Materials and Methods Cell culture Foam cell formation Cellular uptake of DiI-OxLDL In vitro cholesterol efflux assays

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Augustine Webster
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1 Materials and Methods Cell culture Mouse peritoneal macrophages (MPMs) were harvested from adult C57BL/6J mice (Jackson Laboratories, Sacramento, CA) and cultured as previously described. 1 J774.A1, THP-1, and HEK293 cells (American Type Culture Collection, Manassas, VA) were maintained according to the manufacturer s instructions. The differentiation of THP-1 monocytes into macrophages was induced by 167 nmol/l phorbol 12-myristate 13-acetate (Wako, Tokyo, Japan) for 48 hours. 2 Foam cell formation Foam cell formation was assessed by the methods of Oil-red-O and filipin staining which had been established in our laboratory previously. 3 Macrophages were incubated with the vehicle (DMSO), oxidized low-density lipoprotein (OxLDL) (50 g/ml), CoQ10 (Sigma-Aldrich, St Louis, MO) (1, 10, 100 M), or a combination of CoQ10 and OxLDL in RPMI 1640 medium supplemented with 10% FBS for 24 hours. After fixation by 4% paraformaldehyde, cells were stained with 0.5% Oil-red-O and hematoxylin was used as counterstaining. Density of the total cellular lipid content was determined by a microplate reader (absorbance at 540 nm) after alcohol extraction. 4 The total lipid in cells was extracted with hexane/isopropanol (3/2, vol/vol). The lipid extracts were dried under nitrogen flush, and dissolved in a solution with 90% isopropanol and 10% Triton X-100. Total cholesterol content was quantified enzymatically. For filipin staining, macrophages were treated with CoQ10 or DMSO in the presence or absence of OxLDL in RPMI 1640 medium supplemented with 1% Nutridoma for 24 hours. The cells were washed with ice-cold PBS and fixed with paraformaldehyde for 30 min at 4 C, and then maintained in PBS containing 50 µg/ml filipin for 30 min at room temperature. Fluorescent microscopy was explored to visualize cells. 5 Cellular uptake of DiI-OxLDL Macrophages pretreated with CoQ10 (1, 10, 100 M) for 24 hours were incubated with 1,1 -Dioctadecyl-3,3,3 3 -tetramethylindocarbocyanine perchlorate (DiI)-labeled human OxLDL (Intracel Corp, Rockville, Md) in RPMI 1640 medium at 37 C for 4 hours. The cells were then visualized by a fluorescence microscope (Nikon ECLIPSE TI-E). DiI was extracted by isopropanol and the fluorescence was determined at 520/564 nm. 6 In vitro cholesterol efflux assays Assays of in vitro cellular cholesterol efflux have been previously described. 7 Macrophages were labeled with 1.0 Ci/mL 3 H-cholesterol (PerkinElmer, Boston, MA) in RPMI 1640 medium supplemented with or without OxLDL (50 g/ml) for 24 hours. The cells were maintained in media supplemented with 0.1% bovine serum albumin (BSA) in the presence or absence of CoQ10 at the indicated concentrations for 24 hours. Cholesterol efflux was then initiated by medium containing 0.1% BSA in the presence or absence of apolipoprotein A-I (ApoA-I) (15 g/ml) or HDL (100 g/ml) 1

2 for 24 hours. Supernatants were collected after 24 hours and calculated as a percentage of total cell 3 H-cholesterol content (total effluxed 3 H-cholesterol + cell-derived 3 H-cholesterol). Quantitative real-time RT-PCR Total RNA isolation and mrna levels for specific genes in macrophages were performed using a Quantitative real-time polymerase chain reaction (qrt-pcr) assay. Primers sequences used were shown in Table I in the online-only Data Supplement. GAPDH mrna was used as the internal control. Primers specific for mouse and human mir-378, mir-1899, mir-185*, and mir-351 (SABiosciences, San Diego, CA) were utilized and values were normalized to U6 as a house keeping gene. Western blotting Proteins from whole-cell lysates or nuclear extracts were separated by SDS-polyacrylamide gel electrophoresis. Method for Western blot has been previously described. 7 Antibodies against ABCG1 were purchased from Abcam, scavenger receptor class B type I (SR-BI) was from Novus, and -actin was from Cell Signaling Technology. Polyclonal antibodies against ABCA1, LXR, c-jun, c-fos, Elk-1, p-elk-1, c-rel, and histone H1 were form Santa Cruz Biotechnology. Small interfering RNA (sirna) J774.A1 and THP-1 macrophages were transfected with 100 nm ABCG1 or c-jun SMARTpool sirna or sicontrol non-targeting sirna (Dharmacon, Lafayette, CO) at 40-60% confluency. The efficiency of transfection was analyzed utilizing siglo RISC-free non-targeting sirna (Dharmacon). ABCG1 mrna stability assay For mrna stability analysis, macrophages were loaded with OxLDL (50 g/ml) for 24 hours following treatment with CoQ10 (10 M), or the vehicle (DMSO) for another 24 hours. At 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 hours after the addition of actinomycin D (Sigma-Aldrich) (5 g/ml), 7 total RNA from the cells was harvested. ABCG1 mrna levels at indicated time points were quantitated by qrt-pcr and normalized to the GAPDH levels. The remaining mrna was determined by contrast with the expression level of the relevant gene at the zero time point (designated 100%) when actinomycin D was added. GraphPad Prism software version 5.0 was utilized to calculate the transcript half-lives based on a one-phase exponential decay model. mirna microarray analysis J774.A1 macrophages preloaded with OxLDL (50 g/ml) for 24 hours were incubated in medium containing 0.1% BSA and COQ10 (10 M) or the vehicle (DMSO) for another 24 hours. Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA) and mirneasy mini kit (Qiagen, Hilden, Germany). RNA quality and quantity was gauged by a nanodrop spectrophotometer (ND-1000, Nanodrop Technologies), and RNA integrity was determined using gel electrophoresis. The labeling, hybridization and 2

3 scanning of isolated RNA were performed by Exiqon (Denmark) using LNA mircury array (v.16.0). Scanned images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction. The mirna expression levels were modulated with a Fold Change Filtering (treated/control) of 2.0 or more were considered as differentially expressed. mir-378 and anti-mir-378 transfection J774.A1 and THP-1 macrophages loaded with or without OxLDL as well as HEK293 were transfected with miridian mirna mimics (mir-378) or with miridian mirna inhibitors (anti-mir-378) (Dharmacon) utilizing Oligofectamine (Invitrogen) for 48 hours. Transfections of all control samples were performed using a non-targeting negative control mimic sequence (Con mir) or an inhibitor negative control sequence (Con Inh) (Dharmacon) as controls for nonensequence-specific effects in mirna experiments. 3'-UTR vector constructions and luciferase reporter assay The entire 3'-UTRs of mabcg1 (3696 nt, NM_009593) and habcg1 (850 nt, NM_004915) were cloned into EcoRV and Xba1 sites of pgl3 cm generated on the basis of the pgl3-control vector (Promega, Madison, WI), creating the pgl3-m/habcg1-wt constructs. Point mutations in the seed regions of putative mir-378 sites within the 3'-UTRs of m/h ABCG1 were performed utilizing QuikChange Multi Site-Directed Mutagenesis (Stratagene) in accordance with the manufacturer s protocol. Sequencing was used to validate all the constructs. HEK293 cells were plated into 96-well plates and cotransfected with mir-378 or Con mir (Dharmacon), 50 ng of pgl3-wt or pgl3-mutation and 50 ng of prl-tk vector (Promega) using Oligofectamine. Luciferase activity was gauged by the Dual-Luciferase Reporter Assay System (Promega), and prl-tk was used as an internal control. To evaluate the impact of CoQ10, J774.A1 and THP-1 macrophages preloaded with OxLDL (50 g/ml) were cotransfected with pgl3-wt and prl-tk in the presence or absence of CoQ10 (10 M) for 24 hours. The luciferase activity was then measured as above. Chromatin immunoprecipitation (ChIP) The ChIP assay was performed using anti-c-jun antibody (Santa Cruz Biotechnology) and the Magna ChIP-G kit (Millipore) per manufacturer's instruction. Enrichment analysis was carried out using real-time PCR with specific primers. Luciferase reporter constructs and luciferase assay Promoters of mouse and human mir-378 genes were amplified by PCR. The PCR products were separated by agarose gel electrophoresis, and then the DNA fragments isolated and cloned in the restriction enzyme digested pgl3 Basic Vector (Promega) using T4 DNA ligase (Fisher scientific). All constructs were confirmed by sequencing. Mutations were introduced into the AP-1 binding sites using QuikChange site-directed mutagenesis kit (Stratagene). J774.A1 and THP-1 macrophages were transfected with each reporter construct for 24 hours and then treated with CoQ10 for 24 hours in 3

4 the presence or absence of 12-O-tetra-decanoylphorbol-13-acetate (TPA) followed by assessment of luciferase activity. Luciferase activities were measured and normalized to the control -gal level. The luciferase activity of each construct was compared with that of the promoterless pgl3 basic vector. Mouse studies All the experiments involving the use of animals were conducted in accordance with institutional guidelines of Sun Yat-sen University. 4-week old male ApoE / mice (Jackson Laboratories, Sacramento, CA) were weaned and placed on the AIN-93G diet for another 26 weeks, at which point the mice were sacrificed (baseline, n = 12) or subjected to the following experiments. Experiment 1 The ApoE / mice fed with the AIN-93G diet were orally gavaged with CoQ10 (600 mg/kg BW) for 14 days. Half of the mice (n = 12) in 2 groups were intraperitoneally injected with thioglycollate to obtain MPMs on day 10. The residual mice were raised individually in metabolic cages during the following 48 hours, and the efficiency of in vivo macrophage RCT was quantified (see below). Experiment 2 The ApoE / mice were orally gavaged with CoQ10 (600 mg/kg BW), T (10 mg/kg BW 8 ) (Sigma-Aldrich, St Louis, MO), or vehicle (normal saline) for 4 weeks. At sacrifice, mice were fasted overnight, anesthetized with isoflurane, and exsanguinated by cardiac puncture. MPMs, hearts, and thoracic as well as abdominal aortas were collected and stored at -80ºC for further experiments. In vivo macrophage RCT assay Macrophage RCT studies were performed as described previously. 9 In brief, the ApoE / mice were intraperitoneally injected with 3 H-cholesterol-labeled and OxLDL-loaded J774.A1 macrophages. Radioactivities of 3 H-tracer in blood, liver, and feces were then monitored and converted into RCT. Blood parameters measurement Serum total CoQ10 was quantified by high-performance liquid chromatography with electrochemical detection. 10 Plasma TG and total cholesterol (TC), and HDL-C were gauged by commercial kits (BioSino Biotechnology Company, Ltd., Beijing, China). Plasma ApoA-I were measured by immunoturbidimetry. Pooled plasma samples were subjected to fast protein liquid chromatography (FPLC). 7 Individual fractions were assayed for cholesterol content. Fractions contain VLDL and chylomicra; fractions contain IDL and LDL; fractions contain HDL; and fractions contain non-lipoprotein-associated proteins. Statistical analysis Results are presented as mean ± the standard error of the mean (SEM). Data were analyzed by Student s t test or one-way ANOVA coupled with Bonferroni-Dunn post hoc test where are performed using of the SPSS for Windows software (version 13.0; 4

5 SPSS Inc, Chicago, IL). P < 0.05 was considered to be statistical significant. References 1. Wang D, Zou T, Yang Y, Yan X, Ling W. Cyanidin-3-o-beta-glucoside with the aid of its metabolite protocatechuic acid, reduces monocyte infiltration in apolipoprotein e-deficient mice. Biochem Pharmacol. 2011;82: Wang Q, Xia M, Liu C, Guo H, Ye Q, Hu Y, Zhang Y, Hou M, Zhu H, Ma J, Ling W. Cyanidin-3-o-beta-glucoside inhibits inos and cox-2 expression by inducing liver x receptor alpha activation in thp-1 macrophages. Life Sci. 2008;83: Li D, Wang D, Wang Y, Ling W, Feng X, Xia M. Adenosine monophosphate-activated protein kinase induces cholesterol efflux from macrophage-derived foam cells and alleviates atherosclerosis in apolipoprotein e-deficient mice. J Biol Chem. 2010;285: Lu KY, Ching LC, Su KH, Yu YB, Kou YR, Hsiao SH, Huang YC, Chen CY, Cheng LC, Pan CC, Lee TS. Erythropoietin suppresses the formation of macrophage foam cells: Role of liver x receptor alpha. Circulation. 2010;121: Rigamonti E, Helin L, Lestavel S, Mutka AL, Lepore M, Fontaine C, Bouhlel MA, Bultel S, Fruchart JC, Ikonen E, Clavey V, Staels B, Chinetti-Gbaguidi G. Liver x receptor activation controls intracellular cholesterol trafficking and esterification in human macrophages. Circ Res. 2005;97: Li L, Sawamura T, Renier G. Glucose enhances human macrophage lox-1 expression: Role for lox-1 in glucose-induced macrophage foam cell formation. Circ Res. 2004;94: Wang D, Xia M, Yan X, Li D, Wang L, Xu Y, Jin T, Ling W. Gut microbiota metabolism of anthocyanin promotes reverse cholesterol transport in mice via repressing mirna-10b. Circ Res. 2012;111: Levin N, Bischoff ED, Daige CL, Thomas D, Vu CT, Heyman RA, Tangirala RK, Schulman IG. Macrophage liver x receptor is required for antiatherogenic activity of lxr agonists. Arteriosclerosis, thrombosis, and vascular biology. 2005;25: Zhang Y, Zanotti I, Reilly MP, Glick JM, Rothblat GH, Rader DJ. Overexpression of apolipoprotein a-i promotes reverse transport of cholesterol from macrophages to feces in vivo. Circulation. 2003;108: Okamoto T, Fukui K, Nakamoto M, Kishi T, Okishio T, Yamagami T, Kanamori N, Kishi H, Hiraoka E. High-performance liquid chromatography of coenzyme q-related compounds and its application to biological materials. J Chromatogr. 1985;342:

Molecular Medicine. Gut Microbiota Metabolism of Anthocyanin Promotes Reverse Cholesterol Transport in Mice Via Repressing mirna-10b

Molecular Medicine. Gut Microbiota Metabolism of Anthocyanin Promotes Reverse Cholesterol Transport in Mice Via Repressing mirna-10b Molecular Medicine Gut Microbiota Metabolism of Anthocyanin Promotes Reverse Cholesterol Transport in Mice Via Repressing mirna-10b Dongliang Wang, Min Xia, Xiao Yan, Dan Li, Lei Wang, Yuxuan Xu, Tianru