Rapamycin and FTY720 Alleviate Atherosclerosis by Cross ...

Research ArticleRapamycin and FTY720 Alleviate Atherosclerosis by CrossTalk of Macrophage Polarization and Autophagy

Rui-zhen Sun 1 Ying Fan2 Xiao Liang2 Tian-tian Gong1 Qi Wang1

Hui Liu1 Zhi-yan Shan 1 and Lei Lei1

1Department of Histology and Embryology Harbin Medical University 150081 Harbin China2Department of Cardiology First Affiliated Hospital of Harbin Medical University 150081 Harbin China

Correspondence should be addressed to Zhi-yan Shan shanzhiyan1979126com

Received 21 July 2018 Revised 23 October 2018 Accepted 22 November 2018 Published 6 December 2018

Academic Editor Hanrui Zhang

Copyright copy 2018 Rui-zhen Sun et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Foam cell formation and macrophage polarization are involved in the pathologic development of atherosclerosis one of the mostimportant human diseases affecting large and medium artery walls This study was designed to assess the effects of rapamycin andFTY720 (fingolimod) on macrophages and foam cells Mouse peritoneal macrophages were collected and treated with rapamycinand FTY720 to study autophagy polarization and lipid accumulation Next foam cells were formed by oxidizing low-densitylipoprotein to observe changes in lipid accumulation autophagy and polarization in rapamycin-treated or FTY720-treated foamcells Lastly foam cells that had been treated with rapamycin and FTY720 were evaluated for sphingosine 1-phosphate receptor(S1prs) expression Autophagy microtubule-associated protein 1 light chain 3- (LC3-) II was increased and classically activatedmacrophage phenotype markers interleukin- (IL-) 6 cyclooxygenase-2 (COX2) and inducible nitric oxide synthase (iNOS) wereincreased whereas alternatively activatedmacrophage phenotypemarkers transforming growth factor- (TGF-) 120573 arginase 1 (Arg1)and mannose receptor C-type 1 (Mrc1) were decreased by rapamycin in peritoneal macrophages LC3-II was also obviouslyenhanced though polarization markers were unchanged in rapamycin-treated foam cells Moreover lipid accumulation wasinhibited in rapamycin-treated macrophage cells but was unchanged in rapamycin-treated foam cells For FTY720 LC3-II did notchange whereas TGF-120573 Arg1 andMrc1 were augmented and IL-6 was suppressed inmacrophages However LC3-II was increasedand TGF-120573 ARG1 and MRC1 were strikingly augmented whereas IL-6 COX2 and iNOS could be suppressed in foam cellsFurthermore lipid accumulation was alleviated in FTY720-treated foam cells Additionally S1pr1 was markedly decreased in foamcells (P lt 05) S1pr2 S1pr3 S1pr4 and S1pr5 were unchanged in rapamycin-treated foam cells In FTY720-treated foam cells S1pr3and S1pr4 were decreased and S1pr1 S1pr2 and S1pr5 were unchangedTherefore we deduced that rapamycin stimulated classicallyactivatedmacrophages and supressed early atherosclerosis Rapamycinmay also stabilize artery plaques by preventing apoptosis andS1PR1 in advanced atherosclerosis FTY720 allowed transformation of foam cells into alternatively activated macrophages throughthe autophagy pathway to alleviate advanced atherosclerosis

1 Introduction

Atherosclerosis one of the most harmful human diseasesof large and medium artery walls leads to acute myocardialinfarction and sudden death [1] It has been demonstratedthat atherosclerosis involves lipid accumulation andinflammatory infiltration [1] and that macrophages playa crucial role in pathogenesis During the initial phaseof atherosclerosis development circulating monocytesmigrate into the arterial wall via dysfunctional endothelialcells and then differentiate into macrophages [2ndash4] Next

macrophages engulf oxidized low-density lipoprotein (ox-LDL) to digest and transport lipids out of the vascular wall[5] When overloaded with lipid droplets macrophages willtransform into foam cells that initiate plaque formationinside the blood vessels [6] This inflammatory processappears to be a hallmark of atherosclerosis [7ndash9] Thusdecreasing macrophage foam cell formation would be anattractive strategy for reversing atherosclerosis

Macrophage phenotype emerges in response to themicroenvironment in a process referred to as macrophage

HindawiBioMed Research InternationalVolume 2018 Article ID 1010248 9 pageshttpsdoiorg10115520181010248

2 BioMed Research International

activation or polarization [10] Macrophages are either clas-sically activated (M1) or alternatively activated (M2) M1macrophages are activated by treatment with interferon-120574 orlipopolysaccharide whereas M2 macrophages are activatedby treatment with Th2 cytokines interleukin- (IL-) 4 or IL-13 the M2 phenotype switch can be enhanced by IL-10 Earlyin the innate immune response M1 macrophages producereactive oxygen species and proinflammatory cytokines andchemokines to drive inflammation thus they are referredto as ldquokillerrdquo macrophages During the resolution phase ofinflammation M2macrophages scavenge debris and assist inangiogenesis and wound healing thus they are referred toas ldquohealerrdquo macrophages [11] During atherosclerosis devel-opment there is differential polarization of macrophagesthat results in differences in the number and distributionof polarization macrophages within the plaque M1 and M2macrophages link to produce atherosclerotic plaques andthe M2 macrophages can resist foam cell transformation[2] Thus selective removal of macrophages or alteringpolarization status within the plaque may have a role inalleviating atherosclerosis

2-Amino-2-[2-(4-octylphenyl)ethyl]propane-13-diol hydro-chloride (FTY720) also known as fingolimod is an immune-modulating drug used to treat multiple sclerosis andmultipleorgan transplantation It is both a synthetic sphingosine 1-phosphate (S1P) analogue and an S1P receptor modulator[12]The drugmay serve as a functional antagonist or agonistdepending on the S1P receptor subtype and target cell ortissue S1P induces M2 phenotype polarization via IL-4 toprotect against atherosclerosis development [13] Some stud-ies have shown that FTY720 reduces atherosclerosis by sup-pressing monocytemacrophage migration to atheroscleroticlesions [14] Short-term low-dose oral FTY720 has showngreat benefit in inhibiting early development of atheroscle-rosis via induction of regulatory T-cells and inhibition ofeffector T-cell response in apolipoprotein E-deficient micefed a high-cholesterol diet [15] Moreover FTY720 treatmentof low-density lipoprotein receptor- (LDLR-) deficient micefed a cholesterol-rich diet activates M2 phenotype marker IL-4 in peritoneal macrophages to reduce atherosclerotic lesionformation in a dose-dependent manner Concentrations ofproinflammatory cytokines such as tumor necrosis factor-120572IL-6 and IL-12 are also reduced [12] However FTY720 failedto affect atherosclerosis in moderately hypercholesterolemicLDLRminusminus mice [16] Thus some important questions remainregarding how FTY720 affects macrophage function andwhether FTY720 plays a role in alleviating atherosclerosisthrough interaction with foam cells

Autophagy is an evolutionarily conserved physiologicself-protective process Autophagy is classically considered apathway that contributes to cellular homeostasis and adapta-tion to stress [17] Dysfunctional autophagy is associated withsome human diseases A limited number of clinical studieshave shown that autophagy is impaired in the advancedstages of atherosclerosis and that its deficiency induceslethal accumulation of cholesterol crystals and promotesatherosclerosis [18ndash20] Stents eluting the mTOR inhibitoreverolimus selectively clear macrophages by autophagy inrabbit model atherosclerotic plaques to promote stability

without affecting smooth muscle cell viability [21] Sev-eral examinations have also shown that mice with Atg5(an essential autophagy gene) macrophage-specific deletiondevelop plaques characterized by increased apoptosis oxida-tive stress and plaque necrosis [22]These results suggest thatmacrophage autophagy plays an essential but complicatedrole in the pathogenesis of atherosclerosis Moreover invivo experiments have shown that rapamycin an mTORinhibitor reduces macrophage death rate and delays plaqueprogression through autophagy upregulation [23] In vitroexperiments show that rapamycin not only reduces intracel-lular lipid droplet accumulation but also inhibits cell apop-tosis by clearing dysfunctional mitochondria and loweringintracellular reactive oxygen species levels during foam celldevelopment [23] In that study atherosclerosis developmentwas characterized by macrophage autophagy inhibition andchanges to the distribution and rate of macrophage polar-ization [23] Therefore we deduced that selective promotionof macrophage autophagy may reduce M1 macrophagesincrease M2 macrophages and alter macrophage foam cellsto stabilize vulnerable atherosclerotic plaques

Additionally FTY720 can induce autophagy in somecancer cells [24ndash26] However little is known about whetherFTY720 mediates macrophage autophagy and polarizationin atherosclerosis One previous study showed that FTY720stimulated production of 27-hydroxycholesterol an endoge-nous ligand of the liver X receptor to cause liver X receptor-induced upregulation of ATP-binding cassette subfamilyA member 1 (ABCA1) It also conferred atheroprotectiveeffects independent of sphingosine 1-phosphate receptor(S1PR) activation in human primary macrophages [27]Furthermore FTY720 inhibited inflammatory factors inendothelial and vascular smooth muscle cells through S1PR1and S1PR3 and inhibited secretion of monocyte chemotacticprotein 1 (MCP-1) by S1PR3 [14] In vivo and in vitroexperiments indicate that S1P mediates S1PR3 to recruitmonocytesmacrophages and change smooth muscle cells toprotect them from atherosclerosis [28] Although FTY720has a role in lipid metabolism and macrophage migrationto reduce atherosclerosis the drug may act as a functionalantagonist or agonist depending on S1P receptor subtype andcell or tissue target Consequently this study was designedto assess the roles of rapamycin and FTY720 on autophagyand polarization of macrophage cells and foam cells andto explore the S1PR macrophage foam cells to identify newapproaches for reducing atherosclerosis

2 Materials and Methods

21 Animal B6D2F1 mice aged 8 to 10 weeks were pur-chased from Vital River (Beijing China) Animals weretreated according to the Guide for the Care and Use ofLaboratoryAnimals All animal experiments were performedunder the Code of Practice Harbin Medicine UniversityEthics Committees

22 Peritoneal Macrophages and Culture Mouse macro-phages were isolated from the peritoneal cavity of B6D2F1

BioMed Research International 3

mice 3 days after treatment with 3 thioglycolate (T9032-500G Sigma-Aldrich) Macrophages were cultured at 37∘Cin a 5 CO

2humidified incubator Isolated macrophages

were maintained in RPMI 1640 (22400089 Invitrogen) con-taining 10 fetal bovine serum (FBS 04-001-1A BI) and 50120583gmL penicillinstreptomycin (15140-148 Invitrogen) for 24h Next the medium was changed and the cells for RNAor protein analysis were removed 24 h after treatment withFTY720 (SML0700 Sigma-Aldrich) or rapamycin (ab120224Abcam)

23 Immunofluorescence Staining Cells were fixed in 4paraformaldehyde for 20 min at 4∘C Next cells were rinsedwith 025 Triton X-100 in phosphate-buffered saline (PBS)and incubated with 70 alcohol for 5 min before beingincubated with blocking buffer for 30 min at room temper-ature For F480 staining cells were incubated with F480(565612 BD) overnight at 4∘C and then counterstained withHoechst 33342 (14533-100MG Sigma-Aldrich) Lastly cellswere mounted on glass slides and examined using a Nikonmicroscope

24Western Blot Cells were lysed using RIPA buffer (Sigma-Aldrich) containing Halt Protease and Phosphatase InhibitorCocktail (Thermo Fisher Philadelphia PA) for 20 minon ice Samples were incubated at 70∘C for 15 min inNuPAGE sample buffer (Life Technologies) and proteinswere separated on NuPAGE 4 to 12 Bis-Tris gels beforetransfer to PVDF (Life Technologies) for immunoblottingSeveral primary antibodies were used for detection anti-LC3(14600-1-AP Proteintech) anti-TGF-120573 (ab66043 Abcam)anti-IL-6 (21865-1-AP Proteintech) anti-COX2 (12375-1-APProteintech) anti-Arg1 (16001-1-AP Proteintech) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH KC-5G4 KANGCHEN) All secondary antibodies used for visu-alization were either goat anti-mouse or goat anti-rabbit andwere purchased from Abcam Blots were developed withthe SuperSignal West Pico Chemiluminescent Substrate orSuperSignal West Femto Maximum Sensitivity Substrate Kit(Thermo Fisher) and visualized by the ImageQuant LAS4000 biomolecular imager (GE Healthcare Life SciencesPittsburgh PA) Densitometry analysis was completed withthe help of ImageJ software which allows for quantificationof band intensity A rectangle was placed on each band andthe band intensity and background intensity were analyzedQuantification was determined by subtracting band intensityfrom background intensity Protein expression was correctedwith a loading control such as GAPDH by dividing theprotein densitometry value All western blot data is presentedas protein densitometrycontrol protein densitometry

25 Foam Cell Formation Copper ox-LDL (2 mgmL) waspurchased from Peking Union-Biology Co Ltd (China)Mouse peritoneal macrophages were then seeded in a 12-well or 6-well cell culture (Corning) in RPMI 1640 mediacontaining 10 fetal bovine serum (FBS) and allowed toadhere overnight The next day cells were treated with ox-LDL (150 120583gmL) for 48 h

26 Oil Red O Staining Oil Red O stock solution (05) in100 isopropanol was diluted to 60 (volvol) isopropanolusing distilled waterThe solution was then filtered to removeparticulates After incubation with ox-LDL cells were rinsedtwice with PBS and fixed with 4 paraformaldehyde in PBSfor 15 min at room temperature Next cells were rinsed twicewith PBS and stained with a filtered Oil Red O solution for 1h at room temperature They were rinsed with distilled waterand mounted using aqueous mounting media

27 Real-Time Polymerase Chain Reaction RNA samplesfrom differentially treated macrophages were extracted usingTRIzol Reagent (15596026 Invitrogen) cDNA was synthe-sized from mRNA using the High Capacity cDNA ReverseTranscription Kit (AT341-02 TransGen Biotech) Real-timePCR was performed using 1 120583L of cDNA 10 120583L TransStartTM Top Green real time PCR SuperMix (AQ131 TransGenBiotech) and gene-specific primers in a 20 120583L reactionsystem on the CFX96 Real-Time System (Bio-Rad) Specificprimers were obtained from a primer bank for mouse Arg1(forward primer 51015840-CTCCAAGCCAAAGTCCTTAGAGreverse primer 51015840-AGGAGCTGTCATTAGGGACATC)Mrc1 (forward primer 51015840-CTCTGTTCAGCTATTGGA-CGC reverse primer 51015840-GGAATTTCTGGGATTCAGCTT-C) iNOS (forward primer 51015840-GTTCTCAGCCCAACAATA-CAAGA reverse primer 51015840-GTGGACGGGTCGATGTCA-C) IL-6 (forward primer 51015840-CCAAGAGGTGAGTGCTTC-CC reverse primer 51015840-CTGTTGTTCAGACTCTCTCCCT)S1PR1 (forward primer 51015840-ATGGTGTCCACTAGCATCCCreverse primer 51015840-CGATGTTCAACTTGCCTGTGTAG)S1PR2 (forward primer 51015840-ATGGGCGGCTTATACTCA-GAG reverse primer 51015840-GCGCAGCACAAGATGATGAT)S1PR3 (forward primer 51015840-TTTCATCGGCAACTTGGC-TCT reverse primer 51015840-GCTACGAACATACTGCCCTCC)S1PR4 (forward primer 51015840-GTCAGGGACTCGTACCTT-CCA reverse primer 51015840-GATGCAGCCATACACACGG)and S1PR5 (forward primer GCTTTGGTTTGCGCGTGA-G reverse primer 51015840-GGCGTCCTAAGCAGTTCCAG)GAPDH was used as an internal control and each samplewas run in triplicateThe 2-ΔΔCtmethod was used to analyzeqPCR gene expression data Significance was assessed usingtwo-tailed Studentrsquos t-test to compare the levels of DE genesfor different groups (P lt 05)

28 Statistical Analysis Data were expressed as means plusmnstandard deviations and were tested for normality by SPSSStatistics (P gt 05)Then ANOVAwas performed on the datausing SPSS Statistics A value of P lt 05 was considered to bestatistically significant A value of P lt 01 was considered tobe more statistically significant

3 Results

31 Rapamycin Induces M1 Polarization by Increasing Macro-phage Autophagy Peritoneal macrophages were isolated asdescribed previously Immunofluorescence analysis showeda positivity rate of 876 for F480 (Figure 1(a)) To inves-tigate the effect of autophagy on macrophages expressionof autophagic markers LC3I and LC3II in macrophages was


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Figure 1 Rapamycin promoted autophagy in peritoneal macrophages and activated theM1 phenotype (a) Peritoneal macrophages subjectedto immunofluorescence staining by macrophage marker F480 antibody (b) western blot showing LC3 expression in various rapamycin-(Rap-) treated groups relative protein and GAPDH levels shown as histograms (c) real-time PCR showing expression of Arg1 Mrc1 IL-6and iNOS in various rapamycin-treated groups (d) western blot showing expression of IL-6 TGF-120573 COX2 and ARG1 in various rapamycin-(Rap-) treated groups (e) relative protein and GAPDH levels shown as histograms lowastP lt 05 and lowastlowastP lt 01 vs untreated controls Results arerepresentative of 3 independent experiments each experiment was repeated 3 times

analyzed Cells were cultured with or without autophagyactivator rapamycin for 24 h After treatment cells wereharvested and proteins were collected for western blot analy-sis As shown in Figure 1(b) rapamycin increased expressionof LC3-II at different doses Furthermore real-time PCRshowed that IL-6 and iNOS (M1 markers) were increasedwhereas Arg1 and Mrc1 (M2 markers) were decreased(Figure 1(c)) Moreover western blot analysis demonstratedthat IL-6 and COX2 (M1markers) were higher in rapamycin-treated compared with untreated macrophages Meanwhileexpression of TGF-120573 and ARG1 (M2 markers) was lowerin rapamycin-treated compared with untreated macrophages(Figures 1(d) and 1(e))This finding suggested that autophagycould induce M1 polarization

32 Rapamycin Does Not Affect Foam Cell PolarizationMacrophage foam cells indicate advanced stage atheroscle-rosis To generate foam cells macrophages were treated byox-LDL for 48 h Oil Red O staining showed that ox-LDLincreased lipid accumulation and that this accumulation wasdecreased by rapamycin treatment (Figures 2(a) and 2(b)) Todetermine whether autophagy affected polarization of foam

cells rapamycin was used at various doses First westernblot analysis demonstrated that autophagy marker LC3IIwas elevated in the presence of rapamycin Next real-timePCR showed that rapamycin had almost no effect on foamcell polarization (Figure 2(c)) Western blot analysis verifiedthat TGF-120573 ARG1 and COX2 expression were unchangedin rapamycin-treated foam cells IL-6 increased slightly infoam cells treated with 1 120583M rapamycin (Figures 2(d) and2(e)) However lipid accumulation did not differ betweenrapamycin-treated foam cells and ox-LDL-treated foam cells(Figures 2(a) and 2(b))Thus autophagy appeared to have noeffect on foam cell polarization

33 FTY720 Reduces Lipid Accumulation by M2 Polariza-tion of Foam Cells A previous study reported that M2polarization by S1P was responsible for the antiatherogenicproperties of high-density lipoproteins in vivo [13] FTY720was not only an S1P analogue but also an S1P receptormodulator that had contradicting roles in lipid metabolismand macrophage migration in atherosclerotic disease Totest the effect of FTY720 on polarization FTY720-treatedmacrophages and foam cells were used Real-time PCR


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Figure 2 Rapamycin had no effect on foam cell polarization (a) Oil Red O staining demonstrated that rapamycin inhibited macrophagetransformation to foam cells but did not affect foam cells (b) percentage of foam cells with high intensity from 3 experimental iterations 6views were analyzed from 2 randomly selected staining wells lowastP lt 05 and lowastlowastP lt 01 vs ox-LDL (c) real-time PCR showing Arg1 Mrc1 IL-6and iNOS expression for various rapamycin-treated groups (d) western blot showing expression of LC3 IL-6 COX2 TGF-120573 and ARG1 infoam cells treated with various concentrations of rapamycin (e) results shown representative of 3 independent experiments each experimentrepeated 3 times Protein and GAPDH relative levels are shown as histograms P lt 05 and lowastlowastP lt 01 vs untreated controls MΦ macrophagebar 100 120583m

showed that high-dose FTY720 treatment of macrophagesincreased Arg1 and Mrc1 reduced IL-6 and did not changeiNOS However Arg1 and Mrc1 were increased whereasIL-6 and iNOS were decreased in FTY720-treated foamcells (Figure 3(a)) Western blot demonstrated that TGF-120573 and ARG1 expression were increased in both FTY720-induced macrophages and FTY720-induced foam cells how-ever ARG1 expression did not change in FTY720-treated

foam cells (Figures 3(b) and 3(c)) Additionally westernblot was used to verify that IL-6 and COX2 were reducedin FTY720-treated macrophages and foam cells but COX2was not reduced in FTY720-induced macrophages Theseresults indicate that FTY720 could improve M2 polarizationof macrophages and foam cells Furthermore LC3 expressionwas detected in macrophages and foam cells However LC3IIexpression differed in both cell types LC3II expression was


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Figure 3 FTY720 reduces lipid accumulation by M2 polarization of foam cells (a) Real-time PCR showing Arg1 Mrc1 IL-6 and iNOSexpression with various concentrations of rapamycin treatment inmacrophages and foam cells (b)Western blot showing LC3 ARG1 COX2IL-6 and TGF-120573 in macrophages and foam cells treated with various concentrations of rapamycin (c) Results shown representative of 3independent experiments each experiment repeated 3 times Data representative of protein and relative GAPDH are shown as histogramslowastP lt 05 vs untreated controls (d) Oil Red O staining demonstrating that FTY720 reduced lipid accumulation in macrophage foam cellsbar 100 120583m (e) Percentage of foam cells with high intensity from 3 experimental iterations 6 views were analyzed from 2 randomly selectedstaining wells lowastP lt 05 and lowastlowastP lt 01 vs ox-LDL

increased in FTY720-treated foam cells but not in FTY720-treated macrophages (Figures 3(a) and 3(b)) Thus FTY720appeared to transform the polarization of foam cells Addi-tionally lipid accumulation was decreased in FTY720-treatedfoam cells (Figures 3(d) and 3(e)) but not FTY720-treatedmacrophagesThis finding suggests that FTY720 played a keyrole in M2 polarization to alleviate advances in atherosclero-sis autophagy may also promote M2 polarization

34 S1PR Involved in FTY720 and Rapamycin Regulation ofFoam Cells Previous research shows that S1P receptors areinvolved in atherosclerosis possibly through endoplasmicreticulum (ER) stress not PI3Kbeclin1 or mTOR [29] Inthis study S1PR was detected in rapamycin-induced and

FTY720-induced foam cells Real-time PCR showed thatS1pr1 expression was lower in rapamycin-treated comparedwith untreated foam cells (P lt 05 Figure 4(a)) HoweverFTY720 inhibited expression of S1pr3 and S1pr4 in foam cells(Figure 4(a))

Macrophage apoptosis in atherosclerotic plaques mayincrease plaque destabilization Therefore the next investi-gation aimed to determine whether FTY720-mediated M2polarization caused macrophages to resist apoptosis Expres-sion of cleaved-CASPASE 3 was slightly decreased in 5120583M FTY720-mediated foam cells but was unchanged inrapamycin-treated foam cells (Figures 4(b) and 4(c)) Theseresults support the antiapoptotic and atheroprotective effectsof FTY720


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Figure 4 S1PR involvement in FTY720- and rapamycin-regulated foam cells (a) real-time PCR analysis shown for S1pr1-5 expression in foamcells (b) western blot showing apoptosis marker cleaved-CASPASE 3 and total CASPASE 3 (c) data representative of cleaved-CASPASE 3levels relative to total CASPASE 3 levels shown as histograms lowastPlt 05 vs untreated controls Results shown are representative of 3 independentexperiments each experiment was repeated 3 times

4 Discussion

Atherosclerosis is characterized by persistent inflammationof the arterial wallMacrophages themost abundant immunecells in atherosclerotic plaques can be transformed tofoam cells by oxidized low-density lipoprotein (ox-LDL)induction which accelerates atherosclerosis [1 6ndash9] Thusmacrophages and foam cells are crucial in different stagesof atherosclerotic disease and as such are attractive targetsfor therapyMoreover macrophages are functionally complexin their adoption of multiple polarization states based onthe composition of their microenvironment Previous reportsshow that defective phagocytic clearance of macrophagespromotes plaque necrosis and inhibition of autophagy bysilencing ATG5 or other autophagy mediators to enhanceprotective processes in atherosclerosis [22] FurthermoremTOR mediates cross talk of macrophage polarization andautophagy in atherosclerosis [30] However the polariza-tion and autophagy of foam cells in the pathogenesis ofatherosclerosis remain largely unexplained Understandingcross talk of autophagy and polarization regulation is crucialto understanding pathogenesis

In our studies we found that macrophage and foam cellautophagy produce different effects Rapamycin is a recog-nized autophagy activator through its inhibition of mTORsignaling in various cells By increasing LC3II expression wefirst verified the increased autophagy induced by rapamycinin both macrophages and foam cells Furthermore ourfinding that rapamycin inducedM1 polarization by increasingIL-6 iNOS and COX2 expression while decreasing TGF-120573

Arg1 and MRC1 expression in macrophages is consistentwith a previous report [31] However we found no effecton foam cell polarization Moreover Oil Red O stainingshowed decreased lipid accumulation in rapamycin-treatedmacrophages but not rapamycin-treated foam cells Thisfinding suggests that M1 macrophages may inhibit foam cellformation M1macrophages have been previously reported topromote inflammation through direct targeting of the NF-120581Bpathway [32]

Atherosclerosis is a chronic immunoinflammatory dis-ease [1] Here we found that M1 macrophages can enhanceautophagy by inhibiting mTOR signaling a key regulatorof atherosclerosis Thus mTOR depletion stimulates M1macrophages and suppresses early atherosclerosis In thisstudy rapamycin also enhanced autophagy by inhibitingmTOR in foam cells though it did not affect polarizationor alter lipid load In vivo results showed that rapamycinattenuated the macrophage death rate and delayed plaqueprogression through autophagy upregulation [23 33 34]Wang and colleagues previously showed that cholesterolefflux was increased by autophagy in macrophage foam cells[35] Hence rapamycin-induced autophagy in foam cellsdelays intracellular lipid accumulation and may stabilizeartery plaques through prevention of apoptosis and S1PR1 inadvanced atherosclerosis

FTY720 a novel S1P analogue [12] and a potent immuno-suppressive drug acted as a sphingosine kinase (SphK)1 antagonist growth inhibitor and apoptosis inducer invarious human cancer cell lines In this study FTY720 didnot affect autophagy in macrophages but its activity in


suppressing proinflammatory factor IL-6 in macrophageswas consistent with a previous study [36] In animal mod-els FTY720 reduced atherosclerosis by suppressing mono-cytemacrophage migration inducing a regulatory T-cellresponse and inhibiting effector T-cell responses [14 15]Moreover the M1 phenotype marker-IL-6 was inhibitedwhereas the M2 phenotype marker IL-4 was increased inperitoneal macrophage cells from FTY720-treated LDLR-deficient mice fed a cholesterol-rich diet [12] In the presentstudy FTY720 also inhibited M1 phenotype markers IL-6 COX2 and iNOS promoted M2 markers TGF-120573 Arg1and Mrc1 in macrophage foam cells and reduced lipidaccumulation in macrophage foam cells as shown by Oil RedO staining LC3-II was notably increased in FTY720-treatedfoam cells compared with FTY720-treated macrophageswhich have been shown in some cancer studies to inducethe autophagy pathway [24ndash26 37ndash39] Consequently wededuced that FTY720 affected macrophage function andpolarization through constriction of the M1 phenotype andactivation of the M2 phenotype via the autophagy pathway toalleviate advanced atherosclerosis Additionally the findingthat FTY20 inhibited S1PR3 and S1PR4 in macrophagefoam cells was similar to previous reports that FTY20could suppress S1PR1 and S1PR3 to decrease inflammatoryfactors and alleviate atherosclerosis [14 28] It is possiblethat FTY720 suppresses S1PR3 and S1PR4 to reduce lipidaccumulation

5 Conclusion

In summary rapamycin induced autophagy promoted theM1 phenotype suppressed the M2 phenotype and inhibitedfoam cell formation in peritoneal macrophages Additionallyrapamycin activated autophagy in peritoneal macrophagefoam cells but did not affect macrophage polarization orreduce lipid accumulation Thus the mTOR depletion ofmacrophage cells stimulated M1 macrophages and sup-presses early atherosclerosis FTY720 could promote the M2phenotype and suppress the M1 phenotype in peritonealmacrophages and foam cells but it only activated autophagyin peritoneal macrophage foam cells and alleviated lipidaccumulation FTY720 transformation of foam cells into theM2 phenotype occurred through the autophagy pathway andalleviated advanced atherosclerosis

Data Availability

The data used to support the findings of this study areincluded within the article

Conflicts of Interest

All authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Rui-zhen Sun and Ying Fan contributed equally to this study

Acknowledgments

This study was supported by the Nature Scientific Foundationof Heilongjiang Province (H201442 C2018040) and theHeilongjiang Provincial Education Department (12541450)

References

[1] G K Hansson ldquoInflammation atherosclerosis and coronaryartery diseaserdquo e New England Journal of Medicine vol 352no 16 pp 1685ndash1695 2005

[2] K J Moore F J Sheedy and E A Fisher ldquoMacrophages inatherosclerosis a dynamic balancerdquo Nature Reviews Immunol-ogy vol 13 no 10 pp 709ndash721 2013

[3] K Ley Y I Miller and C C Hedrick ldquoMonocyte andmacrophage dynamics during atherogenesisrdquo Arteriosclerosisrombosis and Vascular Biology vol 31 no 7 pp 1506ndash15162011

[4] K J Moore and I Tabas ldquoMacrophages in the pathogenesis ofatherosclerosisrdquoCell vol 145 no 3 pp 341ndash355 2011

[5] M Ouimet and Y L Marcel ldquoRegulation of lipid dropletcholesterol efflux frommacrophage foam cellsrdquoArteriosclerosisrombosis and Vascular Biology vol 32 no 3 pp 575ndash5812012

[6] C Schulz and SMassberg ldquoAtherosclerosis -Multiple pathwaysto lesional macrophagesrdquo Science Translational Medicine vol 6no 239 Article ID 239ps2 2014

[7] J D Plotkin M G Elias A L Dellinger and C L Kepley ldquoNF-120581B inhibitors that prevent foam cell formation and atheroscle-rotic plaque accumulationrdquo Nanomedicine NanotechnologyBiology and Medicine vol 13 no 6 pp 2037ndash2048 2017

[8] I Tabas G Garcıa-Cardena and G K Owens ldquoRecent insightsinto the cellular biology of atherosclerosisrdquoe Journal of CellBiology vol 209 no 1 pp 13ndash22 2015

[9] X Feng Y Yuan C Wang et al ldquoAutophagy involved inlipopolysaccharide-induced foam cell formation is mediated byadipose differentiation-related proteinrdquo Lipids in Health andDisease vol 13 no 1 article no 10 2014

[10] P Murray J Allen S Biswas et al ldquoMacrophage activationand polarization nomenclature and experimental guidelinesrdquoImmunity vol 41 no 1 pp 14ndash20 2014

[11] S B Weisser K W McLarren E Kuroda and L M Sly ldquoGen-eration and characterization of murine alternatively activatedmacrophagesrdquoMethods in Molecular Biology vol 946 pp 225ndash239 2013

[12] J-R Nofer M Bot M Brodde et al ldquoFTY720 a syntheticsphingosine 1 phosphate analogue inhibits development ofatherosclerosis in low-density lipoprotein receptor-deficientmicerdquo Circulation vol 115 no 4 pp 501ndash508 2007

[13] S-J Park K-P Lee S Kang et al ldquoSphingosine 1-phosphateinduced anti-atherogenic and atheroprotectiveM2macrophagepolarization through IL-4rdquoCellular Signalling vol 26 no 10 pp2249ndash2258 2014

[14] P Keul M Tolle S Lucke et al ldquoThe sphingosine-1-phosphateanalogue FTY720 reduces atherosclerosis in apolipoproteinE-deficient micerdquo Arteriosclerosis rombosis and VascularBiology vol 27 no 3 pp 607ndash613 2007

[15] K Huang S Q Li W J Wang et al ldquoOral FTY720 administra-tion induces immune tolerance and inhibits early developmentof atherosclerosis in apolipoprotein E-deficient micerdquo Interna-tional Journal of Immunopathology and Pharmacology vol 25no 2 pp 397ndash406 2012


[16] F Poti S Costa V Bergonzini et al ldquoEffect of sphingosine 1-phosphate (S1P) receptor agonists FTY720 and CYM5442 onatherosclerosis development in LDL receptor deficient (LDL-R--) micerdquoVascular Pharmacology vol 57 no 1 pp 56ndash64 2012

[17] NMizushima andMKomatsu ldquoAutophagy renovation of cellsand tissuesrdquo Cell vol 147 no 4 pp 728ndash741 2011

[18] M M Kockx G R Y De Meyer N Buyssens M W MKnaapen H Bult and A G Herman ldquoCell composition repli-cation and apoptosis in atherosclerotic plaques after 6 monthsof cholesterol withdrawalrdquo Circulation Research vol 83 no4 pp 378ndash387 1998

[19] W Martinet and G R Y de Meyer ldquoAutophagy in atheroscle-rosis a cell survival and death phenomenon with therapeuticpotentialrdquo Circulation Research vol 104 no 3 pp 304ndash3172009

[20] W Li N Sultana N Siraj et al ldquoAutophagy dysfunction andregulatory cystatin C in macrophage death of atherosclerosisrdquoJournal of Cellular and Molecular Medicine vol 20 no 9 pp1664ndash1672 2016

[21] S Verheye W Martinet M M Kockx et al ldquoSelective clear-ance of macrophages in atherosclerotic plaques by autophagyrdquoJournal of the American College of Cardiology vol 49 no 6 pp706ndash715 2007

[22] X Liao J C Sluimer Y Wang et al ldquoMacrophage autophagyplays a protective role in advanced atherosclerosisrdquo CellMetabolism vol 15 no 4 pp 545ndash553 2012

[23] X Liu Y Tang Y Cui H Zhang and D Zhang ldquoAutophagy isassociated with cell fate in the process of macrophage-derivedfoam cells formation and progressrdquo Journal of BiomedicalScience vol 23 no 1 article no 57 2016

[24] N Zhang Y Qi andCWadham ldquoFTY720 induces necrotic celldeath and autophagy in ovarian cancer cells a protective role ofautophagyrdquo Autophagy vol 6 no 8 pp 1157ndash1167 2010

[25] L Zhang H Wang K Ding and J Xu ldquoFTY720 inducesautophagy-related apoptosis and necroptosis in humanglioblastoma cellsrdquo Toxicology Letters vol 236 no 1 pp 43ndash592015

[26] J Li S-WWang D-S Zhang et al ldquoFTY720-induced enhance-ment of autophagy protects cells from FTY720 cytotoxicity incolorectal cancerrdquo Oncology Reports vol 35 no 5 pp 2833ndash2842 2016

[27] T Blom N Back A-L Mutka et al ldquoFTY720 stimulates 27-hydroxycholesterol production and confers atheroprotectiveeffects in human primary macrophagesrdquo Circulation Researchvol 106 no 4 pp 720ndash729 2010

[28] P Keul S Lucke K Von Wnuck Lipinski et alldquoSphingosine-1-phosphate receptor 3 promotes recruitment ofmonocytemacrophages in inflammation and atherosclerosisrdquoCirculation Research vol 108 no 3 pp 314ndash323 2011

[29] S Lepine J C Allegood M Park P Dent S Milstienand S Spiegel ldquoSphingosine-1-phosphate phosphohydrolase-1regulates ER stress-induced autophagyrdquo Cell Death amp Differen-tiation vol 18 no 2 pp 350ndash361 2011

[30] F Tian B-L Yu and J-R Hu ldquoMTOR mediates the cross-talkof macrophage polarization and autophagy in atherosclerosisrdquoInternational Journal of Cardiology vol 177 no 1 pp 144-1452014

[31] X Zhao Y Li and Y Wang ldquoInterleukin-4 regulatesmacrophage polarization via the MAPK signaling pathwayto protect against atherosclerosisrdquo Genetics and MolecularResearch vol 15 no 1 2016

[32] C-P Chang Y-C Su P-H Lee andH-Y Lei ldquoTargetingNFKBby autophagy to polarize hepatoma-associated macrophagedifferentiationrdquo Autophagy vol 9 no 4 pp 619ndash621 2013

[33] L Wang Y Jiang X Song et al ldquoPdcd4 deficiency enhancesmacrophage lipoautophagy and attenuates foam cell formationand atherosclerosis in micerdquoCell DeathampDisease vol 7 ArticleID e2055 2016

[34] R Pakala E Stabile G J Jang L Clavijo and R WaksmanldquoRapamycin attenuates atherosclerotic plaque progression inapolipoprotein E knockoutmice Inhibitory effect onmonocytechemotaxisrdquo Journal of Cardiovascular Pharmacology vol 46no 4 pp 481ndash486 2005

[35] X Wang L Li X Niu et al ldquomTOR enhances foam cellformation by suppressing the autophagy pathwayrdquo DNA andCell Biology vol 33 no 4 pp 198ndash204 2014

[36] H Yu B A Herbert M Valerio L Yarborough L-C Hsu andK M Argraves ldquoFTY720 inhibited proinflammatory cytokinerelease and osteoclastogenesis induced by Aggregatibacter acti-nomycetemcomitansrdquo Lipids in Health and Disease vol 14 no1 article no 57 2015

[37] G Marvaso A Barone and N Amodio ldquoSphingosine analogfingolimod (FTY720) increases radiation sensitivity of humanbreast cancer cells in vitrordquo Cancer Biology amp erapy vol 15no 6 pp 797ndash805 2014

[38] L Zhang K Ding H Wang Y Wu and J Xu ldquoTraumaticBrain Injury-Induced Neuronal Apoptosis is ReducedThroughModulation of PI3K and Autophagy Pathways in Mouse byFTY720rdquoCellular andMolecularNeurobiology vol 36 no 1 pp131ndash142 2016

[39] W-J Leu S P Swain S-H Chan et al ldquoNon-immuno-suppressive triazole-based small molecule induces anticanceractivity against human hormone-refractory prostate cancersThe role in inhibition of PI3KAKTmTOR and c-Myc signalingpathwaysrdquo Oncotarget vol 7 no 47 pp 76995ndash77009 2016

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


activation or polarization [10] Macrophages are either clas-sically activated (M1) or alternatively activated (M2) M1macrophages are activated by treatment with interferon-120574 orlipopolysaccharide whereas M2 macrophages are activatedby treatment with Th2 cytokines interleukin- (IL-) 4 or IL-13 the M2 phenotype switch can be enhanced by IL-10 Earlyin the innate immune response M1 macrophages producereactive oxygen species and proinflammatory cytokines andchemokines to drive inflammation thus they are referredto as ldquokillerrdquo macrophages During the resolution phase ofinflammation M2macrophages scavenge debris and assist inangiogenesis and wound healing thus they are referred toas ldquohealerrdquo macrophages [11] During atherosclerosis devel-opment there is differential polarization of macrophagesthat results in differences in the number and distributionof polarization macrophages within the plaque M1 and M2macrophages link to produce atherosclerotic plaques andthe M2 macrophages can resist foam cell transformation[2] Thus selective removal of macrophages or alteringpolarization status within the plaque may have a role inalleviating atherosclerosis

2-Amino-2-[2-(4-octylphenyl)ethyl]propane-13-diol hydro-chloride (FTY720) also known as fingolimod is an immune-modulating drug used to treat multiple sclerosis andmultipleorgan transplantation It is both a synthetic sphingosine 1-phosphate (S1P) analogue and an S1P receptor modulator[12]The drugmay serve as a functional antagonist or agonistdepending on the S1P receptor subtype and target cell ortissue S1P induces M2 phenotype polarization via IL-4 toprotect against atherosclerosis development [13] Some stud-ies have shown that FTY720 reduces atherosclerosis by sup-pressing monocytemacrophage migration to atheroscleroticlesions [14] Short-term low-dose oral FTY720 has showngreat benefit in inhibiting early development of atheroscle-rosis via induction of regulatory T-cells and inhibition ofeffector T-cell response in apolipoprotein E-deficient micefed a high-cholesterol diet [15] Moreover FTY720 treatmentof low-density lipoprotein receptor- (LDLR-) deficient micefed a cholesterol-rich diet activates M2 phenotype marker IL-4 in peritoneal macrophages to reduce atherosclerotic lesionformation in a dose-dependent manner Concentrations ofproinflammatory cytokines such as tumor necrosis factor-120572IL-6 and IL-12 are also reduced [12] However FTY720 failedto affect atherosclerosis in moderately hypercholesterolemicLDLRminusminus mice [16] Thus some important questions remainregarding how FTY720 affects macrophage function andwhether FTY720 plays a role in alleviating atherosclerosisthrough interaction with foam cells

Autophagy is an evolutionarily conserved physiologicself-protective process Autophagy is classically considered apathway that contributes to cellular homeostasis and adapta-tion to stress [17] Dysfunctional autophagy is associated withsome human diseases A limited number of clinical studieshave shown that autophagy is impaired in the advancedstages of atherosclerosis and that its deficiency induceslethal accumulation of cholesterol crystals and promotesatherosclerosis [18ndash20] Stents eluting the mTOR inhibitoreverolimus selectively clear macrophages by autophagy inrabbit model atherosclerotic plaques to promote stability

without affecting smooth muscle cell viability [21] Sev-eral examinations have also shown that mice with Atg5(an essential autophagy gene) macrophage-specific deletiondevelop plaques characterized by increased apoptosis oxida-tive stress and plaque necrosis [22]These results suggest thatmacrophage autophagy plays an essential but complicatedrole in the pathogenesis of atherosclerosis Moreover invivo experiments have shown that rapamycin an mTORinhibitor reduces macrophage death rate and delays plaqueprogression through autophagy upregulation [23] In vitroexperiments show that rapamycin not only reduces intracel-lular lipid droplet accumulation but also inhibits cell apop-tosis by clearing dysfunctional mitochondria and loweringintracellular reactive oxygen species levels during foam celldevelopment [23] In that study atherosclerosis developmentwas characterized by macrophage autophagy inhibition andchanges to the distribution and rate of macrophage polar-ization [23] Therefore we deduced that selective promotionof macrophage autophagy may reduce M1 macrophagesincrease M2 macrophages and alter macrophage foam cellsto stabilize vulnerable atherosclerotic plaques

Additionally FTY720 can induce autophagy in somecancer cells [24ndash26] However little is known about whetherFTY720 mediates macrophage autophagy and polarizationin atherosclerosis One previous study showed that FTY720stimulated production of 27-hydroxycholesterol an endoge-nous ligand of the liver X receptor to cause liver X receptor-induced upregulation of ATP-binding cassette subfamilyA member 1 (ABCA1) It also conferred atheroprotectiveeffects independent of sphingosine 1-phosphate receptor(S1PR) activation in human primary macrophages [27]Furthermore FTY720 inhibited inflammatory factors inendothelial and vascular smooth muscle cells through S1PR1and S1PR3 and inhibited secretion of monocyte chemotacticprotein 1 (MCP-1) by S1PR3 [14] In vivo and in vitroexperiments indicate that S1P mediates S1PR3 to recruitmonocytesmacrophages and change smooth muscle cells toprotect them from atherosclerosis [28] Although FTY720has a role in lipid metabolism and macrophage migrationto reduce atherosclerosis the drug may act as a functionalantagonist or agonist depending on S1P receptor subtype andcell or tissue target Consequently this study was designedto assess the roles of rapamycin and FTY720 on autophagyand polarization of macrophage cells and foam cells andto explore the S1PR macrophage foam cells to identify newapproaches for reducing atherosclerosis

2 Materials and Methods

21 Animal B6D2F1 mice aged 8 to 10 weeks were pur-chased from Vital River (Beijing China) Animals weretreated according to the Guide for the Care and Use ofLaboratoryAnimals All animal experiments were performedunder the Code of Practice Harbin Medicine UniversityEthics Committees

22 Peritoneal Macrophages and Culture Mouse macro-phages were isolated from the peritoneal cavity of B6D2F1











3 Results



lowast

lowast

lowast

lowast lowast

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowast

Hoechst

Merge

F480

Rapamycin Rapamycin

(a) GAPDH

LC3


GAPDH

TGF-

IL-6Rapamycin(b)

GAPDH

COX2

ARG1

Arg1 Mrc1 IL-6 iNOS


(d)

(c)

(e)


0M 01M 1M 5M0M 1M 5M

Rapamycin0M 1M 5M

Rapamycin0M 1M 5M

Rapamycin0M 1M 5M

0

05

1

15

2

25

Rela

tive e

xpre

ssio

n of

LC3

II

01234567

Rela

tive e

xpre

ssio

n of

IL-6

01234567

Rela

tive e

xpre

ssio

n of

CO

X2

0123456789

10

Rela

tive m

RNA

expr

essi

on

(con

trol

)

0123456789

Rela

tive e

xpre

ssio

n of

ARG

10

02040608

11214

Rela

tive e

xpre

ssio

n of

TG

F-







M+5M rap+ox-LDL

M+ox-LDL

M+ox-LDL +5M rap

M

(a)


M+ox-LDL+Rapamycin

0

20

40

60

80

100

The p

erce

ntag

e of f

oam

cells

in h

igh

inte

nsity


(b)

lowast

lowast

Arg1 Mrc1 IL-6 iNOS


0

02

04

06

08

1

12

14

16

18

Rela

tive m

RNA

expr

essi

on (

cont

rol)

(c)

GAPDH

LC3

+ + +

Rapamycin

ox-LDL

ox-LDL

ox-LDL

-

GAPDH

IL-6

+ + +

Rapamycin -

TGF-

GAPDH

ARG1

+ + +

Rapamycin -

COX2

1M 5M

1M 5M

1M 5M

(d)

lowast

lowast

lowast


ox-LDL+5M Rapamycin


ox-LDL+5M Rapamycin


ox-LDL+5M Rapamycin


ox-LDL+5M Rapamycin


ox-LDL+5M Rapamycin

0

05

1

15

2

25

3

Rela

tive e

xpre

ssio

n of

LC3

II

0

04

08

12

16

Rela

tive e

xpre

ssio

n of

IL-6

0

04

08

12

16

Rela

tive e

xpre

ssio

n of

TG

F-

0

04

08

12

16

Rela

tive e

xpre

ssio

n of

ARG

1

0

05

1

15

2

Rela

tive e

xpre

ssio

n of

CO

X2

(e)





lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast lowast

Arg1 Mrc1 IL-6 iNOS


(a)

(b)

LC-3

GAPDH

+ + +

FTY720

ox-LDL

-

-

GAPDH

TGF-

IL-6

GAPDH

TGF-

IL-6

FTY720

-FTY720

LC-3

GAPDH

+ + +-FTY720

ox-LDL

B

GAPDH

ARG1

COX2

FTY720

GAPDH

ARG1

COX2

+ + +FTY720ox-LDL

(c)

M+ox-LDL

M +FTY720+ox-LDL

M +ox-LDL+FTY720

0

20

40

60

80

100


M+ox-LDL+FTY720

The p

erce

ntag

e of f

oam

cells

in h

igh

inte

nsity

(d)

(e)

0

07

14

21

28

35

Rela

tive e

xpre

ssio

n of

LC3

II

FTY720

FTY720

0

04

08

12

Rela

tive e

xpre

ssio

n of

IL-6

FTY720

0

02

04

06

08

Rela

tive e

xpre

ssio

n of

CO

X2

FTY720

0

05

1

15

2

25

Rela

tive e

xpre

ssio

n of

TG

F-

FTY720


ox-LDL+5M FTY720


ox-LDL+5M FTY720


ox-LDL+5M FTY720


ox-LDL+5M FTY720


ox-LDL+5M FTY720

Arg1 Mrc1 IL-6 iNOS


1M 5M

1M 5M

- 1M 5M- 1M 5M

1M 5M

1M 5M

0

1

2

3

4

5

6Re

lativ

e mRN

A ex

pres

sion

(co

ntro

l)

0

2

4

6

8

10

12

Rela

tive m

RNA

expr

essi

on (

cont

rol)

0

04

08

12

Rela

tive e

xpre

ssio

n of

LC3

II

0M 1M 5M

0M 1M 5M

0M 1M 5M

0M 1M 5M

0M 1M 5M0

08

16

24

32

4

48

Rela

tive e

xpre

ssio

n of

ARG

1

0

05

1

15

2

25

3

35

Rela

tive e

xpre

ssio

n of

ARG

1

lowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

lowastlowastlowast

lowast

BBB0

05

1

15

2

25

Rela

tive e

xpre

ssio

n of

TG

F-

0

08

16

24

32

4Re

lativ

e exp

ress

ion

of C

OX

20

04

08

12

16

Rela

tive e

xpre

ssio

n of

IL-6

lowast







C-CASPASE3

GAPDH

C-CASPASE3

GAPDH


ox-LDL + + + - 1M 5M 1M 5M

005

115

225

335

445

5


tive m

RNA

expr

essi

on (

cont

rol)




CASPASE3 CASPASE3


ox-LDL+5M Rapamycin


ox-LDL+5M FTY720

0123456789

Rela

tive m

RNA

expr

essi

on (

cont

rol)

lowast

lowast lowast

0

02

04

06

08

1

C-CA

SEPA

SE3

TOTA

L CA

SPA

SE3

lowast

lowast

0

04

08

12

16

2

C-CA

SEPA

SE3

TOTA

L CA

SPA

SE3

(a)

(b)

(c)


4 Discussion








5 Conclusion


Data Availability






Acknowledgments


References













































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





























3 Results



lowast

lowast

lowast

lowast lowast

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowast

Hoechst

Merge

F480

Rapamycin Rapamycin

(a) GAPDH

LC3


GAPDH

TGF-

IL-6Rapamycin(b)

GAPDH

COX2

ARG1

Arg1 Mrc1 IL-6 iNOS


(d)

(c)

(e)


0M 01M 1M 5M0M 1M 5M

Rapamycin0M 1M 5M

Rapamycin0M 1M 5M

Rapamycin0M 1M 5M

0

05

1

15

2

25

Rela

tive e

xpre

ssio

n of

LC3

II

01234567

Rela

tive e

xpre

ssio

n of

IL-6

01234567

Rela

tive e

xpre

ssio

n of

CO

X2

0123456789

10

Rela

tive m

RNA

expr

essi

on

(con

trol

)

0123456789

Rela

tive e

xpre

ssio

n of

ARG

10

02040608

11214

Rela

tive e

xpre

ssio

n of

TG

F-







M+5M rap+ox-LDL

M+ox-LDL

M+ox-LDL +5M rap

M

(a)


M+ox-LDL+Rapamycin

0

20

40

60

80

100

The p

erce

ntag

e of f

oam

cells

in h

igh

inte

nsity


(b)

lowast

lowast

Arg1 Mrc1 IL-6 iNOS


0

02

04

06

08

1

12

14

16

18

Rela

tive m

RNA

expr

essi

on (

cont

rol)

(c)

GAPDH

LC3

+ + +

Rapamycin

ox-LDL

ox-LDL

ox-LDL

-

GAPDH

IL-6

+ + +

Rapamycin -

TGF-

GAPDH

ARG1

+ + +

Rapamycin -

COX2

1M 5M

1M 5M

1M 5M

(d)

lowast

lowast

lowast


ox-LDL+5M Rapamycin


ox-LDL+5M Rapamycin


ox-LDL+5M Rapamycin


ox-LDL+5M Rapamycin


ox-LDL+5M Rapamycin

0

05

1

15

2

25

3

Rela

tive e

xpre

ssio

n of

LC3

II

0

04

08

12

16

Rela

tive e

xpre

ssio

n of

IL-6

0

04

08

12

16

Rela

tive e

xpre

ssio

n of

TG

F-

0

04

08

12

16

Rela

tive e

xpre

ssio

n of

ARG

1

0

05

1

15

2

Rela

tive e

xpre

ssio

n of

CO

X2

(e)





lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast lowast

Arg1 Mrc1 IL-6 iNOS


(a)

(b)

LC-3

GAPDH

+ + +

FTY720

ox-LDL

-

-

GAPDH

TGF-

IL-6

GAPDH

TGF-

IL-6

FTY720

-FTY720

LC-3

GAPDH

+ + +-FTY720

ox-LDL

B

GAPDH

ARG1

COX2

FTY720

GAPDH

ARG1

COX2

+ + +FTY720ox-LDL

(c)

M+ox-LDL

M +FTY720+ox-LDL

M +ox-LDL+FTY720

0

20

40

60

80

100


M+ox-LDL+FTY720

The p

erce

ntag

e of f

oam

cells

in h

igh

inte

nsity

(d)

(e)

0

07

14

21

28

35

Rela

tive e

xpre

ssio

n of

LC3

II

FTY720

FTY720

0

04

08

12

Rela

tive e

xpre

ssio

n of

IL-6

FTY720

0

02

04

06

08

Rela

tive e

xpre

ssio

n of

CO

X2

FTY720

0

05

1

15

2

25

Rela

tive e

xpre

ssio

n of

TG

F-

FTY720


ox-LDL+5M FTY720


ox-LDL+5M FTY720


ox-LDL+5M FTY720


ox-LDL+5M FTY720


ox-LDL+5M FTY720

Arg1 Mrc1 IL-6 iNOS


1M 5M

1M 5M

- 1M 5M- 1M 5M

1M 5M

1M 5M

0

1

2

3

4

5

6Re

lativ

e mRN

A ex

pres

sion

(co

ntro

l)

0

2

4

6

8

10

12

Rela

tive m

RNA

expr

essi

on (

cont

rol)

0

04

08

12

Rela

tive e

xpre

ssio

n of

LC3

II

0M 1M 5M

0M 1M 5M

0M 1M 5M

0M 1M 5M

0M 1M 5M0

08

16

24

32

4

48

Rela

tive e

xpre

ssio

n of

ARG

1

0

05

1

15

2

25

3

35

Rela

tive e

xpre

ssio

n of

ARG

1

lowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

lowastlowastlowast

lowast

BBB0

05

1

15

2

25

Rela

tive e

xpre

ssio

n of

TG

F-

0

08

16

24

32

4Re

lativ

e exp

ress

ion

of C

OX

20

04

08

12

16

Rela

tive e

xpre

ssio

n of

IL-6

lowast







C-CASPASE3

GAPDH

C-CASPASE3

GAPDH


ox-LDL + + + - 1M 5M 1M 5M

005

115

225

335

445

5


tive m

RNA

expr

essi

on (

cont

rol)




CASPASE3 CASPASE3


ox-LDL+5M Rapamycin


ox-LDL+5M FTY720

0123456789

Rela

tive m

RNA

expr

essi

on (

cont

rol)

lowast

lowast lowast

0

02

04

06

08

1

C-CA

SEPA

SE3

TOTA

L CA

SPA

SE3

lowast

lowast

0

04

08

12

16

2

C-CA

SEPA

SE3

TOTA

L CA

SPA

SE3

(a)

(b)

(c)


4 Discussion








5 Conclusion


Data Availability






Acknowledgments


References













































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















lowast

lowast

lowast

lowast lowast

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowast

Hoechst

Merge

F480

Rapamycin Rapamycin

(a) GAPDH

LC3


GAPDH

TGF-

IL-6Rapamycin(b)

GAPDH

COX2

ARG1

Arg1 Mrc1 IL-6 iNOS


(d)

(c)

(e)


0M 01M 1M 5M0M 1M 5M

Rapamycin0M 1M 5M

Rapamycin0M 1M 5M

Rapamycin0M 1M 5M

0

05

1

15

2

25

Rela

tive e

xpre

ssio

n of

LC3

II

01234567

Rela

tive e

xpre

ssio

n of

IL-6

01234567

Rela

tive e

xpre

ssio

n of

CO

X2

0123456789

10

Rela

tive m

RNA

expr

essi

on

(con

trol

)

0123456789

Rela

tive e

xpre

ssio

n of

ARG

10

02040608

11214

Rela

tive e

xpre

ssio

n of

TG

F-







M+5M rap+ox-LDL

M+ox-LDL

M+ox-LDL +5M rap

M

(a)


M+ox-LDL+Rapamycin

0

20

40

60

80

100

The p

erce

ntag

e of f

oam

cells

in h

igh

inte

nsity


(b)

lowast

lowast

Arg1 Mrc1 IL-6 iNOS


0

02

04

06

08

1

12

14

16

18

Rela

tive m

RNA

expr

essi

on (

cont

rol)

(c)

GAPDH

LC3

+ + +

Rapamycin

ox-LDL

ox-LDL

ox-LDL

-

GAPDH

IL-6

+ + +

Rapamycin -

TGF-

GAPDH

ARG1

+ + +

Rapamycin -

COX2

1M 5M

1M 5M

1M 5M

(d)

lowast

lowast

lowast


ox-LDL+5M Rapamycin


ox-LDL+5M Rapamycin


ox-LDL+5M Rapamycin


ox-LDL+5M Rapamycin


ox-LDL+5M Rapamycin

0

05

1

15

2

25

3

Rela

tive e

xpre

ssio

n of

LC3

II

0

04

08

12

16

Rela

tive e

xpre

ssio

n of

IL-6

0

04

08

12

16

Rela

tive e

xpre

ssio

n of

TG

F-

0

04

08

12

16

Rela

tive e

xpre

ssio

n of

ARG

1

0

05

1

15

2

Rela

tive e

xpre

ssio

n of

CO

X2

(e)





lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast lowast

Arg1 Mrc1 IL-6 iNOS


(a)

(b)

LC-3

GAPDH

+ + +

FTY720

ox-LDL

-

-

GAPDH

TGF-

IL-6

GAPDH

TGF-

IL-6

FTY720

-FTY720

LC-3

GAPDH

+ + +-FTY720

ox-LDL

B

GAPDH

ARG1

COX2

FTY720

GAPDH

ARG1

COX2

+ + +FTY720ox-LDL

(c)

M+ox-LDL

M +FTY720+ox-LDL

M +ox-LDL+FTY720

0

20

40

60

80

100


M+ox-LDL+FTY720

The p

erce

ntag

e of f

oam

cells

in h

igh

inte

nsity

(d)

(e)

0

07

14

21

28

35

Rela

tive e

xpre

ssio

n of

LC3

II

FTY720

FTY720

0

04

08

12

Rela

tive e

xpre

ssio

n of

IL-6

FTY720

0

02

04

06

08

Rela

tive e

xpre

ssio

n of

CO

X2

FTY720

0

05

1

15

2

25

Rela

tive e

xpre

ssio

n of

TG

F-

FTY720


ox-LDL+5M FTY720


ox-LDL+5M FTY720


ox-LDL+5M FTY720


ox-LDL+5M FTY720


ox-LDL+5M FTY720

Arg1 Mrc1 IL-6 iNOS


1M 5M

1M 5M

- 1M 5M- 1M 5M

1M 5M

1M 5M

0

1

2

3

4

5

6Re

lativ

e mRN

A ex

pres

sion

(co

ntro

l)

0

2

4

6

8

10

12

Rela

tive m

RNA

expr

essi

on (

cont

rol)

0

04

08

12

Rela

tive e

xpre

ssio

n of

LC3

II

0M 1M 5M

0M 1M 5M

0M 1M 5M

0M 1M 5M

0M 1M 5M0

08

16

24

32

4

48

Rela

tive e

xpre

ssio

n of

ARG

1

0

05

1

15

2

25

3

35

Rela

tive e

xpre

ssio

n of

ARG

1

lowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

lowastlowastlowast

lowast

BBB0

05

1

15

2

25

Rela

tive e

xpre

ssio

n of

TG

F-

0

08

16

24

32

4Re

lativ

e exp

ress

ion

of C

OX

20

04

08

12

16

Rela

tive e

xpre

ssio

n of

IL-6

lowast







C-CASPASE3

GAPDH

C-CASPASE3

GAPDH


ox-LDL + + + - 1M 5M 1M 5M

005

115

225

335

445

5


tive m

RNA

expr

essi

on (

cont

rol)




CASPASE3 CASPASE3


ox-LDL+5M Rapamycin


ox-LDL+5M FTY720

0123456789

Rela

tive m

RNA

expr

essi

on (

cont

rol)

lowast

lowast lowast

0

02

04

06

08

1

C-CA

SEPA

SE3

TOTA

L CA

SPA

SE3

lowast

lowast

0

04

08

12

16

2

C-CA

SEPA

SE3

TOTA

L CA

SPA

SE3

(a)

(b)

(c)


4 Discussion








5 Conclusion


Data Availability






Acknowledgments


References













































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















M+5M rap+ox-LDL

M+ox-LDL

M+ox-LDL +5M rap

M

(a)


M+ox-LDL+Rapamycin

0

20

40

60

80

100

The p

erce

ntag

e of f

oam

cells

in h

igh

inte

nsity


(b)

lowast

lowast

Arg1 Mrc1 IL-6 iNOS


0

02

04

06

08

1

12

14

16

18

Rela

tive m

RNA

expr

essi

on (

cont

rol)

(c)

GAPDH

LC3

+ + +

Rapamycin

ox-LDL

ox-LDL

ox-LDL

-

GAPDH

IL-6

+ + +

Rapamycin -

TGF-

GAPDH

ARG1

+ + +

Rapamycin -

COX2

1M 5M

1M 5M

1M 5M

(d)

lowast

lowast

lowast


ox-LDL+5M Rapamycin


ox-LDL+5M Rapamycin


ox-LDL+5M Rapamycin


ox-LDL+5M Rapamycin


ox-LDL+5M Rapamycin

0

05

1

15

2

25

3

Rela

tive e

xpre

ssio

n of

LC3

II

0

04

08

12

16

Rela

tive e

xpre

ssio

n of

IL-6

0

04

08

12

16

Rela

tive e

xpre

ssio

n of

TG

F-

0

04

08

12

16

Rela

tive e

xpre

ssio

n of

ARG

1

0

05

1

15

2

Rela

tive e

xpre

ssio

n of

CO

X2

(e)





lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast lowast

Arg1 Mrc1 IL-6 iNOS


(a)

(b)

LC-3

GAPDH

+ + +

FTY720

ox-LDL

-

-

GAPDH

TGF-

IL-6

GAPDH

TGF-

IL-6

FTY720

-FTY720

LC-3

GAPDH

+ + +-FTY720

ox-LDL

B

GAPDH

ARG1

COX2

FTY720

GAPDH

ARG1

COX2

+ + +FTY720ox-LDL

(c)

M+ox-LDL

M +FTY720+ox-LDL

M +ox-LDL+FTY720

0

20

40

60

80

100


M+ox-LDL+FTY720

The p

erce

ntag

e of f

oam

cells

in h

igh

inte

nsity

(d)

(e)

0

07

14

21

28

35

Rela

tive e

xpre

ssio

n of

LC3

II

FTY720

FTY720

0

04

08

12

Rela

tive e

xpre

ssio

n of

IL-6

FTY720

0

02

04

06

08

Rela

tive e

xpre

ssio

n of

CO

X2

FTY720

0

05

1

15

2

25

Rela

tive e

xpre

ssio

n of

TG

F-

FTY720


ox-LDL+5M FTY720


ox-LDL+5M FTY720


ox-LDL+5M FTY720


ox-LDL+5M FTY720


ox-LDL+5M FTY720

Arg1 Mrc1 IL-6 iNOS


1M 5M

1M 5M

- 1M 5M- 1M 5M

1M 5M

1M 5M

0

1

2

3

4

5

6Re

lativ

e mRN

A ex

pres

sion

(co

ntro

l)

0

2

4

6

8

10

12

Rela

tive m

RNA

expr

essi

on (

cont

rol)

0

04

08

12

Rela

tive e

xpre

ssio

n of

LC3

II

0M 1M 5M

0M 1M 5M

0M 1M 5M

0M 1M 5M

0M 1M 5M0

08

16

24

32

4

48

Rela

tive e

xpre

ssio

n of

ARG

1

0

05

1

15

2

25

3

35

Rela

tive e

xpre

ssio

n of

ARG

1

lowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

lowastlowastlowast

lowast

BBB0

05

1

15

2

25

Rela

tive e

xpre

ssio

n of

TG

F-

0

08

16

24

32

4Re

lativ

e exp

ress

ion

of C

OX

20

04

08

12

16

Rela

tive e

xpre

ssio

n of

IL-6

lowast







C-CASPASE3

GAPDH

C-CASPASE3

GAPDH


ox-LDL + + + - 1M 5M 1M 5M

005

115

225

335

445

5


tive m

RNA

expr

essi

on (

cont

rol)




CASPASE3 CASPASE3


ox-LDL+5M Rapamycin


ox-LDL+5M FTY720

0123456789

Rela

tive m

RNA

expr

essi

on (

cont

rol)

lowast

lowast lowast

0

02

04

06

08

1

C-CA

SEPA

SE3

TOTA

L CA

SPA

SE3

lowast

lowast

0

04

08

12

16

2

C-CA

SEPA

SE3

TOTA

L CA

SPA

SE3

(a)

(b)

(c)


4 Discussion








5 Conclusion


Data Availability






Acknowledgments


References













































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast lowast

Arg1 Mrc1 IL-6 iNOS


(a)

(b)

LC-3

GAPDH

+ + +

FTY720

ox-LDL

-

-

GAPDH

TGF-

IL-6

GAPDH

TGF-

IL-6

FTY720

-FTY720

LC-3

GAPDH

+ + +-FTY720

ox-LDL

B

GAPDH

ARG1

COX2

FTY720

GAPDH

ARG1

COX2

+ + +FTY720ox-LDL

(c)

M+ox-LDL

M +FTY720+ox-LDL

M +ox-LDL+FTY720

0

20

40

60

80

100


M+ox-LDL+FTY720

The p

erce

ntag

e of f

oam

cells

in h

igh

inte

nsity

(d)

(e)

0

07

14

21

28

35

Rela

tive e

xpre

ssio

n of

LC3

II

FTY720

FTY720

0

04

08

12

Rela

tive e

xpre

ssio

n of

IL-6

FTY720

0

02

04

06

08

Rela

tive e

xpre

ssio

n of

CO

X2

FTY720

0

05

1

15

2

25

Rela

tive e

xpre

ssio

n of

TG

F-

FTY720


ox-LDL+5M FTY720


ox-LDL+5M FTY720


ox-LDL+5M FTY720


ox-LDL+5M FTY720


ox-LDL+5M FTY720

Arg1 Mrc1 IL-6 iNOS


1M 5M

1M 5M

- 1M 5M- 1M 5M

1M 5M

1M 5M

0

1

2

3

4

5

6Re

lativ

e mRN

A ex

pres

sion

(co

ntro

l)

0

2

4

6

8

10

12

Rela

tive m

RNA

expr

essi

on (

cont

rol)

0

04

08

12

Rela

tive e

xpre

ssio

n of

LC3

II

0M 1M 5M

0M 1M 5M

0M 1M 5M

0M 1M 5M

0M 1M 5M0

08

16

24

32

4

48

Rela

tive e

xpre

ssio

n of

ARG

1

0

05

1

15

2

25

3

35

Rela

tive e

xpre

ssio

n of

ARG

1

lowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowastlowast

lowastlowastlowast

lowast

BBB0

05

1

15

2

25

Rela

tive e

xpre

ssio

n of

TG

F-

0

08

16

24

32

4Re

lativ

e exp

ress

ion

of C

OX

20

04

08

12

16

Rela

tive e

xpre

ssio

n of

IL-6

lowast







C-CASPASE3

GAPDH

C-CASPASE3

GAPDH


ox-LDL + + + - 1M 5M 1M 5M

005

115

225

335

445

5


tive m

RNA

expr

essi

on (

cont

rol)




CASPASE3 CASPASE3


ox-LDL+5M Rapamycin


ox-LDL+5M FTY720

0123456789

Rela

tive m

RNA

expr

essi

on (

cont

rol)

lowast

lowast lowast

0

02

04

06

08

1

C-CA

SEPA

SE3

TOTA

L CA

SPA

SE3

lowast

lowast

0

04

08

12

16

2

C-CA

SEPA

SE3

TOTA

L CA

SPA

SE3

(a)

(b)

(c)


4 Discussion








5 Conclusion


Data Availability






Acknowledgments


References













































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















C-CASPASE3

GAPDH

C-CASPASE3

GAPDH


ox-LDL + + + - 1M 5M 1M 5M

005

115

225

335

445

5


tive m

RNA

expr

essi

on (

cont

rol)




CASPASE3 CASPASE3


ox-LDL+5M Rapamycin


ox-LDL+5M FTY720

0123456789

Rela

tive m

RNA

expr

essi

on (

cont

rol)

lowast

lowast lowast

0

02

04

06

08

1

C-CA

SEPA

SE3

TOTA

L CA

SPA

SE3

lowast

lowast

0

04

08

12

16

2

C-CA

SEPA

SE3

TOTA

L CA

SPA

SE3

(a)

(b)

(c)


4 Discussion








5 Conclusion


Data Availability






Acknowledgments


References













































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















5 Conclusion


Data Availability






Acknowledgments


References













































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















Rapamycin and FTY720 Alleviate Atherosclerosis by Cross ...

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Transcript of Rapamycin and FTY720 Alleviate Atherosclerosis by Cross ...