D4F alleviates macrophage-derived foam cell apoptosis by inhibiting endoplasmic reticulum...

836 Journal of Lipid Research Volume 56, 2015

Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

This article is available online at http://www.jlr.org

Macrophage apoptosis, a prominent feature of athero-sclerotic plaques, occurs throughout all stages of athero-sclerosis and plays a crucial role in the formation and development of atherosclerotic lesions ( 1 ). Macrophage apoptosis in early lesions, coupled with rapid phagocytic clearance of dead cells (efferocytosis), reduces macro-phage burden and slows lesion progression. Whereas in late lesions, macrophage apoptosis, accompanied by de-fective efferocytosis, promotes the enlargement of the lipid core and results in infl ammation, necrosis, and even plaque rupture, which are identifi ed as the causative pro-cesses in the small percentage of atherosclerotic lesions that cause acute vascular events such as stroke, acute myo-cardial infarction, and sudden coronary death ( 2–4 ). There-fore, it is believed that the suppression of macrophage apoptosis may be a therapeutic implication for combating plaque instability ( 3, 5 ).

An intrinsic pathway mediated by endoplasmic reticulum (ER) stress, mainly involving CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), caspase-12, and c-Jun N-terminal kinase (JNK) signals, has been defi ned

Abstract This study was designed to explore the protec-tive effect of D4F, an apoA-I mimetic peptide, on oxidized LDL (ox-LDL)-induced endoplasmic reticulum (ER) stress-CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) pathway-mediated apoptosis in macro-phages. Our results showed that treating apoE knockout mice with D4F decreased the serum ox-LDL level and apo-ptosis in atherosclerotic lesions with concomitant downregu-lation of cluster of differentiation 36 (CD36) and inhibition of ER stress. In vitro, D4F inhibited macrophage-derived foam cell formation. Furthermore, like ER stress inhibitor 4-phenylbutyric acid (PBA), D4F inhibited ox-LDL- or tu-nicamycin (TM, an ER stress inducer)-induced reduction in cell viability and increase in lactate dehydrogenase leak-age, caspase-3 activation, and apoptosis. Additionally, like PBA, D4F inhibited ox-LDL- or TM-induced activation of ER stress response as assessed by the reduced nuclear translo-cation of activating transcription factor 6 and the decreased phosphorylation of protein kinase-like ER kinase and eu-karyotic translation initiation factor 2 � , as well as the down-regulation of glucose-regulated protein 78 and CHOP. Moreover, D4F mitigated ox-LDL uptake by macrophages and CD36 upregulation induced by ox-LDL or TM. These data indicate that D4F can alleviate the formation and apo-ptosis of macrophage-derived foam cells by suppressing CD36-mediated ox-LDL uptake and subsequent activation of the ER stress-CHOP pathway. —Yao, S., H. Tian, C. Miao, D-W. Zhang, L. Zhao, Y. Li, N. Yang, P. Jiao, H. Sang, S. Guo, Y. Wang, and S. Qin. D4F alleviates macrophage-derived foam cell apoptosis by inhibiting CD36 expression and ER stress-CHOP pathway. J. Lipid Res. 2015. 56: 836–847.

Supplementary key words apolipoprotein A-I mimetic peptide • oxi-dized low density lipoprotein • endoplasmic reticulum stress • C/EBP homologous protein • apoptosis • cluster of differentiation 36

This research was supported by the Taishan Scholars Foundation of the Shan-dong Province (zd056, zd057) and the National Natural Science Foundation of China (81370381, 81202949). The authors declare no confl icts of interest.

Manuscript received 7 October 2014 and in revised form 22 January 2015.

Published, JLR Papers in Press, January 29, 2015 DOI 10.1194/jlr.M055400

D4F alleviates macrophage-derived foam cell apoptosis by inhibiting CD36 expression and ER stress-CHOP pathway

Shutong Yao , 1, * ,† Hua Tian , 1, * Cheng Miao , 1, * Da-Wei Zhang , § Li Zhao , * , ** Yanyan Li , * Nana Yang , * Peng Jiao , * Hui Sang , * ,† Shoudong Guo , * Yiwei Wang , ** and Shucun Qin 2, *

Institute of Atherosclerosis, Key Laboratory of Atherosclerosis in Universities of Shandong,* and College of Basic Medical Sciences, † Taishan Medical University , Taian 271000, China ; Departments of Pediatrics and Biochemistry, § Group on the Molecular and Cell Biology of Lipids, University of Alberta , Edmonton, AB T6G 2S2, Canada ; and Affi liated Hospital of Chengde Medical University,** Chengde Medical University , Chengde 067000, China

Abbreviations: apoE � / � , apoE knockout; ATF6, activating tran-scription factor 6; CD36, cluster of differentiation 36; CHOP, CCAAT/enhancer-binding protein (C/EBP) homologous protein; DAPI, 4 ′ ,6-diamidino-2-phenylindole; eIF2 � , eukaryotic translation initiation factor 2 � ; ER, endoplasmic reticulum; GRP78, glucose-regulated pro-tein 78; IOD, integrated optical density; LDH, lactate dehydrogenase; mAb, monoclonal antibody; MOMA-2, monocyte/macrophage-specifi c antibody; MTT, 3-(4,5-dimethylthiazol-2-y-l)-2,5-diphenyl-2H-tetrazolium bromide; ox-LDL, oxidized LDL; PBA, 4-phenylbutyric acid; p-eIF2 � , phospho-eukaryotic translation initiation factor 2 � ; PERK, double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase; PI, propidium iodide; p-PERK, phospho-double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase; sD4F, scrambled D4F; TC, total cholesterol; TM, tunicamycin; TUNEL, transferase-mediated dUTP nick end-labeling .

1 S. Yao, H. Tian, and C. Miao contributed equally to this work. 2 To whom correspondence should be addressed. e-mail: [email protected]

The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of eight fi gures.

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D4F inhibits macrophage apoptosis by reducing CHOP pathway 837


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Fig. 1. D4F decreases serum ox-LDL level and attenuates macrophage ER stress and apoptosis in atherosclerotic lesions. Male apoE � / � mice were fed a high-fat diet for 8 weeks, and given saline (model group) or 1 mg/kg of D4F (D4F group) per day by intraperitoneal injection during the fi nal 6 weeks. Male C57BL/6J mice were maintained on a normal chow diet as a control group. A: Serum ox-LDL level deter-mined by ELISA assay (n = 8). B: Atherosclerotic lesion formation stained by oil red O. Scale bar = 100 � m, n = 8. C: Cell apoptosis in atherosclerotic lesions under TUNEL staining. Representative fl uorescent images and quantitative data are shown. Red, TUNEL-positive cell; blue, nuclei stained by DAPI. Scale bar = 20 � m, n = 6. D: Immunofl uorescent staining with antibodies against MOMA-2, CHOP, GRP78, and CD36. Scale bar = 20 � m, n = 6. Relative fl uorescence intensity for the expression of CHOP, GRP78, and CD36 (red) in the macrophage-dense areas (green) of the lesions was calculated. E: Western blot analysis of ER stress markers, CD36 and MOMA-2 in aortic arch (n = 4). Data are presented as the mean ± SEM of at least four independent experiments. * P < 0.05, ** P < 0.01 versus control group; # P < 0.05, ## P < 0.01 versus model group.

as the underlying mechanism in macrophage apoptosis. The ER stress-mediated apoptotic pathway is activated when macrophages are exposed to atherogenic factors that trigger ER stress, such as oxidized LDL (ox-LDL), oxidized phospholipids, “free” cholesterol, and hyper-glycemia ( 1, 6 ). CHOP, a specific pro-apoptotic tran-scription factor under condition of ER stress, has been confi rmed to mediate macrophage apoptosis and con-tribute to the instability of atherosclerotic plaques, whereas the defi ciency of CHOP has been demonstrated to protect macrophages from ER stress-mediated apoptosis in vitro and in advanced atherosclerotic plaques of mice ( 7–9 ). Our previous studies have demonstrated that ox-LDL can induce macrophage apoptosis by upregulating CHOP expression during the formation of macrophage-derived foam cells, whereas quercetin protects macrophages from ox-LDL-induced apoptosis by inhibiting CHOP expres-sion ( 10, 11 ). Thus the CHOP-mediated ER stress path-way may be a new therapeutic target for atherosclerosis ( 12, 13 ).

D4F is an apoA-I mimetic peptide that contains four phenylalanine (F) residues and is synthesized from D-amino acids. It does not possess sequence homology to apoA-I, the major lipoprotein component on HDL, but possesses the secondary structural motif of class A amphipathic helices similar to apoA-I ( 14–16 ). Several lines of evidence demonstrate that D4F exhibits an anti-atherogenic effect without changing plasma choles-terol levels in dyslipidemic mouse models ( 17–20 ). It has been reported that D4F can increase paraoxonase activity in HDL, induce pre- � HDL formation, improve HDL-mediated reverse cholesterol transport and anti-infl ammatory properties, and promote endothelial pro-genitor cell proliferation, migration, and adhesion, which are responsible for its anti-atherogenic role ( 21–23 ). In addition, our recent work has shown that D4F is able to inhibit ox-LDL-induced cytotoxicity on human umbilical vein endothelial cells via preventing the downregula-tion of pigment epithelium-derived factor ( 24 ), consis-tent with the report that small dense HDL3 potently protects endothelial cells from ox-LDL-induced apoptosis and that apoA-I is pivotal to such protection ( 25 ). How-ever, little is known of the impact of D4F on macro-phage-derived foam cell apoptosis. In the present study, we investigated the effect of D4F on macrophage apop-totic death, with emphasis on its role in downregulating ER stress-CHOP pathway-mediated apoptosis in vitro and in vivo.

MATERIALS AND METHODS

Reagents Oil red O, tunicamycin (TM), 4-phenylbutyric acid (PBA),

and anti- � -actin antibody were obtained from Sigma-Aldrich (St. Louis, MO). DMEM and DiI-ox-LDL were from Gibco (Rockville, MD) and Xiesheng Biotech (Beijing, China), respectively. Rab-bit antibody against phospho-double-stranded RNA-activated protein kinase-like ER kinase (p-PERK), anti-cluster of dif-feren tiation 36 (CD36) monoclonal antibody (mAb) and rat anti-monocyte/macrophage-specifi c antibody (MOMA-2) anti-body were purchased from Abcam (Cambridge, MA). Rabbit polyclonal antibodies against phospho-eukaryotic translation ini-tiation factor 2 � (p-eIF2 � ), activating transcription factor 6 (ATF6), CHOP, and glucose-regulated protein 78 (GRP78) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Alexa Fluor 594-labeled donkey anti-rabbit and Alexa Fluor 488-labeled donkey anti-rat antibodies were from Molecular Probes (Eugene, OR). SABC-Cy3 immunohistochemistry kits were ob-tained from Boshide (Wuhan, China). Annexin V-FITC apoptosis detection kits were obtained from KeyGEN Biotech (Nanjing, China). Polyvinylidene difl uoride membranes and ECL kits were obtained from Millipore (Bedford, MA) and Thermo Scientifi c Pierce (Rockford, IL), respectively. The terminal deoxynucleoti-dyl transferase-mediated (dUTP) nick end-labeling (TUNEL) as-say kit (In Situ Cell Death Detection kit, TMR red) and caspase-3 activity assay kit were from Roche (Mannheim, Germany) and Beyotime Biotech (Shanghai, China), respectively. Mouse ox-LDL ELISA kits and tissue/cell total cholesterol (TC) assay kits were purchased from Bio-swamp (Shanghai, China) and Apply-gen (Beijing, China), respectively. Real-time PCR reagent kits and lactate dehydrogenase (LDH) assay kits were obtained from Tiangen Biological Chemistry (Beijing, China) and Jiancheng Biotech (Nanjing, China), respectively. D4F (Ac-DWFKAFYDKVAE KFKEAF-NH 2 ) and scrambled D4F (sD4F) (Ac-DWFAKDYFK-KAFVEEFAK-NH 2 ) were synthesized from SciLight Biotechnology (Beijing, China).

Animal protocol Seven-week-old male apoE knockout (apoE � / � ) mice and

C57BL/6J wild-type mice were purchased from Huafukang Bio-Technology Co. (Beijing, China). All animal study protocols were performed in accordance with the national guidelines for the care and use of animals and approved by the laboratory ani-mals’ ethical committee of Taishan Medical University. Sixteen apoE � / � mice were fed a high-fat diet (15.8% fat and 1.25% cho-lesterol) for 8 weeks, and randomly distributed to receive intra-peritoneal injections with either saline (model group, n = 8) or D4F (1 mg/kg per day) (D4F group, n = 8) during the fi nal 6 weeks. Eight male C57BL/6 mice injected intraperitoneally with saline were maintained on a normal chow diet as a control group. The mice were fasted overnight at the end period of the experi-ment and blood samples were collected from their inner canthus


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Fig. 2. D4F reduces ox-LDL-induced intracellular lipid accumulation in RAW264.7 cells. Cells were pre-treated with or without D4F (12.5, 25, and 50 mg/l) or inactive control peptide sD4F (50 mg/l) for 1 h, washed, and then followed by incubation with ox-LDL (100 mg/l) for 24 h. A: The intracellular lipid droplets were stained by oil red O. Representative lipid droplet staining images are shown. Scale bar = 20 � m. B: The average IOD of lipid droplets stained with oil red O from differentiated macrophage foam cells was obtained via checking fi ve fi elds in each con-dition. C: The intracellular TC content was detected using a tissue/cell TC assay kit. Data are expressed as the mean ± SEM of at least four independent experi-ments. * P < 0.05, ** P < 0.01 versus control group; # P < 0.05, ## P < 0.01 versus ox-LDL group.

Fig. 3. Effects of D4F on ox-LDL- or TM-induced cytotoxicity in RAW264.7 cells. RAW264.7 cells were pretreated with or without D4F (12.5, 25, and 50 mg/l), sD4F (50 mg/l), or PBA (5 mmol/l) in DMEM with 1% FBS for 1 h, washed, and then followed by in-cubation with ox-LDL (100 mg/l) for 24 h. Cell via bility (A) and LDH activity in media (B) were mea-sured by MTT assay and a kit, respectively. C: D4F inhibits TM-induced cell death. Cells were pre-treated with D4F (50 mg/l), sD4F (50 mg/l), or PBA (5 mmol/l) for 1 h, washed, and then followed by exposure to TM (4 mg/l) for 24 h. Data are expressed as the mean ± SEM of six independent experiments. * P < 0.05, ** P < 0.01 versus control group; # P < 0.05, ## P < 0.01 versus ox-LDL group; & P < 0.05, && P < 0.01 versus TM group.

for serum preparation. The mice were euthanized by cervical dislocation and hearts, including the aortic root, were perfused with ice-cold PBS, removed transversely, and fi xed in 4% parafor-maldehyde. The hearts were then embedded in OCT compound and frozen at � 80°C. The samples were serially sectioned at 6 � m and sections were collected on glass slides for the subsequent analyses.

Cell culture RAW264.7 macrophages were purchased from the Type Cul-

ture Collection of the Chinese Academy of Sciences (Shanghai, China), maintained in DMEM medium containing 2 mM gluta-mine, 10% FBS, and antibiotics (100 U/ml penicillin and strep-tomycin) in a 37°C humidifi ed incubator containing 5% CO 2 until subconfl uent, and then the medium was replaced with DMEM with 1% FBS.

Isolation and oxidation of LDL Human LDL was isolated from fresh plasma of healthy donors

using sequential ultracentrifugation, and then oxidized with 10 � mol/l CuSO 4 for 18 h at 37°C, as described in a previous study from our group ( 10 ).

Serum ox-LDL level measurement Fasting blood samples from apoE � / � and C57BL/6J mice were

obtained and serum ox-LDL levels were tested by ELISA assays according to the manufacturer’s guidelines.

Oil red O staining To assess the atherosclerotic lesions, serial aortic root cryosec-

tions were stained with oil red O and counterstained with bril-liant green. Atherosclerotic lesions were captured as digital images using a microscope (Olympus, Tokyo, Japan) and total mean lesion area was quantifi ed from fi ve sections for an animal using Image-Pro Plus software (version 6.0; Media Cybernetics, Rockville, MD).

To observe lipid droplets in RAW264.7 macrophages, cells cul-tured on cover glass were fi xed with 4% paraformaldehyde for 20 min, washed with PBS, stained with oil red O (0.5% w/v in 60% isopropanol) for 30 min, and counterstained with hematoxylin for 5 min. The cells were viewed using an Olympus BX51 micro-scope (Olympus), and the lipid droplet content was analyzed us-ing Image-Pro Plus image analysis software 6.0 and expressed as the average value of the integrated optical density (IOD).

Intracellular TC analysis The intracellular TC concentration was measured using a

tissue/cell TC assay kit according to the manufacturer’s instruc-tions and normalized to the level of total cellular protein ( 10 ).

Immunofl uorescence assay For immunofl uorescence staining of atherosclerotic lesions,

serial aortic root cryosections were blocked with 5% normal donkey serum and incubated with the fi rst primary antibodies overnight at 4°C, and then the sections were incubated with a


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mixture of Alexa Fluor 594-labeled donkey anti-rabbit and Alexa Fluor 488-labeled donkey anti-rat antibodies for 1 h. 4 ′ ,6-Diamidino-2-phenylindole (DAPI) was used for counter-staining. Slides were mounted with antifade reagent and viewed using a fl uorescence microscope (Olympus). The mean fl uo-rescence intensity was measured for each corresponding target protein in the macrophage-dense areas using Image-Pro Plus software ( 26 ).

To detect nuclear translocation of ATF6, the treated cells were fi xed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked in 10% goat serum, and then incubated with ATF6 antibody (1:200) overnight at 4°C. Following incubation with sec-ondary antibody, the cells were incubated with SABC-Cy3 com-plexes and DAPI, and then observed using a laser scanning confocal microscope (Bio-Rad Radiance 2100) as described previously ( 11 ).

To measure the expression of CD36, the treated cells were rinsed with PBS, fi xed with 4% paraformaldehyde, blocked with 10% goat serum, and then incubated with CD36 antibody (1:100) overnight at 4°C. Next, the cells were washed and incubated with FITC-conjugated goat anti-rabbit IgG for 30 min at room tem-perature. After incubation with DAPI, the cells were observed us-ing a fl uorescence microscope (Olympus), and CD36 expression was expressed as the mean fl uorescence intensity per cell using Image-Pro Plus software (version 6.0, Media Cybernetics).

Cell viability and LDH assay The viability of the treated cells was evaluated by a colori-

metric assay using 3-(4,5-dimethylthiazol-2-y-l)-2,5-diphenyl-2H-tetrazolium bromide (MTT) as described previously ( 10 ) and expressed as the percentage of the optical density of treated cells relative to that of the untreated control cells (100%).

To further measure the level of cell injury, LDH activity in the media was measured using the assay kit according to the manu-facturer’s instructions.

Cell apoptosis analysis Cell apoptosis was assessed by annexin V-FITC/propidium io-

dide (PI) double-staining assay and TUNEL assay, respectively. In the fi rst assay, cells of each group were harvested, washed twice with ice-cold PBS, resuspended in 500 � l binding buffer contain-ing 5 � l annexin V-FITC and 5 � l PI, and then incubated in the dark at room temperature for 15 min. The apoptosis rates were analyzed using Cell Quest software on a FACScan fl ow cytometer (Becton Dickinson, San Jose, CA).

For the second method, the treated cells on coverslips or aor-tic root cryosections were washed twice with PBS, fi xed with 4% paraformaldehyde for 30 min, and then permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 3 min. Next, cells were incubated with TUNEL reaction mixture for 1 h at 37°C in dark-ness and DAPI for 5 min, respectively. After rinsing three times for 5 min in PBS, the samples were evaluated by fl uorescence microscopy, and the apoptotic cells were calculated as the per-centage of the number of TUNEL-positive cells to total cells.

Measurement of caspase-3 activity Caspase-3 activity was detected with an assay kit according to

the manufacturer’s instructions. Briefl y, the treated cells were harvested, rinsed with PBS, resuspended in lysis buffer, and then incubated on ice for 15 min. The lysate was centrifuged at 16,000 g at 4°C for 15 min. Approximately 70 � l of reaction buffer and 10 � l of caspase-3 substrate were mixed with 20 � l lysate superna-tant, and then incubated in 96-well microtiter plates at 37°C for 2 h. Caspase-3 activity was detected by an Infi nite F200 micro-plate reader (Tecan, Switzerland) at 405 nm and described as a percentage of the control.

Western blot analysis Cellular extracts were obtained by lysing the cells in RIPA buf-

fer containing 1% protease inhibitors, and protein content was detected using a bicinchoninic acid assay. Equal amounts of pro-tein were separated on SDS-PAGE by electrophoresis and then transferred onto polyvinylidene difl uoride membranes. After blocking in 5% nonfat dry milk, the membranes were incubated with primary antibodies overnight at 4°C, washed with Tris-buffered saline containing 0.1% Tween-20, and then incubated with horseradish peroxidase-conjugated IgG for 1 h at room tem-perature. The immunoproteins were visualized by ECL detection system, and the intensities were quantifi ed by Image-Pro Plus software (version 6.0, Media Cybernetics) and normalized to � -actin levels.

Quantitative real-time PCR Total RNA from the treated cells was isolated with Trizol re-

agent (Invitrogen, Carlsbad, CA), and synthesized to the fi rst-strand cDNA using MuLV reverse transcriptase. Primers used in this study were synthesized by Sangon Biotech (Shanghai, China) and the sequences were as follows: 5 ′ -CCACCACACCTGAAAGCAGAA-3 ′ (forward primer) and 5 ′ -GGTGCCCCCAATTTCATCT-3 ′ (reverse primer) for CHOP, 5 ′ -ACATGGACCTGTTCCGCTCTA-3 ′ (forward primer) and 5 ′ -TGGCTCCTTGCCATTGAAGA-3 ′ (reverse primer) for GRP78, 5 ′ -CGGGGACCTGACTGACTACC-3 ′ (forward primer) and 5 ′ -AGGAAGGCT GGAAGAGTGC-3 ′ (reverse primer) for � -actin. Quantitative real-time PCR was performed with SYBR-green PCR master mix kits on a Rotor-Gene Q real-time PCR cycler (Qiagen, Shanghai, China), analyzed using the Rotor-Gene Q software (version 1.7, Qiagen), and then relative mRNA levels were quantifi ed by the 2 – � � Ct method as described previ-ously ( 10 ).

Uptake of Dil-ox-LDL Cells were pretreated with D4F (50 mg/l), inactive control

peptide sD4F (50 mg/l), or anti-CD36 mAb (2 mg/l) for 1 h, fol-lowed by treatment with or without 2 mg/l TM for 4 h, and then incubated with Dil-ox-LDL (50 mg/l) for 4 h. Cells were washed with PBS and lysed with 200 � l lysis buffer. Fluorescence intensity was detected using an Infi nite F200 microplate reader (Tecan, Switzerland), and the data were normalized to the protein con-centration of each sample, as reported previously ( 27 ).

The uptake of Dil-ox-LDL by RAW264.7 cells was further evalu-ated by fl uorescence microscopy. The treated cells were washed with PBS, fi xed in 4% paraformaldehyde, and counterstained with DAPI, and the mean fl uorescence intensity per cell was cal-culated using Image-Pro Plus software (Media Cybernetics).

Statistical analysis Results are expressed as the mean ± SEM. Statistical analysis

was performed by one-way ANOVA with Student-Newman-Keuls test for multiple comparisons and Student’s t -test for comparison between two groups using the SPSS13.0 software for Windows. P values less than 0.05 were considered signifi cant.

RESULTS

D4F attenuates serum ox-LDL level and atherosclerotic lesions in apoE � / � mice

To evaluate the antiatherosclerotic function of D4F in vivo, an experimental atherosclerotic mouse model was established using apoE � / � mice following the method


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Fig. 4. Effect of D4F on ox-LDL- or TM-induced apoptosis in RAW264.7 cells. A: RAW264.7 cells were pretreated with or without D4F (12.5, 25, and 50 mg/l), sD4F (50 mg/l), or PBA (5 mmol/l) in DMEM with 1% FBS for 1 h, washed, and then followed by incubation with ox-LDL (100 mg/l) for 24 h. Cell apoptosis was detected using fl ow cytometry and the total apoptotic cells (early and late-stage apoptosis) were represented by the right side of the panel (annexin V staining alone or together with PI). Cells were pretreated with D4F (50 mg/l), sD4F (50 mg/l), or PBA (5 mmol/l) in DMEM with 1% FBS for 1 h, washed, and then followed by incubation with ox-LDL (100 mg/l) (B) or TM (4 mg/l) (C) treatment for 24 h, and then cell apoptosis was detected by TUNEL assay. Scale bar = 20 � m. D, E: Cells were treated as described in (A) and (C), and then caspase-3 activity was determined by colorimetric assay. Data are expressed as the mean ± SEM of at least four independent experiments. * P < 0.05, ** P < 0.01 versus control group; # P < 0.05, ## P < 0.01 versus ox-LDL group; & P <0.05 versus TM group.

described previously ( 26 ). As shown in Fig. 1A , D4F adminis-tration for 6 weeks signifi cantly reduced the serum ox-LDL level compared with the vehicle-treated model group, al-though there were no signifi cant differences in body weight and serum lipids between the D4F and model groups (sup-plementary Fig. 1A, B). Atherosclerotic plaque formation and apoptosis in the experimental apoE � / � mice were evalu-ated by oil red O staining and TUNEL assay, respectively. As shown in Fig. 1B, C , D4F treatment remarkably attenuated the plaque area and cell apoptosis in the aortic roots of apoE � / � mice compared with the model group.

D4F reduces the upregulation of ER stress markers and CD36 in apoE � / � mice

To elucidate whether D-4F could mitigate ER stress and CD36 upregulation in vivo, we analyzed the expression of ER stress indicators and CD36 in atherosclerotic lesions of apoE � / � mice. Immunofl uorescent and immunohisto-chemical analysis ( Fig. 1D , supplementary Fig. 2) showed a dramatic reduction in the expression of CHOP, GRP78, and CD36 in the macrophage-dense areas of aortic sinuses (shown by staining with monocyte/macrophage-specifi c an-tibody, MOMA-2) in the D4F-treated group when compared with vehicle-treated apoE � / � (model) mice. Western blot analysis of the aortic arch ( Fig. 1E ) also revealed the inhibi-tory effect of D4F treatment on the upregulation of CHOP, GRP78, and CD36. Given the important roles of the ER stress-CHOP pathway in apoptosis, these fi ndings indicate that the reduction of CHOP and GRP78 may contribute to D4F-attenuated apoptosis in atherosclerotic lesions.

D4F attenuates ox-LDL-induced lipid accumulation in RAW264.7 cells

An in vitro ox-LDL-induced macrophage-derived foam cell model was established to further investigate the cyto-protective function of D4F on macrophages. The oil red O staining ( Fig. 2A, B ) and intracellular TC quantitative assay ( Fig. 2C ) revealed that pretreatment of cells with D4F (12.5, 25, and 50 mg/l) remarkably inhibited ox-LDL-induced cellular lipid accumulation in a dose-dependent manner.

D4F suppresses cytotoxicity in RAW264.7 cells induced by ox-LDL or TM

MTT assay revealed that treatment with D4F at 12.5 up to 50 mg/l for 24 h did not remarkably affect cell viability, indicating that there is no signifi cant cytotoxicity of D4F on RAW264.7 cells (data not shown). Treatment of cells with ox-LDL at 100 mg/l for 24 h led to about a 43.9% decrease in cell viability and a dramatic elevation in LDH

leakage in cell media, which were prevented by D4F pre-treatment (12.5, 25, and 50 mg/l) in a dose-dependent manner ( Fig. 3A, B ).

Next, we investigated the relative contribution of ER stress-mediated cell death to the protective effect of D4F. PBA, an ER stress inhibitor, and TM, an ER stress inducer, were used to inhibit and induce ER stress, respectively. As shown in Fig. 3A, B , PBA, like D4F, blocked the reduced cell viability and increased LDH leakage induced by ox-LDL. Conversely, TM, like ox-LDL, decreased cell viability by 52.4%, which was also prevented by D4F pretreatment ( Fig. 3C ). These fi ndings indicate that D4F can reduce ER stress, which may contribute to its inhibitory effect on ox-LDL-induced cytotoxicity.

D4F inhibits apoptosis in RAW264.7 cells induced by ox-LDL or TM

To evaluate the anti-apoptotic effects of D4F, the propor-tion of apoptotic cells was quantifi ed by annexin V-FITC/PI double staining and TUNEL assay. As seen in Fig. 4A, B , ox-LDL treatment for 24 h signifi cantly increased the per-centage of apoptotic cells, which were strongly attenuated by the ER stress inhibitor, PBA, and D4F pretreatment in a dose-dependent manner. In accordance with these fi nd-ings, D4F pretreatment attenuated TM-induced cell apo-ptosis ( Fig. 4C , supplementary Fig. 3). Moreover, the inhibitory effect of D4F on ox-LDL-induced macrophage apoptosis was enhanced by PBA and blocked by TM (sup-plementary Fig. 4A).

The activity of caspase-3, a marker of apoptosis, was also examined to further assess the anti-apoptotic effect of D4F. As shown in Fig. 4D, E , treatment with ox-LDL or TM led to a remarkable activation of caspase-3, which was sig-nifi cantly inhibited by D4F and PBA. Furthermore, PBA enhanced, while TM blocked, the inhibitory effect of D4F on ox-LDL-induced caspase-3 activation (supplementary Fig. 4B). These data suggested that D4F may inhibit ox-LDL-induced macrophage apoptosis via suppressing the ER stress pathway.

D4F attenuates ER stress response in RAW264.7 cells induced by ox-LDL or TM

Given that D4F downregulated the expression of CHOP and GRP78 in atherosclerotic lesions ( Fig. 1 ) and, like PBA, attenuated ox-LDL- and TM-induced cytotoxicity and apo-ptosis in RAW264.7 cells ( Figs. 3, 4 ), we hypothesized that the suppression of the ER stress-CHOP pathway may be one of the underlying mechanisms of the anti-apoptotic effects of D4F. To confi rm the hypothesis, we evaluated the changes


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Fig. 5. D4F inhibits ox-LDL- or TM-induced ER stress response in RAW264.7 cells. A: Cells were pretreated with D4F (50 mg/l), sD4F (50 mg/l), or PBA (5 mmol/l) in DMEM with 1% FBS for 1 h, washed, and then followed by incubation with ox-LDL (100 mg/l) for 24 h, and then immunofl uorescence experiments showed ATF6 visualized by Cy3 labeling (red) and nuclei stained with DAPI (blue). Represen-tative fl uorescent images captured using a laser scanning confocal microscope are shown. Scale bar = 20 � m. B, C: Cells were treated as described in Fig. 4A , and then the protein and mRNA levels of ER stress markers were evaluated by Western blot and quantitative real-time PCR, respectively. D, E: Cells were pretreated with D4F (50 mg/l) or sD4F (50 mg/l) in the presence of TM (4 mg/l) treatment for 24 h, and then ATF6 nuclear translocation and protein levels of ER stress markers were determined by immunofl uorescence experiments and Western blot, respectively. Data are expressed as the mean ± SEM of at least three independent experiments. * P < 0.05, ** P < 0.01 versus control group; # P < 0.05, ## P < 0.01 versus ox-LDL group; & P < 0.05 versus TM group.

in CHOP and its two important upstream molecules, ATF6 and PERK, in vitro. As shown in Fig. 5A–C , ox-LDL resulted in activation of the ER stress-CHOP pathway as deter-mined by nuclear translocation of ATF6 and phosphoryla-tion of PERK and eIF2 � with concomitant upregulation of GRP 78 and CHOP, both at the protein and mRNA levels. However, pretreatment with D4F and PBA signifi cantly in-hibited the ox-LDL-induced ER stress response.

To further demonstrate these fi ndings, we investigated the regulatory effects of D4F on TM-induced ER stress response in RAW264.7 cells. As shown in Fig. 5D, E and supplementary Fig. 5, preincubation with D4F inhibited TM-induced activation of the ER stress-CHOP pathway as assessed by the reduced ATF6 nuclear translocation and the decreased phosphorylation of PERK and eIF2 � and expression of GRP 78 and CHOP. Additionally, the inhibi-tory effect of D4F on ox-LDL-induced CHOP upregulation was signifi cantly enhanced by PBA and blocked by TM (supplementary Fig. 4C).

Similarly, we observed that D4F inhibited ox-LDL-induced apoptosis and CHOP upregulation in mouse peritoneal macrophages (supplementary Fig. 6) and human THP-1-derived macrophages (supplementary Fig. 7).

D4F suppresses ox-LDL uptake and CD36 upregulation in RAW264.7 cells

Approximately 60–70% of macrophage-derived foam cell formation is caused by scavenger receptor CD36-medi-ated ox-LDL uptake, which is a key factor to trigger ER stress response ( 28, 29 ), and our recent study has shown that there is a positive feedback between ER stress and CD36 expression ( 27 ). Moreover, D4F downregulated CD36 expression in atherosclerotic lesions ( Fig. 1 ) and at-tenuated ox-LDL-induced lipid accumulation in RAW264.7 cells ( Fig. 2 ). Therefore, we next explored whether the mechanism underlying the inhibitory effect of D4F on the ER stress-CHOP pathway could be through suppression of CD36 expression. As shown in Fig. 6A, B , similar to anti-CD36 mAb, D4F reduced the uptake of Dil-ox-LDL in RAW264.7 cells and also alleviated the enhancement of ox-LDL uptake induced by TM. Additionally, Western blot and immunofl uorescence results ( Fig. 6C, D ) showed that ox-LDL- or TM-induced CD36 upregulation was remark-ably attenuated by D4F pretreatment.

To further confi rm these fi ndings, we knocked down the expression of CD36 by siRNA and then explored the inhibitory effects of D4F on ox-LDL-induced apoptosis and CHOP upregulation. As shown in supplementary Fig. 8, CD36 siRNA effi ciently reduced the level of CD36. Similar

to D4F, CD36 siRNA treatment inhibited ox-LDL-induced macrophage apoptosis, caspase-3 activation, and CHOP upregulation. However, the inhibitory effects of D4F were not enhanced when CD36 was downregulated by siRNA. These data indicate that D4F may suppress the ox-LDL up-take by macrophages through inhibition of CD36 upregu-lation induced by ox-LDL or TM, subsequently preventing ox-LDL-induced macrophage apoptosis.

DISCUSSION

Macrophage apoptosis has been identifi ed as a promi-nent feature of advanced atherosclerotic lesions and pro-motes infl ammatory response and enlargement of the necrotic cores, which result in plaque rupture and acute vascular events ( 1–5 ). Thus, suppression of macrophage apoptosis may be effective in combating acute atherothrom-botic vascular diseases. In the present study, our results indi-cate for the fi rst time that D4F alleviated the formation and apoptosis of macrophage-derived foam cells by inhibiting CD36-mediated ox-LDL uptake and subsequent activation of the ER stress-CHOP pathway, which was supported by the following observations. First, treating apoE � / � mice with D4F decreased the serum ox-LDL level and suppressed apoptosis in atherosclerotic lesions with concomitant down-regulation of CD36 and inhibition of ER stress, as assessed by diminished CHOP and GRP78 expression in macro-phage-dense atherosclerotic lesions. Second, D4F, like PBA (an ER stress inhibitor), inhibited macrophage-derived foam cell formation and cell injury and apoptosis induced by ox-LDL or TM (an ER stress inducer). Third, like PBA, D4F suppressed the ER stress-CHOP pathway via inhibiting ATF6 and PERK activation on the ER stress models induced by ox-LDL and TM, respectively. Fourth, the inhibitory ef-fects of D4F on ox-LDL-induced macrophage apoptosis, caspase-3 activation, and CHOP upregulation were pro-moted by PBA and blocked by TM. Fifth, D4F mitigated ox-LDL uptake by macrophages and inhibited CD36 up-regulation induced by ox-LDL or TM, while knockdown of CD36 expression by siRNA did not enhance the inhibitory effects of D4F on ox-LDL-induced macrophage apoptosis, caspase-3 activation, and CHOP upregulation.

It is well-known that prolonged and severe ER stress ac-tivates pro-apoptotic signals ( 6, 26 ). CHOP, a signaling mediator that plays a major role in ER stress-associated apoptosis, is upregulated in advanced lesions and plays a crucial role in macrophage apoptosis triggered by oxidative stress and a high level of intracellular cholesterol-induced


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Fig. 6. D4F reduces ox-LDL uptake and CD36 upregulation in RAW264.7 cells. A: Dil-ox-LDL fl uorescence intensity in cells preincubated with D4F (50 mg/l), sD4F (50 mg/l), or anti-CD36 mAb (2 mg/l) in DMEM with 1% FBS for 1 h, washed, and followed by treatment with or without 2 mg/l TM for 4 h, and then incubated with Dil-ox-LDL (50 mg/l) for 4 h. B: Fluorescence microscopy shows Dil-ox-LDL uptake by RAW264.7 cells preincubated with D4F (50 mg/l) in DMEM with 1% FBS for 1 h, washed, and followed by treatment with or without 2 mg/l TM for 4 h, and then incubated with Dil-ox-LDL (50 mg/l) for 4 h. Red staining denotes Dil-ox-LDL fl uorescence and the blue staining denotes nuclei visualized by DAPI (scale bar = 20 � m). C, D: Protein expression of CD36 in RAW264.7 cells pretreated with D4F (50 mg/l) or sD4F (50 mg/l) for 1 h, washed, and followed by treatment with ox-LDL (100 mg/l) or TM (2 mg/l) for 12 h were evaluated by Western blot and immunofl uorescence assay, respectively. Representative fl uorescent images are shown. Green staining denotes CD36 visualized by FITC labeling and the blue staining denotes nuclei visualized by DAPI (scale bar = 20 � m). Data are expressed as the mean ± SEM of at least three independent experiments. * P < 0.05, ** P < 0.01 versus control group; # P < 0.05, ## P < 0.01 versus ox-LDL group; & P < 0.05, && P < 0.01 versus TM group.

prolonged ER stress ( 7, 8, 30 ). CHOP defi ciency reduces macrophage apoptosis and plaque necrosis in advanced atherosclerotic lesions ( 9 ). We have shown that ox-LDL can induce macrophage apoptosis by upregu lating CHOP expression during the formation of macrophage-derived foam cells ( 10, 11 ). In this study, we observed that D4F, like the ER stress inhibitor, PBA, attenuated ox-LDL- and TM (an ER stress inducer)-induced CHOP upregulation, caspase-3 activation, and macrophage apoptosis in several types of macrophages, including RAW264.7 cells, mouse peritoneal macrophages, and human THP-1-derived mac-rophages, and the inhibitory effects of D4F on the ox-LDL-induced macrophage changes above were enhanced by PBA and blocked by TM. Furthermore, the apoptosis and expression of CHOP were reduced in atherosclerotic le-sions of D4F-treated apoE � / � mice. The translocation of ATF6 to the nucleus and phosphorylation of PERK play critical roles in the ER stress-CHOP pathway ( 8, 31, 32 ). Recently, we showed that ATF6 and PERK upregulate CHOP expression and subsequently mediate ox-LDL-induced apoptosis in macrophages ( 10 ). Here, we found that similar to PBA, D4F signifi cantly decreased nuclear translocation of ATF6 and phosphorylation of PERK and eIF2 � in ox-LDL- and TM-treated macrophages. Taken to-gether, these fi ndings indicate that D4F may suppress the activation of the two crucial upstream signals, nuclear translocation of ATF6 and phosphorylation of PERK, thereby inhibiting the ER stress-CHOP pathway.

How does D4F affect the upstream signals of the ER stress-CHOP pathway? Numerous experimental fi ndings, including our own, have revealed that accumulation of intracellular cholesterol is an important inducer of ER stress and macro-phage apoptosis in vivo and in vitro ( 33–35 ). CD36, a class B scavenger receptor, plays a quantitatively crucial role in ox-LDL uptake and cholesterol accumulation in macrophages, whereas suppression of CD36 expression is able to signifi -cantly decrease the ability of macrophages to accumulate ox-LDL and reduce the development of atherosclerosis, suggesting that it could be an important target for therapeu-tic treatment ( 27, 36, 37 ). Our recent study indicated that CD36-mediated ox-LDL uptake in macrophages triggered ER stress response (ATF6, inositol-requiring kinase/endo-nuclease-1, and GRP78), which, in turn, played a critical role in CD36 upregulation and then enhanced the foam cell for-mation by promoting uptake of ox-LDL, indicating that there may be a positive feedback loop between ER stress and CD36 expression ( 27 ). In the present work, we indeed observed

that D4F reduced serum ox-LDL level and CD36 expression in atherosclerotic lesions in apoE � / � mice. In cultured mac-rophages, D4F signifi cantly inhibited the upregulation of CD36 and GRP78 induced by ox-LDL or TM, and attenuated ox-LDL uptake, which was similar to anti-CD36 antibody. Fur-thermore, inhi bition of CD36 expression alone also showed similar effects as D4F. However, we did not observe additive inhibitory effects on ox-LDL-induced macrophage apoptosis, caspase-3 activation, and CHOP upregulation when the cells were treated with both D4F and CD36 siRNA, indicating that D4F and CD36 may work in a sequential rather than parallel pathway. Thus, D4F decreases CD36-mediated ox-LDL up-take and reduces intracellular lipid accumulation, which may inhibit nuclear translocation of ATF6 and phosphorylation of PERK and the subsequent CHOP-mediated macrophage apoptosis.

A clinical trial reveals that oral administration of D4F is safe and well-tolerated, and may improve the HDL anti-infl ammatory index ( 38 ). Thus, the mimetic peptide is increasingly considered as a potential effective treatment for atherosclerosis and related cardiovascular diseases. Our results indicate that D4F can alleviate macrophage-derived foam cell formation and apoptosis by inhibiting CD36-mediated ox-LDL uptake and the subsequent activa-tion of the ER stress-CHOP pathway. However, we have to be aware of the limitations of using in vitro models. Find-ings obtained from cultured cells in our study may not ex-actly represent what happens in vivo. For example, despite the fact that Cu 2+ -ox-LDL is commonly used in macro-phage foam cell study ( 39, 40 ), it is not the same as the ox-LDL in the circulation. Nevertheless, our fi ndings re-veal a potential novel mechanism for the antiatherogenic effects of D4F, which may provide additional support for the future therapeutic use of D4F.

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