Inflammation plays an important role in all stages of atherosclerosis development. Therefore, the use of anti-inflammatory drugs could reduce the risk of major adverse cardiovascular events due to atherosclerosis. Herein, we explored the capacity of fluocinolone acetonide (FA), a glucocorticoid (GC), in modulating foam cell formation and response. Human THP-1 derived foam cells were produced using 100
Atherosclerosis is a chronic artery inflammatory disease, which can lead to fatal acute myocardial infarctions, sudden coronary deaths, and ischemic stroke [
Due to the significant role that inflammatory processes play in the progression of atherosclerosis, several anti-inflammatory drugs that aim to reduce the risk of major adverse cardiovascular events are now being investigated [
Dexamethasone (Dex) is the most common GC used in atherosclerotic treatment. It was shown that Dex at 1 mg/kg daily for 7 days after balloon arterial injury resulted in reduced macrophage accumulation by 96% and 77%, respectively, in the tunica intima and tunica media of arteries in cholesterol fed rabbits [
Fluocinolone acetonide (FA) is a GC primarily used to reduce skin inflammation and relieve itching [
Human monocytic THP-1 cells (ATCC® TIB202™, USA) were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium (ATCC modification; Life Technologies, USA) containing 0.05 mM 2-mercaptoethanol (Sigma Aldrich, USA), 1% Penicillin-Streptomycin Solution (Pan Biotech, Germany), and 10% fetal bovine serum (FBS, Pan Biotech, Germany). The medium was replaced every 2–3 days by centrifuging these suspended cells at 125 g for 7 min. Cultures were maintained in a humidified incubator at 37°C with 5% CO2 and subcultured once cell concentration reaches 8 × 105 cells/mL. THP-1 cells at passages 6–8 were used for all experiments. The monocytes were seeded at a density of 2 × 105 cells/well in 24-well tissue culture plates (TPP, Switzerland) in a differentiation medium, growth medium supplemented with 50 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma Aldrich, USA), for 3 days to obtain macrophages. This differentiation medium contained either 1% or 10% FBS depending on experiments as described in the following sections and are referred to as DM-01 and DM-10, respectively. Subsequently, the macrophages were fed with 100
To understand the role of FBS and OxLDL in foam cell formation, the THP-1 monocytes were suspended in DM-01 or DM-10 for 3 days. The resulting macrophages were then refreshed with the differentiation medium containing the same FBS concentration in the presence or absence of 100
The THP-1 cells were differentiated in DM-10 for 3 days before treatment with DM-10 containing 100
A three-dimensional (3D) foam cell spheroid model was developed using the hanging droplet culture method to further evaluate the efficacy of FA and Dex. After foam cells were formed in DM-10 in a T-75 flask as described above, trypsin (Gibco, USA) was used to detach the cells. The foam cells were then centrifuged at 250 g for 5 min and resuspended to 1.25 × 106 cells/mL. Subsequently, the cells were seeded into Perfecta3D® 96-well hanging drop plate (3D Biomatrix™, USA) and incubated for 4 days at 37°C with 5% CO2. Spheroids formed in this plate were harvested into Corning® Costar® Ultra-Low Attachment well plate (Sigma Aldrich, USA) for subsequent treatments.
The harvested foam cell spheroids were incubated in DM-10 containing either FA or Dex at 0.1 and 1
ORO staining was used to detect the presence of lipid vacuoles in the cells. Details of this assay and the following assays are described in the Supplementary Information. Lipid droplets inside the cells were stained red and observed using an inverted optical microscope (IX53, Olympus, Japan). To quantify the number of cells stained with ORO, 25 images at 400X magnification were taken for each well and cell counting was done manually. The percentage of ORO-stained cells per well was calculated. All the samples were done in triplicate. A cell was considered positively ORO-stained, and therefore a foam cell, when intracellular lipid droplets occupied at least 10% of the cytoplasm space, based on visual inspection by an experienced individual.
To measure OxLDL uptake, cells were cultured on 15 mm glass cover slips (Chemglass Life Sciences, USA) in 24-well tissue culture plates. After fixation, the cells were stained with Rabbit anti-LDL (Copper oxidized) primary antibody (Abcam, United Kingdom) and Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate (Life Technologies, USA), and subsequently mounted with ProLong® Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies, USA). The images were observed and captured using a confocal laser scanning microscope (Leica TCS SP5, Leica Microsystems, Germany).
Cell proliferation was evaluated by quantifying double-stranded DNA (dsDNA) in cell lysates using Quant-iT™ PicoGreen® dsDNA kit (Life Technologies, USA). The amount of dsDNA in the sample was quantified against a calibration curve obtained from dsDNA standards provided in the kit.
CE accumulation in the cells was quantified using Cholesterol/Cholesteryl Ester Quantitation Assay kit (Abcam, United Kingdom). Total and free cholesterol values were calculated against a calibration curve obtained from the supplied cholesterol standards. The amount of CE was determined by subtracting the amount of free cholesterol from the total, and the results were normalized to the dsDNA amount.
To detect secreted cytokines, cell culture supernates were collected and examined using a cytokine array (Proteome Profiler™ Human XL Cytokine Array Kit, R&D Systems, USA) according to the manufacturer’s instructions. Signal intensities were quantified using the ImageQuant software and normalized to the untreated samples.
All the experiments were performed in triplicates, and quantitative results were expressed as mean ± standard deviation (SD). Data were analysed by One-Way and Two-Way ANOVA with post hoc Tukey’s analysis using Origin Pro 2015 (OriginLab, USA). Data presented were representative of 2 or 3 independent experiments. A
The effects of FBS and OxLDL on foam cell formation were characterized using ORO staining as shown in Figure
Different FA/Dex concentrations, 0.1–50
Cell culture supernatants of different samples (1
Chemiluminescent images
Inflammation
Lipid accumulation
Foam cell spheroids of 529 ± 56
LDL uptake by monocyte-derived macrophages is one of the earliest pathogenic events in the nascent plaque. In our study, 5% and 12% of the macrophages were converted to foam cells in DM-01 and DM-10 media, respectively, in the absence of OxLDL (Figure
In the presence of 100
Understanding the uptake of LDL and OxLDL by macrophages and subsequent inflammatory response and lipid droplet accumulation in foam cells are essential to explore the capacity of GCs or other drugs to stop or even reverse the process. Figures
Dex is the most commonly proposed GC for atherosclerotic therapy [
In terms of cytokines release, FA and Dex demonstrated tremendous efficacy in suppressing atherosclerotic inflammation compared to the untreated group (Figure
FA and Dex also revealed their significant importance in decreasing lipid accumulation compared to the control (Figure
The molecular weights of FA and Dex are 452.49 and 392.46 g/mol, respectively. The distribution coefficient, log D, is a measure of the lipophilicity of a drug. It is defined as the ratio between a drug concentration in the organic phase to its concentration in the aqueous phase, at equilibrium of the two immiscible phases, and at an appropriate pH. This value is thus an indirect indication of the solubility of the drug in the membrane and also an indication of the drug’s permeability through lipid bilayers. The log D values of FA and Dex at pH 7.4 are 2.56 and 1.95, respectively [
Although many advantages of GCs have been suggested for atherosclerotic treatment, systemic GC treatment has not been adopted clinically because of side effects such as central obesity, insulin resistance, hyperglycaemia, dyslipidaemia, and hypertension [
Foam cell accumulation is a key event in the early stages of atherosclerotic plaque development. The formation of THP-1 derived foam cells was studied in the absence or presence of OxLDL. Without OxLDL, native LDL present in serum-supplemented culture media could induce a small amount of foam cell formation. With 100
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
This study was supported by the NTU-Northwestern Institute for Nanomedicine (M4081503.F40).
Supplementary Materials are provided in .docx file.