LX Reference Estronca 2012

Materials and Methods

Link to PLoS One

Chemicals and antibodies

Oil-red, Cholesteryl hemisuccinate, Dextran (mol wt 9,000–11,000) were obtained from Sigma. PO-PC and cholesterol were purchased from Avanti Polar Lipids (Alabaster, AL). [3H]-Colesterol was from GE Healthcare. The other chemicals used were of analytical grade from local sources.

Rhodamin-phalloidin, Bodipy 493/503 and FITC-Dextran were purchased from Molecular Probes. DAPI was from Fluka. The anti-mouse (ABL-93) Lamp-2 antibody was from the Developmental Studies Hybridoma Bank, (University of Iowa, Iowa City, IA). Polyclonal guinea-pig anti-ADRP antibody was from Progen Biotechnik (Heidelberg, DE). Secondary antibodies were from Molecular Probes or from Jackson Immunoresearch. Mouse Anti-mouse CD36 (552544) and the negative control mouse anti-mouse IgA (553476) were purchased from BD Pharmigen; rat anti-mouse CD204 (MCA1322) and the negative control rat anti-mouse g2b (MCA1125) were purchased from AbD Serotec. Rabbit anti-mouse SR-BI (NB400-113) and the negative control rabbit anti-mouse IgG (NB810-56910) were purchased from Novus Biologicals.

Preparation of liposomes

Aqueous suspensions of lipids were prepared by mixing POPC and Chs at the desired ratios in an azeotropic mixture of chloroform and methanol. The solvent was evaporated by blowing dry nitrogen over the heated (blowing hot air over the external surface of the tube) solution and then leaving the residue in a vacuum desiccator for at least 12 h at 23°C. The dry residue and the hydration solution (20 mM Hepes, 0.11 M NaCl, 1 mM EDTA, pH 7.4) with or without Dextran in an amount necessary to obtain a density of 1.044 g/ml, were preheated in a water bath at 65°C. The samples were submitted to several cycles of vortex/incubation/mild sonication at 65°C for at least 1 h. The resulting multilamellar vesicle (MLV) suspensions were extruded through two stacked polycarbonate filters (Nucleopore) with a pore diameter of 0.1 µm using a minimum of 6 passes. During the extrusion the water-jacketed extruder (Lipex Biomembranes, Vancouver, British Columbia, Canada) was maintained at 65°C. Final lipid molar ratios of Chs/POPC were: 0/100, 70/30, and 45/55.

LDL isolation, acetylation and Chs incorporation

Human LDL were isolated using a Beckman L80 ultracentrifuge equipped with a 70.1 Ti fixed angle rotor as described previously [46]. LDL were acetylated by repeated additions of acetic anhydride as described [47]. Chs-LDL were prepared by incubating Nat-LDL with Chs-POPC (70:30) liposomes in Hepes buffer containing dextran (density of 1.044 g/ml) overnight at different LDL/Chs ratios at RT without stirring. After incubation the Chs-LDL were re-centrifuged to eliminate the remaining Chs-POPC liposomes and after dialysis they were passed through a 0.2 µm filter. Chs content of Chs-LDL was assessed by measuring the radioactivity of the samples. All stock and working solutions of different LDL models were stored under N2 at 4°C for a maximum of 2 weeks.

Agarose gel electrophoresis

Electrophoresis of LDL preparations was carried out in 0.5% agarose gels in barbital buffer, pH 8.6, at a constant voltage of 75 V for 45 min, and LDL were stained with 0.5% Paragon Blue in 5% acetic acid for 3 min at room temperature. The gel was destained at room temperature with 5% acetic acid, followed by 20% acetic acid, 30% methanol. In each agarose strip, Nat-LDL were used as a control.

Cell culture and incubation with the different LDL models and Chs-POPC liposomes

RAW 264.7 cells (ATCC) were maintained in DMEM supplemented with 10% fetal calf serum, 100 U/ml of penicillin and streptomycin. Cells were grown in a humidified incubator at 37°C under 5% CO2 and used for assays during the exponential growth phase. For lipid droplet staining RAW cells were grown on glass-coverslips or in 24 well-plates for 24 h. They were then incubated with Nat-LDL; Ac-LDL, Chs-LDL, Chs-(Ac-LDL) or Chs-POPC liposomes for different periods of time and at different concentrations, as indicated in the figure legends. After incubation the cells were fixed with PFA.

Immunofluorescence and Confocal microscopy

Cells were grown on 24-well plates and fixed with 4% PFA for 60 min followed by quenching of the aldehyde groups with glycine or ammonium chloride and permeabilization with isopropanol (60%) or saponin (0.1%) for LAMP-2 and ADRP staining, respectively. Cells were then incubated with the primary antibodies for 1 h at room temperature, washed, and finally incubated with the secondary antibodies conjugated with a fluorophore for another 1 h. Antibody dilutions were 1:100 for primary antibodies and 1:500 for secondary antibodies conjugated with Cy3 or 1:200 for secondary antibodies conjugated with Cy5 or Alexa Fluor®594. Fixed samples were analyzed by using the LSM 510 META point-scan confocal laser microscope (Zeiss) with a 63× oil-immersion objective.

Neutral lipid staining and quantification

Neutral lipids were stained either with Oil-Red O (in 60% isopropanol, prepared from a stock solution at 0.4% w/v in 100% isopropanol) for 10 min or with Bodipy 493/503 (diluted 1:1000 in PBS from a saturated ethanolic solution of Bodipy) for 15 min. DAPI at a final concentration of 30 nM was added for 20 min at room temperature to visualize nuclei. Quantification of the number and size of the lipid-rich structures was performed using ImageJ software.

Electron Microscopy

Cells were seeded on glass coverslips and treated the next day. Cells were fixed in 2.5% glutaraldehyde in cacodylate buffer and processed for standard Epon embedding. 70 nm sections were analyzed in a TECNAI12 Biotwin electron microscope equipped with a bottom-mount 4×4 k CCD camera (FEI company).

Toxicity evaluation by the MTT test

Cell viability was measured by the MTT assay as previously described [48].

Mass Spectrometry

Cells were incubated in 6-well plates with Nat-LDL, Ac-LDL, Chs-LDL or Chs-POPC liposomes for 24 and 48 h. After incubations cells were washed three times with 150 mM ammonium bicarbonate and harvested from wells by scraping into 1 ml of ammonium bicarbonate. Protein content of the suspensions was evaluated and aliquots of the cell lysates or Nat-LDL suspensions containing 10 and 1 µg of protein, respectively, were extracted as described previously [49]. For absolute quantification, internal standards were added to the samples prior to lipid extraction. Cholesterol was quantified as described by Sandhoff et al. [50]. Top-down shotgun analysis was performed on a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a TriVersa NanoMate robotic nanoflow ion source (Advion BioSciences, Ithaca, NJ) as described in [33]. Lipids were identified and quantified by LipidXplorer software developed in house [51].

Chs uptake

After incubating the macrophages with 3H-Chs-LDL, 3H-Chs-(Ac-LDL), and 3H-Chs-POPC liposomes with similar radioactivity for different periods of time at 37°C, cell-associated [3H]-Chs was determined after rinsing macrophages with DMEM with 0.5% BSA at pH 3.4 for 10 min on ice, three further rinses with PBS, and a final rinse in distilled water. Macrophages were scraped off into 1 ml distilled water. Then cell samples were assayed for 3H radioactivity and for cellular protein.

For the experiments with the inhibitory antibodies the cells were incubated at 4°C for 15 min with 10 µg/ml of anti-CD36, anti-SR-BI, anti-CD204 and their respective antibody controls; after which 300 µg/ml of 3H-Chs-(Ac-LDL), 3H-Chs-LDL, or 3H-Chs-POPC liposomes (45:55) with equivalent amounts of Chs were added to cells. The cells were then shifted to 37°C for 3 h (in presence of the inhibitory antibodies). After incubation the cells were treated as described above and the radioactivity and the cell protein quantified.

Protein assay

The protein content of cell extracts was measured by the bicinchoninic acid (BCA, Pierce) assay using bovine serum albumin as a standard.

Statistical analysis

Results are expressed as the means ± standard deviations (S.D.). Statistical significance was assessed by the Student t-test (two-tailed). A p value of <0.05 was considered to be statistically significant.