Epigallocatechin-3-gallate Reduces Scavenger Receptor A

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Epigallocatechin-3-gallate Reduces Scavenger Receptor A Expression and Foam Cell Formation in Human Macrophages Sy-Jou Chen, Yung-Hsi Kao, Li Jing, Yi-Ping Chuang, Wan-Lin Wu, Shu-Ting Liu, Shih-Ming Huang, Jenn-Haung Lai, Ling-Jun Ho, Min-Chien Tsai, and Chin-Sheng Lin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05832 • Publication Date (Web): 03 Apr 2017 Downloaded from http://pubs.acs.org on April 4, 2017

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Journal of Agricultural and Food Chemistry

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Epigallocatechin-3-Gallate Reduces Scavenger Receptor A Expression and Foam Cell

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Formation in Human Macrophages

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Sy-Jou Chen1,2, Yung-Hsi Kao3, Li Jing4, Yi-Ping Chuang5, Wan-Lin Wu6, Shu-Ting Liu7,

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Shih-Ming Huang7, Jenn-Haung Lai8, Ling-Jun Ho9, Min-Chien Tsai10, and Chin-Sheng Lin11*

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Center, Taipei, Taiwan, R.O.C.; 2Graduate Institute of Injury Prevention and Control, College of

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Public Health and Nutrition, Taipei Medical University, Taipei, Taiwan, R.O.C.3Department of

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Life Sciences, National Central University, Jhongli, Taoyuan , Taiwan, R.O.C.; 4Department of

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Emergency Medicine, The University of Illinois Hospital &Health Sciences System, Chicago, IL,

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USA; 5Department and Graduate Institute of Microbiology and Immunology, National Defense

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Medical Center; 6Department of Cell Biology and Neuroscience, College of Natural and

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Agricultural Sciences, University of California Riverside, Riverside, California, United States of

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America; 7Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, R.O.C;

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8

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Gung Memorial Hospital, Tao-Yuan, Taiwan, R.O.C.; 9Institute of Cellular and System Medicine,

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National Health Research Institute, Zhunan, Taiwan, R.O.C.;

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National Defense Medical Center, Taipei, Taiwan, R.O.C.; 11Division of Cardiology, Department

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of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan,

Department of Emergency Medicine, Tri-Service General Hospital, National Defense Medical

Division of Allergy, Immunology and Rheumatology, Department of Internal Medicine, Chang

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ACS Paragon Plus Environment

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Department of Physiology,


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R.O.C..

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Total number: 5974 words; 0 tables; 6 figure; 40 pages

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Running Title: Epigallocatechin-3-gallate inhibits foam cell formation

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*

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325, Sec. 2, Cheng-Kung Rd., Neihu 114, Taipei, Taiwan. Tel: 886-2-8792-7160; FAX:

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886-2-6601-2656; E-mail: [email protected]

Address correspondence: Dr. C.-S. Lin, Division of Cardiology, Tri-Service General Hospital, No.

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ABSTRACT

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Foam cells are formed when macrophages imbibe low-density lipoprotein (LDL) through

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scavenger receptors. Here we examined how epigallocatechin-3-gallate (EGCG) influences foam

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cell formation. We found that EGCG dose-dependently reduced oxidized LDL (oxLDL) uptake in

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THP-1 (10 μM, 20.0 ± 0.50, p < 0.05) and primary macrophages (134.6 ± 80.8, p < 0.05), and

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reduced intracellular cholesterol content in these cells, respectively (10 μM, 32.6 ± 0.14, p < 0.05;

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31.7 ± 1.26, p < 0.05). EGCG treatment decreased scavenger receptor A expression, but not the

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expression of CD36 or of reverse cholesterol transporters. Moreover, EGCG stimulated

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translocation of the p50 and p65 subunits of NF-κB, and enhanced NF-κB DNA-binding activity,

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thus suppressing SR-A promoter activity. EGCG’s suppression of SR-A expression was blocked

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by the NF-κB inhibitor Bay. The present findings suggest that EGCG regulates NF-κB activity,

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and thus suppresses SR-A expression, oxLDL uptake, and foam cell formation.

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KEYWORDS: atherosclerosis, foam cells, epigallocatechin-3-gallate, oxidized low-density

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lipoprotein, macrophage

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INTRODUCTION

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Characteristics of atherosclerosis include chronic inflammation, lipid accumulation, and foam cell

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formation within the intimal layer of medium and large arteries.1,2 Reducing foam cell formation

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is a potential strategy for atherosclerosis prevention or reduction. Macrophages can transform into

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foam cells through accumulation of excessive cholesterol, which further prompts the release of

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proinflammatory chemokines and cytokines and further activation of vascular endothelial cells.3,4

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Macrophages ingest oxidized low-density lipoprotein (oxLDL) or acetylated LDL mainly through

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scavenger receptors (SRs) on their plasma membranes. CD36 and class A SR (SR-A) are the two

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main SRs responsible for oxLDL internalization in macrophages. Excessive intracellular

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cholesterol can be removed by reverse cholesterol transporters (RCTs),4–6 including ATP-binding

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cassette transporter A1 (ABCA1) and G1 (ABCG1), which mediate cellular cholesterol efﬂux.

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Epigallocatechin-3-gallate (EGCG), the main catechin in green tea (Camellia sinensis),

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possesses multiple important biological properties with both antioxidant and nonantioxidant

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functions.7,8 Based on its potent antioxidant effects and anti-inflammatory nature, EGCG has been

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intensely investigated as a cancer-preventive agent. EGCG also shows clinically beneficial effects

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against diabetes, obesity, stroke, Alzheimer’s disease, and Parkinson’s disease.8–10 In vivo studies

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reveal that EGCG is inversely associated with atherosclerosis development and progression in

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atherogenic animals,11–13 likely due to EGCG’s antioxidant, antiendothelial dysfunction,

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anti-inflammatory, and antithrombogenic properties.14

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In studies of human endothelial cells, Yamagata et al. demonstrated that EGCG modulates 4


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tumor necrosis factor alpha (TNFα)-induced monocytic cell adhesion, autophagy, and apoptosis

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by affecting LC3, as well as processes related to vascular cell adhesion protein-1 (VCAM1).15

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Other investigations of endothelial cells reveal that EGCG inhibits AhR-regulated genes [e.g.,

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cytochrome P450 1A1 (Cyp1A1), monocyte chemotactic protein-1 (MCP-1), and VCAM-1] and

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induces antioxidant enzymes regulated by Nrf2, which ultimately protects against vascular

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inflammatory responses induced by polychlorinated biphenyl, such as atherosclerosis.16 In

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hypercholesterolemic rat livers, EGCG significantly reduces the expression of TNFα-mediated

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nuclear factor of activated T-cells, indicating its capacity to inhibit hepatic steatosis and influence

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early atherosclerotic events.17 Prior studies have also examined EGCG’s anti-inflammatory effects

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on macrophages. In endotoxin-stimulated macrophages, EGCG stimulates autophagy and reduces

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the cytoplasmic proinflammatory mediator high mobility group box 1 protein, which is beneficial

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against several inflammatory diseases, including sepsis, cancer, and atherosclerosis.18 These

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results elucidate the molecular mechanisms underlying EGCG’s protective roles in

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atherosclerosis.

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Although prior studies of tea catechin show its antiartherogenic efficacy and indicate its

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potential to inhibit foam cell formation processes, scarce data are available regarding EGCG’s

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effects on foam cell development and the mechanisms underlying this process. To delineate these

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effects, we assessed how cholesterol uptake and efflux impacted human THP-1 macrophages and

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primary macrophages, and examined the potential underlying mechanisms.

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MATERIALS AND METHODS

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Chemicals. EGCG of >98% purity was isolated using a Sephadex LH-20 column, and

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characterized based on absorption at 280 nm, high-performance liquid chromatography, and

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nuclear magnetic resonance spectroscopy as previously described.19,20 We purchased

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1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocyanine perchlorate (DiI)-labeled oxidized LDL

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(DiI-oxLDL) from Intracel (Frederick, MD). We obtained an Amplex Red Cholesterol assay kit

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from Molecular Probes (Eugene, OR), anti-CD14 microbeads from Miltenyi Biotec (Auburn, CA),

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macrophage-colony stimulating factor (M-CSF) from R&D systems (Minneapolis, MN), and

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Bay11-7085 from Merck Millipore. Unless otherwise specified, all other reagents were purchased

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from Sigma-Aldrich Chemical Company (St. Louis, MO).

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Cell Culture. The human monocytic cell line THP-1 was obtained from the Bioresource

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Collection and Research Center (Hsinchu, Taiwan). These cells were grown in RPMI 1640

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medium with the addition of 10% fetal bovine serum (FBS), streptomycin (100 µg/mL), and

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penicillin (100 U/mL). To promote differentiation into macrophages, the THP-1 cells were

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incubated with 100 ng/mL PMA for 3 days at a density of 1 × 106 cells/mL.21

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Primary Macrophage Establishment from Human Peripheral Blood Monocytes. CD14+

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monocytes were positively selected using a MACS cell isolation kit (Miltenyi Biotech, Auburn,

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CA), and were used to establish human primary macrophages.21 Using the Ficoll–Hypaque

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technique, a diluted buffy coat suspension was layered from an anticoagulated blood sample. After

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several washes and centrifugation, the peripheral blood mononuclear cells were collected and were

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incubated for 15 min at 4–8 °C with anti-CD14 microbeads. Next, the CD14+ cells were isolated

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via magnetic separation with a MACS cell isolation column. We cultured these CD14+ monocytes

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at 1 × 106 cells/mL density in a RPMI 1640 medium containing 10% FBS and 50 ng/mL M-CSF.

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Before performing additional experiments, the cells were cultured for 6–7 d, with culture medium

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replacement every two days.

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Assessment of EGCG’s Nonspecific Cytotoxicity. As an indicator of plasma membrane

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damage and cell death, we measured lactate dehydrogenase (LDH) release using an assay kit

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(Roche, Indianapolis, IN) in accordance with the manufacturer’s instructions. The cytotoxicity

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percentage was calculated as follows: ([sample value − med ium control]/[high control − medium

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control]) × 100. The sample values were the absorbance value averages from triplicate

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measurements with the indicated doses of EGCG-treated THP-1 macrophage supernatants,

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following subtraction of background control absorbance values. As a medium control, we used

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untreated cell culture supernatants, and the control average absorbance values were calculated

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similarly. As a high control, we used equal amounts of 1% Triton X-100-treated cells.

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We performed the tetrazolium salt 3-[4,5-dimethylth-iazol-2-yl]-2,5-diphenyl tetrazolium

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bromide (MTT) assay as previously described.22,23 Macrophages were incubated for 24 h at a

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density of 1 × 106 /mL in a 100-µL volume with or without EGCG. We then added 100 µL MTT

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(5 mg/mL in H2O) for an additional 3.5-h incubation at 37 °C, followed by the addition of 100 µL

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dimethylformamide. Finally, we measured the dissolved reduced MTT crystal content at 570 nm

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with an ELISA reader (Dynatech, Chantilly, VA).

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DiI-oxLDL Uptake Assay. We incubated THP-1 and primary macrophages with DiI-oxLDL

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for 24 h at 37 °C in RPMI 1640 medium supplemented with 10% FBS. We used flow cytometry to

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analyze DiI-oxLDL uptake.24,25

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Cholesterol Efflux. THP-1 macrophages in 12-well plates (1 × 106 cells/well) were incubated

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with 0.5 μCi/mL 3H-cholesterol (Perkin Elmer) at 37 °C for 24 h. The cells were then washed

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twice with PBS, and then treated for 24 h with indicated doses of EGCG. Treated cells were then

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incubated for another 2 h in medium containing 2% fatty acid-free BSA (FAFA, Sigma) as well as

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2 μM acetyl-coenzyme A acetyltransferase inhibitor. Next, we added 50 μg/mL human apoA1 or

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HDL, and the cells were incubated for another 6 h. Finally, the cells and medium were collected,

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and we measured the radioactivity. Values are expressed as the percentage of total 3H-cholesterol

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content in the cell (total effluxed 3H-cholesterol + cell-associated 3H-cholesterol).

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Intracellular Cholesterol Assay. We quantified the total intracellular cholesterol content of

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macrophages using a previously described fluorometric method.24 Macrophages at a density of 2.5

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× 105/mL were fixed in 2% (v/v) paraformaldehyde for 15 min. We then washed the cells with

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PBS, and incubated them for 30 min at 4 °C with 300 μL 3:2 (v/v) hexane:isopropanol to extract

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cellular lipids. Next, the organic solvents were evaporated using a speed vacuum (Thermo Fisher,

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Chicago, IL), and the extracted lipids were dissolved in the dilution buffer supplied in the

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cholesterol assay kit. We incubated 50 μL of the diluted extracted lipids with 50 μL Amplex Red

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assay reagent and cholesterol esterase at 37 °C in the dark for 30 min. Finally, we determined total

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cholesterol content by measuring the fluorescence intensity with excitation and emission

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wavelengths of 544 nm and 584 nm, respectively, using a BioTek microplate reader (Bio-Tek

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Instruments, Inc., Winooski, VT, USA). Total cholesterol level are reported as µg total cholesterol

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per mg protein.

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Oil Red O Staining. After washing with PBS, THP-1 and primary macrophages were fixed

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via incubation with 10% formalin for 1 h.21 Next, the macrophages were removed from the

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formalin, treated for 5 min with 60% isopropyl alcohol, and then stained for 10 min with 0.2% Oil

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Red O solution (Sigma-Aldrich) in 60% isopropyl alcohol. Prior to use, the Oil Red O solution

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was run through a 0.22-μm filter. The cells were washed 4–5 times to remove excess stain, and

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then incubated for 1 min with hematoxylin. Finally, the cells were washed and then examined by

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confocal microscopy (Zeiss LSM780).

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Western Blotting. We performed enhanced chemiluminescence (ECL) western blotting

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(Amersham) as previously described.26 The treated cells were washed, and then resuspended in

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lysis buffer, followed by protein concentration determination. We loaded equal quantities of whole

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cellular extracts into 8–10% sodium dodecyl sulfate-polyacrylamide gels for electrophoresis. Then

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the separated proteins were transferred to a nitrocellulose membrane, which was blocked for 1 h

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with TBS-T with 5% nonfat milk. The membrane was next incubated overnight at 4 °C with

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primary antibodies against SR-A, CD36, total ERK, JNK, p38 (Santa Cruz Biotechnology), actin

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(Chemicon, Temecula, CA), ABCG1 (Novus Biologicals, Littleton, CO), and ABCA1 (Abcam,

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Cambridge, UK), or phosphorylated ERK, JNK, and p38 (Cell Signaling, Danvers, MA). The next

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day, the membrane was washed with TBS-T buffer, and then incubated for 1 h with conjugated

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secondary antibodies (diluted 1:5000) in blocking buffer. Finally, the membrane was thoroughly

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washed, soaked in a substrate reagent, and exposed to X-ray film.

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Quantitative Reverse Transcription Polymerase Chain Reaction. We performed

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quantitative reverse transcription polymerase chain reaction (qRT-PCR) using SYBR Green

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Master Mix (Applied BioSystems, Foster City, CA) in accordance with the manufacturer’s

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instructions. Treated cells were lysed with Trizol (Invitrogen, Carlsbad, CA). Then total RNA was

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isolated, and reverse transcribed to cDNA, which was used for PCR. Briefly, we amplified 10 ng

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cDNA in a 20-μL total volume including 1× Master Mix and gene-specific primers at a final

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concentration of 100 nM. Reactions involved 40 cycles of denaturation at 95 °C, and annealing

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and extension at 60 °C, in an ABI 7300 real-time PCR system (Applied BioSystems, Foster City,

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CA). Data are presented as expression of the gene of interest relative to the expression of an

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internal

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2−ΔΔCT method.27 In accordance with our previous studies,21,28 the following sequences were used:

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SR-A, 5ʹ -TTTGATGCTCGCTCAATGACA-3ʹ and 5ʹ-GCTGCCACTATTCCAATGAGAG-3ʹ;

control

gene

(GAPDH)

using

the

comparative CT method,

10


also

called

the

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CD36, ʹ 5 -GGCTGTGACCGGAACTGTG-3ʹ

and

5ʹ-TTCTGTGCCTGTTTTAACCCAA-3ʹ;

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GAPDH, ʹ5 -ATGGGGAAGGTGAAGGTCG-3ʹ and 5ʹ-TAAAAGCAGCCCTGGTGACC-3ʹ;

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ABCG1, 5'-CCAGAAGTCGGAGGCCATC-3' and 5'-AAGTCCAGGTACAGCTTGGCA-3'; and

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ABCA1,

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5'-TGTCCTCATACCAGTTGAGAGAC-3'.

5'-GGTGATGTTTCTGACCAATGTGA-3'

and

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Nuclear Extract Preparation. We prepared nuclear extracts as previously described.29 Cells

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were collected, suspended in 70 µL buffer A [l0 mM HEPES, pH 7.9; 10 mM KCl; 1.5 mM

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MgC12; 1 mM dithiothreitol (DTT); 1 mM PMSF; and 3.3 µg/mL aprotinin], and incubated for 15

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min at 4 °C with gentle vortexing. We next centrifuged the resultant swollen cells for 3 min at

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15,000 rpm, and collected the supernatants, which contained the cytoplasmic extract. We

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subsequently washed the pelleted nuclei in 70 µL buffer A, and centrifuged them for another 20

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min at 15,000 rpm. We next resuspended these nuclear pellets in 25 µL buffer C (20 mM HEPES,

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pH 7.9; 1.5 mM MgCl2; 420 mM NaCl; 0.2 mM EDTA; 1 mM DTT; 0.5 mM PMSF; 25%

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glycerol; and 3.3 µg/mL aprotinin) and incubated the mixture for 20 min at 4 °C with occasional

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vigorous vortexing. Finally, we centrifuged these mixtures for 20 min at 15,000 rpm, and collected

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the supernatants, which contained the nuclear extract.

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Electrophoresis Mobility Shift Assay. As probes, we used DNA oligonucleotides that

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contained the NF-κB binding siteʹ (5 -AGTTGAGGGGACTTTCCCAGGC-3ʹ) and were

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radiolabeled with [γ-32P] ATP using T4 kinase (Promega, MI, USA). The previously prepared

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nuclear extracts were mixed with binding buffer [10 mM Tris-HCl, pH 7.5; 50 mM NaCl; 1 mM

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EDTA; 1 mM DTT; 5% glycerol; and 2 µg poly(dI-dC)] and incubated for 20 min at 4 °C. Next, 5

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µg of nuclear extract in binding buffer was incubated with the radiolabeled NF-κB probes, and

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this reaction mixture was incubated for 20 min at room temperature. Following this reaction time,

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the mixture was run in a 6.6% nondenaturing polyacrylamide gel at constant voltage using 0.5×

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Tris-borate/EDTA as the electrophoresis buffer. Finally, we dried the gel and exposed it to X-ray

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film at −80 °C overnight.

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Transfection and Reporter Assay. CMV expression plasmid DNAs for NFκB p65 and p50

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were purchased

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reporter hSR.A(−1000/+122)-LUC

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(5′-hSR.A 5′-ccgCTCgAgATCATTATAGAGAGAACAGGAGT-3′

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and 3′-hSR.A 5′-CCCAAgCTTACTTCTTTCGTCCTAAAGAAAGC-3′)

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basic-LUC reporter plasmid via the XhoI and HindIII restriction sites. Cells in 24-well plates were

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transfected with jetPEI (PolyPlus-transfection, Illkirch, France) following the manufacturer’s

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protocol. Luciferase activity measurements were attained from two transfected sets using the

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Promega Luciferase Assay kit (Promega), and results were numerically expressed in relative light

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units as the mean ± standard deviation as previously described.30

from

Panomics,

Inc.

by inserting

(CA,

USA). We

constructed

the

the

appropriate

PCR

fragments

into

the pGL3

214

Statistical Analysis. Comparisons between multiple groups were performed using one-way

215

analysis of variance (ANOVA) with Bonferroni post-hoc tests. Comparisons between two groups

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were performed using Student's t-tests. A p value of

Epigallocatechin-3-gallate Reduces Scavenger Receptor A

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