Cobalt Chloride Induces Macrophage Foam Cell Formation: A Chemical Hypoxia Model for Anti-Atherosclerotic Drug Screening

Abstract

Macrophages would engulf circulating oxidized (ox)- low-density lipoprotein and form lipid droplet-laden foam cells. Macrophage foam cells are considered an important therapeutic target of atherosclerosis. The aim of the study was to investigate a hypoxic foam cell model for anti-atherosclerotic drug screening using the chemical hypoxia-mimicking agent cobalt chloride (CoCl₂). The oil red O stating results showed that treatment with CoCl₂ could induce lipid accumulation and lead to cell transformation to spindle-shaped and lipid-rich foam cells in RAW 264.7 macrophages. Incubation with 150 μM CoCl₂ for 24 h significantly increased the area of intracellular lipid droplets in macrophages, compared with the control group. Our findings indicate that CoCl₂-triggered macrophage foam cells should be a potential in vitro hypoxia model for atherosclerosis drug discovery.

Introduction

Macrophages, a kind of phagocytes, are myeloid immune cells and involved in inflammatory processes of atherosclerosis.¹ Accumulation of cholesterol ester and low-density lipoprotein (LDL) in the intima of arteries would induce monocyte recruitment and differentiation through penetration of dysfunctional endothelium.² The monocytes differentiate into macrophages and express scavenger receptors that mediate the internalization of chylomicron remnant and modified LDL, and consequently become cytoplasmic lipid droplet-laden foam cells.^2
–4 In addition, macropinocytosis in macrophages is the other pathway to modulate uptake of LDL and foam cell formation in atherosclerosis.⁵

Oxidized (Ox)- and minimally modified (mm)-LDLs play an important role in macrophage foam cell formation and subsequent fatty streak formation derived from accumulation of lipid and foam cell debris.⁶ Activation of inflammatory signaling pathways in macrophage foam cells lead to more cell recruitment and LDL oxidation, and promote the progression of the atherosclerotic plaque.²

Macrophage foam cells are reported as a therapeutic target in preventing cardiovascular diseases.⁷ Inhibiting lipid droplet formation in macrophage and inducing removal of foam cells could reduce inflammatory responses and ameliorate atherosclerosis.^7,8 Recent study demonstrates that macrophage foam cell-targeting immunization improved atherosclerotic lesion in the whole aorta and the aortic root with enhanced lesion stability in apolipoprotein E-deficient (ApoE ^−/−) mice.⁸ Regulating macrophage foam cell formation should be related to the progress of atherosclerotic lesions.

Hypoxia, presented in atherosclerotic plaques in humans and animal models, is considered related to the pathogenesis of atherosclerosis.^9,10 Hypoxia would induce the formation of lipid droplets in macrophages and increased local inflammation, and trigger atherosclerotic lesion progression.⁹ It is also demonstrated that chronic hypoxia would accelerate the development of atherosclerosis in ApoE ^−/− mice.¹¹ The hypoxic macrophage foam cells should play a crucial role in the development of atherosclerosis.

Cobalt chloride (CoCl₂) is one of the most commonly used hypoxia models in cell culture, through inducting expression of hypoxia-inducible factors (HIFs)-1α and 2α under normoxic conditions.¹² However, the effect of CoCl₂ on lipid accumulation in macrophages remained unclear. The purpose of this study was to perform a hypoxic macrophage foam cell model for atherosclerosis drug discovery by using the chemical hypoxia agent CoCl₂. We hypothesized that CoCl₂ would induce lipid droplet and foam cell formation in macrophages, as well as in response to low oxygen condition.

Materials and Methods

Cell Culture

The murine macrophage cell line, RAW 264.7, was purchased from the American Type Culture Collection (Manassas, VA) and cultured as described in our earlier study.¹³ RAW 264.7 macrophages were maintained in cell culture medium composed of Dulbecco's modified Eagle medium (Invitrogen Life Technologies, Carlsbad, CA), 10% fetal bovine serum (Invitrogen), 100 unit/mL of penicillin (Invitrogen), and 100 μg/mL of streptomycin (Invitrogen) at 37°C in a humidified incubator with 5% CO₂.

Oil Red O Staining

The protocol of oil Red O staining was described in our earlier study.¹⁴ In brief, oil red O powder (Sigma-Aldrich, St. Louis, MO) was dissolved in 2-propanol (Kanto Chemical Corporation, Tokyo, Japan) as 0.5% solution, and then diluted to 0.3% solution with distilled H₂O. The oil red O solution was filtered through a 0.22 μm filter before use. RAW 264.7 macrophages were seeded on poly-l-lysine solution (Sigma-Aldrich)-coated coverslips in a 6-well plate (3 × 10⁵ cells/well; total 2 mL) overnight. After treatment, the adhered cells on coverslips were washed with phosphate-buffered saline (PBS) briefly, and 1 mL of 0.15% glutaraldehyde (Sigma-Aldrich) was added to each well for 10 min. After washing with PBS three times, the fixed cells were stained with 0.5 mL 0.3% oil red O solution for 5 min. The stained cells on coverslips were washed with PBS for another three times. Finally, the coverslips were mounted and observed by using Nikon epifluorescence microscope (Nikon, Tokyo, Japan). The experiments were repeated three times (n = 3), and the representative data are shown.

Quantification of Lipid Droplets

Quantification of cytoplasmic lipid droplets was measured based on a free and open source software ImageJ (Wayen Rasband, U.S. National Institutes of Health; Bethesda, MD; https://imagej.nih.gov/ij/index.html). At first, open the image file and select “Adjust” from the “Image” menu. Find the option “Color Threshold” and choose “red” in the “Threshold color” dynamic drop-down menu. Modify hue, saturation, brightness, and the other conditions to make sure that selection of intracellular lipid droplets is suitable under the model “Select” and “Filtered.” After correction of lipid droplet selection, turn on “Analyze Particles…” from the “Analyze” menu. Select size, circularity, and the other display options, and then output the data for statistical analysis.

Statistical Analysis

The data are presented as mean ± standard deviation. The statistical significance of differences between groups was analyzed using two-tailed unpaired Student's t-test by Microsoft Office Excel software (Microsoft Corp., Redmond, WA). The differences with a p < 0.05 were considered significant.

Results

CoCl₂ Induced Lipid Droplet Formation in RAW264.7 Macrophages

To investigate the effect of CoCl₂ on foam cell formation in macrophages, RAW264.7 macrophages were incubated with 300 μM CoCl₂ for 24 h and then stained with oil red O. The results showed that treatment with CoCl₂ led to cell transformation to spindle-shaped and accumulation of oil red O-labeled lipid droplets in macrophages (Fig. 1). The results of oil immersion microscopy confirmed that incubation with CoCl₂ increased cytoplasmic lipid droplets surrounding the nucleus of macrophages (Fig. 2). Treatment with 150 and 300 μM CoCl₂ for 24 h both markedly increased the density of intracellular lipid droplets in macrophages (Fig. 3). Our findings suggest that CoCl₂ could induce lipid droplet and foam cell formation in macrophages.

Fig. 1.

CoCl₂ induced lipid accumulation and cell transformation in RAW264.7 macrophages. RAW264.7 macrophages were treated with 300 μM CoCl₂ for 24 h and stained with oil red O. (A) Control group (scale bar = 20 μm). (B) CoCl₂-treated group (scale bar = 20 μm). The dark arrow indicates macrophages foam cells. Color images are available online.

Fig. 2.

Accumulation of intracellular lipid droplets in CoCl₂-treated RAW264.7 macrophages observed by using oil immersion microscopy. RAW264.7 macrophages were treated with 300 μM CoCl₂ for 24 h and stained with oil red O. (A) Control group (scale bar = 20 μm). (B) CoCl₂-treated group (scale bar = 20 μm). The dark arrow indicates macrophages foam cells. Color images are available online.

Fig. 3.

Dose-dependent treatment with CoCl₂ triggered lipid droplet formation in RAW264.7 macrophages. RAW264.7 macrophages were treated with 150 or 300 μM CoCl₂ for 24 h and stained with oil red O. (A) Control group (scale bar = 20 μm). (B) The 300 μM CoCl₂-treated group (scale bar = 20 μm). (C) The 150 μM CoCl₂-treated group (scale bar = 20 μm). Color images are available online.

CoCl₂ Increased the Number and Size of Intracellular Lipid Droplets in RAW264.7 Macrophages

To quantify the effect of CoCl₂ on foam cell formation, the intracellular lipid droplets were selected and measured by digital image processing software ImageJ (Fig. 4). The results of quantification of lipid droplets in Figure 4 showed that treatment with 150 and 300 μM CoCl₂ for 24 h increased almost 40- and 38-folds of total area of cytoplasmic lipid droplets in macrophages, respectively. Incubation with 150 and 300 μM CoCl₂ both significantly elevated the averaged number and area of cytoplasmic lipid droplets compared with the control group, but there were no significant differences between 150 and 300 μM CoCl₂-treated macrophages (Figs. 5 and 6). Moreover, the quantified results showed that treatment with 150 μM CoCl₂ could significantly increase the lipid droplet area in RAW264.7 macrophages compared with the control group (Table 1). It supposes the proatherosclerotic effect of CoCl₂ on lipid droplet formation in macrophages.

Fig. 4.

Selection of cytoplasmic lipid droplets in CoCl₂-induced macrophage foam cells using the ImageJ software. (A and B) Control group. (C and D) The 150 μM CoCl₂-treated group. (E and F) The 300 μM CoCl₂-treated group. Color images are available online.

Fig. 5.

Quantification of lipid droplets in Figure 4 revealed that treatment with CoCl₂ increased the averaged number of cytoplasmic lipid droplets in RAW264.7 macrophages. ***p < 0.001 compared with the control group. ^### p < 0.001 compared with the control group. Color images are available online.

Fig. 6.

Quantification of lipid droplets in Figure 4 revealed that treatment with CoCl₂ increased the averaged area of cytoplasmic lipid droplets in RAW264.7 macrophages. ***p < 0.001 compared with the control group. ^### p < 0.001 compared with the control group. Color images are available online.

Table 1.

Quantification of Cobalt Chloride-Induced Lipid Droplet Accumulation in RAW264.7 Macrophages

Conc.	0 μM				150 μM
Conc.	Total area	Selected LD area	Selected LD area per cell	%	Total area	Selected LD area	Selected LD area per cell	%
Exp. 1	4900654	40	1.48	100	4787347	2240	58.95	3979
Exp. 2	4888279	90	1.88	100	4737459	2454	90.89	4847
Exp. 3	4891023	104	3.71	100	4766815	3779	114.52	3083
Mean ± SD	4893319 ± 6499	78.0 ± 33.6	2.36 ± 1.19	100	4763874 ± 25074	2824.3 ± 833.7^**	88.12 ± 27.89^**	3970

RAW264.7 macrophages were treated with or without 150 μM CoCl₂ for 24 h and stained with oil red O. After quantification, data represent the mean ± SD of three independent experiments. ^** p < 0.01 compared with the control group. CoCl₂, cobalt chloride; Conc., concentration; Exp., experiment; LD, lipid droplet; SD, standard deviation.

Discussion

This study is the first one that demonstrates and quantifies CoCl₂-mediated lipid droplet formation in macrophages. In summary, we found that the chemical hypoxia agent CoCl₂ triggered lipid droplet formation and cell transformation in RAW264.7 macrophages. Treatment with CoCl₂ induced morphologic and physiologic changes in macrophages, and stimulated cell transformed to lipid-rich foam cells. The quantified results showed that incubation of CoCl₂ led to a significant increase of the number and size of cytoplasmic lipid droplets in macrophage foam cells.

Treatment with CoCl₂ triggered cell transformation and lipid droplet formation in M1/M2 RAW264.7 macrophages. It is known that alternative stimulation into M2 macrophages results in increased foam cell formation by inducing scavenger receptor expression.¹⁵ CoCl₂ exposure enhances alternative M2 activation in resting murine J774A.1 macrophages, and remodel the classical M1 phenotype of macrophage activation by changing the balance of inducible nitric oxide synthase, nicotinamide adenine dinucleotide phosphate oxidase 2, and interleukin-6.¹⁶ Moreover, cobalt (II) ions modulate macrophage migration and cytoskeleton organization, and lead to induction of podosome formation and enhancement in macrophage spreading, adhesion, and inhibition of migration, which could induce prolonged immune cell retention and consequent chronic inflammation.¹⁷ Collectively, incubation of CoCl₂ would induce macrophage transformation into foam cells accompanied by accumulation of intracellular lipid droplets and inflammatory response.

CoCl₂-stimulated macrophage foam cells should be a potential hypoxic cell model applied in anti-atherosclerotic drug screening. It is reported that treatment with 100 μM CoCl₂ results in marked expression of HIF-1α protein, a subunit of a heterodimeric transcription factor HIF-1, in the nuclei of RAW264 macrophages.¹⁸ HIF-1 exerts its effect on the pathogenesis of atherosclerosis, and should be a therapeutic target on the disease development.¹⁹ Taken together, these studies imply that not only accumulation of intracellular lipid droplets but also stimulation of HIF-1 expression in CoCl₂-mediated macrophage foam cells have therapeutic values for atherosclerosis drug discovery. In addition, it is investigated that foam cell formation in vivo converts macrophages to a profibrotic phenotype, which could contribute to atherosclerotic plaque stability, especially in early lesions of atherosclerosis that majorly consists of cholesterol-engorged macrophages but not vascular smooth muscle cells.²⁰ The in vivo results suggest that the candidates that could attenuate CoCl₂-induced macrophage foam cells may prevent the progression of atherosclerosis.

CoCl₂-mediated macrophage foam cell formation is similar to that induced by ox-LDL as shown in our previous study.¹⁴ It is reported that hypoxia would contribute markedly to lipid metabolism in atherosclerotic lesions.²¹ Hypoxia enhances lipid uptake into RAW 264.7 macrophages and regulates the expression of ox-LDL receptors, which may affect lipid droplet formation in macrophages.²² In THP-1 cell line-derived and human monocyte-differentiated macrophages, hypoxia causes an accumulation of triglyceride-containing cytosolic lipid droplets and conversion of macrophages into foam cells.^23,24 Theses results suppose that macrophages may accumulate oil red O-stained lipid droplets containing triacylglycerol in response to CoCl₂ exposure. However, it is demonstrated that incubation of a small volume of Watanabe heritable hyperlipidemic rabbit serum could induce foam cell formation with an increase of cellular content of cholesteryl ester in J774A.1 macrophages.²⁵ It suggests that CoCl₂-mediated lipid accumulation may derive from supplement of serum in the cell culture medium. The components of lipid droplets in CoCl₂-induced macrophage foam cells need to be further investigated.

There are several studies investigating the regulatory mechanism of foam cell formation in macrophages. It is reported that peroxisome proliferator-activated receptor (PPAR)-α and γ agonists, but not the PPAR-β agonist, inhibit macrophage foam cell formation in vivo through distinct ATP-binding cassette subfamily A member 1-independent pathways.²⁶ On the contrary, cluster of differentiation (CD) 146, expressed on the macrophages in human and mouse atheroma, could trigger macrophage activation by driving the internalization of scavenger receptor CD36 during lipid uptake, subsequently promote macrophage foam cell formation and retention during atherosclerosis.²⁷

Our findings suggest that CoCl₂-induced macrophage foam cells should be as a surrogate target for rapid screening of anti-atherosclerotic effects in response to small molecule drugs. The in vitro hypoxic macrophage-derived foam cell model combined with high content screening technology may provide more probability on atherosclerosis drug discovery.

Footnotes

Acknowledgment

The authors thank Prof. Shwu-Fen Chang for offering RAW 264.7 macrophage cell lines.

Disclosure Statement

No competing financial interests exist.

Funding Information

This work was partly funded by a grant from Putian University (Grant No. 2019114).

ABBREVIATIONS USED

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