The in vitro effect of myeloperoxidase oxidized LDL on THP-1 derived macrophages

Abstract

Cardiovascular diseases (CVDs) linked to atherosclerosis remains the leading cause of death worldwide. Atherosclerosis is primarily caused by the accumulation of oxidized forms of low density lipoprotein (LDL) in macrophages (MΦs) in the subendothelial layer of arteries leading to foam cell and fatty streak formation. Many studies suggest that LDL that is modified by myeloperoxidase (MPO) is a key player in the development of atherosclerosis. MΦs can adopt a variety of functional phenotypes that include mainly the proinflammatory M1 and the anti-inflammatory M2 MΦ phenotypes which are both implicated in the process of atherogenesis. In fact, MΦs that reside in atherosclerostic lesions were shown to express a variety of phenotypes ranging between the M1- and M2 MΦ types. Recently, we pointed out the involvement of MPO oxidized-LDL (Mox-LDL) in increasing inflammation in MΦs by reducing their secretion of IL-10. Since little is known about Mox-LDL-mediated pro-atherosclerostic responses in MΦs, our study aimed at analyzing the in vitro effects of Mox-LDL at this level through making use of the well-established model of human THP-1-derived Mφs. Our results demonstrate that Mox-LDL has no effect on apoptosis, reactive oxygen species (ROS) generation and cell death in our cell model; yet, interestingly, our results show that Mox-LDL is significantly engulfed at a higher rate in the different MΦ subtypes supporting its key role in foam cell formation during the progression of the disease as well as previous data that were generated using another primary MΦ cell model of atherosclerosis.

Keywords: Atherosclerosis; Mox-LDL; THP-1; foam cells; macrophages.

Figure 1.

Effect of Mox-LDL treatment on TNF-α release by THP-1-derived M0, M1 and M2-MΦs. TNF-α levels were assayed by ELISA in the culture supernatants of untreated and Mox-LDL-treated (at a concentration of 100 μg/ml for 24 h) M0-, M1-, M2-Mφs. Bar graphs presenting mean values of TNF-α levels (of 3 independent experiments n = 3; with each condition performed in duplicate). Error bars represent SEM.

Figure 2.

Effect of Mox-LDL treatment on ROS generation in THP-1 derived M0, M1 and M2-MΦs. Different THP-1-derived MΦ types were cultured in 6-well plates and left untreated or treated with 100 µg/ml of Mox-LDL for 24 h. Cells were then harvested, labeled with H2DCFDA and analyzed via flow cytometry. Bar graph presenting the geometric MFI values (mean ± SEM) of DCF-stained MΦs (of 3 independent experiments n = 3; with each condition performed in duplicate). MFI, mean fluorescent intensity; H2DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; DCF, 2,7-dichlorofluorescein.

Figure 3.

Effect of Mox-LDL treatment on apoptosis and viability in M0, M1, and M2 MΦs. Different MΦ types were cultured in 6-well plates and left untreated or treated with 100 µg/ml of Mox-LDL for 24 h. Apoptosis was then assessed by Annexin V/ PI apoptosis assay followed by flow cytometric analysis. (a) Representative flow cytometry dot plots and (b) bar graphs representing the % (mean ± SEM) of viable, early apoptotic, late apoptotic and dead subpopulations with untreated and Mox-LDL-treated THP-1 derived MΦ types (of 4 independent experiments n = 4; with each condition performed in duplicate).

Figure 4.

Effect of Mox-LDL treatment on lipid uptake in THP-1-derived MΦ types: M0, M1 and M2. Different THP-1 derived MΦ types were cultured in 6-well plates and left untreated or treated with 100 µg/ml of either native LDL or Mox-LDL for 24 h. Cells were then harvested and lipid uptake was assessed by Oil Red O staining and quantified via spectrophotometry analysis at 492 nm. Bar graph presenting the OD values (mean ± SEM) of Oil Red O stained MΦs (of 3 independent experiments n = 3; with each condition performed in duplicate). One-way ANOVA followed by Tukey's multiple comparison post hoc test was used to calculate statistical significance. * P < 0.05; ** P < 0.01.