophages

As described previously, THP-1 and RAW264.7 were labeled with 22-NBD-cholesterol

–

1

(

2.5 mg·L ) for 24 h in laser-scanning confocal microscope (LSCM) dishes, and cellular

cholesterol efflux was induced by exposure to C-TPP-P12 for 12 h. The fluorescent images of

cells were photographed in track 1 (22-NBD-cholesterol, Ex = 488 nm, Em = 500–550 nm,

green), track 2 (C-TPP-P12, Ex = 515 nm, Em = 640–730 nm, red), and the bright-field via

LSCM (LSM710, Zeiss).

2

.9 Animal experiments

.9.1 Animal management

All experiments involving animals were performed according to protocols approved by

the Animal Care and Use Committee of Lanzhou University. Mice were purchased from

Beijing Charles River Laboratories, maintained in a temperature- and light-controlled facility,

and fed specific pathogen-free chow. ApoE⁻^/⁻mice and background wild-type C57BL/6J

mice (RRID:IMSR_JAX:000664) 20 ± 1 g, 10 weeks were used for the in vivo experiments.

Background wild-type C57BL/6J mice (n=10, 5 females and 5 males) were fed a commercial

basal diet and apoE⁻^/⁻mice (n = 60, 30 females and 30 males) were fed a high-fat diet (0.5%

cholesterol and 10% fat). After 4 weeks, apoE⁻^/⁻mice were divided into six groups. Low and

–

1

high doses (10 and 20 mg·kg , respectively) of P12 were injected intraperitoneally into mice

three times per week for 12 weeks. Saline, L-4F (10 mg·kg ), and apoA-I221–240 (10 mg·kg^–¹)

were used as background, positive, and negative controls, respectively. After 16 weeks, mice

were anesthetized with isoflurane and killed via cervical dislocation, after an overnight fast.

Plasma samples, organs, and tissues were then collected.

–1

Other batches of wild-type C57BL/6J (n=20, male) and apoE⁻^/⁻(n = 20, male) mice were

fed using the aforementioned method for the following four supplementary experiments: the

determination of plasma apoA-I levels after P12 injection, plasma clearance of P12, the

effects of 1 week of P12 exposure on blood lipid levels, and the effect of 2 weeks of P12 on

cholesterol excretion (five C57BL/6J and five apoE⁻^/⁻mice for each experiment).

In all experiments, animals were randomly assigned to treatment groups. The numbers of

mice in each group are designated in the individual figures. The sample size in each group

was determined according to previous studies with similar experimental protocols. The

studies were blinded for treatment assignment and outcome assessment. Animal studies are

reported in compliance with the ARRIVE guidelines (Kilkenny, Browne, Cuthill, Emerson &

Altman, 2010) and with the recommendations made by the British Journal of Pharmacology.

2

.9.2 Histopathological examination

Paraffinized sections of the aortic arch were stained with hematoxylin and eosin. The

images were captured using a CX21 microscope (Olympus, Japan). The atherosclerotic lipid

plaque and relative thickness of the arterial wall were quantified via computer image analysis

using Adobe Photoshop CC 2017 software (RRID: SCR_014199).

2

.9.3 Serum lipid profiles, turbidity, inflammatory factors, and lipid peroxidation

Serum levels of total cholesterol (TC), LDL-C, HDL-C, triglyceride (TG), and C-reactive

protein (CRP) were determined using biochemical kits according to the manufacturer’s

instructions on an automatic biochemical analyzer (Hitachi, Japan). Serum IL–6, monocyte

chemoattractant protein-1 (MCP-1), and TNF-α levels were measured using ELISA assay kits

according to the manufacturer’s protocol. Serum turbidity was represented as the absorbance

as measured using a multimode reader at 430 nm. Serum malondialdehyde (MDA) and

superoxide dismutase (SOD) levels were determined in all mice using biochemical kits

according to the manufacturer’s instructions.

2

.9.4 Plasma clearance of P12 in C57BL/6J and apoE⁻^/⁻mice

Both wild-type C57BL/6J (n = 5) and apoE⁻^/⁻mice (n = 5) were intravenously

−

1

administered 10 mg·kg P12. P12 levels in plasma were measured using HPLC at 0, 1, 5, 10,

1

5, 30, 60, 90, 120, 180, 240, 360, 480, 720, 1440, and 2880 min after injection. The half-life

1/2) of P12 in plasm were calculated in C57BL/6J and apoE⁻^/⁻mice using Microsoft Excel

019 (Microsoft Excel, RRID: SCR_016137).

(t

2

.9.5 Effect of P12 on plasma apoA-I levels

Plasma apoA-I levels in wild-type C57BL/6J (n=5) and apoE⁻^/⁻mice (n=5) were

−

1

measured before and after the intravenous injection of P12 (10 mg·kg ) using an apoA-I

assay kit.

2

.9.6 Regulative effect of 1 week of P12 exposure on blood lipids

Five new male apoE⁻^/⁻mice were maintained on their diets for 1 week after 4 months of

feeding with the high-fat diets. Five new male wild-type C57BL/6J mice were used as the

−

1

−1

control group. They received intraperitoneal injections of P12 (10 mg·kg ·day ). Serum TC,

LDL-C, HDL-C, and non-HDL-C levels were measured in both mouse groups before and

after daily P12 administration.

2

.9.7 Effect of P12 on cholesterol excretion for two weeks

After 4 months of feeding, an additional five male apoE⁻^/⁻mice fed with aforementioned

high-fat diets and five male wild-type C57BL/6J mice fed with basal diets were maintained

on their respective diets for 2 additional weeks. In the first week, feces were collected from

mice in each group. In the second week, mice received intraperitoneal injections of P12 (10

−

1

−1

mg·kg ·day ). Feces were collected for measuring of cholesterol levels. To extract the

cholesterol, 1 g of feces powder was soaked in 5 mL of chloroform and shaken 10 min on a

vortex mixer. After filtering samples, non-cholesterol impurities in chloroform were extracted

using water, and cholesterol in chloroform was measured using a cholesterol kit according to

the manufacturer's instructions.

2

.10 In vivo mechanism experiments

.10.1 Lipoprotein analysis by size exclusion chromatography

Fresh blood was drawn from apoE⁻^/⁻mice into tubes containing potassium EDTA, and

plasma was separated immediately by centrifugation at 500 × g for 30 min at 4°C. In total, 5

–

1

μL of peptides (P12 and C-TPP-P12, final concentration = 0.25 mg·mL ) were mixed with

5 μL of mouse plasma and incubated at 37°C for 2 h. Size exclusion chromatography was

4

performed at ambient temperature using a Superose 6 column (10 × 1000 mm) on a semi-

preparative HPLC system (Waters). A 200 µL aliquot of the peptide/plasma mixture was

–

1

injected per run and eluted in PBS (pH= 7.4) with 1 mM EDTA at a flow rate of 1 mL·min .

–

1

A total of 60 fractions (1.5 mL·fraction ) were directly collected into microtiter plates for

further analysis. The TC content in each fraction was measured using a cholesterol kit

according to the manufacturer's instructions and FI represented the C-TPP-P12 concentration

in each fraction. Saline was used as a control.

2

.10.2 Western blot quantification of mouse preβ-HDL levels

The western blotting procedures and analysis were performed according to the BJP

guidelines (Alexander et al., 2018). Peptide solution (5 μL) was mixed with 45 μL of apoE⁻^/⁻

mouse plasma, incubated at 37°C with shaking at 300 rpm for 2 h, and then subjected to one-

dimensional, non-reducing, anti-mouse apoA-I Western blotting. Briefly, samples were

diluted 1:1 with Tris-glycine native sample buffer, and 4–20% native polyacrylamide gel

electrophoresis as performed at 90 V for 10 min and at 150 V for 1 h. Proteins were

transferred to a PVDF membrane (0.45 μm) overnight (200 mA at 4°C). After transfer, the

membrane was blocked with 6% lipid-free milk and washed three times with Tris-buffered

saline with Tween-20 (10 mM Tris, pH 7.4, 150 mM NaCl, and 0.1% Tween-20). Rabbit anti-

mouse apoA-I antibody (1:500) and HRP-conjugated goat anti-rabbit antibody (1:1000) were

used as the primary and secondary antibodies, respectively. Blots were developed using the

HRP substrate luminol reagent and scanned using a Typhoon 9400 Imager (GE Healthcare,

Piscataway, NJ, USA). Preβ-HDL was identified on the basis of its electrophoretic mobility,

and the band was quantified using Adobe Photoshop CC 2017 software (Adobe Photoshop,

RRID:SCR_014199). The results were expressed as the gray value representing the signal

intensity of the band.

2

.11 Data and analysis

The data and statistical analysis comply with the recommendations of the British Journal

of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018).

All experiments were randomized and blinded. The sample size in each group was

determined based on our previous studies with similar experimental protocols. At least five

samples per group (n = 5) were included in the statistical analysis, where n is the number of

independent values. Date for animals in which relative arterial wall thickness could not be

measured because of the rupture of arterial plaque were excluded from the analysis. A normal

probability plot was used to examine data distributions. Statistical analyses were performed

using GraphPad Prism 5 software (GraphPad Prism, RRID: SCR_002798), and data are

presented as mean ± SEM unless otherwise stated. Student’s t-test was used for comparisons

between two groups. For comparisons of three or more groups, one-way ANOVA followed by

Dunnett’s post-hoc test was used for normal distributions with one variable. The significance

level was set at P < .05.

2

.12 Materials

2-NBD-cholesterol, DMEM, RPMI-1640, and FBS were purchased from Thermo Fisher

2

Scientific (MA, USA). Serum TC, LDL-C, HDL-C, TG, and CRP assay kits were purchased

from Maccura Biotechnology Co., Ltd. (Sichuan, China). IL-6, MCP-1, and TNF-α assay kits

were purchased from Cloud-Clone Corp (Beijing, China). 9-cis-retinoic acid, TO-901317,

cAMP, MDA, and SOD were purchased from Sigma-Aldrich (St. Louis, MO, USA). A

cholesterol assay kit was purchased from Beijing Apllygen Gene Technology Co. LTD

(Beijing, China). Rabbit anti-mouse apoA-I antibody (Proteintech Cat# 14427-1-AP, RRID:

AB_2056524) and HRP-conjugated goat anti-rabbit antibody (Proteintech Cat# SA00001-2,

RRID: AB_2722564) were purchased from Proteintech Group, inc. (IL, USA).

2

.13 Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries at

http://www.guidetopharmacology.org (IUPHAR/BPS Guide to Pharmacology,

RRID:SCR_013077), the common portal for data from the IUPHAR/BPS Guide to

Pharmacology (Harding et al., 2018), and are permanently archived in the Concise Guide to

Pharmacology 2017/18 (Alexander, Christopoulos et al., 2017; Alexander, S. P. H., Fabbro et

al., 2017; Alexander, Peters et al., 2017).

3

. Results

.1 Secondary structure and retention time was influenced by hydrophobicity

Changes in the secondary structures of the new AMPs were observed with the increases in

hydrophobicity. In aqueous medium, apoA-I221–240 and P1–P10 did not form α-helices,

whereas L-4F and P11–P14 had stable α-helical conformations. Interestingly, P15–P17 had β-

sheets conformations. In the simulated cell membrane environment of 50% TFE, apoA-I221–

2

40, L-4F, and P1–P14 had α-helical structures, whereas P15–P17 had α-helical and β-sheet

structures (Figure 2a, 2b, and S3).

The hydrophobicity and hydrophobic moment of P1–P17 had a linear relationship (Figure

2

c). The retention time (t

R

) of these new peptides were measured using the analytical HPLC.

of P1–P12 gradually increased, whereas those of P12–P17

The result illustrated that t

R

gradually decreased. However, the theoretical value of hydrophobicity was increased from P1

to P17 (Figure 2d).

3

.2 The lipid affinity of P12 ensures its cholesterol efflux activity

Macroscopically, the DMPC emulsion is dissolved by sufficiently hydrophobic AMPs

into a clear liquid. Of the studied AMPs, apoA-I221–240 and P1–P8 induced no changes of the

DMPC emulsion within 90 min. However, P9–P11, and L-4F quickly dissolved in DMPC

within 10 min. Meanwhile, the emulsion became gradually clear in the presence of P12–P17,

but the emulsion became completely clear after 90 min only following exposure to P12

(Figure 2e).

3

.3 P12, a new peptide with excellent activity and low toxicity, was identified

In RAW264.7 cells, apoA-I221–240 and P1–P5 did not induce cholesterol efflux, and their

intracellular cholesterol efflux rates were less than 2% at concentrations of 1–200 μM. L-4F

and P6–P17 displayed concentration-dependent cholesterol efflux activity. When the

concentration exceeds the safe limit, P13–P17 could damage the membrane of RAW264.7 cells,

leading to cholesterol leakage from cells and consequent changes of the dose-effect curves

(Table 2, Figure 3a, S4, and S5).

Then, the cytotoxicity of these new mimetic peptides were determined in RAW264.7

cells. The results illustrated that apoA-I221–240 and P1–P12 had no cytotoxic effects. P13 had

significant cytotoxicity at concentrations of 100 and 200 μM, resulting in cell viability rates

of less than 80%. P14 and P15 exhibited cytotoxic effects at 20 and 5 μM, respectively. P16,

P17, and L-4F had concentration-dependent cytotoxic effects in RAW264.7 cells (Table 2,

Figure 3b).

The influence of new mimetic peptides on mouse erythrocytes in comparison with their

parent peptide and L-4F is presented in Table 2 and Figure S6. After 12 h, apoA-I221–240, P1–

P13, and L-4F induced slight hemolysis, and the hemolytic rate was less than 2% at a

concentration of less than 200 μM. P14 was hemolytic at concentrations exceeding 200 μM

(hemolytic rate >2%). P15–P17 induced obvious hemolysis at 20 μM, and the hemolytic rate

approached 8% at 200 μM. Based on these results, P12, with good activity and no toxicity,

was screened for further study.

3

.4 In vitro mechanism

.4.1 P12 can promote cholesterol efflux in macrophages

P12 more strongly induced cholesterol efflux in THP-1 cells than in RAW264.7 cells

(

Figure 4a).

The results of cholesterol efflux determination using flow cytometry are shown in Figure

b. Compared with the findings in of PP (apoA-I221–240), both P12 and L-4F significantly

4

promoted the intracellular cholesterol efflux at 10 µM in both RAW264.7 (efflux rates of 3.2

and 4.56%, respectively) and THP-1 cells (efflux rates of 5.5 and 9.89%, respectively).

ApoA-I221–240 did not display distinctive cholesterol efflux activity in either cell line (efflux

rates of 0.18 and 0.59%, respectively, at 10 µM).

3

.4.2 P12 induced cholesterol efflux through ABCA1

P12 promotes cholesterol efflux through ABCA1. After treatment with 9-cis-retinoic acid

and TO-901317, P12 increased cholesterol efflux in RAW264.7 cells by 0.40% at 1 μM and

.02% at 10 μM. Meanwhile, and Probucol suppressed the cholesterol efflux induced by P12.

1

Similar results were obtained for L-4F. ABCA1-dependent cellular cholesterol efflux was

more strongly induced by 9-cis-retinoic acid and TO-901317 in THP-1 cells than in

RAW264.7 cells. P12 increased cholesterol efflux by 1.53% at 1 μM and 1.92% at 10 μM in

THP-1 cells. Meanwhile, in the presence of Probucol, P12-induced cellular cholesterol efflux

was reduced by 1.75% at 1 μM and 2.18% at 10 μM (Figure 4c).

In both RAW264.7 and THP-1 cells, P12-induced cholesterol efflux was obviously

enhanced by treatment with cAMP. Unexpectedly, L-4F was cytotoxic in THP-1 cells, leading

to cholesterol release at concentrations exceeding 10 μM (Figure 4d). It is shows that P12 has

stronger ABCA1-dependent cholesterol efflux activity than L-4F.

3

.4.3 P12 promoted cholesterol efflux by acting on the cell membrane

To further confirm the acting site of P12, the fluorescence of 22-NBD-cholesterol and

P12 labeled with C-TPP were examined in RAW264.7 and THP-1 cells. As shown in Figure

e, 22-NBD-cholesterol had a strong fluorescent signal throughout RAW264.7 and THP-1

4

cells excluding the nucleus at 24 h, but C-TPP-P12 fluorescence was only observed outside

the cell or on the cell membrane. Moreover, THP-1 cells exhibited much greater membrane

fluorescence for C-TPP-P12 than RAW264.7 cells. This result was consistent with the results

in Figure 4a and b.

3

.5 Animal experiment

.5.1 P12 suppressed the abnormal thickening of the arterial wall

The typical pathological characteristics of atherosclerosis were confirmed in apoE⁻^/⁻mice

(Figure S9). The therapeutic effect of P12 is presented in Figure 5a and b. The statistical

analysis illustrated that the relative intimal thickening and plaque size of each sample were

significantly decreased by P12. The relative thickness of the arterial wall in male apoE⁻^/⁻

mice was reduced by L-4F, their parent peptide, and low- and high-doses of P12 by 80.24,

4

5.48, 90.26, and 94.46%, respectively, and by 69.64, 39.50, 84.88, and 92.04%, respectively,

in female apoE⁻^/⁻mice.

.5.2 P12 has lipid-lowering, anti-inflammatory and anti-oxidative effects in apoE⁻^/⁻

mice

3

Serum turbidity and MDA, TG, TC, HDL-C, and LDL-C levels were significantly higher

in apoE⁻^/⁻mice than in wild-type mice after 4 months of feeding. P12 significantly prevented

lipid accumulation and peroxidation in blood vessels. SOD levels were increased by P12 in

apoE⁻^/⁻mice. Serum HDL-C levels were higher in un-treatment apoE⁻^/⁻mice than in wild-type

mice after 4 months. HDL-C levels were increased by high doses of P12 in apoE⁻^/⁻mice (P

<

.05). TC, TG, and LDL-C levels were dramatically reduced by P12 in apoE⁻^/⁻mice (P < .05).

After 4 months of feeding. the gender differences in serum lipids (TG, TC, HDL-C, and LDL-

C) were obvious. Serum lipids levels were lower in female mice than in male mice. Serum TG

and TC levels were more strongly lowered by P12 in male mice than in female mice. P12 had

no significant effect on serum HDL-C levels in female or male mice, whereas it reduced LDL-

C levels in female and male mice by the same extent (Figure 5b and c).

After high-fat diet feeding, serum CRP, IL-6, MCP-1, and TNF-α levels were

significantly higher in apoE⁻^/⁻mice than wild-type mice (P < .05). Meanwhile, P12

significantly suppressed these inflammatory indices. The apoA-I221–240 had no cholesterol

efflux activity in vitro and no satisfactory lipid-lowering activity in vivo, although it

–

1

exhibited favorable anti-inflammatory effects. CRP levels were close to 0 mg·L in wild-

–

1

−/−

type mice compared with more than 150 mg·L in apoE mice. Serum CRP levels in

apoE⁻^/⁻mice were reduced by approximately 70% by P12 treatment compared with those in

un-treatment apoE⁻^/⁻mice. After 4 months of feeding. the gender differences in serum

inflammatory factor levels were extremely obvious. Excluding CRP, serum inflammatory

factor levels (IL-6, MCP-1, and TNF-α) were lower in all of male mice than in all of female

mice (Figure 5d).

3

.5.3 P12 has a short half-life in wild-type C57BL/6J and apoE⁻^/⁻mice

The plasma clearance curve and fitted half-life of P12 are presented in Figure 6a. No

significant difference was observed in the plasma clearance rate of P12 between apoE⁻^/⁻and

C57BL/6J mice (P = 0.9404, t-test). However, the t1/2 of P12 was longer in apoE⁻^/⁻mice

(3.09 h) than in C57BL/6J mice (2.40 h). After intravenous injection, P12 remained in the

plasma approximately 25 h.

3

.5.4 P12 can slowly increase plasma apoA-I levels in apoE⁻^/⁻mice within 24 h

As shown in Figure 6b, plasma apoA-I were lower in apoE⁻^/⁻mice than in C57BL/6J

mice. P12 had no effect on plasma apoA-I in C57BL/6J mice. After the intravenous injection

of P12, plasma apoA-I in apoE⁻^/⁻mice displayed a downward trend from 0 to 2.5 h and an

upward trend from 2.5 to 24 h. However, after the intravenous injection of P12 for 24 h,

plasma apoA-I levels were increased in apoE⁻^/⁻mice by 0.094 g·L^–¹.

3

.5.5 P12 decreased blood lipid levels and increased cholesterol excretion on apoE⁻^/⁻

mice

P12 had indistinctive effects on serum lipids levels in wild-type C57BL/6J mice and

HDL-C levels in apoE⁻^/⁻mice after 7 days of treatment, whereas the AMP significantly

reduced serum TC, LDL-C, and non-HDL-C levels in apoE⁻^/⁻mice (Figure 6c). Similarly,

P12 had no effect on cholesterol excretion in wild-type mice within 7 days whereas its

treatment increased the cholesterol excretion in apoE⁻^/⁻mice by 76.8% (Figure 6d).

3

.6 P12 can bind to plasma HDL, and transform α-HDL into preβ-HDL

The results in Figure 7 illustrated that the peak HDL area was smaller in plasma treated

with P12 than in plasma incubated with saline. The peak HDL area was significantly

increased by incubation with C-TPP-P12 and plasma. The increased peak fluctuation of the

red line was the absorbance of C-TPP-P12 (Figure 7a). As shown in Figure 7b, most of the

cholesterol in saline-treated plasma was concentrated in HDL, whereas the cholesterol

content of HDL was significantly reduced in P12-treated plasma. As shown in Figure 7b and

c, P12 reduced TC content. C-TPP-P12 was concentrated in the peak period of HDL, which

fully indicated that C-TPP-P12 can bind to mature α-HDL.

As shown in Figure 7d and e, P12 converted α-HDL to preβ-HDL, and the conversion

rates increased with increasing P12 concentrations. The preβ-HDL conversion rates in the

–

1

presence of 125, 250, 500, and 1000 μg·mL of P12 were 8.42, 17.48, 38.16, and 40.57%,

–

1

respectively. Conversely, the preβ-HDL conversion rate in the presence of 500 μg·mL L-4F

was 35.12%. Interestingly, the parent peptide apoA-I221–240 was also linked to a high preβ-

–

1

HDL conversion rate at 500 μg·mL (12.36%).

4

. Discussion

This study was aimed to design new short apoA-I mimetics possessing the biological

functions of apoA-I. Considering that AMPs exert anti-atherosclerotic effects through RCT,

the effect of hydrophobicity on their lipid affinity and ability to transport cholesterol is a

primary factor of consideration (Datta et al., 2001; Van Lenten et al., 2009). In this study,

fragment 221–240 of apoA-I were used as the parent peptide because it has an essential

sequence for cholesterol efflux and contains the ABCA1 binding domain (Chroni, Koukos,

Duka & Zannis, 2007). In order to maintain the original sequence of apoA-I221–240 as much as

possible, the novel AMPs were designed using a strict amphiphilic construct. The

hydrophobic surface is beneficial for affinity with the fatty chain of phospholipids. The

positively charged amino acids at the amphiphilic junction facilitate them binding to the

negatively charged phospholipid head, and the negatively charged amino acids in the middle

of the hydrophilic surface can balance the positive charge and stabilize ABCA1.

This study found that new AMPs induced cholesterol efflux when the hydrophobic value

was ≥ 0.260 (calculated using HeliQuest), whereas their cytotoxicity was observed at values

of ≥ 0.548. Furthermore, when their hydrophobic value was ≥ 0.699, AMPs induced slight

hemolysis at high concentration. According to the in vitro mechanism studies, the possible

explanations of the aforementioned results are as follows. 1) Hydrophobicity affects the

secondary structure of new AMPs. As hydrophobicity increased, the secondary structure of

new AMPs changed from disordered structure (P1–P10) to an α-helical (P11–P14) or β-sheet

(P15–P17) structure. In line with this finding, prior research uncovered that AMPs must have

an amphipathic α-helical structure to induce cholesterol efflux (Beaufils et al., 2007). In this

study, P11–P14 always maintained a stable α-helical structure whether in an aqueous or the

simulated biofilm environment, leading to their stronger activity and weaker cytotoxicity

compared with the other AMPs. 2) Hydrophobicity affects lipid affinity and amphipathicity

(represented by the hydrophobic moment) of AMPs. With an increasing hydrophobicity,

AMPs exhibit increasing ability to dispersion DMPC vesicles. The result indicated that P9–

P11 had good lipid affinity and appropriate amphipathicity (in line with prior findings that the

moment balance between hydrophobic and hydrophilic face is important for lipid

interactions) (Natarajan, Forte, Chu, Phillips, Oram & Bielicki, 2004), thereby explaining

their ability to rapidly bind to micron-DMPC vesicles in water and then disperse them into

nanovesicles. Although lipid affinity is the basis of their cholesterol efflux activity, P12 more

strongly induced cholesterol efflux than P9–P11 despite its weaker lipid affinity. This finding

also suggested that other factors, such as ABCA1 dependence, affect the cholesterol efflux

activity of AMPs in addition to lipid affinity and amphipathicity (Yao, Gordon, Barochia,

Remaley & Levine, 2016).

Several studies reported that apoA-I can promote the dephosphorylation of the PEST

sequence of ABCA1, reduce the calpain-mediated degradation of ABCA1 and allow permit

greater ABCA1 expression on the cell membrane surface, leading to the enhancement of

cholesterol efflux from cells (Arakawa, Hayashi, Remaley, Brewer, Yamauchi & Yokoyama,

2

004; Martinez, Agerholm–Larsen, Wang, Chen & Tall, 2003). ApoA-I promotes cholesterol

efflux by binding to ABCA1 rather than impairing cell membranes. ABCA1 overexpression

can enhance the cellular binding of apoA-I (Jin et al., 2016; Xie, Zhao & Li, 2010). The

process of cholesterol efflux through ABCA1 is mild, and it does not impair cell membranes,

leading to a smooth dose-effect curve. Similarly, our results indicated that P12 had a nearly

smooth dose-effect curve and functioned together with ABCA1. LSCM also confirmed that

P12 acted only on the cytomembrane surface without impairing cellular integrity.

Numerous studies have confirmed that the function of HDL is more important to anti-

atherosclerotic effects than its quantity (Chapman, Orsoni, Robillard, Therond & Giral, 2018;

Tsompanidi, Brinkmeier, Fotiadou, Giakoumi & Kypreos, 2010). In this study, P12

observably regressed atherosclerotic plaques in apoE⁻^/⁻mice, although serum HDL-C levels

did not fluctuate after the 12 weeks of P12 treatment. A possible mechanism for P12-induced

plaque suppression might be the promotion of HDL function. Moreover, P12 can slowly

increase the concentration of apoA-I within 24 h. These findings illustrate that P12 can

increase the ability of HDL to block the accumulation of harmful lipids in the body.

−

1

–1

Unexpectedly, the hypolipidemic effect of P12 was better at 10 mg·kg than at 20 mg·kg of

P12, suggesting that excessive P12 affected some functions of HDL. In addition to RCT,

HDL also prevents atherosclerosis through anti-inflammatory effects. Interestingly, apoA-

I221–240 had excellent anti-inflammatory effects in atherosclerotic mice, suggesting that P12

retained the anti-inflammatory properties of its parent peptide. The findings indicate that P12

can potentially treat atherosclerosis by lowering blood lipid levels, promoting cholesterol

excretion, suppressing inflammation, and reducing lipid peroxidation. In addition, gender

differences lead to differences in blood lipids, lipid peroxidation and inflammatory factors

were observed.

As mentioned previously, HDL consists of nascent HDL particles (pre β-HDL, size

approximately 7.2 nm) and mature HDL particles (α-HDL, size approximately 12.2 nm)

according to its electrophoretic mobility (Tsompanidi, Brinkmeier, Fotiadou, Giakoumi &

Kypreos, 2010). Compared with α-HDL, preβ-HDL is a cholesterol-unsaturated moiety

containing less cholesterol and cholesterol ester content. When preβ-HDL loads cholesterol to

saturation, it becomes α-HDL. Thus, preβ-HDL has a stronger ability to carry and load

cholesterol than α-HDL, making it a key molecule for the HDL function (Hafiane & Genest,

2

015). Our results illustrated that P12 promoted the function of HDL remodeling α-HDL into

preβ-HDL. Further analysis indicated that P12 can bind with the phospholipids of HDL to

promote RCT, thereby improving its efficiency.

Overall, as a novel developed AMP with an amphipathic α-helical structure, P12 has

stronger ABCA1-dependent cholesterol efflux activity and lower cytotoxicity than L-4F, and

it can significantly suppress the formation of atherosclerotic plaque by promoting the

cholesterol efflux from the plaques through ABCA1 and combine with cholesterol-saturated

α-HDL particles in the plasma to jointly carry cholesterol to the liver. Furthermore, P12 can

also convert α-HDL into nascent preβ-HDL particles, which can more efficiently transport

cholesterol in the atherosclerotic plaques to the liver and remove excess cholesterol from the

arteries. Moreover, P12 can alleviate atherosclerosis through anti-inflammatory and

antioxidant effects in apoE⁻^/⁻mice.

Therefore, P12 is a promising candidate peptide for development as a new generation

atheroprotective agent for cardiovascular disease. P12 represents a new technical innovation

that positions us to respond to the“statins-resistance” without contributing to the associated

side effects and mortality epidemics or losing the required mildly and effectively anti-

atherosclerotic effect.

Acknowledgements

This work was financially supported by the funding from the National Natural Science

Foundation of China (81273440) and (81773564).

Conflict of Interest

The authors declare no conflicts of interest.

Author contributions

S.G. performed the experiments and data processing. L.W., G.B. and B.L. did the peptides

identification and modification. C.Z. participated in peptides purification. X.C. and X.O.

participated in data statistical analysis. X.O. and S.G. participated in animal studies. X.C.

Y.Z. and H.L. did the manuscript reading and correction. J.N. and S.G. conceived, supervised,

and funded the work. J.N. and S.G. wrote the paper. All authors reviewed the manuscript.

Declaration of transparency and scientific rigour

This Declaration acknowledges that this paper adheres to the principles for transparent

reporting and scientific rigour of preclinical research as stated in the BJP guidelines for

Design & Analysis, Immunoblotting and Immunochemistry, and Animal Experimentation and

as recommended by funding agencies, publishers, and other organizations engaged with

supporting research.

ORCID

Sanhu Gou https://orcid.org/0000-0003-3231-7427

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Article Doi

A novel apoA-I mimetic peptide suppresses atherosclerosis by promoting physiological HDL function in apoE?/? mice

DOI: 10.1111/bph.15213

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Article abstract of DOI:10.1111/bph.15213

Full text of DOI:10.1111/bph.15213

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