E17241 As a Novel ABCA1 (ATP-Binding Cassette Transporter A1) Upregulator Ameliorates Atherosclerosis in Mice

Arteriosclerosis, Thrombosis, and Vascular Biology

ORIGINAL RESEARCH E17241 as a Novel ABCA1 (ATP-Binding Cassette Transporter A1) Upregulator Ameliorates Atherosclerosis in Mice

Yanni Xu (许艳妮) , Chang Liu (刘畅), Xiaowan Han (韩小婉) , Xiaojian Jia (贾晓健) , Yongzhen Li (李永瑧) , Chao Liu (刘超), Ni Li (李霓), Lunming Liu (刘伦铭), Peng Liu (刘鹏), Xinhai Jiang (姜新海), Weizhi Wang (王伟志), Xiao Wang (王潇), Yining Li (李依宁), Mingzhu Chen (陈明华), Jinque Luo (罗金雀), Xuan Zuo (左璇), Jiangxue Han (韩江雪), Li Wang (王丽), Yu Du (杜郁) , Yang Xu (徐扬), Jian-Dong Jiang (蒋建东), Bin Hong (洪斌) , Shuyi Si (司书毅)

OBJECTIVE: Reverse cholesterol transport, removing excess cholesterol from peripheral tissues, is an important therapeutic target for atherosclerosis treatment. In this study, we propose a new small molecule, E17241, that may be used to treat atherosclerosis by promoting reverse cholesterol transport via ABCA1 (ATP-binding cassette transporter A1) upregulation. APPROACH AND RESULTS: E17241 (4-(1,3-dithiolan-2-yl)-N-(3-hydroxypyridin-2-yl)benzamide) was first identified as an ABCA1 upregulator using a cell-based reporter assay. E17241 significantly increases the mRNA and protein expression levels of ABCA1 in both hepatic cells and macrophages. It promotes cholesterol efflux to apo AI in macrophage cells, and this effect depends on ABCA1. It also decreases total cholesterol content in Ox-LDL (oxidized low-density lipoprotein) loading macrophage cells. E17241 treatment increases the content of 3H-labeled cholesterol in the feces of male C57BL/6J mice intraperitoneally injected with 3H-cholesterol-labeled macrophage J774 cells, indicating that it could promote in vivo

Downloaded from http://ahajournals.org by on January 31, 2021 macrophage reverse cholesterol transport. Compared with the western diet group (western diet–fed male ApoE−/− mice), the E17241 group (western diet+E17241 treatment) shows decreased plasma cholesterol, liver cholesterol, and triglyceride levels, with increased fecal cholesterol content. Importantly, E17241 reduces atherosclerotic lesion areas in the en face aorta and aortic sinus while increasing ABCA1 protein levels in both liver and macrophages. Human proteome microarray, coimmunoprecipitation, and other assays demonstrate that PKCζ (protein kinase C zeta) is a binding target of E17241, and this small molecule increases ABCA1 expression in macrophages via the PKCζ-NR (nuclear receptor) pathway. CONCLUSIONS: E17241 may be developed as a new lead or drug candidate for the treatment of atherosclerosis by upregulating ABCA1.

Key Words: atherosclerosis ◼ cardiovascular disease ◼ cholesterol ◼ liver ◼ macrophages

therosclerosis is a major cause of cardiovascu- balance.1–3 Therapeutic strategies designed to promote lar disease. Atherosclerotic lesions develop when macrophage RCT might prevent the progression of the imbalance between deposition and removal of atherosclerosis.2,3 A 1 arterial cholesterol occurs. Reverse cholesterol trans- ABCA1 (ATP-binding cassette transporter A1), a port (RCT) pathway transfers accumulated cholesterol membrane protein of the human transporter sub-family from peripheral cells such as macrophages to the liver ABCA, mediates the efflux of cellular cholesterol to lipid- for excretion, which is important to maintain cholesterol poor apo AI and plays an important role in cholesterol

Correspondence to: Jian-Dong Jiang, PhD, Institute of Medicinal Biotechnology, CAMS&PUMC, 1 Tiantan Xili, Beijing 100050, China, Email [email protected]; or Bin Hong, PhD, Institute of Medicinal Biotechnology, CAMS&PUMC, 1 Tiantan Xili, Beijing, 100050, China, Email [email protected]; or Shuyi Si, PhD, Institute of Medicinal Biotechnology, CAMS&PUMC, 1 Tiantan Xili, Beijing, China, Email [email protected] *These authors contributed equally to this work. The Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/ATVBAHA.120.314156. For Sources of Funding and Disclosures, see page xxx. © 2021 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at www.ahajournals.org/journal/atvb

Arterioscler Thromb Vasc Biol. 2021;41:00–00. DOI: 10.1161/ATVBAHA.120.314156 April 2021 1 Downloaded from http://ahajournals.org by on January 31, 2021 31, January on by http://ahajournals.org from Downloaded Original Research - AL sis. the foamcellformationinhumancoronaryatherosclero- smooth musclecellsinarterialplaquealsocontributesto the progressionofatherosclerosis. processandpotentially target forpromoting prevent RCT from peripheralcellsandisconsideredtobeapromising in thedifferent includingcholesterol stepsofRCT efflux tissues, andprematurecoronaryatherosclerosis. protein) deficit,build-upof cholesterol intheperipheral (high-densitylipo- that ischaracterized bysevereHDL lead toTangier disease,anautosomalrecessivedisorder 2-yl)-N-(3-hydroxypyridin-2-yl)benzamide), is actually a rosis. The proposedmolecule,E17241 (4-(1,3-dithiolan- upregulatory drugcandidatetotreatatheroscle- ABCA1 homeostasis andRCT. Xu etal atherosclerosis. foam cellformationandincreasesthesusceptibilityto marrow–derived cellsleadstoenhancedmacrophage 2 cholesterol efflux toapoAI. phages fromAbca1-knockout mice substantiallyreduce phenotype ofTangier diseasepatients, knockout micehaveaphenotypesimilartotheclinical WD WB VLDLs TC SR-BI SIRT1 RT-PCR RCT PKC Ox-LDL ORO NR ND LDL-C IDLs HDL-C HDL CMC-Na CLA-1 BMDMs AST ALT AKT ABCA1 Nonstandard AbbreviationsNonstandard andAcronyms This workaimsatdevelopinganew, highlyefficacious 9 Based on these studies, ABCA1 playsapivotalrole Basedonthesestudies,ABCA1 April 2021

Western diet Western blot very-low-density lipoproteins total cholesterol scavenger receptorclassBtypeI sirtuin 1 real-time polymerasechain reaction reverse cholesterol transport protein kinaseC oxidized low-densitylipoprotein Oil RedO Nuclear receptor diet normal laboratory low-density lipoproteincholesterol intermediate-density lipoproteins cholesterol HDL high-density lipoprotein sodium carboxymethylcellulose analogous-1 II LIMP and CD36, human homologofSR-BI, marrow–derivedmacrophages bone aspartate transaminase alanine aminotransferase PKB protein Ser/Thr kinaseB,alsonamed ATP-binding cassettetransporterA1 8 Reduced ABCA1 expression ReducedABCA1 inintimal 3,4 MutationsintheAbca1gene 7 Deletion of ABCA1 in bone inbone DeletionofABCA1 3,10 6 andmacro- 2021;41:00–00. DOI: 10.1161/ATVBAHA.120.314156Arterioscler ThrombVascBiol. 2021;41:00–00. DOI: 5

Abca1- and UseCommittee oftheInstituteMedicinal Biotechnology. accordance with the regulations of the Institutional Animal Care Animal careandexperimental procedureswereperformedin tein], Trophic AnimalFeed High-tech, Co,Ltd,Nantong,China). 19.8% pro- carbohydrate, [21% fat,0.2%cholesterol, 49.1% Biotechnology, Co,Ltd)orawestern diet(WD,no.TP26300 [Beijing] SPF no.Co60, diet (ND, were fed anormallaboratory Technology, Co,Ltd(Beijing,China).Allofthemiceorhamsters were purchased fromBeijingVital Animal RiverLaboratory male goldenSyrianhamsters,andKunming micethat and inwestern diet (WD)–induced male ApoE mechanisms ofE17241 areinvestigatedinvitro,ex vivo, any biological activity. The antiatherosclerotic effects and small moleculethathasnotpreviouslyreportedtohave C57/BL6J ApoE In vitroassessmentswerecarriedouton8-weekoldmale Animals lipofectamine 2000,andlipofectamine RNA Biology, Co,Ltd(Beijing,China), and22-NBD-cholesterol, dized low-densitylipoprotein)wereprovidedbyPeking Union- andEnamineLtd(Kyiv,MO) Ukraine),respectively. (oxi Ox-LDL - and E17241Go6983 were purchased from Sigma (St. Louis, Reagents mL G418. µg/ and600 medium (Hyclone)supplementedwith10%FBS were maintainedinRoswellPark MemorialInstitute1640 Meanwhile, the stably transformed ABCA1p-LUC HepG2 cells and J774 (Hyclone). DMEM cellswerekeptin10%FBS were maintainedinourlaboratory. The HepG2,RAW264.7, J774, andstablytransformedABCA1p-LUC HepG2cells HepG2, RAW264.7 (murineperitonealmacrophagecells), Cell Cultures from thecorrespondingauthoruponreasonablerequest. The datathatsupportthefindingsofthisstudyareavailable METHODS MATERIALS AND from Invitrogen. and malehamsters. • • • • receptor) pathway. (nuclear ζ (proteinkinaseCzeta)andNR the PKC E17241 proteinexpression regulatesABCA1 via ApoE aorta atheroscleroticlesionsinwestern diet–fed E17241 obviouslyamelioratesthedevelopmentof reverse cholesterol transport. E17241 significantly promotesin vivomacrophage (ATP-binding cassettetransporterA1)upregulator. idin-2-yl)benzamide) is shown to be an ABCA1 E17241 (4-(1,3-dithiolan-2-yl)-N-(3-hydroxypyr- 11 −/− mice. E17241 AmelioratesAtherosclerosis inApoE −/− mice,maleandfemale C57/BL6J mice, Highlights MAX wereobtained −/− −/− mice Mice 11

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3 14 Mice -AACA −/− μg/mL, ′, reverse: (Thermo (Thermo MAX April 2021 -GGTGTGAACC Briefly, RAW264.7 RAW264.7 Briefly, 13 -CCTGTTGCTGTAGCC -GAGTCCTTCCACGATAC ′); mouse ABCA1 (forward: , reverse: 5′ , reverse: ′ , reverse: 5′ using the following primer sequences: human using the following primer sequences: human 14 E17241 Ameliorates Atherosclerosis in ApoE in Atherosclerosis Ameliorates E17241 g/mL, sc-8393;μg/mL, PKC(1 Cruz), δ Santa ); and mouse GAPDH (forward: 5′ ′ (1 α The antibodiesABCA1The analysis include used for this (2 μg/ mL, NB400-105; Novus Biologicals), PKC kinase (protein C) siRNA Assay cells plated in 6- or 96-well RAW264.7 plates were transfected Santa Cruz), with scrambled siRNA (20 nmol/L, sc-37007; ABCA1 siRNA (20 nmol/L, Guangzhou GenePharma, Co, Ltd, China), PKCζ siRNA (20 nmol/L, sc-36254), or SIRT1 siRNA Santa Cruz) using RNA (20 nmol/L, sc-40987, For quantitative RT-PCR analysis, the cells were treated with analysis, the cells quantitative RT-PCR For for 24 hours. Then, at the indicated concentrations E17241 the total RNA from cells and tissues using the was extracted QIAGEN RNeasy Hilden, Germany) and TRIzol Mini kit (Qiagen, analysis RT-PCR quantitative the Finally, respectively. (Invitrogen), was performed WB Analysis reported method. WB analysis was based on a previously Santa sc-8402; Santa Cruz), PKCζ (0.02 μg/mL, sc-17781; Cell Cruz), P-PKCPKCζ (phosphorylated ζ; 1:1000, no. 9378, P-AMPKα (1:1000, no. 2531, Cell Signaling Technology), SIRT1 (sirtuin 1; 1:1000, ab12193; Signaling Technology), Abcam), SR-BI (scavenger receptor class B type I; 1 μg/ NB400-104,mL, Ab217318; 0.5 μg/mL, Biologicals; Novus images of Abcam), and β-actin (A5441, 1:10000; Sigma). The system 5200 Tanon the using recorded were cells analyzed the Co, Ltd, Shanghai, China). Science & Technology, (Tanon Ox-LDL Loading Experiment Loading Ox-LDL according was carried out Ox-LDL loading cell experiment et al. by Feng to the method described Chain Polymerase Quantitative Real-Time Reaction Analysis Cruz) or ABCA1 siRNA (20 nmol/L, siG160928115822, the Then, hours. 6 for China) Ltd, Co, GenePharma, Guangzhou treated with 22-NBD-cholesterolcells were (10.0 and E17241 µmol/L), efflux assay. the cholesterol as in ABCA1 (forward: 5′-GGTGGTGTTCTTCCTCATTACT-3 5′-CCGCCTCACATCTTCATCTT-3 5’-CACATCCTC reverse: 5’-GGGTGGTGTTCTTCCTCATTAC-3’, human GAPDH (forward: 5′ ATCCTCGTCATTC-3’); GCAACTCCCACTCTTC-3 GTATT-3 ′ ATGAGAAGTATGA-3 ′). CAAAG-3 macrophages or BMDMsmacrophages in 96 were seeded plates or 12-well incubation with Ox-LDL(Costar), followed by (at the final the concentration of 60 Subsequently, µg/mL) for 24 hours. (2.0 or 10.0 µmol/L) for 24 E17241 cells were treated with fixed and stained with Oil Red O (ORO).hours, then they were content of the incubated cholesterol Otherwise, the intracellular assay using the cholesterol and determined cells was extracted China). To Inc, Beijing, Technologies, kit (no. E1015, Applygen protein concentrations were cholesterol, assess the content of total amount using the BCAThe measured kit (Pierce). assay in μmol/g protein. is expressed of intracellular cholesterol , β / was car- 11 cells/mL) and °C for another 6 As for the ABCA1 As for the 11,12 it was generated using the ABCA1-the using generated it was 13 were performed using HepG2 cells. E17241 HepG2 cells. E17241 were performed using 14,15 , and RXR. pBIND-NR-LBD, The GLA4-pGL4- δ / γ / Femur and tibia bones old were isolated from 8-week Femur α 16 For ABCA1 siRNA assay, RAW264.7 cells were pretrans- RAW264.7 ABCA1 siRNAFor assay, ried out using ABCA1p-LUC HepG2 cells, whereas the ABCA1 HepG2 cells, whereas ried out using ABCA1p-LUC DR4 mutation plasmid and pBIND-NR-LBD/GAL4 chimera reporter assays male or female C57/BL6J mice. Marrow was flushed out with mice. C57/BL6J male or female cold PBSsyringe containing a 5 mL and 2% heat-inactivated μm FBS cells were passed through a 70 (3–5 mL/mouse). The lysis buf- volumes of red blood cell cell strainer (Costar). Three Co, Ltd) (no. R1010, Beijing Solarbiofer Science &Technology to remove red were added and incubated on ice for 10 minutes rpm for 5 the cells were spun down at 1000 blood cells. Then PBS+2%FBScold with washed were pellets cell The minutes. BMDMin resuspended and mL) (20–50 medium growth (DMEM10% FBS,with supplemented M-CSF (monocyte-col- no. 315-02), and ony stimulating factor; 10 ng/ml, PeproTech, seeded finally were isolated cells The penicillin-streptomycin). in 60 96 mm or well tissue culture plates (2×10 changed into fresh BMDMchanged growth medium on day 3. On day mature BMDMs to the indicated medium for were changed 7, Ox-LDL efflux, or uptake assay, blot (WB), cholesterol Western respectively. was added to the cells at specific concentrations, and 24 hourswas added to the cells cells was detected. activity of these the luciferase later, Cholesterol Efflux Assay Cholesterol BMDMsor cells RAW264.7 96-well in seeded were black-bot - with the cells were equilibrated Then, tom plates (PerkinElmer). 22-NBD-cholesterol of 5.0 µmol/L) (at the final concentration they were washed and incubated for 24 hours. Subsequently, (0, 0.4, 2.0, and 10.0 µmol/L). After with or without E17241 24 hours, apo AI was added at the final concentration of 10 μg/mL, and the samples were incubated at 37 6 hours. The cells were fixed and washed 3× with 3× fixed and washed were cells PBS.The 6 hours. Nuclei were counterstained with 40,6-diamidino-2-phenylindole (no. 62248, Fisher Scientific) for 10 minutes before Thermo being imaged or analyzed by a High Content Analysis System average fluorescence amount The CA). Fremont, (PerkinElmer, is normalized as fluorescence per of intracellular cholesterol amount cholesterol fluorescence final intracellular the cell. Then after subtracting blank well. The is expressed sample for each percentage ratio of 22-NBD-cholesterol efflux was calculated based on the following equation: 100×(fluo- sample, for each rescence of 22-NBD only−fluorescence of sample)/(fluorescence of 22-NBD only). Santa scrambled siRNA with fected (20 nmol/L, sc-37007, luc, and ABCA1-promoter-pGL3 plasmids were constructed previously reported methods. as per the PPAR Isolation of Bone Marrow–Derived Marrow–Derived Isolation of Bone Macrophages isolation of boneThe marrow–derived macrophages (BMDMs) described by Ying were carried out according to the method et al. Transfection and Luciferase Assay and Luciferase Transfection NRsThe include LXR described herein (nuclear receptors) α promoter-pGL3 verified by plasmid as a template and DNA ABCA1 transcriptional activity assay sequencing. The DR4 plasmid, mutation Arterioscler Thromb Vasc Biol. 2021;41:00–00. DOI: 10.1161/ATVBAHA.120.314156 Xu et al Xu

Downloaded from http://ahajournals.org by on January 31, 2021 Downloaded from http://ahajournals.org by on January 31, 2021 31, January on by http://ahajournals.org from Downloaded Original Research - AL ing effects ofestrogenonatherosclerosis development. were usedinthisexperiment toavoidthepotentialconfound- vious study. to establishanatheroscleroticmodel,asdescribedinapre- American HeartAssociationStatement. atherosclerosis, we strictly abided by the guidelines of the stored in20%sucrose.Inthisexperimental studyofmouse Before analysis,theorganswereplacedin4%formalinand and hearts(withupperaorticroot)oftheinvestigatedmice. −20 lected fromthemousecages,dried,weighed,andstoredat μ fecesTC (expressed as micromoles/L per microgram feces; Beijing, China).The amountofcholesterol insamplesofmice cial kits(no.E1015,no.E1013,ApplygenTechnologies, Inc, in theliverweredeterminedusingappropriatecommer- The cholesterol andtriglyceridecontents(μ terol, triglyceride,quantitativeRT-PCR, andWBanalyses. snap-frozen inliquidnitrogen,andstoredat−80 (Hitachi 71800, Chiyoda,Japan).The liverswereharvested, Science, Inc,China)andanautomaticbiochemical analyzer corresponding commercialkits(BiosinoBiotechnology and in thecollectedbloodsamplesweremeasuredusing ferase), AST (aspartatetransaminase),creatinine,andurea cholesterol), glucose,ALT (HDL HDL-C (alanineaminotrans- (TC), triglyceride,LDL-C (low-densitylipoproteincholesterol), they wereeuthanized.The levelsofplasmatotalcholesterol were takenfromtheretro-orbitalplexus offastedmicebefore day for10weeks. or E17241 wereadministeredtothemicebygavage,oncea (0.5%) with12miceineach group.CMC-Na 0.5% CMC-Na), andE17241-H (fed WD,50mg/kgin kg in0.5%CMC-Na), E17241-L (fedWD (fed WD,25mg/ WD,0.5%CMC-Na), sodium[CMC-Na]), 0.5%carboxymethylcellulose (fed ND, The mice were randomly divided into 4 groups, including ND ApoE Atherosclerosis AnalysisofApoE described above. WB analysisorcholesterol efflux assaywasthenperformedas incubated withE17241 attheindicatedconcentrations,and incompletemedium.Afterward, thecellswere with 10%FBS Fisher Scientific).Sixhourslater, themediumwas exchanged Xu etal 4 models exhibit variations in atherosclerosis, male ApoE or hematoxylin and eosin for imaging using Nikon DS-U3. The or hematoxylin andeosinforimagingusingNikon DS-U3. Germany). Thereafter, thecutsampleswerestainedwithORO cross-sections usingacryostat (Leica, Microsystems,Wetzlar, Torrance, CA) thensectioned into serial 10 μm aortic sinus in optimumcuttingtemperature compound(SakuraFinetek, tic area(plaque/total).Asforthe hearts,theywereembedded to assesstheratioofatherosclerotic lesionareatototalaor- An Image J Graphic Analysis System was used camera (Sony). a LeicaS8APO microscope(Wetzlar, Germany)andadigital stainingandmicroscopicanalysisusing fat, followedbyORO face aortasweresplitlongitudinallytoremovetheadventitial stereomicroscope (Wetzlar, Germany). Subsequently, theen and leftsubclavianarterywerecapturedwithaLeica EZ4W tic arch withbrachiocephalic artery, leftcommoncarotidartery, M/mg feces) wasalsodetermined.These sampleswerecol- M/mg Atherosclerotic lesionanalysiswasconductedontheaortas Upon theterminationofthisgavageperiod,bloodsamples April 2021 °C beforeanalysis. −/− mice kept on a normal or awestern diet were used 14 Consideringthatthesexes ofhumanoranimal 21 Imagesoftheaor- −/− Mice mol/g protein) °C forcholes- 2021;41:00–00. DOI: 10.1161/ATVBAHA.120.314156Arterioscler ThrombVascBiol. 2021;41:00–00. DOI: −/− mice 17–19 14,20

μ Co., Ltd.). able enzymatickit(no.7020, BeijingLeadman Biochemistry μ The concentrationofcholesterol ineach elutedfraction(500 ataconstantflowrateof0.25mL/min. were elutedwithPBS and fractionedbyAKTA Healthcare).Then, they Explorer(GE Healthcare) column(GE GL a SuperoseIncrease610/300 liquid scintillationcounter. and fecal cholesterol analyses were then performed using a Plasma,liver, were anesthetizedandperfusedwithcoldPBS. hours later, medium), and 48 Memorial Institute 1640 they Plasma Lipoprotein Profiles Image Jsoftware. stained wasdeterminedusingtheNationalInstitutesofHealth andthepercentageoflesionareathatwaspositively FV3000) obtained with a laser scanning confocal microscope (Olympus, for10minutes.Imageswere 40,6-diamidino-2-phenylindole minutesat37 for 30 Abcam) orgoatanti-rabbitCy3(1:200,no.ab97075; Abcam) (1:200,no.ab150113; goat anti-mouseAlexa Fluor 488® were thenfluorescentlylabeledusingsecondaryantibodies Abcam)at4 (1:100, no.ab201340; NovusBiologicals)andCD68 no.NB400-105; (1:600, ABCA1 against cross-sections wasperformedusingprimaryantibodies Immunofluorescence doublestainingassayforaorticsinus Immunofluorescence Assay Eight-week oldmaleApoE ApoE Isolation ofPeritoneal Macrophages From 3×10 neally injected with 3H-cholesterol-labeled J774 cells (about study. macrophageRCT 48-hour The micewereintraperito- The treatmentwascontinuedforanother2daysduringthe group) bygavage,oncedailyfor14days(n=6pergroup). (control with E17241 (50mg/kgperday)or0.5%CMC-Na Eight-week oldmaleC57/BL6J micewereadministered Mice C57/BL6J In Vivo Macrophage RCT StudiesConductedon liquid chromatography (FPLC). Plasma lipoproteinprofilesweredeterminedbyfastprotein Institutes ofHealthImageJsoftware. aortal sinus cross-sections were measured with the National sinus fromeach mouse.The atheroscleroticlesionareasofthe averaged from6serialsectionsspanning≈200 hematoxylin andeosinstaining lesionsizeinaorticsinuswas lets were washed with cold PBS andthenusedforWBassay.lets werewashed withcoldPBS cells werespundownat1000 rpm for5minutes.The cellpel - bated onicefor10minutestoremove redbloodcells.Then the tion syringe.Redbloodcelllysis buffer wasaddedandincu - the peritonealmacrophageswere collectedusinga5mLinjec- and intraperitoneallyinjectedwith 5mLsalinesolution.Then sclerosis analysis,n=8pergroup). The micewereanesthetized WD,E17241-L,4 groups(ND, andE17241-H asinathero- L) pooledfromeach groupofApoE L) wasdeterminedbyEnvisionusingacommerciallyavail- 6 cellscontaining1.3×10 −/− Mice E17241 AmelioratesAtherosclerosis inApoE °C. Nucleiwerecounterstained with 14 −/− micewererandomlydividedinto 6 22,23 cpmin0.50mLRoswellPark The plasmasamples(150 °C overnight.The sections −/− micewereloadedon μm oftheaortic −/− Mice Original Research - AL 5 2C Mice value −/− 50 April 2021 , MW=318.42 2 S 2 O it has been shown 2 11 N 14 H 15 mol/L, E17241 remarkably enhances remarkably µmol/L,E17241 E17241 Ameliorates Atherosclerosis in ApoE in Atherosclerosis Ameliorates E17241 for 2 groups or Kruskal-Wallis tests for multiple U for 2 groups or Kruskal-Wallis that E17241 (Figure 1A, C that E17241 g/mol) has a significant effect on ABCA1 expression. on ABCA1 g/mol) has a significant effect in Figure 1B confirm that E17241 results illustrated The of the human ABCA1 gene stimulates the expression the EC in a dose-dependent manner and that in ABCA1p-LUC HepG2 cells is 0.28 µmol/L. HepG2 cells is in ABCA1p-LUC Simi- on ABCA1 in were observed of E17241 lar effects cells as well. In short, E17241 and HepG2 RAW264.7 the mRNA upregulates significantly lev- protein and els of ABCA1 in both hepatic (Figure 1C and 1E) and 1D and 1F). In addition, the cells (Figure RAW264.7 was also evalu- on ABCA1 expression of E17241 effect BMDMsin mice. ated C57/BL6J female and male from significantly upregu- E17241 results showed that The lates protein levels of ABCA1 in both male and female BMDMs our data show (Figure 1G and 1H). In summary, expression significantly increases ABCA1 that E17241 vivo. in vitro and ex and 2D), which indicates that the efficiency of E17241 E17241 of efficiency the that indicates which 2D), and efflux is mainly dependent on in promoting cholesterol also enhances the efflux E17241 ABCA1. Furthermore, to apo AI in both BMDMsof cholesterol male and female in a dose-dependent manner (Figure 2E and 2F). Effect of E17241 on the Cholesterol Efflux in Efflux on the Cholesterol Effect of E17241 RCT Macrophage and In Vivo Macrophages in implicated molecules protein key the of one ABCA1is It promotes the RCT process by facili- the RCT pathway. cholesterol from macrophage foam tating the efflux of cells, thereby inhibiting the progression of atherosclero- ABCA1increases expression, E17241 that Knowing sis. efflux of this molecule in regulating cholesterol the effect results illustrated in was tested in vitro and in vivo. The Figure 2A and 2B demonstrate that at concentrations of 10.0 and 2.0, 0.4, This cells. cholesterol to apo AI in the efflux of RAW264.7 down of is significantly weakened upon knocking effect the ABCA1 gene by ABCA1-specific siRNA (Figure of normality of data was tested using a normality and lognor- using a normality and of data was tested of normality for testing the test was used Brown-Forsythe The mality test. - groups were ana between variances. Differences equality of or 1-way group comparisons) Student t test (for 2 lyzed using Dunnett by followed comparisons) group multiple (for ANOVA hoc post Bonferroni by followed ANOVA or 2-way test hoc post and lognormality and equal vari- test. If data failed normality were further analyzed using the Mann- ance tests, the results Whitney by Dunnett post hoc test. P<0.05 group comparisons, followed significant. is considered statistically RESULTS In Vitro Expression on ABCA1 Effect of E17241 Using the human ABCA1 assay as a promoter-reporter high-throughput screening model, 2.2 E-10, ≤ mol/L) was 20 000 individual °C for 15 minutes. The °C for 15 minutes. The by Wayen Biotechnologies Inc Biotechnologies by Wayen 24 1.4. The complete raw data of human 1.4. The 000 rpm and 4 1.4, and ratio ≥ ≥ mol/L) 450 nm using a microplate reader. was measured at Statistical Analysis 8 was used for statistical analyses and figure Prism GraphPad as mean±SEM.plotting. All data are expressed An assumption PKC activity was assessed using the PKCkinase activity assay kit solid- based on a United Kingdom) that is Cambridge, (ab139437, phase ELISA. A specific synthetic peptide was utilized as a substrate for PKC, and a polyclonal antibody was used to recognize the absorbance of samplesphosphorylated form of this substrate. The (0, 0.01, 0.1, 1, 10 treated with varying concentrations of E17241 µ PKC Activity Assay Coimmunoprecipitation Assay incubated cells were plated in a 100 mm dish and RAW264.7 cells hours. The or Biotin for 12 with 50 μM Biotin-E17241 were washed twice with PBS and lysed on ice for 1 hour with 1 Then, the lysates mL Pierce IP Scientific). (Thermo lysis buffer were centrifuged at 12 human GST- and His-tagged full-length proteins were obtained full-length proteins and His-tagged human GST- Microarray Institutions Protein Hopkins Medical from the Johns Core (CDI Laboratories,proteomic analyses Inc). Microarray below, the procedure detailed according to were performed and the data were processed Human Microarray Proteomic Analysis Proteomic Human Microarray comprised of ≈ chips binding microarray Protein Arterioscler Thromb Vasc Biol. 2021;41:00–00. DOI: 10.1161/ATVBAHA.120.314156 (Shanghai, China). Briefly, HuProt proteome microarrays were HuProt (Shanghai, China). Briefly, for pH=7.5) (5% BSA buffer in 1×TBST, with blocking blocked then washed with 1× TBST1.5 hour at room temperature, for (10 µ buffer in blocking 5 minutes. Biotin-E17241 proteome microarrays at room blocked then incubated with the washedwere microarrays the Thereafter, hour. 1 for temperature time, and Cy5-Streptavidin for 5 minutes each 3× with TBST, Cy5- with incubation Following dilution). (1:1000 added was were again washed with TBSTStreptavidin, the microarrays they were spun dry for 2 min- time). Finally, (3×, 5 minutes each GenePix4000B. GenePix Axon an with scanned then utes The data extract to used was Instruments) (Axon software 6.0 Pro ratio signal-to-noise from the recorded microarray images. The (SNR) SNRs protein. The was calculated for each of microarrays as to referred are without Biotin–E17241 and with incubated SNR(+) and SNR(−), and ratio signifies SNR(+)/ respectively, SNR(−). mean value SNR The represents the signal of the pro- cutoff values of candidates were set to P tein. The SNR(+) proteome microarray are publicly available at the protein micro- proteome microarray are publicly available array database (PMDE245, http://www.proteinmicroarray.cn/). supernatants were collected and the protein concentrations supernatants were collected and the protein (Pierce). acid assay kit were determined using a bicinchoninic as input sample, Fifty microliters of the suspension were used whereas the remaining 950 μL were added to Streptavidin Scientific) as coimmunoprecipitation magnetic beads (Thermo interacting proteins sample, to capture biotin or biotin-E17241 by input sample was precipitated overnight. The on a rotator, centrifugation at 4000×g for 5 minutes and washed 4× with to remove nonspecifically The cell lysis buffer bound proteins. was analyzed by WB. protein complex Biotin-E17241 Xu et al Xu

Downloaded from http://ahajournals.org by on January 31, 2021 Downloaded from http://ahajournals.org by on January 31, 2021 31, January on by http://ahajournals.org from Downloaded Original Research - AL lesterol efflux (invitro, ex vivo,andinvivo),itsrole in Considering theeffects ofE17241 inpromotingcho- Male ApoE Effect ofE17241 Treatment onLipidProfiles in recovery of show thatE17241 treatmentsignificantlyincreasesthe on maleC57/BL6J mice.The obtainedresults(Figure 2K) only(Figure 2Hthrough2J). group treatedwithOx-LDL compared with the and female BMDMs, male BMDMs, total cholesterol loadingRAW264.7, contentinOx-LDL the quantitative results suggest that E17241 reduces macrophage foamcellformation(Figure 2G).Inaddition, induced alsoinhibitstheOx-LDL (2.0 and10.0µmol/L) proteinlevelsinHepG2cells(n=4;E analyses ofABCA1 levels inHepG2andRAW264.7 cells(n=4–5).E mRNA real-timepolymerasechain reactionanalysesofABCA1 Quantitative A Figure 1.EffectofE17241 onABCA1 (ATP-binding cassettetransporterA1)expression. Xu etal 6 promotes cholesterol efflux both invitro, promotes cholesterolex vivo,andinvivo. efflux in vivo.Overall,ourdatashowsthatE17241 significantly excretion, thereby enhancingmacrophage-to-feces RCT proves thatE17241 markedlyincreasesfecal 3H-tracer recovery wereobservedintheplasmaorliver. This group. Meanwhile,noappreciabledifferences in3H-tracer and female BMDMs (n=3;H and femaleBMDMs expressed as mean±SEM andcomparedwith vehicle (E17241expressed asmean±SEM at0μ , ChemicalstructureofE17241. B The in vivo macrophage experimentRCT was performed Based ontheOx-LDL-stained imagingresults,E17241 April 2021 3 H-tracer TC infeces, compared with the control −/− Mice ), respectively. E , Dependence of ABCA1 expression in ABCA1p-LUC HepG2cellsonE17241 expressioninABCA1p-LUC , DependenceofABCA1 concentration(n=3).C – H , Representative immunoblotsarepresented,andthelevelsnormalizedtoβ , Representative ), RAW264.7 cells(n=5;F 2021;41:00–00. DOI: 10.1161/ATVBAHA.120.314156Arterioscler ThrombVascBiol. 2021;41:00–00. DOI: M). The data wereanalyzedby1-wayANOVA.M). The data *P due to elevated VLDL (very-low-densitylipoprotein) and due toelevated VLDL the WDgroupshowedanincrease inTC, which ismainly group, was determinedbyFPLC. Comparedwith theND cose metabolism. indicates thatitmighthave aregulatoryeffect onglu- mouse model.Briefly, maleApoE formation was also investigated, using an atherosclerotic andinatheroscleroticlesion regulating lipidmetabolism ApoE ment significantlylowerstheserumglucoselevelin and triglyceride(Figure 3A).Inaddition,E17241 treat- has no significant effectHDL-C, onthelevels of LDL-C, decreases theconcentrationofTC inthe plasma, butit Figure 3A). Meanwhile,E17241 treatmentsubstantially group; ide levels were decreased (compared with the ND the WDgroup;however, andtriglycer- theplasmaHDL-C LDL-C levelswerefoundtobesignificantlyincreasedin (Data IAintheDataSupplement).The plasma TC and weight, comparedwiththeWDgroup no increaseinbody treatment, theE17241-L andE17241-H miceshowed as describedinthemethodssection.After 10weeksof or westerndietsweretreatedwithvehicleE17241 The lipoproteinprofileofpooledplasmas ineach group ), male bone marrow–derived macrophages (BMDMs; n=3;G ), malebonemarrow–derivedmacrophages(BMDMs; −/− mice (Data IB intheDataSupplement ), which mice(DataIB E17241 AmelioratesAtherosclerosis inApoE <0.05, **P −/− micekeptonnormal – -actin. H , Western blot <0.01, ***P C – H , Data are are , Data – D <0.001. −/− , Mice ), ), Original Research - AL 7 Mice −/− April 2021 E17241 Ameliorates Atherosclerosis in ApoE in Atherosclerosis Ameliorates E17241 H-tracer in the plasma, liver, and feces was detected (n=6). All data are shown as mean±SEM;H-tracer in the plasma, liver, (A, E–F, 3 Effect of E17241 on cholesterol efflux in vitro, ex vivo, and in vivo. efflux on cholesterol Figure 2. Effect of E17241 cells to apo AI. n=4. B, Representative efflux from RAW264.7 image of 22-NBD on cholesterol (green) labeled cholesterol A, Effect of E17241 with and 40,6-diamidino-2-phenylindole (DAPI) staining cells administered with or without apo AI and E17241 (blue) for nuclei in RAW264.7 magnification (×40). cassette transporter A1)-specific siRNA Scale bar=50 μm. C–D, Cholesterol efflux assay in with ABCA1 (ATP-binding efflux from on cholesterol cells (C), and representativemacrophage RAW264.7 immunoblots are presented (D). n=3–4. E–F, Effect of E17241 male and female bone marrow–derived macrophages (BMDMs) to apo AI. n=4. G, Representative Oil Red O–stained images are shown in Ox- cells. Scale bar=50LDL macrophage foam cell formation in RAW264.7 low-density lipoprotein)–induced μm. H–J, Effect of E17241 (oxidized on Ox-LDL cells (H), male BMDMs loading experiment in RAW264.7 I), and female BMDMs ( was intracellular cholesterol The (J), respectively. transport in male on macrophage reverse cholesterol extracted, determined, and expressed in μmol/g protein. n=3–4. K, Effect of E17241 of distribution C57BL/6J mice. The ) 2-way ANOVA analysis; ( K) Student t test; *P<0.05, **P<0.01, ***P<0.001. C) 2-way ANOVA analysis; ( and H–J) 1-way ANOVA Arterioscler Thromb Vasc Biol. 2021;41:00–00. DOI: 10.1161/ATVBAHA.120.314156 Xu et al Xu

Downloaded from http://ahajournals.org by on January 31, 2021 Downloaded from http://ahajournals.org by on January 31, 2021 31, January on by http://ahajournals.org from Downloaded Original Research - AL mice (n=12).A ###P with eithervehicleorE17241. Scalebar=100μ cage werepooledtogether).F 12 pergroup.D Xu etal Figure 3.EffectofE17241 onthelipidlevelsinplasmaandliverofwestern diet(WD) 8 levels inApoE A fecal cholesterol. laboratory diet (ND) group.n=12fortheother groups.C laboratory diet(ND) plasma cholesterol indifferenttypesoflipoproteinswasdeterminedafterseparationby fastproteinliquidchromatography (FPLC). n=10fornormal , Plasma total cholesterol (TC),, Plasmatotal LDL-C (high-densitylipoproteincholesterol), (low-densitylipoproteincholesterol), andtriglyceride(TG) HDL-C April 2021 <0.01 vs ND group;*P <0.01 vsND −/− – , Representative image of Oil Red O (ORO)–stained or hematoxylin and eosin (H&E)–stained liversofApoE orhematoxylin andeosin(H&E)–stained imageofOilRedO(ORO)–stained , Representative mice(n=12).B F . Data were statistically analyzed by 1-way ANOVA analysis, and the values are expressed as mean±SEM. #P analyzed by 1-wayANOVA werestatistically analysis,andthevalues areexpressedasmean±SEM. . Data <0.05, **P , PlasmaALT levelsinApoE urea,and creatinine(CRE) (alanineaminotransferase),AST transaminase), (aspartate , Plasmacholesterol distributioninApoE <0.01, ***P m. E <0.01 vs WD group. VLDL indicatesvery-low-densitylipoprotein. <0.01 vsWDgroup. VLDL , Fecal cholesterol (expressedasμ , TC andTG levelsintheliversofApoE 2021;41:00–00. DOI: 10.1161/ATVBAHA.120.314156Arterioscler ThrombVascBiol. 2021;41:00–00. DOI: −/− mice.Plasmawaspooledfromeach group,andthedistribution of M/mg feces)inApoE M/mg E17241 AmelioratesAtherosclerosis inApoE −/− mice(withorwithoutE17241 treatment,n=10- –induced maleApoE −/− mice. n=3 (feces from the same mice.n=3(fecesfromthesame −/− −/− mice administered miceadministered <0.05, ##P miceandon −/− <0.01, <0.01, Mice −/−

Original Research - AL

9 −/− −/− Mice −/− mice. In Data Sup- Data −/− April 2021 mice. in the Data Sup- −/− mice, whereas E17241 E17241 whereas mice, −/− in the Data Supplement). Considering 4G and 4H). Notably, E17241-H signifi- E17241-H Notably, 4H). and 4G 4I and 4J). These data demonstrate that that demonstrate data These and 4J). 4I E17241 Ameliorates Atherosclerosis in ApoE in Atherosclerosis Ameliorates E17241 ). The results show that ABCA1 Data Supplement). The uorescence staining method was used to method was used staining Immunofluorescence detect ABCA1 protein expression in the atherosclerotic the atherosclerotic in expression ABCA1protein detect of ABCA1 specificity antibodysinus. The was proved and immunohistochemistry using immunofluorescence livers from male ABCA1 on hearts and staining assay addition, WB results show that E17241 noticeably addition, WB results show that E17241 level of ABCA1 protein in the increases the expression of ApoE liver tissues and peritoneal macrophages E17241 Binding to Protein Kinase C (PKC) Binding to Protein E17241 the E17241, target of binding first the identify To syn- was (Biotin–E17241) conjugate E17241-biotin the Data Supplement). in thesized and used (Data VA results illustrated in Figure 5A demonstrate that like The ABCA1upregulates activity Biotin–E17241 E17241, to a similar degree. Based on human proteome microarray analysis (Figure 5B), 819 candidates of specific Effect of E17241 on Plasma Lipid Profiles and Profiles on Plasma Lipid Effect of E17241 in Hyperlipidemic Excretion Fecal Cholesterol Golden Hamsters diet to a western kept on were hamsters Male golden treat- E17241 establish a hyperlipidemic hamster model. levels, and LDL-C ment appreciably lowers the plasma TC the in IVA (Data group WD the with compared it does not significantly influence the plement). However, concentrations of HDL-C and triglyceride. Interestingly, golden of E17241-treated content in the feces the TC that recorded for hamsters was found to be higher than promotes indicates that E17241 the WD group, which in hyperlipidemic golden ham- excretion cholesterol fecal sters (Data IVB and urea levels were not altered AST, that the serum ALT, administration (Data IVC upon E17241 is not obvi- plement), it may be concluded that E17241 towards the investigated model. Overall, the ously toxic lipid serum the enhances E17241 that suggest results cholesterol efflux in hyperlipidemic profile and promotes golden hamsters. and WT hamsters simultaneously (Data IIIC simultaneously and WT hamsters and IIID in the lower in the WD group than positive area is significantly NDthe in ApoE male of group (Figure mice the in expression ABCA1protein increase could E17241 macrophages and in the liver of ApoE treatment significantly increases ABCA1treatment significantly protein level in lesions compared with that of the WD the atherosclerotic (Figure group cantly increases ABCA1 intensity relative to fluorescent CD68,in atherosclerotic lesions a macrophage marker, 4H), which WD (Figure of mice fed compared with that increases macrophage treatment indicates that E17241 of ApoE in the plaques ABCA1 expression

4 or 4 ) than 2 m μ

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μ 4 to 29.81±2.15×10 < µ

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4 Mice −/− - group, by decreas with the WD mice, compared −/− The hematoxylin and eosin–staining analysis show and eosin–staining analysis hematoxylin The The TC content of livers in the WD group was also also was the WD group of livers in content TC The E17241 treatment reduces the areas of these lesions E17241 from 46.06±2.60×10 dosages of E17241 (Figure 4E and 4F). dosages of E17241 or 22.35±2.36×10 As shown in Figure 4A and 4B, WD-fed mice exhibit en face aorta many more lesions (11.18%) in the whole than ND-fed the ORO- mice (2.40%; P<0.01). However, in E17241-treated stained en face lesions’ percentage and 5.13% for E17241-H) for E17241-L groups (5.76% group (11.18%, are substantially less than that in the WD treated P<0.05; Figure 4A and 4B). In addition, E17241 as compared mice display smaller lesions in aortic arch with that of WD group mice (Data IIIA and IIIB in the Data Supplement). that there are more lesions in the aortic sinuses of the WD group (46.06±2.60×10 Effect of E17241 on Atherosclerotic Lesions in Lesions on Atherosclerotic Effect of E17241 Male ApoE IDLlipoprotein)/LDL (intermediate-density (Fig- levels content in decreases the TC treatment ure 3B). E17241 ApoE ing the levels of VLDL-C (VLDL cholesterol) and IDL/ and (VLDL cholesterol) of VLDL-C ing the levels 3B). (Figure LDL-C the ND higher than that in found to be much group triglyceride lev- and increased TC (Figure 3C). The (Fig- be decreased by E17241 els in WD group may the ORO-positiveure 3C). Moreover, the lipid area in WD smaller than that in the group is much E17241 and eosin staining group (Figure 3D). Hematoxylin improv- of capable is treatment E17241 that shows the (Figure 3D). Remarkably, ing liver morphology mice of E17241-treated the feces content in TC than that of WD group mice, was found to be higher cholesterol increases fecal indicating that E17241 in WD-inducedexcretion ApoE in those of the ND group (7.26±1.12×10 In addition, it significantly decreases plasma ALT and and plasma ALT In addition, it significantly decreases urea and creatinine but does not affect levels AST that at the investigated dosage, suggests levels. This or nephrotoxic is not appreciably hepatotoxic E17241 assay shows that intra- acute toxicity (Figure 3F). The (1000 doses gastric administration of single E17241 the death of any mg/kg) for 7 days does not lead to (Data not obviously toxic is E17241 mice. Therefore, II). in the Data Supplement ND group (7.26±3.88×10 28.33±2.29×10 4C and 4D). The ORO-staining (Figure 4C and 4D). The ages of E17241 analysis also shows that there are more lesions in the aortic sinuses of the WD group (42.00±3.16×10 and 4F). E17241 treatment reduces the areas of these and 4F). E17241 lesions from 42.00±3.16×10 Arterioscler Thromb Vasc Biol. 2021;41:00–00. DOI: 10.1161/ATVBAHA.120.314156 Xu et al Xu

Downloaded from http://ahajournals.org by on January 31, 2021 Downloaded from http://ahajournals.org by on January 31, 2021 31, January on by http://ahajournals.org from Downloaded Original Research - AL A analyzed withImageJsoftware(n=10–12). C lesionswerequantitatively enfaceaorta betweenthetwoblack imagesofORO-stained linesontherulerrepresents 1mm.Thedistance digital expressed as mean±SEM. expressed asmean±SEM. and thelevelsare normalizedtoβ Figure 4.EffectofE17241 onatheroscleroticplaquelesionsinmaleApoE Xu etal 10 ORO-stained (E ORO-stained phenylindole staining (blue) for nuclei, in aortic sinus lesions. Scale bar=500 μ (blue)fornuclei,inaorticsinuslesions.Scalebar=500 phenylindole staining (ATP-binding40,6-diamidino-2- ofABCA1 (green),with immunofluorescencestaining CD68 Representative cassettetransporterA1;red)and protein levelsintheliversandperitoneal macrophagesofApoE fluorescent intensityinaorticsinus plaquesofApoE andB April 2021 , Representative en face view of Oil Red O (ORO)–stained aortas taken fromApoE taken aortas enfaceviewofOilRed O(ORO)–stained , Representative andF ) aortic sinus cross-sections taken fromApoE ) aorticsinuscross-sectionstaken ### P <0.001 vs normal laboratory diet (ND) group;*P <0.001 vsnormal laboratorydiet(ND) -actin (n=6-8).B,D,F, H,I,and J – F , Respective images and quantitative analysisofhematoxylin andeosin(H&E;C , Respectiveimagesandquantitative −/− miceadministeredvehicleorE17241 (n=10-12).I 2021;41:00–00. DOI: 10.1161/ATVBAHA.120.314156Arterioscler ThrombVascBiol. 2021;41:00–00. DOI: −/− micetreatedwithvehicleorE17241. immunoblotsareshown, Representative , Data were statistically analyzed by1-wayANOVA, werestatistically andthe values are , Data −/− miceadministeredvehicleorE17241. μ n=10-12.Scalebar=500 m. <0.05, **P H , ABCA1 fluorescent intensity and ABCA1 relative to CD68 CD68 relativeto fluorescentintensity andABCA1 , ABCA1 −/− mice. <0.01, ***P −/− E17241 AmelioratesAtherosclerosis inApoE miceadministeredwithvehicleorE17241. The <0.001 vswestern diet(WD)group. – J , Western blot analysisoftheABCA1 andD ) or ) or −/− m. Mice G , Original Research - AL β μ, 11 / Mice −/− ζ, PKC April 2021 δ, PKC (PPARs), LXRα (PPARs), δ / β, PKC γ / mice or golden hamsters. mice or golden hamsters. −/− mice. It also improves dyslipid- −/− mice and male hyperlipidemic golden golden male hyperlipidemic mice and −/− E17241 Ameliorates Atherosclerosis in ApoE in Atherosclerosis Ameliorates E17241 -NR pathway. -mediated E17241-induced ABCA1 expression ABCA1 expression E17241-induced -mediated ζ ζ -NR pathway. The PKC family is composed of >10 The ζ-NR pathway. To elucidate the mechanism of E17241 activity, the activity, of E17241 elucidate the mechanism To hamsters. Importantly, E17241 effectively reduces en reduces en effectively E17241 hamsters. Importantly, lesions in WD-fedface and aortic sinus atherosclerotic atherosclerotic ApoE emia in the investigated animal models. Based on the on the Based animal models. investigated emia in the presents no E17241 assay, results of the acute toxicity admin- when intragastrically effects obvious in vivo toxic not does it Moreover, mg/kg. 1000 of dose at a istrated plasma the in levels urea and AST, ALT, the increase ApoE of E17241-treated (LXRs), and RXR indicates that at (Figure 6E). This upregulates E17241 the investigated concentrations, by activating NR.ABCA1 expression PKC partici- significantly as evidenced by the pates in this effect, activation upon the addi- reduced E17241-induced tion of Go6983 we may conclude (Figure 6E). Thus, via the regulates ABCA1 expression that E17241 PKC DISCUSSION ABCA1 promotes As a key transporter protein in RCT, thus, protects against atheroscle- efflux and cholesterol signifi- it was shown that E17241 rosis. In this study, ABCA1 of expression increases the macrophage cantly of E17241 effect both vivo, and in vivo. This in vitro, ex metabolism,allows it to regulate cholesterol as evi- cholesterol in denced by the enhanced efflux of both and BMDMs. macrophages RAW264.7 Our results also in vivo mac- treatment promotes indicate that E17241 levels in cholesterol rophage RCT and increases fecal bothApoE male To examine this hypothesis, the role of NR this hypothesis, examine in the To PKC results show obtained The was investigated. effect of the DR4that the mutation in the ABCA1 element in of E17241 the effect region eliminates promoter ABCA1triggering (Figure 6D). activity transcriptional indeed, are, receptors nuclear indicates that This of ABCA1 levels the expression involved in increasing results of pBIND-NR-LBD/ Based on the by E17241. acti- clearly E17241 assay, reporter chimera GAL4 α vates NRs, PPAR as such Overall, our data suggest that E17241 is capable of is capable of Overall, our data suggest that E17241 and regulating the metabolism in RCT, of cholesterol thus, it has great potential for treating atherosclerosis by upregulating ABCA1. direct binding protein of this small molecule was identi- combina- in microarray proteomic human the using fied obtained tion with immunoprecipitation/WB analysis. The results indicate that PKCζ is a major target of E17241- PKCa inhibitor of use The ABCA1induced expression. cells further demonstrates PKCζ in RAW264.7 blocks via the promotes ABCA1 expression that E17241 PKC members, including PKCα, PKC

26,27 5G) or a 5G) or To identify To mice (Fig- −/− 27–29 5F presents images of images presents 5F δ, and PKCζ, are implicated δ, and PKCζ, among others. Figure is the target protein of E17241 is the target protein of E17241 25,26 β, PKC , also named PRKCZ),ζ, also named SIRT1 some and 5G and 5H, the ABCA1 transcription upregu- ABCA1transcription the and 5H, 5G ζ and SIRT1 taken from the same location blocks siRNA, the influence of E17241 in increasing the ζ siRNA, the influence of E17241 ζ (PKC - ABCA1 in regulating expres of E17241 effect The Knowing that PKCζ Knowing PKC in the microarray. sion via PKCζ or SIRT1 As shown was assessed further. Figure in in the is significantly decreased lating activity of E17241 of a SIRT1presence inhibitor EX527 (Figure broad-spectrum PKC inhibitor Go6983 (Figure 5H and the addi- ). Moreover, in the Data Supplement Data VC in aug- tion of Go6983 of E17241 eliminates the effect the menting ABCA1 protein levels (Figure 5J). However, use of the SIRT1 specific siRNA the pro- to knockdown E17241-induced tein has no obvious influence on the increase in ABCA1Data (Figure 5I and Data VD in the 5K in Figure the results presented Supplement). Finally, increases PKCa activity in demonstrate that E17241 it regulates ABCA1 Therefore, dose-dependent manner. PKC, through not SIRT1.protein expression - ure 6C). Overall, these results confirm that the mecha is triggered by nism of ABCA1 upregulated by E17241 the activation of the PKCζ pathway. and that ABCA1 expression is regulated by NR,by regulated is ABCA1expression that and is it suggested that PKC might be related to NR activity. Effect of E17241 in Regulating ABCA1 Protein Protein ABCA1 in Regulating Effect of E17241 via the PKCζ-NRExpression Pathway PKC members, includ- family is composed of various The ing PKCα, PKC in the regulation of ABCA1 expression. According to previous publications, some of these mem- PKCα, PKCbers, namely, other genes are found to be included in lipid or glucose included in lipid or are found to be other genes metabolism–related as insulin signaling pathways, such and response to leptin, Signaling pathway, FoxO pathway, the Data Supplement). Among these so on (Data VB in candidates, PKCζ and SIRT1 potential for showed great since both of ABCA1 by E17241, induced upregulation of with the regulatory mechanism them are associated ABCA1expression. E17241-binding proteins were identified, as shown identified, as shown proteins were E17241-binding 5C. KEGGin Figure GO pathway and pro- biological pro- the results of human used to analyze cess were kinase Protein 5D and 5E). (Figure teome microarrays C the PKC member that E17241 interacts with, immuno- the PKC member that E17241 obtained The performed. was analysis precipitation/WB the biotin group, results demonstrate that compared with interacts with PKCζ, but not with PKCα biotin-E17241 down by PKCζ is knocked or PKCδ (Figure 6A). When PKC level of ABCA1 protein is markedly reduced (Figure 6B). obviously increases the phosphor- E17241 Furthermore, the level of ylation of PKCζ (P-PKCζ), without affecting total PKCζ protein in the livers of ApoE Arterioscler Thromb Vasc Biol. 2021;41:00–00. DOI: 10.1161/ATVBAHA.120.314156 Xu et al Xu

Downloaded from http://ahajournals.org by on January 31, 2021 Downloaded from http://ahajournals.org by on January 31, 2021 31, January on by http://ahajournals.org from Downloaded Original Research - AL ANOVA meansnosignificance. analysis;*P<0.05,*****0.001,NS (G,HandI)2-wayANOVA analysis;(JandK)1-way areexpressed asmean±SEM; activity(3independentexperiments).Alldata on PKC onE17241-induced (10µmol/L) expression inRAW264.7 cells.n=3-4. H, EffectofE17241 ABCA1 inhibitorGo6983 the effectofPKC immunoblotsregarding immunoblotsareshown,andthelevels arenormalizedtoβ-actin.n=3.J,Representative experiments). Representative (3independent siRNA proteinexpression levelsinRAW264.7 cellstreated withorwithoutE17241ABCA1 orSIRT1 andscrambledsiRNA (sirtuin1)inhibitiononE17241-induced transcriptionalactivity. (proteinkinaseC)andSIRT1 ≥ 1.4.GandH,EffectsofPKC ABCA1 n=4.I , Xu etal proteins intheproteomemicroarray. The cutoffofthecandidateswassetatPvalue ≤2.2×10 biologicalprocessanalysisoftheresultshumanproteomemicroarrays.F, pathway andGO were probedwithBiotin-E17241, C,The bindingproteinsfoundbyBiotinorBiotin-E17241. followedbyCy5-labeledstreptavidin. D, KEGG HepG2cells.B,Schematic oftheprocedureusedtodetectBiotin-E17241ABCA1p-LUC binding proteins.Humanproteomemicroarrays (ATP-bindingA, DependenceofABCA1 cassettetransporterA1)expressionontheconcentrationsofBiotin,E17241, orBiotin-E17241 in Figure 5.IdentificationofE17241 bindingproteinsbyhumanproteome microarrays. 12 April 2021 2021;41:00–00. DOI: 10.1161/ATVBAHA.120.314156Arterioscler ThrombVascBiol. 2021;41:00–00. DOI: E17241 AmelioratesAtherosclerosis inApoE −10 Representative imagesofE17241-interacting Representative , signal-to-noise ratio (SNR) (+)≥1.4,andratio , signal-to-noiseratio(SNR) −/− Mice Original Research - AL 13 It 30 Mice −/− Finally, Finally, 28 April 2021 E17241 Ameliorates Atherosclerosis in ApoE in Atherosclerosis Ameliorates E17241 apo AI activates PKCα signaling to phosphorylate and by TLR2 (Toll-like receptor 2) in RAW264.7 cells. receptor 2) in RAW264.7 (Toll-like by TLR2 has also been shown that LDL induces the expression expression LDLthe that induces shown been also has PKCABCA1the of activating ζ-Sp1-LXR/RXR by signaling cascade in mouse macrophage cells. Mean- 27 mice treated with vehicle or E17241. Representative immunoblots are shown, and mice treated with vehicle or E17241. −/− ζ) and PKC livers of ApoE protein levels in the controls the ABCA1 expression triggered ABCA1PKCthe expression while, controls η . Previously, it has been shown that some of and PKCη. Previously, - these members are capable of regulating ABCA1 expres PKCδ and PKCζ mediate the ABCA1 sion. In particular, induced by 22-(R)-HCexpression and 9CRA. ζ (protein cassette transporter A1) protein expression via the PKC (ATP-binding in regulating ABCA1 Figure 6. Effect of E17241 kinase C zeta)–NR (nuclear receptor) pathway. blot (WB; 5 independent experiments). cells by coimmunoprecipitation/Western binding proteins in RAW264.7 A, Identification of E17241 B, ABCA1 protein IP. input; lane 3: biotin immunoprecipitation (IP); lane 4: Biotin-E17241 Lane 1: biotin input; lane 2: Biotin-E17241 and scrambled siRNA or PKCζ siRNA cells treated with or without E17241, (3 independent experiments). expression level in RAW264.7 blot analysis of the P-PKCζ (phosphorylated Representative immunoblots are shown, and the levels are normalized to β-actin. C, Western PKC 0.001, E17241 are expressed as mean±SEM***P<0.001, E17241 the levels are normalized to β-actin. Values and statistically analyzed by 1-way ANOVA. ABCA1treatment vs western diet group (n=6). D, Effect of DR4 element mutation in the ABCA1 promoter region on E17241-induced transcriptional activity in HepG2 cells. n=4. B and D, Data and the values are expressed as were statistically analyzed by 2-way ANOVA, NRmean±SEM. activation in HepG2 cells. n=3. The *P<0.05. **P<0.01, ***P<0.001. E, Effect of PKC inhibition on E17241-induced at the same vs E17241+Go6983 data the values are expressed as mean±SEM. and were statistically analyzed by 2-way ANOVA, E17241 ***P<0.001. NS indicates not significant. concentration of E17241, Arterioscler Thromb Vasc Biol. 2021;41:00–00. DOI: 10.1161/ATVBAHA.120.314156 Xu et al Xu

Downloaded from http://ahajournals.org by on January 31, 2021 Downloaded from http://ahajournals.org by on January 31, 2021 31, January on by http://ahajournals.org from Downloaded Original Research - AL stream genes.For example, regulatestheSR-BI, LXR ζ through PKC treatment maybedevelopedinthefuture. studies, anewtherapeuticapproach toatherosclerosis PKC are neededtoestablishadefinite correlationbetween lin secretion. tolerance ismarkedlyimpairedduetodefective insu- β fibers ofhigh-fatdiet–fed mice. lism andtheglucoseuptakeinadultskeletalmuscle the - cholesterol of can ABCA1 disturb both metabo and transfer tothebile. cholesterol uptakeintheliver leading toenhancedHDL stabilize ABCA1. Xu etal 14 transport, lating insulin secretion and insulin-stimulated glucose PKC ApoE decreases theserumglucoselevelinatherosclerotic graphic abstract. and themolecularmechanism diagramisshowninthe expressionulates ABCA1 ζ viathePKC obtained results,itmaybeconcludedthatE17241 reg- ance. specifically, suffer fromliver-impairedglucoseintoler- was demonstratedthatmicelacking Abca1,globallyor atherosclerosis andtype2diabetes.Inotherstudies,it lesterol imbalanceisrelatedtothedevelopmentof and markedly reduces plasma HDL levels. and markedlyreducesplasmaHDL clearance in thetransgenicmiceacceleratesHDL-C uptake intheliver, andhepaticoverexpression ofSR-BI receptor playsakeyroleinmediatingselectiveHDL-C asthefunctionalHDL in theDataSupplement).SR-BI analogous-1) invitroandvivo(DataVIAthroughVID II andLIMP CD36, (humanhomologofSR-BI, CLA-1 E17241 canalsoincreasetheexpression ofSR-BI/ GLUT4 (glucosetransporter4). GLUT4 pathway. Together, ourdata indicatethatE17241 has expressionABCA1 ζ-NR-ABCA1 proceedsviathePKC to determinethatthemechanism of E17241-induced binding proteinsofthissmall molecule,wewereable olism in vitro, ex vivo, and invivo. By identifying the direct macrophage cholesterol efflux,andregulate lipidmetab- can significantly increase ABCA1 expression, promote byE17241.metabolism exact mechanism of co-regulation of lipid and glucose Further studiesareneededtoassessthemetabolism. E17241 mighthaveaneffect inregulatingtheglucose and glucose homeostasis, and it is speculated that cholesterol playsacriticalroleinboth studies, ABCA1 treatment in WD-induced ApoE treatment inWD-induced explain afterE17241 alack ofanincrease inHDL-C -cells, cholesterol homeostasis is altered, andglucose Our resultsshowthatE17241 activatesNRs In additiontoitseffect oncholesterol efflux,E17241 In conclusion,wehavedemonstratedthatE17241 and human metabolic diseases. Based on these ζ and human metabolic ζ April 2021 35,36 −/− pathwaysthatplayanimportantroleinregu- mice. Previously, ithasbeenshownthatcho- InthecaseofspecificAbca1inactivationin 37–39 35 aswellinpromotingthetranslationof This orPI3K/ istiedtothePI3K/AKT that might regulate some other down- 29 Despitethesereports,morestudies 32 Inthisstudy, wefoundthat 40,41 −/− mice.Basedonthe 42 Reducingthelevel Basedonthese 33,34 -NR pathway,-NR This might 2021;41:00–00. DOI: 10.1161/ATVBAHA.120.314156Arterioscler ThrombVascBiol. 2021;41:00–00. DOI: 31

tion Project (H2019205318),andBeijingNaturalScienceFoundation (7162129). 2018ZX09735001-002-001), Beijing-Tianjin-Hebei BasicResearch Coopera- ogy ofChina(2018ZX09711001-003-006, 2018ZX09711001-007-002, and Science andTechnology MajorProject oftheMinistryScienceandTechnol- (81573482, 81973328, 81473214 81621064, theNational and81402929), I2M-1-011, 2016-I2M-2-002),NationalNaturalScienceFoundation ofChina InnovationFund CAMS forMedicalSciences(2016- Funds (2019-RC-HL-009), This work was supported by the grants from FundamentalCAMS Research Sources ofFunding ration ofthisarticle. We thankLetPub (www.letpub.com) for itslinguistic assistance during the prepa- Acknowledgments Health Center, ShenzhenUniversityHealthScienceCenter, China. Shenzhen, PR Zhejiang Province, China. Beijing,China(X.H.,N.L.,J.-D.J.). CAMS&PUMC, tive SubstanceandFunction ofNaturalMedicines,InstituteMateriaMedica, J.L., X.Z.,J.H.,L.W., Y.D., Y.X., ofBioac- J.-D.J.,B.H.,S.S.).StateKey Laboratory China (Y.X., C.L.,X.H.,X.Jia,Y.L., C.L.,N.L.,L.L.,P.L., X.Jiang,W.W., X.W., Y.L., M.C., Beijing, of MedicalSciences&Peking UnionMedicalCollege(CAMS&PUMC), Microbial Drug Screening, Institute of Medicinal Biotechnology, Chinese Academy ofBiotechnology Key ofAntibiotics,NationalCenterforNew Laboratory NHC Affiliations Received July23,2019;acceptedDecember27, 2020. ARTICLE INFORMATION date forthetreatmentofatherosclerosis. great potentialasanewhigh-qualityleadordrugcandi- REFERENCES None. Disclosures 9. 8. 6. 5. 4. 3. 2. 1. 7. Current addressforX.Jia:ShenzhenKangning Hospital&ShenzhenMental Current addressforL.Liu:ZhejiangChineseMedicalUniversity, Hangzhou, 1551–1559. doi:10.1161/CIRCULATIONAHA.113.005015 macrophage-like cells inhumanatherosclerosis.Circulation . 2014;129: tribution ofintimalsmoothmusclecells tocholesterol accumulationand AbrahamT,Allahverdian S,ChehroudiAC, McManusBM, Francis GA.Con- 10.1073/pnas.092327399 doi: recruitment intotissues.ProcNatlAcadSciUSA . 2002;99:6298–6303. controlssusceptibilitytoatherosclerosisandmacrophage kocyte ABCA1 Van AmersfoortES,Christiansen-Weber TA, Fung-Leung WP, etal.Leu- Kaminskivan Eck WE,OrsóE,RotheG,Twisk M,BosIS, J,Böttcher A, doi:10.1074/jbc.M00340720028640. cholesterol effluxfromvariouscelllines.JBiolChem.2000;275:28634– TheFrancone correlationofATP-binding OL. levelswith cassette1mRNA RoyerLJ,McNeishJ, StoudtG,HoppeKL, Bortnick AE,RothblatGH, doi:10.1038/72869 Abc1-deficient mice.NatGenet.2000;24:192–196. from golgi to plasma membrane is defective in tangier disease patients and Götz A,ChambenoitO,Diederich W, LangmannT, etal.Transport oflipids Orsó E, Broccardo C, Kaminski WE, Böttcher A, Liebisch G, Drobnik W, doi:10.1038/11921 1999;22:352–355. tions inthegeneencodingATP-binding cassettetransporter1.NatGenet. Duverger N,Denèfle Brewer HB, P, et al. Tangier diseaseiscausedbymuta- DeleuzeJF,Rust S,RosierM,Funke H,RealJ,AmouraZ,PietteJC, doi:10.1191/1358863x05vm593ra Vasc Med.2005;10:109–119. andatherosclerosis. ABCA1 S,Albrecht C,DaviesAH,GibbsRG. Soumian 2224. doi:10.1172/JCI32057 rophage reversecholesterol transportinvivo.JClin Invest . 2007;117:2216– promotemac- butnotSR-BI, Rader DJ. andABCG1, MacrophageABCA1 Wang Ranalletta M, Fuki IV, X, Collins HL, Billheimer JT, Tall Rothblat GH, AR, doi:10.1111/j.1463-1326.2008.01012.x Obes Metab. 2009;11:534–543. Tan KC. Reversecholesterol transportintype2diabetesmellitus.Diabetes 10.1161/CIRCULATIONAHA.104.475715 doi: the regressionofatherosclerosis?Circulation.2006;113:2548–2555. Cuchel M, Rader DJ. Macrophage reverse cholesterol transport: key to E17241 AmelioratesAtherosclerosis inApoE −/− Mice

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Arterioscler Thromb Vasc Biol. 2021;41:00–00. DOI: 10.1161/ATVBAHA.120.314156 Xu et al Xu

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