Microrna-144 Silencing Protects Against Atherosclerosis in Male, but Not Female Mice

Arteriosclerosis, Thrombosis, and Vascular Biology

TRANSLATIONAL SCIENCES MicroRNA-144 Silencing Protects Against Atherosclerosis in Male, but Not Female Mice

Joan Cheng,* Angela Cheng,* Bethan L. Clifford, Xiaohui Wu, Ulf Hedin, Lars Maegdefessel, Nathalie Pamir, Tamer Sallam, Elizabeth J. Tarling,† Thomas Q. de Aguiar Vallim†

OBJECTIVE: Atherosclerosis is a leading cause of death in developed countries. MicroRNAs act as fine-tuners of gene expression and have been shown to have important roles in the pathophysiology and progression of atherosclerosis. We, and others, previously demonstrated that microRNA-144 (miR-144) functions to post-transcriptionally regulate ABCA1 (ATP binding cassette transporter A1) and plasma HDL (high-density lipoprotein) cholesterol levels. Here, we explore how miR- 144 inhibition may protect against atherosclerosis.

APPROACH AND RESULTS: We demonstrate that miR-144 silencing reduced atherosclerosis in male, but not female low-density lipoprotein receptor null (Ldlr−/−) mice. MiR-144 antagonism increased circulating HDL cholesterol levels, remodeled the HDL particle, and enhanced reverse cholesterol transport. Notably, the effects on HDL and reverse cholesterol transport were more pronounced in male mice suggesting sex-specific differences may contribute to the effects of silencing miR-144 on atherosclerosis. As a molecular mechanism, we identify the oxysterol metabolizing enzyme CYP7B1 (cytochrome P450 enzyme 7B1) as a miR-144 regulated gene in male, but not female mice. Consistent with miR-144-dependent changes in CYP7B1 activity, we show decreased levels of 27-hydroxycholesterol, a known proatherogenic sterol and the endogenous substrate for CYP7B1 in male, but not female mice.

CONCLUSIONS: Our data demonstrate silencing miR-144 has sex-specific effects and that treatment with antisense Downloaded from http://ahajournals.org by on October 19, 2020 oligonucleotides to target miR-144 might result in enhancements in reverse cholesterol transport and oxysterol metabolism in patients with cardiovascular disease.

VISUAL OVERVIEW: An online visual overview is available for this article.

Key Words: antisense oligonucleotides ◼ atherosclerosis ◼ cardiovascular disease ◼ cholesterol ◼ microRNA

icroRNAs (miRs) are small noncoding RNAs, translation.1,2 Several miRs have been described to regu- ≈20 to 22 nucleotides in length, that target sets late lipid metabolism and cholesterol homeostasis.3–9 Mof genes in complimentary pathways to regu- We and others, previously demonstrated that miR-144 late gene expression. Generally, miRNAs regulate gene functions to posttranscriptionally target ABCA1 (ATP-bind- expression posttranscriptionally by base-pair binding to ing cassette transporter A1). We identified miR-144 as an target mRNAs. Typically, when miRNAs bind in near-per- FXR (farnesoid X receptor)-induced miR in hepatocytes that fect complementarity to their target mRNAs, they trigger reduced ABCA1 protein levels and activity, reduced plasma mRNA degradation by Ago2 (argonaute 2) cleavage.1,2 HDL (high-density lipoprotein) cholesterol levels and in vitro, When miRNAs bind with imperfect or partial comple- decreased cholesterol efflux from primary mouse hepato- mentarity they impair mRNA stability and inhibit protein cytes to Apo-AI (apolipoprotein A1).5 In a parallel submission,

Correspondence to: Elizabeth J. Tarling, PhD, UCLA Division of Cardiology, Department of Medicine, A2-237 CHS, 650 Charles E Young Dr S, Los Angeles, CA 90095, Email [email protected]; or Thomas Q. de Aguiar Vallim, PhD, Departments of Biological Chemistry and Medicine, A2-237 CHS, UCLA Division of Cardiology, 650 Charles E Young Dr S, Los Angeles, CA 90095, Email [email protected] *These authors contributed equally to this article as first authors. †These authors contributed equally to this article as senior authors. The online-only Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/ATVBAHA.119.313633. For Sources of Funding and Disclosures, see pages 423 and 424. © 2019 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at www.ahajournals.org/journal/atvb

412 February 2020 Arterioscler Thromb Vasc Biol. 2020;40:412–425. DOI: 10.1161/ATVBAHA.119.313633 TRANSLATIONAL SCIENCES - AL

/− 15 − 413 ) mice. −/− February 2020 February mice (The Jackson mice (The /− − Highlights Silencing miR-144 Protects Against Atherosclerosis Against Protects miR-144 Silencing - cause of death in devel is a leading Atherosclerosis oped countries. MicroRNAs expression gene are fine-tuners of roles in pathophysiology of shown to have important atherosclerosis. that silencing microRNA-144 demonstrate We mice. in male, but not female, reduces atherosclerosis metabolizingoxysterol identify the enzyme We p450 enzyme 7b1) as a (cytochrome CYP7B1 microRNA- in male, but 144-regulated target gene mice. not female, that targeting microRNA-144Our data demonstrate in reverse cholesterol might result in enhancements metabolismtransport and oxysterol with in patients cardiovascular disease. In the current study, we demonstrate that silencing miR- we demonstrate that silencing In the current study, • • • • • Laboratory) maintained on a 12 were on a C57BL6/J background hour/12 hour light/dark cycle with unlimited access to food and All atherosclerosis studies adhered to the American Heart water. atherosclerosis studies. Association guidelines for experimental Antagonism of miR-144 increased circulating plasmacirculating increased miR-144 of Antagonism HDL the HDL levels, remodeled cholesterol particle, and treat- anti–miR-144 of effects the Notably, RCT. enhanced ment on HDL and RCT were more pronounced in male to may contribute differences suggesting sex-specific mice miR-144 on atherosclerosis devel- of silencing the effects miR-144 inducedopment. Indeed, we show that silencing metabolizing hepatic levels of the oxysterol enzyme CYP7B1 7B1) in male mice. One conse- P450 enzyme (cytochrome - 27-hydroxycholes quence was a decrease in the levels of and a known terol, the endogenous substrate for CYP7B1 with male mice,proatherogenic sterol. In contrast, compared endogenous hepatic Cyp7b1 mRNA4-fold lower levels are by silencing mice and these levels are unaffected in female data demonstrate that there are different miR-144. These mice and of silencing miR-144 in male and female effects that silencing miR-144 may be a possible therapeutic inter- vention for cardiovascular disease. For chronic miR-144 silencing atherosclerosis studies, male Ldlr miR-144 silencing atherosclerosis studies, chronic For diet containing 21% fat and mice were maintained on a Western Mice, Diets, and Treatments 12-week-old Ldlr Male and female, METHODS that support the findings of this study are available data The from the corresponding authors on reasonable request. performed in vivo in the context of atherosclerosis. We, of atherosclerosis. We, performed in vivo in the context silencing miR-144therefore, set out to determine whether in vivo would lead to a reduction in atherosclerosis. notbut in male, area reduced atherosclerotic lesion 144 receptor null (Ldlr low-density lipoprotein female

11–13 It is well 10 used an agomir to over- 14 14 (apolipoprotein e null) mice was

−/− ATP binding cassette transporter A1 cassette transporter binding ATP transporter G1 binding cassette ATP acetylated LDL Surgery Trial Asymptomatic Carotid Biobank of Karolisnka Endarterectomies enzyme 7B1 P450 cytochrome high-density lipoprotein receptor low-density lipoprotein major urinary protein platelet factor transport reverse cholesterol serum amyloid A -HDL, of HDL, the precursor and in the identified miR-144 as an LXR identified miR-144 as an (liver X 4

In a previous study Hu et al 13 Nonstandard Abbreviation and Acronyms Nonstandard Abbreviation Based on these prior observations, we speculated that 2’F/MOE 2’fluoro/methoxyethyl ABCA1 ABCG1 AcLDL ACST APO apolipoprotein BiKE CYP7B1 HDL Ldlr microRNA-144 miR-144 MUP PF RCT SAA express miR-144 and demonstrated that increased levels miR-144 and express bothdecreased miR-144 of macrophage ABCA1 levels efflux in vitro. Consistent with these results,and cholesterol agomir treatment of Apoe elevated levels of miR-144 in macrophage foam cells due to increased concentrations of LXR agonists might limit mac- ABCA1 by restricting capacity efflux cholesterol rophage of also hypothesized that long-term silencing We activity. miR-144 would result in an increase in hepatic expression of ABCA1 protein, increased plasma HDL lev- cholesterol transport (RCT). els and in enhanced reverse cholesterol - might acceler changes further predicted that such We atherosclerotic lesions ate the regression of preexisting no and attenuate the development of disease. Importantly, studies using antagomirs to silence miR-144 have been Ramirez et al appreciated that ABCA1 plays a key role both the hepatic in generation of pre-β sterol-loaded macrophages. cholesterol from efflux of shown to result in a decrease in both plasma HDL levels and transport and, in addition, acceleratedreverse cholesterol progression of atherosclerosis. Biol. 2020;40:412–425. DOI: 10.1161/ATVBAHA.119.313633 Arterioscler Thromb Vasc receptor)-induced miR in macrophages and demonstratedreceptor)-induced miR in macrophages silencing of miR-144 decreased or or that overexpression to Apo-AI in vitro respectively, efflux, increased cholesterol macrophage cells. in Huh7 hepatoma and J774 Macrophages constitutively express scavenger receptorsMacrophages constitutively express cholesterol- of oxidized/modified uptake facilitate the that LDL efflux of cellu- (low-density lipoprotein). Subsequent rich the formation of cholesterol-loaded to prevent lar cholesterol 2 sterol transport- foam cell formation is dependent on the binding cassette transporter ers ABCA1 and ABCG1 (ATP G1). Cheng et al et Cheng

Downloaded from http://ahajournals.org by on October 19, 2020 Downloaded from http://ahajournals.org by on October 19, 2020 19, October on by http://ahajournals.org from Downloaded Translational Sciences - AL Ldlr a weekthereafterfor4weeks.Atherosclerosis studiesinfemale thefirstweek,spaced2daysapart,andthen once volume ofPBS Mice received2injectionsof10mg/kganti-miRoranequivalent miR oligonucleotide,or2′ n=19–21mice/group),2′ no treatment(PBS; Teklad) andfurtherrandomizedinto3treatmentgroups;vehicle/ modified mouse/rat diet catalog number 7013, Harlan (NIH31 dietfor4weeks groups ofmicewereswitched to normallaboratory euthanized immediately, asBaseline(n=23).The remaining3 diet-feeding, micewererandomized into4groups.Onegroupwas for16weeks.After 16weeksofWesterndiet (asdescribedabove) data-development acquisition,aspreviouslydescribed. trap mass spectrometer (Fusion, Thermo Electron Corp.) using andpositiveionmassspectra wereacquiredwithanorbi- ESI, digestswereintroducedintothegasphaseby kV setting.HDL (Michrom BioResources,Inc)ata10mL/minflowrateand1.4 under liquidnitrogenandstoredat−80C. medium andfrozenimmediately, andlivertissuewassnap-frozen tion. Aorticrootswereembeddedinoptimalcuttingtemperature For all studies, mice were fasted overnight before euthaniza- University ofCalifornia,SanDiegoLipidomicsCore. 414 and 2mmofthoracic aorta)werehomogenized andtotalRNA Liver tissueandaorta (comprisingthe ascending aorta, aortic arch, Chain Reaction Polymerase Isolation andQuantitative RNA viously described. spectradetectedforaproteinaspre- total numberofMS/MS Proteins werequantifiedusingpeptidespectramatches: the against a mouse UniProt database as previously described. spectrawerematched usingtheCometsearch engine MS/MS andQuantification Protein Identification described. 0.15×50 mm;Michrom BioResources,Inc),aspreviously a C18trapcolumn(Paradigm PlatinumPeptide Nanotrap, (1µgprotein)wereinjectedonto and trypticdigestsofHDL (10µgprotein)wasisolatedaspreviouslydescribed, HDL Mass Spectrometry) Analysis Electron SprayIonization-MassSpectrometry/ (Liquid Chromatography- LC-ESI-MS/MS ously described. cholesterol weredeterminedasprevi- Plasma totalandHDL Plasma LipidAnalysis sclerosis studies,maleLdlr (Regulus Therapeutics; ASO antisenseoligonucleotides modified) phosphorothioatebackbone kg anti–miR-1442′ or 10 mg/ mice/group), or an equivalent volume of vehicle/PBS, were injectedintraperitoneallywith10mg/kgcontrol(n=8–11 0.2% cholesterol (Research DietsD12079B) for16weeks.Mice Cheng etal HDL particlesfromindividualmice. HDL spectra match to compare the relative protein composition of each sample,wereusedtocalculateanormalizedpeptide normalized tototalpeptidespectramatches forpeptidesfrom −/− mice were performed as described above as in male mice. asinmalemice. micewereperformedasdescribedabove February 2020 18 ESI was performed usingaCaptiveSpray source ESI 16 Plasmasterolanalysiswasperformedatthe 18 Peptide spectra matches for each protein, fluoro/methoxyethyl-modified (2′ F/MOE anti–miR-144 oligonucleotide. anti–miR-144oligonucleotide. F/MOE −/− ). For acutemiR-144silencingathero- mice were maintainedonaWestern F/MOE controlanti- F/MOE Arterioscler ThrombVasc 10.1161/ATVBAHA.119.313633 DOI: Biol. 2020;40:412–425. 17 18 F/MOE- 18 18

µCi/mL were incubated with 37.5 and5 (acetylated LDL) µg/mLacLDL radioactivity injectedatbaseline. plasma, liver, andfeces wascalculatedasapercentageoftotal to radioactivity determinedbyliquid scintillationcounting.RCT isopropanol (3:2)anddriedovernight.Lipidsweresolubilized rifice, liver sampleswere collectedand incubated with hexane/ an aliquotwasremovedforliquidscintillationcounting.At sac- afterwhich injection andhomogenizedovernightin50%NaOH, time point.Feces hoursaftermacrophage werecollectedfor48 was usedforliquidscintillationcountingimmediatelyateach hoursateuthanization.Analiquotofplasma tion, andafter48 orbital bleedingat6hoursand24aftermacrophageinjec- to determinebaselineradioactivity. Bloodwasobtainedbyretro- an aliquotofcellswascountedusingaliquidscintillationcounter Before injection, anti–miR-144 for 4 weeks, as described above. Western controlanti-miRor dietandtreatedwitheitherPBS, injected intraperitoneallyintoindividuallyhousedmicefed a Forest University), (1:1000;agiftfromDrJohnParks, toABCA1 Wakeantibodies to nitrocellulose.Membraneswereincubatedovernightwith gel(BioRad)andtransferred was separatedonanSDS-PAGE [phenylmethylsulfonylfluoride; Sigma]).Protein (25μg) PMSF leupeptin [Sigma];2µg/mLaprotinin200µmol/L tablet [Roche]; 25µg/mLcalpaininhibitor[Sigma];10 EDTA-free protease inhibitor cocktail mix (1 Complete MINI radioimmunoprecipitation assaybuffer, withproteaseinhibitor Liver tissue was homogenized, and protein was extracted using Western Blotting the procedure outlined in the American Heart Association the procedure outlined in the American Heart Association Atherosclerotic lesionanalysiswasperformed accordingto Atherosclerotic Lesion Analysis mice aspreviouslydescribed. Resident peritonealmacrophageswerepreparedfromC57BL/6J In Vivo RCT Assay normalized to36b4mRNA. exon-exon andareavailableonrequest.Resultswere boundaries pooledfromallsamples.Primers weredesignedacross cDNA mined froma3-logserialdilutionsstandardcurvemade using anefficiencycorrectedmethod,andwasdeter- mastermix (Roche). Relativegene expression wasdetermined titative polymerase chain reaction machine and Lightcycler480 real-timequan- expression determinedusing a Lightcycler480 andgene wasreversetranscribedasabove, 500 ngtotalRNA (Applied Biosystems).For allothergeneexpression analysis, 202 primers tohsa-miR-144-3pandnormalizedSnoRNA real-time quantitativepolymerasechain reactionusingspecific Kit (Applied Biosystems), and miR-144 was detected by Taqman ReverseTranscriptasescribed usingtheHighCapacitycDNA wasreversetran- measurements,100ngtotalRNA For miRNA extracted usingQIAzolreagent (InvitrogenLife Technologies). Cells were resuspended in ice-cold PBS, and 1×10 Cells wereresuspendedinice-coldPBS, Russell, UTSouthwestern). Cell Signaling),andCYP7B1 (1:1000;agiftfromDrDavid Imager (GE Healthcare). Imager (GE ies (1:10 - (horseradish peroxidase)-conjugated secondaryantibod HRP 3 H-cholesterol for24hours,aspreviouslydescribed. 000; GE Healthcare) and visualized using an AI600 Healthcare)andvisualizedusinganAI600 000; GE Silencing miR-144Protects AgainstAtherosclerosis 19 PDI (proteindisulfideisomerase; 1:1000; PDI 20 3,21 Proteins weredetectedwith For assays,macrophages RCT 6 cells were cellswere 3,21

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5 415 Agostino- values for normally values for values for nonnor- values for February 2020 February . Anti–miR-1441A). online-only Data Supplement).online-only mice (n=10 mice/group) a Western mice (n=10 mice/group) a Western <0.05 was considered significant, and sta- <0.05 was considered −/− Silencing miR-144 Protects Against Atherosclerosis Against Protects miR-144 Silencing -F/MOE anti–miR-144 compound to silence miR-144. ′ test or a 1-way ANOVA with either Tukey or Sidak post hoc or Sidak either Tukey with 1-way ANOVA test or a diet for 16 weeks to promote atherosclerotic disease anddiet for 16 weeks to promote atherosclerotic (PBS),concurrently treated mice with either vehicle con- anti-miR, or anti–miR-144 (Figure trol treatment did not significantly affect gain orbody weight levels (Figure IA and IBtotal plasma cholesterol in the online-only Data Supplement). RNA the livers analysis of was effectively of these mice demonstrated that miR-144 the anti–miR-144silenced in the animals treated with compound (Figure 1B). As we previously reported, mRNA but levels of the miR-144 target Abca1 were unchanged, mRNA levels of miR-144 target genes Nfe2l2 and Rarb were significantly increased in mice treated with anti– miR-144 (Figure IC). in the online-only Data Supplement Chronic treatment with anti–miR-144 did not cause any - as measured by plasma aspartate aminotransfer toxicity enzyme levels (Fig- ase and alanine aminotransferase IDure IE through in the ABCA1 protein levels were significantly increased (Fig- ure 1C), and consistent with increased ABCA1 levels and plasma HDL levels were increased activity, cholesterol 20% with anti–miR-144 treatment (Figure 1D). En face analysis of Sudan IV–stained aortae revealed a significant 35% decrease in lesion area in mice receiving anti– miR-144 (Figure 1E and 1F). Oil red O staining of aortic root sections also demonstrated reduced lipid accumula- tion and lesion area in animals treated with anti–miR-144 our data demonstrates that (Figure 1G and 1H). Thus, Here, we fed Ldlr Here, we fed To test the hypothesis that long-term silencing of miR- test the hypothesis that long-term silencing of To we used a144 would protect against atherosclerosis, 2 figure legends. Normality was determined using D′ determined using Normality was figure legends. testing. P normality Shapiro-Wilk and Pearson Student using either a 2-tailed data were calculated distributed t legends. P indicated in the figure analysis as using the Mann-Whitney data were calculated mally distributed with Dunn multiple com- test rank-sum test or the Kruskal-Wallis parisons testing. A P shown as described in the figure legends. tistical significance is Study Approval by the Office of Animal were approved All animal experiments Animal Care and Use Oversight and the Institutional Research Angeles. All of California Los Committee at the University consent according to the Declaration patients provided written using human samples were approved of Helsinki. Experiments and the and Stockholm in Munich by the local ethics committee Board. Angeles Institutional Review University of California Los RESULTS Silencing of miR-144 Long-Term Chronic of Atherosclerosis Attenuates Progression compound previously showed that this anti–miR-144 We in vivo in C57BL6/J silences miR-144 mice. effectively Plaque vul- 27 stenosis at the Lesion size was Lesion 24 22,23 The Taqman High Capacity Taqman The or patients enrolled in the 25 25 Symptoms of plaque instability 26 For BiKE qualifying without samples, patients For 29 Briefly, each entire aorta (from the ascending entire each Briefly, Hearts embedded in optimal cutting temperature in optimal Hearts embedded 15 22,23 as well as the American Heart Association classifica- as well as the American Heart Association 28 In total, n=10 plaques from symptomatic (Ruptured), 30 were defined as transitory ischemic attack, minor stroke, and minor stroke, and attack, were defined as transitory ischemic in the BiKE attack) amaurosis fugax (retinal transitory ischemic plaque was Biobank, retrieved Vascular cohort. In the Munich were snap-frozen divided into 3 to 6 segments (3–4 mm) which and stored at −80C or fixed in formalin (4%) overnight, decalci- (Sigma), embedded fied with ethylenediaminetetraacetic acid stained with hema- in paraffin, and sectioned. Sections were and eosin as well as Elastica van Gleeson. toxylin were serially sectioned through the aortic root (10 µm) from thethe aortic root (10 µm) sectioned through were serially stained aortic sinus. Slides were aortic segment to the ascending - or used for immunohis O for lesion quantification with Oil Red previously described. analysis as tochemical Statistics Graphpad soft- Statistical analysis was performed using Prism are mean±SEM All results or ±SD,the as stated in (V7.0). ware Analysis of Human Carotid Plaques Analysis of Human Carotid samples were Human atherosclerotic carotid artery lesion carotid endarterec- obtained from patients who underwent tomy surgery for high-grade (>50% NASCET [North American Symptomatic Carotid Endarcterectomy Trial]) statement. n=10 plaques from asymptomatic (Stable) patients, and n=10 For plaques from healthy controls were used in this study. Biobank samples, RNA from was extracted Vascular Munich caps fibrous stable with patients) female 5 male, (5 paques 10 and 10 plaques with unstable/ruptured fibrous caps (5 male, 5 RNA carotid artery plaque and Whole patients). isola- female tion were performed as described. cDNA Transcription Kit (Thermo Fisher Scientific) was used for used was Scientific) Fisher (Thermo Kit cDNATranscription cDNA synthesis, and primer assays for human miR-144, U6, Fisher Scientific) were used to GAPDH, (Thermo and CYP7B1 levels. in expression detect changes measured with ImageProPlus software by averaging 6 sections software by with ImageProPlus measured of the lesion size/coverage in the aorta. Quantification of each asperformed was analysis face en by aorta descending entire described. symptoms within 6 months before surgery were categorized as categorized were surgery months before 6 within symptoms asymptomatic and indication for carotid endarterectomy based (Asymptomatic Carotid Surgery on results from the ACST Trial). Department of Vascular Surgery, Karolinska University Hospital, Karolinska Surgery, Department of Vascular Sweden and were enrolled in the BiKEStockholm, (Biobank Endarterectomies), of Karolisnka nerability was assessed according to the Rothewell & Regrave nerability was assessed according to the criteria, Munich Vascular Biobank. Vascular Munich tion after Stary. aorta to the iliac bifurcation) was harvested and fixed by 4% by fixed and harvested was bifurcation) the iliac to aorta opened longitudi- was aorta The paraformaldehyde overnight. with Sudan IV and 3 low power photo- nally and was stained (from the correspond to the aortic arch micrographs taken that intercostal), the thoracic segment (from theaortic root to the first mesenteric artery), and the abdominal seg- first intercostal to the total area of the artery bifurcation. The ment ending at the iliac positive(staining lesion the to corresponding area the and aorta software in using ImageProPlus with Sudan IV) were determined together of the 3 sections and the values routinely added each to determine the percent lesion coverage. Biol. 2020;40:412–425. DOI: 10.1161/ATVBAHA.119.313633 Arterioscler Thromb Vasc Cheng et al et Cheng

Downloaded from http://ahajournals.org by on October 19, 2020 Downloaded from http://ahajournals.org by on October 19, 2020 19, October on by http://ahajournals.org from Downloaded Translational Sciences - AL 416 of atherosclerosisintervention.MaleLdlr Therefore, we turned to a moreclinically relevant model and preventfurtherdevelopmentareclinicallyattractive. rotic disease. As such strategies that can reverse disease and patientstypicallypresentwithestablishedatheroscle- Cardiovascular diseaseisthenumberonecauseofdeath Atherosclerosis Reduces Acute InhibitionofmiR-144 opment ofatherosclerosis. blocking miR-144 partially protects mice from the devel- Cheng etal ure 2C and2D),confirming effective miR-144silencing. 15%, respectively)inmicetreated withanti–miR-144(Fig- cholesterol levelswereincreased(45%and plasma HDL online-only DataSupplement ). protein ABCA1 levels and inthe throughIIE notransferase enzymelevels(FigureIIA levels, orplasmaaspartateaminotransferase/alanine ami- weight, total plasma cholesterol,body hepatic miR-144 levels(Figure 2B)butdidnotsignificantlyeffect ment withanti–miR-144for4weekseffectively reduced anti-miR, oranti–miR-144for4weeks(Figure 2A).Treat- and dividedinto3groupstreatedwitheithervehicle,control further developmentandprogressionofatherosclerosis ing animals were then switched to a chow diet to attenuate animals wereeuthanized establish atheroscleroticlesions,afterwhich onegroupof 14 mice/group)werefed aWestern dietfor16weeksto A, Malelow-densitylipoproteinreceptornull(Ldlr treatmentattenuatesatherosclerosisprogression. (miR-144) Figure 1.Anti–microRNA-144 determined by1-wayANOVA. *P<0.05,**<0.01,and***p<0.001. significancewas Statistical arepresentedasmean±SEM. aorticrootsections(20sections/mouse,n=4mice/group).Data Oil RedO-stained Scalebar, withOilRedO.Originalmagnification×50. 200.H,Quantificationof G, Aorticrootlesionsfrommicedescribedin(A)werestained (n=8–10 mice/group)after16wkofWestern barsindicatethemeanandindividualsymbolsmice. dietfeeding.Horizontal F,Quantificationofenfacelesionareamice after 16wk(w16)ofWestern enfaceaortas. SudanIV–stained dietfeeding.E,Representative ofthestudy(d4;day4)and (high-densitylipoprotein)cholesterol levelsatthestart binding cassettetransporterA1)protein.D,PlasmaHDL polymerase chain reaction.C,Western (ATP n=5mice/group)andquantification(n=10ofhepaticABCA1 blot(representative injection frequency. Redarrowsindicatebloodcollection.B,HepaticexpressionofmiR-144quantifiedby Taqman real-timequantitative oligonucleotides atadoseof10mg/kg(n=10mice/group).Bluediamondsindicate 2′fluoro/methoxyethyl′F/MOE) (2 anti-miR oranti–miR-144 February 2020 ( Figure 2A; baseline).Allremain- −/− −/− ) mice were fed a Western diet for 16 wk and treated with either vehicle (PBS), control ) micewerefedaWestern dietfor16wkandtreatedwitheithervehicle(PBS), mice(n=12– mRNA Abca1 mRNA Arterioscler ThrombVasc 10.1161/ATVBAHA.119.313633 DOI: Biol. 2020;40:412–425. resenting increased efferocytosis. We found increased associated/free apoptoticcells, withhighervaluesrep- a macrophage(free).Dataare presentedastheratioof (indicative of an efferocytic event) or not associated with 3-positive) thatareeitherassociated withamacrophage 3) andquantifiedforapoptotic cells(cleavedcaspase apoptosis (cleavedcaspase 3) andmacrophages(Mac- formed astandardassaywherelesionsarecostainedfor was associated with changes in efferocytosis we per- and 2H).To determineifthisdecreaseinapoptoticcells apoptotic cellscomparedwithcontrolmice(Figure 2F lesions ofmicetreatedwithanti–miR-144hadfewer pase 3activation by staining for cleaved caspase 3. The by measuringthepercentageoflesioncellswithcas- rocytosis ofapoptoticcells.We assayedapoptoticcells core formationisdefective phagocyticclearance or effe- amounts ofcelldeath,andamajorcausenecrotic (Figure 2F and2G). treated withanti–miR-144alsohadreducedlesionarea aorticrootlesionsdemonstratedthatanimals O-stained 4 weeksonthechow diet(Figure 2E).AnalysisofOilred orcontrol anti-miR-treated miceafter sis insaline/PBS expected, therewasnofurtherincreaseinatherosclero- with vehicleorthecontrolanti-miR(Figure 2E).As 35% smallerlesionsascomparedwith animals treated atherosclersosis, as anti–miR-144-treated mice had Advanced atheroscleroticlesionshavesignificant Treatment withanti–miR-144significantlyreduced Silencing miR-144Protects AgainstAtherosclerosis TRANSLATIONAL SCIENCES - AL 417 We, there- We, 3 February 2020 February Silencing miR-144 Protects Against Atherosclerosis Against Protects miR-144 Silencing ). Thus, our results our Thus, Supplement). Data online-only It has been previously demonstrated that systemically Supplement) were significantly decreased, suggesting that these infiltrating macrophages are M2 macrophages. delivered 2′-F/MOE tis- anti-miR compounds can reach as the aorta. such sues other than the liver, fore, analyzed miR-144 in the aortae of mice treated as in Figure 2 and observed significant silencing of III(Figure anti–miR-144 with treated mice in miR-144 the in suggest that anti–miR-144 is not only being targeted within atherosclerotic but also in macrophages in liver, data suggest that treatment with these plaques. Together online- ) mice were fed a Western diet for 16 wk. They were then switched to a regular chow to a regular chow then switched were diet for 16 wk. They ) mice were fed a Western −/− Cd206, and Il10 were and Tnfa (Figure IIH in the online-only Data Il6, Il1b, diet and treated for 4 wk with either vehicle (PBS), anti-miR or anti–miR-144 control (2′F/MOE) 2′fluoro/methoxyethyl oligonucleotides at Red arrows indicate blood collection. B, Hepatic diamonds indicate injection frequency. a dose of 10 mg/kg (n=18–20 mice/group). Blue blot (representative n=5 Taqman real-time quantitativechain reaction. C, Western polymerase expression of miR-144 from mice quantified by D, Plasma HDL binding cassette transporter A1) protein. of hepatic ABCA1mice/group) and quantification (n=10 mice/group) (high- (ATP after 4 wk of treatment. E, Representative levels density lipoprotein) cholesterol en face aortae and quantification of en face lesion area of diet) and after 4 wk of the indicated treatment. Horizontal indicate the mean, bars of Western mice (n=18–20 mice/group) at baseline (16 wk F, Aortic root lesions were stainedand individual symbols indicate individual mice. caspase 3 (a with Oil Red O and antibodies for Cleaved indicate white dotted line demarcates aortic lesion area. Gray arrowheads macrophages). The marker for apoptosis) and Mac-3 (a marker for 200 mm macrophage-associated apoptotic cells. Original magnification ×50.free apoptotic cells and white arrowheads indicate Scale bar, G, Quantification of Oil Red O-stained(Oil Red O), 125 mm (immunohistochemistry). n=4 mice/ aortic root sections (20 sections/mouse, group). H, Aortic root sections were stained total for Cleaved caspase 3 and DAPI. The of Cleaved caspase 3-positive cells were number 36±3.2, 32±1.4, 12±1.1 for PBS, Data control anti-miR, and anti–miR-144-treated as the percentage are presented mice, respectively. of positive cells of total nuclei. I, Aortic root sections were costained cleaved caspase 3-positive cell was for Mac-3 and cleaved caspase 3. Each with a macrophage, and the datadetermined to be either associated or not associated macrophage-associated are presented as the ratio of and 127±2.8 total cells. The (Mac-associated) apoptotic cells to free apoptotic number of Mac-3-positive cells were 142±4.3, 157±3.9, for PBS, Horizontal control anti-miR, and anti–miR-144-treated bars indicate the mean, and individual symbols indicate mice, respectively. of MIR-144 in healthy arteries (normal; n=7) or carotid arteries from patients with individual mice. J, Relative expression levels (fold change) atherosclerosis (plaque; n=7) from patients enrolled in the BiKE of Karolinska (Biobank K, Relative expression levels (fold Endarterectomies). of MIR-144 in human atherosclerotic carotid artery lesions from asymptomatic (Stable;change) n=7) vs symptomatic (ruptured; n=7) patients enrolled in the BiKE. Data are presented as mean±SEM. (C–F, I–K) or Students t Statistical significance was determined by 1-way ANOVA test (L–M). *P<0.05, **P<0.01, and ***P<0.001. Figure 2. Anti–microRNA-144 (miR-144) accelerates regression of atherosclerosis. treatment A, Male low-density lipoprotein receptor null (Ldlr ). Steady-state mRNASteady-state Data Supplement). only for levels the M2 macrophage markers Arg1, significantly elevated (Figure IIG in the online-only Data mark- Supplement), and levels of the M1 macrophage ers efferocytosis in lesions of mice treated with anti– in lesions of mice efferocytosis for the miR-144 (Figure 2I), suggesting one mechanism smaller lesion area and decreased numbers of apoptotic Macrophage infiltration, cells is improved efferocytosis. as assessed by quantifying CD68 transcript abundance in the aortas, was greater in anti–miR-144-treated ani- IIF(Figure controls with compared as mals the in Biol. 2020;40:412–425. DOI: 10.1161/ATVBAHA.119.313633 Arterioscler Thromb Vasc Cheng et al et Cheng

Downloaded from http://ahajournals.org by on October 19, 2020 Downloaded from http://ahajournals.org by on October 19, 2020 19, October on by http://ahajournals.org from Downloaded Translational Sciences - AL protective. Huetal (50%; Figure 3D).This assayfollowsthemovementof 3C), andfeces Figure Figure 3B),liver(80%; (30%; RCT toplasma miR-144 hadsignificantlyincreased assay(Figure 3A).Micetreatedwithanti– in vivoRCT anti–miR-144 leads to increased RCT, we performed an the protein composition of HDL particles, weperformed the proteincomposition ofHDL levelsinmicetreatedwithanti–miR-144. HDL r cholesterol concentrations (Figure 3H; with plasma HDL dent increaseineffluxcapacity wassignificantlycorrelated lated fromcontrolmice.Additionally, themiR-144-depen - capacity (Figure that silencingmiR-144significantlyincreasedsterolefflux with either anti–miR-144 or control anti-miR, and we show frommicetreated (Figure 3G).WeABCG1 isolatedHDL overexpressing inducible forms of (Figure ABCA1 (babyhamsterkidneyfibroblast) cells (Figure 3E) orBHK functional assaysusingradiolabeledJ774 macrophages steroleffluxcapacity capacity, weperformedinvitroHDL with anti–miR-144haveenhancedcholesterol efflux miR-144 oligonucleotides. tion hasnotbeenaffected bypriortreatmentwithanti– been loadedwithcholesterol invitroandwhosefunc- levels in hypercholesterolemic invivo.ToRCT determinewhethertheincreasedHDL to reducemiR-144levelsanddeterminingchanges in the converse,thatis,treatmentofmicewithantagomir (20%). However, therearenopriorstudiesreporting toplasma(15%),liver(25%),andfecesdecreased RCT cholesterol levels by 25% and decreased plasma HDL of miR-144tosupraphysiologiclevelsusinganagomir 418 3 miR-144 in hypercholesterolemic The dataofFigures 1and2demonstratethatsilencing ParticleInVivo Remodels theHDL EnhancesRCT and Silencing miR-144 differences inplaqueremodelinghumans. reduced miR-144expression levelsareassociatedwith atic humancarotid plaques (Figure 2K),suggesting that reduced inplaquesfromasymptomaticversussymptom- agonists.MiR-144levelswerealsosignificantly of LXR increased macrophagefoamcellsandlevels levels tobeelevatedinhumanplaquesconsistentwith sis andcontrolarteries(Figure 2J).We foundmiR-144 of miR-144inhumanpatientswithcarotidatherosclero- human atheroscleroticplaques,wemeasuredthelevels observed inmicetreatedwithanti–miR-144. which likelycontributestothereducedatherosclerosis rophages with enhanced efferocytic ability in the aortae, anti–miR-144 resultedinmoreanti-inflammatorymac- Cheng etal =0.43, H-cholesterol fromwildtypemacrophagesthathave To determine whetheranti-miR144treatmentaffected To particlesfrommicetreated addresswhetherHDL To determine whether miR-144 levels are different in February 2020 P =0.03), consistent with the observed increased =0.03), consistentwiththeobserved increased 3E through 3G), compared with HDL iso- 3E through 3G),compared with HDL 14 demonstratedthatoverexpression Ldlr Ldlr −/− mice treated with −/− mice is athero- Arterioscler ThrombVasc 10.1161/ATVBAHA.119.313633 DOI: Biol. 2020;40:412–425. 3F) or 3F) or 27-hydroxycholesterol to7α,27-dihydroxycholesterol, Cyp7b1 isadirecttargetofmiR-144 (Figure 5E). a concentration-dependent manner, demonstrating that to identifydifferentially expressed proteins. anti–miR-144 (Figure isolated frommicetreatedwitheithercontrolanti-miRor unbiased shotgun mass spectrometric analysis of HDL and 27-hydroxycholesterol hasbeenshowntopotently Abca1. luciferase reportergene,aswehavedone previouslyfor the 3′UTR (3untranslatedregion)downstreamofa whether control anti-miR-treatedlivers(Figure 5D).To determine ers ofanimalstreatedwithanti–miR-144,compared that CYP7B1 proteinlevelswerealsoelevated intheliv- (Figure 5C)andWesternmRNA blottinganalysisshows confirmed amiR-144-dependentinductioninCyp7b1 real-time quantitativepolymerasechain reactionanalysis genes wasCyp7b1 (blue dot/arrow;Figure 5A). Indeed, pathways (Figure both significantlyinducedandrepressed identified way analysisofthemostdifferentially regulatedgenes control anti-miRandanti–miR-144(Figure 5A).Path- expression analysisfromtheliversofmicetreatedwith in atherosclerosis regression, we performed global gene To betterunderstandthemoleculartargetsofmiR-144 Pathways ofNovelmiR-144–Regulated Identification vated APOA2. increases in cholesterol efflux to particles containing ele- Data Supplement),consistentwithpreviouslyobserved (r=0.65, of APOC4 P=0.04; Figure 4E)andinverselycorrelatedwithlevels nificantly correlatedwiththelevelsofAPOA2 (r=0.68, HDL-dependent cholesterol effluxcapacitywassig- let factor)levelsweredecreased(Figure 4Band4D). (plate- andPF4 ure 4B through4D),whereasAPOC4 and SAA1(serumamyloidA)levelswereincreased(Fig- MUP20, (majorurinaryprotein),MUP6, APOA2, MUP2 mice treatedwithanti–miR-144(Figure 4Athrough4D). isolatedfrom differentially expressed (P<0.05)onHDL 144 (Figure 4Aand4B).We identified7proteinsas wasunaffected bymiR- the majorconstituentofHDL, vivo (Figure 3). RCTincreased fluxof pathwayin cholesterol throughthe terol effluxfromcells,afindingthatmaycontributetothe particlethatismoreefficientatpromoting choles- HDL that silencingmiR-144resultsintheproductionofan Data Supplement).Taken together, these datasuggest (r=0.26, correlate withlevelsofSAA1(r=0.37, particlesdidnot HDL cholesterol effluxcapacityofthe response proteinsSAA1andSAA2(Figure 4D),the CYP7B1/oxysterol 7α -hydroxylase metabolizes 5 MiR-144wasabletoreduce luciferase activityin Cyp7b1 isadirecttargetofmiR-144,wecloned =0.46; Figure IIIB and IIIC intheonline-only andIIIC FigureIIIB P=0.46; 31–33 Silencing miR-144Protects AgainstAtherosclerosis Despiteincreasesintheacutephase 5B). One of the most highly induced 0.04; Figure IIIA intheonline-only P=0.04; FigureIIIA 4A). Spectral counting was used P=0.3) orSAA2 18 APOA1, 34

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419 20,34 , 20,34,37 February 2020 February H-cholesterol tracer levels. H-cholesterol 3 , Fecal , Fecal which prompted us to investigate which , The relationship of plasma HDL, The cholesterol 20,34 Silencing miR-144 Protects Against Atherosclerosis Against Protects miR-144 Silencing , Macrophage cholesterol efflux capacity (cAMP-, Macrophage cholesterol <0.01. ) mice (n=10 mice/group) treated with either vehicle −/− ) ABCG1 (ATP binding cassette transporter G1)-specific ) ABCG1 (ATP H-cholesterol-labeled, acLDLH-cholesterol-labeled, (acetylated low-density lipoprotein)- decreased circulating levels of 27-hydroxycholesterol decreased circulating levels of 27-hydroxycholesterol function in the can also (Figure 5F). Hepatic CYP7B1 alternative pathway for the synthesis of bile acids. whether there may be sex-specific effects of the regula- of effects whether there may be sex-specific previous studies tion of Cyp7b1 by miR-144. These However, mice treated with anti–miR-144 had no sig- mice treated with anti–miR-144 However, in total biliary bile acid levels (Figure nificant differences together, Data Supplement). Taken IVB in the online-only our data suggest that silencing miR-144 also induces Cyp7b1 mRNA activity, resulting in increased CYP7B1 proathero- the of levels circulating decreasing thereby 27-hydroxycholesterol. genic oxysterol, Antagonism of miR-144 Alters Oxysterol Metabolism to in a Sexually Dimorphic Manner Against Atherosclerosis Protect in male expressed Cyp7b1 is known to be differentially mice, and female 3 <0.05 and **P , Serum HDL obtained was by polyethylene glycol (PEG) precipitation of G ) and blood collected at 6, 24, and 48 hours post-injection of radiolabeled – H-cholesterol tracer relative to that of the totalH-cholesterol cpm tracer injected±SEM. B 3 H-cholesterol tracer levels after 48H-cholesterol hours. D 3 , Hepatic We hypothe- We 35,36 ) ABCA1 (ATP binding cassette transporter A1)-specific cholesterol efflux (mifepristone-stimulatedcholesterol transporter A1)-specific binding cassette ) ABCA1BHK (baby (ATP 5G), suggesting that local effects effects local that suggesting 5G), , Data are expressed as the percentage of the D – H-cholesterol distribution in plasma. C H-cholesterol 3 B ). Statistical significance was determined by 1-way ANOVA. *P ). Statistical by 1-way ANOVA. significance was determined G – Ldlr , Mice were treated as in Figure 2. Male low-density lipoprotein receptor null ( D – Time course of Time sized that increases in hepatic CYP7B1 enzyme activity in sized that increases in hepatic CYP7B1 mice treated with anti–miR-144 would result in reduced - 27-hydrocho oxysterol, proatherogenic the of levels lesterol. Consistent with this hypothesis, we show that levels were decreased plasma 27-hydroxycholesterol 34% in mice treated with anti–miR-144, compared with those injected with PBS or control anti-miR (Figure 5F). were specific to 27-hydroxycholesterol, changes These with anti–miR-144 did not change as other oxysterols online-only in the treatment (Figure 5F and Figure IVA Cyp7b1 Data Supplement). Macrophages also express so we analyzed and produce 27-hydroxycholesterol Cyp7b1 mRNAtreated with aortae of mice levels in the observed significantly increased anti–miR-144. We treated isolated from mice in aortae Cyp7b1 expression with anti–miR-144, compared with mice treated with the (Figure anti-miR control of miR-144 on macrophages may also contribute to the Feces were collected continuously from 0 to 48Feces hours after injection. E ApoB-containing serum from mice treated as in Figure 3A. E lipoproteins from plasma-derived stimulated J774 macrophages), (F stimulated J774 efflux (mifepristone-stimulated cholesterol BHK (baby hamster kidney fibroblast) cells minus BHK cells incubated in medium alone) were measured after a 4 hours incubation with serum HDL from mice treated as in Figure 3A. H (2.8% v/v) isolated concentration and HDL Pearson correlation. Data efflux capacity was quantified by presented as mean±SEM. are Horizontal the bars indicate mean (E hamster kidney fibroblast) cells minus BHK incubated in medium alone) and (G cells exacerbate atherosclerosis progression. exacerbate Figure 3. Anti–microRNA-144 (miR-144) transport (RCT) and HDL increases reverse cholesterol treatment (high-density lipoprotein) efflux capacity. A (PBS), control anti-miR or anti–miR-144 subcutaneously were injected with before euthanization (A loaded resident peritoneal macrophages 2 days macrophages. Biol. 2020;40:412–425. DOI: 10.1161/ATVBAHA.119.313633 Arterioscler Thromb Vasc Cheng et al et Cheng

Downloaded from http://ahajournals.org by on October 19, 2020 Downloaded from http://ahajournals.org by on October 19, 2020 19, October on by http://ahajournals.org from Downloaded Translational Sciences - AL male establish atherosclerosis (Figure6A),aswedidwith fed female Ldlr ences intheroleofmiR-144andatherosclerosis,we effects of estrogen. To determine potential sex differ - protected, aneffect thatisattributedtotheprotective dramatic effects ondiseaseandthatfemales areoften mice (FigureVC intheonline-onlyDataSupplement ). genes intheliversofmalemicecomparedwithfemale onstrates thatCyp7b1isamongthemostupregulated over 100strainsofgeneticallyuniquein-bredmice,dem- 420 gene familiesinmouseliver enzymes asamongthemosthighlysexually dimorphic sexes haspreviouslyidentifiedcytochrome P450 analysis ofgeneexpression insomatictissuesbetween terol in male and female mice. Indeed, comprehensive also differences inthegenerationof27-hydroxycholes- 27-hydroxycholesterol levels,suggestingthatthereare mice, andlossofCyp7b1ledtoagreaterincreasein 2- to 3-fold higher in malemicecompared with female have demonstratedthathepaticCyp7b1expression is significancewasdeterminedby1-wayANOVA. Statistical *P arepresentedasmean±SEM. Data relative abundancecalculatedasZscoresgeneratedfromadjustedspectralcounts.B spectralcounts).A number ofpeptidesidentifiedforagivenproteinnormalizedtototal isolated byultracentrifugationfrommice(n=5–6mice/group)treatedasinFigure 2.Proteinswerequantifiedspectralcounting(total (liquid chromatography-electron sprayionization-massspectrometry/massspectrometry)analysisofproteinson HDL LC-ESI-MS/MS (high-densitylipoprotein)particle. remodelstheHDL (miR-144) Figure 4.SilencingmicroRNA-144 Cheng etal control anti-miR, or anti–miR-144 for4weeks. Basal gression ofatherosclerosisand treatedwitheithersaline, to achow diettoattenuatefurtherdevelopment andpro- diet (baseline),andtheremaining animalswereswitched of animalswereeuthanized after 16weeksofWestern (apolipoprotein) levels on HDL and macrophage efflux capacity of HDL particleswasquantifiedby Pearsoncorrelation. HDL andmacrophage effluxcapacityof (apolipoprotein) levelsonHDL It iswellappreciatedthatsex differences canhave Ldlr February 2020 −/− miceinFigure 2A.As 2, onegroup −/−

mice a Western dietfor16weeksto 38 andanalysisofthelivers Arterioscler ThrombVasc 10.1161/ATVBAHA.119.313633 DOI: Biol. 2020;40:412–425. 20% increase protein ABCA1 levels (Figure treated with anti–miR-144, we observed amore modest tively). Further, whenweanalyzedtheliversoffemale mice and50%,respec- 80%, observed inmalemice(30%, changes weresignificantlylessrobust thatthose Figure 6F), andfeces (31%;Figure 6G).Notably, these toplasma(20%;Figure 6E),liver(33%; increased RCT In female mice, weobservedthatsilencingmiR-144 controlanti-miR,oranti–miR-144). with vehicle[PBS], this timeinfemale Ldlr lesion area, werepeated analysis,but our invivo RCT cholesterol levels, butnochange inatherosclerotic HDL online-only DataSupplement). no change in totalcholesterol levels(FigureVA inthe rable tothatseeninmalemice(15%;Figure 2D),with cholesterol by13%(Figure 6D),compa - plasma HDL in female mice(Figure 6C).SilencingmiR-144increased had nosignificanteffect onatheroscleroticlesionburden 0.19; Figure 6B).Surprisingly, anti–miR-144treatment els werealmost2-foldhigherinfemale mice(0.37versus howeverresidual miR-144 lev- seen in male mice (80%), 75% reductioninmiR-144levels,andcomparabletothat Silencing miR-144waseffective infemale micewith differentially regulatedinmalesandfemales (Figure 6B). compared withmales,suggestingmiR-144itselfmaybe levels ofmiR-144were50%elevatedinfemale mice, Because weobservedsignificantincreasesinplasma – , Heatmapofdifferentiallyexpressedproteins,with D , Quantification of representative HDL proteins. HDL , Quantificationofrepresentative Silencing miR-144Protects AgainstAtherosclerosis <0.05. −/− E mice(treatedasinFigure 2 , RelationshipbetweenAPOA2 6H), which TRANSLATIONAL SCIENCES - AL

421 , , Validation , Validation Consistent with Consistent with February 2020 February 26 , Differentially expressed male mice significantly inhibits the −/− Silencing miR-144 Protects Against Atherosclerosis Against Protects miR-144 Silencing 6N), patients (Figure 6N), in female but not 6M), 2K, expression of MIR- the human plaque data in Figure 2K, expression 144 was significantly reduced in stable plaques in both patients (Figure 6K and 6L). CYP7B1 male and female levels were increased in stable plaques in male patients male patients plaques in in stable were increased levels (Figure suggesting that levels of CYP7B1 may be important in data, plaque composition and stability in humans. These mice, sug- together with the data in male and female of in miR-144 regulation differences gest that the sex responsible oxysterol pathways is efflux and cholesterol on atherosclerosis observed with in part for the effects mice. silencing miR-144 in male versus female progression and promotes regression of atherosclerosis. , blue- and black-boxed genes), suggesting genes), black-boxed and blue- Supplement, in males and miR-144 is not only regulated differently sex. genes in either different but may also target females, in Cyp7b1 in determine whether these differences To patients, we atherosclerosis are also observed in human from male obtained stable and ruptured plaque samples carotid endarterectomy patients undergoing and female Biobank. Vascular from the Munich DISCUSSION In the current article, we have demonstrated that silencing miR-144 in Ldlr , Plasma concentrations of 24-, 25-, and 27-hydroxycholesterol. G , Plasma concentrations of 24-, 25-, and 27-hydroxycholesterol. ABC indicates ATP-binding cassette; and OHC; cassette; <0.001. ABC hydroxycholesterol. indicates ATP-binding -fold changes indicates the change in expression after comparison of anti–miR-144 indicates the change -fold changes Cyp7b1, and Cyp8b1 as measured by real-time quantitative polymerase chain untranslated region) reporter plasmid was determined 48 untranslated region) reporter plasmid was determined h after transfection and 2 UTR (3′ ′ Cyp7a1, <0.01, and ***P Cyp27a1, <0.05, **P online-only Data Supplement). Several -galactosidase activity. The activity of the reporter plasmid in the absence of a miRNA activity of the reporter The overexpression plasmid was independently -galactosidase activity. , Luciferase activity of the Cyp7b1 3 , Western blot (representative n=5 mice/group) and quantification (n=10 mice/group) of hepatic CYP7B1 (cytochrome p450CYP7B1 (cytochrome enzyme blot (representative n=5 mice/group) and quantification (n=10 mice/group) of hepatic , Western D In agreement with previous studies, hepatic Cyp7b1 In agreement with previous studies, , RNA-sequencing on livers of mice treated with either control anti-miR or anti–miR-144 was performed in Figure 2. Gene expression as Expression of Cyp7b1 mRNA in the aortae of mice treated as in Figure 2. Data are presented as mean±SEM. Statistical significance was *P determined by 1-way ANOVA. profiling was performed in triplicate for each condition. Log profiling was performed in triplicate for each Identification of novel pathways regulated by microRNA-144Figure 5. Identification of novel pathways regulated (miR-144) in atherosclerosis. A with control anti-miR treatment. Red=significantly down-regulated genes, blue=significantly up-regulated genes. B with control anti-miR treatment. Red=significantly 7b1) protein. E (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment using gene-set and KEGGgenes were associated with biological functions of Genes and Genomes) pathway enrichment Encyclopedia (Kyoto analysis and GO annotations. processes up-regulated pathways. C Red=significantly down-regulated pathways, blue=significantly of hepatic mRNA of Abcb11, expression reaction. normalized to β set to 100%. Results are representative of 3 independent experiments. F was significantly less than we observed in male mice was significantly less than we observed these (50% Figure 2C versus 20% Figure 6H). Together, through the data suggest that reduced levels of flux RCT in of changes pathway could contribute to the absence mice treated with anti–miR-144. lesions in female higher in the livers of PBS-treated was 3-fold expression mice (Figure 6I). In con- male mice compared with female mRNAof Cyp7b1 the induction trast to in anti– observed miR-144-treated male mice, hepatic and aorta Cyp7b1 mRNA mice treated in female levels were unchanged with anti–miR-144 (Figure 6I and Figure VD in the online- only Data Supplement). Consistent with the absence of was no effect there in Cyp7b1 expression, any changes levels (Figure 6J), on plasma 27-hydroxycholesterol (Figure VB in the online-only Data other oxysterols Supplement), or bile acid levels (Figure VE in the online- determine whether silencing only Data Supplement). To dimorphic regulates other sexually miR-144 differentially hepatic genes, we overlayed our miR-144 dif- expressed - genes (from Figure 5) with all sex expressed ferentially livers ually dimorphic genes between male and female in the VC (Figure other sexually dimorphic genes were also significantly other sexually regulated by silencing miR-144 (including differentially in the online-only Data Elovl3 and Slco1a1; Figure VC Biol. 2020;40:412–425. DOI: 10.1161/ATVBAHA.119.313633 Arterioscler Thromb Vasc Cheng et al et Cheng

Downloaded from http://ahajournals.org by on October 19, 2020 Downloaded from http://ahajournals.org by on October 19, 2020 19, October on by http://ahajournals.org from Downloaded Translational Sciences - AL increased inflammation, of SAA1andSAA2inplasma havebeenassociatedwith mice treatedwithanti–miR-144. Althoughincreasedlevels levels were significantly induced in the livers of mRNA the significantlyregulatedproteins, onlySaa1andSaa2 isolated frommicetreatedwithanti–miR-144.Of in HDL fied numerousproteinsthat weredifferentially regulated Usingproteomics,weidenti- composition ofplasmaHDL. treatment hadsignificanteffects onthepropertiesand pathwaytobeexcretedthrough theRCT inthefeces. isbyincreasingfluxof miR-144 improvesRCT cholesterol these datasuggestthatonemechanism bywhich silencing and cholesterol excretion fromthebody. Taken together, the liverthatareconsistentwithenhancebiliarysecretion cant changes inAbcb11/ treated withmiR-144.Additionally, weobservedsignifi- 422 cells, vitro cholesterol effluxstudieswithHuh7, J774, andTHP-1 Previous studieswithantagomirsofmiR-144focusedonin Cheng etal using anagomirtooverexpress miR-144,whereHuetal 3H-cholesterol. These data areinagreementwithstudies invivo,resultinga50%increasefecalenhances RCT in vivo.We showforthefirsttimethatsilencingmiR-144 observed the converse decrease in RCT inApoe observed theconversedecreaseinRCT radiolabeled macrophages. Data are expressed as the percentage ofthe areexpressedasthepercentage radiolabeled macrophages.Data hpost-injectionof low-density lipoprotein)-loadedresidentperitonealmacrophages2daysbeforeeuthanizationandbloodcollectedat6,24,48 treated with either vehicle (PBS), control anti-miR or anti–miR-144 were injected subcutaneously with wereinjectedsubcutaneously controlanti-miR oranti–miR-144 treated witheithervehicle(PBS), Figure 2 andfemalemicefrom6A,quantifiedbyRT-qPCR. J (ATPhepatic ABCA1 bindingcassettetransporterA1)proteinfrommicetreated asinFigure 6A.I hafterinjection.H were collectedcontinuouslyfrom0to48 E A doesnotprotectagainstatherosclerosisinfemalemice. (miR-144) Figure 6.SilencingmicroRNA-144 individual symbolsindicatemice. D of mice(n=13–18mice/group)atbaseline(16wkWestern barsindicatethemean, and diet)andafter4wkoftheindicatedtreatment.Horizontal polymerasechain reaction(RT-qPCR).time quantitative C in Figure2formalemice.B 2′ controlanti-miRoranti–miR-144 vehicle (PBS), Vascular Biobank. Data are presented as mean±SEM. Statistical significancewasdeterminedby1-wayANOVA. Statistical *P Vascular arepresentedasmean±SEM. Biobank.Data MIR144 ( , Time courseof , Female low-densitylipoproteinreceptornull(Ldlr We alsoreportforthefirsttimethatanti–miR-144 10 February 2020 anddidnotanalyzeeffects oncholesterol efflux K andL ) andCYP7B1( 3 H-cholesterol distributioninplasma.F , HepaticexpressionofmiR-144inmalemicefromFigure 2andfemale6A,quantifiedby Taqman real- 39 HDL particles containing SAA1 particlescontaining SAA1 HDL Bsep andCyp7a1expression in M andN ) expressionlevels(foldchange) inhumanatheroscleroticarterylesionsfrompatientstheMunich , Plasma HDL cholesterol levelsinmicetreatedasA , PlasmaHDL fluoro/methoxyethyl (2′ −/− ) micewerefedaWestern dietfor16wkand thensubsequentlytreatedfor4wkwitheither , Hepatic Arterioscler ThrombVasc 10.1161/ATVBAHA.119.313633 DOI: Biol. 2020;40:412–425. , Representative Sudan IV–stained en face aortas and quantification of en face lesion area andquantificationofenfacelesionarea enfaceaortas SudanIV–stained , Representative , Western n=5mice/group)andquantification(n=10of blot(representative −/− mice mice 3 H-cholesterol tracer levels after 48 h.G H-cholesterol tracerlevelsafter48 , Plasmaconcentrationsof24-,25-,and27-hydroxycholesterol. K 14

F/MOE) oligonucleotides at a dose of 10 mg/kg (n=13–18 mice/group) as oligonucleotides atadoseof10mg/kg(n=13–18mice/group)as F/MOE) 3 H-cholesterol tracer relative to that of the total cpm tracer injected±SEM. cpmtracerinjected±SEM. H-cholesterol tracerrelativetothatofthetotal are wellestablished. anti–miR-144 treatment.The protectiveeffects ofAPOA1 with anti–miR-144andAPOA1 levelswereunchanged by particlesisolated frommicetreated was increasedinHDL observedinmicetreatedwithanti–miR-144.APOA2RCT cleared bytheliverandmayexplain, inpart,theincreased in micetreatedwithanti–miR-144maybepreferentially rapidly fromthecirculation, and SAA2havebeendemonstratedtobeclearedmore increased atherosclerosis particlescontainingAPOA2 and HDL efflux capacityof observed with some groups reporting reduced cholesterol are lesswellunderstood,andconflictingresultshavebeen better effluxcapacity. with APOA2 are actually smaller particles and have HDL atherosclerotic lesionsinfemale Ldlr observed thatsilencingmiR-144 didnothaveaneffect on not infemale mice. We were surprised and intriguedto motes regression of atherosclerosis in male mice but tially removedfromthecirculation. that ismoreefficientat cholesterol efflux andpreferen- particle isbygeneratinganHDL silencing improvesRCT in vivosuggestthatonemechanism bywhich miR-144 protein compositionandcholesterol effluxinvitroand We alsoshowedthatantagonizingmiR-144 pro- Silencing miR-144Protects AgainstAtherosclerosis in male mice from , HepaticexpressionofCyp7b1inmalemicefrom . E 42–44 – 3 32,33 H-cholesterol-labeled, acLDL (acetylated (acetylated H-cholesterol-labeled, acLDL G , Female Ldlr , Fecal However, theeffects ofAPOA2 Taken together, ourdataonHDL 45,46 40,41 and others reporting that andothersreportingthat 3 H-cholesterol tracerlevels.Feces suggesting that the HDL suggestingthattheHDL <0.05 and***P −/− mice (n=10 mice/group) mice(n=10mice/group) −/− mice.We initially <0.001. – N , Relative , Relative TRANSLATIONAL SCIENCES - AL 423 Therefore, it Therefore, 51 February 2020 February the miR response response miR the share - for techni Han thank Tieyan ) mice used in Figure 1. We −/− Silencing miR-144 Protects Against Atherosclerosis Against Protects miR-144 Silencing , 2019; accepted November 15, 2019. cal assistance with histological analysis of en face lesions. We thank Dr Jake Lusis thank Dr Jake Lusis cal assistance with histological analysis of en face lesions. We withassisting for Pan Calvin and panel diversity mouse hybrid the to access for HMDP thank Regulus Therapeutics (hybrid mouse diversity panel) analyses. We for the gift of Tang and Chongren Vaisar thank Tomas for anti-miR compounds. We binding BHKABCA1 (ATP overexpressing fibroblast) cells kidney (baby hamster binding cassette transporter G1). cassette transporter A1) and ABCG1 (ATP will require extensive further studies to determine if any if determine to studies further extensive require will to the also contribute mechanisms of these additional observed with anti–miR-144 reduction in atherosclerosis - in atheroprotec differences the treatment. Additionally, and the data mice and female tion observed in male may have important implications for from human plaques as a potential therapeutic in humans. silencing miR-144 suggest that data our when taken together, Nonetheless, oligonucleotides to target miR- treatment with antisense RCToxysterol in and enhancements in result might 144 metabolism in patients with cardiovascular disease. Received August 30 of binding sites for a specific miR within a given 3′UTR miR within a given sites for a specific of binding other miR-144 targets of relative expression and (2) the UTR. This binding in their 3′ for the miR that can compete endog- by competing be further complicated may also RNAs,enous that transcripts influence genes and can the miR target element with other by competing for miR binding. each INFORMATIONARTICLE Affiliations - Depart Chemistry (J.C., A.C., T.Q.d.A.V.), the Department of Biological From Biology Insti- Molecular T.Q.d.A.V.), E.J.T., T.S., ment of Medicine (B.L.C., X.W., Comprehensive Cancer Center (E.J.T., and Johnsson T.Q.d.A.V.), E.J.T., tute (T.S., Medi- Angeles; Department of Molecular University of California Los T.Q.d.A.V.), Institute, Karolinska (L.M.), Medicine of Department and (U.H.) Surgery and cine Klinikum and Endovascular Surgery, Solna, Sweden; Department of Vascular Germany (L.M.); and Department University Munich, der Isar—Technical rechts Univer- Sciences & Health Oregon Institute, Cardiovascular Knight Medicine, of (N.P.). Portland sity, Acknowledgments J. conceived and designed the research. de Aguiar Vallim and T.Q. E.J. Tarling E.J. Tarling, L. Maegdefessel, N. Pamir, Cheng, A. Cheng, B.L. Clifford, X. Wu, Sallam provided and T. X. Wu conducted experiments. de Aguiar Vallim and T.Q. atherosclerotic lesions. assistance in conducting histological analysis of en face provided access to human samples from the Bio- U. Hedin and L. Maegdefessel Biobank, and L. Vascular Munich and the Endarterectomies bank of Karolinska reaction analysis of hu- polymerase chain performed quantitative Maegdefessel performed proteomic and in vitro analyses man carotid artery plaques. N. Pamir of HDL (high-density lipoprotein) particles. J. Cheng, A. Cheng, B.L. Clifford, N. performed data de Aguiar Vallim and T.Q. E.J. Tarling L. Maegdefessel, Pamir, wrote and edited the article. All de Aguiar Vallim and T.Q. analysis. E.J. Tarling authors reviewed the article. Funding of Sources (NIH)Health of Institutes National by is supported HL118161 grants E.J. Tarling Sallam is supported by NIHand HL136543; T. grant HL139549; de Agu- and T.Q. NIHby supported is DK112119and bypart in HL122677 and grants Vallim iar was also supported by the Uni- HL028481 de Aguiar Vallim and DK102559. T.Q. Science Insti- Angeles (UCLA)versity of California, Los Clinical and Translational tute (UL1TR000124);and the American Heart Association (SDG18440015). E.J. also supported by the UCLA/University were de Aguiar Vallim of and T.Q. Tarling Center (DK063491).Dr Peter thank California, San Diego Diabetes Research We Tontonoz also thank Dr Peter Edwards for his guidance and input on this article. We and sug- and members of the UCLA Unit for feedback Research Atherosclerosis lipo- low-density for Navab Mohamad and Fogleman Alan Drs thank We gestions. protein receptor null (Ldlr The specific The At least 4 miRs At 4–6,8,10 48–50 adding a further layer of regulation 5,10 Often thought of as rheostats that fine-tune Often thought of as rheostats that 47 miRs can regulate hundreds, if not thousands of of thousands not if hundreds, regulate can miRs contributions of each individual miR in regulating ABCA1 contributions of each specific will be determined by the abundance of each tis- miR and the abundance of ABCA1 itself in different that we and others, have demonstrated sues. Additionally, different under stimuli different by regulated is miR-144 cellular contexts, have been reported to regulate ABCA1, including miR- 144, in multiple tissues and cell types. profiled miR-144 levels to determine whether the anti– determine whether miR-144 levels to profiled miR-144 in female at silencing was effective miR-144 of in the levels a significant reduction observed mice. We mice to that seen in male that was comparable miR-144, female reduction in (75% anti–miR-144 treated with 80% versus mice Interestingly, mice). male in reduction were 1.3-fold higher in control relative miR-144 levels and after controls, with male livers compared female there was 2-fold more residual miR- miR-144 silencing also livers. We livers compared with male 144 in female on ABCA1 of miR-144 silencing determined the effect and observed a mice livers of female protein levels in the female in ABCA1 protein hepatic in increase significant was however this effect mice treated with anti–miR-144, mice (50% mice compared with male smaller in female versus 20% increase in female increase in male mice in the regulation of ABCA1 may difference mice). This livers in female be due to the increased residual miR-144 observed smaller after treatment with anti–miR-144. The increase in ABCA1 protein levels also translated into a ver- smaller increase in RCT to plasma (20% in females sus 30% in versus 50% in males), liver (33% in females versus 80% in females (31% in males) males), and feces with anti–miR-144. mice treated in female genes. to this system. Similar to Abca1, the 3′UTR of Cyp7b1 is also large and is, therefore, likely subject to extensive posttranscriptional regulation. Indeed, the Cyp7b1 3′UTR and miR-26, harbors miR-758, binding sites for miR-33, miRs that have been shown to regulate Abca1 and lipid metabolism. regulation of Abca1 and/or Cyp7b1 by The miR-144 will likely also be influenced by (1) the number gene expression, miRs frequently regulate sets of genes miRs frequently regulate sets of genes gene expression, understand better To in common pathways or processes. silencing of following expression in gene the changes analy- expression miR-144, we performed global gene resulted in signifi- sis and show that silencing miR-144 (a total of 152 gene expression in hepatic cant changes genes), and likely also genes in regulated differentially Fig- in shown (as wall artery the including tissues, other ures II online-only Data Supplement). These and V in the of silencing or indirect effects could be direct changes miR-144. As we have mentioned above, a given miR can genes (miR-144 be predicted to target several hundred has over 1400mRNA predicted targets), and at least of mRNAs have at least one, if not several, predicted 70% miR binding sites in their 3′UTR. Biol. 2020;40:412–425. DOI: 10.1161/ATVBAHA.119.313633 Arterioscler Thromb Vasc Cheng et al et Cheng

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