Phenylarsine oxide inhibits the accumulation of lipid droplets in macrophages

Intracellular accumulation of esterified cholesterol-containing lipid droplets, which leads to the formation of foam cells associated with related inflammatory reactions, is the core pathological event in atherosclerosis [ 1, 2] . Cardiovascular and cerebrovascular diseases caused by atherosclerosis remain the leading causes of human death and disability after decades of clinical application of highly effective lipid-lowering treatment [ 3, 4] . Clearly, lowering lipids alone or even in combination with some potential anti-inflammation resolution is far from enough to prevent and treat atherosclerosis-induced vascular disease, demanding new approaches and/or therapeutic targets for anti-atherosclerosis.

Three types of cells are involved in the occurrence of atherosclerosis, i.e, endothelial cells, macrophages, and arterial medial smooth muscle cells. However, the most critical processes in pathogenesis are mediated by macrophages. The formation of foam cells is a cornerstone during the development of atherosclerosis [5]. Because of increased lipid uptake and cholesterol esterification and insufficient cholesterol efflux, excessive cholesteryl ester can accumulate in macrophages [6]. When the cholesterol ester in macrophages exceeds 50% of the total cholesterol, macrophages turn into macrophage-derived foam cells. Intracellular degradation of lipids, including esterified cholesterol, involves lipid autophagy (lipophagy) which is impaired in atherosclerosis [ 7, 8] , and an increase in autophagy-lysosomal biogenesis reduces atherosclerosis [8]. Interestingly, our previous study showed that both genetic and chemical (phenylarsine oxide, PAO) inhibition of phosphatidylinositol-4 kinase type III alpha (PI4KIIIα) can upregulate autophagy and restore autophagic flux [9].

To investigate whether inhibition of PI4KIIIa with PAO can reduce the accumulation of or facilitate the degradation of cholesterol ester-containing lipid droplets in foam cells, THP-1 cells were cultured in RPMI 1640 (Gibco, Carlsbad, USA) supplemented with 10% FBS (Gibco) and then differentiated into macrophage cells by using 100 nM phorbol 12-myristate 13-acetate (PMA, Sigma–Aldrich, St Louis, USA) for 72 h. The THP-1-derived macrophage cells were cultured in serum-free medium without (Control) or with oxidized low-density lipoprotein cholesterol (ox-LDL) for 24 h and then cultured in ox-LDL-containing complete medium without (Model) or with PAO (Nuo-Beta Pharmaceutical Technology, Shanghai, China) at various concentrations (PAO-treated groups) for 48 h, except that control cells were cultured in pure complete medium. After the treatment, cells in each group were washed once with PBS, fixed with 4% paraformaldehyde for 30 min, and washed twice with PBS. The intracellular lipid droplets were detected using an Oil red O (ORO) staining kit (Beyotime, Beijing, China) according to the manufacturer’s instructions. As shown in Figure 1A, the ORO staining in model cells was much stronger than that in control cells, while the ORO staining in PAO-treated cells was significantly reduced in a dose-dependent manner and even became weaker than that in control cells when the PAO concentration was 60 nM.

Figure 1 .

Effects of PAO on ox-LDL-induced intracellular lipid droplets and internalized Dil-ox-LDL in macrophage cells

(A) Representative images showing the ORO staining of lipid droplets in ox-LDL-treated macrophage cells without and with PAO co-treatment at various concentrations. Scale bar: 100 μm. (B) Representative confocal imaging of internalized Dil-ox-LDL in macrophage cells without and with PAO treatment at various concentrations for 1 h. Magnification fold: 100×. Experiments in both (A) and (B) were repeated independently at least three times. In both (A) and (B), either ox-LDL or Dil-ox-LDL was included in the culture medium in all groups except the control group.

To investigate whether the PAO-produced reduction in intracellular lipid droplets could be ascribed to a change in the processing of internalized ox-LDL, macrophage cells were first cultured in serum-free medium containing 40 μg/mL Dil-ox-LDL (Guangzhou Yiyuan, Guangzhou, China) for 6 h and then treated without or with PAO at different concentrations for 1 h. After the treatment, cells in each group were washed twice with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature, washed twice with PBS, stained with DAPI at room temperature for 10 min, and then washed twice with PBS again before imaging. Intracellular fluorescence intensity was detected using a laser confocal microscope. Figure 1B showed that PAO markedly reduced intracellular Dil-ox-LDL in a dose-dependent manner. Therefore, PAO reduced intracellular ox-LDL or cholesterol ester accumulation by a mechanism subsequent to ox-LDL internalization.

To test whether the effect of PAO on intracellular cholesterol esters is associated with an upregulation of autophagy, the expression level of LC3-II, a core mediator and the most widely used biomarker of autophagy activity, was examined in the macrophage cells in the control group and PAO-treated group by western blot analysis and fluorescent immunostaining. THP-1 cells were lysed in RIPA buffer (Beyotime) supplemented with 1 mM PMSF (Beyotime) and centrifuged at 14,000 g for 10 min at 4°C, and the resultant supernatants were collected and subjected to western blot analysis using primary antibodies against LC3 (1:1000 dilution; CST, Beverly, USA) and GAPDH (1:1000 dilution; Beyotime) and an HRP-conjugated secondary antibody (1:1000 dilution; Beyotime). For immunofluorescent staining, THP-1 cells were washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 30 min, washed twice with PBS again, followed by incubation with anti-LC3 antibody (1:100 dilution; CST) overnight and then incubated with Alexa Fluor 488 secondary antibody (Beyotime) for 1 h. PBS was used to wash the cells three times. Finally, the cells were stained with DAPI (Beyotime). Images were captured using a confocal microscope. Indeed, PAO moderately and significantly increased LC3-II level in a dose-dependent manner, which was visualized by both immunoblotting and immunostaining ( Figure 2A–C).

Figure 2 .

PAO upregulates autophagy and blockage of autophagy abolishes the effect of PAO on ox-LDL-induced accumulation of lipid droplets in macrophage

(A) Representative images of western blots showing LC3II expression. (B) Densitometry values of the western blots were normalized to GAPDH expression and presented as the relative intensity. Data are presented as the mean±SEM and the statistical significance of the differences was evaluated by Student’s t-test using SPSS 23.0 (n=3, * P<0.05). (C) Cells were fixed and incubated with anti-LC3II antibody. The images were captured with a confocal microscope. The presented images were from at least three independent experiments. Magnification fold: 200×. (D) Representative images showing the ORO staining of lipid droplets in macrophage cells without (control) and with ox-LDL-treatment in the absence (Model) and presence of PAO alone (60 nM PAO) or PAO and 3-MA together (3-MA). Scale bar: 100 μm. In (A‒D), ox-LDL was included in the culture medium in all groups except the control group.

Since autophagy is involved in the degradation of intracellular cholesterol esters, we further explored the possibility that PAO-induced upregulation of autophagy contributes to the PAO-induced reduction of cholesterol esters in macrophages by combined use of PAO and 3-methyladenine (3-MA; MCE, Shanghai, China), a selective inhibitor of Vps34 required for autophagy initiation. The results showed that combination of PAO and 5 mM 3-MA abolished the effect of PAO (60 nM) on ORO staining in ox-LDL-treated macrophage cells ( Figure 2D).

In summary, PAO, as a selective inhibitor of PI4KIIIα, dramatically reduces cholesterol ester accumulation in macrophage cells at low concentrations possibly via the upregulation of autophagy-related processing of ox-LDL. This study suggests that PI4KIIIα and its inhibitors are potential new therapeutic targets and drugs, respectively, for preventing and treating atherosclerosis and atherosclerosis-induced diseases.

COMPETING INTERESTS

The authors declare that they have no conflict of interest.

Funding Statement

This work was supported by the grants from the National Natural Science Foundation of China (Nos. 81771416 and 8197100).